5-Brom-2-[(3,4,5-trifluorphenoxy)-difluormethyl]-1,3-difluorbenzene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction (OECD 421, RL1), rat (m/f), NOAEL = 600 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-15 to 2018-08-23

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: 339 - 409 g (mean: 361.36 g, ± 20% = 289.09 – 433.63 g)
females: 205 - 250 g (mean: 229.53 g, ± 20% = 183.62 – 275.43 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of 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.
The animal study was authorized by local government under file no. 55.2-1-54-2532.0-31-2014.

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). Acidification of the water applies to the drinking water for mice and rats only. The purpose of the acidification is to provide additional protection against microbial contamination. This is a standard veterinary practice at BSL and should not be associated with any risk for long-term studies.
- Animals were housed in groups of 5 animals / sex / cage in IVC cages (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.
- 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

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed showing 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.
Each animal was marked with its identification number by individual ear tattoo or tail marking.

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Remarks:

Specifications provided by the supplier Manufacturer: Manufacturer:colorcon, Batch No.: DT409094, Physical State: solid, Storage Conditions: at room temperature, Expiry Date: 11 February 2019

Details on exposure:

Preparation of the Test Item Formulations

The vehicle was selected in consultation with 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 homogenized using an ultra turrax for 5 min.
 
Based on the results of stability testing (Eurofins Munich Study No. 176269), the test item formulations were prepared at least once every 10 days within the stability time frame as given by Eurofins Munich Study No. 176269. The prepared formulation was stored at 2-8 °C. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.


Experimental Groups and Doses

According to the results of a previous dose range finding study (BSL Munich Study No. 176267, non GLP) and 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 30 mg/kg bw/ day (Male No.:11-20/ Female No.:51-60)
MD 120 mg/kg bw/ day (Male No.:21-30/ Female No.:61-70)
HD 600 mg/kg bw/ day (Male No.:31-40/ Female No.:71-80)

The highest dose level was chosen based on the maximum feasible concentration in the vehicle of 60 mg/mL giving a homogeneous suspension and on the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a 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 were administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 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:3 mg/mL; MD: 12 mg/mL; HD: 60 mg/mL

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female) wherever 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. 176269). Study pre start stability analysis was included on the samples from high dose and low dose group. Prestart homogeneity investigation included the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
Since the test item was not homogenous according to Eurofins Study No. 176269, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 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. 176270) 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.
The phase plan was amended to the study plan. The results are reported in the appendix of the final 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 was completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 29 August 2017
Study Initiation Date: 15 December 2017
Amendment to Study Plan: 09 January 2018
Delivery of Animals: 14 December 2017
Acclimatisation Period: 14 December 2017 - 27 December 2017
Experimental Starting Date: 27 December 2017
Treatment Period: 02 January 2018 – 24 February 2018
Necropsies: 22 January 2018, 30-31 January 2018, 12-13 February 2018, 20-25 February 2018
Experimental Completion Date: 25 February 2018
Completion Date of Delegated Phase (Histopathology): 13 August 2018
Completion Date of Delegated Phase (Formulation Analysis): 22 May 2018

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

120 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

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
Based on the results of a previous dose range finding study (BSL Munich Study No. 176267, non GLP) and in consultation with the sponsor, dose levels had been selected for this study. The highest dose level was chosen based on the maximum feasible concentration in the vehicle of 60 mg/mL giving a homogeneous suspension and on the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a 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. Any animals prematurely sacrificed were weighed prior to the sacrifice.
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 were recorded.
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 male pups was counted on PND 12.


From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, 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 day 13 pups were assessed for serum levels for thyroid hormones (T4). 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. The two pups per litter were female pups to reserve male pups for nipple retention evaluations, when possible. 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
Males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using an anesthesia (ketamine/xylazin).
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle for animals no. 41, 46, 48, 54, 56, 57, 67, 70 and 75 to 80.
Non-pregnant females were sacrificed on study day 26 from the day of mating or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which includes 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 female 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 from all male and female animals were preserved in 4 % neutral-buffered formaldehyde except for testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol: all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix, vagina and thyroid/parathyroid glands.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination ((adrenal glands, all gross lesions, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole, rectum, spleen, stomach, testes, thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus with cervix, vagina)).
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females with the exception of pups of dam no. 45 (control group), 58 (LD group), 61, 64 (MD group) and 72 (HD group) were preserved for potential histopathological examination.


Organ Weights
The wet weight of the reproductive organs (epididymides,testes, ovaries, uterus with cervix, prostate, seminal vesicles and coagulating glands) of all sacrificed adult males and females from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females with the exception of pups of dam no. 45 (control group), 58 (LD group), 61, 64 (MD group) and 72 (HD group) were preserved. Weight of thyroid/parathyroid glands was measured after fixation.


Histopathology
A full histopathology was carried out on the preserved organs and tissues 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 vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the 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 MD animals.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in the HD group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
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 will be performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation will be 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 were performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analysed 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 1.1.3 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:

mortality observed, non-treatment-related

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):

no effects observed

Description (incidence and severity):

see Details on results P0

Food efficiency:

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

Histopathological findings: non-neoplastic:

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone (T4) analysis and Thyroid/parathyroid gland weight: see Details on results P0

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

Clinical Observations
In the female groups, animal no. 71 (HD) was found with moderate piloerection, moderately reduced spontaneous activity, stilted gait, sunken flank and abnormal breathing on gestation day 5 and was therefore euthanized in moribund condition. Histopathologically, pericardial inflammation and mediastinal inflammation at the thymus indicated gavage error.
Moving the bedding was seen for animal no. 36 in the male HD group from day 3-9 during mating/postmating phase. No further clinical findings were recorded for any animal in the male groups. Regurgitation of test item was found on gestation day 15 for LD animal no. 58 and on day 4 for MD animal no. 62. Furthermore, moving the bedding was seen on PND 2 for HD animal no. 74. The clinical signs moving the bedding or regurgitation were observed immediately after the dose administration and therefore considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
Two females were seen with alopecia on the head (HD group, animal no. 79 and 80) from premating to gestation phase and in one control animal at the abdomen and both fore and hind limbs while gestation and lactation. Alopecia observed in the control and HD group is not considered to be related to test item treatment.
Vaginal bleeding on gestation day 14 in one control animal was considered to be an incidental finding due to absence of any other clinical findings during the entire study period.

Mortality
Animal no. 71 in the female HD group was sacrificed on gestation day 5 in moribund condition. Histopathologically, pericardial inflammation and mediastinal inflammation at the thymus indicated gavage error.
No further mortalities were recorded at the other dose levels.


Body Weight Development
Regarding mean body weight gain, a statistical significant moderate decrease was found for male animals in the HD group from beginning of mating/postmating phase to premating/terminal sacrifice, but no dose dependency was observed.
Furthermore, no statistically significances in mean body weight development occurred in all male groups. Mean values in all dose groups were comparable to control group, whereas in the HD group the mean body weight was slightly lower than in control group, but no tendency to dose relation was seen in LD and MD group. 
No statistical significant changes in mean body weight or body weight gain were found for all female dose groups during premating, gestation an lactation phase. Body weight development of LD, MD and HD group was comparable to control group.
Under consideration of the lack of dose dependency in males or females and in absence of test item related clinical findings the statistical significances in body weight gain are assumed not to be an adverse effect of toxicological relevance of test item treatment.


Food Consumption
The test item had no effect on food consumption in this study. No statistical significant changes were seen during the entire study period for male and female groups. Mean food intake of male and female groups was in a normal range of variation throughout the entire treatment period of this study and is comparable to control group.


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. Mean values of dose groups were comparable to mean control values.

Pathology- Macroscopic Findings
There were no macroscopic findings recorded at necropsy for all animals in all groups.
For HD animal no. 71, which was euthanized in moribund condition, the abdominal cavity was filled with fluid. Pericardial inflammation and mediastinal inflammation at the thymus observed at histopathological evaluation indicated gavage error.


Histopathology
At histopathological evaluation, there was no histological lesion that distinguished controls from test item-treated animals. There was no change at all noted during sperm staging. The stages were complete and avoid of any degenerative change.
Pathology findings were not seen in reproductive organs from non-mating male no. 16 and 18, and female no. 56 and 58 from LD group.
There were no gross lesions or histology findings that could be related to treatment with the test item. Therefore, the NOAEL may be established at 600 mg/kg bw.

Thyroid Hormone (T4) Analysis and Thyroid/Parathyroid Gland Weight
Mean values for male thyroxine (T4) were markedly and statistically significantly lower in the MD and HD group (23.78% and 59.41% 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.
Additionally, mean organ weight of thyroid/parathyroid for adult males and females was without statistical significance in all dose groups and mean absolute and relative organ weights for LD, MD and HD were comparable to control group, the statistical significant decrease of mean values for thyroxine is considered not to be an adverse effect of the test item.

Estrous Cycles
The test item had no biologically significant effect on the estrous cycle analyzed during 2 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 were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to the highest dose tested.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed on reproductive performance up to the highest dose tested.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

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:

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone (T4) analysis and thyroid gland weight: see Details on results F1;
Pre- and Post-Natal Data: see Details on results F1;
Reproductive Indices: see Details on results F1

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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. On the day of necropsy swelling on snout of pup no. 12 from dam no. 61 (MD group) was noted. This single finding is not considered to be test item related.

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 (4.79%) and HD group (3.47%). These mortalities were attributed to one missing pup of animal no. 52 and 3 pups of animal no. 53. A loss of 5 pups was observed for animal no. 76 (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 single HD animal are considered to be not related to treatment with test item.
Litter Data
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 changes noted for litter data and litter data parameters were comparable with control data.

Litter Weight Data
There were no effects on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13. There was no statistically significant change in dose groups compared to corresponding controls.
 

Anogenital Distance and Nipple Retention
The mean absolute and relative anogenital distance of male and female pups on PND 0 was marginally but statistically significantly lower in the HD group (2.42 mm and 1.31 for males, 1.12 mm and 0.62 mm for females) when compared to the control group (2.70 mm and 1.46 for males, 1.24 mm and 0.69 mm). Mean Pup weight, cube root of pup weight and nipple retenteion were comparable to control mean value for all dose groups. Under consideration that all parameters for all male and female dose groups were within the historical range, the statistical significanct findings are not considered to be an adverse effect of the test item.

Thyroid Hormone (T4) Analysis and Thyroid/Parathyroid Weight
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, mean values for pup thyroxine (T4) were markedly and statistically significantly lower in the MD and HD group (16.62% and 54.98% 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
Additionally, under consideration that thyroid weight of male and female pups is without statistical significance or test item related effect, the statistical significant decrease of mean values for pup thyroxine is considered not to be an adverse effect of the test item.


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 pre implantation loss was found for LD group. This increase is considered to be based on animal no. 56, for which a pre implantation loss of 100 % was found. 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. However, a slightly reduced fertility index (number of females pregnant / No. of females copulated X 100) of 80 % was observed in the LD group compared to 100 % in the control group. In the absence of dose response dependency, the finding was not considered to be of toxicological relevance.
A lower viability index (PND 0-4) for dams no. 52, 53 and 76 were recorded as several pups were not found or found dead after littering. These values are in the normal range of biological variation and without dose dependency and not considered to be test item related.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to the highest dose tested.

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 with dose levels of 30, 120, and 600 mg/kg body weight/day the following conclusion can be made:
No adverse signs of general systemic toxicity were found up to dose levels of 600 mg/kg bw/d. Therefore, the NOAEL for general toxicity is considered to be 600 mg/kg body weight/day.
A marginally but statistically significantly lower mean absolute and relative anogenital distance for male and female pups when compared to controls was not considered to be an adverse effect of the test item. Furthermore, the statistically significant decreased thyroxine mean values for males and pups were not considered to be an adverse finding. Therefore, the NOAEL for this reproduction/developmental toxicity screening study is considered to be 600 mg/kg bw/day.

Executive summary:

Summary

The aim of this study was to assess the possible effects of the test material on male and female fertility and embryofetal development after repeated dose administrationinWistarrats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of up to 63 days, 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 were completed. Animals of an additional control group were handled identically as the dose groups but received0.5% aqueous hydroxypropyl methylcellulose(Methocel^®K4M Premium), 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 animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                  0 mg/kg bw /day

Low Dose:            30 mg/kg bw/day

Medium Dose:     120 mg/kg bw/day

High Dose:           600 mg/kg bw/day

The test item formulations were prepared within ten days, based on the stability time frame as given by Eurofins Munich Study No. 176269. The test item was emulsified inhydroxypropyl methylcellulose(Methocel^®K4M Premium)and administered daily during14 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 dosedfor 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministrationvolume was 10 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals 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 to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards 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, on day 4 and day 13 post-partum. 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 thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment 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 carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues (adrenal glands, testes, epididymides, ovaries, oviducts, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland) was performed on high dose and control animals, in non pregnant female animals and male mating partners of the 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.All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

One animal in the female HD group (animal no. 71) was sacrificed on gestation day 5 in moribund condition. The animal was found with moderate piloerection, moderately reduced spontaneous activity, stilted gait, sunken flank, abnormal breathing and fluid filled abdominal cavity. Histopathologically, pericardial inflammation and mediastinal inflammation at the thymus indicated gavage error. No further mortalities were recorded at the other dose levels

There were no clinical findings that could be attributed to test item treatment. Moving the bedding observed for animal no. 36 in the male and animal no. 74 in the female HD group or regurgitation of test item for LD animal no. 58 and MD animal no. 62 were considered to be a sign of local reaction or discomfort to the test item rather than a systemic adverse effect and has no toxicological relevance.

Single findings like alopecia recorded for two females in the HD group and one female in the control group and vaginal bleeding in one control animal are considered to be an incidental finding.

Regarding mean body weight gain, a statistical significant moderate decrease was found for male animals in the HD group from beginning of mating/postmating phase to premating/terminal sacrifice, but no dose dependency was observed. No further statistical significances were found for male and female dose groups. Under consideration of the lack of dose dependency in males or females and in absence of test item related clinical findings the statistical significances in body weight gain are assumed not to be an adverse effect of toxicological relevance of test item treatment.

The test item had no effect on food consumption in this study. No statistical significant changes were seen during the entire study period for male and female groups.

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.

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 changes noted for litter data and litter data parameters were comparable with control data.

There were no effects on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13. There was no statistically significant change in dose groups compared to corresponding controls.

There were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

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.

The mean absolute and relative anogenital distance of male and female pups on PND 0 was marginally but statistically significantly lower in the HD group (2.42 mm and 1.31 for males, 1.12 mm and 0.62 mm for females) when compared to the control group (2.70 mm and 1.46 for males, 1.24 mm and 0.69 mm). Mean Pup weight, cube root of pup weight and nipple retenteion were comparable to control mean value for all dose groups. Under consideration that all parameters for all male and female dose groups were within the historical range, the statistical significant findings are not considered to be an adverse effect of the test item.

No statistical significant or test item related effects were found for thyroid gland weight for male and female pups. However, man values for males and pup thyroxine (T4) were markedly and statistically significantly lower in the MD and HD group (adult male-thyroxine: 23.78% and 59.41% below control, pup-thyroxine: 16.62% and 54.98% 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. Additionally, under consideration that thyroid weight of male and female pups is without statistical significance or test item related effect and furthermore, mean organ weight for thyroid/parathyroid for adult males and females was without statistical significance in all dose groups and mean absolute and relative organ weights for LD, MD and HD were comparable to control group, the statistical significant decrease of mean values for male and pup thyroxine is considered not to be an adverse effect of the test item.

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 recorded at necropsy for all animals in all groups. For animal no. 71, which was euthanized in moribund condition, the abdominal cavity was filled with fluid

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. Mean values of dose groups were comparable to mean control values.

At histopathological evaluation, there was no histological lesion that distinguished controls from test item-treated animals. There was no change at all noted during sperm staging. The stages were complete and avoid of any degenerative change. Pathology findings were not seen in reproductive organs from non-mating male no. 16 and 18, and female no. 56 and 58 from LD group. There were no gross lesions or histology findings that could be related to treatment with the test item. Therefore, the NOAEL may be established at 600 mg/kg bw.

Regarding Dose Formulation Analysis, nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 20 % and all samples were homogenous, as COV was below or equal 15 %.

Conclusion

On the basis ofthis reproduction/ developmental toxicity screening test with the test item in male and femaleWistarrats with dose levels of 30, 120, and 600 mg/kg body weight/day the following conclusion can be made:

No adverse signs of general systemic toxicity were found up to dose levels of 600 mg/kg bw/d. Therefore, the NOAEL for general toxicity is considered to be 600 mg/kg body weight/day.

A marginally but statistically significantly lower mean absolute and relative anogenital distance for male and female pups when compared to controls wasnot considered to be an adverse effect of the test item. Furthermore, the statistically significant decreased thyroxine mean values for males and pups were not considered to be an adverse finding. Therefore,the NOAEL for this reproduction/developmental toxicity screening study is considered to be 600 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Species:

rat

Quality of whole database:

OECD Guideline study under GLP conditions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Oral route (OECD 421)

On the basis of this reproduction/ developmental toxicity screening test with the test item in male and femaleWistarrats with dose levels of 30, 120, and 600 mg/kg body weight/day the following conclusion can be made:

No adverse signs of general systemic toxicity were found up to dose levels of 600 mg/kg bw/d. Therefore, the NOAEL for general toxicity is considered to be 600 mg/kg body weight/day.

A marginally but statistically significantly lower mean absolute and relative anogenital distance for male and female pups when compared to controls wasnot considered to be an adverse effect of the test item. Furthermore, the statistically significant decreased thyroxine mean values for males and pups were not considered to be an adverse finding. Therefore,the NOAEL for this reproduction/developmental toxicity screening study is considered to be 600 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification,Lab elling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.