Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-01-24 to 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-24 to 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 349 - 388 g; (mean: 368.23 g, ± 20 % = 294.58 – 441.87 g)
females: 204 - 243 g; (mean: 220.05 g, ± 20 % = 176.04 – 264.06 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities.
Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage (type IV, polysulphone cages) during the premating period for both males and females and
during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept
individually during gestation/lactation period and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels were provided for all males and for females until GD 18
- Nesting material were provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Number and Sex of the Animals
100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage were made. No animal showed pathological signs before the first administration.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
Each animal was marked with its identification number by individual ear tattoo or tail marking.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Preparation of the Test Item Formulation
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate
final concentration of the test item. The formulation was placed into ultrasonic bath for approximately 30 minutes at approximately 40 °C
and finally vortexed and/or stirred until visual homogeneity was achieved. The HD formulation was heated in an incubator (approximately 40 °C)
directly before application for 15 (+/- 5) minutes. Based on the results of stability testing (Eurofins Munich Study No. 177722), the test item formulations
were prepared once every 10 days (within stability time frame as given by Eurofins Munich Study No. 177722). The prepared formulations were
stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.


Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females,
i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

C 0 mg/kg bw/ day (Male No.: 1-10/ Female No.: 41-50)
LD 100 mg/kg bw/ day (Male No.: 11-20/ Female No.: 51-60)
MD 300 mg/kg bw/ day (Male No.: 21-30/ Female No.: 61-70)
HD 1000 mg/kg bw/ day (Male No.: 31-40/ Female No.: 71-80)

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.


Administration of Doses
The test item and vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Concentration: C: 0 mg/mL; LD:25 mg/mL; MD: 75 mg/mL; HD: 250 mg/mL


Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating.
If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm
was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability
and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 177722).
Study pre start stability analysis was included on the samples from high dose and low dose group and the investigation will be made
for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose
and low dose groups. Since the test item was shown to be homogenous according to Eurofins Study No. 177722 (after at least 30 min without stirring),
samples were collected during the study for the investigation of homogeneity and only samples were taken for substance
concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study
(gestation / lactation) from all groups (16 samples). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL).
The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 177723) and until then stored under appropriate
conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females,
i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period
and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.
Frequency of treatment:
7 days/week
Details on study schedule:
Arrival of the Test Item: 23 October and 09 November 2017
Study Initiation Date: 24 January 2018
Amendment to Study Plan: 08 February 2018
Delivery of Animals: 25 January 2018
Acclimatisation Period: 25 January 2018 – 30 January 2018
Experimental Starting Date: 30 January 2018
Treatment Period: 13 February 2018 – 08 April 2018
Necropsies: 13 - 14 March 2018 and 03 - 09 April 2018
Experimental Completion Date: 10 April 2018
Completion Date of Delegated Phase (Histopathology): to follow
Completion Date of Delegated Phase (Formulation Analysis): to follow
Study Completion Date: to follow


Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days
before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study.
Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.
Control animals:
yes, concurrent vehicle
Details on study design:
Justification for Dose Selection
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
TThe highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for
morbidity and mortality except on weekends and public holidays when observations were made once daily. Clinical observations included spontaneous activity, lethargy, recumbent position,
convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.
Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.


Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy,
females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups.
All animals were weighed at termination. Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration.
Food consumption was not measured during the mating period in males and females and the post-mating period in males.


Clinical Biochemistry
From all adult males at termination blood samples were collected wherever possible from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).












Oestrous cyclicity (parental animals):
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity.
Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
As a part of histopathology the following sperm parameters were examined: a detailed qualitative examination of the testes was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Litter observations:
The duration of gestation was recorded and is calculated from day 0 of the pregnancy.
Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing.
In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded. On PND 12 pups of animal no. 56 were not observed.  
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation
of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in all male pups were counted on PND 12, with the exception of pups of animal no. 56.

From 2 female pups/litter on day 4 (in case the number of pups allowed) after birth, from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination,
blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males
were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups were done if deemed necessary.
Pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments.
If possible the two pups per litter were female pups to reserve male pups for nipple retention evaluations except in the event that removing these pups leaves no remaining females for assessment at termination.
No pups were eliminated when litter size dropped below 8 pups. If there was only one pup available above a litter size of 8, only one pup was sacrificed.

Postmortem examinations (parental animals):
Pathology
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on the respective PND 13
along using an anaesthesia (ketamine/xylazin) with the exception of animal no. 59 which was sacrificed on PND14. All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Non-pregnant females were sacrificed on study day 26 from the day of mating or from the last day of the mating period.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. If appropriate and possible, the number of corpora lutea and implantation sites
was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.
Special attention was paid to the organs of the reproductive system. The following tissues (all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole,
testes, uterus with cervix, vagina, thyroid/parathyroid glands) from all male and female animals were preserved in 4 % neutral-buffered formaldehyde
except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved in 4 % neutral-buffered formaldehyde.


Organ Weights
The wet weight of the reproductive organs (epididymides, testes, ovaries, uterus with cervix, prostate, seminal vesicles and coagulating glands,
thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females), was weighed after fixation) of all sacrificed adult males and females from each group were recorded as soon as possible. Paired organs were weighed together. Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved.
Weight of thyroid/parathyroid glands was measured after fixation.


Histopathology
A full histopathology was carried out on the preserved organs and tissues (all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole,
testes, uterus with cervix, vagina, thyroid/parathyroid glands) of all animals of the control and high dose groups which were sacrificed at the end of the treatment period.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicles with coagulating glands) were trimmed, embedded into paraffin,
cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals.
Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
Additionally, thyroid glands were evaluated histopathologically from all males and dams and one PND 13 pup per gender per litter in control and HD group, respectively.
Any gross lesion macroscopically identified was examined microscopically in all animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal,
Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten,
Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice
adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.



Postmortem examinations (offspring):
All surviving pups were killed by cervical dislocation on PND 13. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values o
f dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically
analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test
or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Reproductive indices:
The reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups were compared to control group indices.
Offspring viability indices:
see Reproductive Indices
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Mortality:
no mortality observed
Description (incidence):
see Details on results P0
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
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:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
see Details on results P0
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see Details on results P0
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
see under Histopathology, in Details on results P0
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Precoital interval and duration of gestation: see Details on results P0;
Pre-and postnatal data: see Details on results F1;
Reproductive indices: see Details on results F1
Mortality
No mortalities were observed during the study period in any of the groups.

Clinical Observations
There were no clinical signs of toxicological relevance.
Moving the bedding was seen in 8/10 female animals of the HD group transiently during lactation period and also in 3/10 females during the
gestation period, when this was also seen in 3/10 MD females. This clinical sign was observed immediately after the dose administration
and therefore is considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and is not toxicological relevant.
Moving the bedding was not observed in male animals of the dose groups. A single MD male animal no. 21 was observed salivating on the
last 2 treatment days. Single findings, i.e. abnormal breathing, nasal discharge, piloerection, reduced spontaneous activity were observed
transiently in single or few animals and are not assumed to be signs of systemic toxicity.


Body Weight Development
The test item had no effect on body weight or body weight gain in both sexes except during the first mating/post mating week,
when there was reduction in body weight gain in males, though there was no statistical significance. As there was no dose dependency
in males and in absence of test item related clinical signs, this reduction is assumed not to be an adverse effect or treatment related.

Food Consumption
The test item had no effect on food consumption in this study. No considerable difference in food intake was measured between
dose groups and control group throughout the treatment period in either gender. A very slight but statistically significantly higher food consumption
of female animals of the LD and HD group during the second week of gestation (approx. 14 % above controls) is not considered toxicologically relevant.

Estrous Cyclicity
The test item had no biologically significant effect on the estrous cycle analyzed during the two weeks premating period after the
first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence
of cycle stages between the dose groups and the control group.

Precoital Interval and Duration of Gestation
There was no test item related or statistically significant effect observed on the duration of precoital interval and the duration of
gestation in the dose groups when compared to the control group.

Pre- and Post-Natal Data
There were no test item treatment related effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0, PND 4
and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group. High mean post implantation
loss was found for LD group. This increase is considered to be based on animal no. 54, for which a post implantation loss of 85 % was found.
This is considered to be an incidental finding and not test item dependent. No dose dependency or statistically significant effects were observed.
All pre and post-natal data parameters including pre and post implantation loss were within the range of biological variation and historical control data.

Reproductive Indices
There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups
when compared to the control group.
A slightly lower viability index (PND 0-4 & 4-13) for dams no. 54, 55 (LD group) and 80 (HD group) was recorded as several pups were not
found missing or dead after littering. These values are in the normal range of biological variation, without dose dependency and are not considered
to be test item related.


Thyroid Hormone (T4) Analysis
No statistical significant or test item related effects were found for thyroid gland weight for male and female pups. Mean values were comparable
to control data and were within the range of biological variation and historical control data. However, the mean values for males and pup thyroxine (T4)
were moderately and statistically significantly decreased in the LD, MD and HD group (adult male-thyroxine: 27.42 %, 40.66 % and 43.79 % below control,
pup-thyroxine: 42.57 %, 61.99 % and 62.67 % below control) than in the control group, but all mean group values for T4 were within the range of
biological variation and historical control data. In addition, there were no statistically significant changes in thyroid/parathyroid gland weight of male
and female pups or test item related effect. The mean absolute and relative organ weights for LD, MD and HD were comparable to control group,
the statistical significant decrease in mean values of male and pup thyroxine is considered to be an non-adverse effect of the test item.
Finally, histopathologically no abnormalities were detected in thyroid glands of parent (male and female) animals and PND 13 pups.

Pathology
There were no macroscopic findings observed in any of the dose groups at necropsy, except few incidental findings of abnormal color-red in right
testes in 1 control male, small-left testes & epididymis and enlarged spleen in LD, small thyroid gland in MD male and left ovaries with single cyst
with fluid in HD group. They are not considered to be treatment related findings.

Organ Weight
In males and female, there were no statistically significant differences in the absolute and relative organ weights of all dose groups when
compared to the control group, except in LD female where the mean weight of ovaries was statistically significant increased (p<0.05).
Mean values of dose groups were comparable to mean control values.

Histopathology
At histopathological evaluation, there was no histological lesion that distinguished controls from test item-treated animals.
There was no change noted during sperm staging that were indicative for test item-related alterations. The stages were complete
and avoid of induced degenerative changes. The test item caused no histological changes indicating toxicity in reproductive organs.
There were no gross and histopathological findings that could related to treatment effects, therefore the NOEL may be established for
the reproductive system at 1000 mg/kg bw/day. There were no changes noted in thyroid glands in the parent and second generation.


Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study
weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 89.6 % and 100.7 % of
the nominal value, between 95.2 % and 97.3 % for the MD dose group and between 96.0 % and 102.1 % of the nominal value for HD dose group.
The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 94.5 %, 96.2 %, and 99.6 % of the nominal
concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance
criterion of 10 %. However one sample (no. 6, LD week 3) did not meet this criterion with a recovery of 89.6 %.

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no relevant effects observed up to 1000 mg/kg bw/d
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pup external findings: see Details on results F1
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pup external findings: see Details on results F1
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Litter Observation
There were no test item treatment related effects on litter data including total number of pups born, number of male pups, number of female pups,
sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio
on PND 4 and PND 13. There were no statistically significant differences noted for litter data as the values of all parameters were comparable between
dose groups and control group.

Litter Weight Data
There was a statistically significant reduction in pup mean weight observed on PND 0 (12 % below controls) and PND 4 (21 % below controls)
in the HD group only. Furthermore the mean total litter weight of the HD group was slightly, though not statistically significantly lower than in the control
group on PND 4 and 13 (18 and 16 % below controls, respectively). However, as these effects were very slight and not associated with other findings,
they are not assumed to be toxicologically relevant.

Pup Survival Data
There were no statistical significant effects on the survival of the pups from PND 1 through PND 13 in all dose groups when compared to control group,
however mortality was found on PND 0-4 for LD (4.44%) and HD group (10.48%). These mortalities were attributed to 7 missing pups of animal no. 54
and 6 pups from animal no. 55. A loss of 3 pups was observed for HD animal no. 80 (PND 0-13). No further mortality occurred from PND 4-13.
Due to the lack of dose dependency, the findings on pup survival in two LD animals and one HD animal are considered to be not related to
treatment with test item.

Anogenital Distance and Nipple Retention
The mean absolute and relative anogenital distance of male pups was marginal but statistically significantly lower in the HD group (2.42 mm (p<0.05)
and 1.34 for males, respectively) when compared to the control group (2.62 mm and 1.41, respectively) and there were no statistical significance
changes in female pups in both mean absolute and relative anogenital distance. Nipple retention in treated groups was found to be comparable with control.
All parameters for male and female dose groups were within the historical range, the statistical significant findings are not considered to be an
adverse effect.

Thyroid Hormone (T4) Analysis
No statistical significant or test item related effects were found for thyroid gland weight for male and female pups. Mean values were comparable
to control data and were within the range of biological variation and historical control data. However, the mean values for males and pup thyroxine (T4)
were moderately and statistically significantly decreased in the LD, MD and HD group (adult male-thyroxine: 27.42 %, 40.66 % and 43.79 % below control,
pup-thyroxine: 42.57 %, 61.99 % and 62.67 % below control) than in the control group, but all mean group values for T4 were within the range of
biological variation and historical control data. In addition, there were no statistically significant changes in thyroid/parathyroid gland weight of male
and female pups or test item related effect. The mean absolute and relative organ weights for LD, MD and HD were comparable to control group,
the statistical significant decrease in mean values of male and pup thyroxine is considered to be an non-adverse effect of the test item.
Finally, histopathologically no abnormalities were detected in thyroid glands of parent (male and female) animals and PND 13 pups.


Pup External Findings
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.
Few incidences of pale, small pups in LD, 2 small pups at PND0 in MD and 1 small pup at PND0, 2 pups with dehydration at PND4 and hind limb
absent in 1 pup at PND0 in HD. On the day of necropsy, few occurrences of incidental findings like alopecia in 2-3 pups of LD and MD and 5 pups
with underdeveloped, small/little growth of coat in 5 HD pups were observed. These findings are not considered to be test item related.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
On the basis of this reproduction/developmental toxicity screening test with the test material in male and female Wistar rats at the dose levels of 100, 300, and 1000 mg/kg/day the following conclusions can be made:
There were no adverse signs of general systemic toxicity and anatomic pathology found up to the dose levels of 1000 mg/kg/day.
Therefore, the NOEL of the test material in this study is considered to be 1000 mg/kg body weight/day.
Executive summary:

The objective of this study was to assess the possible effects of the test material on reproduction and development, after repeated dose administrationinWistarrats and also intended to get information atan early stage of assessing the toxicological properties of the test material.

The test item was administered daily in graduated doses to 3 groups, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females rats, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days. Control group rats were handled similar to that of the dose groups but receivedcorn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats.Before dosing, all females were screened for two weeks for regular estrous cyclicity and rats (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study. Control, low, mid and high dose group rats received the dose at 0, 100, 300 and 1000 mg/kg/day respectively, as repeated dose at the dose volume of 4 mL/kg.

The test item formulation was prepared once every 10 days. The test item was dissolved incorn oiland administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements.Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 75 mg/mL and 250 mg/mL in study weeks 1, 3, 5 and in the last week of the study.

All rats were observed on each day for signs of toxicity during the study period. All rats were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment, both male and female rats were mated (1:1) for a maximum period of 14 days and on subsequent days (morning) the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted, sexed and litters weighed within 24 hours of parturition (PND 0), on PND 4 and PND 13. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroxine (T4).

The males were sacrificed after completion of the mating period on day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post-natalday 4 or 13 and those found dead, were examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues (all gross lesions, epididymides, ovaries, prostate and seminal vesicle with coagulating gland, testes, uterus with cervix, vagina andthyroid/parathyroid glands) was performed on high dose and control animals. Thyroid/parathyroid glands were also evaluated from one PND 13 pup per gender and litter in control and HD group.

Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicles with coagulating glands) were examined in control and HD animals and in non-pregnant female animals of the LD and MD animals.

Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in the mating partners of the non-pregnant female LD and MD animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Summary Results

Dose formulation analysis for nominal concentration revealed that the mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 94.5 %, 96.2 %, and 99.6 % of the nominal concentration, respectively.

There were no test item related mortalities observed in this study.

There were no test item related clinical findings observed during the study, except few incidences of alopecia, regurgitation of vehicle in control, abnormal breathing, nasal discharge, moving the bedding, piloerection, reduced spontaneous activity and salivation. All these signs were not related to treatment and considered to be asign of local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

The test item had no effect on body weight or body weight gain in both sexes.

The test item had no effect on food consumption of the animals in both sexes.

The test item had no biologically significant effect on estrous cycle analysed during the 2 weeks premating period and after the administration in the dose groups when compared to the controls.

There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group.

There were no test item related effects on litters which includes total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13. There were no statistically significant changes noted for litters and all parameters were found to be comparable to control.

The total number of pups on PND 0, 4 and 13, the number of males and females on PND 0, 4 and 13 were found to be comparable with control. The sex ratio was slightly lower in the HD group, however this effect is not considered as toxicologically relevant.

There was a statistically significant reduction in pup mean weight observed on PND 0 and PND 4 in the HD group only. Furthermore the mean total litter weight of the HD group was slightly lower than the control on all recorded days (PND 0, 4 and 13). These effects are not considered toxicologically relevant.

There was no test item related effector statistically significant effectsobserved on the duration of pre-coital interval and the duration of the gestation in all dose groups compared with the control.

There were no test item treatment related effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0, PND 4 and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group.

There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group.There were no statistical significant effects on the survival of the pups from PND 1 through PND 13 in all dose groups when compared to control group.

There were no toxicologically relevant changes in mean absolute and relative anogenital distance, pup nipple retention on PND12, mean pup weight. These parameters were within the historical range and a statistically significantly lower anogenital distance in male pups of MD and HD group is not considered to be adverse.

No statistical significant or test item related effects were found for thyroid gland weight of male and female pups. However, mean values of males and pup thyroxine (T4) were moderately and statistically significantly lower in the LD, MD and HD group than in the control group, however, all mean group values for T4 were within the range of biological variation and historical control data.

No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. There were no macroscopic findings observed in any of the dose groups at necropsy, except few incidental findings of abnormal red color in right testes in 1 control male, small left testes & epididymis and enlarged spleen in LD, small thyroid gland in MD male and left ovaries with single cyst with fluid in HD group. They are not considered to be treatment related findings.

In males and female, there were no statistically significant differences in the absolute and relative organ weights of all dose groups when compared to the control group except in LD female where the mean weight of ovaries was statistically significantly increased. Mean values of the dose groups were comparable to mean control values.

The test item did not produced any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. There was no change noted during sperm staging that were indicative for test item-related alterations. The stages were complete and avoid of induced degenerative changes. There were no changes noted in thyroid glands in the parent and second generation.

Conclusion

On the basis of this reproduction/developmental toxicity screening test with the test material in male and femaleWistarrats at the dose levels of 100, 300, and 1000 mg/kg/day the following conclusions can be made:There were no adverse signs of general systemic toxicity and anatomic pathology found up to the dose levels of 1000 mg/kg/day.Therefore, the NOEL of the test material in this study is considered to be 1000 mg/kg body weight/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2020

Materials and methods

Test material

Constituent 1
Chemical structure
Reference substance name:
608-148-3
EC Number:
608-148-3
Cas Number:
279246-65-0
Molecular formula:
C18H32
IUPAC Name:
608-148-3
Test material form:
solid: crystalline

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
There were no adverse signs of developmental toxicity found up to the dose levels of 1000 mg/kg bw/d in the Reproduction/Developmental Toxicity Screening Study (OECD TG 421). Based on this screening study, the NOAEL for developmental toxicity is considered to be 1000 mg/kg bw/d.