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Effects on fertility

Description of key information

DRF study according to OECD 421


The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development (LPT, 2020). Furthermore, this study was used as dose range finding study for the OECD 443 study (LPT, 2021). The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day. In the high dose group, mortality was observed (two females died at GD 20 or GD 21, respectively). Furthermore, clinical signs, i.e. salivation, reduced motility and yellow discoloured urine were noted in both sexes at intermediate and/or high dose levels. However, no influence was noted on the fertility of the female animals (oestrus cycle, fertility index, gestation index, pre-coital time and gestation length) in any of the dose groups. 


For general toxicity the NOAEL was considered to be 100 mg/ kg b.w./ day, based on clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day.


EOGRTS study according to OECD 443


In a final decision on a compliance check dated 21 December 2018 (Decision number CCH-D-2114453561-52-01/F) ECHA requested to perform an extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:


- At least two weeks premating exposure duration for the parental (P0) generation;


-  Dose level setting shall aim to induce some toxicity at the highest dose level;


-  Cohort 1A (Reproductive toxicity);


-  Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;


-  Cohorts 2A and 2B (Developmental neurotoxicity).


 


Based on the concern regarding reproductive and developmental toxicity as found in juvenile male rats in the Pubertal Development and Thyroid function assay (Marty et al., 2011), this OECD 443 study aimed to provide additional information on sexual function and fertility in P0 and F1 Generation and on development and toxicity of the offspring from birth until adulthood due to pre- and postnatal and adult exposure in the F1 generation.


In contrast to the observed effects in Marty et al., 2011, in the present extended one-generation reproductivity study, the seminal vesicle weight, the serum level of testosterone and the preputial separation were not affected in the male animals of the F1 Generation by the extended treatment. For vaginal opening, a statistically significant delay was noted in the high dose group (600 mg test item/kg b.w./day) when considering all F1 females of Cohorts 1A, 1B and 2A combined.


However, the mean body weight of the juvenile rats at the time point of vaginal opening was also reduced (not significant) when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment.


Therefore, no delay in sexual maturation was considered and no other test item- induced effect was found indicating reproductive and developmental toxicity.


Taken all findings together, a primary affectation of the endocrine system was therefore excluded. Only the increased liver weights and the centrilobular hepatocellular hypertrophy were signs for treatment-related hepatic enzyme induction which subsequently leads to adverse effects by exceeding the maximal tolerated dose (Hall et al., 2012), as observed in the Pubertal Development Assay (Marty et al., 2011) and the carcinogenicity study (NTP, 1986).    


The No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the F0 and in the adult animals of Cohort 1A, 1B and 2A was determined to be 65 mg/kg/day based on the reduced body weight, increased organ weights, histopathological changes, as well as the biochemical and urine changes in the high and/or intermediate dose group.


The No Observed Adverse Effect Level (NOAEL) for reproduction was considered to be above 600 mg test item/kg/day in F0 and F1 Generation Cohort 1B.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June 2019 to October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This OECD 421 study is used as dose range finding study for the OECD 443 study (LPT, 2021).
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
This OECD 421 study is used as dose range finding study for the OECD 443 study (LPT, 2021).
Species:
rat
Strain:
other: CD/ Crl:CD(SD)
Details on species / strain selection:
For this study CD rats bred by Charles River Laboratories Germany GmbH were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age: Group 1 to 4: 76 days, Group 5: 71 days
- Body weight (at 1st administration): Males: 426.9 g - 459.2 g, Females: 257.0 g - 277.4 g
- Housing: With the exception of the mating period (see Section 3.1.10 'Mating procedure'),
the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx.18 cm
The animals were placed in the animal room as follows:
Male animals: On one side of the room with an empty row between groups.
Female animals: On the other side of the room with an empty row between groups.
- Diet (ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Air changes per hour: 15 to 20
- Photoperiod: 12 hours dark/12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item formulations were prepared for up to seven (7) administration days in advance and were administered orally at a constant administration volume/kg b.w. once daily. The test item was diluted in the vehicle to the appropriate concentrations. Between administrations, the test item formulations were stored at 2°C to 8°C. Prior to each administration, the test item formulations were allowed to warm up to room temperature for at least 30 minutes and stirred on a magnetic stirrer for approximately 3 minutes to ensure homogeneity. The amount of test item was adjusted the animals current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
Details on mating procedure:
Sexually mature male and female rats were paired monogamously: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or two weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD 0) was considered to be the day on which sperm was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, two (2) aliquots of approximately 3 mL each were taken at the following times and stored at -20°C ± 10% until analysis at LPT:
- At start of the treatment period (first dosing day): Analysis of concentration and homogeneity,
- At start of administration, during (middle) administration and before administration to the
last animal of each dose level group (3 samples / dose level group; groups 2, 3, 4). Number of samples: 9 (18 aliquots)
- At start of the treatment period of group 5 (first dosing day): Analysis of concentration and homogeneity At start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples / dose level group; group 5). Number of samples: 3 (6 aliquots)
Towards the end of the treatment period (when the majority of animals was dosed):
Analysis of concentration During treatment always before administration to the last animal/dose level group (1 sample / dose level group; groups 2, 3, 5). Number of samples: 3 (6 aliquots)
Sum of all samples: 15 (30 aliquots)
The samples were labelled with the study number, species, type of sample, concentration, aliquot number, test day, sampling time and date.
The samples were analysed at LPT using a validated HPLC-UV method. The following parameters were determined: Linearity, Accuracy, Precision, Sensitivity, Specificity, Stability. The test item formulation analysis for the concentration and homogeneity revealed concentrations that ranged between 102.4% and 109.7% of the nominal concentration indicating correctly prepared and homogeneous test item formulations.
Duration of treatment / exposure:
The study animals were treated during the following periods:
Males: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 33 at maximum) and during the post-mating period until test day 43 (one day before sacrifice on test day 44).
Females: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 41 at maximum) and during the lactation period until test day 64 to 68 (corresponding to lactation day 13). The last dosing was always one day before
sacrifice.
Frequency of treatment:
Once daily
Details on study schedule:
- Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study
- Prior to the start of treatment and at least once daily, all animals were observed for signs of functional/behavioural toxicity. Oestrus cyclicity is evaluated followed by daily monitoring of vaginal smears from beginning of treatment until evidence of mating
- On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period. After counting on PND 4, the litters were adjusted to 10 pups per litter (if possible, 5 pups per sex and litter) by eliminating (culling) surplus pups using a randomization scheme
-The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males: On test day 44
Pups: On lactation day 13
Dams (surviving dams): Between lactation day 14 and 16
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose group (this dose was
terminated after 3 treatments (males) or 1
treatment (females))
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities. The dose levels of this study were selected by the Sponsor based on available toxicological data. In the 90-day and the 16-day gavage studies with the test item, the NOAEL was considered to be 500 mg/kg b.w. (both NTP, 1986). In the 16-day study, at the high dose of 1000 mg/kg b.w., only decreased body weights (13.7% for males, 6.7% for females) were observed. One female rat that received 1000 mg/kg b.w. died in the 90-day gavage study. Final mean body weights for rats were not clearly related to dose in this study. Therefore, 1000 mg/kg b.w. was selected as
the highest dose level in this OECD 421 study.

At 1000 mg test item/kg b.w./day, the female animal no. 73 was found dead after the first administration. Hence, the high dose group (1000 mg test item/kg b.w./day) was terminated immediately for the females and after the third administration for the males and 750 mg test item/kg b.w./day was selected as the new high dose level.

Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals that died prematurely were necropsied as soon as possible after exitus. In the case of prematurely sacrificed animals, blood sampling for Thyroid hormone determination was performed.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness. Any signs of illness or reaction to treatment were recorded for each individual animal. Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset,intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

MORTALITY
Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.

BODY WEIGHT
The adult animals were weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights were recorded. The report includes weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
For the female animals the body weights on the following days are given in the report:
Pre-mating period :Test days 15, 22, 29
Gestation period:Gestation days 0, 7, 14, 20
Lactation period: Lactation days 1, 4, 13

FOOD AND DRINKING WATER CONSUMPTION
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined.
Food residue (or total food left) was weighed and recorded as follows:
Study period Males Females
Pre-mating period TD 22 and TD 29 TD 22 and TD 29
Mating period None None
Gestation period Not applicable GD 7, GD 14 and GD 20
Lactation period Not applicable LD 1, LD 8 and LD 13

Drinking water consumption was monitored daily by visual appraisal throughout
the study.

REPRODUCTIVE PARAMETERS
Pre-coital time and gestation length
- number of pregnant females
- duration of pre-coital time
- gestation length
(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore).
Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

T4 DETERMINATION
Blood samples were taken under isoflurane anaesthesia and processed for serum always at the same time of the day, if possible, as scheduled below. Blood sampling was performed randomised from the parental animals.
Oestrous cyclicity (parental animals):
During the 14-day pre-exposure period (TD 1 to TD 14), the oestrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a regular oestrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomisation process on test day 14 that was performed to allocate the female animals to the test groups of the main study. The female animals that were used for the main study revealed 2 or 3 typical 4 to
5 day cycles during the 14-day pre-exposure period. After the allocation of the animals to the study groups and the start of treatment, the oestrus cycle was further monitored during the pre-mating period and the mating period until a positive mating sign was noted. Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy.
Sperm parameters (parental animals):
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of all males of groups 1 and 5.
Litter observations:
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups. Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.
Number of pups absolute
- at birth (alive and dead)
- after 4 and 13 days of life
Number of pups per dam
- at birth (alive and dead)
- after 4 and 13 days of life
Number of male and female pups
- at birth
- after 4 and 13 days of life
Number of stillbirths
- absolute
- per dam
Number of pups with malformations
- absolute
- per dam

- Ano-genital distance:
On post-natal day 4 before litter adjustment, the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalized to the pup size (i.e. the cube root of body weight).

- Litter adjustment on PND 4:
After counting on PND 4, the litters were adjusted to 10 pups per litter (if possible, 5 pups per sex and litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.

- Blood sampling for thyroid hormone (T4) determination
On PND 4 and on PND 13, blood samples for T4 hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection.
Blood samples were taken under isoflurane anaesthesia and processed for serum always at the same time of the day, if possible, as scheduled below. For the blood withdrawal of the pups, the litter sequence was determined by randomization of the dams; pups were selected from the dams in ascending order: On LD 4, the first male and female pup per dam selected for culling were used, if possible. On LD 13, the first male and female pup per dam were used.

- Male nipples counting
Nipples were counted in all male pups on PND 13 (shortly before scheduled
sacrifice).
Postmortem examinations (parental animals):
GROSS NECROPSY
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males: On test day 44
Dams (surviving dams): Between lactation day 14 and 16

DISSECTION OF ADULT ANIMALS
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. During necropsy, the number of implantation sites was recorded in the female animals. Apparently, non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulphide for about 10 minutes to stain possible implantation sites in the
endometrium according to SALEWSKI.
The organ weights below of all animals were determined before fixation. Paired organs were weighed individually and identified as left or right.
Organs to be weighed - adult animals: Epididymis (2), Thyroid (1) (including parathyroids), Ovary (2), Prostate and seminal vesicles with coagulating glands, as a whole, Testicle (2), Uterus including cervix

The organs or parts thereof of all adult animals were preserved in the appropriate fixative:
Fixative: modified Davison's solution
-Epididymis (2)
-Testicle (2)

Fixative: 7% neutral buffered formalin
- Gross lesions observed
- Ovary and oviduct (2)
- Prostate
- Seminal vesicles with coagulating glands
- Thyroid including parathyroids
- Uterus with cervix

HISTOPATHOLOGY
Histopathological examination was performed on the organs listed below of all adult animals of the control group and the high dose group (groups 1 and 5).
Sections to be prepared and evaluated - groups 1 and 5 adults:
Organ HE-stained sections PAS-stained sections
Epididymis 2 (left and right) 1
Ovary 2 (left and right) none
Testicle 2 (left and right) 1
The histotechnique was performed at LPT.
Histopathological evaluation was performed by AnaPath GmbH, AnaPath Services according to all relevant AnaPath SOPs.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of all males of groups 1 and 5.
Postmortem examinations (offspring):
Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
The thyroid of 1 male and 1 female pup from each litter was preserved in 7% formalin. Thyroid weight was determined after fixation.
Statistics:
The statistical evaluation of the parametrical values was done by Provantis(Provantis® integrated preclinical software, version 10.2.1.0, Instem LSS Ltd) using the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT's test (p ≤ 0.01 and p ≤ 0.05).

The following statistical methods were used for data not captured by Provantis software (reproductive data): Homogeneity of variances was test using the BARTLETT's test. In case of homogeneity, intergroup comparison was performed by the DUNNETT's test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group was performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
Statistically significantly different data are indicated in the summary tables and
in the tables of the result sections.
Reproductive indices:
The following indices were calculated for each group:
Female Fertility Index [%] = (Number of pregnant rats/ Number of rats used) x 100
The female fertility index reflects the total number of dams that had achieved
pregnancy, including dams, which delivered at term, aborted or had fully resorbed
litters.
Gestation Index [%] = (Number of dams with live pups/ Number of pregnant rats) x 100
For each litter and group the following indices were determined:
Birth Index [%] = (Total number of pups born (alive + dead)/ Number of implantation scars) x 100
Live Birth Index [%] = (Number of pups alive on day 0/1 of lactation/ Total number of pups (alive + dead)) x 100
Viability Index [%] pre-cull = (Number of pups alive on day 4 (pre cull)/ Number of pups alive on day 0/1) x 100
Viability Index [%] post cull = (Number of pups alive on day 13/ Number of pups alive on day 4 (post cull)) x 100
Post-implantation loss [%] = (Implantations - living fetuses/ Implantations) x 100
Offspring viability indices:
Birth and live birth index, post-impantation loss and viability indices of the pups
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Males:
In the control group, the male animal no. 2 was noted with a haemorrhagic canthus on TD 27 and TD 28. This observation was considered to be spontaneous.
In the low dose group (100 mg test item/kg b.w./day), slight salivation was noted for 5 male animals on 5 (no. 27), 2 (no. 25) or 1 (nos. 22, 23 and 24) test days between TD 27 and TD 43. Salivation started immediately to 5 minutes after administration and lasted until 20 to 60 minutes after administration. Due to the low severity, the low number of days observed and the short duration salivation was considered to be not test item-related.
At 300 mg test item/kg b.w./day, slight to moderate salivation, slightly reduced motility, a haemorrhagic nose/snout were noted as well as yellow discoloured faeces and urine.
As salivation, reduced motility and the discolouration of the urine was noted inalmost all animals of the intermediate dose group and were noted with increased incidence, severity and number of days in the high dose group, these clinical signs were considered to be test item-related.
In the high dose group (750 mg test item/kg b.w./day), slight to severe salivation, moderately reduced motility, yellow discoloured urine and pale faeces were noted for the male animals,
Salivation, reduced motility and yellow discoloured urine were noted in almost all male high dose animals with an increased severity for salivation and reduced motility in comparison to the intermediate dose group. As also for reduced motility and the discolouration of the urine the number of observed days per animal was increased compared to the intermediate dose group, salivation, reduced motility and the yellow discoloured urine were considered to be test item-related.
The male animals of group 4 (1000 mg test item/kg b.w./day) displayed moderate salivation, moderately reduced motility on TD 16 and severe salivation and severely reduced motility on TD 17 and also yellow discoloured urine was noted on TD 16 and 17 for all male animals. Additionally, the male animal no. 65 was noted with a haemorrhagic nose/snout.
Group 4 was terminated for the male animals on TD 18 due to pronounced signs of toxicity in form of moderate to sevee salivation and moderately to severel reduced motility and on TD 16 for the female animals due to one premature death of one female animal for animal welfare reasons.
- Start and duration of clinical signs (Males):
At 300 mg test item/kg b.w./day, salivation started immediately to 5 minutes after administration and ended between 20 and 60 minutes after administration. Reduced motility started immediately to 5 minutes after administration and ended 20 to 60 minutes after administration or 2 to 6 hours after administration. For the animals of the high dose group (750 mg test item/kg b.w./day), salivation was observed immediately to 5 minutes after administration and ended 20 to 60 minutes after administration. Reduced motility started 5 to 20 minutes after test item administration and lasted for the first administration days consistently during the day. Thereafter, reduced motility ended 6 to 24 hours after administration and in the further course of the study, reduced motility ended 2 to 6 hours after administration.
In group 4 (1000 mg test item/kg b.w./day), salivation and reduced motility started immediately to 5 minutes after test item administration and ended 2 to 6 hours after test item administration.
- Females (Pre-mating period, gestation period, lactation period):
No alterations of the behaviour, external appearance or the faeces were noted in the female animals of the control group. In the low dose group (100 mg test item/kg b.w./day), 6 of 10 females were noted with slight to moderate salivation.
At 300 mg test item/kg b.w./day, 8 of 10 females displayed slight to severesalivation and 3 of 10 animals were noted with slightly reduced motility.
Due to the predominantly low severity, the low number of animals that were affected and as reduced motility was observed for only one day per animal, reduced motility was considered to be not test item-related. However, as salivation was noted for almost all test item-treated animals and was observed for a high number of days for some animals, salivation was considered to be test item-related. In the high dose group (750 mg test item/kg b.w./day), slight to severe salivation was noted for 7 of 9 females in the pre-mating/mating period and in all female animals in the gestation and lactation period. Moderately reduced motility and yellow discoloured urine were noted in all females in all three periods of the study. Due to the high incidence during all three study periods and the high number of days the alterations were observed, salivation, reduced motility and the yellow discoloured urine were considered to be test item-related.
- Start and duration of clinical signs (Females): In the low and intermediate dose group (100 or 300 mg test item/kg b.w./day), salivation started immediately to 5 minutes after administration and ended 20 to 60 minutes after administration for the female animals. For the female animals dosed with 750 mg test item/kg b.w./day, during the premating/ mating, gestation and lactation period, salivation started immediately to 5 minutes after administration and lasted until 20 to 60 minutes after administration.
Reduced motility started on TD 15 of the pre-mating/mating period immediately to 5 minutes after administration and lasted consistently during the day until TD 19. From TD 20 to TD 22 reduced motility started immediately to 5 minutes after administration and ended 6 to 24 hours after administration. From TD 23 until the end the pre-mating/mating period, reduced motility was noted immediately to 5 minutes after administration until 2 to 6 hours after administration. The duration of reduced motility in the gestation and lactation period for the high dose group was also observed immediately to 5 minutes after administration until 2 to 6 hours after administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
No prematurely deceased male or female animals were noted in the control group and in the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
In the high dose group (750 mg test item/kg b.w./day), two female animals (nos. 96 and 100) were found dead during their gestation day 21 or 20. Necropsy revealed for dam no. 96 a slightly enlarged spleen and for no. 100 thickened thyroids (left and right) and spongeous and dark-red discoloured tissue in the upper thorax region. Additionally, both animals were noted with a discharge of yellowish liquid from the vagina. On the day before and on the day of its death, dam no. 96 was noted with piloerection, slightly laboured breathing and lethargy. Moderate salivation, slightly or moderately reduced motility and yellow discoloured urine were noted on nearly all gestation days. Dam no. 100 as well displayed slightly to moderately reduced motility and yellow discoloured urine on all or several gestation days.
While the death of dam no. 96 was considered to be test item-related, the death of dam no. 100 was deemed not to be directly related to the test item.
The female animal no. 73 of the prematurely terminated group 4 (1000 mg test item/kg b.w./day) was found dead in the morning of TD 16, i.e. after the first administration. Hence, the female animals of group 4 were immediately terminated.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
In the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day),
no test item-related changes in body weight were noted for the male animals in
comparison to the control group.
At 750 mg test item/kg b.w./day, a reduction was noted for the body weight
during the whole treatment period from test day 15 until test day 43 (at maximum
13.7% below the value of the control group on test day 43, statistically significant
for all evaluated test days at p ≤ 0.01). This distinctly reduced body weight noted
for the male animals of the high dose group was considered to be test itemrelated.
No test item-related changes in the body weight gain were noted between the
control group and the low and intermediate dose groups (100 or 300 mg test
item/kg b.w./day).
According to the reduced body weight of the males dosed with 750 mg test
item/kg b.w./day, also the body weight gain was decreased throughout the
treatment period.
Female animals:
For the females, no test item-related influence was noted on the body weight of
the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day) in
comparison to the control group.
At 750 mg test item/kg b.w./day, in the gestation period, a decreased body
weight was noted for the GD 14 and GD 20 (6.4% and 13.0% below the value of
the control group, statistically significant at p ≤ 0.01 on GD 20). In the lactation
period, the decreased body weight was still present at LD 1 (9.3% below the
value of the control group, statistically significant at p ≤ 0.01). Thereafter, the
body weight recovered and no statistically significant decrease was noted on LD 4
and LD 13 (7.9% and 2.5% below the value of the control group). The distinctly
decreased body weight during the gestation period was considered to be test item related.
Also, the body weight of the pups and the litter weights on LD 1 were decreased. Therefore, the
reduced female body weights at the end of the gestation period and at the
beginning of the lactation period were considered to be not only due to a reduction
in the body weight of the parental female animals but also partly due to the
reduced litter weights. Accordingly, no influence on the body weight gain of the female animals was
noted after dosing of 100 or 300 mg test item/kg b.w./day during the pre-mating,
gestation or lactation period in comparison to the control group.
In the high dose group (750 mg test item/kg b.w./day), the decreased body weight
noted for the gestation period led to a decrease in body weight gain during the
gestation period (32.2% body weight in group 5 compared to 53.2% body weight
gain in the control group). Therefore, also the decreased body
weight gain during the gestation period was considered to be test item-related.

Males and females:
No test item-related influence on the body weight at autopsy was noted for the male and female animals of the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day) in comparison to the control group.
In the high dose group (750 mg test item/kg b.w./day), a reduction was noted for the male and female animals (13.9% or 10.7% below the value of the control group, for both statistically significant at p ≤ 0.01). As the reduction for the male and female animals is in accordance with the decreased body weight, the body weight at autopsy was considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
During the pre-mating period (TD 15 to TD 29) no test item-related difference in
food consumption was noted between the male rats of the control group and
those of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
Statistically significantly decreased values for the food consumption were noted in
the test week 3 (TD 15 to TD 22) for the low and intermediate dose groups (7.7%
or 11.1% below the value of the control group, statistically significant at
p ≤ 0.05 or p ≤ 0.01).
However, as the food consumption recovered in test week 4 for the low and
intermediate dose group and no statistically significant difference was noted for
the high dose group in both test weeks, the reduced food consumption was
considered to be not test item-related.
The relative food consumption during the pre-mating period is given in Figure 2-1
as the mean daily values per group in test week 3 (TD 15 to TD 22) and test week
4 (TD 22 to TD 29).
Females:
No test item-related differences in food consumption were noted between the
female rats of the control group and the female rats of the dose groups (100, 300
or 750 mg test item/kg b.w./day) during the pre-mating, the gestation and the
lactation period.
Statistically significant differences for the food consumption were noted between
the female animals of the control group and the females of the dose groups. However, these differences in food consumption were consideredto be not test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual appraisal revealed no test item-related changes in the consumption of
drinking water for the male and female rats treated with 100, 300 or 750 mg test
item/kg b.w./day.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
T4 Determination- Males:
No test item-related changes were noted for the T4 levels of the male animals of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
In the low dose group (100 mg test item/kg b.w./day), slightly increased T4 levels were noted (12.1% above the value of the control group, not statistically significant). However, as no statistical significance and no dose response-relationship was present, the slightly increased T4 levels were considered to be spontaneous. All individual values except from 1 control animal (animal no. 5, 38.9 nmol/L) and two animals of group 5 (animals no. 85 and 86, 41.7 and 42.8 nmol/L, respectively) are within the range of the LPT background data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation and reduced motility were noted for the male animals and salivation was noted for the female animals.
At 750 mg test item/kg b.w./day, salivation, and reduced motility was noted for the male and female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females:
Histopathological examinations were performed on testes, epididymides and ovaries of
animals from Groups 1 and 5 (0 and 750 mg/kg bw/day, respectively). No treatment-related
microscopic changes that could be attributed to treatment with the test item were observed in
these organs of any animals (including survivors and decedents) examined. All recorded findings were deemed to be incidental or were within the range of normal background alterations that may be recorded in animals of this strain and age.
All findings were rated grade 1 (Minimal / very few / very small) and included mononuclear cell focus/foci, interstitium, unilateral or bilateral, grade 1 in the epididymides of animals from the control group and the high dose group and sertoli cell vacuolation, focal/sporadic (single cell),
unilateral, grade 1 in the testes of animals from the control group and the high dose group.
In conclusion, the histopathology revealed that the test item produced no morphological
evidence of toxicological properties in testes, epididymides and ovaries of adult animals
receiving 750 mg/kg bw/day of the test item.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the dose groups (100, 300 or 750 mg test item/kg b.w./day). None of the females showed a complete oestrus cycle during the mating period.
Deviations from the typically expected sequence of oestrus cycles were noted in the form of longer sequences (more than five consecutive dioestrus stages). Such elongated dioestrus sequences were noted for one female each in the control and the high dose group and for two females of the low dose group. However, these elongated dioestrus sequences were not considered to be test item-related, as the incidences were only small, one control animal was also affected and no influence on the reproductive outcome was observed.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence on the fertility index of the female rats was noted for any of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
All female rats except animal no. 98 were successfully mated (confirmed by sperm detection; for animal no. 98 the sperm detection gave a false-positive result). However, one female animal of the high dose group did not become pregnant, leading to a fertility index of 89% for the female animals of the high dose group.
The occurrence of one non-pregnant female at the high dose level was considered to be spontaneous and not test item-related.
Males and females:
No premature death was noted for the male and female animals of the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
At 750 mg test item/kg b.w./day, two female animals (nos. 96 and 100) were found dead on GD 20 or GD 21.
At 1000 mg test item/kg b.w./day, the female animal no. 73 was found dead after the first administration. Hence, the high dose group (1000 mg test item/kg b.w./day) was terminated immediately for the females and after the third administration for the males.

General toxicity-
Parental male and female animals:
Two female animals dosed with 750 mg test item/kg b.w./day were found dead on GD 20 or GD 21, respectively.
In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation, reduced motility and yellow discoloured urine were noted for the male animals and salivation was noted for the female animals.
At 750 mg test item/kg b.w./day, salivation, reduced motility and yellow discoloured urine was noted for the male and female animals.
In the high dose group (750 mg test item/kg b.w./day), reductions were noted for the body weight and the body weight gain.
No test item-related influence was noted on the food consumption.
No test item-related pathologic findings were observed during macroscopic post mortem examination the male and female animals.
Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related changes.
No influence on the T4 levels were noted for the male animals.

Reproductive toxicity-
Parental females:
No influence was noted on the fertility of the male and female animals in any of the dose groups.
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal behaviour was noted for the pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the dose groups (100, 300 or 750 mg test item/kg b.w./day).
Furthermore, the reproductive indices as the birth index, the live birth index and the percentage of post-implantation loss revealed no test item-related differences between the control group and the dose groups.
Pre-cull period:
No difference between the control group and the dose groups (100, 300 or 750 mg test item/kg b.w./day) was noted for the viability index (group level) between lactation days 1 and 4. The viability index from LD 0/1 to LD 4 ranged between 93.1% (low dose group) and 99.3% (control group).

Post-cull period:
No test item-related influence on the viability index was noted for the dose groups (100, 300 or 750 mg test item/kg b.w./day) after culling on LD 4. One dead pup each was noted in the low and intermediate dose group between LD 5 and LD 13.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day) on LD 1, LD 4 and LD 13.
In the high dose group (750 mg test item/kg b.w./day), a reduced body weight was noted for the male and female pups on LD 1, LD 4 and LD 13 (between 18.2% and 21.9% below the value of the control group, statistically significant for all values at p ≤ 0.01). The distinctly decreased body weight was noted on LD 1, LD 4 and on LD 13 and therefore, was considered to be test item-related.
One runt each was noted in the control group and in the low dose group. The single occurrence of one runt in the control group and in the low dose group was considered to be spontaneous.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
T4 determination:
No test item-related changes were noted for the T4 levels of the male and female pups of the dose groups (100, 300 or 750 mg test item/kg b.w./day) on LD 13.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the ano-genital distance of the male and the female pups of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
A reduction of the absolute ano-genital distance was noted for male and female pups of the high dose group (14.0% and 11.9% below the value of the control group, statistically significant for the male animals at p ≤ 0.01). However, as no difference was noted for the relative ano-genital distance, the reduced absolute ano-genital distance was due to the reduced body weight of the pups. Therefore, the test item was considered to have no influence on the ano-genital distance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (100, 300 or 750 mg test item/kg b.w./day).
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100, 300 or 750 mg test item/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
REPRODUCTIVE PARAMETERS No test item-related influence was noted on the number of implantations, the number of resorptions, the number of live born pups and the ratio of male to female pups for any of the dose groups.
PUP BODY WEIGHT In the high dose group (750 mg test item/kg b.w./day), a reduced body weight was noted for the male and female pups on LD 1, LD 4 and LD 13 (between 18.2% and 21.9% below the value of the control group, statistically significant for all values at p ≤ 0.01)

LITTER WEIGHT In the high dose group (750 mg test item/kg b.w./day), reduced litter weights were noted for the male and the female litters on the lactation days 1, 4 and 13 (between 12.3% and 22.6% below the value of the control group, statistically significant for the female litters and for the male and female litters combined on LD 13 at p ≤ 0.01).
EXTERNAL EXAMINATION The external macroscopic examination of the pups after sacrifice revealed no gross abnormalities.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity

Text table 3‑1:      Group size and dose levels.

Group

test item dose #

[mg/kg b.w./day, p.o.]

Number and

sex of animals

Animal number[1]

1

0

(vehicle control)

10

10

m

f

1 - 10

11 - 20

2

100

(low dose group)

10

10

m

f

21 - 30

31 - 40

3

300

(intermediate dose group)

10

10

m

f

41 - 50

51 - 60

4[2]

1000

(high dose group)

10

10

m

f

61 - 70

71 - 80

5

750

(high dose group)

10

10

m

f

81 - 90

91 - 100

#:       no correction factor was used.

m:     male

f:       female

 

[1]             Including spare animals; 8 female animals per group were evaluated.

[2]             All group 4 females were terminated on 20 June 2019 on TD 16, i.e. after the first administration. All group 4 males were terminated on 22 June 2019 on TD 18, i.e. after the third administration.

 

Text table 4‑1:      Reproductive outcome of the female animals per group.

test item

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

Group 4 #1

1000 mg/kg

Females started dosing

10

10

10

10

10

Females used for pairing

10

10

10

10

n.a.

Females excluded

0

0

0

1 #2

n.a.

Females mated #3

10

10

10

9

n.a.

Females not pregnant

0

0

0

1

n.a.

Females pregnant #4

10

10

10

8

n.a.

Prematurely deceased females

0

0

0

2 #5

1

Females with litters #6

10

10

10

6

n.a.

Females that delivered only stillbirth

0

0

0

0

n.a.

Females with live born pups

10

10

10

6

n.a.

Females surviving the lactation period until scheduled sacrifice

10

10

10

6

n.a.

n.a.:   not applicable

#1:     Group 4 was terminated on TD 16 (females), i.e. after the first administration, or TD 18 (males), i.e. after the third administration due to signs of pronounced toxicity and a premature death (for details see Section 4.2 'Mortality' and 4.3 'Clinical signs'). No data of group 4 are available except for mortality and clinical signs.

#2:     In group 5, for animal no. 98 sperm detection was false-positive and animal no. 98 was erroneously considered to be mated. Therefore, it was excluded from the study (see Section 2.12 'Study Plan deviations').

#3:     Number of animals at start of the pre-mating and mating period.

#4:     Number of evaluated animals at start of the gestation period.

#5:     In group 5, the female animal no. 96 was found dead on GD 21 and the female animal no. 100 was found dead on GD 20.

#6:     Number of evaluated animals at start of the lactation period.

 

Text table 4‑2:      Changes in behaviour, external appearance or faeces noted for male animals of group 3.

Observations in group 3

(300 mg test item/kg b.w./day)

Observation

Affected males

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to moderate)

9 of 10

TD 27 - TD 43

4 - 15

Motility reduced

(slight)

10 of 10

TD 16 - TD 41

10 - 22

Discolouration, urine

(yellow)

8 of 10

TD 20 - TD 24

5

Discolouration, faeces

(yellow) #

10 of 10

TD 16 - TD 17

2

Haemorrhagic nose/snout #

1 of 10

(no. 42)

TD 27

1

#:       Considered to be spontaneous due to low number of affected animals and/or low number of days observed.

 

Text table 4‑3:      Changes in behaviour, external appearance or faeces noted for male animals of group 5.

Observations in group 5

(750 mg test item/kg b.w./day)

Observation

Affected males

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to severe)

9 of 10

TD 21 - TD 44

2 - 21

Motility reduced

(slight to moderate)

10 of 10

TD 15 - TD 43

29

Discolouration, urine

(yellow)

10 of 10

TD 28 - TD 38

11

Discolouration, faeces

(pale) #

1 of 10

(no. 81)

TD 21 - TD 22

2

#:       Considered to be spontaneous due to low number of affected animals and low number of days observed.

 

Text table 4‑4:      Changes in behaviour, external appearance or faeces noted for female animals of group 2.

Test item-related observations in group 2

(100 mg test item/kg b.w./day)

 

Affected females per period #1

 

Incidence

Observation

Pre-mating/

Mating

Gestation

Lactation

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to moderate)

6 of 10

6 of 10

1 of 10

TD 27 - LD 4

1 - 11

 

Text table 4‑5:      Changes in behaviour, external appearance or faeces noted for female animals of group 3.

Observations in group 3

(300 mg test item/kg b.w./day)

 

Affected females per period #1

 

Incidence

Observation

Pre-mating/

Mating

Gestation

Lactation

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to severe)

8 of 10

10 of 10

7 of 10

TD 27 - LD 13

4 - 27

Reduced motility (slight) #²

3 of 10

0 of 10

0 of 10

TD 27

1

#1:         The number of females examined per period is given in Section 4.1.

#²:     Considered to be not test item-related due to low number of affected animals and low number of days observed.

 

Text table 4‑6:      Changes in behaviour, external appearance or faeces noted for female animals of group 5.

Test item-related observations in group 5

(750 mg test item/kg b.w./day)

 

Affected females per period #1

 

Incidence

Observation

Pre-mating/

Mating

Gestation

Lactation

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to severe)

7 of 9

8 of 8

6 of 6

TD 21 - LD 13

5 - 29

Reduced motility (slight to moderate)

9 of 9

8 of 8

6 of 6

TD 15 - LD 13

35 - 53

Discolouration of urine (yellow)

9 of 9

8 of 8

6 of 6

TD 28 - LD 13

17 - 35

Lethargy #²

none

1 of 8

none

GD 20 - GD 21

2

Piloerection #²

none

1 of 8

none

GD 20 - GD 21

2

Laboured breathing

(slight) #2

none

1 of 8

none

GD 20 - GD 21

2

#1:         The number of females examined per period is given in Section 4.1.

#²:     Lethargy, piloerection and laboured breathing were noted only for one animal on the two days before its premature death. Hence, these observations were considered to be pre-mortal symptoms.

 

Text table 4‑7:      Body weight gain during the treatment period (TD 15 - TD 43) - male animals.

Body weight gain - Males #

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

(TD 15 to TD 43)

+15.8 %

+14.0 %

+12.2 %

+7.2 %

 

Text table 4‑8:      Body weight gain of the female animals during the pre-mating/mating, gestation and lactation period.

Body weight gain - Females

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

pre-mating period

(TD 15 to TD 29)

+0.5%

+1.7%

+2.6%

+3.6%

gestation period

(GD 0 to GD 20)

+53.2%

+50.8%

+45.2%

+32.2%

lactation period

(LD 1 to LD 13)

+9.4%

+11.2%

+10.8%

+17.6%

 

Text table 4‑9:      Statistically significant differences for the food consumption that were considered to be not test item-related.

Period

Ref. table no.

Group

Test week

Increase  ­

Decrease ¯

(% change to control)

Statistical significance

Reason

Pre-mating period

5-2

3

TD 15 - TD 22

¯ (-12.3%)

p £ 0.01

A

5

TD 15 - TD 22

¯ (-16.1%)

p £ 0.01

A

Gestation period

5-3

3

GD 0 - GD 7

¯ (-11.1%)

p £ 0.01

A, B

5

GD 0 - GD 7

­ (+18.8%)

p £ 0.01

A, B

5

GD 14 - GD 20

­ (+34.7%)

p £ 0.01

A, B

A:      No statistically significant difference in the following test weeks.

B:      No dose response-relationship present.

 

Text table 4‑10:    Stage of the oestrus cycle at necropsy (LD 14).

Stage of oestrus at necropsy #

Group 1

Control

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

750 mg/kg/day

Pro-oestrus

-

1 of 10

-

-

Oestrus

-

-

-

-

Metoestrus

1 of 10

-

1 of 10

-

Dioestrus

9 of 10

9 of 10

9 of 10

6 of 6

- :

not detected

#:

The non-pregnant and the excluded animals were not considered in this table.

 

Text table 4‑11:    Comparison of the T4 levels of the male animals per group with the LPT background data.

Parameter

Values from this study #2

Mean values per group ±SD

(range of individual values)

LPT Background Data #1

obtained from the control groups of 11 OECD 422 / 421 studies performed at LPT from 2016 - 2018

Males

(mean value from 110 control males ± SD

(range of the individual values)

T4 levels

- males -

(nmol/L)

Group 1

51.97 ± 7.20

(38.9 - 59.5)

67.62 ± 13.98

(42.9 - 114.8)

Group 2

58.25 ± 6.74

(48.5 - 69.0)

Group 3

51.55 ± 6.23

(43.7 - 62.9)

Group 5

52.59 ± 7.55

(41.7 - 66.9)

#1:     Data not audited by QAU.

 

Text table 4‑12:    Mean length and mean number of oestrus cycles.

Parameter

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

Pre-mating: Test day 15 (start of treatment) until pairing (test day 29)

Mean cycle length [days]

4.08

4.19

4.30

4.46

Number of cycles

2.1

2.4

2.1

2.0

 

Text table 4‑13:    Fertility indices per group.

Group / Dose level

Fertility index [%]

Pregnant rats / rats used

Group 1

(Control)

100

10 / 10

Group 2

(100 mg/kg)

100

10 / 10

Group 3

(300 mg/kg)

100

10 / 10

Group 5

(750 mg/kg)

  89 #

  8 /   9 #

#:       Including the two pregnant females that were found dead on GD 20 or GD 21.

 

Text table 4‑14:    Gestation indices per group.

Group / Dose level

Gestation index %

Dams with live pups / pregnant rats

Group 1

(Control)

100

10 / 10

Group 2

(100 mg/kg)

100

10 / 10

Group 3

(300 mg/kg)

100

10 / 10

Group 5

(750 mg/kg)

100 #

6 / 6 #

#:       Excluding the two pregnant females that were found dead on GD 20 or GD 21.

 

Text table 4‑15:    Litter weight of the male and female pups per dam on LD 1, LD 4 and LD 13.

Litter weight for the male and female pups combined

Group

Parameter

LD 1

LD 4

LD 13

Group 1

control

mean per dam

99.5 g

140.6 g

292.3 g

difference to control

n.a.

n.a.

n.a.

Group 2

100 mg/kg b.w./day

mean per dam

102.6 g

134.2 g

273.8 g

difference to control

+3.1%

-4.6%

-6.3%

Group 3

300 mg/kg b.w./day

mean per dam

92.5 g

127.2 g

269.9 g

difference to control

-7.0%

-9.5%

-7.7%

Group 5

750 mg/kg b.w./day

mean per dam

83.8 g

111.4 g

235.7 g **

difference to control

-15.8%

-20.8%

-19.4%

n.a.:   not applicable

**:     Statistically significant at p ≤ 0.01 (Dunnett test)

 

Text table 4‑16:    % difference of the T4 levels of the pups in comparison to the control group.

Pup T4 level

% difference to the control group

Group 2

Group 3

Group 5

Male pups

LD 13

-3.0

-2.4

-8.1

Female pups

LD 13

+3.5

-1.6

-3.8

Male + Female pups

LD 13

+0.1

-2.0

-6.0

 

Tabular summary report of effects on reproduction development (Annex 3 of OECD method 421)

OBSERVATIONS

VALUES

Dosage (mg/kg)

0 (control)

100

300

750

Pairs started (N)

10

10

10

10

Oestrus cycle (mean length / number of animals with elongated dioestrus periods per group )

4.1 / 1

4.2 / 2

4.3 / 0

4.5 / 1

Females showing evidence of copulation (N)

10

10

10

10

Females achieving pregnancy (N)

10

10

10

8

Conceiving days 1 - 5 (Pre-coital interval) (N)

10

10

10

5

Conceiving days ≥ 5 (N)

0

0

0

1

Pregnancy ≤ 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

3

6

6

6

Pregnancy ≥ 23 days (N)

7

4

4

0

Dams with live young born (N)

10

10

10

6

Dams with live young at day 4 pp (N)

10

10

10

6

Implants/dam (mean)

15.6

17.0

16.3

17.3

Live pups/dam at birth (=lactation day 1) (mean)

14.6

16.0

14.7

15.2

Live pups/dam at day 4 (mean)

14.5

14.9

14.5

14.5

Sex ratio (m/f) at birth (=lactation day 1) (mean)

1.09

0.95

1.10

1.02

Sex ratio (m/f) at day 4 (mean)

1.10

0.94

1.13

1.02

Litter weight at birth (=lactat. day 1) (mean, g)

99.53

102.61

92.53

83.77

Litter weight at day 4 (mean, g)

140.59

134.19

127.24

111.37

Pup weight at birth (=lactat. day 1) (mean, g)

6.852

6.428

6.377

5.542

Pup weigh t a t the time of AGD

measurement (lactation day 4)
(mean males , mean females,  g)

10.035 /

9.657

9.244 /

8.699

9.141 /

8.668

7.984 /

7.540

Pup AGD on the same post-natal day,

lactation day 4 (mean m ales , mean females , note PND)

35.75 /

17.14

PND 4

34.54 /

16.77

PND 4

36.21 /

17.62

PND 4

30.76 /

15.24

PND 4

Pup weight at day 4 (mean)

9.853

8.973

8.905

7.747

Pup weight at day 13 (mean)

29.539

27.975

27.242

23.962

Male pup nipple retention at day 13 (mean)

0.26

0.02

0.32

0.40

ABNORMAL PUPS

Dams with 0

10

10

10

6

Dams with 1

0

0

0

0

Dams with ≥ 2

0

0

0

0

LOSS OF OFFSPRING

Pre-natal (implantations minus live births or sum of resorptions and stillbirths)

Females with 0

3

 

 

2

2

 

3

1

2

Females with 1

4

5

5

1

Females with 2

3

1

2

2

Females with ≥ 3

0

1

2

1

Post-natal (prematurely deceased pups between PND 0/1 and PND 13))

Females with 0

9

5

8

2

Females with 1

1

4

1

4

Females with 2

0

0

1

0

Females with ≥ 3

0

1

0

0

Conclusions:
The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day. For general toxicity the NOAEL was considered to be 100 mg/ kg b.w./ day, based in clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day. The NOAEL for reproductive toxicity is considered to be 300 mg/ kg bw/ day based on the reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity. The NOAEL for adverse effects on the reproductive parameters of the parental females is considered to be above 750 mg/ kg bw/day.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day.

 

General toxicity

Parental male and female animals

Two female animals dosed with 750 mg test item/kg b.w./day were found dead on GD 20 or GD 21, respectively.

In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation, reduced motility and yellow discoloured urine were noted for the male animals and salivation was noted for the female animals.

At 750 mg test item/kg b.w./day, salivation, reduced motility and yellow discoloured urine was noted for the male and female animals.

In the high dose group (750 mg test item/kg b.w./day), reductions were noted for the body weight and the body weight gain.

No test item-related influence was noted on the food consumption.

No test item-related pathologic findings were observed during macroscopic post mortem examination the male and female animals.

Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related changes.

No influence on the T4 levels were noted for the male animals.

 

Reproductive toxicity

Parental females

No influence was noted on the fertility of the male and female animals in any of the dose groups.

 

 

Pups

No influence was noted of the pre-natal development (number of implantations, number of resorptions or number of live born pups) at all dose groups.

For the post-natal development, changes in form of a reduced body weight and accordingly in form of a reduced litter weight were noted for the pups of the high dose group (750 mg test item/kg b.w./day). This is considered to be a secondary effect of the reduced body weight of the dams.

No influence on the T4 levels was noted for the male and female pups.

No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.

 

The following no-observed-adverse-effect levels (NOAEL) were established:

General toxicity (parental animals)

 

 

NOAEL.....................................................................

100 mg test item/kg b.w./day, p.o.

Based on clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day p.o.

 

Reproductive toxicity

 

 

 

a) adverse effects on the reproductive parameters of the parental females

NOAEL......................................................................

above 750 mg test item/kg b.w./day, p.o.

 

 

b) adverse effects on pre- and postnatal development

NOAEL.....................................................................

300 mg test item/kg b.w./day, p.o.

Based on the reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity.

Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.02.2020 to 15.03.2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to the ECHA final decision on a compliance check from 2018-12-21 (Decision number CCH-D-2114453561-52-01/F) the study design of this EOGRTS according to OECD 443 is as follows:

Based on Article 41 of Regulation (EC) No 1907/2006 (the REACH Regulation), ECHA requests you to submit information on:

- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (Developmental neurotoxicity).
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted June 25, 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The study design is based on final decision on a compliance check from ECHA dated 21 Decemberl 2018 (Decision number CCH-D-2114453561-52-01/F). An Extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows, was requested:
- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (Developmental neurotoxicity).

The aim of the study is to evaluate the pre- and postnatal effects of the test item on development as well as systemic toxicity in pregnant and lactating females and young and adult offspring.
According to the final decision, a possible mode of action related to endocrine disruption should be clarified: Therefore, possible endocrine disruptor effects
of the test item will be examined by using appropriate laboratory methods (e.g. T4 and TSH hormone ELISA). Furthermore, based on findings reported by Marty et al. (2011), further examinations will be conducted to investigate pubertal development of males, i.e. determination of serum testosterone levels in all male F1 animals on PND 53 and weight of the seminal vesicles including coagulating glands in male animals of F1 generation cohort 2A. In addition, the study will provide and/or confirm information about the effects of a test item on the integrity and performance of the adult male and female reproductive systems.

Additionally, effects on developmental neurotoxicity will be evaluated.

Furthermore, liver weight will also be determined for all F1 cohort 2A animals to establish a timeline for the possible decrease in liver weight.
Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies and required by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: F0: 70 weeks
- Number of parental animals:
Pre-exposure period
120 female animals were evaluated in order to yield 96 females with a regular estrous cycle for the main study.
Main study
192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation.
- Weight at study initiation: Males: 386.2 g - 494.3 g, Females: 226.9 g - 287.8 g
- Housing: With exception of the mating period, the animals are kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages are cleaned and changed once a week. The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet: ssniff® R/M-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest), ad libitum with the exception of the night before the day of blood withdrawal for laboratory examinations. Food residue was removed and weighed. Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL (see Appendix 3: 'Limitation for Contaminants in the Diet'). Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water: Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001 ]. In addition, drinking water samples taken at the test institute are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001, Anlage 1' [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C
- Humidity (%): 55% ± 10%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- Route of administration: Oral, via gavage
- Frequency of administration: Once daily
- Vehicle: corn oil
Administration volume: 5 mL/kg b.w.
Dosages: 0, 65, 200, 600 mg/kg bw/d
Selection of route of administration: According to international guidelines.
Details on mating procedure:
Sexually mature male and female rats of the F0 Geneartion and F1 Generation Cohort 1B of the same dose group were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug.
The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from their male partner after 2 weeks without further opportunity for mating.
In case of an insufficient number of males, for example due to male death before mating with its female partner, males that had already mated were paired with a second female such that all females were paired.

Establishment of F2 generation using Cohort 1B (potential reproductive toxicity):
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Sexually mature male and female rats of the F1 Generation Cohort 1B of the same dose group were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug.
The day of conception (day 0 of gestation or GD 0) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from their male partner after 2 weeks without further opportunity for mating.
In case of an insufficient number of males, for example due to male death before mating with its female partner, males that had already mated were paired with a second female such that all females were paired.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The administration formulations were prepared for up to 7 administration days in advance and were administered orally at a constant application volume of 5 mL/kg b.w. once daily.
The test item was diluted in the vehicle to the appropriate concentrations. Following the last dosing on each administration day, the test item formulations were stored at 2 - 8°C. Prior to the first administration of each administration day, the test item formulations were allowed to warm up to room temperature for at least 30 minutes and stirred for approximately 3 minutes to ensure homogeneity. The amount of the test item was adjusted to the animal's current body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.


For the analysis of the test item-vehicle formulations, two aliquots of approximately 3 mL each were taken at the following times and stored at -20°C ± 10% until analysis.
The samples were labelled with study number, test species, generation, cohort no., type of sample, aliquot number, group no., concentration, sampling time and date.
The samples were analysed according to a method validated in a seperate study (“Validation of an analytical method for the determination and quantification of 3,5,5-trimethylcyclohex-2-enone in liquid formulations with HPLC-UV detection”).

At start of the treatment period of the F0 animals (1st dosing day): Analysis of concentration and homogeneity, at the start of administration, during (middle) administration and before administration to the last animal of each dose group. (3 samples/dose group). At a time when most F0 females have delivered: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At termination of the F0 dosing period at a time when the majority of animals is dosed: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At start of the treatment period of the F1 animals (1st dosing day): Analysis of concentration and homogeneity. At the start of administration, during (middle) administration and before administration to the last animal of each dose group (3 samples/dose group). At termination of Cohort 1A at a time when the majority of animals is dosed: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At termination of Cohort 1B at a time when the majority of animals is dosed: Analysis of concentration. During treatment always before administration to the last animal/dose group (1 sample/dose group).

The results of the test item formulation analysis ranged between 98.5% and 106.3% of the nominal concentration, indicating correctly prepared and homogenized test item vehicle mixtures.
Duration of treatment / exposure:
The animals were treated with the test item during the following periods:
F0 GENERATION:
Males: 2 weeks prior to mating, during the mating period and at least until weaning of the F1 Generation (up to and including the day before sacrifice).;
Females: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).

F1 GENERATION:
F1 PUPS: Until weaning, the pups were indirectly exposed to the test item through the breast milk.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed individually via gavage.
COHORT 1A: The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 89 to 94).
COHORT 1B: The male and female animals were dosed until sacrifice of their F2 Pups up to and including the day before sacrifice (males and females: PNDs 119 to 152).
COHORT 2A: Up to and including the day before sacrifice between PNDs 77 and 81.
COHORT 2B: Cohort 2B animals were indirectly exposed to the test item through the breast milk until sacrifice on PND 21.

F2 GENERATION:
F2 PUPS: F2 pups were indirectly exposed to the test item through the breast milk until sacrifice on PND 21.
Frequency of treatment:
once daily
Details on study schedule:
PARENTAL ANIMALS, PRE-EXPOSURE:
120 female animals were evaluated in order to yield 96 females with a regular estrous cycle for the main study.
MAIN STUDY:
192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation.

F1 GENERATION:
A few days before the pups from those dams with the earliest litter date had reached postnatal day 21 (PND 21) pups from all available litters were randomly selected for the F1 Generation using Provantis . When all selected pups had reached postnatal day 21, all the selected pups were transferred to their respective Cohort of the F1 Generation and the F1 Generation started with test day 1.
Obvious runts, i.e. pups with a body weight less than 70% of litter mean on PND21, would have been exchanged subsequently. However, no runt was noted on PND 21.

F2 GENERATION:
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures were imilar to those for the parental animals (F0 generation).

The animals were treated with the test item during the following periods:
F0 Generation:
- Males: 2 weeks prior to mating, during the mating period and at least until weaning of the F1 Generation (up to and including the day before sacrifice).
- Females: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).
F1 Generation:
- F1 Pups: Until weaning, the pups were indirectly exposed to the test item through the breast milk.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed individually via gavage.
- Cohort 1A: The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 89 to 94).
- Cohort 1B: The male and female animals were dosed until sacrifice of their F2 Pups up to and including the day before sacrifice (males and females: PNDs 119 to 152).
- Cohort 2A: Up to and including the day before sacrifice between PNDs 77 and 81.
- Cohort 2B: Cohort 2B animals were indirectly exposed to the test item through the breast milk until sacrifice on PND 21/22.
F2 Generation:
- F2 Pups: F2 pups were indirectly exposed to the test item through the breast milk until sacrifice on PND 21.

DURATION OF STUDY:
- at least 5 adaptation days of the F0 generation
- F0 (parental generation):
2 weeks pre-exposure
2 weeks pre-mating
2 weeks mating
3 weeks pregnancy
3 weeks lactation
- F1 Cohort 1A: approx. 10 weeks
- F1 Cohort 1B: approx. 10 weeks
2 weeks mating for establishing of F2 3 weeks pregnancy
3 weeks lactation
- F2 Generation: lactation until PND 21
- F1 Cohort 2A: approx. 10 weeks
- F1 Cohort 2B: lactation until PND 21/22
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
65 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
F0 generation: 20 m/f per group
F1 generation Cohorts 1A + 1B: 20 m/f per group
F1 generation Cohorts 2A + 2B: 10 m/ f per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study were selected in agreement with the Sponsor based on available toxicological data and the results of a 16-day repeated dose toxicity study in rats (conducted 1986), a 90-day repeated dose toxicity study in rats (conducted 1986) as well as an OECD 421 study in rats (LPT Study No. 37311, 2020). Additionally, the results and the study design of the Pubertal Development Assay (Marty et al., 2011) were taken into account. In the 16-day and the 90-day study, dose levels of 125, 250, 500 and 1000 mg/kg b.w. per day were employed. In the Pubertal Development Assay, dose levels of 50, 200 and 800 mg/kg b.w. per day were selected. In the OECD 421 study, dose levels of 100, 300, 750 and 1000 mg/kg b.w. per day were used.
In the 90-day and the 16-day gavage studies with the test item, the NOAEL was considered to be 500 mg/kg b.w. (both NTP, 1986). In the 16-day study at the high dose of 1000 mg/kg b.w., only decreased body weights were observed. One female rat that received 1000 mg/kg b.w. died in the 90-day gavage study. Final mean body weights for rats were not clearly related to dose in this study. In the Pubertal Development Assay, increased liver weights, reduced seminal vesicle weights, decreased serum level of testosterone and delayed preputial separation were observed at 800 mg/kg b.w. These findings are considered to be signs of hepatic enzyme induction prior to puberty that results in a greater metabolism rate of testosterone. The thus reduced testosterone level consequently leads to a subsequent delay in the maximum luteinizing hormone (LH) release and attainment of sufficient testosterone levels to drive puberty onset and accessory sex tissue size, e.g. seminal vesicles (Marty et al., 2018). It is assumed that prepubertal animals are particularly sensitive to this mode of action because opposite to adult males, they possess a positive feedback loop where higher levels of testosterone are stimulatory, potentiating the pituitary’s response to GnRH (Gonadotropin releasing-hormone), resulting in maximum LH release that further increases testosterone secretion (Marty et al., 2018, Stoker et al., 2000).
In the OECD 421 study, female animals treated with 1000 mg/kg b.w. had to be sacrificed after the first dosing and male animals after the third dosing due to animal welfare reasons. Two female animals dosed with 750 mg/kg b.w. were found dead on GD 20 and GD 21. Both male and female animals treated with 750 mg/kg b.w. showed slight to severe salivation, slightly to moderately reduced motility and yellow discoloured urine. Furthermore, a reduction in body weight was noted for the male animals during dosing period, while female animals showed a reduced body weight between GD 14 and GD 20. Accordingly, also the body weight gain was reduced for male and female animals during the respective periods. Reduced body weights were also noted at necropsy for both male and female animals. Therefore, the results of the above-mentioned historical studies could not completely be reproduced under the test conditions of the DRF OECD 421 study (LPT Study No. 37311, 2020).
For the pups of animals treated with 750 mg/kg b.w. reduced weights were noted for male and female animals on lactation days 1, 4 and 13.
Male animals treated with 300 mg/kg b.w. showed slight to moderate salivation, a slightly reduced motility and yellow discoloured urine, while slight salivation was observed in females treated with 300 mg/kg b.w.
Based on these findings, doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death. Taking all study results into account and considering the extended exposure time of the F0 and F1 Generation within the OECD 443 study, 600 mg/kg b.w. per day were considered the maximum tolerated dose for the F0 Generation of the OECD 443 study, with no systemic effects expected in the F1 Generation. Furthermore, 600 mg/kg b.w. per day were selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).

- Fasting period before blood sampling for clinical biochemistry: the night before the day of blood withdrawal for laboratory examinations
Positive control:
no
Parental animals: Observations and examinations:
In order to derive the NOAELs the following results from the F0 generation + F1 generation Cohorts 1A, 1B and 2A were used. For an easier understanding these results are included under "parental animals" even though only the F0 and F1 Cohort 1B animals were used to establish a following generation.
CLINICAL SIGNS:
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal.

All animals:
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness. Any signs of illness or reaction to treatment were recorded.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
In case signs of toxicity occurred the frequency of observations was increased.
In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

F0 and F1 Generation (Cohort 1B) after weaning:
A more detailed examination of all F0 and F1 Cohort 1B animals was conducted on a weekly basis. F0 animals were examined once before the first test item treatment on test day 14 to allow for within-subject comparisons. Thereafter, the examination was performed weekly until termination. The F1 animals of Cohort 1B were examined weekly after weaning until termination.
Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

MORTALITY:
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.
If necessary, these provisions allowed to record any premortal symptoms in detail and a post mortem examination to be carried out during the working period of a day.

BODY WEIGHT:
P0: The body weight of the animals was recorded as follows:
Pre-mating period: Daily, starting on the first day of dosing i.e. test day 15*
Mating period: Daily* (males, no weights of female animals reported)
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 1, 4, 7, 14, 21
Post-mating period: Males: Daily*, Females: See gestation and lactation period
*: Weekly values will be reported

Schedule for body weight recordings of offspring animals:
F1/ F2: Lactation period: PND 1, 4, 7, 14, 21
After weaning: Daily, starting on PND 22*
F1 Cohort 1B animals only
Mating period: Daily* (males, no weights of female animals reported)
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 1, 4, 7, 14, 21
Post-mating period: Males: Daily*, Females: See gestation and lactation period
*: Weekly values will be reported
In addition, all animals will be weighed at sacrifice.

FOOD AND WATER CONSUMPTION:
P0: Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days that are listed below.
Pre-mating period: weekly
Mating period: none
Gestation period (females): GD 0, 7, 14, 21
Lactation period (females): PND 1, 7, 14, 21
Post-mating period: Males: Weekly*, Females: See gestation and lactation period
*: Starting on a suitable day after the mating period to consolidate all male animals (test day 35)

F1: Food consumption of offspring animals
Cohort 1B: Mating period: none
Gestation period (females): GD 7, 14, 21
Lactation period (females): PND 1, 7, 14, 21
Post-mating period: Males: Weekly* Females: See gestation and lactation period
*: Starting on a suitable day after the mating period to consolidate all male animals
Water consumption is monitored by visual appraisal daily throughout the study.

SEXUAL MATURATION:
F1 Cohort 1A, 1B and 2A:
F1 animals (Cohorts 1A, 1B, 2A) were evaluated daily for balano-preputial separation or vaginal opening, which indicate sexual maturity of the animals. The genitals were examined for any abnormalities. The body weight was recorded at the time point of balano-preputial separation or vaginal opening. The results were evaluated individually for each cohort as well as for all cohorts combined.

TESTOSTERONE DETERMINATION:
F1 Cohorts 1A, 1B, 2A: In order to investigate possible effects of the test item in progression of male sexual maturation, a sufficient amount of blood was taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, as scheduled below to obtain 2 x 100 µL serum. Blood was collected on the same post-natal day for all animals. Blood was collected on PND 53 +/- 1. After collection, the blood was processed for serum and serum samples were divided into aliquots and stored at 20 °C ± 10% at the test facility until analysis using ELISA. The testosterone ELISA (commercial kit 'Testosterone ELISA Kit' , cat. no. RE52151; lot no. ETE131, Instrument: Tecan Sunrise) was conducted by the test facility.

URINALYSIS:
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
Time of urine collection: At the end of dosing period (prior to blood withdrawal)
Animals:
F0 Generation: 10 males and 10 females randomly selected from each group
F1 Generation - Cohort 1A: 10 males and 10 females randomly selected from each group
The following parameters were determined by using the instruments given below:
Parameters/ Unit/ Instrument:
- volume/ mL/ Graduated vessel
- pH/ non-dimensional/ Digital pH meter, type WTW InoLab pH 720
- specific gravity/ g/mL/ Kern Refractometer, type ORA 2PA; Sample compared with water (nominal value of 1.000)

The following examinations were also performed using Combur 9 Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine: protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
Epithelial cells
Leucocytes
Erythrocytes
Organisms
Further constituents (i.e. sperm, casts)
Crystalluria
The frequency of the above parameters was recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.

REPRODUCTIVE PERFORMANCE - F0 and F1 Cohort 1B animals
The reproductive parameters and reproductive indices listed below were determined to evaluate the reproductive performance.
Reproductive parameters
- stages of the oestrous cycle
- pre-coital time
- number of pregnant females
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4, 7, 14 and 21
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4, 7, 14, 21
Number of stillbirths
- per group
- per dam
Number of pups with malformations (see Appendix 4)
- per group
- per dam

s. also reproductive indices
Oestrous cyclicity (parental animals):
Vaginal lavages were taken and the oestrous cycle stages were determined at the following time points:
Animals/ Monitoring schedule
- F0 main study animals/ 14 days pre-exposure period to select 96 main study animals with regular oestrous cycles (4 to 5 days per cycle) and during 2 weeks of pre-mating until evidence of mating.
- F1 animals, Cohort 1A/ Start after onset of vaginal patency until first appearance of cornified cells.
Two weeks starting around PND 75.
- All adult F0 and F1 animals/ On the day of sacrifice, shortly before necropsy.
Sperm parameters (parental animals):
F0 and F1 Generation Cohort 1A: One epididymis and one testis of all F0 and F1 Cohort 1A males were used for the sperm count. The sperm viability was determined and the sperm morphology was examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Litter observations:
EXAMINATION OF THE PUPS (F1 and F2 Generation):
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (i.e. body weight less than 70% of mean litter weight) and the presence of gross abnormalities.
Abnormal behaviour or changes in the external appearance of the pups noted during the daily cage side inspections were recorded.
The following examinations/observations were done for the offspring:

COUNTING, SEXING AND WEIGHING:
Live pups were counted, sexed and weighed on post-natal days (PND) 1, 4, 7, 14 and 21.

ANO-GENITAL DISTANCE:
On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalised to the cube root of body weight.

LITTER ADJUSTMENT:
After counting on PND 4 (lactation day 4), the litters were adjusted to 10 pups per litter (5 pups/sex/litter) by eliminating (culling) surplus pups using a randomisation scheme generated by Provantis® .
Selective elimination of pups, e.g. based upon body weight was not appropriate. In case of unequal gender distribution, a partial litter size adjustment was performed (e.g. 6 male and 4 female pups).

NIPPLES/ AREOLAE COUNTING:
Nipples/areolae were counted in all male pups on PND 13.

SEXUAL MATURATION:
F1 animals (Cohorts 1A, 1B, 2A) were evaluated daily for balano-preputial separation or vaginal opening, which indicate sexual maturity of the animals. The genitals were examined for any abnormalities. The body weight was recorded at the time point of balano-preputial separation or vaginal opening. The results were evaluated individually for each cohort as well as for all cohorts combined.
Balano-preputial separation:
During this examination the time point of the onset of the function of the preputial glands was determined.
A soft pressure was exerted against the root of the penis. If this led to the observation of small drops of secretion from the preputial glands on both sides of the foreskin, the postnatal day of this observation was determined as the time point of the onset of preputial gland function.

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
Pups, 2 surplus pups per litter, all litters, if possible, PND 4, non-fasted, T4 only
Pups, 2 surplus pups per litter, all litters, non-fasted, T4 and TSH

FOOD CONSUMTION OF OFFSPRING ANIMALS:
Cohort 1A/ Cohort 2A: starting after weaning: weekly

TESTOSTERONE DETERMINATION:
F1 Cohort 1A/ 2A:In order to investigate possible effects of the test item in progression of male sexual maturation, a sufficient amount of blood was taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, as scheduled below to obtain 2 x 100 µL serum. Blood was collected on PND 53 +/- 1 for all animals. After collection, the blood was processed for serum and serum samples were divided into aliquots and stored at 20 °C ± 10% at the test facility until analysis using ELISA. The testosterone ELISA (commercial kit 'Testosterone ELISA Kit' , cat. no. RE52151; lot no. ETE131, Instrument: Tecan Sunrise) was conducted by the test facility.

URINALYSIS:
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
Time of urine collection: At the end of dosing period (prior to blood withdrawal)
Animals:
F0 Generation: 10 males and 10 females randomly selected from each group
F1 Generation - Cohort 1A: 10 males and 10 females randomly selected from each group
The following parameters were determined by using the instruments given below:
Parameters/ Unit/ Instrument:
- volume/ mL/ Graduated vessel
- pH/ non-dimensional/ Digital pH meter, type WTW InoLab pH 720
- specific gravity/ g/mL/ Kern Refractometer, type ORA 2PA; Sample compared with water (nominal value of 1.000)

The following examinations were also performed using Combur 9 Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine: protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
Epithelial cells
Leucocytes
Erythrocytes
Organisms
Further constituents (i.e. sperm, casts)
Crystalluria
The frequency of the above parameters was recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.

NEUROLOGICAL SCREENING OF F1 COHORT 2A ANIMALS:
- PND 24 (±1): Auditory startle response
- PND 63-75: Functional observational battery and motor activity

- Auditory startle response: Each animal was placed in a container and observed for Preyer's reflex in response to a sound stimulus. Each sessions consisted of 50 trials. The mean response amplitude on each block of 10 trials (total: 5 blocks of 10 trials) was determined, with test conditions optimised to produce intra-session habituation.
The day of testing was counterbalanced across treated and control groups.

OBSERVATIONAL SCREENING
- Righting reflex: The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; for this outcome (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
- Body temperature: An electronic probe thermometer with a blunt probe was used to take the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
- Salivation: Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.
- Startle response: With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, whereby a zero (0) was recorded in the blank space of the scoring sheet. If there was no response, a plus sign (+) was recorded.
- Respiration: While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
- Mouth breathing: Rats are normally obligatory nose-breathers. Each animal was observed whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded; mouth breathing was documented by a plus sign (+).
- Urination: When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyurea) with 3 being normal.
- Convulsions: If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.
- Pilo-erection: The fur of the animal's back was observed whether it was raised or elevated. In the normal animal no pilo-erection should be observed and a score of zero (0) was recorded. If pilo-erection was present, a plus sign (+) was recorded.
- Diarrhoea: In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state is for there to be none (0); in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
- Pupil size: It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
- Pupil response: The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign (-) was recorded in the blank space.
- Lacrimation: The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. If a discharge was present, a plus sign (+) was recorded.
- Impaired gait: The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).
- Stereotypy: Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns occurring out of context and with an abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).
- Toe pinch: The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.
- Tail pinch: The toe pinch procedure was utilised with the animal's tail instead of its hind limb and was graded on the same scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.
- Wire manoeuvre: The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
- Hind leg splay: The hind paws were marked with ink using an ink pad. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured (in cm).
- Positional passivity: The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
- Tremors: Periods of continued fine movements, usually starting in the limbs and perhaps limited to them. The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
- Positive geotropism: The animal was placed on the inclined (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.
- Limb rotation: One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.
- Auditory function: Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+); if there was no response a zero (0) was recorded.
- Posture: While in the cage, the animal body position was assessed and classified as either (A) asleep, i.e. lying on the side up or crouching, (S) sitting or normally standing, (R) rearing, i.e. standing on the hindlimbs, (H) hunchback, i.e. the back is rounded, even when walking, and the animal appears to be holding its stomach, (L) lying on the side with limbs in the air, (F) flattened, i.e. the animal is spread out with the abdomen pressed to the floor, or (C) catalepsy, i.e. the animal is in a cataleptic-like state. Only (S), (A) or, if a mild degree, (R) is considered normal.
- Palpebral closure: While in the cage, the closure of the eyes of the animal was assessed and graded on a scale from 1 (open eyes) to 3 (eyes completely shut).
- Vocalisation: While in the cage, occurrence of spontaneous vocalisation was scored with 1 point. Absence of vocalisation, considered to be normal, was scored with 0 points.
- Arousal: Outside of the cage, animal arousal was graded on a scale from 1 (decreased arousal) to 3 (increased arousal) with 2 being normal exploratory behaviour.
- Bizarre behaviour: Outside of the cage, each animal was observed for occurrence of abnormal / bizarre behaviour, e.g. squirming, running backwards. Absence of abnormal behaviour was rated zero (0), for any bizarre behaviour a score of 1 point was recorded. The type of bizarre behaviour would have been documented. However, no abnormal behaviour was noted.
- Stains: The appearance of the animals was assessed and graded on a scale from 0 (no stains) and 1 (minor stains) to 3 (marked stains).
- Ease of removal / Handling: The reaction of the animal to being removed from its cage and the ease of handling was scored with 1 (does not resist / very easy to handle), 2 (squeaks and / or exhibits mild resistance / very easy to handle), 3 (animal freezes and dose not move, becomes rigid in hand) or 4 points (very difficult; animal struggles, squirms, appears distressed, attempts to bite). Two (2) is considered normal.
- Muscle tone: While held in hand, the muscle tone of the animal was assessed and graded on a scale from 1 (soft; no resistance) to 3 (very hard; high resistance) with 2 being normal.
- Approach response: The tip of a dull object was slowly moved towards the front of the animal and hold at a distance of 2 to 3 cm for 4 seconds. The reaction of the animal was recorded on a scale from 1 (reduced response) to 3 (increased response) with 2 (animal slowly approaches, sniffs and pulls back) being normal.
- Touch response: The animal was gently touched on the abdomen with a dull object, and the reaction of the animal was scored with 1 (no reaction), 2 (slowly retreats (normal)), 3 (twitches (muscular tenseness)) or 4 points (exhibits violent overreaction such as jumping, biting, squeaking or attacking).
- Body weight: The weight of the animals on the day of the observational screening was recorded.

FUNCTIONAL TESTS
- Grip strength: Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. It was continued to pull the animal along the trough until the hind limbs grasped the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.
- Locomotor activity: The motility was measured using the TSE InfraMot system . The infrared sensor was placed on the cage and any movements were measured for a duration of 12 min by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.
The treatment groups were counter-balanced across devices and across test times to avoid confounding by circadian rhythm of activity.
Postmortem examinations (parental animals):
LABOBRATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
EDTA anticoagulant (whole blood): for haematological investigations
Citrate anticoagulant (plasma): for coagulation tests
LiHeparin anticoagulant (plasma): for clinical chemistry tests

Sampling time: at necropsy
Animals: 10 males and 10 females randomly selected from each F0 group.

HAEMATOLOGY:
The following parameters were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter (in blood)/ Unit
- Haemoglobin content (HGB)/ mmol/L
- Erythrocytes (RBC)/ 10^6/µL
- Leucocytes (WBC)/ 10^3/µL
- Differential blood count :
- relative/ %
- absolute/ 10^3/µL
- Reticulocytes (Reti)/ %
- Haematocrit value (HCT)/ %
- Platelets (PLT)/ 10^3/µL
- Mean corpuscular volume (MCV)/ fL
- Mean corpuscular haemoglobin (MCH)/ fmol
- Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
The following parameters were determined (instrument: Amax Destiny Plus™, TCoag Deutschland GmbH, 32657 Lemgo, Germany):
Parameter (in plasma)/ Unit
- Prothrombin time (PT)/ sec
- Activated partial thromboplastin time (aPTT)/ sec

BIOCHEMICAL PARAMETERS:
The following parameters were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany):
Parameter (in serum)/ Unit
- Albumin/ g/L
- Bile acids/ µmol/L
- Bilirubin (total)/ µmol/L
- Cholesterol (total)/ mmol/L
- Creatinine/ µmol/L
- Glucose/ mmol/L
- Protein (total)/ g/L
- Blood urea nitrogen (BUN)/ mmol/L
- Calcium/ mmol/L
- Chloride/ mmol/L
- Potassium/ mmol/L
- Sodium/ mmol/L
- Alanine aminotransferase (ALAT)/ U/L
- Alkaline phosphatase (aP)/ U/L
- Aspartate aminotransferase (ASAT)/ U/L
- Lactate dehydrogenase (LDH)/ U/L
by substraction:
- Globulin/ g/L
by calculation (non-dimensional):
- Albumin/globulin ratio
- Sodium/Potassium ratio
- BUN/creatinine ratio

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of test day 79/85 (at sacrifice) (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals)
10 males and 10 females randomly selected from each F0 group
Blood samples were processed for serum, divided into aliquots and stored at -20 °C ± 10% at the test institute until analyses using commercial ELISA kits as follows:
Parameter (in serum)/ Type of ELISA kit/ Instrument
- Total thyroxine (T4)/ T4 ELISA Kit Cat. no. RE55261, IBL Lot nos. 304K030, 304K050, 304K090/ Tecan Sunrise
- Thyroid-stimulating hormone (TSH)/ Rat TSH ELISA Kit Cat. no. RE45021, IBL Lot nos. V051, V055/ Tecan Sunrise
The LODs (limit of detection) and LLOQs (lower limit of quantification) for each parameter are given below:
LODs and LLOQs for thyroid hormones:
Thyroid hormone/LOD /LLOQ
Total thyroxine (T4)/ 8.0 nmol/L/ 25.0 nmol/L
Thyroid-stimulating hormone (TSH)/ 0.081 ng/mL/ 2.5 ng/mL

GROSS NECROPSY:
On the day of necropsy, vaginal lavages of the adult animals (F0 and F1 Generation) were obtained and examined to determine the stage of oestrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanised by carbon dioxide (CO2) inhalation and then exsanguinated by cutting the aorta abdominalis.
F0: Males, all, After weaning of F1 Generation, TD 85-86, full necropsy
F0: Females, After weaning of F1 Generation, TD 77-79, full necropsy
F1 Cohort 1B: shortyl after weaning of the F2 animals, PND 120 - 153, full necropsy

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or premature death during the study, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS:
F0 generation:
During necropsy, the number of implantation sites in the uteri of the female animals was recorded and used to evaluate reproductive performance.
Apparently non-pregnant uteri of the F0 animals were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI
The following organs of the male and female F0 animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right:
- Adrenal gland (2)
- Ovary (2)
- Prostate (dorsolateral and ventral parts combined)
- Brain
- Oviducts (2)
- Seminal vesicles with coagulating glands
- Epididymis (2)
- Spleen
- Pituitary
- Heart
- Testis (2)
- Uterus incl. cervix
- Kidney (2)
- Thyroid, including parathyroids (after fixation) (1, left)
- Liver
- Thymus
Weighing before fixation except for thyroids. Paired organs were weighed individually and identified as left or right.

The following organs or parts thereof obtained from the male and female animals of the F0 Generation were preserved in an appropriate fixative:
Davidson’s solution:
- Eye with optic nerve (2)
modified Davidson's solution
- Epididymis (1)#
- Testis (1)#
7% buffered formalin
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches; Swiss roll method)
- Spleen
- Stomach
- Intestine, large (colon, rectum)
- Thyroid (2, incl. parathyroids)
- Kidney and ureter (2)
- Thymus
- Liver
- Trachea (incl. larynx)
- Lungs (with mainstem bronchi and bronchioles)
- Urinary bladder
- Mammary gland
- Uterus (incl. cervix)
- Muscle (skeletal)
- Vagina
- Nerve (sciatic)
- Vas deferens
- Oesophagus
# The second epididymis and testis were not preserved but used for the spermiogram
Any other organs displaying macroscopic changes were also preserved. In addition, sperm viability and morphology were evaluated for all male F0 animals and bone marrow smears were prepared for selected F0 animals (same as used for laboratory examinations)

F1 generation Cohort 1B:
The following organs of male and female F1 Cohort 1B animals were weighed before fixation except for the vagina and the vas deferens. Paired organs were weighed individually and identified as left or right.
Organ/ Weigh/ Fixative/ Block
Endocrine system
- Adrenal gland (2)/ Yes/ 7% formalin/ No
- Pituitary/ Yes/ 7% formalin/ Yes
- Thyroid (2) (including parathyroids)/ 1, post-fixation/ 7% formalin/ No
Reproductive system
- Epididymis (2)/ Yes/ Modified Davidson's/ Yes
- Ovary (2)/ Yes/ 7% formalin/ Yes
- Prostate /Yes/ 7% formalin/ Yes
- Seminal vesicles with coagulating glands/ Yes/ 7% formalin/ Yes
- Testicle (2)/ Yes/ Modified Davidson's/ Yes
- Uterus (including oviducts and cervix)/ Yes/ 7% formalin/ Yes
- Vagina/ No/ 7% formalin/ Yes
- Vas deferens/ No/ 7% formalin/ No
The adrenals, thyroids and vas deferens were not processed to block stage as this was not required by the current OECD TG 443 (paragraph 67) adopted 25 June 2018.
In the case of test item-related changes in the corresponding organs of the F0 and F1 Cohort 1A animals, the Study Monitor would have been given sufficient notice before the respective organs of F1 Cohort 1B animals were sectioned and examined histopatho-logically. However, histopathological examination of the F1 Cohort 1B animals was not necessary.

BONE MARROW:
During dissection, fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of 10 male and 10 female F0 and F1 Generation Cohort 1A animals and stained according to PAPPENHEIM. The myeloid:erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.

HISTOPATHOLOGY:
Blood smears were prepared from all samples during the haematological examination and were stored for possible histopatho-logical examination. However, they were examined and evaluated only depending on necropsy findings and upon agreement with the study monitor.
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F0 Generation: 20 randomly selected animals/sex/group
A full histopathology was also performed for all deceased or prematurely sacrificed animals from the F0 Generation.
The organs listed above were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the selected control and high dosed animals of groups 1 and 4 (i.e. 20 male animals/group) of the F0 Generation following H&E and PS staining.
In addition, histopathological evaluation was performed on H-E-stained sections of the following organs of groups 2 and 3 of the F0 Generation:
Organ/ Number and sex of animals
- Adrenal gland (2)/ 20 males/group = 40 males + 20 females/group = 40 females
- Kidney (2)/ 20 males/group = 40 males
- Liver/ 20 males/group = 40 males + 20 females/group = 40 females
- Thyroid (incl. parathyroids) (2)/ 20 males/group = 40 males + 20 females/group = 40 females

F1 Cohort 1B animals:
In the case of test item-related changes in the corresponding organs of the F0 and F1 Cohort 1A animals, the Study Monitor would have been given sufficient notice before the respective organs of F1 Cohort 1B animals were sectioned and examined histopathologically. However, histopathological examination of the F1 Cohort 1B animals was not necessary.

Details of the Histology processing and the Histopatholigical evaluation could be found in the histopathological report attached to this study endpoint record.
Postmortem examinations (offspring):
LABOBRATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
EDTA anticoagulant (whole blood): for haematological investigations
Citrate anticoagulant (plasma): for coagulation tests
LiHeparin anticoagulant (plasma): for clinical chemistry tests

Sampling time: at sacrifice
Animals: 10 males and 10 females randomly selected from each F1 cohort 1A group.

HAEMATOLOGY:
The following parameters were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter (in blood)/ Unit
- Haemoglobin content (HGB)/ mmol/L
- Erythrocytes (RBC)/ 10^6/µL
- Leucocytes (WBC)/ 10^3/µL
- Differential blood count :
- relative/ %
- absolute/ 10^3/µL
- Reticulocytes (Reti)/ %
- Haematocrit value (HCT)/ %
- Platelets (PLT)/ 10^3/µL
- Mean corpuscular volume (MCV)/ fL
- Mean corpuscular haemoglobin (MCH)/ fmol
- Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
The following parameters were determined (instrument: Amax Destiny Plus™, TCoag Deutschland GmbH, 32657 Lemgo, Germany):
Parameter (in plasma)/ Unit
- Prothrombin time (PT)/ sec
- Activated partial thromboplastin time (aPTT)/ sec

BIOCHEMICAL PARAMETERS:
The following parameters were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany):
Parameter (in serum)/ Unit
- Albumin/ g/L
- Bile acids/ µmol/L
- Bilirubin (total)/ µmol/L
- Cholesterol (total)/ mmol/L
- Creatinine/ µmol/L
- Glucose/ mmol/L
- Protein (total)/ g/L
- Blood urea nitrogen (BUN)/ mmol/L
- Calcium/ mmol/L
- Chloride/ mmol/L
- Potassium/ mmol/L
- Sodium/ mmol/L
- Alanine aminotransferase (ALAT)/ U/L
- Alkaline phosphatase (aP)/ U/L
- Aspartate aminotransferase (ASAT)/ U/L
- Lactate dehydrogenase (LDH)/ U/L
by substraction:
- Globulin/ g/L
by calculation (non-dimensional):
- Albumin/globulin ratio
- Sodium/Potassium ratio
- BUN/creatinine ratio

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below:
- 10 males and 10 females randomly selected from each F1 cohort 1A group
- F1 pups (2 surplus pups per litter, all litters if possible) on PND 4 (T4 only)
- F1 pups (2 surplus pups per litter, all litters) on PND 22
- F2 pups 10 males an 10 females randomly selected at sacrifice PND 21
Blood samples were processed for serum, divided into aliquots and stored at -20 °C ± 10% at the testing facility until analyses using commercial ELISA kits as follows:
Parameter (in serum)/ Type of ELISA kit/ Instrument
- Total thyroxine (T4)/ T4 ELISA Kit Cat. no. RE55261, IBL Lot nos. 304K030, 304K050, 304K090/ Tecan Sunrise
- Thyroid-stimulating hormone (TSH)/ Rat TSH ELISA Kit Cat. no. RE45021, IBL Lot nos. V051, V055/ Tecan Sunrise
The LODs (limit of detection) and LLOQs (lower limit of quantification) for each parameter are given below:
LODs and LLOQs for thyroid hormones:
Thyroid hormone/LOD /LLOQ
Total thyroxine (T4)/ 8.0 nmol/L/ 25.0 nmol/L
Thyroid-stimulating hormone (TSH)/ 0.081 ng/mL/ 2.5 ng/mL

GROSS NECROPSY:
On the day of necropsy, vaginal lavages of the adult animals (F0 and F1 Generation) were obtained and examined to determine the stage of oestrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanised by carbon dioxide (CO2) inhalation and then exsanguinated by cutting the aorta abdominalis.
The pups were euthanised by decapitation (PND 4) or by carbon dioxide (CO2) inhalation (PND 21/22).
A gross or full necropsy of the animals of the F0 and F1 animals was carried out. At gross necropsy, the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighing of selected organs.
Blood samples for determination of haematological and biochemical parameter as well as for thyroid hormone determination were taken.
The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:
F1: "surplus" pups, on PND 4, gross necropsy
F1: "surplus" pups, on PND 22, gross necropsy including assessment of reproductive organs (10 ‘surplus’ pups/sex/group: Gross necropsy and partial organ/tissue preservation)
F1 Cohort 1A: at the end of the dosing period (PND 90-95), full necropsy
F1 Cohort 2A: after behavioural testing, around PND 75
F1 Cohort 2B: on PND 21/ 22, full necropsy
F2 all pups: on PND 21, Gross necropsy including assessment of reproductive organs
Animals that were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.

NECROPSY OF DEAD OR SURPLUS PUPS
External inspection for gross abnormalities:
Dead pups and culled F1 Pups on PND 4 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
External and internal inspection for gross abnormalities:
Pups not selected for the F1 Cohorts were sacrificed on PND 21/22 and examined macroscopically for any abnormalities or pathological changes.

DISSECATION OF ALL ADULT ANIMALS:
At the time of sacrifice or premature death during the study, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS:
Weighing / Preservation of organs - 'Surplus' F1 and F2 pups:
Ten 'surplus' F1 and F2 pups per sex and group were used for organ weighing and preservation.
The following organs/tissues of the F1 and F2 pups were weighed and/or preserved in 7% formalin:
- Brain#
- Spleen#
- Gross Abnormalities
- Thymus#
- Identified target organs
# Organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.

Weighing / Preservation of organs - F1 Generation Cohort 1A:
The following organs of all adult male and female F1 Cohort 1A animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.
Weighing of the following organs/tissues#1
- Adrenal gland (2)
- Lymph node (1, mesenteric)
- Spleen
- Brain
- Ovary (2)
- Testis (2)
- Epididymis (2)
- Oviducts (2)
- Thymus
- Heart
- Pituitary
- Uterus incl. cervix
- Kidney (2)
- Prostate (dorsolateral and ventral parts combined)
- Liver
- Seminal vesicles with coagulating glands
- Lymph node (1, cervical)
- Thyroid incl. parathyroids (after fixation) (1, left)
#1 Weighing before fixation except for thyroids. Paired organs were weighed individually and identified as left or right.
#2 Lymph nodes of 10 animals/sex/group for Cohort 1A were weighed (1 animal per litter, all litters represented by at least 1 F1 animal; randomly selected).

The following organs or parts thereof of all adult male and female animals of the F1 generation Cohort 1A were preserved in an appropriate fixative:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: modified Davidson’s solution
- Epididymis (1)#1
- Testis (1)#1
Fixative: 7%buffered formalin
- Adrenal gland (2)
- Oesophagus
- Bone
- Ovary (2)
- Bone marrow (os femoris)
- Oviduct (2)
- Brain (cerebrum, cerebellum, pons)
- Pituitary
- Gross lesions observed
- Prostate
- Heart (3 levels: right and left ventricle, septum)
- Seminal vesicles with coagulating glands
- Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches; Swiss roll method)
- Spinal cord (3 sections)
- Spleen#3
- Intestine, large (colon, rectum)
- Stomach
- Kidney and ureter (2)
- Thyroid (2, incl. parathyroids)
- Liver
- Thymus
- Lungs (with mainstem bronchi and bronchioles)
-Trachea (incl. larynx)
- Lymph node (1, cervical)#2
- Urinary bladder
- Lymph node (1, mesenteric)#2
- Uterus (incl. cervix)
- Mammary gland
- Vagina
- Muscle (skeletal)
- Vas deferens
- Nerve (sciatic)
#1 The second epididymis and testis were not preserved but used for the spermiogram.
#2 Lymph nodes were preserved for all Cohort 1A animals, however, histopathology was only carried out for 10 animals/sex/group (1 animal per litter, all litters represented by at least 1 F1 animal; randomly selected).
#3 For 10 animals/sex/group of Cohort 1A, randomly selected (same as selected for laboratory examination): One half of the spleen was preserved for histopathological evaluation, the second half was used for splenic lymphocyte subpopulation analysis.

Any other organs displaying macroscopic changes were also preserved. In addition, sperm viability and morphology were evaluated for all male animals of F1 Cohort 1A and bone marrow smears were prepared for selected animals of F1 Cohort 1A animals.

Weighing / Preservation of organs - F1 Generation Cohorts 2A, 2B:
The following organs of male and female F1 Cohort 2A and 2B animals listed below were weighed before fixation and/or preserved. Paired organs were identified as left and right.
Weighing and preservation of organs/tissues
Cohort 2A:
7% buffered formalin
- Brain
- Nerve (sciatic) #
- Liver
- Seminal vesicle, incl. coagulating glands
- Muscle (skeletal) #
- Spinal cord (3 sections) #
- Davidson's solution
- Eye with optic nerve and retina (2) #
Cohort 2B:
7% buffered formalin
- Brain
# Organ weight was not determined
Liver and seminal vesicles with coagulating glands were weighed and preserved from all animals of Cohort 2A in order to verify findings of the Pubertal Developmental Assay (Marty et al. (2011); (see Section 3.7). Animals of Cohort 2A were used as their age at dissection was closest to the age of the animals examined by Marty et al. (2011).

BONE MARROW:
During dissection, fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of 10 male and 10 female F1 Generation Cohort 1A animals and stained according to PAPPENHEIM. The myeloid:erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.

PHENOTYPIC ANALYSIS OF SPLEEN CELLS:
The spleens of selected male and female F1 Cohort 1A animals were split in two halves. The portion of the spleen not preserved for histopathology was minced using a mechanic dissociator to prepare single cell suspensions.
The following parameters were determined in the samples by using the instruments given below:
Instrument: MACSQuant® Analyzer 10/16, Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, 51429 Bergisch Gladbach, Germany
- CD4+ T-Lymphocytes
- CD8+ T-Lymphocytes
- Pan-T-lymphocytes (CD3+)
- B-lymphocytes (CD45RA+)
- Natural killer cells (CD161+)
Evaluation was performed by the test institute.

HISTOPATHOLOGY:
Blood smears were prepared from all samples during the haematological examination and were stored for possible histopatho-logical examination. However, they were examined and evaluated only depending on necropsy findings and upon agreement with the study monitor.
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F1 Generation (Cohort 1A): 20 randomly selected animals/sex/group
A full histopathology was also performed for all deceased or prematurely sacrificed animals from the F1 Generation Cohort 1A.
The organs listed above were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the selected control and high dosed animals of groups 1 and 4 (i.e. 20 male animals/group) of the F1 Generation Cohort 1A following H&E and PS staining.
In addition, histopathological evaluation was performed on H-E-stained sections of the following organs of groups 2 and 3 of the F1 Generation Cohort 1A:
Organ/ Number and sex of animals
- Adrenal gland (2)/ 20 males/group = 40 males + 20 females/group = 40 females
- Kidney (2)/ 20 males/group = 40 males
- Liver/ 20 males/group = 40 males + 20 females/group = 40 females
- Thyroid (incl. parathyroids) (2)/ 20 males/group = 40 males + 20 females/group = 40 females

F1 Cohort 2A + 2B animals - Developmental neurotoxicity:
Neurohistopathological evaluation was performed of the organs/tissues listed below of the F1 Cohort 2A and 2B animals of groups 1 and 4.
Cohort 2A:
- Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem, and cerebellum)
- Eye with optic nerve (2)
- Muscle (skeletal)
- Nerve (sciatic)
- Spinal cord (3 sections)
Cohort 2B:
- Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem, and cerebellum)

Histology processing included:
- H-E, Fluoro-Jade and Luxol Fast Blue / Chresylviolet staining for brain tissue;
- H-E staining for all other organs listed above
Histopathological evaluation included:
- alterations in the gross size or shape of the olfactory bulbs, cerebrum or cerebellum;
- alterations in the relative size of various brain regions, including decreases or increases in the size of regions resulting from the loss or persistence of normally transient populations of cells or axonal projections (e.g. external germinal layer of cerebellum, corpus callosum);
- alterations in proliferation, migration and differentiation, as indicated by areas of excessive apoptosis or necrosis, clusters or dispersed populations of ectopic, disoriented or malformed neurons or alterations in the relative size of various layers of cortical structures;
- alterations in patterns of myelinations, including an overall size reduction or altered staining of myelinated structures;
- evidence of hydrocephalus, in particular enlargement of ventricles, stenosis of the cerebral aqueduct and thinning of the cerebral hemispheres;
- morphometric (quantitative) evaluation: linear or areal measurement of the following major brain regions: cerebral cortex, mid-brain (thecum, tegmentum and cerebral peduncles), brain stem and cerebellum.
In addition, the organs listed of all F1 Cohort 2A group 1 and 4 animals were examined histopathologically after preparation of paraffin sections and H-E staining in order to verify the findings of the Pubertal Development Assay (Marty et al., 2011).

Organs to be histopathologically evaluated - F1 Cohort 2A:
- Liver
- Seminal vesicles with coagulating glands

Details of the Histology processing and the Histopatholigical evaluation could be found in the histopathological report attached to this study endpoint record.
Statistics:
DATA ACQUISITION
The following data were captured or calculated by the departmental computerised system Provantis: Clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters.
Data maintained on paper (e.g. pup data, ELISA experiments) were entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.
STATISTICS
The statistical evaluation of the parametrical values was done by Provantis® using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
For the statistical evaluation of the histopathological data FISHER's exact test was used.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to 1 may occur caused by rounding.
Significantly different data are indicated in the summary tables and the result tables.
Reproductive indices:
F0 GENERATION:
For each F0 group the gestation index is determined:
Gestation Index = (Number of dams with live pups/ Number pregnant rats) x 100
Fertility Index female [%] = (Number of pregnant rats with verified copulation/ Number of rats with verified copulation) x 100

F1 GENERATION COHORT 1B:
For each F1 Cohort 1B group the gestation index is determined:
Gestation Index = (Number of dams with live pups/ Number pregnant rats) x 100
Fertility Index female [%] = (Number of pregnant rats with verified copulation/ Number of rats with verified copulation) x 100
Offspring viability indices:
F0 GENERATION: For each litter and group the following indices are determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars) x 100
Live Birth Index = (Number of pups born alive on day 0/1/ Total number born (live + dead)) x 100
Viability Index pre-select= (Number of pups alive on day 4 (pre-select)/ Number of pups live on day 0/1) x 100
Viability Indexpost select= (Number of pups alive on day 21 (post-select)/ Number of pups live on day 0/1) x 100
Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100

F1 GENERATION COHORT 1B:
For each litter and group the following indices are determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars) x 100
Live Birth Index = (Number of pups born alive on day 0/1/ Total number born (live + dead)) x 100
Viability Indexpre-select= (Number of pups alive on day 4 (pre-select)/ Number of pups live on day 0/1) x 100
Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in behaviour, the external appearance or the faeces that were considered to be adverse were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

Male animals:
Salivation was noted for all male animals of the dose groups and in some cases, the saliva was red discoloured. Salivation started immediately to 5 minutes or 20 to 60 minutes after dosing and was not noted anymore after 60 minutes post dosing at maximum. A dose-dependent increase of the severity and the number of observations was noted. Therefore, salivation was considered to be test item-related. However, as salivation was only noted for at maximum one hour post administration, the transient observation of salivation was considered to be not adverse.
Reduced motility was noted for all male animals of the intermediate dose group at start of the dosing period on TD 15 and for all high dose males on TD 15 and TD 16. Additionally, one animal was noted with reduced motility on TD 51 and TD 54. Reduced motility started 20 to 60 minutes after dosing and lasted up to 60 minutes after dosing. As reduced motility was with two exceptions only noted on the first day(s) at start of dosing, reduced motility was considered to be test item-related, but not adverse.
Red discoloured urine was noted for one animal each of the low and intermediate dose group. In detail, the low dose male no. 58 was noted with red discoloured urine on TD 56 and TD 65 and the intermediate dose male no. 108 was noted with red discoloured urine on 15 days between TD 29 and TD 86. In addition, in the high dose group, all male animals displayed dark yellow discoloured urine on 4 consecutive days starting one day after start of administration (TD 16 to TD 19). As the dark yellow discolouration of the urine was noted for all high dose males, the dark yellow discoloured urine was considered to be test item-related. However, as the dark yellow discolouration of the urine was not noted after TD 19 anymore, these transient observations were considered to be not adverse. The single occurrences of red discoloured urine noted for one low dose male and one intermediate dose male was considered to be not test item-related.
The observations of piloerection, diarrhoea, increased water consumption, pultaceous faeces and haemorrhagic canthus were only noted for up to two days for single animals and therefore were considered to be spontaneous.

Female animals:
Nearly all female animals of all dose groups displayed salivation including one intermediate dose female that was noted with red saliva for three days in the pre-mating period. In the female animals, a dose-dependent increase was noted for the severity and the number of days salivation was observed. However, salivation was noted for only up to one hour post administration and therefore, salivation was considered to be test item-related but not adverse.
Dark yellow discoloured urine was noted for all high dose females during the test days 16 to 19. As all female high dose animals displayed dark yellow discoloured urine, this observation was considered to be test item-related. However, as the observation was noted in all animals for the same 4 consecutive days, dark yellow discoloured urine was considered to be not adverse.
Reduced motility was observed on TD 15 for all females of the intermediate dose group. In the high dose group, reduced motility was noted between TD 15 and TD 21 for all females and for two females for one day each on GD 19 or GD 20. As all female animals displayed reduced motility, the observation was considered to be test item-related. However, due to the occurrence for only up to three days per animal at maximum, reduced mobility was considered to be not adverse. Reduced motility started 20 to 60 minutes after administration and lasted not longer than 60 minutes after administration.
Increased water consumption and diarrhoea were noted only for a few days for a low number of animals and therefore, were considered to be spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males and females
No test item-related premature death was noted for the dose groups (65, 200 or 600 mg test item/kg b.w./day) and the control group.
However, the male control animal no. 9 was found dead in the morning of TD 35. Necropsy revealed oedematous lungs, white deposits and approximately 3 mL of a turbid liquid in the thorax and enlarged adrenals. Histopathological examination of animal no. 9 revealed inflammation of the oesophagus, the larynx and of the tissue surrounding the thymus. In the lungs, alveolar oedema, congestion and alveolar macrophages were noted, while the adrenal glands showed diffuse cortical hypertrophy. The premature death of animal no. 9 was considered to be due to a misgavage. The enlarged adrenal glands were considered to be a stress-related change.
Furthermore, two prematurely deceased males were noted for the high dose group (nos. 161 and 166): Animal no. 161 was found dead during TD 45 and displayed piloerection and moderate salivation in the days prior to its death. During necropsy, oedematous lungs were observed, as well as the thorax filled with 2 mL of a clear and viscous liquid and the neck region being gelatinous. During the histopathological examination, inflammation was noted for the oesophagus and the surrounding tissue, the trachea, the larynx and the surrounding tissue of the thymus. Further, alveolar oedema and congestions were observed for the lungs, as well as centrilobular hepatocellular hypertrophy in the liver and hyaline droplets in the kidneys. Animal no. 166 was found dead during TD 36. Necropsy revealed the left lobe of the lungs being oedematous and dark-red discoloured, the thorax was filled with approximately 2 mL of a viscous liquid and the liver was enlarged. Histopathological examination of the animal revealed pathological changes of the larynx (inflammation), the lungs (alveolar oedema, congestion), the thymus (atrophy), liver (centrilobular hepatocellular hypertrophy, focal necrosis and congestion), the thyroid glands (follicular cell hypertrophy) and the kidneys (hyaline droplets). While hepatocellular hypertrophy was a treatment-related change observed in this study, it was not considered to have contributed to the death of animal no. 166.
In conclusion, the deaths of the animals nos. 161 and 166 were also considered to be not test item-related but due to a misgavage.
None of the female animals died prematurely.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day), a reduced body weight was noted from TD 36 until TD 84 before sacrifice on TD 85/86 (at maximum 7.9% below the value of the control group on TD 84, statistically significant at p ≤ 0.05 or 0.01). This constantly lower body weight was considered to be test item-related and adverse.
In consequence to the reduced body weight for the high dose group, also the body weight gain from TD 15 to TD 84 was decreased for the high dose males.

Females:
No test item-related changes in body weight were noted for the female rats between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), a transiently reduced body weight was noted at the end of the gestation period on GD 21 and at the beginning of the lactation period on LD 1 (8.1% and 6.1% below the value of the control group on GD 21 and LD 1, statistically significant at p ≤ 0.05 and 0.01), respectively. However, as the body weight recovered during the lactation period, the transiently reduced body weight was considered to be not test item-related but spontaneous.
In accordance with the body weight, also no changes were noted for the female body weight gain of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

Body weight at autopsy
Males and females:
No test item-related differences for the body weight at autopsy of the male and female animals were noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day) as well as for the female high dose group (600 mg test item/kg b.w./day).
For the male high dose group (600 mg test item/kg b.w./day), a decreased body weight at autopsy was observed (9.1% below the value of the control group, statistically significant at p ≤ 0.01), which is in accordance with the test item-related decrease of the body weight noted between TD 36 and TD 84. Therefore, also the decreased body weight at autopsy was considered to be test item-related and adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
No adverse differences in food consumption were noted between the control male animals and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Reductions in food consumption were noted for the low, intermediate and high dose groups in the first week of dosing (8.4%, 11.5% and 15.9% below the value of the control group, respectively, statistically significant at p ≤ 0.01). Further decreased values for the food consumption were observed for the low dose group between TD 22 and TD 29 and between TD 56 and TD 63 (15.7% and 6.5% below the value of the control group, respectively, statistically significant at p ≤ 0.01). However, as the male food consumption recovered thereafter and no dose response-relationship was present, the reduced food consumption was considered to be test item-related but not adverse.
However, for the high dose males, an increased food consumption was noted from TD 49 onwards until TD 84, one day before termination of the F0 males (at maximum 9.2% above the value of the control group, statistically significant at p ≤ 0.05 or 0.01). Nevertheless, an increased food consumption in the high dose group was considered to be not toxicologically relevant.
No food intake of male animals was recorded during the mating period as both sexes were housed together.

Females: Pre-mating, gestation and lactation period
No test item-related differences in female food consumption were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A reduced food consumption was noted for the female animals of the dose groups at start of the dosing. In detail, a decreased food consumption was noted for all dose groups between TD 15 and TD 22, for the low and intermediate dose groups between TD 22 and TD 29 and again for all dose groups after mating between GD 0 and GD 7, ranging from 5.5% to 23.0% below the value of the control group (statistically significant at p ≤ 0.05 or 0.01). Thereafter, the food consumption recovered and no difference between the dose groups and the control group was noted in the third week of gestation and the lactation period. Therefore, the transiently reduced food consumption was considered to be test item-related but not adverse.
No food intake of female animals was recorded during the mating period as both sexes were housed together.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females: No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day).
Slight statistically significant differences in comparison to the control group were noted for the MCHC of the male high dose animals. In addition, for the female animals a slight but statistically significant reduction for the number of eosinophilic granulocytes in the differential blood count for the high dose animals was noted. However, both these changes were considered to be spontaneous, as no dose response-relationship was noted, no changes were noted for the other gender, and the values were within the range of the test institute´s background data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females:
No test item-related differences for the examined biochemical parameters were noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day), increased levels were noted for globulin, for total cholesterol and for total protein in both sexes. The increased levels for globulin and total protein of the male high dose animals were above the 95%-confidence interval of the test institute´s background data. Furthermore, the increases were also noted for the male and female animals of Cohort 1A. Therefore, the increased levels of globulin, total cholesterol and total protein were considered to be test item-related and adverse.
Endocrine findings:
no effects observed
Description (incidence and severity):
Males and females: No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Statistically significant differences for the T4 and TSH serum levels were noted for the male and female animals. The T4 serum levels of the male and female low and intermediate dose animals were outside 5% to 95% confidence interval of the test institute´s background data but no dose-dependence relationship was present. The decreased TSH levels of the female low and intermediate dose animals were within the 5% to 95% confidence interval of the test institute´s background data and no dose-response relationship was present. Therefore, the changes for the serum thyroid hormone levels in the low and intermediate dose groups were considered to be not test item-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females: No test item-related differences were noted between the control group and the male animals of the low dose group (65 mg test item/kg b.w./day) and for the females of any dose group for the examined urinalysis parameters.
For the male intermediate and high dose animals (200 or 600 mg test item/kg b.w./day), a statistically significantly decreased pH was noted (6.0% and 5.7% below the value of the control group, respectively; p ≤ 0.01). The values for the male intermediate and high dose animals were below the 5% to 95%-confidence interval of the test institute´s background data and a decreased pH was also noted for the male intermediate and high dose animals of Cohort 1A. Therefore, the decreased pH of the urine of the male intermediate and high dose animals was considered to be test item-related and adverse
Additionally, erythrocytes in the urine were noted. In the male animals, erythrocytes were observed for either one or two of 10 animals of the control group (animal no. 3), the low dose group (animals nos. 52 and 58) and the high dose group (animals nos. 152 and 164). For the female animals, erythrocytes in the urine were noted for 1 of 10 low dose animals (animal no. 78) and for 1 of 10 intermediate dose animal (animal no. 139). However, as no dose-response relationship was present for the male and female animals, the findings of erythrocytes in the urine was considered to be not test item-related but spontaneous. While erythrocytes could be observed microscopically, their concentration was too low to have an impact on the visual appearance of the urine, hence no red discolouration of the urine was observed.
All other parameters evaluated (e.g. presence of leucocytes, crystalluria, etc.) did not show any difference between the control group and the dose groups in any sex.
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females:
Test item-related but non-adverse findings were observed for the following organs: In the adrenal glands, an increased incidence and severity of fine vacuolation was noted in the zona fasciculata of male and female animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day). In the thyroid glands, a slightly increased incidence of follicular cell hypertrophy was noted for the intermediate and high dose males and females. These findings were test item -related, but of adaptive nature and not adverse.
Furthermore, findings of centrilobular hepatocellular hypertrophy were noted with increasing incidence and severity in the livers of the males of all dose groups (65, 200 or 600 mg test item/kg b.w./day) and of the female animals of the intermediate and high dose groups. For the male animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day), a dose-dependently increased incidence was noted for hyaline droplets in the proximal tubular cells of the kidneys. As the effects at the highest dose level of 600 mg test item/kg b.w./day indicate towards previously reported changes seen in carcinogenicity studies, the findings were considered to be adverse at the high dose level and thus, the histomorphological no-observed-adverse-effect-level (NOAEL) could be established at a dose level of 200 mg test item/kg b.w./day.
Details can be found in the Histopathology Phase Report.

For male and female reproductive organs, no test item-related changes were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Bone marrow: Males and females: No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group of both sexes.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Exposure period:
After the allocation of the animals to the test groups and the start of treatment on test day 15, the oestrous cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign (verification of copulation) was noted.
No test item-related differences were noted for the mean length and the mean number of oestrous cycles per dam during the pre-mating period between the female animals of the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
A statistically significantly increased mean cycle length of 4.5 days in comparison to 4.3 days in the control group was noted at the high dose level (600 mg test item/kg b.w./day). However, as no dose-response relationship was present the increased oestrous cycle length in the pre-mating period was considered to be not test item-related. In addition, the difference to the control group was only marginal over all groups.
In addition, a reduced number of oestrous cycles was noted for the period between start of dosing on TD 15 and start of pairing on TD 29 for the high dose group (1.4 cycles in the high dose group compared to 2.2 cycles in the control group). However, as this was due to the not test item-related increase in oestrous cycle length, also the reduced number of oestrous cycles was considered to be not test item-related.

Stage of estrous cycle at necropsy: No test item-related differences were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) in the distribution of the stages of the estrous cycle. A slightly decreased number of females in the metoestrus stage and an increased number of females in the dioestrus stage were noted for the females of the high dose group based on the samples obtained by vaginal lavage at necropsy. However, the results of the histopathologic examination showed very similar rates for the different oestrous cycle stages, with most animals being in the stage of “lactational dioestrus” as specified by the histopathologist.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm number:
No test item-related difference was noted between the rats of the control group and the rats dosed with 65, 200 or 600 mg test item/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.

Sperm motility:
No test item-related differences were noted between the rats of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.

Sperm morphology:
The examination of spermatozoa from the epididymal cauda revealed no increased numbers of spermatozoa with a malformation in the dose groups (65, 200 or 600 mg test item/kg b.w./day) in comparison to the control group.
In detail, banana-like sperm heads were noted for three males of the control group, for two males of the low dose group and for three males of the intermediate dose group.

Additionally, all male animals did inseminate their female partner.
Reproductive performance:
no effects observed
Description (incidence and severity):
Female fertility: No test item-related influence on the fertility index was noted for the female rats of the treatment groups (65, 200 or 600 mg test item/kg b.w./day).
One female each of the control group (no. 36) and the low (no. 92) and intermediate dose group (no. 140) and 2 animals at the high dose group (nos. 177 and 185) did not become pregnant. This is in the range of normal biological variability.

Gestation index: No difference was noted for the gestation index between the females of the control group and the females dosed with 65, 200 or 600 mg test item/kg b.w./day).
All pregnant females delivered live pups.

Pre-coital time: No test item-related differences were noted for the length of the pre-coital time between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).

Gestation length: No test item-related differences were noted for the length of the gestation period between the rats of the control group and those of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly shortened gestation length was noted for the dams of the high dose group (2.1% below the value of the control group, p ≤ 0.01). However, a slightly shorter gestation length was considered to be of no toxicological relevance.
General toxicity
Parental male and female animals (F0 Generation)
No deaths that were considered to be test item-related were noted at any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Test item-related but non-adverse changes for the behaviour and the external appearance were noted in form of salivation for the male and female animals of all dose groups and in the form of reduced motility for the male intermediate and high dose animals.
A test item-related decrease was noted for the body weight of the male animals of the high dose group (600 mg test item/kg b.w./day) from TD 36 until sacrifice.
No adverse differences in food consumption were noted between the control group and the dose groups.
No test item-related influences were noted for the male and female animals on food consumption, drinking water consumption, the haematological parameters, the levels of the thyroid hormones and the sperm parameters.
During urinalysis, a decreased pH was observed for the male animals of the intermediate and high dose animals (200 or 600 mg test item/kg b.w./day).
For the biochemical parameters, increased levels of globulins, total cholesterol and total protein were noted for the male and female high dose animals (600 mg test item/kg b.w./day).
During the macroscopic examination, enlarged adrenals were noted for one intermediate dose female and 4 high dose females. This was in accordance with the increased weights of the left and right adrenals of the male and female high dose animals. In addition, the weights of the liver were increased in the male and female high dose animals as well as the weights of the left and right kidneys for the male and female intermediate and high dose animals (200 or 600 mg test item/kg b.w./day).
No changes were noted during the examination of the bone marrow.
During histopathological examination, increased incidences of pathological changes were noted for the kidneys (increased hyaline droplets in the proximal tubular cells) of the male animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day). Further pathological changes considered to be related to the test item were noted for the liver (increased centrilobular hepatocellular hypertrophy) of the males of all dose groups and for the intermediate and high dose females. As the effects at 600 mg/kg b.w./day indicate towards previously reported changes in carcinogenicity studies, the findings were considered to be adverse at the high dose level.
Further findings were noted for the thyroid glands (increased follicular cell hypertrophy) and adrenal glands (increased fine vacuolation) of the male and female intermediate and high dose animals. These findings were considered to be test item related but not adverse. No test item-related observations were noted for the reproductive organs during the histopathological examination of the male and female animals of any dose group.

Reproductive toxicity
Parental females
No influences were noted on the number or length of the oestrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity (F0)
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity (F0)
Effect level:
65 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B: Males:
Males
No test item-related changes of behaviour, external appearance or the faeces that were considered to be adverse were noted for the male animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Transient changes in behaviour or external appearance were noted for the control group and for the high dose group. In detail, the male control animal no. 367 was observed with a hemorrhagic nose/snout on TD 108. Furthermore, in the high dose group, three male animals (nos. 483, 488 and 494) displayed reduced motility on TD 15 and animal no. 494 additionally, showed an increased water consumption on TD 105. The high dose animal no. 498 was noted with breathing sounds and gasping on TD 90 and with piloerection on TD 94 and 95. Due to the low number of observations, the above-mentioned changes were considered to be not test item-related.
In addition to these single occurrences, also in Cohort 1B salivation was noted in all male animals of any dose group with a dose-dependent increase in severity and number of days with salivation per animal. For two animals each of the intermediate dose group (nos. 446 and 456) and the high dose group (nos. 491 and 492) the saliva was occasionally red discoloured. However, as salivation for the male animals of any dose group was noted in almost all cases not longer than 60 minutes post administration, salivation was considered to be test item-related but not adverse.
Females:
No test item-related observations considered to be adverse were noted for the female animals of any dose group (65, 200 or 600 mg test item/kg b.w./day).
Single occurrences of reduced motility were noted during the pre-mating/mating period for 4 of 20 high dose group females and in the lactation period for 2 of 19 females for one or two days per animal.
Salivation was noted for 5 of 20 control females during the pre-mating/mating period for one or two test days per animal. In the low dose group, salivation was observed for all animals in the pre-mating/mating period and for 7 or 10 of 18 animals in the gestation and lactation period. Furthermore, all female animals of the intermediate and high dose group displayed salivation in all study periods (see Text Table 9 2 on the following page). However, as also for the female animals the salivation was noted for not longer than 60 minutes post administration, the test item-related salivation was considered to be not adverse.
Start and duration of observations
With the exception of two observations of salivation for male animals that were noted before start of application, all observations were noted immediately after application or within 5 minutes after application. The duration of salivation was for one male animal consistently during the day. For one male animal, salivation ended one to two hours and for another animal 6 to 24 hours after application. All other observations of salivation ended 5 to 20 minutes or 20 to 60 minutes after application for the male and female animals. The observation of gasping for one male animal appeared immediately to 5 minutes after administration and disappeared 6 to 24 hours after administration. Reduced motility was noted for the male and female animals from immediately to 5 minutes after application until 5 to 20 minutes or 20 to 60 minutes after application.
Detailed clinical observations
The detailed clinical observations were performed once weekly.
Males:
No observations were noted during the detailed clinical observations for the male animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Females:
No observations were noted during the detailed clinical observations for the female animals of the low dose group (65 mg test item/kg b.w./day).
In the control group and in the intermediate dose group (200 mg test item/kg b.w./day) the female animals nos. 396 and 466 were noted with a loss of the tail tip from test week 1 or test week 9 onwards, respectively. In addition, the female animal no. 514 of the high dose group (600 mg test item/kg b.w./day) was noted with a wound in the neck region (approximately 4 mm in diameter) in test week 9. The single occurrences of a missing tail tip and a wound in the neck region noted in the intermediate or high dose groups are considered to be spontaneous.

COHORT 1A: Males and females:
No adverse changes in behaviour, the external appearance or the faeces that were considered to be adverse were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Single occurrences of changes of behaviour or external appearance were noted for a few animals of different groups. In detail, piloerection was noted for the male animal no. 211 of the control group, for the male animal no. 298 of the intermediate dose group on TD 2 and 3 and for the female animal no. 231 of the control group on TD 2 to 7. Furthermore, reduced motility was noted in the high dose group for the male animal no. 339 and for the female animals nos. 341 and 352 on TD 15 or on TD 15 and 16. All changes were regarded to be spontaneous.
Moreover, salivation was noted for all male and female animals of all dose groups and additionally, for the female animal no 236 of the control group on TD 44 .
Salivation started immediately to 5 minutes after administration and ended 5 to 20 or 20 to 60 minutes after administration. Reduced motility was observed immediately to 5 minutes after administration and lasted not longer than until 20 minutes after administration. As salivation was noted with increasing severity and with an increasing number of days per animal, salivation was considered to be test item-related. However, as salivation was noted at maximum up to 60 minutes post administration, salivation was considered to be not adverse.

COHORT 2A: Males and females: No adverse changes in the behaviour, the external appearance or the faeces were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Salivation was noted for all male and female animals of any dose group with a slight increase in severity and with an increasing number of days per animal. For one high dose male and one intermediate dose female, salivation was red discoloured on three days each. However, salivation was also noted for one male animal and for 6 of 10 female animals of the control group (see Text Table 10 1).
In addition, reduced motility was noted for one high dose female on one test day.
As salivation ended 60 minutes after administration at the latest and was also noted for male and female animals of the control group, salivation was considered to be test item-related but not adverse.
Start and duration of observations
Salivation started always immediately to 5 minutes after administration and ended 60 minutes after administration at maximum. Reduced motility started immediately to 5 minutes after administration and ended 20 to 60 minutes after administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
COHORT 1B: Males and females: Males and females
No premature death was noted for the female animals of any group and for the male animals of the control group and the low and high dose group (65 or 600 mg test item/kg b.w./day).
In the intermediate dose group (200 mg test item/kg b.w./day), the male animal no. 457 was found dead in the morning of TD 74. Necropsy revealed a reddened thymus and dark-red discoloured lungs. No changes were noted for the behaviour or the external appearance of animal no. 457. Therefore, the death of the animal was considered to be not test item-related but most likely due to a misgavage.

COHORT 1A: Males and females: No premature death was noted for the female animals of all groups and no premature death was noted for the male animals of the control group and the low and high dose groups (65 or 600 mg test item/kg b.w./day).
In the intermediate dose group (200 mg test item/kg b.w./day), the male animal no. 283 was found dead in the morning of TD 37 and male no. 291 was found dead during TD 16. Necropsy revealed emphysematous lungs for both animals. During histopathological examination of animal no. 283, inflammation and blood in the lumen was noted for the lungs and centrilobular hepatocellular hypertrophy and congestion for the liver. For animal no. 291, pathological changes were noted for the trachea and larynx (inflammatory/mucuos exudate and blood in the lumen), the liver (centrilobular hepatocellular hypertrophy), the testes (no complete maturity and less elongated /mature spermatids in comparison to the normal status of testes in the corresponding age) and for the epididymides (oligospermia). The death of the both animals was considered to be most likely due to a misgavage.

COHORT 2A and 2B: Males and females: No test item-related premature death was noted for the male animals and no prematurely deceased female animal was observed in any dose group (65, 200 or 600 mg test item/kg b.w./day).
The male control animal no. 522 was found dead during TD 42. External examination revealed a haemorrhagic nose/snout and the lungs were noted to be oedematous and dark-red discoloured. Furthermore, the male animal no. 567 of the intermediate dose group (200 mg test item/kg b.w./day) was found dead during TD 43. During necropsy, emphysematous lungs were noted. The histopathological examination of the lungs and/or the liver of the two prematurely deceased animals revealed no pathological changes for animal no. 522 and for animal no. 567, alveolar haemorrhage was noted for the lungs and centrilobular hepatocellular hypertrophy for the liver. The premature deaths of both animals were considered to be most likely due to misgavage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
Males:
Body weight
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day), a constantly decreased body weight was noted from TD 8 until TD 119 (at maximum 14.8% below the value of the control group, statistically significant from TD 15 to TD 119 at p ≤ 0.01).
Body weight gain
In accordance with the lower body weight of the high dose males, also the body weight gain was decreased in comparison to the control group. The decreased body weight gain for the intermediate dose group was due to a slightly higher body weight on TD 1 and a lower body weight on TD 119. However, the reduced body weight gain noted for the male animals of the intermediate dose group was considered to be not test item as no influence was noted on the body weight.
Females:
Body weight
No test item-related changes in body weight were noted for the female rats between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Body weight gain
Also for the body weight gain, no test item-related changes were noted for the female animals of all dose groups (65, 200 or 600 mg test item/kg b.w./day).
Body weight at autopsy:
Males and females
No test item-related differences were noted between the control group and the female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day) and the male low and intermediate dose animals (65 or 200 mg test item/kg b.w./day).
The male animals of the high dose group (600 mg test item/kg b.w./day) were noted with a decreased body weight at autopsy (14.9% below the value of the control group, statistically significant at p ≤ 0.01). As this is in accordance with the test item-related decreased body weight from TD 8 onwards, also the decreased body weight at autopsy of the male high dose animals was considered to be test item-related and adverse.

COHORT 1A: Males:
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In accordance with the reduced body weights of the F1 pups of the high dose males (600 mg test item/kg b.w./day), also after transfer into Cohort 1A, the body weight of the male high dose animals remains decreased (at maximum 11.7% below the value of the control group, statistically significant between TD 8 and 64 at p ≤ 0.05 or 0.01). Body weight gain: No differences in body weight gain were noted between the control group and the low and intermediate dose groups.

Females:
No test item-related changes in body weight and body weight gain were noted for the female rats between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day). In the high dose group, a reduced body weight was noted on TD 1, TD 8 and TD 15 (12.8%, 13.1% and 8.2% below the value of the control group, statistically significant on TD 8 at p ≤ 0.01 and on TD 15 at p ≤ 0.05). This was in accordance with the reduced body weight of the F1 pups throughout the lactation period. However, after TD 15 the body weight recovered and ranged between TD 22 and TD 64 between 4.7% and 0.8% of the control value. Therefore, the transiently reduced body weight between TD 1 and TD 15 was considered to be not test item-related but due to the already lower body weight before transfer into Cohort 1A.
Body weight gain: The increased body weight gain of the high dose females in comparison to the control group was due to a lower body weight of the high dose group on TD 1 and the recovery until TD 64. Therefore, the increased body weight gain of the high dose group was considered to be not test item-related.
Body weight at autopsy: Males and females:
No test item-related influence on the body weight at autopsy were noted for the female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day) and for the male animals of the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day). A decreased body weight at autopsy was noted for the male animals dosed with 600 mg test item/kg b.w./day (11.9% below the value of the control group, statistically significant at p ≤ 0.01). The distinctly decreased body weight at autopsy was considered to be test item-related and adverse.

COHORT 2A: Males: No test item-related differences in comparison to the control group was noted for the body weight and the body weight gain of the male animals of the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), a reduced body weight compared to the control group was noted from TD 2 until necropsy on TD 56. In detail, the difference between the control group and the intermediate dose group ranged between 8.5% and 12.0% below the value of the control group (statistically significant from TD 36 until TD 56 at p ≤ 0.05). In the high dose group, the decrease in body weight ranged from 6.6% to 13.5% below the value of the control group and was statistically significant from TD 29 until TD 56 at p ≤ 0.05 or 0.01. Body weight gain: In accordance with the decreased body weight, also the body weight gain of the male high dose animals was decreased in comparison to the control group. The body weight gain of the intermediate dose group however, appeared to be only slightly decreased when compared to the control group. This was due to the low body weight of the intermediate dose group on TD 2. Females: No test item-related differences for the body weight of the female animals was noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), a decreased body weight was noted from TD 2 until necropsy on TD 56. On TD 2, the body weight of the high dose females was 20.5% below the value of the control group (statistically significant from TD 2 to TD 36 at p ≤ 0.01) and the difference decreased until TD 56 to 6.0% below the value of the control group. Due to the high difference in the beginning of the Cohort 2A and the constantly decreased values in comparison to the control group, the lower body weight of the high dose group was considered to be test item-related. Body weight gain:
The body weight gain of the female animals of the dose groups from TD 2 to TD 56 was higher in comparison to the control group. This was due to the fact that the difference between the control group and the dose groups on TD 2 was higher than the difference on TD 56. Therefore, the increased body weight gain of the females of the high dose group was considered to be not test item-related.
Body weight at autopsy: Males and females: No test item-related differences for the body weight at autopsy were noted between the control group and male and female animals of low dose animals (65 mg test item/kg b.w./day) and for the intermediate dose females (200 mg test item/kg b.w./day).
Although not statistically significant, the decreased body weight at autopsy for the male animals dosed with 200 mg test item/kg b.w./day (9.5% below the value of the control group) was considered to be test item-related due to distinct difference and as the body weight on TD 56 was test item-related decreased.
In the high dose group (600 mg test item/kg b.w./day), statistically significantly decreased body weights at autopsy were observed for the male and the female animals (12.6% or 6.8% below the value of the value of the control group, p ≤ 0.05 or 0.01). These distinctly decreased body weights at autopsy were considered to be test item-related and adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
COHORT 1B: Males: No test item-related difference in the relative food consumption was noted between the male animals of the control group and all dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly decreased food consumption was noted between TD 92 and TD 99 for the low dose group (5.7% below the value of the control group, p ≤ 0.05) and a statistically significantly increased food consumption was noted for the intermediate dose group between TD 99 and TD 106 (6.8% above the value of the control group, p ≤ 0.01). Furthermore, a statistically significantly increased food consumption was noted for the high dose group between TD 50 and TD 113 (at maximum 14.0% above the value of the control group, statistically significantly at p ≤ 0.01). However, an increased relative food consumption was not considered to be test item-related but was most likely due to the decreased body weight of the male high dose animals.
Females: No test item-related difference in food consumption was noted between the female animals of the control group and all the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly increased food consumption was noted for the intermediate and high dose females between TD 64 and TD 71 (22.5% or 14.4% above the value of the control group, p ≤ 0.01). However this transiently increased food consumption was considered to be not test item-related.

COHORT 1A: Males and females
No test item-related difference in food consumption was noted between the male and female animals of the control group and in the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly increased relative food consumption was noted for the male animals of the intermediate dose group from TD 36 to TD 43 (6.0% above the value of the control group, p ≤ 0.01). In addition, also for the male and female high dose animals, an increased food consumption was noted between TD 29 and TD 64 or between TD 15 and TD 64 (at maximum 13.0% or 8.9% above the value of the control group, with the exception for the female animals between TD 43 and TD 50 statistically significant at p ≤ 0.05 or 0.01). However, an increased relative food consumption was considered to be not test item-related and is considered to be not toxicologically relevant.

COHORT 2A: Males and females
No test item-related difference for the food consumption was noted between the male and female animals of the control groups and those of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Single occurrences of a statistically significantly increased food consumption were noted for the male and female high dose animals and a statistically significantly decreased food consumption was noted for the low dose males. However, these statistically significant differences were only transient and were therefore considered to be not test item-related but spontaneous.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
COHORT 1A: Males and females: No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day).
The statistically significantly decreased concentrations in the female animals for the haemoglobin (HGB) of the intermediate dose group and for the red blood cells (RBC) of all dose groups were considered to be not test item-related as no dose-response relationship was present and the means were within the 5% to 95% confidence interval of the test institute´s background data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1A: Males and females: No test item-related differences for the examined biochemical parameters were noted between the control group and the low and intermediate dose group (65 or 200 mg test item/kg b.w./day).
Statistically significantly increased levels of globulin, total cholesterol and total protein were noted for the male and/or female animals of the intermediate dose group. However, all values of the above mentioned parameters were within the 5% to 95%-confidence interval of the test institute´s background data and no difference was noted for the F0 generation. Hence, the increased levels of globulin, total cholesterol and total protein were considered to be not test item-related.
In the high dose group (600 mg test item/kg b.w./day) of both sexes, the levels of globulin, total cholesterol and total protein were even further increased but were also still within the range of the test institute´s background data range. However, the increases were statistically significantly different from the control group for the male and female high dose animals for all parameters and were also noted for the male and female high dose animals of the F0 Generation. Therefore, the increased levels of globulin, total cholesterol and total protein were considered to be test item-related and adverse.
Endocrine findings:
no effects observed
Description (incidence and severity):
COHORT 1B: Testosterone level analysis: No test item-related differences were noted between the male animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) for the serum levels of testosterone in the male Cohort 1B animals at PND 53. Therefore, the effect observed in Marty et al., 2011, i.e. a decreased serum level of testosterone, was not established.

COHORT 1A: Testosterone hormone level analysis: No test item-related differences for the serum testosterone were noted for the male Cohort 1A animals dosed with 65, 200 or 600 mg test item/kg b.w./day) compared to the males of the control group at PND 53. Thus, the effect observed in Marty et al., 2011, i.e. a decreased serum level of testosterone, was not established.
Thyroid hormone levels: Males and females: No statistically significant differences for the levels of the examined thyroid hormones T4 and TSH were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).

COHORT 2A: Testosterone level analysis: No test item-related differences for the serum levels of testosterone of the male animals were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day). Thus, the effect observed in Marty et al., 2011, i.e. a decreased serum level of testosterone, was not established.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1A: Males and females
No test item-related changes for the urine parameters were noted for the male and female animals of the low dose group (65 mg test item/kg b.w./day).
In the intermediate dose group (200 mg test item/kg b.w./day), statistically significant reductions were noted for the pH values of the male animals (5.3% below the value of the control group, p ≤ 0.05). Additionally, even more pronounced reductions were noted for the pH values of the male as well as the female animals (10.3% or 8.5% below the value of the control group, p ≤ 0.01) in the high dose group (600 mg test item/kg b.w./day).
The urine pH of the male and female high dose animals was outside the 5% to 95%-confidence interval of the test institute´s background data, whereas the pH of the male intermediate dose animals was within this range. However, the decreased urine pH of the males of both the intermediate and the high dose group was considered to be test item-related as a dose-response relationship was present and the urine pH was also decreased for the male intermediate and high dose animals of the F0 generation. Also, the decreased urine pH of the female high dose animals was considered to be test item-related and adverse.

For 1 of 10 male control animals (no. 218) erythrocytes were noted in the urine, as well as for 2 of 10 low dose males (nos. 250 and 252) and for 2 of 10 males of the intermediate dose group (nos. 282 and 294). None of the female animals of the control group and the dose groups were observed with erythrocytes in the urine. Therefore, and as no dose response-relationship was present, the findings of erythrocytes in the urine of the low and intermediate dose males were considered to be not test item-related but spontaneous.
In addition, no differences between the control group and the dose groups were noted for the other evaluated parameters in any sex.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B: Organ weights (relative and absolute):
Males:
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the male animals of the the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), statistically significantly increased relative organ weights were noted for the adrenal glands. The weights of the adrenals were within the 5% to 95% confidence interval of the test institute´s background data. However, as the female intermediate and high dose animals of Cohort 1B, the high dose animals of Cohort 1A and the intermediate and high dose animals of the F0 Generation also displayed test item-related increased adrenal weights, the increased weights of the adrenal glands in the high dose males of Cohort 1B were considered to be test item-related and adverse. However, no test item-related decrease in the weight of the seminal vesicles was notes as observed in Marty et al., 2011.
Further organ weights of the high dose males that were statistically significantly increased in comparison to the male animals of the control group are listed in Text Table 9-10 which can be found in the attached document. However, for most of these, no dose response-relationship was present and the group mean values at the high dose level were within the background range. Therefore, the increases were considered to be not test item-related but due to the reduced body weight at autopsy.
Females:
No test item-related differences for the examined absolute and relative organ weights were noted between the female animals of the control group and the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), increased values were noted for the relative and absolute weights of the left and right adrenals. An increase in the adrenal weights were noted also for the Cohort 1B males and for the Cohort 1A males and females dosed with 600 mg test item/kg b.w./day as well as for the males and females in the intermediate and high dose group of the F0 Generation. Therefore, the increased weights of the adrenal glands for the intermediate and high dose Cohort 1B females were considered to be test item-related.
Additionally, statistically significantly increased relative and absolute weights were noted for the thyroids of the intermediate dose females. However, as no dose response-relationship is present, the increased thyroid weights were considered to be not test item-related.

COHORT 1A: Organ weights (relative and absolute): Males: No test item-related differences for the absolute and relative weights of the examined organs were noted between the male animals of the control group and the low dose group (65 mg test item/kg b.w./day).
Although, statistically significant, the increased kidney weights noted for the male low dose animals was considered to be not test item-related as the difference (4.8% or 3.9% above the value of the control group, for the left or right kidney) was only slight.
In the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day), increased relative weights were noted for the left and right kidney and the liver of the male animals. The weights were within the 5% to 95% confidence interval of the test institute´s background data. However, as the increased weights showed a dose response-relationship and were also noted for the female animals of Cohort 1A as well as for the F0 animals, the increased organ weights were considered to be test item-related and adverse.
Furthermore, increased weights were noted for the left and right adrenals of the male high dose animals. As the adrenal weights were increased also for the female Cohort 1A animals as well as for the F0 animals, the increased weights for the left and right adrenal were also considered to be test item-related and adverse.
In addition, a decreased absolute weight was noted for the heart of the high dose group. This distinctly lower weight of the heart in the high dose group was also considered to be test item-related and adverse. Furthermore, statistically significant increases were noted for the relative weights of the brain, left and right epididymis and the left and right testis of the male high dose animals. However, as no dose response-relationship was present, the increased relative organ weights were considered to be not test item-related, but due to the reduced body weight at autopsy.
However, no test item-related decrease in the weight of the seminal vesicles was noted as observed in Marty et al., 2011
Females: No test item-related differences for the organ weights were noted between the female animals of the control group and the low dose group (65 mg test item/kg b.w./day).
Statistically significantly increased absolute and relative weights were noted for the liver of the female low dose animals (15.7% or 11.9% above the value of the control group, p ≤ 0.05). However, as no pathologic changes for the liver were noted during macroscopical post mortem examination and no influence on the liver of the low dose group were noted for the F0 Generation and the Cohorts 1B and 2A, the increased liver weights for the low dose females were considered to be not test item-related.
For the female animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day), increased values were noted for the absolute and relative liver weights. Furthermore, increases were noted for the weights of the left and right adrenal glands and the left and right kidneys of the high dose group.
As the increased weights were also noted for the male animals of Cohort 1A and for the F0 Generation, for the female animals the increased weights of the above mentioned organs were also considered to be test item-related and adverse.

COHORT 2A: Males and females: No test item-related differences for the absolute and relative organ weights were noted between the male and female animals of the control group and the low dose group (65 mg test item/kg b.w./day). No test item-related decrease in the weight of the seminal vesicles was noted for Cohort 2A. The effect observed in Marty et al., 2011 was thus not established.
Statistically significantly increased relative weights were noted for the livers of the male and female intermediate and high dose animals (200 or 600 mg test item/kg b.w./day) and for the brains of the high dose males. For the males however, no noteworthy differences were noted for the absolute weights of the livers of the intermediate dose group and for the brains of the high dose group. Therefore, the statistically significantly increased relative organ weights for the livers of the intermediate dose males and the brains of the high dose males were considered to be not test item-related but due to a reduced body weight at autopsy of the animals.
For the male and female high dose animals however, distinct differences were also noted for the absolute liver weights and statistically significantly increased absolute and relative liver weights were also noted for the male and female high dose animals of the F0 Generation and Cohort 1A of the F1 Generation. Therefore, the increased liver weights for the male high dose animals and the female intermediate and high dose animals were considered to be test item-related and adverse.

COHORT 2B: Males and females: In Cohort 2B, no differences were noted for the absolute brain weights of male and female animals between the control group and any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
COHORT 1B: Males and females: No observations that were considered to be test item-related were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

COHORT 1A: Males and females: No pathologic changes that were considered to be test item-related were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly increasing number of the thymus being reddened was noted for the male animals of the dose groups (2, 3 or 4 findings for the low, intermediate and high dose group, respectively. However, as no test item-related changes were noted during microscopic examination of the thymus of the high dose animals, the reddened thymuses were considered to be not test item-related.

COHORT 2A and COHORT 2B:
At termination of Cohort 2A animals (PND 77 to 81) and Cohort 2B animals (PND 21 to 22), necropsy revealed no pathological changes for the male and female animals of the control group and of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1A: Males and females: In parallel to the animals of the F0 Generation, also for the Cohort 1A male and female animal of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day), test item-related but non-adverse findings were observed for the, thyroid glands and adrenal glands. In detail, for the thyroid glands, an increased incidence of follicular cell hypertrophy was noted for the male and female animals of the high dose group. Also, increases of the incidence and, in case of the female animals, the severity, were observed for the adrenal glands in form of increased fine vacuolation for the male and female high dose animals. These findings were test item-related, but of adaptive nature and not adverse.
Dose-dependently increased incidences and severities that could be attributed to the test item were observed in the liver (centrilobular hepatocellular hypertrophy) of the males and females dosed with 65, 200 or 600 mg test item/kg b.w./day.
Additionally, for the male animals, additional findings in form of a dose-dependently increased incidence of hyaline droplets in the proximal tubular cells in the kidneys were noted in all dose groups (65, 200 or 600 mg test item/kg b.w./day).
As the effects at 600 mg/kg b.w./day indicate towards previously reported changes in carcinogenicity studies, the findings were considered to be adverse at high dose level and thus, the histomorphological no-observed-adverse-effect-level (NOAEL) could be established at 200 mg test item/kg b.w./day.
Male reproductive organs: No test item-related changes were noted. Female reproductive organs - including quantitative examination of ovaries: No test item-related changes were noted for the examined reproductive organs and in the number of follicles and corpora lutea during the quantitative examination of the ovaries.

COHORT 2A: Histopathological examinations were conducted on organs of the male and female Cohort 2A animals of the control group and the high dose group (600 mg test item/kg b.w./day). Pathological changes were observed in form of centrilobular hepatocellular hypertrophy in the livers of 10 male and 10 female high dose animals compared to none of the male and female control animals. As this effect indicates towards changes previously reported in carcinogenicity studies, this finding was considered to be adverse and thus, the histomorphological no-observed-adverse-effect-level (NOAEL) could be established at 200 mg test item/kg b.w./day.
During histopathological examination of the seminal vesicles and the coagulating glands of the male control and high dose Cohort 2A animals, no abnormalities were noted.

COHORT 2A and 2B: Neurohistopathology: The histopathological evaluation of the brains was performed for the male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day) from PND77 to 81 (Cohort 2A) or from PND21 to 22 (Cohort 2B).
The image analysis and the histomorphometry of the brains of the male and female Cohort 2A and 2B animals revealed no test item-related differences between the control animals and the animals of the high dose group.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
COHORT 1A: Lymphocyte typing in spleen: Males and females: No statistically significant differences were noted in the proportion of the examined lymphocyte subtypes in the male and female animals between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Bone marrow: Males and females: No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (600 mg test item/kg b.w./day) of both sexes.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
COHORT 1B: Stage of estrous cycle at necropsy: No test item-related differences were noted between the female animals of the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day) in the distribution of the stages of the estrous cycle.
A slightly decreased number of females in the metestrus stage and an increased number of females in the diestrus stage were noted for the females of the high dose group. However, these slightly differences were considered to be spontaneous.

COHORT 1A: The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 50 and 63 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day). Stage of the estrous cycle at necropsy: No test item-related differences were noted between the female animals of the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day) in the distribution of the stages of the estrous cycle.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
COHORT 1A: Examination of the sperm number, viability and morphology: No test item-related difference was noted between the rats of the control group and the rats treated with 65, 200 or 600 mg test item/kg b.w./day for the sperm number, the viability and morphology.
Reproductive performance:
no effects observed
Description (incidence and severity):
COHORT 1B: Female fertility: No test item-related influence on the fertility index was noted for the female rats of the treatment groups (65, 200 or 600 mg test item/kg b.w./day).
One female each of the control and the low and intermediate dose group (nos. 395, 423 and 470) did not mate with its male partner within the 14-day mating period and one female each of the low and intermediate dose group (nos. 428 and 462) did not become pregnant. For females nos. 478 (group 3) and 520 (group 4), the presence of sperm was not noticed, hence these animals were pregnant and littered without a verified copulation.
However, these numbers are within the range of normal biological variability.

Gestation index: No test item-related influence on the gestation index was noted for the female rats of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Dams nos. 382 and 511 of the control group or the high dose group lost all of their implantations. However, these numbers are within the range of normal biological variation.

Pre-coital time and gestation length: No test item-related influence was noted on the pre-coital time and for the gestation length for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly and not statistically significantly shorter pre-coital time was noted for the females of the high dose group (2.1 days in the high dose group compared to 3.0 days in the control group, not statistically significant). However, as no dose response-relationship was present, the slightly shorter pre-coital time was considered spontaneous.
COHORT 1A:
General toxicity
No test item-related premature deaths were noted for the male and female animals of any dose group (65, 200 or 600 mg test item/kg b.w./day).
Test item-related but non-adverse changes in the behaviour and the external appearance were noted in form of salivation for the male and female animals of all dose groups.
A test item-related reduction in body weight, the body weight gain and for the body weight at autopsy was noted for the male high dose animals (600 mg test item/kg b.w./day).
No influence was noted for the male and female animals on the food consumption and the haematological parameters.
A decreased pH of the urine was noted for the male and female animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day) and increased serum levels of globulin, total cholesterol, and total protein for the male and female high dose animals (600 mg test item/kg b.w./day).
No influence was noted on the proportion of the lymphocyte subtypes in the spleen of both sexes.
No test item-related changes were noted during the macroscopic examination and the examination of bone marrow for the male and female animals of all dose groups.
Test item-related increased weights were observed for the adrenal glands, the kidneys and the liver of the male and female animals of the high and/or intermediate dose groups (200 or 600 mg test item/kg b.w./day). Additionally, a decrease in the weight of the heart was noted for the male high dose animals (600 mg test item/kg b.w./day).
Histopathological changes that could be attributed to the test item were observed in the kidneys (increased hyaline droplets in the proximal tubular cells) for the males and in the liver (increased centrilobular hypertrophy) for the male and female animals of all dose groups (65, 200 or 600 mg test item/kg b.w./day). As the effects at 600 mg test item/kg b.w./day indicate towards changes previously reported in carcinogenicity studies, the findings were considered to be adverse at high dose.
Further pathological changes were noted for the thyroid glands (increased follicular cell hypertrophy) and the adrenal glands (increased fine vacuolation) of the male and female high dose animals. These findings were considered to be test item-related but not adverse.
The detailed histopathological examination of testes and epididymides and the quantitative examination of primordial and small growing follicles and the corpora lutea did not reveal any test item-related changes or differences.

Reproductive toxicity
No influence was noted on the serum levels of testosterone of the male animals and the serum levels of the thyroid hormones of the male and female animals of any dose group.
Furthermore, no differences between the control group and the dose groups were observed for the sperm parameters of the males and the oestrous cycles between TD 50 and TD 63 of the females.
No influence on the sexual maturation was noted during evaluation of the balano-preputial separation of the male animals of Cohort 1A or all F1 animals.
Additionally, no statistically significant differences were noted for the day of vaginal opening, the body weight on the day of vaginal opening, the day of appearance of cornified cells in the vaginal smear and the period between the day of vaginal opening and the appearance of cornified cells for Cohort 1A females only. However, a slight but not statistically significant delay in the day of vaginal opening was noted for the high dose group (600 mg test item/kg b.w./day) for Cohort 1A females. This was also noted for the females of Cohort 1B and Cohort 2A, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when Cohorts 1A, 1B and 2A females of appropriate age were included in the comparison.
However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not statistically significant) in Cohort 1A and when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment. Taking all findings together, the delay in vaginal opening was considered to be not adverse.

COHORT 1B
This part of Study 37312 reports the effects of the test item 3,5,5-trimethylcyclohex-2-enone on the development of the F1 male and female animals of Cohort 1B after weaning on lactation day 21 until necropsy and on the development of the F2 pups until lactation day 21.
General toxicity
No test item-related premature death was noted for the male and female animals of Cohort 1B of any dose group (65, 200 or 600 mg test item/kg b.w./day).
Test item-related but non-adverse salivation was noted for the male and female animals of all dose groups.
The detailed clinical observations of the male and female animals of Cohort 1B revealed no test item-related findings.
A reduced body weight was noted for the male high dose animals (600 mg test item/kg b.w./day) from TD 8 until TD 119. Accordingly, a reduced body weight of the high dose males was also observed at autopsy.
No test item-related differences between the male and female animals of the control group and the dose groups were noted for the food consumption.
Increased weights were noted for the adrenal glands of the female intermediate and high dose animals (200 or 600 mg test item/kg b.w./day) and of the male high dose animals (600 mg test item/kg b.w./day).

COHORT 2A and COHORT 2B
This part of Study 37312 reports the influence of the test item 3,5,5-trimethylcyclohex-2-enone on the neurological development of the male and female animals in the adulthood (Cohort 2A) and in the early lifetime (Cohort 2B).
General toxicity
No test item-related premature deaths were noted for the male and female animals of Cohort 2A and Cohort 2B.
Test item-related but non-adverse salivation was noted for the male and female Cohort 2A animals of all dose groups.
A reduction in body weight, body weight gain and consequently in the body weight at autopsy was noted for the male animals of the intermediate and high dose groups and for the female animals of the high dose group of Cohort 2A.
In Cohort 2A, no test item-related differences were noted for the food consumption.
No test item-related pathological changes were observed in any dose group of Cohorts 2A and 2B.
Increased liver weights were noted for the male animals of the high dose group and the female animals of the intermediate and high dose groups of Cohort 2A.
No test item-related difference was noted for the brain weights of male and female animals of Cohort 2A and Cohort 2B.
The histopathological examinations revealed test item-related changes in the liver in form of centrilobular hepatocellular hypertrophy in the male and female animals of the high dose group (600 mg test item/kg b.w./day) of Cohort 2A. As this effect indicates towards changes previously reported in carcinogenicity studies, this finding was considered to be adverse.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity (F1)
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity (F1)
Effect level:
65 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups: No test item-related changes of behaviour or external appearance were noted for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day) during the lactation period.
From LD 13 or 14 until necropsy of the pups on LD 22 or 23, changes in the external appearance of the pups in form of thin fur were noted for all remaining pups of one litter each of the control group (dam no. 39) and the high dose group (dam no. 192), and for all remaining pups of two litters of the low dose group (dams nos. 77 and 91). As also one litter of the control group was noted with thin fur and no dose-response relationship was present, the observations of thin fur were considered to be not test item-related.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 pups: Viability index - Pre-and post-cull period: No test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
In the high dose group, a slightly increased number of prematurely deceased pups in comparison to the control group was noted: In the pre-cull period, 12 prematurely deceased pups in 22 litters were noted for the high dose group compared to 5 prematurely deceased pups in 23 litters in the control group. In the post-cull period, 8 prematurely deceased pups in 22 litters were noted for the high dose group whereas in the control group, there were 3 deceased pups in 23 litters. However, the resulting values for the viability indices of the high dose group for the pre-cull period (96.6%) and the post-cull period (96.4%) were only slightly below the values of the control group (98.6% or 98.7%). Therefore, the slightly increased numbers of prematurely deceased pups in the pre- and post-cull period were considered to be not test item-related but to be within the normal variability.

Number of live pups: No test item-related differences were noted for the mean number of live pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) during the lactation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 pups: No test item-related influence on the body weight of pups was noted for the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day), reduced body weights for the male and female pups were noted throughout the lactation period from LD 1 until LD 21 (at maximum 13.5% below the value of the control group for the female pups on LD 1, statistically significant at p ≤ 0.01). Furthermore, the means of the male and female litters per dam combined were only marginally above the 5% percentile of the test institute´s background data for the LD 1, LD 4 and LD 7 and were below the 5% to 95% confidence interval on LD 14 and LD 21.
Hence, the distinctly and constantly reduced pup body weights that were partly outside the 5% to 95% confidence interval of the test institute´s background data were considered to be test item-related.
Runts: One runt each was noted for the control group and at the low and high dose level (65 or 600 mg test item/kg b.w./day). The single occurrences of one runt per group were regarded to be spontaneous
Litter weight: No test item-related influence on the litter weight was noted for the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In accordance with the reduced pup body weights for the dams dosed with 600 mg test item/kg b.w./day, also the weight of the male and female litters combined were decreased (at maximum 12.3% below the value of the control group on LD 1, statistically significant at p ≤ 0.01). The reduction of the litter weight was considered to be test item-related.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
F1 pups: Thyroid hormone determination: T4 levels: No test item-related differences between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the T4 serum level on LD 4 and LD 22 for the male and female pups.
Statistically significantly decreased T4 serum levels were noted for the male pups of the intermediate dose group and for the male and female pups of the high dose group. However, the mean values of the T4 levels on LD 4 for the male and female pups were within the 5% to 95% confidence interval of the test institute´s background data and no statistically significant difference was observed for the T4 serum levels on LD 22. Hence, the decreased T4 levels on LD 4 were considered to be not test item-related.
TSH level: No statisctically significant differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the TSH level on LD 22 for the male and female pups.
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B: Males
No test item-related differences between the control group and the test item-treated groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the time point of balano-preputial separation. Thus, the effect observed in Marty et al., 2011, i.e. a delay in balano-preputial separation, was not established.
A slight but statistically significant decrease was noted for the body weight at balano-preputial separation for the male animals of the high dose group (8.8% below the value of the control group, p ≤ 0.05). This was due to the test item-related decreased body weight of the animals on LD 21.
Females
No statistically significant differences between the control group and the dose groups (65 or 200 mg test item/kg b.w./day) were noted for the body weight at the time point of vaginal opening.
A statistically significantly delayed time point of vaginal opening was noted for the females of the high dose group (600 mg test item/kg b.w./day) (5.7% above the value of the control group, p ≤ 0.05). A similar tendency could also be observed in the Cohorts 1A and 2A, leading to a statistically significant difference between the control group and the high dose group when the females of the Cohorts 1A, 1B and 2A were combined. However, the mean body weights at the time point of vaginal opening of the juvenile female rats of the high dose group were also reduced (not statistically significant) when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment. Taking all findings together, the delay in vaginal opening for the high dose group was considered to be test item-related but not adverse.

COHORT 1A: Males:
No test item-related differences between the control group and the test item-treated groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the time point of balano-prepuital separation and the body weight at the time point of balano-prepuital separation. Thus, the effect observed in Marty et al., 2011, i.e. a delay in balano-preputial separation, was not established.
A statistically significantly lower body weight at the time point of balano-preputial separation was noted for the high dose animals. However, this was not due to an influence on the sexual maturation but due to the slightly earlier time point of balano-preputial separation and the lower body weight already in the lactation period of the high dose animals.
Additionally, the summarised comparison all F1 males of the appropriate age (Cohorts 1A, 1B and 2A) revealed the same result: No statistically significant difference was noted for the time point of the balano-preputial separation between the control group and the dose groups. For the body weight at the time point of the balano-preputial separation a statistically significant decrease was noted in the high dose group (9.9% below the value of the control group, p ≤ 0.01).
Females
No statistically significant differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the body weight at the time point of vaginal opening, although a slightly reduced body weight was noted for the high dose females. However, this was due to the reduced body weight noted already during the gestation period and after transfer into Cohort 1A rather than an influence on the sexual maturation of the female animals. Furthermore, no difference was noted for the appearance of cornified cells, that marks the entry into the oestrus stage of the oestrous cycle for the first time in their life.
No statistically significant differences between the control group and the dose groups were observed for the day of vaginal opening for the Cohort 1A females only. However, a slight delay in the day of vaginal opening was noted for the high dose group. This was also noted for the Cohort 1B and for Cohort 2A, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when all F1 females of appropriate age were included in the comparison (i.e. Cohorts 1A, 1B and 2A).
Additionally, the number of female animals with a day of vaginal opening above the 5% to 95% confidence interval of the test institute´s background data increased with the dose level.
However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not statistically significant) at the high dose in Cohort 1A and when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the high dose treatment. Taking all findings together, the delayed day of vaginal opening for the F1 females of the high dose group (600 mg test item/kg b.w./day) was considered to be test item related but not adverse.

COHORT 2A: Males:
No influence was noted on the time point of the balano-preputial separation of the male animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day). Thus, the effect observed in Marty et al., 2011, i.e. a delayed balano-preputial separation, was not established.
A slightly lower body weight was noted for the body weight of the high dose males at balano-preputial separation. However, this was due to the test item-related reduction of the body weight already during lactation period.
Females:
No statistically significant differences were noted for the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear, and the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear for Cohort 2A females of all dose groups (65, 200 or 600 mg test item/kg b.w./day).
No influence was noted on the time point of vaginal opening of the female animals of the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose females (600 mg test item/kg b.w./day), a slight but statistically not significant delay was observed for the day of vaginal opening (8.1% above the value of the control group). This was also noted for the females of Cohort 1A and Cohort 1B, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when Cohort 1A, 1B and 2A females of appropriate age were included in the comparison. However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not statistically significant) in Cohort 2A and when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment. Taking all findings together, the delay in vaginal opening for the F1 females of the high dose group was considered to be test item related but not adverse.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 pups: No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 pups: No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), a statistically significant increase was noted for the number of dams with nipple-bearing male pups and also for the number of nipples per pup. In detail, 0.9 male pups with nipples per dam each were noted in the intermediate or high dose group in comparison to 0.2 pups per dam in the control group (p ≤ 0.05). Furthermore, the number of nipples per pup was increased for the intermediate and high dose group (0.44 or 0.46 nipples per pup) when compared to the male pups of the control group (0.11 nipples per pup, statistically significant at p ≤ 0.05). The increase of these two parameters also led to an increase in the number of nipples per dam (2.2 for the intermediate or high dose group compared to 0.6 nipples on male pups per dam of the control group, statistically significant at p ≤ 0.05). However, the toxicological relevance of the increased number of pups with nipple retention for the intermediate and the high dose group is unclear but limited by the following facts:
1) the finding is isolated as no influence was noted on a related endpoint (e.g. ano-genital distance)
2) no dose response relationship was noted for the number of male pups with nipples per dam and for the number of nipples per dam
3) no influence was noted on the male pups of the F2 Generation of any dose group
4) for all calculated mean values, the standard deviation is higher than the mean value, indicating large variety
5) the investigation of this end point within this study type was only introduced in 2015, so that the data base of historical control data is limited as well as the interpretation of the described effects.
Based on the above mentioned considerations, the observation was considered to be spontaneous and no test item related.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F1 pups: No statistically significant differences for the examined absolute organ weights were noted for the F1 pups between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1 pups: Macroscopic internal and/or external examinations were performed on animals found dead during the lactation period, at litter adjustment on LD 4 or at termination of the lactation period between LD 22 and 24. During these macroscopic post mortem examinations, no gross abnormalities (e.g. malformations or variations) were noted for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
For the control pup no. 35-11 necropsy after terminal sacrifice on LD 22 revealed inflated stomach and intestines, a thymus being reduced in size and a reddened brain. These single occurrences for one control pup were considered to be spontaneous.
Histopathological findings:
no effects observed
Description (incidence and severity):
Neurohistopathology - Cohort 2A and Cohort 2B: The histopathological evaluation of the brains was performed for the male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day) from PND77 to 81 (Cohort 2A) or from PND21 to 22 (Cohort 2B).
The image analysis and the histomorphometry of the brains of the male and female Cohort 2A and 2B animals revealed no test item-related differences between the control animals and the animals of the high dose group.
Other effects:
no effects observed
Description (incidence and severity):
F1 pups: Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Male to female ratio of the pups: No test item-related influence on the male to female ratio was noted for all dose groups (65, 200 or 600 mg test item/kg b.w./day).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurological screening - Cohort 2A:
Auditory startle response: Males and females
No test item-related difference was noted between the male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) for their auditory startle response.
When the last block of trials for a group was compared to the respective first one, the male and female control animals as well as the animals of the dose groups showed a slight habituation between the blocks.
Observational screening - Cohort 2A: Males and females
No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faces were noted for the male and female animals of all treatment groups (65, 200 or 600 mg test item/kg b.w./day). Furthermore, no test item-related differences were noted with regards to the body temperature.
A statistically significantly increased hind-leg splay was noted for the male and female animals at the intermediate and/or high dose level. However, all individual values of the male animals were within the test institute´s background range, while for the female animals three values (animals no. 539 (control group) as well as nos. 551 and 556 (group 2)) were lower than the historical background data. Additionally, the mean values of the control group were rather low compared to the historical background data for both sexes. Hence, the changes in the hind-leg splay were considered to be spontaneous.
Functional tests - Cohort 2A: Males and females
Grip strength
No test item-related differences between the male and female animals of the control group and those of the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the grip strength of the fore- and hind-limbs.
A statistically significantly increased grip strength was noted for the fore-limbs of the male animals of the low and high dose groups (37.0% or 32.8% above the value of the control group, respectively; statistically significant at p ≤ 0.01). However, as no dose response-relationship was present and no differences were noted for the grip strength of the hind-limbs, the increased fore-limb grip strength for the male low and high dose animals was considered to be not test item-related.
Spontaneous motility: No statistically significant differences between the male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the spontaneous motility.
Developmental immunotoxicity:
not examined
F1 pups:
No test item-related effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post-implantation loss).
For the post-natal development, a decrease in body weight and accordingly a decrease in litter weight was noted for the pups of the high dose group (600 mg test item/kg b.w./day) during the lactation period.
No test item-related effect was noted on the postnatal development of the pups regarding the viability indices (pre- and post-cull) and the ano-genital distance.
No effect that was considered to be test item-related was noted for the nipple retention of the male pups. The increased number of pups with nipples in the intermediate and high dose group were considered to be spontaneous.
The examination of the thyroid hormone levels on lactation days 4 and 22 revealed no test item-related differences.
The gross inspection (internal and/or external) of the pups at necropsy revealed no changes for the pups for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
No test item-related influence was noted on the organ weights from pups sacrificed after weaning.

COHORT 1B: Reproductive toxicity
No test item-related influence was noted for the sexual maturation of the male animals of all dose groups and also for the testosterone levels in the serum of the male animals of all dose groups. No statistically significant differences were noted for the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear and the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear for Cohort 1B females only. A statistically significantly delayed time point of vaginal opening was noted for the females of the high dose group (600 mg test item/kg b.w./day) of Cohort 1B females. A similar tendency could also be observed in the Cohorts 1A and 2A, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when females of appropriate age of Cohorts 1A, 1B and 2A were included in the comparison. However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not statistically significant) when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment. Taking all findings together, the delay in vaginal opening was considered to be not adverse.
No influences were noted for the reproduction parameters (indices for fertility and gestation, pre-coital time, gestation length).

COHORT 2A and 2B: Reproductive toxicity
No test item-related differences were noted for the testosterone levels of the male animals at PND 53 in any dose group (65, 200 or 600 mg test item/kg b.w./day).
No test item-related influence was noted on the day of balano-preputial separation and the body weight on the day of balano-preputial separation for the male animals.
No statistically significant differences were noted for the day of vaginal opening, the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear as well as the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear for Cohort 2A females only. A slight but statistically not significant delay in the day of vaginal opening was noted for the high dose group (600 mg test item/kg b.w./day) of Cohort 2A females. This was also noted for the females of Cohort 1A and 1B, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when Cohorts 1A, 1B and 2A females of appropriate age were included in the comparison. However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not statistically significant) in Cohort 2A and when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment. Taking all findings together, the delay in vaginal opening was considered to be not adverse.

Neurological development (Cohort 2A and 2B)
During neurological screening no test item-related influences were noted on the male and female Cohort 2A animals of any dose group.
No test item-related differences between the control groups and the high dose group animals were noted during neurohistopathological examination of Cohorts 2A and 2B.
Key result
Dose descriptor:
NOAEL
Remarks:
Prenatal development (conceptus to birth)
Generation:
F1
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Postnatal development (pup)
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Neurological development
Generation:
F1
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F2 pups: Pre- and post-cull period: No test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
Number of live pups: No test item-related differences were noted for the mean number of live pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) during the lactation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2 pups: No test item-related difference for the body weight of the male and female F2 pups was noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), a constant decrease of the pup body weights in comparison to the control was noted on the investigated days during the lactation period. In detail, slightly lower but not statistically significantly different pup body weights were noted on LD 1 for males as well as females (3.1% below the value of the control group for the male and female pups each). At later time points, the difference in body weight between the pups of the control group and the high dose group increased. Accordingly, a statistically significantly decreased body weight of the male and female high dose pups was noted on LD 14 (10.8% or 11.4% below the value of the control group, p ≤ 0.01) and on LD 21 (11.2% or 11.6% below the value of the control group, p ≤ 0.01). Therefore, the reduced body weights of the F2 pups were considered to be test item-related.
Runts: One runt was noted for the low dose group (65 mg test item/kg b.w./day). The single occurrence of one runt was regarded to be spontaneous.
Litter weight: No test item-related difference for the litter weight was noted between the control group and the low and intermediate dose group (65 or 200 mg test item/kg b.w./day).
Due to the decreased body weight of the pups of the high dose group (600 mg test item/kg b.w./day), also the weight of the high dose litters was decreased before culling (12.1% or 13.6% below the value of the control group for the male and female litters combined on LD 1 or LD 4, both statistically significant at p ≤ 0.05). Decreased litter weights in the high dose group were also noted after culling on LD 14 and LD 21 (11.6% or 11.8% below the value of the control group for the male and female litters combined, both statistically significant at p ≤ 0.01).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F2 pups: Thyroid hormone determination: T4 level: No test item-related differences were noted for the serum T4 levels of the pups at sacrifice between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Decreased T4 levels were noted for the pups of the intermediate dose group. However, as no dose response relationship was present, the decreased serum T4 levels for the intermediate dose group were considered to be not test item-related.
TSH level: No test item-related differences were noted for the serum TSH levels of the pups at sacrifice between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F2 pups: No test item-related differences for the ano-genital distance on LD 4 were noted between the pups of the control group and the pups of the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F2 pups: No test item-related influence was noted on the nipple retention on PND 13 for the male pups of the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F2 pups: No test item-related differences were noted for the examined organ weights of the pups in the F2 Generation between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Decreased spleen weights were noted for the male and female pups of the high dose group (24.2 % or 24.7 % below the control, statistically not signiifcant or p ≤ 0.05).
The reduced spleen weight was due to the reduced pup body weight at autopsy and the reduced live body weight on PND 21 and not due to an adverse effect of the test item on the spleen.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F2 pups: No pathological changes were noted for pups that were found dead during the lactation period and also no pathological changes were noted during necropsy of the pups in any dose group (65, 200 or 600 mg test item/kg b.w./day).
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
F2 pups: Pre-and postnatal development: Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly higher value was noted for the post-implantation loss in the high dose group (12.5% for the high dose group compared to 5.8% in the control group) leading to a lower birth index (87.5% in the high dose group in comparison to 94.9% in the control group). However, as no statistically significant difference and no dose response-relationship were present and also no influence on the post-implantation loss was noted for the F0 Generation, the slightly increased post-implantation loss was considered to be not test item-related.
Male to female ratio of the pups: No test item-related influence on the ratio of male to female pups was observed for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
F2 pups – pre- and postnatal development
Also, no test item-related difference was noted for the pre-natal development (numbers of implantation sites, resorptions, post-implantation loss, pups born).
For the post-natal pup development, a reduction was noted on the body weight of the F2 pups and consequently on the weight of the litters of the high dosed animals (600 mg test item/kg b.w./day). No test item-related differences between the control group and the dose groups were noted for the viability of the pups, the ano-genital distance, the numbers of nipples of the male pups and for the T4 and TSH levels.

F2 pups – examination at necropsy
Necropsy of the pups on LD 4 or LD 21 revealed no pathologic changes and no test item-related differences for the organ weights of the F2 pups.
Key result
Dose descriptor:
NOAEL
Remarks:
Prenatal development (conceptus to birth)
Generation:
F2
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Postnatal development (pup)
Generation:
F2
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Reproductive effects observed:
no

Please refer to result tables attached under "Overall remarks, attachments".

Conclusions:
Based on the concern regarding reproductive and developmental toxicity as found in juvenile male rats in the Pubertal Development and Thyroid function assay (Marty et al., 2011), this study aimed to provide additional information on sexual function and fertility in P0 and F1 generation and on development and toxicity of the offspring from birth until adulthood due to pre- and postnatal and adult exposure in the F1 generation.
In contrast to the observed effects in Marty et al., 2011, in the present extended one-generation reproductivity study, the seminal vesicle weight including coagulating glands, the serum level of testosterone and the preputial separation were not affected in the male animals of the F1 Generation by the extended treatment. For vaginal opening, a statistically significant delay was noted in the high dose group (600 mg test item/kg b.w./day) when considering all F1 females of Cohorts 1A, 1B and 2A combined.
However, the mean body weight of the juvenile rats at the time point of vaginal opening was also reduced (not significant) when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment.
Therefore, no delay in sexual maturation was considered and no other test item- induced effect was found indicating reproductive and developmental toxicity.
Taken all findings together, a primary affectation of the endocrine system was therefore excluded. Only the increased liver weights and the centrilobular hepatocellular hypertrophy were signs for treatment-related hepatic enzyme induction which subsequently leads to adverse effects by exceeding the maximal tolerated dose (Hall et al., 2012 ), as observed in the Pubertal Development Assay (Marty et al., 2011) and the carcinogenicity study (NTP, 1986).
Executive summary:

Conclusion


The aim of the study was to evaluate the effects of the test item at dose levels of 65, 200 or 600 mg/kg b.w./day on the reproductive toxicity of the F0 Parental Generation and the F1 Cohort 1B as offspring as well as F1 Parental Generation. Further, general toxicity and developmental toxicity of the F1 and F2 Generation from weaning until adulthood (OECD 443) including effects on neurological development were evaluated.


Triggered by findings in a screening assay (Pubertal Development assay, Marty et.al, 2011), particular focus was put on the analysis of reproductive organs and tissues as well as the pubertal development of male rats, i.e. seminal vesicle weight, serum level of testosterone and balano-preputial separation as markers for sexual maturation. Due to the extended treatment period, including the treatment between PND 23-53, this extended one-generation reproductive toxicity study especially aimed to investigate the concern in relation to reproductive/developmental toxicity in juvenile rats in more detail.


 


Maximum tolerated dose


Based on the findings in the OECD 421 study (see Section 3.7), doses of 1000 and 750 mg test item/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death.


Taking all study results into account and considering the extended exposure time of the F0 and F1 Generation within the OECD 443 study (compared to the short-term exposure time in OECD 421), 600 mg test item/kg b.w. per day were considered the maximum tolerated dose for the F0 Generation of the OECD 443 study, with no systemic effects expected in the F1 Generation. Furthermore, 600 mg test item/kg b.w. per day have been selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).


For more details, please refer to Section 3.7 ‘Rationale for dose selection’.


 


General and reproductive toxicity


General toxicity (F0 Generation and Cohorts 1A, 1B and 2A)


No test item-related mortality was noted for any of the dose groups. Test item-related but non-adverse salivation was noted for all animals of all dose groups (65, 200 or 600 mg test item/kg b.w./day).


A test item-related and adverse decreased body weight was noted for the males of the F0 Generation and the F1 Cohort 1A and 1B dosed with 600 mg test item/kg b.w./day as well as the males of the F1 Cohort 2A dosed with 200 or 600 mg test item/kg b.w./day. No test item-related changes in body weight were noted for the female animals.


No influence was noted on food consumption, serum T4 and TSH levels, haematological parameters and the ratios of the lymphocyte sets in the spleen. Additionally, no test-item related influence was noted on the serum testosterone levels. Thus, the finding in the Pubertal Development assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


The evaluation of the clinical biochemical parameters revealed increased serum levels of globulin, total cholesterol and total protein for the male and female high dose animals of the F0 Generation and for the male and female intermediate and high dose animals of the F1 Cohort 1A (200 or 600 mg test item/kg b.w./day). These effects were considered to be test item-related and adverse.


During urinalysis, a lower pH was observed for the male animals of the intermediate and high dose group of the F0 generation as well as the female and male animals of the F1 Cohort 1A.


For the intermediate and high dose females of the F0 Generation (200 or 600 mg test item/kg b.w./day), macroscopic findings were noted during necropsy, i.e. enlarged adrenals. This observation was in accordance with the increased absolute and relative adrenal glands weight of the female as well as male animals of the intermediate and/or high dose group of the F0 Generation and the F1 Cohorts 1A and 1B (200 or 600 mg test item/kg b.w./day).


Furthermore, increased weights were noted for the kidneys and livers of both sexes of the intermediate and/or high dose group of the F0 Generation and the F1 Cohorts 1A. Additionally, a decreased heart weight was noted for the high dose males of Cohort 1A. These effects were considered as test-item related. However, a treatment-related reduced weight of seminal vesicles in juvenile rats as observed in the Pubertal Development assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


The histopathological evaluation revealed test-item related changes in the liver (at 65 and /or 200  and 600 mg test item/kg b.w./day), the adrenal glands and the thyroid glands (at 200 and 600 mg test item/kg b.w./day) of both sexes of the F0 Generation and the F1 Cohort 1A. Additionally, the treatment-related finding in the liver was also observed in both sexes (at 600 mg test item/kg b.w./day) from F1 Cohort 2A. Furthermore, histopathological changes were detected in the kidneys of males from F0 Generation and F1 Cohort 1A at 65 and/or 200 and 600 mg test item/kg b.w./day.


The microscopic finding in the liver tissue, i.e. centrilobular hepatocellular hypertrophy (observed with dose-dependent increases in incidence and/or severity) was considered to have contributed to the increased liver weights in each generation and cohort. Meanwhile, in any generations or cohorts, there were no further indicators of liver injury.


Therefore, for the duration of the dose administered under the circumstances of this study, this change was considered to be of metabolic nature and of adaptive character like an enzyme induction, and not adverse.


However, when liver responses exceed adaptive changes, e.g. by chronic exposure, toxicity and hepatocarcinogenicity occur (Hall et al., 2012) as observed in male mice after 2 years exposure with 500 mg test item/kg b.w./day in the carcinogenicity study (NTP, 1986).


Taking into account the effects observed in previous studies, i.e. in NTP, 1986 and Marty et al., 2011, the findings in the liver are considered potentially adverse because it is assumed that longer or higher dosages would eventually lead to a failure of adaptive mechanisms.


The microscopic finding in the kidneys, i.e. the enhanced hyaline droplet accumulation observed in the proximal tubular cells, was treatment-related. The change was considered to be induced by the alpha2µ-globin associated mechanism. Thus, the effect noted was considered as adverse. However, this male rat-specific process that is driven by an overload of synthetic protein derived from hyperfunction of the liver is assumed to be not relevant to humans.


In the adrenal glands, diffusely increased fine vacuolation in the zona fasciculata was noted in the histopathological examination. These changes were considered to have contributed to the increased adrenal weights in both F0 and F1 Cohort 1A generations and the grossly enlarged adrenal glands that were recorded in the F0 generation. Diffuse fine (microvesicular) vacuolation is a normal pattern of the adrenal glands, and enhanced vacuolation was considered to be indicative of increased cholesterol utility. In the meantime, there were no further indicators of functional impairment (e.g. increase in coarse (macrovesicular) vacuolation) and tissue injury (e.g. necrosis, inflammation) in either generations. Thus, the increase in fine vacuoles with no toxicologically relevant histomorphological abnormalities was considered to be of adaptive nature and hence, deemed not to be adverse.


The microscopic finding in the thyroid glands revealed a slight increase in the incidence of follicular cell hypertrophy. This was thought to be the change elicited as a secondary consequence of hepatic enzyme induction (Gopinath et al., 2014) and hence, deemed not to be adverse.


No test item-related changes were noted for the reproductive organs of the male and female animals. Especially, no abnormalities were found in the seminal vesicles of male animals. Thus, the finding described by Marty et al., 2011 was not established in this higher tier study with an extended treatment period.


Additionally, no lesions were noted during the neurohistomorphological examinations of the Cohorts 2A and 2B.


 


Reproductive toxicity (F0 Generation and Cohort 1B)


No test item-related influence was noted on the reproductive performance of the parental animals (number and length of oestrous cycles, fertility and gestation index, pre-coital time and gestation length).


The test item did not affect the prenatal development of the pups (number of resorptions, stillbirths and live born pups).


A reduction was noted for the body weight of the male and female F1 and F2 pups of the high dose group (600 mg test item/kg b.w./day) and consequently, the weights of the high dose litters. The distinctly and constantly reduced pup body weights were considered to be test item-related because the means of the male and female litters per dam combined were partly (i.e. on LD 14 and LD 21) outside the 5% to 95% confidence interval of the test institute´s background data.


The other parameters of post-natal pup development (viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


 


Developmental toxicity (F1 - Cohorts 1A, 1B and 2A)


During their post-weaning development no test item-related prematurely deceased animals were noted in any dose group.


The adult F1 animals of all dose groups displayed non-adverse post-dosing salivation and the body weight was decreased for the high dose males of the Cohorts 1A and 1B, as well for the intermediate and high dose males and the high dose females of Cohort 2A. No influence was noted on the food consumption, on the haematological parameters investigated and on the serum levels of the thyroid hormones T4 and TSH of the male and female animals of the dose groups. Furthermore, the serum testosterone level of the males at PND 53 was not changed. Regarding the biochemical parameters and the urinalysis, reductions of the serum levels of globulin, total cholesterol and total protein and a lower pH of the urine were observed for the male and female intermediate and high dose animals of Cohort 1A. These effects were considered to be test item-related and adverse. Macroscopic post mortem examination revealed no test item-related pathological changes.


Increased organ weights were noted for the adrenal glands (high dose animals of Cohort 1A, high dose males and intermediate and high dose females of Cohort 1B), for the kidneys (intermediate and high dose animals of Cohort 1A) and for the liver (intermediate and high dose animals of Cohort 1A and high dose males and intermediate and high dose females of Cohort 2A). Additionally, a decreased absolute heart weight was noted for the male high dose animals of Cohort 1A. Histopathological examinations of the reproduction organs of the high dose animals of Cohort 1A revealed no pathological changes.


For the sexual maturation, the time point of balano-preputial separation of the male animals did not differ between the control group and the dose groups in the Cohorts 1A, 1B and 2A. Thus, the finding in the Pubertal Development Assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


A statistically significant delay for the time point of vaginal opening for the female animals was noted for the high dose group (600 mg test item/kg b.w./day) when comparing all F1 females of the Cohorts 1A, 1B and 2A combined with the control group. However, no statistically significant differences between the control group and the dose groups were observed for the day of vaginal opening for the Cohort 1A females and the Cohort 2A females, respectively when the Cohorts were considered separately.


Additionally, the mean body weight of the juvenile rats at the time point of vaginal opening was also reduced (not statistically significant) in all Cohorts (except of Cohort 1B) as well as when comparing Cohort 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment, indicating that there is no delay in sexual maturation. Consequently, this finding was considered as not adverse.


 


Neurotoxicity (F1 - Cohorts 2A and 2B)


The neurological screening of the animals of Cohort 2A between PND 58 and 67 revealed no test item-related differences between the dose groups and the control group. Also, the examination of the brain weights on PND 21/22 (Cohort 2B) or between PND 77 to 81 (Cohort 2A) showed no test item-related difference between the control group and the dose groups.


 


Overall Conclusion


Based on the concern regarding reproductive and developmental toxicity as found in juvenile male rats in the Pubertal Development and Thyroid function assay (Marty et al., 2011), this study aimed to provide additional information on sexual function and fertility in the F0 and F1 generation and on the development and toxicity of the offspring from birth until adulthood due to pre- and postnatal and adult exposure in the F1 generation.


In contrast to the effects observed in Marty et al., 2011, in the present extended one-generation reproduction study, the seminal vesicle weight, the serum level of testosterone and the preputial separation were not affected in the male animals of the F1 Generation by the extended treatment. For vaginal opening, a statistically significant delay was noted in the high dose group (600 mg test item/kg b.w./day) when considering all F1 females of Cohorts 1A, 1B and 2A combined.


However, the mean body weight of the juvenile rats at the time point of vaginal opening was also reduced (not statistically significant) when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment.


Therefore, no delay in sexual maturation was considered and no other test item-induced effect was found indicating reproductive and developmental toxicity.


Taking all findings together, a primary affectation of the endocrine system was therefore excluded. Only the increased liver weights and the centrilobular hepatocellular hypertrophy were signs for a treatment-related hepatic enzyme induction, which subsequently leads to adverse effects by exceeding the maximal tolerated dose (Hall et al., 2012) as observed in the Pubertal Development Assay (Marty et al., 2011) and the carcinogenicity study (NTP, 1986).


 


The following no-observed-adverse-effect levels (NOAEL) were established for the parental animals of the F0 Generation and the F1 Generation:


F0 Generation:


General toxicity


NOAEL                                       65 mg/kg b.w./day


Based on the reduced body weight, increased organ weights (adrenal glands, kidney and liver), histopathological changes (in kidney and liver) as well as the biochemical and urine changes in the high and/or intermediate dose groups of the F0 animals.


 


Reproductive toxicity


NOAEL                                     600 mg/kg b.w./day


 


F1 Generation:


General toxicity (Cohorts 1A, 1B and 2A)


NOAEL                                       65 mg/kg b.w./day


Based on the reduced body weight, increased organ weights (adrenal glands, kidney, liver and heart), histopathological changes (in kidney and liver) as well as the biochemical and urine changes in the high and/or intermediate dose groups of the F1 Cohort 1A, 1B and 2A animals.


 


Developmental toxicity (Cohorts 1A, 1B and 2A)


Adverse effects on pre- and postnatal development



  1. Adverse effects on prenatal development (conceptus to birth)


NOAEL                         600 mg/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                         200 mg/kg b.w./day


Based on the reduced body weight of the male and female pups.


Neurological developmental toxicity (Cohorts 2A and 2B)


NOAEL                                     600 mg/kg b.w./day


 


Reproductive toxicity


NOAEL                                     600 mg/kg b.w./day


 


F2 Generation:


Developmental toxicity


Adverse effects on pre- and postnatal development



  1. Adverse effects on prenatal development (conceptus to birth)


NOAEL                         600 mg/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                         200 mg/kg b.w./day


Based on the reduced body weight of the male and female pups of the F2 Generation.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

DRF study according to OECD 421


The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development (LPT, 2020). Furthermore, this study is used as dose range finding study for the ongoing OECD 443 study (LPT, 2021). The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day.


 


General and reproductive toxicity


General toxicity (Parental animals)


For general toxicity the NOAEL was considered to be 100 mg/ kg b.w./ day, based on clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day.


Two female animals dosed with 750 mg test item/kg b.w./day were found dead on GD 20 or GD 21, respectively. Furthermore, reduced body weight and reduced body weight gain, salivation, reduced motility and yellow discoloured urine were noted for the male and female animals at 750 mg test item/kg b.w./day.


In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation, reduced motility and yellow discoloured urine were noted for the male animals and salivation was noted for the female animals.


No test item-related influence was noted on the food consumption.


No test item-related pathologic findings were observed during macroscopic post mortem examination the male and female animals.


Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related changes.


No influence on the T4 levels were noted for the male animals.


 


Reproductive toxicity (Parental females)


No influence was noted on the fertility of the female animals (oestrus cycle, fertility index, gestation index, pre-coital time and gestation length) in any of the dose groups.


Thus, the No Observed Adverse Effect Level (NOAEL) for reproduction was considered to be above 750 mg test item/ kg bw/day.


 


EOGRTS study according to OECD 443


The aim of the study was to evaluate the effects of the test item at dose levels of 65, 200 or 600 mg/kg b.w./day on the reproductive toxicity of the F0 Parental Generation and the F1 Cohort 1B as offspring as well as F1 Parental Generation. Further, general toxicity and developmental toxicity of the F1 and F2 Generation from weaning until adulthood including effects on neurological development were evaluated.


Triggered by findings in a screening assay (Pubertal Development assay, Marty et.al, 2011), particular focus was put on the analysis of reproductive organs and tissues as well as the pubertal development of male rats, i.e. seminal vesicle weight, serum level of testosterone and preputial separation as marker for sexual maturation. Due to the extended treatment period, including the treatment between PND 23-53, this extended one-generation reproductive study especially aimed to investigate the concern in relation to reproductive/developmental toxicity in juvenile rats in more detail.


 


Maximum tolerated dose


Based on the findings in the OECD 421 study (as described above), doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death.


Taking all study results into account and considering the extended exposure time of the F0 and F1 Generation within the OECD 443 study (compared to the short-term exposure time in OECD 421), 600 mg/kg b.w. per day were considered the maximum tolerated dose for the F0 Generation of the OECD 443 study, with no systemic effects expected in the F1 Generation. Furthermore, 600 mg/kg b.w. per day have been selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).


 


General and reproductive toxicity


General toxicity (F0 Generation and Cohorts 1A, 1B and 2A)


No test item-related mortality was noted for any of the dose groups. Test item-related but non-adverse salivation was noted for all animals of all dose groups (65, 200 or 600 mg test item-related/kg b.w./day).


A test item-related and adverse decreased body weight was noted for the males of the F0 Generation and the F1 Cohort 1A and 1B dosed with 600 mg test item/kg b.w./day as well as the males of the F1 Cohort 2A dosed with 200 or 600 mg test item/kg b.w./day. No test item-related changes in body weight were noted for the female animals.


No influence was noted on food consumption, serum T4 and TSH levels, haematological parameters and the ratios of the lymphocyte sets in the spleen. Additionally, no test-item related influence was noted on the serum testosterone levels. Thus, the finding in the Pubertal Development assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


The evaluation of the clinical biochemical parameters revealed increased serum levels of globulin, total cholesterol and total protein for the male and female high dose animals of the F0 Generation and for the male and female intermediate and high dose animals of the F1 Cohort 1A (200 or 600 mg test item/kg b.w./day).


During urinalysis, a lower pH was observed for the male animals of the intermediate and high dose group of the F0 Generation as well as the female and male animals of the F1 Cohort 1A.


For the intermediate and high dose females of the F0 Generation (200 or 600 mg test item/kg b.w./day), macroscopic findings were noted during necropsy, i.e. enlarged adrenals. This observation was in accordance with the increased absolute and relative adrenal glands weight of the female as well as male animals of the intermediate and/or high dose group of the F0 Generation and the F1 Cohorts 1A and 1B (200 or 600 mg test item/kg b.w./day).


Furthermore, increased weights were noted for the kidneys and livers of both sexes of the intermediate and/or high dose group of the F0 Generation and the F1 Cohorts 1A . Additionally, a decreased heart weight was noted for the high dose males of Cohort 1A. These effects were considered as test-item related. However, a treatment-related reduced weight of seminal vesicles in juvenile rats as observed in the Pubertal Development assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


The histopathological evaluation revealed test-item related changes in the liver (at 65 and /or 200  and 600 mg test item/kg b.w./day), the adrenal glands and the thyroid glands (at 200 and 600 mg test item/kg b.w./day) of both sexes  of the F0 Generation and the F1 Cohort 1A. Additionally, the treatment-related finding in the liver was also observed in both sexes (at 600 mg test item/kg b.w./day)  from F1 Cohort 2A. Furthermore, histopathological changes were detected in the kidneys of males from F0 Generation and F1 Cohort 1A at 65 and/or 200  and 600 mg test item/kg b.w./day.


As the effects at 600 mg/kg b.w./day indicate towards previously reported changes in carcinogenicity studies, the findings were considered to be adverse at high dose and thus, the histomorphological no-observed-adverse-effect-level (NOAEL) could be established at 200 mg/kg bw/day.


No test item-related changes were noted for the reproductive organs of the male and female animals. Especially, no abnormalities were found in the seminal vesicles of male animals. Thus, the finding in Marty et al. 2011, was not established in this higher tier study with an extended treatment period.


Additionally, no lesions were noted during the neurohistomorphological examinations of the Cohorts 2A and 2B.


Taken all findings together, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the F0 and in the adult animals of Cohort 1A, 1B and 2A was determined to be 65 mg/kg/day based on the reduced body weight, increased organ weights, histopathological changes, as well as the biochemical and urine changes in the high and/or intermediate dose group.


 


Reproductive toxicity (F0 Generation and Cohort 1B)


No test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).


Thus, the No Observed Adverse Effect Level (NOAEL) for reproduction was considered to be above 600 mg test item/kg/day in F0 and F1 Generation Cohort 1B.

Effects on developmental toxicity

Description of key information

OECD 414, Fischer F344 rats and mice (Exxon, 1984)


In two guideline comparable inhalation teratogenicity studies with rats and mice, the NOAECs for maternal toxicity were 289 mg/m3 (based on < 7 % reductions in body weight gains) (Exxon, 1984). During the conduct of the dose range finding study in rats there was one instance of exencephaly noted in one late resorption at 144 ppm (Exxon, 1984). During the conduct of the dose range finding study in mice there were three instances of exencephaly noted in mouse fetuses: in a late resorption in one litter and two alive fetuses in a second litter (Exxon, 1984). Based on the observations made in these studies the authors did not believe that this anomaly is related to the test item. Within the framework of the dose levels and test methods used, it is concluded, that the test item was not teratogenic or fetotoxic in rats and mice.


  


OECD 414, Fischer F344 rats (Vivotecnia, 2020)


In order to clarify the relevance of exencephalies observed in two different species, including in Fischer rats (Exxon, 1984), a full prenatal developmental toxicity study was performed in Fischer F344 rats by the inhalation route according to OECD 414 (Vivotecnia, 2020).


There was no effect of the test item exposure at any level on pregnancy outcome, in terms of pre- or post-implantation loss, litter weight or the incidence of fetal abnormalities and variants. Therefore, a NOAEL for developmental toxicity was determined to be >= 151 ppm (0.86 mg/L). Foetal body weight was reduced in dosed groups, and even though these differences become statistically significant, they were below 5% for groups B and C and considered to be within a normal biological variation. However, the decreased on foetal body weight when exposed at 151 ppm (0.86 mg/L) was 13.6% both statistically and biologically significant. Therefore, a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).


 


DRF study according to OECD 421


The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development (LPT, 2020). Furthermore, this study is used as dose range finding study for the ongoing OECD 443 study (LPT, 2021). The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day.


No influence was noted of the prenatal development (number of implantations, number of resorptions or number of live born pups) at all dose groups.


For the postnatal development, changes in form of a reduced body weight and accordingly in form of a reduced litter weight were noted for the pups of the high dose group (750 mg test item/kg b.w./day). This was considered to be a secondary effect of the reduced body weight of the dams.


No influence on the T4 levels was noted for the male and female pups.


No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.


 


EOGRTS study according to OECD 443


The aim of the study was to evaluate the effects of the test item at dose levels of 65, 200 or 600 mg/kg b.w./day on the reproductive toxicity of the F0 Parental Generation and the F1 Cohort 1B as offspring as well as F1 Parental Generation. Further, general toxicity and developmental toxicity of the F1 and F2 Generation from weaning until adulthood including effects on neurological development were evaluated.


The test item did not affect the prenatal development of the pups (number of resorptions, stillbirths and live born pups). A reduction was noted for the body weight of the male and female high dose F1 and F2 pups (600 mg test item/kg b.w./day) and consequently, the weights of the high dose litters. The distinctly and constantly reduced pup body weights were considered to be test item-relate because the means of the male and female litters per dam combined were partly (i.e. on LD 14 and LD 21) outside the 5% to 95% confidence interval of the historical background data.


The other parameters of post-natal pup development (viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


Thus, the No Observed Adverse Effect Level (NOAEL) for prenatal development (conceptus to birth) was considered to be above 600 mg test item/kg bw/day.


The No Observed Adverse Effect Level (NOAEL) for postnatal development (pup) was considered to be 200 mg test item/kg bw/day based on the reduced body weight of the male and female pups. As no further postnatal effects were observed as well as no subsequent effects on reproductive performance, the substance must not be classified regarding developmental toxicity according to GHS criteria.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-10-22 to end of December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without restriction
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
no
Remarks:
All techniques and procedures were performed according to the standard operating procedures.Instruments and facilities are routinely supervised and validated according to GLP guidelines.
Limit test:
no
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj) and Wistar (Crl:Wi(Han))
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj) and Wistar (Crl:Wi(Han))
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Source Wister: Charles River C/Argenters 7, Local AB 08290 Cerdanyola del Vallés Barcelona – Spain
- Age: 10 - 11 weeks on arrival
- Body weight on day 1 of gestation: Fischer 344: 165.0 - 196.4 g, Wistar: 225.7 - 285.1 g
- Animals per cage (before / after distribution): 5 (1 after distribution and before mating)
- Bedding material: Souralit C16 / 11 (irradiated), Souralit, Spain
- Diet: ad libitum, Global diet, Harlan Teklad Station Road Blackthorn, Bicester Oxon, OX25 1TP United Kingdom
- Water: ad libitum, Tap water
- Acclimatisation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 30 - 70 %
- Light cycle: 12:12 – 07.00 to 19.00 CET
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past, nose-only exposure system. Exposure chambers type EC-FPC-232
(anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes were used.
- Method of holding animals in test chamber: The rats were individually exposed in glass tubes matching their size.
- Source and rate of air: Compressed air.
The flow of air at each tube was between 0.5 to 1 L/min, which was sufficient to minimize re-breathing of the test item vapour as it is more than twice the respiratory minute volume of rats.
- Method of conditioning air:
- System of generating particulates/aerosols: Vapors of test item were generated using a VOC (Volatile organic compound) vapor generator
(TSE-systems).
- Temperature, humidity, pressure in air chamber:
Mean chamber temperature was maintained within the recommended range of 22±3°C over the entire exposure period.
Mean chamber relative humidity was maintained within the a range of 14-20% over the entire exposure period, excluding chamber corresponding
to sham air exposed groups (A and D) on day 1 of exposure which showed a relative humidity of 9%.
Throughout the exposure period the oxygen concentration was maintained above 19% (values ranged between 20.8-20.9%) while the CO2
concentration remained below 1% (0.04-0.09%).
- Air flow rate: The airflow rate was maintained at 25 L/min for sham air and low dose exposure chambers over the 18 days of exposure. The
airflow rate for high dose exposure chambers (days 1 and 2 of exposure) was maintained at 50 L/min.
- No diet or drinking water was available during exposure.


TEST ATMOSPHERE
- Brief description of analytical method used:
Vapour samples were captured in a solvent trap containing a defined volume of methanol (cooled with ice) positioned in an empty port of the
inhalation chamber. Based on the results from the technical trials, an additional second solvent trap might be placed immediately after the first
solvent trap to ensure that all test item was captured during sampling. The sampling flow rate matched the air flow rate per port. The defined volume of methanol in the solvent trap was refilled to the start volume after sampling completion. Aliquots of samples of the resulting solution of test item
in methanol were appropriately labelled and stored at 2° - 8°C until analysis or shipment. Test item concentrations were analyzed using an analytical method validated in compliance to GLP at Swiss BioQuant AG

VEHICLE (if applicable)
- Concentration of test material in vehicle: Technical trials were performed without animals and conducted before the animal phase of the study to
establish the conditions for vapour generation and sampling, which included:
- Determination of the target concentration and/or technical limit. Several tests were performed to establish the highest stable vapour
concentration achievable that could be maintained at least for 6 hours. Vapor concentration was aimed to 2.0 mg/L in order to compare the results with a previous non-GLP reproduction toxicity study. A stable respirable vapour at a maximum concentration of 1.25 mg/L could be achieved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical determination of the vapour concentration was performed thrice during each day of exposure for each test item group. Additional samples
were analysed if considered necessary.
The quantification of test item was performed by column separation with reversed-phase chromatography followed by detection with UV.


Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was
observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The
day of mating was designated day 0 post coitum (GD0).
Male rats of the same source and strain were used only for mating. These male rats are in the possession of Vivotecnia and are not considered part of the test system.
Duration of treatment / exposure:
- Duration of dosing: From day 6 post coitum (implantation) to day 20 post coitum
- Dosing regimen: 6h/day, Groups B and E were exposed together on the same exposure chamber. The same applies to Groups C and F.
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 21
No. of animals per sex per dose:
6 females per group, 18 pregnant females per strain were distributed in 3 groups
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for target concentration selection:
The target concentrations were selected based on toxicity information available and the technical limit for the maximal vapour concentration.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
The animals were examined twice daily for mortality and morbidity.
Any clinical signs, discomfort and mortality were recorded in accordance with the humane endpoints guidance document of the OECD.
Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Where the condition of an animal required premature sacrifice, it was euthanized by CO2 asphyxiation..

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations in response to treatment were performed on all animals once daily, approximately 1h after exposure.
Any visible clinical signs, discomfort and mortality were recorded.
Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.

BODY WEIGHT: Yes
Body weight was recorded daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
Food was weighed on days 0, 3, 6, 9, 12, 15, 18 and 21 post coitum.
Data were presented in terms of consumption per animal per day for the periods (days 0 – 3), (3 – 6), (6 – 9), (9 – 12), (12 – 15), (15 – 18) and
(18 – 21).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Gross necropsy:
At the scheduled necropsy (day 21 post coitum) females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
All females were examined, either at the scheduled necropsy or during the study if death occurs.
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora
lutea count and position of fetuses in the uterus, was performed and the data recorded.
The uteri (and contents) of all females with live fetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites

Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight taken.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if
considered relevant.
Animals dying during the study or sacrificed for animal welfare reasons were also subjected to a gross necropsy. Similarly, nasal cavity, trachea,
larynx and lungs from animals showing relevant signs of irritation during exposure (i.e nasal bleeding) were collected and preserved in fixation
medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams were instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral
phosphate buffered 4% formaldehyde solution for possible further examinations.


Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents,
corpora lutea count and position of fetuses in the uterus, was performed and the data recorded.
The uteri (and contents) of all females with live fetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites

Fetal examinations:
Fetal Pathology
Fetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually, and examined for gross external abnormalities.External examination of foetuses was performed at the test facility. After external examination, foetuses were assigned with a number and sacrificed
by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). Approximately one half of the foetuses (foetuses with odd numbers) from each litter were fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the fetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed
examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of
glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol
(ca.94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed
through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining
ossified bone) and aqueous glycerin for preservation and storage [see literature (2)]. The skeletons were examined and all abnormal findings and
variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib
cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Peer review of at least 10% of the fetal examinations was performed. Fetuses with abnormalities were photographed, when considered appropriate.
The foetal pathology results were provided to the study director by e-mail and a pathology phase report was sent to the study director upon
completion of the study for inclusion in the final report
Statistics:
Provided that the sample size was considered adequate, the following parameters were subject to statistical analysis:
- Body weight
- Food consumption
Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Dunn’s post test
Test of equal variance No Dunn’s post test
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Over the first two days of exposure period (corresponding to gestation days 6 and 7), results obtained were indicative of excessive toxicity in animals exposed to the high concentration (1.25 mg/L) of test item (Group C, F344 strain and Group F, Wistar strain). As a result, 4 females from group C in addition to 4 females from group F were found dead or sacrificed for humane reasons over this period.
On the basis of these results, it was decided for animal welfare reasons to decrease the target concentration of both experimental groups (Groups C
and F). Thereby, from GD 8 to GD20 the 2 surviving females from each group (Group C, animals ID 17 and 18 and Group F, animals ID 35 and 36) were exposed to the low concentration (0.85 mg/L) being administered as well to groups B (Strain F344) and E (Strain Wistar).
Dose descriptor:
NOAEL
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Basis for effect level:
other: developmental toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
see below
Abnormalities:
not specified
Developmental effects observed:
not specified

Maternal data

Maternal mortality

 

Strain

Group

Treatment

Target concentration (mg/L)

Animal ID

Exposure day 1

Exposure day 2

GD8

GD6

GD7

F344

C

Isophorone

1.25

13

FD 1h post dose

 

 

14

 

S pre-dose

 

15

S 5 hs exposure

 

 

16

FD 6 hs exposure

 

 

Wistar

F

Isophorone

1.25

31

 

 

S pre-dose

32

 

 

S pre-dose

33

S 1h post dose

 

 

34

 

 

S pre-dose

FD: Found dead. S: Humane sacrifice

 

Pregnancy rates

F344 Strain: The pregnancy rate for control animals (Group A) was 100%. In the case of the group B (0.85 mg/L), 1/6 animals showed no implantation sites upon c-section, being pregnancy rate 83.3%. Finally, pregnancy rate achieved by the 2 surviving animals of group C (1.25/0.85 mg/L) was 100%.

Wistar Strain: Pregnancy rate achieved in the three experimental groups was 100%.

Clinical signs and observations

F344 Strain: The main and more frequent clinical signs present in animals belonging to all experimental groups were the presence of chromodacryorrhea (eye red secretion, or Harderian gland secretion) piloerection and wet fur immediately after the end of the daily exposure period. The incidence of these clinical signs was slightly higher in test item exposed groups (B and C, 0.85 mg/L) when compared to control animals from this strain.

Wistar Strain: The main clinical signs evident in females belonging to group D (Wistar rats, control group) from GD6 to GD13, comprised the presence of piloerection, chromodacryorrhea together with dirty fur. From GD14 onwards of exposure period, the only remarkable observation found was the presence of dirty fur. Regarding females belonging to group E (test item, 0.85 mg/L) and F (test item, 1.25/0.85 mg/L), piloerection, chromodacryorrhea and dirty fur were recorded over the entire exposure period. The incidence of these signs observed in animals from group E was slightly higher than the observed in the sham control group.

Comparison of findings in both strains: When comparing clinical signs observed in sham control animals from both strains, F344 rats seemed to be more sensitive to the stress induced by the experimental procedures, since the incidence of the main clinical signs observed over the exposure period (piloerection, chromodacryorrhea and dirty fur) was slightly higher in F344 strain.

Maternal body weight and body weight-gain

F344 Strain: No statistically significant differences were observed among groups in body weights and body weight gains prior to the start of the exposures (GD 0-6). Thereafter, over the early gestation period (GD6 -12) body weight loss was observed in the two experimental groups exposed to test item (Group B, 0.85 mg/L and C 1.25/0.85 mg/L), without statistically significant differences being observed with respect to control group.

Thereafter from GD9 to GD21 in the case of animals from group B (0.85 mg/L) maternal body weight gain was still significantly reduced when compared to control animals. As a result, mean body weight gain for the overall exposure period (GD6-GD21) was statistically lower in animals from group B (reaching a body weight gain of -10.13%) when compared to control group (which achieved a body weight gain of 23.18%). This effect was considered to be attributable to maternal toxicity, leading to effects on embryonic development.

Regarding animals from group C (1.25/0.85 mg/L), lower body weight gain was noted for the remainder exposure period (GD9-GD21) for the two surviving animals compared to control group. Hence, mean body weight gain for the overall exposure period (GD6-GD21) in animals from group C reached nearby the 66% of the achieved by control animals. Given to the limited group size in group C statistical analysis could not be performed.

Wistar Strain: Maternal body weight and weight changes were equivalent across all groups prior to the start of exposures (GD0-GD6) as well as over the exposure period (GD6-GD21), with no statistically significant differences being found among the experimental groups.These results were in accordance with c-section observations, which revealed a 100% of dams with viable foetuses in control and test item exposed groups G (Group B, 0.85 mg/L and C 1.25/0.85 mg/L).

Comparison of findings in both strains: When comparing body weight changes of control animals (Group A, strain F344 and group D, strain Wistar) a similar trend was observed in both strains over the entire observation period. In particular, control animals from both strains exhibited body weight gain prior to the start of the exposures (GD 0-6), reaching on GD6 a 5% and 12% increase with respect GD0 in F344 and Wistar control rats, respectively.

Initiation of exposure led to a decrease of body weight in both strains of similar magnitude. Thus, from GD6 to GD9 a body weight drop nearby a 4.3% was observed in animals belonging to control group A (strain F344). Similarly, over this period (GD6-GD9) animals from control group D (strain Wistar) exhibited a body weight loss up to a 3%.

Thereafter, from GD9 to GD21 body weight increase was observed in both experimental groups, being slightly higher in Wistar rats in terms of percentage of increase across the exposure period (16% F344 control rats vs 25% Wistar control rats), as well as over the entire gestation period (23.2% F344 control rats vs 38.74% Wistar control rats).

Food consumption

F344 Strain: Over the pre-exposure period (GD0-GD6) food consumption was similar across all the experimental groups. Afterwards, in general terms, food consumption of females exposed to test item

(Group B, 0.85 mg/L and group C, 1.25/0.85 mg/L) was slightly lower than the observed from sham control F344 rats (Group A) over the entire exposure period. This effect was more remarkable from GD6 to GD9, stage at which animals from test item exposed groups exhibited a decline in food intake up to a 29.5% when compared to control group. Thereafter, from GD9 to GD 21, the drop of food consumption in females exposed to test item (Groups B and C) ranged from 10% to 17%. This effect did not reach statistical significance.

Wistar Strain: Over the pre-exposure period (GD0-GD6) food consumption was similar in control (Group D) and test item exposed (Group B, 0.85 mg/L and group C, 1.25/0.85 mg/L) Wistar rats. Thereafter, exposure to test item did not seem to have an impact in food consumption of any experimental group. Only animals from group E (test item, 0.85 mg/L) exhibited a transient and statistically significant decline in food consumption from GD15 to GD18 (12% respect to control group).

Comparison of findings in both strains: According to the results disclosed in this section, animals from F344 strains seemed to be more sensitive to the exposure to test item in terms of food consumption. This lower food consumption values in F344 strain correspond with body weight effects in these groups together with c-section findings.

Female necropsy data and pregnancy outcome

Macroscopic observations No test item related macroscopic observations were recorded in any animal from any experimental group upon macroscopic necropsy performed on GD21.

Uterine implantation data

F344 Strain: Mean number of corpora lutea was similar across groups. Concerning mean implantation sites and pre-implantation loss, similar results were observed in control animals and in the two animals from group C surviving up to GD21. In particular mean number of implantation sites per animal observed in group A were 10.8, while in group C (1.25/0.85 mg/L) mean number of implantation sites per animal was 10.0. These results involved a mean percentage of pre-implantation loss of 4.7% in control animals (Group A) and equal to 30.5% in group C (1.25/0.85 mg/L).

In the case of group B (0.85 mg/L), mean percentage of pre-implantation loss was 42.4%, being remarkably higher than the 4.70% recorded in control animals. It should be noted that one female belonging to this group (ID11) showed a 100% of pre-implantation loss together with the circumstance that within group variability was large. Accordingly, this effect did not achieve statistical significance.

In terms of post-implantation losses, a clear treatment related effect was observed in groups B (0.85 mg/L) and C (1.25/0.85 mg/L). In particular, mean post-implantation losses per animal in group B was 84.4% while in the case of group C this value reached a 63.7%. These values were remarkably higher than the 17.52% of mean post-implantation losses per animal found in the control group, with no statistically significant differences being observed.

It must be pointed out that, while in control group the major part of the post-implantation losses were classified as early or embryonic resorptions (11/12 losses) in the case of test item exposed animals, most of these losses took place at an earlier stage. In particular, in group B (0.85 mg/L) 24 of the 27 post-implantation losses recorded were defined as empty implantation site, meaning a very early resorption or aborted implantation. Concerning group C, animal ID 17 showed a 100% of post-implantation losses, all of them considered empty implantation sites, whereas animal ID 18 showed 3 post-implantation losses (from a total of 11 implantation sites), being the 3 described as embryonic resorptions.

All together, these circumstances led to a statistically significant decrease in the number of viable foetuses recorded in animals from group B (0.85 mg/L) when compared to control group. In particular, a total of 56 live foetuses were observed upon c-section in animals from group A, while in the case of group B only 1 female presented viable fetuses (a total of 5).

Wistar Strain: Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation losses were similar across all groups. Moreover, post-implantation losses and litter sizes were similar when comparing test item exposed and control animals.

Comparison of findings in both strains: In general terms, comparison of data from control groups showed a similar reproductive performance for both strains concerning to mean pre-implantation losses per animal. Thereafter, F344 females showed a worse reproductive performance compared to Wistar rats, when considering the mean percentage of post-implantation losses per animal (17.52% in F344 strain versus 6.56% in Wistar strain). On the other hand, litter size was higher in Wistar rats (12.8 fetus per litter) than in F344 strain (8.8 fetus per litter).

These results are in agreement with the published literature, reporting relatively poor reproductive performance of the F344 strain (inbred strain) when compared to outbred strains such as Wistar rats, known for their better reproductive capabilities. Therefore, F344 strain would not be the appropriate model of choice for prenatal developmental toxicity studies.

Gravid uterine weight data

F344 Strain: Upon c-section the 6 females belonging to control group (Group A) were determined to be gravid. By contrast, in the case of group B (Test item, 0.85 mg/L) from the 6 animals surviving until GD21 only 1 (ID7) was found to be gravid on GD21 and showed viable fetuses. Finally, regarding from the two females from group C (Test item 1.25/0.85 mg/L) surviving the entire observation period, only one of them (ID18) was determined to be gravid and showed viable festuses.

Therefore, on the basis of the limited group size available, no remarkable differences were found in gravid uterine weight between control and test item exposed groups or neat terminal body weight. A decline in neat body weight gain from GD6 to GD21, was found in all experimental groups, ranging between -7.7% to -9.5%.

Wistar Strain: Upon c-section conducted on GD21, the 6 females from groups D (Control group) and group E (Test item, 0.85 mg/L) were determined to be gravid. In addition, the two surviving females from group F (Test item, 1.25/0.85 mg/L) well found as well to be gravid.

Gravid uterine weight, neat terminal body weight together with neat body weight gain was very similar among the three experimental groups, with no statistically significant differences being observed. A decline in neat body weight gain from GD6 to GD21, was found in all experimental groups, ranging between - 4.2 % to - 4.9%.

Comparison of findings in both strains: Control animals from both strains showed a decrease in neat body weight gain from GD6 to GD21, being this effect more noticeable in F344 rats. In particular, over the exposure period F344 control females exhibited a neat body weight gain up to a -9.4%, while in Wistar rats neat body weight gain was - 4.2%.

Conclusions:
Fischer 344 strain:
Based on the results of this study, a no observed-adverse-effect level (NOAEL) for maternal effects and embryo-fetal development could not be established for this strain.
Wistar strain:
On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects and embryo-fetal development was evident at 0.85 mg/L (150 ppm).
Strain comparison:
- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development
toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among
others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was
discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long
lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).
- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from
gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and
post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.
Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on
embryo-fetal development observed in F344 rats.
- Results of the present study together with the information existing in the published literature, provide clear evidence that F344
rats are not a suitable strain for prenatal developmental toxicity studies.
Executive summary:

Objective

In a first instance this study was designed to assess the potential effects of the test item on pregnancy and embryo-fetal development in F344 and Wistar rats, following daily inhalation exposure (nose-only) at concentrations of 0.85 (150) and 1.25 mg/L (220 ppm), during 6 hours per day, from implantation to one day prior to scheduled caesarean section (day 6 to day 20 post-coitum, inclusive). Therefore this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.

Study Design

For these purposes, 18 pregnant females per strain were distributed in 3 groups (6 animals per group). F344 rats were allocated into groups A-C. Animals from group A were exposed to filtered air (Group A, sham controls), whereas animals allocated in group B were exposed to the low concentration of the test item (0.85 mg/L) and animals from group C were exposed to the high concentration

(1.25 mg/L).

Similarly, Wistar rats were allocated into groups D-F. Group D served as sham control, while animals from groups D

and E were exposed to the low (0.85 mg/L) and high (1.25 mg/L) doses of test item, respectively.

Over the first two days of exposure (Gestation day 6-7), the high concentration of the test item was reduced from 1.25 mg/L to 0.85 mg/L (low concentration) due to unscheduled mortality and adverse clinical signs observed at this exposure concentration in both strains.

All animals were observed twice daily for mortality and clinical observations. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a cesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Study Results

F344 strain

Over the first two days of exposure (GD6-GD7), 4 females from the high concentration group (Group C, 1.25 mg/L) were found dead (2/4) or sacrificed for humane reasons (2/4). Thus, only 2/6 animals from this group survived until the end of the exposure period, involving exposure to a concentration of 1.25 mg/L from gestation day (GD) 6 to GD 7 and to a concentration of 0.85 mg/L thereafter, from GD8 to GD20.

Exposure related clinical signs (such as piloerection and wet fur) were found in all experimental groups, which a slight higher incidence in test item treated animals. Over the early pregnancy (GD6-GD12) no statistically significant differences were observed in body weight or body weight gain between sham control and test item exposed animals. Thereafter, from GD9 to GD121 a statistically significant body weight reduction was observed in females from group B (0.85 mg/L). This effect was considered to be attributable to maternal toxicity, leading to effects on embryonic development.

In general terms, food consumption of test item exposed females tended to be slightly lower of the observed for sham control animals over the entire observation period. Nevertheless, these differences did not achieve statistical significance.

No test item related macroscopic findings were noted in animals from group B and C. A test item related effect was observed regarding pre and post-implantation losses in animals from both experimental groups (0.85 and 1.25 / 0.85 mg/L), which showed remarkably higher percentages of losses with respect to control group.

Upon c-section (GD21) 5/6 females from group B and 2/2 females from group C showed implantation sites and/or viable fetuses. On the basis of the limited group size available, no remarkable differences were found in gravid uterine weight or in neat body weight gain (GD6-GD1) when comparing test item treated animals with sham control groups.

Finally, there was no effect of exposures in fetal weight at GD21 and no external, visceral or skeletal abnormalities or variations were noted.

Wistar strain

Over the first two days of exposure (GD6-GD7), 4 females from the high concentration group (Group F, 1.25 mg/L) were sacrificed for humane reasons. Thus, only 2/6 animals from this group survived until the end of the exposure period, involving exposure to a concentration of 1.25 mg/L from gestation day (GD) 6 to GD 7 and to a concentration of 0.85 mg/L thereafter, from GD8 to GD20.

No test item exposure related effects were observed on maternal observations, gestational parameters or fetal evaluations.

Conclusion

Taken together the results disclosed in the present report, it can be concluded that under these experimental conditions:

F344 strain

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did induce maternal toxicity (statistically significant body weight loss).

- Accordingly, a no observed-adverse-effect level (NOAEL) for maternal effects could not be established in this study.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from gestation day 6

to gestation day 20 led to an exposure related effect on pregnancy outcome, in terms of pre and post-implantation loss.

These effects are considered to be due to overt maternal toxic effects. On the basis of the very limited analysis performed under

the conditions of this study, due to the low number of foetuses available in females exposed to test item vapour by inhalation

at 0.85 mg/L, exposure was not associated with any effect on fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, a NOAEL for embryo-fetal effects could not be established for this strain.

Wistar strain

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did not induce maternal toxicity and did not have any effect in pregnancy outcome,

in terms of pre- or post-implantation loss, fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects

and embryo-fetal development was evident at 0.85 mg/L (150 ppm).

Strain comparison

- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development

toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among

others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was

discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long

lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).

- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from

gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and

post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.

Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on

embryo-fetal development observed in F344 rats.

- Results of the present study together with the information existing in the published literature, provide clear evidence that F344

rats are not a suitable strain for prenatal developmental toxicity studies.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-07-23 to 2018-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
no
Remarks:
All techniques and procedures were performed according to the standard operating procedures.Instruments and facilities are routinely supervised and validated according to GLP guidelines.
Limit test:
no
Specific details on test material used for the study:
Physical state (at 20ºC): Neat Liquid
Purity / composition: ≥98.0 to ≤ 99.6%
Molecular weight: 138.20 g/mol
Colour: Clear
Storage conditions: Room temperature (ca. 23ºC); protected from light
Expiry date: July, 2019
Handling conditions: Personnel will wear gloves and auto-ventilated equipment equipped with filters appropriate vapours
Other: Sensitive to air, highly volatile, odour of peppermint.
Avoid inhalation and contact with eyes and skin. Test item may cause irritation of respiratory tract, eyes and skin
The neat substance without solvent will be tested
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj)
Remarks:
Although the F 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specificaly asked for their use in order to compare the results with previous non-GLP reproduction toxicity studies, in which this strain was used
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj)
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Age: 9 - 10 weeks on arrival; 12 - 13 weeks on first item exposure
- Animals per cage (before / after distribution): Up to 5 (1 after distribution and before mating)
- Bedding material: Souralit C16 / 11 (irradiated), Souralit, Spain
- Diet: ad libitum, Global diet, 2914C, Envigo Teklad
- Water: ad libitum, Tap water
- Acclimatisation period: at least 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 °C - 24.6° C
- Humidity (%): 28 - 61 %
- Light cycle: 12 hours light, 12 hours dark
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Preparation of the exposure system
Previous to exposure onset, a complete setup of the system and flow/pressure characterization was performed according to the corresponding internal SOPs. The tasks were the following:
- Making an outline of each line including identification of the corresponding equipment
- Leak test through the vapour conveyor glass tubes
- Checking of working air temperature and relative humidity
- Flow of working air entering the machine and internal pressure thereof
- Pump status: measuring puff volume, pump settings
- Theoretical calculations regarding dilution and Venturi tubes to be used
- Vacuum system checking in sampling ports: capillary installed, vacuum pressure measurement, sampling flow
- Exposure test: Determination of pressure inside the inhalation chamber versus room pressure and difference of pressure between both chamber compartments. Determination of aerosol flow in different positions of the chamber randomly selected: six ports from the high level, six ports from the mid level and six ports from the low level. Calculation of coefficient of variation (%CV) within each level and across the three levels. Within each level, the CV will not be higher than 5%, whereas the CV among the three levels will not exceed 10%.
- All these determinations were also performed in the sham exposed group with the exception of the flow through chamber.

Test item Vapour generation and characterisation
- A vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).
- The test item was placed inside the generator vessel in liquid state. It was heated at a controlled temperature of approximately 55ºC by means of a recirculating water bath equipped with a temperature probe to generate a vapour phase.
- In addition, the liquid was stirred by means of a magnetic stirrer in order to increase the evaporation.
- At the same time, a source of compressed air at a controlled temperature was delivered into the VOC generator. This compressed air was mixed with the vapour phase of test item extracting the formulation of the product in a controlled amount of air (L/min) and concentration.
- The vapour was conveyed via glass tubing from the generator to the exposure chambers.
- The flow rate through the exposure chamber was adjusted as necessary.
- In order to prevent exposure of the animals to test item droplets from condensation, a filter will be placed immediately before the exposure chamber. For sham exposure, filtered, compressed fresh air will be provided to the exposure chamber.

Exposure conditions
- Inhalation exposure was performed using a flow-past, nose-only exposure system.
- The animals were confined separately in restraint glass tubes matching their size which are positioned radially around the exposure chamber.
- The exposure system ensures a uniform distribution and provides a constant flow of test material to each exposure tube.
- The flow of air at each tube was within 1.0 ± 0.5 L/min.
- The position of the rats in each group within the exposure chamber was changed daily according to an insertion schedule.
- Sham-exposed rats were exposed to filtered, conditioned air, the exposure conditions being the same as those for the test item-exposed rats.
- Exposure chambers type EC-FPC-232 (anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes) were used. The rats were individually exposed in glass tubes matching their size.
- Before starting the inhalation period, the animals were not adapted to the glass tubes to avoid pre-implantation losses due to the stress caused by the procedure itself.
- The temperature of the test atmosphere in the exposure chamber was maintained at 22 ± 5 °C and the relative humidity as required by experimental conditions.
- Air flow per animal was monitored regularly.
- No diet or drinking water was available during exposure.
- The time spent during animal shifting was not included within the exposure time.
- Before the animal exposures, technical trials were performed in order to set-up the conditions.
- A vapour was generated for up to 6 hours for two consecutive days. During this period, the concentrations as well as the temperature and relative humidity of the chamber were monitored.
- The set-up conditions and results were similar to those obtained during the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Characterisation of test atmosphere
In order to characterize the test atmospheres and to check the reproducibility of aerosol generation, several parameters were determined at defined intervals.
Determination of the nominal vapour concentration
The test item usage was determined daily by volume difference in the vapour generator before and after each exposure. The weight used, calculated by means of the specific gravity, was then divided by the total air-flow feeding the vapour generator to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.
Determination of test item concentration
Test item vapour concentration was determined three times a day on each day of exposure for each test item group by high performance liquid chromatography (HPLC) after collecting the vapour in a solvent trap containing 50 mL of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. The analytical method was validated afterwards in Vivotecnia study B-02791. Sampling time was 5 minutes at a flow rate of approximately 1 L/min. Trap volume was determined before and after sampling in order to evaluate discrepancies in the final volume. Aliquots of the samples were analysed freshly immediately after the sampling process.
The solvent trap capacity was evaluated in Vivotecnia study number N-02130 by sampling the vapour in two in-line solvent traps. No test item was detected in the second trap. Thus, the capacity of one single trap was considered adequate at the concentrations performed in this study.
Chromatographic method see "other information on materials and methods".
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum (GD 0).
Male rats of the same source and strain were used only for mating. These male rats were not considered as part of the study.
Only animals that meet the inclusion criteria will be recruited for the study and distributed into experimental groups.
Duration of treatment / exposure:
Duration of dosing: Daily from day 6 post coitum (implantation, GD 6) to day 20 post coitum (GD 20)
Dosing regimen: 6h/day
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 21
Dose / conc.:
0 mg/L air
Remarks:
Control Group (Group A), Sham filtered air
Dose / conc.:
0.1 mg/L air
Remarks:
Low Group, 17 ppm (Group B)
Dose / conc.:
0.3 mg/L air
Remarks:
Mid Group,, 50 ppm (Group C)
Dose / conc.:
0.85 mg/L air
Remarks:
High Group, 150 ppm (Group D)
No. of animals per sex per dose:
8 females per group, 32 pregnant females were distributed into four experimental groups. One control group and three different dose groups (low, mid and high vapour concentration).
Control animals:
yes, concurrent vehicle
Details on study design:
- Introduction and study outline
The objective of this dose range finding (DRF) study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20). Therefore, this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.
For those purposes, 32 pregnant female rats were distributed into four experimental groups (A to D). Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A; control) 6 hours/day from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20).
Animals from group A were exposed to filtered compressed fresh air and served as control and animals from groups B to D were exposed to diluted vapour from the test item at nominal concentrations of 0.1, 0.3 and 0.85 mg/L air, respectively. The vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems). In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.
All animals were observed twice daily for mortality and clinical observations before and within 1 hour after exposure. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a caesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations

- Rationale for selection of species / strain: Although the Fisher 344 strain is normally discouraged in reproductive toxicity studies (OPPTS 870.6300), the authorities specifically asked for their use in both the DRF and main studies in order to compare the results with previous non-GLP reproduction toxicity studies, in which the Fischer 344 strain was used.

- Rationale for target concentration selection: The target concentrations were selected by the sponsor according to previous prenatal development toxicity studies (Exxon, 1984 and VVT, 2016) available for this test item.

- Inclusion criteria
Only animals that meet the inclusion criteria will be recruited for the study and distributed into experimental groups.
Inclusion criteria: All animals were subjected to individual veterinary examinations. Only animals in good health and with pregnancy status confirmed were included in the study
Stratification method: Animals were randomly distributed by means of the body weight stratification method at least 2 days before first administration

- Determination of the temperature
The temperature in the chamber was measured hourly during exposure using a thermohygrometer. The target range is 22±5ºC.
- Determination of the relative humidity
The relative humidity in the chamber was measured hourly during exposure using a thermohygrometer.
- Determination of oxygen and CO2 concentration
The oxygen concentration of the test atmosphere was measured hourly during each day of exposure using an appropriate device. The oxygen and CO2 concentrations were maintained above 19% and below 1% respectively, during the exposure period.
- Airflow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure using the pressure difference over a Venturi tube. Air flow per animal was monitored regularly (at least 3 times per group and day) during the daily exposure period.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Experimental data concerning body weight, general clinical signs, organ weights and gross necropsy findings (dams) and ovaries and uteri examination were recorded through Provantis 8.5.2.1TM (Instem LSS Ltd., Staffordshire, United Kingdom) preclinical software.

Mortality and premature sacrifice
All animals were examined daily for mortality and morbidity. Any clinical signs, discomfort and mortality were recorded in accordance with the human endpoints guidance document to the OECD (ENV/JM/MONO (2000)7).


DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations were recorded before and within 60 minutes after finishing exposure in all animals.
Any visible clinical signs, discomfort and mortality was recorded in accordance with the humane endpoints guidance document of the OECD (ENV/JM/MONO(2000)7). Observations included changes in skin, eyes, and mucous membranes. Alterations in respiratory pattern or behaviour as well as changes in posture and response to handling and the presence of abnormal movements were also recorded.


BODY WEIGHT: Yes
All animals were weighed on day GD0, every 3 days during the inhalation period and on day 21 post coitum.
Additional body weights were obtained as necessary for animal welfare reasons.


FOOD CONSUMPTION: Yes
Food consumption was recorded in all animals on GD 0, 3, 6, 9, 12, 15, 18 and 21 post coitum. The consumption was calculated by cage, estimating the average food intake for each animal.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Gross necropsy
At the scheduled necropsy (GD 21 post coitum) females were sacrificed by CO2 asphyxiation and the foetuses were removed by caesarean section.
All females were examined at the scheduled necropsy. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count, implantations sites and position of foetuses in the uterus, was performed and the data recorded. The uteri (and contents) of all females with live foetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight was recorded.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant. Similarly, nasal cavity, trachea, larynx and lungs from animals showing relevant signs of irritation during exposure (i.e nasal bleeding) were collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams were weighted and instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral phosphate buffered 4% formaldehyde solution for possible further examinations.
Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count, implantations sites and position of foetuses in the uterus, was performed and the data recorded. The uteri (and contents) of all females with live foetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
Fetal Pathology
Foetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually and examined for gross external abnormalities. External examination of foetuses was performed at Vivotecnia. After external examination, foetuses were assigned with a number, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). Approximately one half of the foetuses (foetuses with odd numbers) from each litter were fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the foetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissues were preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol (ca.94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage . The skeletons were examined, and all abnormal findings and variations were recorded. The assessment included,but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Foetuses with abnormalities were photographed, when considered appropriate. The foetal pathology phase report is attached to this study endpoint record.
Statistics:
Provided that the sample size was considered adequate, the following parameters will be subject to statistical analysis:
- Body weight
- Food consumption

Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Kruska-Wallis Dunn's post test
Test of equal variance No Kruska-Wallis Dunn’s post test
A value of P < 0.05 will be considered statistically significant
Indices:
DRF, Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical sign present in animals belonging to all experimental groups, including sham, was piloerection. This sign is commonly observed in nose-only inhalation studies and, since it is equally present in the control group, it can be considered not to be caused by the test item.
Low dose group showed punctual wet fur at the beginning of the exposure. Additionally, incidental bleeding in vagina was observed on isolated days of animals belonging to groups B – D (< 2 animals per group). Since both signs were single observations (< 2 and not in consecutive days) and there was no dose-dependent relationship, these observations are considered incidental and probably not test item related.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during exposure. All animals survived until the schedule necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences were found in mean body weight among the four experimental groups neither during the exposure period nor at the end of the study.

No statistically significant differences were found in mean body weight gain among the four experimental groups neither during the exposure period nor at the end of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar among the four experimental groups (no significant differences) over the course of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistical differences were recorded neither in Terminal body weight nor in gravid uterus weight. The adjusted body weight to gravid uterus was also similar.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant macroscopic abnormalities related to the exposure to the test item were observed.
Red spots in the uterus were observed for dams 11 and 15 (group B) as well as dams 23 and 24 (Group C). However, this finding could be considered incidental and therefore not related to test item exposure since it was not observed in any of the dams belonging to the high dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
An increase trend was however observed in pre-implantation loss and post-implantation loss (not statistically significant in any case) for dams of groups C and D. Although it cannot be ruled out a potential test item effect, since this effect was more relevant for pre-implantation loss than for post-implantation loss, it is thought that it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item .
After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight decrease in the number of foetuses alive in both mid and high-dose groups. In addition, the litter weight was also low in these groups, but it is mainly related with the litter size as the mean of foetal weight was similar in all groups. Similar trend with no statistically significant data was found after the Main study.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Caesarean section (C-section) on day 21.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females in the groups were pregnant. No abortions were recorded.
Other effects:
not examined
Details on maternal toxic effects:
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
An increase trend was however observed in pre-implantation loss and post-implantation loss (not statistically significant in any case) for dams of groups C and D. Although it cannot be ruled out a potential test item effect, since this effect was more relevant for pre-implantation loss than for post-implantation loss, it is thought that it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item.
After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.
Key result
Dose descriptor:
NOAEL
Remarks:
Taking into account all observations in this DRF study, for maternal effects, NOAEL could not clearly achived based on the small size of the groups.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of the exposures on foetal weight at GD 21.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight decrease in the number of foetuses alive in both mid and high-dose groups. In addition, the litter weight was also low in these groups, but it is mainly related with the litter size as the mean of foetal weight was similar in all groups. Similar trend with no statistically significant data was found after the Main study.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical differences were found in litter weight. There was a slight decrease in the number of foetuses alive in both mid and high-dose groups. In addition, the litter weight was also low in these groups, but it is mainly related with the litter size as the mean of foetal weight was similar in all groups. Similar trend with no statistically significant data was found after the Main study.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were noted during external examination of foetuses at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was observed during the skeletal examination at any dose level and no test item-related findings were noted.
The alterations observed during the skeletal examination of foetuses were assigned to
- bone ossification stage/supernumerary ribs, which were related to a change from the expected ossification state of rat bones in this stage.
- bone or cartilage variations, which were used to describe structural changes (as changes in shape or size).

Ossification stage/supernumerary ribs
Fusion of zygomatic arch was observed in 2 foetuses in 2 litters in group C and in 1 foetus in 1 litter in group D, this finding was isolated and did not have any dose-related pattern.
In group D, there was a slightly increased incidence of litters with Thoracic ribs/supernumerary one rudimentary (5 litters in comparison with 3 litters in group A). Due to low incidence and the absence of a dose-dependency, this finding was considered to be incidental.
All remaining findings recorded were observed in structures known to be the most likely to show variable stages of ossification at this post coital age in rat. Therefore, there was no test item effect on the stage of development at any dose level.

Bone and cartilage variations:
Sternebra bipartite ossification was observed only in one foetus of group D, therefore it was considered to be incidental and not to be test item-related effect.
Costal cartilage 11 interrupted, long or supernumerary site was observed in group B (5 foetuses in 4 litters), in group C (3 foetuses in 2 litters) and in group D (8 foetuses in 6 litters). This finding was considered not to be test item related but a result of biological variability, since no corresponding or further ribs showed any comparable findings.
All remaining variations were comparable to the concurrent control and they were commonly seen in rats at this post coital age, therefore they were considered to be incidental and not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:
42% examined foetuses in 88% litters in Sham filtered air
48% examined foetuses in 100% litters in Isophorone (Group B)
57% examined foetuses in 88% litters in Isophorone (Group C)
50% examined foetuses in 88% litters in Isophorone (Group D)

No test item-related findings were noted.
Malformation such kidney and ureter absent was found in one foetus in group A and in one foetus in group B. It was considered to be incidental and not test item related, as the incidence was similar in both groups.
Moderate urinary bladder distended was observed in 2 foetuses in 2 litters of group C and in 2 foetuses in 2 litters of group D. Due to the low and similar incidence in both treated groups, this finding was considered not to be test item-related.
The remaining variations observed were isolated incidences and did not indicate any test item effect. Thus, they were considered not to be test item-related.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
- No statistically significant differences on reproductive parameters were observed, although a trend in percentage of pre and post implantation loss was recorded in the high dose group. However, due to the very low number of animals examined in this dose group, a scientifically valid interpretation of these results is not possible. After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.
- Although a test item related effect cannot be ruled out, as this effect was more relevant for pre-implantation loss than for post-implantation loss, it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item.
- No effects on external, visceral or skeletal abnormalities or variations were noted.
Key result
Dose descriptor:
NOAEL
Effect level:
0.84 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Key result
Developmental effects observed:
no

Terminology Used in the Assessment of the Data

Term

Description

Empty Implantation Site:

Very early resorption or aborted implantation

Early Resorption:

Amorphous mass being resorbed

Late Resorption:

Clearly defined foetal body being resorbed

Dead Foetus:

Appearance of live foetus at C-section but without induced respiration or movement

Live Foetus:

Breathing and/or moving foetus

Malformation:

A structural change in a foetus that would probably impair its health or development

Variation:

A foetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development

 Calculations

Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices

Pre implantation loss [%]       =       Corpora lutea (per group) - Implantations (per group) / Corpora lutea (per group)  x 100

Post implantation loss [%]       =       Implantations (per group) - living fetuses (per group) /  Implantations (per group)  x 100

             

Uterine and implantation data

Sex :Females

Group A

(Control)

Group B

(0.1 mg/L)

Group C

(0.3 mg/L)

Group D

(0.85 mg/L)

Number with Foetuses

8

8

8

7

Number of Corpora Lutea

Sum

112

111

107

97

Mean

14.0

13.9

13.4

13.9

SD

2.1

1.9

1.4

1.7

Number of Implantations

Sum

105

100

95

74

Mean

13.1

12.5

11.9

10.6

SD

2.0

2.1

2.2

3.1

% Pre-implantation Loss

Mean

5.9

9.9

10.6

23.4

SD

8.8

9.8

17.0

21.4

Early resorption

Sum

7

3

10

1b

Mean

0.9

0.4

1.3

0.1

SD

0.8

0.5

2.8

0.4

Late resorption 

Sum

0

0

0

1b

Mean

0

0

0

0.1

SD

0

0

0

0.4

Number of Dead Foetuses

Sum

0

0

0

0

Mean

0

0

0

0

SD

0

0

0

0

Number of Live Foetuses

Sum

98

96

82

67

Mean

12.25

12.00

10.25

9.57

SD

2.25

2.20

2.87

4.31

%Post-Implant Loss

Mean

6.85

4.16

12.61

12.61

SD

6.52

4.63

20.51

22.93

Live Foetus %Implant

Mean

93.2

95.8

87.4

87.4

SD

6.5

4.6

20.5

22.9

Litter size / foetal data

Sex :Females

Group A (Control)

Group B

(0.1 mg/L)

Group C

(0.3 mg/L)

Group D

(0.85 mg/L)

 

n

8

8

8

7

Total live Foetuses

Sum

98

96

82

67

Mean

12.25

12.00

10.25

9.57

SD

2.25

12.00

10.25

9.57

Live male foetuses

Sum

43

54

42

29

Mean

5.38

6.75

5.25

4.14

SD

1.92

2.55

2.05

2.41

Live female foetuses

Sum

55

42

40

38

Mean

6.88

5.25

5.00

5.43

SD

2.53

2.12

1.85

2.57

% Male foetuses

Mean

44.44

55.73

50.95

43.34

Litter weight

Mean

57.92

56.74

48.94

47.14

SD

10.33

9.11

11.82

20.67

Mean foetal weight (M+F)

Mean

4.74

4.75

4.84

4.95

SD

0.30

0.33

0.31

0.24

Mean foetal weight (M)

Mean

4.89

4.87

4.91

4.93

SD

0.28

0.34

0.31

0.33

Mean foetal weight

(F)

Mean

4.62

4.58

4.75

4.95

SD

0.28

0.31

0.33

0.27

Placenta weight

Mean

0.52

0.49

0.49

0.53

SD

0.07

0.06

0.06

0.10

Test atmosphere related parameters

Test item concentration in vapour

Throughout the study period, the variation of the mean test item concentrations with respect to the target values was within the ± 11% and the Relative Standard Deviation (RSD) on study means was 25%, 12% and 11% in groups B, C and D, respectively.

The deviations from target concentration on the daily individual atmosphere determinations in Groups C and D were < ±20% accepted criteria according the OECD guidelines except for 3 and 2 not consecutive days, respectively, when deviation from target was always < 24%.

Therefore, even though the deviations from target concentration on the daily individual atmosphere determinations were above the ±20% accepted criteria (according the OECD guidelines), the results obtained did not impact the objective of the study.

 

Summary of the mean test item concentrations (mg/L air) in test atmosphere:

 

Group

Exposure

Target test item conc.

(mg/L air)

Test item vapour concentration (mg/L air)

Mean

SD

RSD (%)[DS1] 

% Deviation from target

A

Air

-

-

-

-

-

B

Low dose

0.1

0.11

0.03

25

11

C

Mid dose

0.3

0.31

0.04

12

2

D

High dose

0.85

0.84

0.09

11

0.7

 

Nominal concentration

The nominal concentration was calculated from the daily consumed test item and the air flow feeding the vapour generator and thus it was determined for the overall inhalation system. Under consideration of the respective dilution air flow at every chamber, an efficiency of approximately 50 % or higher could be estimated for all groups. Overall, this value is within the expected range for this type of atmosphere generation, especially taking into account the actual concentrations determined at the representative sampling ports for every group.

Temperature and relative humidity

Mean values for exposure temperature were between 23.4 and 23.5 ºC for all groups. Mean Relative humidity was in the range 14.5 – 17.6 % for the different groups. These values are below the target range for husbandry. The difference was considered due to technical reasons. However, it was considered not to have affected the outcome of the study. 

Oxygen and carbon dioxide (CO2)

Mean oxygen concentration was 20.9 % for all groups. Carbon dioxide was always < 1% (mean concentration of 0.04 % for all groups).

Flow per animal

The flow per animal was between 0.5 and 1.5 L/min, which is sufficient to avoid re-breathing as it was more than twice the minute volume of rats.

Conclusions:
It was considered that the determination of a no observed-adverse-effect level (NOAEL) embryo-foetal development was suggested at (0.84 mg/L (149 ppm). However, for maternal effects, NOAEL could not clearly be achieved.
Executive summary:

Introduction and study outline

The objective of this dose range finding (DRF) study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20). Therefore, this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.

For those purposes, 32 pregnant female rats were distributed into four experimental groups (A to D). Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A; control) 6 hours/day from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20).

Animals from group A were exposed to filtered compressed fresh air and served as control and animals from groups B to D were exposed to diluted vapour from the test item at nominal concentrations of 0.1, 0.3 and 0.85 mg/L air, respectively. The vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).

In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.

All animals were observed twice daily for mortality and clinical observations before and within 1 hour after exposure. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a caesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Results

The composition and characteristics of test atmospheres were stable throughout the study period in Groups C and D. There was a slightly higher variation in Group B. The mean concentrations obtained deviated < 11% from the respective target.

Concentrations of 0.11, 0.31 and 0.84 mg/L air corresponding to 20, 54 and 149 ppm were achieved in Groups B, C, and D, respectively.

 

Maternal toxicity

None of the animals died during exposure.

The most frequent clinical sign present in animals belonging to all experimental groups, including sham, was piloerection. This sign is commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item.

No statistically significant differences were found in mean body weight/body weight gain and food consumption among the four experimental groups neither during the exposure period nor at the end of the study.

No relevant necropsy findings related to the test item exposure were recorded for the dams. There were no effects of the exposure on the pregnancies and/or gravid uterus weight. There were no statistically significant differences in pre- and post-implantation losses, however, a trend was observed especially for pre-implantation loss (%) where a 4-fold increase was recorded for dams of the high dose group ( 149 ppm, 0.84 mg/L) comparted to control group.

Post-implantation loss (%) was higher for mid and high-dose group compare to control group. In addition, the number of live foetuses was smaller in the mid- and high dose groups if compare to the control.

With regards to litter size, it was similar in all the groups.

Foetal toxicity

There was no effect of the exposures on foetal weigh at GD 21. Finally, no external, visceral or skeletal abnormalities or variations were noted.

Conclusions

No statistically significant differences on reproductive parameters were observed, although a trend in percentage of pre and post implantation loss was recorded in the high dose group. However, due to the very low number of animals examined in this dose group, a scientifically valid interpretation of these results is not possible. After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.

Although a test item related effect cannot be ruled out, as the observed effect was more relevant for pre-implantation loss than for post-implantation loss, it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item.

No effects on external, visceral or skeletal abnormalities or variations were noted. Therefore, under the conditions described for this DRF study, the test item did not reveal any teratogenic potential up to and including the highest dose level of 149 ppm (0.84 mg/L).

On the basis of these results and under the conditions of this study, it was considered that the determination of a no observed-adverse-effect level (NOAEL) embryo-foetal development was suggested at 149 ppm (0.84 mg/L). However, for maternal effects, NOAEL could not clearly be achieved.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-12 to 2019-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Physical state (at 20ºC): Neat Liquid
Purity / composition: ≥98.0 to ≤ 99.6%
Molecular weight: 138.20 g/mol
Colour: Clear
Storage conditions: Room temperature (ca. 23ºC); protected from light
Expiry date: July, 2019
Handling conditions: Personnel will wear gloves and auto-ventilated equipment equipped with filters appropriate vapours
Other: Sensitive to air, highly volatile, odour of peppermint.
Avoid inhalation and contact with eyes and skin. Test item may cause irritation of respiratory tract, eyes and skin
The neat substance without solvent will be tested
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj)
Remarks:
Although the F 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specificaly asked for their use in order to compare the results with previous non-GLP reproduction toxicity studies, in which this strain was used
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj)
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Age: 8 - 13 weeks on arrival; 11 - 12 weeks on first item exposure
- Animals per cage (before / after distribution): Up to 5 (1 after distribution and before mating)
- Bedding material: Sodispan (SR-CHOPO-T) (autoclavable)
- Diet: ad libitum, Global diet, 2914C, Envigo Teklad
- Water: ad libitum, Tap water
- Acclimatisation period: at least 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 24.6° C
- Humidity (%): 28 - 61 %
- Light cycle: 12 hours light, 12 hours dark
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Preparation of the exposure system
Previous to exposure onset, a complete setup of the system and flow/pressure characterization was performed according to the corresponding internal SOPs. The tasks were the following:
- Make an outline of each set up including identification of the corresponding equipment.
- Leak test through the atmosphere conveyor glass tubes.
- Identification of each group (name, dose, total number of animals, total flow of vapour/air).
- Check air temperature and relative humidity.
- Theoretical calculations regarding dilution.
- Check vacuum system in sampling ports: capillary installed, vacuum pressure measurement, sampling flow.
- Exposure test: determination of pressure inside the inhalation chamber versus room pressure and difference of pressure between both chamber compartments. Determination of atmosphere flow and vapour concentration at different positions of the chambers was obtained to confirm a homogenous distribution of the vapour.
- Six ports from the high level, six ports from the mid level and six ports from the low level. Calculation of coefficient of variation (%CV) within each level and across the three levels. Within each level, the CV was not higher than 5%, whereas the CV among the three levels will not exceed 10%.


Test item generation
- A vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).
- The test item was inside the generator vessel in liquid state.
- It was heated at a controlled temperature by means of a water bath equipped with a temperature probe to generate a vapour phase.
- At the same time, a source of compressed air at a controlled temperature was delivered into the VOC generator. This compressed air was mixed with the vapour phase of test item extracting the formulation of the product in a controlled amount of air (L/min) and concentration.
- The vapour was conveyed via glass tubing from the generator to the exposure chambers.
- The flow rate through the exposure chamber was adjusted as necessary.
- In order to prevent exposure of the animals to test item droplets from condensation, a filter was placed immediately before the exposure chamber.
- For sham exposure, filtered, compressed fresh air was provided to the exposure chamber.


Exposure conditions
- Inhalation exposure was performed using a flow-past, nose-only exposure system.
- The animals were confined separately in restraint glass tubes matching their size which are positioned radially around the exposure chamber.
- The exposure system ensures a uniform distribution and provides a constant flow of test material to each exposure tube.
- The flow of air at each tube was between 0.5 to 1.5 L/min, which was sufficient to minimize re-breathing of the test item vapour as it is more than twice the respiratory minute volume of rats.
- The position of the rats in each group within the exposure chamber was changed daily according to an insertion schedule.
- Sham-exposed rats were exposed to filtered, conditioned air, the exposure conditions being the same as those for the test item-exposed rats.
- Exposure chambers type EC-FPC-232 (anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes) were used. The rats were individually exposed in glass tubes matching their size.
- Before starting the inhalation period, the animals were not adapted to the glass tubes to avoid pre-implantation losses due to the stress caused by the procedure itself.
- The temperature of the test atmosphere in the exposure chamber was maintained at 22 ± 5 °C and the relative humidity as required by experimental conditions.
- Air flow per animal was monitored regularly.
- No diet or drinking water was available during exposure.
- The time spent during animal shifting was not included within the exposure time.

Technical trials
- Technical trails were performed without animals and conducted before the animal phase of the study to establish the conditions for vapour generation and sampling.
- A vapour was generated for up to 6 hours for four days.
- During this period, the concentrations as well as the temperature and relative humidity of the chamber were monitored.
- The set-up conditions and results were similar to those obtained during the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Characterisation of test atmosphere
In order to characterize the test atmosphere and to check the reproducibility of aerosol generation, several parameters were determined at defined intervals.
- Determination of the nominal vapour concentration
The test item usage was determined daily by weighing the amount in the syringe pump and adjacent lines before and after each exposure to determine the quantity of test item used. The weight used was then be divided by the total air-flow volume to give the nominal concentration. The nominal concentration for different groups was calculated from the nominal concentration of high concentration chamber and the dilution factor for each group, respectively. These data were used for the purpose of monitoring the performance of the generation system.
- Analytical determination of the vapour concentration
Analytical determination of the vapour concentration was performed three times on each day of exposure for each test item group (groups B - D). Additional samples were analysed if considered necessary.
Vapour samples were captured in a solvent trap containing a defined volume of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. Based on the results from the N-02130 study previously conducted at Vivotecnia, no additional solvent trap was necessary to place immediately after the first solvent trap, since it was demonstrated that all test item was expected to be captured during sampling. The sampling flow rate matched the air flow rate per port. The defined volume of methanol in the solvent trap was refilled to the start volume after sampling completion. Aliquots of samples of the resulting solution of test item in methanol were appropriately labelled and stored ca. 5°C until analysis.
Test item concentrations were analysed at Vivotecnia using an HPLC analytical method transferred by Swiss BioQuant AG during a previous study conducted at Vivotecnia. Samples of test item trapped in methanol were analysed within 3 days after sampling. The analytical method was validated at Vivotecnia in the study B-02791 .
Test item vapour concentration was determined three times a day on each day of exposure for each test item group by high performance liquid chromatography (HPLC) after collecting the vapour in a solvent trap containing 50 mL of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. The analytical method was validated afterwards in Vivotecnia study B-02791. Sampling time was 5 minutes at a flow rate of approximately 1 L/min. Trap volume was determined before and after sampling in order to evaluate discrepancies in the final volume. Aliquots of the samples were analysed freshly immediately after the sampling process.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which mating was detected was designated gestation day 0 (GD0).
Male rats of the same source and strain were used only for mating. These male rats were in the possession of Vivotecnia and were not considered part of the test system.
Duration of treatment / exposure:
Duration of dosing: Daily from day 5 post coitum (implantation, GD 05) to day 19 post coitum (GD 19)
Dosing regimen: 6h/day
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 20
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group (Group A), Sham filtered air
Dose / conc.:
17 ppm (nominal)
Remarks:
Low dose group (Group B)
Dose / conc.:
53 ppm (nominal)
Remarks:
Mid dose group (Group C)
Dose / conc.:
150 ppm (nominal)
Remarks:
High dose group (Group D)
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group (Group E), Pregnancy reference group
No. of animals per sex per dose:
24 pregnant female rats per dose
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
The present study was designed to assess the potential effects of the test item 3,5,5-trimethylcyclohex-2-enone (CAS 78-59-1) on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration through the inhalation route (nose-only) from day GD 05 to GD 19 (the day prior to Caesarean section). This study has helped to demonstrate any dose-related response and to establish the no-observed adverse effect level (NOAEL).
For this purpose, 120 pregnant female rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section).
All animals were observed once daily for mortality and clinical observations. Body weight and food consumption were recorded at appropriate intervals. On gestational day 20, a caesarean section was performed on each female and selected organ were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Rationale for target concentration selection: T
he target concentrations were selected by the sponsor according to two previous dose range finding prenatal development toxicity studies (N-02669, VVT 2019 and N-02130, VVT 2016) available for this test item.
Within these dose range finding studies, mortality was observed at 1.25 mg/L (220 ppm), while exposure at 0.85 mg/L (150 ppm) did induce maternal toxicity (statistically significant body weight losses) in one of the dose range finding studies. Based on these results and due to the fact that the observed effects at 220 ppm shows a very steep dose-response curve of the test substance, 150 ppm was chosen as highest dose concentration for the OECD 414 main study.
This concentration level is slightly above 144 ppm, which was requested by ECHA in order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon, 1983). In these prior studies, isolated instances of exencephaly were noted in rat and mice foetuses at 144 ppm.

Inclusion criteria: All animals were subjected to individual veterinary examinations. Only animals in good health and with pregnancy status confirmed were included in the study
Stratification method: Animals were randomly distributed by means of the body weight stratification method at least 2 days before first administration

- Determination of the temperature
The temperature in the chamber was measured hourly during exposure using a thermohygrometer. The target range is 22±3ºC.
- Determination of the relative humidity
The relative humidity (%) in the vapour was measured hourly in each group during each day of exposure using a thermohygrometer.
- Determination of oxygen and CO2 concentration
The oxygen concentration of the test atmosphere was measured hourly during each day of exposure using an appropriate device. The oxygen and CO2 concentrations were maintained above 19% and below 1% respectively, during the exposure period.
- Airflow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure. The actual airflow rate was monitored hourly in each group during each exposure. Additional measurements were performed if considered necessary.
Maternal examinations:
Experimental data concerning, body weight, food consumption, general clinical signs, thyroid weight, gross necropsy findings (dams) and ovaries and uteri examination were recorded through Provantis 8.5.2.1TM (Instem LSS Ltd., Staffordshire, United Kingdom) preclinical software.

CAGE SIDE OBSERVATIONS: Yes
The animals were examined once daily by cage side observation for mortality and morbidity. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Where the condition of an animal required premature sacrifice, it was euthanized by a sodium pentobarbital overdose. Any decision regarding premature sacrifice were made after consultation with the Study Director (or a nominated person) and with the study monitor, if possible.

DETAILED CLINICAL OBSERVATIONS: Yes
On exposure days, clinical observations were recorded before exposure, just after exposure and 1h after exposure. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Any visible clinical signs, discomfort and mortality was recorded in accordance with the humane endpoints guidance document of the OECD (ENV/JM/MONO(2000)7). Observations included changes in skin, eyes, and mucous membranes. Alterations in respiratory pattern or behaviour as well as changes in posture and response to handling and the presence of abnormal movements were also recorded.

BODY WEIGHT: Yes
Body weight were recorded on gestation day 0 (GD0), at least every 3 days and on GD20. In addition, body weight after sacrifice was also recorded.
Additional body weights were obtained as necessary for animal welfare reasons.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food was weighed on GD 0, 3, 5, 8, 11, 14, 17 and 20. Food consumption was calculated on these defined intervals by cage, estimated the daily food intake for each animal.
Data was presented in terms of consumption per animal per day for the periods (days 0 – 3), (3 – 5), (5 – 8), (8 – 11), (11 – 14), (14 – 17) and (17 – 20).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Gross necropsy
At the scheduled necropsy (GD20) females were sacrificed by CO2 asphyxiation and the foetuses were removed by caesarean section. For all animals the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs examined. Organs or tissues showing any macroscopic abnormalities were removed and fixed in appropriate fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites and position of foetuses in the uterus.
- Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight were recorded. From all dams, the weight of the thyroid gland was recorded and preserved in 4 % neutral-buffered formaldehyde for histopathological assessment. Weight of gravid uteri including the cervix was recorded.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams will be instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral phosphate buffered 4% formaldehyde solution for possible further examinations.

- Endocrine disrupter relevant endpoints
Concentration of Thyroid hormones, including T4, T3 and thyroid-stimulating hormone (TSH), were measured by the appropriate analytical method from all animals. Blood samples were collected from the abdominal aorta at the end of the study in non-treated tubes and serum samples were processed following the appropriate SOP.
Serum samples of all animals were examined at the end of treatment for an evaluation of test item-related effects on thyroid hormones at BSL BIOSERVICE Scientific Laboratories Munich GmbH (delegated phase). After arrival of serum tubes (a total of 360 tubes = 3 aliquots per animal and sampling a two-times 30µl and one-time 100µl) shipped on dry ice, tubes was stored at < -70°C.
Parameter Units
T3 pg/mL or ng/mL
T4 pg/mL or ng/mL
TSH pg/mL or ng/mL
Hormone determination was performed on the MagPIX, Luminex or on the DRG:HYBRID-XL Analyzer, DRG.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
For pregnant females the following observations were made:

- Number of corpora lutea
- Number and distribution of implantations in each uterine horn, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
- Gravid uterus weight: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day GD 20 to enable the calculation of the corrected body weight

- Placental weight
The live foetuses and their placentae were removed.


Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight were recorded. From all dams, the weight of the thyroid gland was recorded and preserved in 4 % neutral-buffered formaldehyde for histopathological assessment. Weight of gravid uteri including the cervix was recorded.
Fetal examinations:
Foetal Pathology
Foetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually and examined for gross external abnormalities. External examination of foetuses was performed at Vivotecnia. The anogenital distance (AGD) was measured in all live rodent foetus. After external examination, foetuses were assigned with a number, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique)]. Approximately one half of the foetuses (foetuses with odd numbers) from each litter will be fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the foetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissues were preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol (ca. 94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined, and all abnormal findings and variations were recorded. The assessment included but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Foetuses with abnormalities were photographed, when considered appropriate.

The foetal pathology phase report is attached to this endpoint study record.

Terminology Used in the Assessment of the Data
Term Description
Empty Implantation Site: Very early resorption or aborted implantation
Early Resorption: Amorphous mass being resorbed
Late Resorption: Clearly defined foetal body being resorbed
Dead Foetus: Appearance of live foetus at C-section but without induced respiration or movement
Live Foetus: Breathing and/or moving foetus
Malformation: A structural change in a foetus that would probably impair its health or development
Variation: A foetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development


Calculations
Comparison Group A and E, against Groups B, C and D.
Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means.
The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Pre-implantation loss (%) = (# of corpora lutea –# of implantation sites) x 100/ no. corpora lutea

Post-implantation loss (%) = (# of implantation sites – # of live foetuses) x 100/ #. of implantation sites

Statistics:
Provided that the sample size was considered adequate, the following parameters will be subject to statistical analysis:
- Body weight
- Body weight change
- Gravid uterine weight
- Body weight change corrected for gravid uterine weight
- Food consumption
- Absolute and relative organ weights
- T3, T4 and TSH levels
- Anogenital distance statistically evaluated by gender

Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Kruska-Wallis Dunn's post test
Test of equal variance No Kruska-Wallis Dunn’s post test
A value of P < 0.05 will be considered statistically significant
Indices:
Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Historical control data:
Although the Fisher 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specifically asked for their use in both the DRF and main studies in order to compare the results with previous non-GLP reproduction toxicity studies, in which the Fischer 344 strain was used.
Due to the very limited number of historical control data on Fischer 344 rats this OECD 414 study was performed using two control groups and three dose-level groups in order to receive scientifically meaningful results.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical signs present in animals belonging to all experimental groups, including sham, were chromorrhinorrea, chromodacryorrhea and wet fur. These signs are commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item. In addition, it was observed loss of stability at the beginning of the studio in animals exposed to the high dose, however this sign disappeared after the first days of exposure.
Additionally, incidental bleeding through the vagina was observed from days 12 to 16 of gestation in animals belonging to groups A, B, C and D. Even though the number of females with occasional bleeding was higher in the high dose group, these signs were single observations (< 2 and not in consecutive days) and there was not a dose-dependent relationship. Therefore, these observations were considered incidental and probably not test item related.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during exposure. All animals survived until the schedule necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The exposure regimen resulted in only transient reductions in maternal weight in group D,151 ppm (approximately 3.5% reduced from Control Group A and 12.3% reduced from Pregnancy Reference Group E) on day 17 of Gestation.
No statistically significant differences were found in mean body weight gain among the four experimental groups neither during the exposure period nor at the end of the study. Body weight gain was reduced at GD 14-17, but this difference was not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was decreased between the days 5 and 8 of the exposure period in Group D (151 ppm) when compared to Group Control A (up to approximately 15%) and Pregnancy Reference Group E (30.9%), p<0.01. Even though it cannot be ruled out a test item effect on food consumption at the beginning of the study, after these transient reductions there were a similar consumption among the five experimental groups (no significant differences) at the end of the study. Therefore, the variation on food consumption at the beginning of the exposure was considered incidental and devoid of any toxicology effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistical differences were recorded neither in terminal body weight nor in gravid uterus weight when compare the Group Control A with the exposed groups. The adjusted body weight to gravid uterus was also similar among exposed groups at concentrations of 18, 55 and 151 ppm, respectively. However, when compared Group E pregnancy reference group with both Control A and exposed Groups, there were some statistically significant increased. Nevertheless, these differences were considered to be related with the procedure itself rather than with the exposure.
Uterine weight in Group C (55 ppm) and D (151 ppm) was reduced (96% of the control group value) but this reduction was not considered adverse because the differences were slight, within normal animal variation and there were no statistically significant differences between the treated groups and control group.

Absolute and relative organ weights of thyroid gland were similar among all experimental groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No relevant macroscopic abnormalities related to the exposure to the test item were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Under the conditions of this study all animals survived their scheduled period and there were no necropsy findings or histology changes in the thyroid glands that could be related to the treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Endocrine disrupter relevant endpoints
The serum levels of total T3 were elevated in dosed groups C and D (exposed to 55 and 151 ppm 0.86 mg/L of test Item) when compared to Control Group A. This elevation was not due to enhanced T4 to T3 conversion. In addition, when compared with Reference Group E, only difference with Group D became significant. Individual data showed a great variability between animals from the same group. Taking into consideration that not differences were observed in TSH and T4 levels, the values were considered incidental findings and devoid of toxicological significance or not treatment-related. When compared with historic control data from BSL Bioservice, thyroid hormone levels were within the normal range.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase on post-implantation loss (not statistically significant in any case) was recorded for dams of groups C (55 ppm) and D (151 ppm). In addition, 2 dams from group D (151 ppm) presented total resorption. These differences were not significant and were considered not test item related. In addition, the post-implantation losses (%) where within the observed range (6.86%-18.46%) from vivotecnia historical control data in Fischer 344 sham filtered exposed.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
A total of two pregnant dams with total resorption were recorded in the high dose group (151 ppm ).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
A total of two pregnant dams with total resorption were recorded in the high dose group (151 ppm ).
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no significant decrease in the number of foetuses alive in any of the dose groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Caesarean section (C-section) on day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
From the 120 dams enrolled in the study, 117 were pregnant.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 151 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: no adverse and statistically significant maternal toxicity was observed, in addition no differences on reproductive parameters were observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were some marginal differences of the exposures on foetal weight at GD 20 post coitum between control group A and exposed groups B (18 ppm) and C (55 ppm). The very marginal (3-5%) difference while achieving statistical significance in total foetal, male and female weights, was considered to be within the normal biological variation and therefore, devoid of toxicological significance or not treatment-related. On the other hand, the statistical difference in foetal body weight when compared to controls at the high dose-level showed a decrease of 13.6%, consequently a test item effect on foetal weight in the high dose group D cannot be excluded.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of foetuses alive were similar among dose groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male foetuses were also similar between all dose groups.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical differences were found in litter weight, although a decreased of 9% was observed in group D (151 ppm) compared with Control group A.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were noted during external examination of foetuses at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was observed during the skeletal examination at any dose level and no test item-related findings were noted.
The alterations observed during the skeletal examination were assigned to
• bone ossification stage/supernumerary ribs, which were related to a change from the expected ossification state of rat bones in this stage.
• bone or cartilage variations, which were used to describe structural changes (as changes in shape or size).
The type and frequencies of recorded variations were similar at all dose levels, in the control groups, group A (Sham filtered air) and group E (pregnancy reference).

- Ossification stage/supernumerary ribs
Ossification stage of foetal skeleton and number of supernumerary ribs showed no indication of any test item-related effect at any dose level.
At dose levels of 55 ppm (group C) and 151 ppm (group D), a higher number of incomplete ossified supraoccipital bone was observed when calculated either on a foetus or a litter basis. At dose levels of 18 ppm and 151 ppm a higher number of non-ossified caudal vertebrae was observed when calculated either on a foetus or a litter basis. For both alteration no dose dependency was noted, therefore these findings were considered not to be test item related but a result of biological variability.

- Bone and cartilage variations:
Bone and cartilage variations showed no indication of any test item-related effect at any dose level.
Among bone and cartilage variations, an increase in the incidence of sternebrae offset ossification sites was noted at 18 ppm and 55 ppm when compared to the incidence in pregnancy reference (group E) (20% and 22% respectively, versus 4% in the pregnancy reference group). Whereas, no significant difference was observed when compared to the concurrent control (Sham filtered air). Furthermore, this finding was noted only in 2% of foetuses of group D (151 ppm) when compared to the concurrent control (Sham filtered air), as no dose-dependency was noted, this finding was considered to be incidental and not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:

33% examined foetuses in 78% litters in Sham filtered air (Group A)
41% examined foetuses in 88% litters in 3,5,5-trimethylcyclohex-2-enone (Group B,18 ppm)
35% examined foetuses in 96% litters in 3,5,5-trimethylcyclohex-2-enone ( Group C, 55 ppm)
35% examined foetuses in 75% litters in 3,5,5-trimethylcyclohex-2-enone ( Group D 151 ppm)
53% examined foetuses in 96% litters in Pregnancy Reference (Group E)

No test item related findings were noted in any treated group.

Malformation such as renal pelvis dilated severe was observed in group B (litter 31, foetus 356) in one of 143 foetuses examined. Since this alteration occurred only in one foetus, it was considered to be incidental and not test item related.
The overall incidences of foetuses or litters with any remaining finding were isolated incidences and did not indicate any test item effect. Thus, they were considered not to be test item-related. The incidence of one single variations in form of thymus long and testis malpositioned were increased on foetal and litter basis when compared to group A (Sham filtered air) and they were comparable to those observed in group E (pregnancy reference). These specific findings were known to be normally associated with a slight immaturity of development; therefore, they were considered to be not related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
No differences were observed in anogenital distance for both females and males.
Details on embryotoxic / teratogenic effects:
Taking into account the test item effect on foetal body weight, a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L). However, under the conditions of the study the test item did not reveal any teratogenic potential up to and including the highest dose level (NOAEL, teratogenic > 151 ppm).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 151 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Dose descriptor:
NOAEL
Effect level:
55 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Under the condition of this inhalation OECD 414 study in Fischer F344 rats the test item did not reveal any teratogenic potential up to and including the highest dose level of 151 ppm (0.86 mg/L).
Key result
Developmental effects observed:
no

Summary Foetal External examinations

No treatment-related findings were noted during external examination of foetuses at any exposure level.

The table that summarizes the macroscopic findings observed at necropsy (only foetuses with findings are included in the table) is attached to this endpoint study record.

Inhalation Technical Data: Tables see attached document to this endpoint study record

In-Life Data: Tables: see attached document to this endpoint study record

Test atmosphere related parameters

 Test item concentration in vapour

Throughout the study period, the variation of the mean test item concentrations with respect to the target values was within the ± 3% and the Coefficient of Variation (CV) on study means was 8%, 10% and 7% in groups B, C and D, respectively.

The deviations from target concentration on the daily individual atmosphere determinations in Groups B, C and D were <± 20% accepted criteria according the OECD guidelines except for 2 not consecutive days, in group C, when deviation from target was always < 22%.

Therefore, even though the deviations from target concentration on the daily individual atmosphere determinations were punctually above the ± 20% accepted criteria (according the OECD guidelines), the results obtained did not impact the objective of the study.

Summary of the mean test item concentrations (mg/L air) in test atmosphere:

Group

Exposure

Target test item conc.

Test item vapour concentration (mg/L air)

(mg/L air)

Mean

SD

RDS (%)

% Deviation from target

A

Air

-

-

-

-

-

B

Low dose

0.1

0.10

0.01

8

2

C

Mid dose

0.3

0.31

0.03

10

3

D

High dose

0.85

0.86

0.06

7

1

 

Nominal concentration

The nominal concentration was calculated from the daily consumed test item and the air flow feeding the vapour generator and thus it was determined for the overall inhalation system and for each exposure concentration. Overall, this value is within the expected range for this type of atmosphere generation, especially taking into account the actual concentrations determined at the representative sampling ports for every group.

 

Temperature and relative humidity

Mean values for exposure temperature were between 21.78 and 21.55 °C for all groups. Mean relative humidity was in range 6.25 - 7.23 % for the different groups. These values are below the target range for husbrandy. The difference was considered due to technical reasons. However, it was considered not to have affected the outcome of the study.

   

Oxygen and carbon dioxide (CO2)

Mean oxygen concentration was 20.9 % for all groups. Carbon dioxide was always < 1 % (mean concentration of 0.04 % for all groups).

Flow per animal

The flow rate per animal was between 0.5 and 1.5 L/min, which is sufficient to avoid re-breathing as it was more than twice the minute volume of rats.

Conclusions:
Under the conditions of the inhalation OECD 414 study with Fischer rats a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L).
The test item did not reveal any teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L) and therefore, the NOAEL for developmental effects was determined to be >= 151 ppm (0.86 mg/L). As an effect on foetal body weight at 151 ppm (0.86 mg/L), a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).
Executive summary:

Introduction and study outline

The objective of this developmental toxicity study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) during the period of organogenesis, from gestation day GD05 to day GD 19 (the day prior to Caesarean section).

Therefore, this study aimed to characterize maternal and foetal toxicity at the exposure levels tested and todetermine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

 

For this purpose, 120 pregnant female rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Group Ewas included to be able to identify the possible findings related with the inhalation procedure itself, as the stress of the procedure could affect the pregnancy outcome and related variables. Therefore, differences between group E and groups A, B, C and D are just related with the procedure itself and not with the test item exposure. Furthermore, Group E served as second control group. Animals were exposed to diluted vapour from the test item (groups B to D) at nominal concentrations of 17, 53 and 150 ppm (0.1, 0.3 and 0.85 mg/L air, respectively), respectively, or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section).

The vapourwas generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).

In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.

 

All animals were observed three times daily for mortality and clinical observations before, just after and within 1 hour after exposure. Body weight and food consumption were recorded at appropriate intervals.

On gestational day 20, a caesarean section was performed on each female. The animals were subjected to a macroscopic postmortem evaluation and corpora lutea/implantation data were recorded and the gravid uterus and thyroid gland were weighed. The foetuses were examined for externally visible abnormalities, sexed and weighed. Approximately one-half of the fetuses (alternating fetuses) in each litter were examined for soft-tissue abnormalities using a microdissection procedure. The other half of the fetuses were then stained with Alizarin Red S and examined for skeletal abnormalities and ossification variations.

 

Results

The composition and characteristics of test atmospheres were stable throughout the study period in Groups A, B, C and D. The mean concentrations obtained deviated 3% from the respective target. The overall mean (± standard deviation) analytical exposure concentrations for the respective exposure groups were as follows: 0.10 ± 0.1, 0.31 ± 0.031, and 0.86 ± 0.05 mg/L corresponding to 18, 55 and 151 ppm.

Maternal toxicity

None of the animals died during exposure.

The most frequent clinical signs present in animals belonging to all experimental groups, including sham, were chromorrhinorrea, chromodacryorrhea and wet fur. These signs are commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item. In addition, it was observed loss of stability at the beginning of the study in animals exposed to the high dose, however this sign disappeared after the first days of exposure.

The exposure regimen resulted in only transient reductions in maternal weight in high dose group D (approximately 3.5% reduced from Control Group A and 12.3% reduced from Pregnancy Reference Group E).Body weight gain was also reducedat GD 14-17, but this difference was not statistically significant. Food consumption was decreased at the beginning of the exposure in Group D (151 ppm) when compared to Group Control A (up to approximately 15%) and Pregnancy Reference Group (30.9%) between the day 5 and 8 of the exposure period. However, the Pregnancy Reference Group E, had a significantly increased in food consumption when compare with all the exposed groups including sham filtered control group A. Consequently, the difference with group E were considered to be related with the exposure procedure and not with the test item.

The Reference Pregnancy Group when compared with the test item exposure groups, showed differences in Body weight, Bodyweight gain and food consumption, however there were also differences when compare the Control Group A with the Pregnancy Reference Group E. Therefore, it was considered that the differences in Body weight, Bodyweight gain and food consumption with group E were related with the stress of the procedure itself and not with the exposure to the test item.

The serum levels of total T3 were elevated in dams from groups C and D (exposed to 55 and 151 ppm of test Item), this elevation was not due to enhanced T4 to T3 conversion. In addition, no differences were observed in thyroid weight and after the histopathology evaluation and when compare with historic control data from BSL Bioservice the T3 levels were within the normal range.

No relevant necropsy findings related to the test item exposure were recorded for the dams. There were no effects of the exposure on the pregnancies and/or gravid uterus weight. There were no statistically significant differences in pre- and post-implantation losses, even though, post-implantation loss (%) was higher for mid and high-dose group compared to control group, where a 2-fold increase was recorded for dams of group D (high dose, 151 ppm) compared to control group. These differences were not significant and were considered not test item related. In addition, two dams from group D had complete resorption which increased the total percentage of post-implantation losses for the group. In addition, the post-implantation losses (%) where within the observed range (6.86%-18.46%) in Fischer 344 sham filtered exposed from vivotecnia historical control data.

Foetal toxicity

There was no foetal death recorded during the study and the sex ratio for live fetuses were unaffected by treatment.

The number of total and live foetuses was similar among all groups. With regards to litter size, it was similar in all the groups. Even though litter weight was smaller in the group D (151 ppm), approximately 9% smaller than the control group A, this difference was not statistically significant. On the other hand, Fetal body weight per litter (male, female, or total) was significantly reduced in all the exposed groups, these differences were below 5% for groups B (18 ppm) and C (55 ppm) and considered devoid of any toxicological significance, but a reduction of 13.6% on foetal body weight, when exposed at 151 ppm (Group D), was both statistically and biologically significant.

Finally, no external, visceral or skeletal abnormalities or variations were noted, and it was concluded that no test item related findings were noted in any treated group, neither during skeletal examination, nor during visceral examination. Therefore, the test item did not reveal any teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L). As an effect on foetal body weight at 151 ppm (0.86 mg/L), a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L).

Conclusion

    

-   No adverse and statistically significant maternal toxicity was observed during the study, in addition no differences on reproductive parameters were observed. Only transient reductions in maternal weight gain on GD17 and food consumption between day 5 and 8 of gestation and an incidental increased on T3 serum levels. It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L).

 

-        There was no effect of the exposures at any level on pregnancy outcome, in terms of pre- or post-implantation loss or the incidence of fetal abnormalities and variants. Therefore, under the conditions of this study, the test item did not reveal teratogenic potential up to and including the dose level and the NOAEL for developmental effects was determined to be >= 151 ppm (0.86 mg/L).

Nevertheless, taking into account the test item effect on foetal body weight, a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-12-21 to 1984-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Teratogenicity Test
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Charles River Labs (Kingston Facility)
- Age at study initiation: 9 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days
- Housing: animals were contained in a 12.9 m3 stainless steel and glass chamber and exposed either to conditioned air or to an atmosphere of
isophorone suspended in air
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: no vehicle
- Concentrations: 0 / 25 / 50 / 115 ppm (corresponds to 0, 144, 289 and 664 mg/m3)
- Type or preparation of particles: vapour
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were routinelly sampled on approximately an hourly basis by gas chromatograpgy.
Study mean concentrations as determined by GC were: group I = 0 ppm, group II = 27 ppm, group III = 52 ppm, group IV = 111 ppm
Details on mating procedure:
MATING PROCEDURES
Virgin female CD-1 mice (approximately 9 weeks of age)  were paired with similar males for mating. Females were confirmed to have mated by
observation of a copulatory plug in the vagina or by observation  of sperm in a vaginal rinse. Confirmed mated females were assigned to groups
randomly.
Duration of treatment / exposure:
6th to 15th day of gestation
Frequency of treatment:
6 h/d
Duration of test:
Mice were sacrified by methoxyflurane inhalation and examined by gross necropsy on the 18th day of gestation.
No. of animals per sex per dose:
22 female mice
Control animals:
other: yes, concurrent conditioned air
Details on study design:
Sex: female
Duration of test: section on the 18th d of gestation
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weight gain: each 3rd day
- Food consumption: not determined
- Clinical observations: each 3rd day

Ovaries and uterine content:
- Examination of uterine content: identified as live fetuses, dead  fetuses, late resorptions, and early resorptions at end of study (day 18  of gestation).
  The uterus of each animal was stained in 10 % aqueous ammonium sulfide and further examined for confirmation of implantation  sites. 
Corporalutea were counted.
Fetal examinations:
- Examination of fetuses: Live and dead fetuses were weighed, examined  externally for gross abnormalities, and crown-rump distances were  
determined.  One half of the fetuses from each litter were decapitated, the heads were preserved and subsequently sectioned and examined, viscera of these fetuses were examined by the Staples technique. All fetuses were eviscerated and processed for skeletal staining . Only those fetuses which
had not been decapitated were examined for skeletal malformations and ossification  variations.

Statistics:
STATISTICAL METHODS: 
- Bartlett's test of homogeneity of variance: body weight, body weight  change, number of implantation sites, ratio of live fetuses to  implantation 
sites, ratios of resorptions to implant sites, malformations  per litter.
- Kruskal-Wallis test if variances were not equivalent.
- Standard nested analysis of variance for fetal weights.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: no mortalities
- Number pregnant per dose level: 22
- Number of resorptions: no statistically significant differences between  treated and control
- Number of implantations: no statistically significant differences  between treated and control
- Number of corpora lutea: no statistically significant differences  between treated and control
- Duration of Pregnancy: not statistically significant differences  between treated and control
- Body weight: reduced in day 18 mice in 664 mg/m3 dose group (-5.6 %,  corrected for uterine weight)
- Clinical signs: Unremarkable
Dose descriptor:
NOAEL
Effect level:
289 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL DATA: 
- Litter size and weights: no statistically significant differences  between treated and control
- Number viable: no statistically significant differences between treated  and control
- Sex ratio: no statistically significant differences between treated and  control
- Grossly visible abnormalities: no statistically significant differences  between treated and control
- External abnormalities: no statistically significant differences  between treated and control
- Soft tissue abnormalities: no statistically significant differences  between treated and control
- Skeletal abnormalities (control, 144, 289, 664 mg/m3): 24/106, 26/120,  31/111, 36/110   
no statistically significant differences between treated and control. 
- Other examinations: Three instances of exencephaly were noted in mouse  fetuses (no data on doses). These effects are regarded as not to be  
compound-related by the authors.
Dose descriptor:
NOAEL
Effect level:
>= 664 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

NOAEL (maternal): based on reduced body weight




Conclusions:
In this inhalation teratogenicity study with mice, isophorone elicited a clinical effect in the pregnant dams in the form of lower body weights (664 mg/m3, day 18 of gestation). During the conduct of the probe study there were three instances of exencephaly noted in mouse fetuses. Based on the
observations made in this study the authors do not believe that these anomalies were related to the test material.
Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in mice.
Executive summary:

Pregnant CD-1 mice were treated daily for 6 hours by inhalation (whole body) with isophorone from day 6 to day 15 of gestation to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (conditioned air control), 144, 289 and 664 mg/m3, respectively (22 animals per dose level).

Body weight was reduced in mice of the highest dose group (gestation day 18, corrected for uterine weight: -5.6 %). Additionally, in one animal a dose related increase in alopecia was observed, as well as a discoloration of the cervical and aogenital region. Adverse effects on the fetuses were not observed.

Therefore, under the conditions of this inhalation teratogenicity study with mice, the NOAEL for maternal toxicity was determined to be 289 mg/m3 (based on < 6 % reduction in body weight gain). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-12-21 to 1984-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Method: guideline comparable inhalation teratogenicity test
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: 11 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days
- Housing: animals were contained in a 12.9 m3 stainless steel and glass chamber and exposed either to conditioned air or to an atmosphere of
isophorone suspended in air
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: no vehicle
- Concentrations: 0 / 25 / 50 / 115 ppm (corresponds to 0, 144, 289 and 664 mg/m3)
- Type or preparation of particles: vapor
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were routinelly sampled on approximately an hourly basis by gas chromatograpgy.
Study mean concentrations as determined by GC were: group I = 0 ppm, group II = 27 ppm, group III = 52 ppm, group IV = 111 ppm

Details on mating procedure:
MATING PROCEDURES: 
Virgin female Fischer rats (approximately 11 weeks of  age) were paired with similar males for mating. Females were confirmed to  have mated by 
observation of a copulatory plug in the vagina or by  observation of sperm in a vaginal rinse. Confirmed mated females were assigned to groups
randomly.
Duration of treatment / exposure:
6th to 15th day of gestation
Frequency of treatment:
6 h/d
Duration of test:
Rats were sacrified by methoxyflurane inhalation and examined by gross necropsy on the 20th day of gestation.
No. of animals per sex per dose:
22 female rats
Control animals:
other: yes, concurrent conditioned air
Details on study design:
Sex: female
Duration of test: section on the 20th d of gestation
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weight gain: each 3rd day
- Food consumption: 3 day intervals
- Clinical observations: each 3rd day

Ovaries and uterine content:
- Examination of uterine content: identified as live fetuses, dead  fetuses, late resorptions, and early resorptions at end of study
(day 20  of gestation). The uterus of each animal was stained in 10 % aqueous  ammonium sulfide and further examined for confirmation of 
implantation  sites. Corpora lutea were counted.
Fetal examinations:
Examination of fetuses: Live and dead fetuses were weighed, examined  externally for gross abnormalities, and crown-rump distances were  
determined. One half of the fetuses from each litter were decapitated, the heads were preserved and subsequently sectioned and examined, viscera
of these fetuses were examined by the Staples technique. All fetuses were eviscerated and processed for skeletal staining . Only those fetuses which
had not been decapitated were examined for skeletal malformations and ossification  variations.
Statistics:
STATISTICAL METHODS: 
- Bartlett's test of homogeneity of variance: body weight, body weight  change, food consumption, number of implantation sites,  ratio of live 
fetuses to implantation sites, ratios of resorptions to implant sites,  malformations per litter.
- Kruskal-Wallis test if variances were not equivalent.
- Standard nested analysis of variance for fetal weights.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: no mortalities
- Number pregnant per dose level: 22
- Number of resorptions: no statistically significant differences between  treated and control groups
- Number of implantations: no statistically significant differences  between treated and control groups
- Number of corpora lutea: no statistically significant differences  between treated and control groups
- Duration of Pregnancy: no statistically significant differences between  treated and control groups
- Body weight: reduced in days 12 (-6.1 %) and 15 (-6.8 %) rats in 664  mg/m3 dose group
- Food/water consumption: reduced food consumption in 664 mg/m3 dose group
- Clinical signs: alopecia and cervical or anogenital staining (each  dose-related).
Dose descriptor:
NOAEC
Effect level:
289 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL DATA: 
No statistically significant differences between treated and control  groups:
- Litter size and weights
- Number viable
- Sex ratio
- Grossly visible abnormalities
- External abnormalities
- Soft tissue abnormalities
- Skeletal abnormalities
Dose descriptor:
NOAEC
Effect level:
>= 664 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

NOAEL (maternal): based on reduced body weight, clinical signs are not considered

Conclusions:
In this inhalation teratogenicity study with rats, isophorone elicited a clinical effect in the pregnant dams in the form of decreased food consumption
(664 mg/m3, days 6-20 and 0-20), lower body weights (664 mg/m3, days 12 and 15 of gestation), and dose related increases in alopecia and
staining of the cervical and anogenital areas.
During the conduct of the probe study there was one instance of exencephaly noted in a rat fetus. Based on the observations made in this study the
authors do not believe that this anomaly was related to the test material.
Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in rats.
Executive summary:

Pregnant Fischer 344 rats were treated daily for 6 hours by inhalation (whole body) with isophorone from day 6 to day 15 of gestation to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (conditioned air control), 144, 289 and 664 mg/m3, respectively (22 animals per dose level).

There was a significant reduction in food consumption of rats of the highest dose group. Body weight was also reduced in animals of the highest dose group (gestation day 12: -6.1%; gestation day 15: -6.8%). Additionally, a dose related increase in alopecia was observed, as well as a discoloration of the cervical and aogenital region. Adverse effects on the fetuses were not observed.

Therefore, under the conditions of this inhalation teratogenicity study with rats, the NOAEC for maternal toxicity was determined to be 289 mg/m3 (based on < 7% reduction in body weight gain). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
Klimisch 1
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Exxon, 1984 and 1983 (rat and mice)


In the first inhalation teratogenicity study with rats, the test item elicited a clinical effect in the pregnant dams in the form of decreased food consumption (111 ppm (640 mg/m3), days 6-20 and 0-20), lower body weights (111 ppm 640 mg/m3, days 12 and 15 of gestation), and dose related increases in alopecia and staining of the cervical and anogenital areas. During the conduct of the DRF study (Exxon, 1983) there was one instance of exencephaly noted in a rat fetus at 144 ppm. Based on the observations made in this study the authors do not believe that this anomaly was related to the test material. Within the framework of the dose levels and test methods used, it is concluded, that the test item was not teratogenic or fetotoxic in rats (Exxon, 1984).


In the second inhalation teratogenicity study with mice the test item elicited a clinical effect in the pregnant dams in the form of lower body weights (111 ppm (640 mg/m3), day 18 of gestation). During the conduct of the DRF study (Exxon, 1983) there were three instances of exencephaly noted in mouse fetuses at 144 ppm. Based on the observations made in this study the authors do not believe that these anomalies were related to the test material. Within the framework of the dose levels and test methods used, it is concluded, that the test item was not teratogenic or fetotoxic in mice (Exxon, 1984).


 


DRF for OECD 414 (inhalation), F344 and Wistar rats (Vivotecnia, 2016)


According to the decision on substance evaluation dated 17 June 2015 (SEV-D-2114303205-67-01/F; “original decision”) the registrants were requested to perform a pre-natal developmental toxicity study (test method: EU B.31./OECD 414 as limit test) in Fischer 344 rats by the inhalation route at doses above 144 ppm (maximum tolerable and attainable concentration) on order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon ,1983).


The registrants decided to perform a dose-range finding study in advance to estimate the maximum tolerable and attainable concentration as requested in the ECHA decision. Based on occurrence of exencephalies in the two preliminary prenatal developmental toxicity studies at 144 ppm (Exxon, 1983 and 1984), the registrants decided to conduct the DRF study with target concentrations of 150 and 220 ppm. The estimation for the technical limit for the maximal vapour concentration is based on the physico-chemical properties of the test material. The actual technical limit was conducted to be at 220 ppm during technical trails and was used at high dose. Due to the unsuitability of F344 rats as well as due to the absence of historical control data, Wistar rats were also tested in the DRF study in order to be able to generate scientifically established results. (Remark: Due to the fact that no historical controls are available F344 rats and the differences in timing of developmental events compared to strains like Wistar which are commonly tested in developmental/reproductive toxicity studies, the assessment of results is hardly possible. These two points do not allow to differentiate between strain specific and/or incidental findings and substance related effects. For this reason, EPA guideline explicitly points out that Fischer 344 strain should not be used for developmental studies (see EPA Health effects Test Guidelines OPPTS 870.6300, August 1998). Therefore, in this study Wistar rats were used for quality control and comparison of findings in both strains and with available historical control data from Wistar rats.) The legal basis for this test strategy is the ECHA Factsheet "Follow up to dossier evaluation decisions p.2; ECHA-13 -FS-05".


Within this DRF study a high and promptly mortality was observed in F344 rats at the high dose (220 ppm) as well as a statistically significant lower body weight and body weight gain was observed at low dose (150 ppm) when compared to air-treated F344 rats (control group). Therefore, both concentrations were associated with overt maternal toxicity in F344 rats but no major toxicity was observed in Wistar rats. For animal welfare reason the high dose exposure (220 ppm) was stopped for both strains. However, this means that 150 ppm is the maximum tolerated concentration while 220 ppm is the maximum attainable concentration for F344 rats. No test item-related abnormalities were noted during the visceral or skeletal examinations of fetuses of both treated strains (F344 and Wistar rats).


Due to the overt maternal toxicity reproductive effects (post implantation loss) were observed in F344 rats but not in Wistar rats. No test-item related abnormalities were noted during the visceral or skeletal examinations of foetuses of both strains. In so far, the results of the Exxon studies were not reproducible. In particular against the background of all problems and difficulties with Fisher rats and due to animal welfare reasons, at this time, the registrants decided not to carry out a main OECD 414 study with the F344 strain because no scientifically based results could be generated. Additionally, no developmental effects were reported at lower concentration (111 ppm) in the Exxon study (Exxon, 1984).


 


DRF for OECD 414 (inhalation), F344 rats (Vivotecnia, 2019)


Based on the decision from ECHA of 10 April 2018 as a response to the updated dossier and the decision SEV-D-2114303205-67-01/F (“original decision”), ECHA came to the conclusion that the original decision was not met, and the registrants are still required to provide a full prenatal developmental toxicity study (according to OECD 414) in F344 rats by the inhalation route.


For this, a further DRF study was performed in Fischer 344 rats. 32 pregnant females were distributed into four experimental groups (A to D). Based on the results of the DRF in 2016 (Vivotecnia, 2016) animals were exposed to diluted vapour from the test item (groups B to D) in concentrations of 20, 54 and 149 ppm or to filtered compressed fresh air (Group A; control) 6 hours/day from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20). Within this DRF study, no statistically significant differences on reproductive parameters were observed, although a trend in percentage of pre- and post implantation loss was recorded in the high dose group. However, due to the very low number of animals examined in this dose group, a scientifically valid interpretation of these results was not possible. After the main study (Vivotecnia, 2020) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses. Although a test item related effect cannot be ruled out, as this effect was more relevant for pre-implantation loss than for post-implantation loss, it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item. No effects on external, visceral or skeletal abnormalities or variations were noted. Therefore, under the conditions described for this DRF study, the test item did not reveal any teratogenic potential up to and including the highest dose level of 149 ppm (0.84 mg/L). On the basis of these results and under the conditions of this study, it was considered that the determination of a no observed-adverse-effect level (NOAEL) embryo-foetal development was suggested at 149 ppm (0.84 mg/L). However, for maternal effects, NOAEL could not clearly be achieved.


 


OECD 414 (inhalation), F344 rats (Vivotecnia, 2020)


In the main OECD 414 inhalation study 120 pregnant female F344 rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Group E was included to be able to identify the possible findings related with the inhalation procedure itself, as the stress of the procedure could affect the pregnancy outcome and related variables. Therefore, differences between group E and groups A, B, C and D are just related with the procedure itself and not with the test item exposure. Furthermore, due to the lack on historical control data, Group E served as second control group. Animals were exposed to diluted vapour from the test item (groups B to D) at concentrations of 18, 55 and 151 ppm (0.1, 0.31 and 0.86 mg/L air, respectively), respectively, or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section). The target concentrations were selected by the registrants according to the two previous dose range finding prenatal development toxicity studies (Vivotecnia 2016 and 2019): Within these dose range finding studies, mortality was observed at 220 ppm), while exposure at 150 ppm did induce maternal toxicity (statistically significant body weight losses) in one of the dose range finding studies. Based on these results and due to the fact that the observed effects at 220 ppm show a very steep dose-response curve of the test substance, 150 ppm was chosen as highest dose concentration for the OECD 414 main study. This concentration level is slightly above 144 ppm, which was requested by ECHA in order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon, 1983). In these prior studies, isolated instances of exencephaly were noted in rat and mice foetuses at 144 ppm.


 


Maternal toxicity


Maternal toxicity was determined by the occurrence of a decrease in net body, weight gain, decrease in food intake and evaluation of intrauterine parameters. Summarizing the results of this study it can be concluded that no relevant clinical signs or necropsy findings related to the test item exposure were recorded for the dams. In addition,only transient reductions were observed for the dosed group D (151 ppm) compared to control group A for body weight/body weight gain and food consumption. Food consumption between gestation day 5 and 8 day decreased in 14.5% (p<0.01) when compared to sham air exposed control at the high dose-level (151 ppm). Nevertheless, no other difference in food consumption was observed over the study period. On the other hand, body weight on day 17 of gestation was reduced in group D (151 ppm) by 3.5% (p<0.05) when compared to control group A. However, on day 20 of gestation no difference was observed.


The serum levels of total T3 were elevated in dosed groups C and D (exposed to 55 and 151 ppm of test Item) when compared to Control Group A. This elevation was not due to enhanced T4 to T3 conversion. In addition, no differences were observed in thyroid weight and after the histopathology evaluation. When compared with Reference Group E, only difference with Group D became significant. Individual data showed a great variability between animals from the same group. Taking into consideration that not differences were observed in TSH and T4 levels,the values were considered incidental findings and devoid of toxicological significance or not treatment-related.


There was no effect of the exposures on the pregnancies. No statistical differences were observed in pre- and post-implantation losses and litter size were similar in all exposed and control groups.


It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects was evident at 151 ppm (0.86 mg/L).


 


Foetal toxicity


There was no effect of the test item exposure on litter weight or litter size at GD 20. Although foetal body weight per litter (male, female, or total) was significantly reduced in all the exposed groups, this difference was lower than 5% for groups B (18 ppm) and C (55 ppm) and therefore was considered to be within the normal biological variation. Foetal body weight in group D (151 ppm) was 13.6% lower than in group A and statistically significant, therefore a test item effect at 151 ppm (0.86 mg/L) cannot be ruled out.


Finally, no external, visceral or skeletal abnormalities or variations were noted, therefore it was concluded that no test item related findings were noted in any treated group. Therfore, the test item did not reveal teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L). However, considering the test item effect on foetal body weight, a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).


 


Accordingly, it can be concluded:


- Exposure of pregnant rats to atmospheres of 0 (Air Control), 18 ppm (0.10 mg/L), 55 ppm (0.31 mg/L) or 151 ppm (0.86 mg/L) 6 hours/day on gestation days 5-19, resulted in only transient reductions in maternal weight gain and food consumption during the exposure period, and an incidental increased on T3 serum levels. These changes were almost negligible at 55 ppm and most noticeable at 151 ppm. It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L) 


- There was no effect of the test item exposure at any level on pregnancy outcome, in terms of pre- or post-implantation loss, litter weight or the incidence of fetal abnormalities and variants. Therefore, a NOAEL for developmental toxicity was determined to be >= 151 ppm (0.86 mg/L). Foetal body weight was reduced in dosed groups, and even though these differences become statistically significant, they were below 5% for groups B and C and considered to be within a normal biological variation. However, the decreased on foetal body weight when exposed at 151 ppm (0.86 mg/L) was 13.6% both statistically and biologically significant. Therefore, a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).


- As no test-item related abnormalities were noted during the visceral or skeletal examinations of foetuses of Fischer 344 rats up to and including the dose level of 151 ppm, the observed exencephalies in the two preliminary prenatal developmental toxicity studies (Exxon, 1983 and 1984) cannot be confirmed in any of the three prenatal toxicity studies (Vivotecnia, 2016, 2019 and 2020).


 


DRF study according to OECD 421


Developmental toxicity (pups)


No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the dose groups (100, 300 or 750 mg test item/kg b.w./day).


Furthermore, the reproductive indices as the birth index, the live birth index and the percentage of post-implantation loss revealed no test item-related differences between the control group and the dose groups.


No difference between the control group and the dose groups (100, 300 or 750 mg test item/kg b.w./day) was noted for the viability index (group level) between lactation days 1 and 4. The viability index from LD 0/1 to LD 4 ranged between 93.1% (low dose group) and 99.3% (control group).


No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 750 mg test item/kg b.w./day).


In the high dose group (750 mg test item/kg b.w./day), a reduced body weight was noted for the male and female pups on LD 1, LD 4 and LD 13 (between 18.2% and 21.9% below the value of the control group, statistically significant for all values at p ≤ 0.01). The distinctly decreased body weight was noted on LD 1, LD 4 and on LD 13 and therefore, was considered to be test item-related.


In the high dose group (750 mg test item/kg b.w./day), reduced litter weights were noted for the male and the female litters and for the weights of the male and female litters combined on the lactation days 1, 4 and 13 (statistically significant for the female litters and for the male and female litters combined on LD 13 at p ≤ 0.01).


No test item-related differences were noted between the control group and the dose groups (100, 300 or 750 mg test item/kg b.w./day) for the number of live pups per dam on the lactation days 1, 4 or 13.


A reduction of the absolute ano-genital distance was noted for male and female pups of the high dose group (14.0% and 11.9% below the value of the control group, statistically significant for the male animals at p ≤ 0.01). However, as no difference was noted for the relative ano-genital distance, the reduced absolute ano-genital distance was due to the reduced body weight of the pups. Therefore, the test item was considered to have no influence on the ano-genital distance.


No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (100, 300 or 750 mg test item/kg b.w./day).


No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100, 300 or 750 mg test item/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.


No test item-related changes were noted for the T4 levels of the male and female pups of the dose groups (100, 300 or 750 mg test item/kg b.w./day) on LD 13.


 


EOGRTS study according to OECD 443


Developmental toxicity (F1 - Cohorts 1A, 1B and 2A)


Based on the concern regarding reproductive and developmental toxicity as found in juvenile male rats in the Pubertal Development and Thyroid function assay (Marty et al., 2011), this OECD 443 study aimed to provide additional information on sexual function and fertility in P0 and F1 generation and on development and toxicity of the offspring from birth until adulthood due to pre- and postnatal and adult exposure in the F1 generation.


Therefore, the test animals were exposed via the oral route (by gavage) with 65, 200 or 600 mg test item/kg b.w./day in the extended one-generation reproductive toxicity study. The dose levels were selected base on the findings in the DRF OECD 421.


In the present OECD 443 study, the test item did not affect the prenatal development of the pups (number of resorptions, stillbirths and live born pups).


Thus, the No Observed Adverse Effect Level (NOAEL) for prenatal development (conceptus to birth) was considered to be above 600 mg test item/kg bw/day.


A reduction was noted for the body weight of the male and female high dose F1 and F2 pups (600 mg test item/kg b.w./day) and consequently, the weights of the high dose litters. The distinctly and constantly reduced pup body weights were considered to be test item-related.


The other parameters of post-natal pup development (viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


During their post-weaning development no test item-related prematurely deceased animals were noted in any dose group.


The adult F1 animals of all dose groups displayed non-adverse post-dosing salivation and the body weight was decreased for the high dose males of the Cohorts 1A and 1B, as well for the intermediate and high dose males and the high dose females of Cohort 2A. No influence was noted on the food consumption, on the haematological parameters investigated and on the serum levels of the thyroid hormones T4 and TSH of the male and female animals of the dose groups. Furthermore, he serum testosterone levels at PND 53 of the males was not changed. Regarding the biochemical parameters and the urinalysis, reductions of the serum levels of globulin, total cholesterol and total protein and a lower pH of the urine were observed for the male and female intermediate and high dose animals of Cohort 1A. Macroscopic post mortem examination revealed no test item-related pathological changes.


Increased organ weights were noted for the adrenal glands (high dose animals of Cohort 1A, high dose males and intermediate and high dose females of Cohort 1B), for the kidneys (intermediate and high dose animals of Cohort 1A) and for the liver (intermediate and high dose animals of Cohort 1A and high dose males and intermediate and high dose females of Cohort 2A). Additionally, a decreased absolute heart weight was noted for the male high dose animals of Cohort 1A. Histopathological examinations of the reproduction organs of the high dose animals of Cohort 1A revealed no pathological changes.


For the sexual maturation, the time point of balano-preputial separation of the male animals did not differ between the control group and the dose groups in the Cohorts 1A, 1B and 2A. Thus, the finding in the Pubertal Development Assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


A statistically significant delay for the time point of vaginal opening for the female animals was noted for the high dose group (600 mg test item/kg b.w./day) when comparing all F1 females of the Cohorts 1A, 1B and 2A combined with the control group. However, no statistically significant differences between the control group and the dose groups were observed for the day of vaginal opening for the Cohort 1A females and the Cohort 2A females, respectively when the Cohorts were considered separately.


Additionally, the mean body weight of the juvenile rats at the time point of vaginal opening was also reduced (not significant) in all Cohorts (except of Cohort 1B) as well as when comparing Cohort 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment indicating that there is no delay in sexual maturation. Consequently, this finding was considered as not adverse.


The No Observed Adverse Effect Level (NOAEL) for postnatal development (pup) was considered to be 200 mg test item/kg bw/day based on the reduced body weight of the male and female pups. As no further postnatal effects were observed as well as no subsequent effects on reproductive performance, the substance must not be classified regarding developmental toxicity according to GHS criteria.


 


Neurotoxicity (F1 - Cohorts 2A and 2B)


The neurological screening of the animals of Cohort 2A between PND 58 and 67 revealed no test item-related differences between the dose groups and the control group. Also, the examination of the brain weights on PND21/22 (Cohort 2B) or between PND77 to 81 (Cohort 2A) showed no test item-related difference between the control group and the dose groups.

Justification for classification or non-classification

Based on the result of all available studies the test substance is not classified with regard to reproduction and development toxicity according to the criteria of CLP Regulation 1272/2008. Especially, as the observed reduction of pup weights has no subsequent effects on reproductive performance and the further development of the animals, the test item must not be classified regarding developmental toxicity according to GHS criteria.

Additional information