Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Type of information:
experimental study
Remarks:
Study started in February 2020. All available information is submitted in this updated dossier by 4 January 2021 as requested in a decision on a cc from ECHA (Decision number CCH-D-2114453561-52-01/F). Draft report will be expected in March 2021.
Adequacy of study:
key study
Study period:
Study started in February 2020. Draft report is expected in March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to the ECHA final decision on a compliance check from 2018-12-21 (Decision number CCH-D-2114453561-52-01/F) the study design of this EOGRTS according to OECD 443 is as follows:

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

- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (Developmental neurotoxicity).

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011
Reference Type:
other: study plan plus first results
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

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

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

Additionally, effects on developmental neurotoxicity will be evaluated.

Furthermore, liver weight will also be determined for all F1 cohort 2A animals to establish a timeline for the possible decrease in liver weight

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5,5-trimethylcyclohex-2-enone
EC Number:
201-126-0
EC Name:
3,5,5-trimethylcyclohex-2-enone
Cas Number:
78-59-1
Molecular formula:
C9H14O
IUPAC Name:
3,5,5-trimethylcyclohex-2-enone
Test material form:
liquid
Details on test material:
3,5,5-trimethylcyclohex-2-enone from Evonik, Batch: 20012404

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies and required by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: F0: 12-13 weeks
- Number of parental animals:
Pre-exposure period:
At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study:
192 animals (96 males and 96 females)
A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.
- Weight at study initiation: Details will be stated in the raw data and the report
- Fasting period before study:
- Housing: With exception of the mating period, the animals are kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm at a room temperature of 22°C ± 3 °C (maximum range) and a relative humidity of 55% ± 10% (maximum range).
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages are cleaned and changed once a week.
The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
- Diet: ssniff® R/M-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest), ad libitum with the exception of the night before the day of blood withdrawal for laboratory examinations
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C
- Humidity (%): 55% ± 10%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle
IN-LIFE DATES: From: To: *

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- Route of administration: Oral, via gavage
- Frequency of administration: Once daily
- Vehicle: corn oil
Administration volume: 5 mL/kg b.w.
Dosages: 0, 65, 200, 600 mg/kg bw/d
Selection of route of administration: According to international guidelines.
Details on mating procedure:
Sexually mature male and female rats of the same dose group are paired randomly monogamously, i.e. 1 male and 1 female animal are placed in one cage during the dark period. The female is placed with the same male until evidence of mating is observed or two weeks have elapsed. If mating has not occurred after 2 weeks, the animal will be separated without further opportunity for mating.
The females are examined each morning for the presence of sperm. The day of conception (day 0 of gestation) is considered to be the day on which sperm is found. If there are insufficient males, for example due to male death before pairing, then male(s) which have already mated may be paired with a second female such that all females are paired.

Establishment of F2 generation using Cohort 1B (potential reproductive toxicity):
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures will be similar to those for the parental animals (F0 generation); sibling mating will be avoided. Reproductive performance of F1 animals will be assessed according to section 4.10 and animals of the F2 generation will be assessed in the same way as F1 animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item formulations will be prepared for up to seven (7) administration days in advance and will be administered orally at a constant application volume/kg b.w. once daily. The test item will be diluted in the vehicle to the appropriate concentrations. Following the last dosing on each administration day, the test item formulations will be stored at 2 – 8°C. Prior to the first administration of each administration day, the test item formulations will be allowed to warm up to room temperature for at least 30 minutes and stirred on a magnetic stirrer for approximately 3 minutes to ensure homogeneity. The amount of the test item will be adjusted to the animal's current body weight daily.
The control animals receive the vehicle at the same administration volume daily in the same way.
The homogeneity and concentration of the test item formulations will be monitored.

For the analysis of the test item formulations, two (2) aliquots of approximately 3 mL each are taken as scheduled and stored at -20°C ± 10% until analysis at LPT.
At start of the treatment period of the F0 animals (1st dosing day): Analysis of concentration and homogeneity, at the start of administration, during (middle) administration and before administration to the last animal of each dose group. (3 samples/dose group). At a time when most F0 females have delivered: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At termination of the F0 dosing period at a time when the majority of animals is dosed: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At start of the treatment period of the F1 animals (1st dosing day): Analysis of concentration and homogeneity. At the start of administration, during (middle) administration and before administration to the last animal of each dose group (3 samples/dose group). At termination of Cohort 1A at a time when the majority of animals is dosed: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At termination of Cohort 1B at a time when the majority of animals is dosed: Analysis of concentration. During treatment always before administration to the last animal/dose group (1 sample/dose group).
Duration of treatment / exposure:
The study animals will be treated during the following periods:
F0 ANIMALS: Males: 2 weeks prior to mating, during the mating period, and at least until weaning of the F1 generation (up to and including the day before sacrifice). Females: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).
F1 ANIMALS: Until weaning, F1 animals are indirectly exposed to the test item through the breast milk. After weaning, dosing will continue in the same way as for the parental generation.
COHORT 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 91).
COHORT 1B: Until sacrifice of the F2 generation (up to and including the day before sacrifice, i.e. F2 PND 21).
COHORT 2A: Up to and including the day before sacrifice, i.e. around PND 75 (after behavioural testing).
COHORT 2B: Until sacrifice at PND 21/22, Cohort 2B animals are indirectly exposed to the test item through the breast milk.
F2 ANIMALS: Until sacrifice at PND 21, F2 animals are indirectly exposed to the test item through the breast milk.
Frequency of treatment:
once daily
Details on study schedule:
PARENTAL ANIMALS, PRE-EXPOSURE:
At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
MAIN STUDY:
192 animals (96 males and 96 females), a sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.

F1 GENERATION:
At weaning, around PND 21, the pups from all available litters per group are randomly selected for further examinations (Cohorts 1A, 1B, 2A and 2B). Obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) will be excluded.

F2 GENERATION:
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures will be similar to those for the parental animals (F0 generation); sibling mating will be avoided. Reproductive performance of F1 animals will be assessed according to section 4.10 and animals of the F2 generation will be assessed in the same way as F1 animals

The study animals will be treated during the following periods:
F0 ANIMALS: Males: 2 weeks prior to mating, during the mating period, and at least until weaning of the F1 generation (up to and including the day before sacrifice). Females: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).
F1 ANIMALS: Until weaning, F1 animals are indirectly exposed to the test item through the breast milk. After weaning, dosing will continue in the same way as for the parental generation.
COHORT 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 91).
COHORT 1B: Until sacrifice of the F2 generation (up to and including the day before sacrifice, i.e. F2 PND 21).
COHORT 2A: Up to and including the day before sacrifice, i.e. around PND 75 (after behavioural testing).
COHORT 2B: Until sacrifice at PND 21/22, Cohort 2B animals are indirectly exposed to the test item through the breast milk.
F2 ANIMALS: Until sacrifice at PND 21, F2 animals are indirectly exposed to the test item through the breast milk.

DURATION OF STUDY:
- at least 5 adaptation days of the F0 generation
- F0 (parental generation):
2 weeks pre-exposure
2 weeks pre-mating
2 weeks mating
3 weeks pregnancy
3 weeks lactation
- F1 Cohort 1A: approx. 10 weeks
- F1 Cohort 1B: approx. 10 weeks
2 weeks mating for establishing of F2 3 weeks pregnancy
3 weeks lactation
- F2 Generation: lactation until PND 211
- F1 Cohort 2A: approx. 10 weeks
- F1 Cohort 2B: lactation until PND 21/22
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
65 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
F0 generation: 20 m/f per group
F1 generation Cohorts 1A + 1B: 20 m/f per group
F1 generation Cohorts 2A + 2B: 10 m/ f per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on available toxicological data and the results of a 16-day repeated dose toxicity study in rats (conducted 1986), a 90-day repeated dose toxicity study in rats (conducted 1986) as well as an OECD 421 study in rats (LPT Study No. 37311, 2020). Additionally, the results and the study design of the Pubertal Development Assay (Marty et al., 2011) were taken into account. In the 16-day and the 90-day study, dose levels of 125, 250, 500 and 1000 mg/kg b.w. per day were employed. In the Pubertal Development Assay, dose levels of 50, 200 and 800 mg/kg b.w. per day were selected. In the current OECD 421 study, dose levels of 100, 300, 750 and 1000 mg/kg b.w. per day were used.

In the 90-day and the 16-day gavage studies with the test item, the NOAEL was considered to be 500 mg/kg b.w. (both NTP, 1986). In the 16-day study, at the high dose of 1000 mg/kg b.w., only decreased body weights were observed. One female rat that received 1000 mg/kg b.w. died in the 90-day gavage study. Final mean body weights for rats were not clearly related to dose in this study. In the Pubertal Development Assay, there are indications of a mode of action related to endocrine disruption based on the effects observed at 800 mg/kg b.w., i.e. reduced seminal vesicle weights, reduced serum testosterone, increased age at preputial separation).
In the current OECD 421 study, female animals treated with 1000 mg/kg b.w. had to be sacrificed after the first dosing and male animals after the third dosing due to animal welfare reasons. Two female animals dosed with 750 mg/kg b.w. were found dead on GD 20 and GD 21. Both male and female animals treated with 750 mg/kg b.w. showed slight to severe salivation, slight to moderately reduced motility and yellow discoloured urine. Furthermore, a reduction in body weight was noted for the male animals during the dosing period, while female animals showed a reduced body weight between GD 14 and GD 20. Accordingly, also the body weight gain was reduced for male and female animals during the respective periods. Reduced body weights were also noted at necropsy for both male and female animals. Therefore, the results of the above-mentioned historical studies could not completely be reproduced under the test conditions of the current DRF OECD 421 study (LPT, 2020). For the pups of animals treated with 750 mg/kg b.w. reduced body weights were noted for male and female animals on lactation days 1, 4 and 13.
Male animals treated with 300 mg/kg b.w. showed slight to moderate salivation, a slightly reduced motility and yellow discoloured urine, while slight salivation was observed in females treated with 300 mg/kg b.w.

Based on the findings in these studies, doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death. Taking all study results into account and considering the extended exposure time of the F0 and F1 generation within the OECD 443 study, 600 mg/kg b.w. per day was considered the maximum tolerated dose for the F0-generation of the OECD 443 study, with no systemic effects to be expected in the F1-generation. Furthermore, 600 mg/kg b.w. per day have been selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).

- Fasting period before blood sampling for clinical biochemistry: the night before the day of blood withdrawal for laboratory examinations
Positive control:
no

Examinations

Parental animals: Observations and examinations:
F0 generation + F1 generation Cohort 1B
CLINICAL SIGNS:
- All animals
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded.Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness.Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed will be recorded. In addition, animals will be checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals will be checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Dated and signed records of appearance, change, and disappearance of clinical signs will be maintained on clinical history sheets for individual animals.
- F0 animals and F1 animals Cohort 1B after weaning
Additionally, a more detailed examination of all F0 and F1 Cohort 1B animals is conducted on a weekly basis. F0 animals will be examined once before the first exposure (to allow for within-subject comparisons) and weekly thereafter until termination. F1 animals will be examined weekly after weaning until termination. Detailed clinical observations will be made in all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded.

MORTALITY:
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m. Premortal symptoms will be recorded in detail; as soon as possible after exitus, a post mortem examination will be performed. In the case of prematurely sacrificed animals, laboratory examinations will be performed, if possible.

BODY WEIGHT:
P0: The animals will be weighed and the weights recorded as scheduled.
Pre-mating period: Daily, starting on the first day of dosing*
Mating period: Daily*
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 4, 7, 14, 21
Post-mating period: Males: Daily*, Females: See gestation and lactation period
*: Weekly values will be reported

Schedule for body weight recordings of offspring animals:
F1/ F2: Lactation period: PND 1, 4, 7, 14, 21
After weaning: Daily, starting on PND 22*
F1 Cohort 1B animals only
Mating period: Daily*
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 4, 7, 14, 21
Post-mating period: Males: Daily*, Females: See gestation and lactation period
*: Weekly values will be reported
In addition, all animals will be weighed at sacrifice.

FOOD AND WATER CONSUMPTION:
P0: Food residue is weighed and recorded as follows:
Pre-mating period: weekly
Mating period: none
Gestation period (females): GD 7, 14, 21
Lactation period (females): PND 1, 7, 14, 21
Post-mating period: Males: Weekly*, Females: See gestation and lactation period
*: Starting on a suitable day after the mating period to consolidate all male animals

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

SEXUAL MATURATION:
F1 Cohort 1B: daily until achieved

TESTOSTERONE DETERMINATION:
F1 Cohort 1B: In order to investigate possible effects of the test item on progression of male sexual maturation, a sufficient amount of blood will be taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, if possible, as scheduled below to obtain 2 x 100 μL serum. Blood will be collected on PND 53 +/- 1.

URINALYSIS:
Urine samples are collected from animals fasted overnight at the following times and the parameters listed below are determined.
At the end of the F0 dosing period: 10 males and 10 females randomly selected from each F0 group (animals also selected for laboratory examinations).
The urine is collected for 16 hours in a URIMAX funnel cage. The collection of urine will be terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination.
Parameters: volume, pH, specific gravity
Analytes: protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite
microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Organisms, Further constituents (i.e. sperm, casts), Crystalluria

REPRODUCTIVE PERFORMANCE - F0 animals
Evaluation/parameters:
- Number of pregnant females
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites:
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups absolute:
- at birth (alive and dead)
- after 4 days of life, after 21 days
Number of pups per dam:
- at birth
- after 4 days of life, after 21 days
Number of male and female pups:
- at birth
- after 4 days of life, after 21 days
Number of stillbirths:
- absolute
- per dam
Number of pups with malformations:
- absolute
- per dam

REPRODUCTIVE PERFORMANCE - F1 Cohort 1B animals
Evaluation/parameters:
- Number of pregnant females
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites:
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups absolute:
- at birth (alive and dead)
- after 4 days of life
Number of pups per dam:
- at birth
- after 4 days of life
Number of male and female pups:
- at birth
- after 4 days of life
Number of stillbirths:
- absolute
- per dam
Number of pups with malformations:
- absolute
- per dam

s. also reproductive indices
Oestrous cyclicity (parental animals):
Vaginal smears will be taken and the oestrus cycle stages will be determined at the following time points:
F0 ANIMALS: 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days).
During 2 weeks of premating until evidence of mating and at sacrifice.
F1 ANIMALS, COHORT 1A: Start after onset of vaginal patency until first appearance of cornified cells. Two weeks starting around PND 75 and at sacrifice.
F1 ANIMALS, COHORT 1B: Starting from the time of pairing until mating evidence is detected and at sacrifice.
F1 ANIMALS, COHORT 2A: at sacrifice
Sperm parameters (parental animals):
all F0 males + all F1 Cohort 1A males: One epididymis and one testicle will be used for the sperm count. The sperm viability is determined and the sperm morphology is examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Litter observations:
LITTERING:
Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts5 and the presence of gross abnormalities. Based on these parameters, the reproductive performance of the dams will be evaluated. The pups will be examined as described below. Any abnormal behavior will be recorded.

COUNTING, SEXING AND WEIGHING:
Live pups will be counted and sexed. Live pups are weighed on PND 1 and regularly thereafter.
Schedule for body weight recordings of offspring animals:
F1/ F2: Lactation period: PND 1, 4, 7, 14, 21
After weaning: Daily, starting on PND 22*

ANO-GENITAL DISTANCE:
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups will be determined using a scale.

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

SEXUAL MATURATION:
Only F1 animals (Cohort 1A and 2A) are evaluated daily for balano-preputial separation or vaginal patency before expected achievement of these endpoints to detect if sexual maturation occurs early. Any abnormalities of the genitals are recorded. Sexual maturity of the F1 animals is compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Pups, 2 surplus pups per litter, all litters, if possible, PND 4, non-fasted, T4 only
Pups, 2 surplus pups per litter, all litters, non-fasted, T4 and TSH

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

TESTOSTERONE DETERMINATION:
F1 Cohort 1A/ 2A: In order to investigate possible effects of the test item on progression of male sexual maturation, a sufficient amount of blood will be taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, if possible, as scheduled below to obtain 2 x 100 μL serum. Blood will be collected on PND 53 +/- 1.

URINALYSIS:
Urine samples are collected from animals fasted overnight at the following times and the parameters listed below are determined.
At the end of the F1 cohort 1A dosing period: 10 males and 10 females randomly selected from each F1 cohort 1A group (animals also selected for laboratory examinations).
The urine is collected for 16 hours in a URIMAX funnel cage. The collection of urine will be terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination.
Parameters: volume, pH, specific gravity
Analytes: protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite
microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Organisms, Further constituents (i.e. sperm, casts), Crystalluria

NEUROLOGICAL SCREENING OF F1 COHORT 2A ANIMALS:
- PND 24 (±1): Auditory startle response
- PND 63-75: Functional observational battery and motor activity
functional tests: Grip strength, Locomotor activity
Postmortem examinations (parental animals):
LABOBRATORY EXAMINATIONS:
Blood samples will be taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight as scheduled. The blood samples collected will be divided into tubes as follows:
EDTA anticoagulant (whole blood): for haematological investigations
Citrate anticoagulant (plasma): for coagulation tests
LiHeparin anticoagulant (plasma): for clinical chemistry tests

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

HAEMATOLOGY:
The parameters listed below will be determined:
- Differential blood count (Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells will be simultaneously quantified during measurement of the differential blood count.), relative
- Differential blood count (absolute)
- Erythrocytes (RBC)
- Leucocytes (WBC)
- Haematocrit value (HCT)
- Haemoglobin content (HGB)
- Platelets (PLT)
- Reticulocytes (RET)
- Mean corpuscular volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
Following the haematological examinations using the ADVIA system, blood smears will be prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
Prothrombin time (PT)/ Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY:
- Sodium
- Potassium
- Calcium
- Chloride
- Albumin
- Total bilirubin
- Total cholesterol
- Glucose
- Total protein
- Blood urea (BUN)
- Creatinine
- Alanine amino-transferase (ALAT/GPT)
- Alkaline phosphatase (aP)
- Aspartate aminotransferase (ASAT/GOT)
- Bile acids
- Lactate dehydrogenase (LDH)
- Sodium/Potassium ratio
- Globulin
- Albumin/globulin ratio
- BUN/creatinine ratio

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples will be taken at sacrifice. The blood is processed for serum.
10 males and 10 females randomly selected from each F0 group

GROSS NECROPSY:
For adult F0 and F1 females a vaginal smear taken on the day of sacrifice shortly before necropsy will be examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals will be euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled:
F0: Males, all, after weaning of the F1 animals, full necropsy
F0: Females,shortly after weaning of the F1 animals, full necropsy
F1 Cohort 1B: shortyl after weaning of the F2 animals, full necropsy

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
All superficial tissues will be examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.
Organ weights of selected organs will be determined for each generation and cohort. Organ weights of the animals which die or are sacrificed prematurely will be recorded but not included in the mean value comparison.


ORGAN WEIGHTS:
F0 generation:
The number of implantation sites will be recorded and used to evaluate reproductive performance (s. reproductive indicies).

The weights of the following organs of all adult F0 animals will be determined before fixation, where applicable. Thyroid weight will be determined after fixation. Paired organs will be weighed individually and identified as left or right:
- Adrenal gland (2)
- Testicle (2)
- Brain
- Thyroid (1) (including parathyroid, post-fixation)
- Epididymis (2)
- Thymus
- Heart
- Prostate (dorsolateral and ventral parts combined)
- Kidney (2)
- Seminal vesicles with coagulating glands
- Liver
- Pituitary
- Ovary (2)
- Uterus including cervix
- Oviducts (2)
- Identified target organs
- Spleen
The following organs or parts thereof of all adult male and female animals of the F0 generation will be preserved in an appropriate fixative:
Davidson’s solution:
- Eye with optic nerve (2)
modified Davidson’s solution:
- Epididymis (1)
- Testicle (1)
The second epididymis and testicle will not be preserved but used for the spermiogram
7% buffered formalin:
- Adrenal gland (2)
- Oviducts
- Bone
- Pituitary
- Bone marrow (os femoris)
- Prostate
- Brain (cerebrum, cerebellum, pons)
- Seminal vesicles with coagulating glands
- Gross lesions observed
- Spinal cord (3 sections)
- Heart (3 levels: right and left ventricle, septum)
- Spleen
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Stomach
- Intestine, large (colon, rectum)
- Thyroid (2) (including parathyroids)
- Kidney and ureter (2)
- Thymus
- Liver
- Trachea (including larynx)
- Lungs (with mainstem bronchi and bronchioles)
- Urinary bladder
- Mammary gland
- Uterus (including cervix)
- Muscle (skeletal)
- Vagina
- Nerve (sciatic)
- Vas deferens
- Oesophagus
- Identified target organs
- Ovary (2)

F1 generation Cohort 1B:
Determination of organ weight and preservation of the F1 Cohort 1B animals is restricted to the organs listed below:
- Adrenal gland (2)
- Pituitary
- Thyroid (2) (including parathyroids)
- Epididymis (2)
- Ovary (2)
- Prostate
- Seminal vesicles with coagulating glands
- Testicle (2)
- Uterus (including oviducts and cervix)
- Vagina
- Vas deferens
- Identified target organs

BONE MARROW:
During dissection fresh bone marrow will be obtained from the os femoris (3 airdried smears/animal) from 10 male and 10 female randomly selected F0 animals per group and stained according to PAPPENHEIM. The myeloid: erythroid ratio will be determined by cell differentiation (counting of 200 nuclei-containing cells) for animals of groups 1 and 4.

HISTOPATHOLOGY:
The blood smears prepared from all animals during the haematological examination will be available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Study Monitor.
Full histopathology will be performed on the preserved organs of F0 animals: 20 randomly selected animals/sex/group of groups 1 and 4 and of all deceased or prematuraly sacrificed animals
The organs listed in above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged. In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined.
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of the selected F0 males of groups 1 and 4 following H-E and PAS staining.

F1 Cohort 1B animals:
In the case of test item-related changes in the corresponding organs of the F0 and F1 Cohort 1A animals, the Study Monitor will be given sufficient notice before the respective organs of F1 Cohort 1B animals are sectioned and examined histopathologically.

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

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

HAEMATOLOGY:
The parameters listed below will be determined:
- Differential blood count (Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells will be simultaneously quantified during measurement of the differential blood count.), relative
- Differential blood count (absolute)
- Erythrocytes (RBC)
- Leucocytes (WBC)
- Haematocrit value (HCT)
- Haemoglobin content (HGB)
- Platelets (PLT)
- Reticulocytes (RET)
- Mean corpuscular volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
Following the haematological examinations using the ADVIA system, blood smears will be prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
Prothrombin time (PT)/ Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY:
- Sodium
- Potassium
- Calcium
- Chloride
- Albumin
- Total bilirubin
- Total cholesterol
- Glucose
- Total protein
- Blood urea (BUN)
- Creatinine
- Alanine amino-transferase (ALAT/GPT)
- Alkaline phosphatase (aP)
- Aspartate aminotransferase (ASAT/GOT)
- Bile acids
- Lactate dehydrogenase (LDH)
- Sodium/Potassium ratio
- Globulin
- Albumin/globulin ratio
- BUN/creatinine ratio

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples will be taken at sacrifice. The blood is processed for serum.
- 10 males and 10 females randomly selected from each F1 cohort 1A group
- F1 pups (2 surplus pups per litter, all litters if possible) on PND 4 (T4 only)
- F1 pups (2 surplus pups per litter, all litters) on PND 22
- F2 pups 10 males an 10 females randomly selected at sacrifice PND 21

GROSS NECROPSY:
The animals will be euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled:
F1: "surplus" pups, on PND 4, gross necropsy
F1: "surplus" pups, on PND 22, gross necropsy including assessment of reproductive organs (All F1 “surplus” pups from PND 22 will be subject to a gross necropsy, however only a limited number of pups will be selected for tissue preservation)
F1 Cohort 1A: at the end of the dosing period (PND 91), full necropsy
F1 Cohort 2A: after behavioural testing, around PND 75
F1 Cohort 2B: on PND 21/ 22, full necropsy
F2 all pups: on PND 21 (Depending on data collected during the study, dissection of F2 pups may be scheduled for PND 4 upon agreement with the Sponsor), Gross necropsy including assessment of reproductive organs

DISSECATION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
All superficial tissues will be examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.
Organ weights of selected organs will be determined for each generation and cohort. Organ weights of the animals which die or are sacrificed prematurely will be recorded but not included in the mean value comparison.

ORGAN WEIGHTS:
The following organs or parts thereof of all adult male and female animals of the F1 generation Cohort 1A will be preserved in an appropriate fixative:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: modified Davidson’s solution
- Epididymis (1)
- Testicle (1)
Fixative: 7% buffered formalin
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Spleen
- Intestine, large (colon, rectum)
- Stomach
- Kidney and ureter (2)
- Thyroid (2) (including parathyroids)
- Liver
- Thymus
- Lungs (with mainstem bronchi and bronchioles)
- Trachea (including larynx)
- Lymph node (1, cervical)
- Urinary bladder
- Lymph node (1, mesenteric)
- Uterus (including cervix)
- Mammary gland
- Vagina
- Muscle (skeletal)
- Vas deferens
- Nerve (sciatic)
- Identified target organs
- Oesophagus

Determination of organ weight and preservation of the F1 Cohort 2A and 2B animals is restricted to the organs listed below:
Cohort 2A:
- Brain
- Eye with optic nerve and retina (2)
- Liver
- Muscle (skeletal)
- Nerve (sciatic)
- Seminal vesicles with coagulating glands
- Spinal cord (3 sections)
Cohort 2B:
- Brain
Liver and seminal vesicles with coagulating glands will be weighed and preserved from all animals of Cohort 2A in order to verify findings of the Pubertal Developmental Assay (Marty et al. (2011); see section 2.10). Animals of Cohort 2A will be used as there age at dissection is closest to the age of the animals examined by Marty et al. (2011).

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

PHENOTYPIC ANALYSIS OF SPLEEN CELLS:
After weighing, spleens of selected Cohort 1A animals will be split in two halves. The portion of the spleen not preserved for histopathology will be minced using a mechanic dissociator to prepare single cell suspensions. Samples will then be used for the following examinations:
- Splenic lymphocyte subpopulation analysis via FACS using the MACSQuant® Analyzer 10/1614
CD4+ T-Lymphocytes
CD8+ T-Lymphocytes
Pan-T-lymphocytes (CD3+)
B-lymphocytes (CD45RA+)
Natural killer cells (CD161+)

HISTOPATHOLOGY:
Full histopathology will be performed on the preserved organs of F1 animals: groups 1 and 4 of Cohort 1A.
The organs listed above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined.
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of all F1 Cohort 1A males of groups 1 and 4 following H-E and PAS staining.
Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea will be performed on one ovary of the F1 Cohort 1A females of groups 1 and 4. Therefore, the ovary will be processed as follows:
- Ten (10) 2-4 μm step sections with 50 μm steps in between;
- Each slide will be labelled with the slide number to follow the sequence.
In case of test item-related changes in group 4, the Sponsor will be given sufficient notice before the corresponding organs of the F1 Cohort 1A of the intermediate/low dose level groups (group 2 and 3) are sectioned and examined histopathologically.

Neuro-histopathology of the F1 Cohort 2A and 2B group 1 and 4 animals is restricted to the following organs:
Cohort 2A:
- Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum)
- Eye with optic nerve and retina (2)
- Muscle (skeletal)
- Nerve (sciatic)
- Spinal cord (3 sections)
Cohort 2B:
-Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum)
Organs and tissues demonstrating treatment-related changes will be also examined for the animals in the lower dose groups.
In addition, the liver as well as the seminal vesicles with coagulating glands of all F1 Cohort 2A group 1 and 4 animals will be examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining in order to verify the findings of the Pubertal Development Assay (Marty et al. (2011)).
Organs and tissues demonstrating treatment-related changes will be also examined for the animals in the lower dose groups.

Details of the Histology processing and the Histopatholigical evaluation could be found in the Study Plan attached to this study endpoint record.
Statistics:
A) The following settings will be used for the statistical evaluation of the parametrical values captured by (Provantis®15 Integrated preclinical software, Instem LSS Ltd.): Homogeneity of variances and normality of distribution will be tested using the BARTLETT’s and SHAPIRO-WILK test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values will be performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group will be made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

B) The following statistical methods will be used for data not captured by Provantis software (reproductive data). For the parametric values (number of pups etc.), homogeneity of variances will be tested using the BARTLETT test. In case of homogeneity, intergroup comparison will be performed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group will be performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01). For the non parametric values (for example the indices of reproduction or the survival rates), the statistical analysis will be performed using the FISHER's exact test (n < 100) or the chi2-test with YATES' correction for continuity (n ≥ 100) at significance levels of p ≤ 0.05 and p ≤ 0.01.

All data are evaluated statistically in this manner. Individual results which differ significantly from those of the control group will be indicated in the tables of the report.
The mean values and standard deviations will be calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
F0 GENERATION:
For each F0 group the gestation index is determined:
Gestation Index = (Number of litters with live pups/ Number pregnant) x 100
Fertility Index female [%] = (Number of pregnant rats/ Number of females used) x 100

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

F1 GENERATION COHORT 1B:
For each litter and group the following indices are determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars) x 100
Live Birth Index = (Number of pups born alive on day 0/1/ Total number born (live + dead)) x 100
Viability Indexpre-select= (Number of pups alive on day 4 (pre-select)/ Number of pups live on day 0/1) x 100
Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: in-life phase completed; draft report not yet available, expected in March 2021

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: in-life-phase completed; draft report not yet available, expected in March 2021

Target system / organ toxicity (P1)

Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: in-life-phase completed; draft report not yet available, expected in March 2021

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Results: F2 generation

Effect levels (F2)

Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: in-life-phase completed; draft report not yet available, expected in March 2021

Target system / organ toxicity (F2)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The OECD 443 study started in Februar 2020 and the in-life-phase is completed.
The first draft report of the study is expected to be available in March 2021.
Executive summary:

The OECD 443 study started in Februar 2020 and the in-life-phase is completed. The first draft report of the study is expected to be available in March 2021.