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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
Adequacy of study:
key study
Study period:
start in February 2020; final report awaiting, the dossier will be updated after the final report is received, data are from 2nd draft report.
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:
publication
Title:
Unnamed
Year:
2018
Report date:
2018
Reference Type:
publication
Title:
Unnamed
Year:
2012
Report date:
2012
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C
- Humidity (%): 55% ± 10%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

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 F0 Geneartion and F1 Generation Cohort 1B of the same dose group were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug.
The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from their male partner after 2 weeks without further opportunity for mating.
In case of an insufficient number of males, for example due to male death before mating with its female partner, males that had already mated were paired with a second female such that all females were paired.

Establishment of F2 generation using Cohort 1B (potential reproductive toxicity):
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. 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 administration formulations were prepared for up to 7 administration days in advance and were administered orally at a constant application volume of 5 mL/kg b.w. once daily.
The test item was diluted in the vehicle to the appropriate concentrations. Following the last dosing on each administration day, the test item formulations were stored at 2 - 8°C. Prior to the first administration of each administration day, the test item formulations were allowed to warm up to room temperature for at least 30 minutes and stirred for approximately 3 minutes to ensure homogeneity. The amount of the test item was adjusted to the animal's current body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.


For the analysis of the test item-vehicle formulations, two aliquots of approximately 3 mL each were taken at the following times and stored at -20°C ± 10% until analysis.
The samples were labelled with study number, test species, generation, cohort no., type of sample, aliquot number, group no., concentration, sampling time and date.
The samples were analysed according to a method validated in LPT Study No. 37532 (“Validation of an analytical method for the determination and quantification of 3,5,5-trimethylcyclohex-2-enone in liquid formulations with HPLC-UV detection”).

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

The results of the test item formulation analysis ranged between 98.5% and 106.3% of the nominal concentration, indicating correctly prepared and homogenized test item vehicle mixtures.
Duration of treatment / exposure:
The 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:
120 female animals were evaluated in order to yield 96 females with a regular estrous cycle for the main study.
MAIN STUDY:
192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation.

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

F2 GENERATION:
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures 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 for this study were selected in agreement with the Sponsor based on available toxicological data and the results of a 16-day repeated dose toxicity study in rats (conducted 1986), a 90-day repeated dose toxicity study in rats (conducted 1986) as well as an OECD 421 study in rats (LPT Study No. 37311, 2020). Additionally, the results and the study design of the Pubertal Development Assay (Marty et al., 2011) were taken into account. In the 16-day and the 90-day study, dose levels of 125, 250, 500 and 1000 mg/kg b.w. per day were employed. In the Pubertal Development Assay, dose levels of 50, 200 and 800 mg/kg b.w. per day were selected. In the OECD 421 study, dose levels of 100, 300, 750 and 1000 mg/kg b.w. per day were used.
In the 90-day and the 16-day gavage studies with the test item, the NOAEL was considered to be 500 mg/kg b.w. (both NTP, 1986). In the 16-day study at the high dose of 1000 mg/kg b.w., only decreased body weights were observed. One female rat that received 1000 mg/kg b.w. died in the 90-day gavage study. Final mean body weights for rats were not clearly related to dose in this study. In the Pubertal Development Assay, increased liver weights, reduced seminal vesicle weights, decreased serum level of testosterone and delayed preputial separation were observed at 800 mg/kg b.w. These findings are considered to be signs of hepatic enzyme induction prior to puberty which results in a greater metabolism rate of testosterone. The thus reduced testosterone level consequently leads to a subsequent delay in the maximum luteinizing hormone (LH) release and attainment of sufficient testosterone levels to drove puberty onset and accessory sex tissue size, e.g. seminal vesicles (Marty et al., 2018). It is assumed that prepubertal animals are particularly sensitive to this mode of action because opposite to adult males, they possess a positive feedback loop where higher levels of testosterone are stimulatory, potentiating the pituitary’s response to GnRH (Gonadotropin-releasing-hormone), resulting in maximum LH release which further increases testosterone secretion (Marty et al., 2011, Stoker et al., 2000).
In the OECD 421 study, female animals treated with 1000 mg/kg b.w. had to be sacrificed after the first dosing and male animals after the third dosing due to animal welfare reasons. Two female animals dosed with 750 mg/kg b.w. were found dead on GD 20 and GD 21. Both male and female animals treated with 750 mg/kg b.w. showed slight to severe salivation, slightly to moderately reduced motility and yellow discoloured urine. Furthermore, a reduction in body weight was noted for the male animals during dosing period, while female animals showed a reduced body weight between GD 14 and GD 20. Accordingly, also the body weight gain was reduced for male and female animals during the respective periods. Reduced body weights were also noted at necropsy for both male and female animals. Therefore, the results of the above-mentioned historical studies could not completely be reproduced under the test conditions of the DRF OECD 421 study (LPT Study No. 37311, 2020).
For the pups of animals treated with 750 mg/kg b.w. reduced weights were noted for male and female animals on lactation days 1, 4 and 13.
Male animals treated with 300 mg/kg b.w. showed slight to moderate salivation, a slightly reduced motility and yellow discoloured urine, while slight salivation was observed in females treated with 300 mg/kg b.w.
Based on these findings, doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death. Taking all study results into account and considering the extended exposure time of the F0 and F1 Generation within the OECD 443 study, 600 mg/kg b.w. per day were considered the maximum tolerated dose for the F0 Generation of the OECD 443 study, with no systemic effects expected in the F1 Generation. Furthermore, 600 mg/kg b.w. per day 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 Cohorts 1A, 1B and 2A
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 after weaning
Additionally, a more detailed examination of all F0 and F1 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 1A, 1B and 2A:
F1 animals (Cohorts 1A, 1B, 2A) were evaluated daily for balano-preputial separation or vaginal opening, which indicate sexual maturity of the animals. The genitals were examined for any abnormalities. The body weight was recorded at the time point of balano-preputial separation or vaginal opening. The results were evaluated individually for each cohort as well as for all cohorts combined.

TESTOSTERONE DETERMINATION:
F1 Cohorts 1A, 1B, 2A: In order to investigate possible effects of the test item 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 and 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, 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

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: 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 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 given sufficient notice before the respective organs of F1 Cohort 1B animals are sectioned and examined histopathologically. However, histopathological examination of the F1 Cohort 1B animals was not necessary.

Details of the Histology processing and the Histopatholigical evaluation could be found in the histopathological report attached to this study endpoint record.
Postmortem examinations (offspring):
LABOBRATORY EXAMINATIONS:
Blood samples 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 histopathological report 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 dams with live pups/ Number pregnant rats) x 100
Fertility Index female [%] = (Number of pregnant rats with verified copulation/ Number of rats with verified copulation) x 100

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in behaviour, the external appearance or the faeces that were considered to be adverse were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

Salivation was noted for all male and nearly all female animals of the dose groups and in some cases, the saliva was red discoloured. A dose-dependent increase of the severity and the number of observations was noted. However, as salivation was only noted for at maximum one hour post administration, the transient observation of salivation was considered to be not adverse.

Dark yellow discoloured urine was noted for all high dose males and females during the test days 16 to 19. This observation was considered to be test item-related but not adverse as it was noted in all animals but only for the same 4 consecutive days.
The low dose male animal no. 58 was noted with red discoloured urine on TD 56 and TD 65 as well as the intermediate dose male no. 108 between TD 29 and TD 86. However, the single occurrences of red discoloured urine were considered to be not test item-related.


Reduced motility was observed on TD 15 for all males and females of the intermediate dose group, and for all high dose males on TD 15 and TD 16 and all high dose females between TD 15 and TD 21. Additionally, one high dose male was noted with reduced motility on TD 51 and TD 54 and two high dose females for one day each on GD 19 or GD 20. Reduced motility started 20 to 60 minutes after dosing and lasted up to 60 minutes after dosing. The observation was considered to be test item related but not adverse.

Piloerection was noted for the high dose males nos. 159 and 165 on TD 43. These single occurrences were considered to be spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related premature death was noted for the dose groups (65, 200 or 600 mg test item/kg b.w./day) and the control group. However, the male control animal no. 9 was found dead in the morning of TD 35. Necropsy revealed edematous lungs, white deposits and approximately 3 mL of a turbid liquid in the thorax and enlarged adrenals. Histopathological examination of animal no. 9 revealed inflammation of the esophagus, the larynx and of the tissue surrounding the thymus. In the lungs, alveolar edema, congestion and alveolar macrophages and in the adrenal glands, diffuse cortical hypertrophy were noted.The premature death of animal no. 9 was considered to be due to a misgavage. Furthermore, two prematurely deceased males were noted for the high dose group (nos. 161 and 166): Animal no. 161 was found dead during TD 45 and it displayed piloerection and moderate salivation in the days prior to its death. During necropsy, edematous lungs were observed, as well as the thorax filled with 2 mL of a clear and viscous liquid and the neck region being gelatinous. During the histopathological examination, inflammation was noted for the esophagus and the surrounding tissue, the trachea, the larynx and the surrounding tissue of the thymus. Further, alveolar edema and congestions were observed for the lungs, as well as centrilobular hepatocellular hypertrophy in the liver and hyaline droplets in the kidneys. Animal no. 166 was found dead during TD 36. Necropsy revealed the left lobe of the lungs being edematous and dark-red discoloured, the thorax was filled with approximately 2 mL of a viscous liquid and the liver was enlarged. Histopathological examination of the animal revealed pathological changes of the larynx (inflammation), the lungy (alveolar edema, congestion), the thymus (atrophy), liver (centrilobular hepatocellular hypertrophy, focal necrosis and congestion), the thyroid glands (follicular cell hypertrophy) and for the kidneys (hyaline droplets). Hepatocellular hypertrophy was the treatment-related change in this study, however, not considered to have contributed to the animal’s death. The deaths of the animals nos. 161 and 166 were considered to be not test item-related but due to a misgavage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day), a reduced body weight was noted from TD 36 until TD 84 before sacrifice on TD 85/86 (at maximum 7.9% below the value of the control group on TD 84, statistically significant at p ≤ 0.05 or 0.01). This constantly lower body weight was considered to be test item-related.
In consequence to the reduced body weight for the high dose group, also the body weight gain from TD 15 to TD 84 was decreased for the high dose males.

Females: No test item-related changes in body weight were noted for the female rats between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), a transiently reduced body weight was noted at the end of the gestation period on GD 21 and at the beginning of the lactation period on LD 1 (8.1% and 6.1% below the value of the control group on GD 21 and LD 1, statistically significant at p ≤ 0.05 and or 0.01), respectively. However, as the body weight recovered during the lactation period, the transiently reduced body weight was considered to be not test item-related but spontaneous.
In accordance with the body weight, also no changes were noted for the body weight gain of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: No adverse differences in food consumption were noted between the control male animals and in the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Reductions in food consumption were noted for the low, intermediate and high dose groups in the first week of dosing (8.4%, 11.5% and15.9% below the value of the control group, respectively, statistically significant at p ≤ 0.01). Further decreased values for the food consumption were observed for the low dose group between TD 22 and TD 29 and between TD 56 and TD 63 (15.7% and 6.5% below the value of the control group, respectively, statistically significant at p ≤ 0.01). However, as the male food consumption recovered thereafter and no dose response-relationship was present, the reduced food consumption was considered to be test item-related but not adverse.
However, for the high dose males, an increased food consumption was noted from TD 49 onwards until TD 84, one day before termination of the F0 males, (at maximum 9.2% above the value of the control group, statistically significant at p ≤ 0.05 or 0.01). Nevertheless, an increased food consumption in the high dose was considered to be not toxicologically relevant.
Females: No test item-related differences in female food consumption were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A reduced food consumption was noted for the female animals in the dose groups at start of the dosing. In detail, a decreased food consumption was noted for all dose groups between TD 15 and TD 22, for the low and intermediate dose groups between TD 22 and TD 29 and again for all dose groups after mating between GD 0 and GD 7 ranging between 5.5% and 23.0% below the value of the control group (statistically significant at p ≤ 0.05 or 0.01). Thereafter, the food consumption recovered and no difference between the dose groups and the control group was noted in the third week of gestation and the lactation period. Therefore, the transiently reduced food consumption was considered to be test item-related but not adverse. No food intake of female animals was recorded during the mating period as both sexes were housed together.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females: No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day).
Slight statistically significant differences in comparison to the control group were noted for the MCHC of the male high dose animals. Also for the female high dose animals a slight but statistically significant reduction of the number of eosinophilic granulocytes in the differential blood count was noted. However, these changes were considered to be spontaneous, as no dose response-relationship was noted, no changes were noted for the other gender, and the values were within the range of the Provivo background data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females:
No test item-related differences for the examined biochemical parameters were noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day) of both sexes, increased levels were noted for globulin, for total cholesterol and for total protein. The increased levels for globulin and total protein of the male high dose animals were above the 95%-confidence interval of the Provivo background data. Furthermore, the increases were also noted for the male and female animals of Cohort 1A. Therefore, the findings were considered to be test item-related and adverse.
Endocrine findings:
no effects observed
Description (incidence and severity):
Males and females: No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Statistically significant differences for the T4 and TSH serum levels were noted for the male and female animals. The T4 serum levels of the male and female low and intermediate dose animals were outside 5% to 95% confidence interval of the Provivo background data but no dose-dependence relationship was present. The decreased TSH levels of the female low and intermediate dose animals were within the 5% to 95% confidence interval of the Provivo background data and no dose-response relationship was present. Therefore, the changes for the serum thyroid hormone levels in the low and intermediate dose groups were considered to be not test item-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females: No test item-related differences were noted between the control group and the male animals of the low dose group (65 mg test item/kg b.w./day) and for the females of any dose group for the examined urinalysis parameters.
For the male intermediate and high dose animals (200 or 600 mg test item/kg b.w./day), a statistically significantly decreased pH was noted (6.0% and 5.7% below the value of the control group, respectively, p ≤ 0.01). The values for the male intermediate and high dose animals were below the 5% to 95%-confidence interval of the Provivo background data and a decreased pH was also noted for the male intermediate and high dose animals of Cohort 1A. Therefore, the decreased pH of the urine of the male intermediate and high dose animals was considered to be test item-related and adverse.
Additionally, erythrocytes in the urine were noted. In the male animals, erythrocytes were observed for either one or two of 10 animals of the control group (animal no. 3), the low dose group (animals nos. 52 and 58) and the high dose group (animals nos. 152 and 164). For the female animals, erythrocytes in the urine were noted for 1 of 10 low dose animals (animal no. 78) and for 1 of 10 intermediate dose animal (animal no. 139). However, as no dose-response relationship was present for the male and female animals, the findings of erythrocytes in the urine was considered to be not test item-related but spontaneous. While erythrocytes could be observed microscopically, their concentration was too low to have an impact on the visual appearance of the urine, hence no red discolouration of the urine was observed.
All other parameters evaluated (e.g. presence of leucocytes, crystalluria, etc.) did not show any difference between the control group and the dose groups in any sex.
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females: Test item-related but non-adverse findings were observed for the following organs: In the adrenal glands, an increased incidence and severity of fine vacuolation was noted in the zona fasciculata of male and female animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day). In the thyroid glands, a slightly increased incidence of follicular cell hypertrophy was noted for the intermediate and high dose males and females.
Furthermore, findings of centrilobular hepatocellular hypertrophy were noted with increasing incidence and severity in the livers of the males of all dose groups (65, 200 or 600 mg test item/kg b.w./day) and of the female animals of the intermediate and high dose groups. For the male animals of the intermediate and high dose groups (200 or 600 mg test item/kg b.w./day), a dose-dependently increased incidence was noted for hyaline droplets in the proximal tubular cells of the kidneys. As the effects at 600 mg/kg b.w./day indicate towards previously reported changes in carcinogenicity studies, the findings were considered to be adverse at high dose.
For male and female reproductive organs, no test item-related changes were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Bone marrow: Males and females: No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group of both sexes.

Reproductive function / performance (P0)

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

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

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

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

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

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

Gestation length: No test item-related differences were noted for the length of the gestation period between the rats of the control group and those of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly shortened gestation length was noted for the dams of the high dose group (2.1% below the value of the control group, p ≤ 0.01). However, a slightly shorter gestation length was considered to be of no toxicological relevance.

Details on results (P0)

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

Reproductive toxicity: No influences were noted on the number or length of the estrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity (F0)
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity (F0)
Effect level:
65 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B: Males: No test item-related changes of behaviour, external appearance or the faeces that were considered to be adverse were noted for the male animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Transient changes in behaviour or external appearance were noted for the control group and for the high dose group. In detail, the control male animal no. 367 was observed with a hemorrhagic nose/snout on TD 108. Furthermore, in the high dose group, three male animals (nos. 483, 488 and 494) displayed reduced motility on TD 15 and animal no. 494 additionally, showed an increased water consumption on TD 105. The high dose animal no. 498 was noted with breathing sounds and gasping on TD 90 and with piloerection on TD 94 and 95. Due to the low number of observations, the above mentioned changes were considered to be not test item-related.
In addition to these single occurrences, also in Cohort 1B, salivation was noted in all male animals of any dose group with a dose-dependent increase in severity and number of days with salivation per animal. For two animals each of the intermediate dose group (nos. 446 and 456) and the high dose group (nos. 491 and 492) the saliva was occasionally red discoloured. However, as salivation for the male animals of any dose group was noted in almost all cases not longer than 60 minutes post administration, salivation was considered to be test item-related but not adverse.
Females: No test item-related observations considered to be adverse were noted for the female animals of any dose group (65, 200 or 600 mg test item/kg b.w./day).
Single occurrences of reduced motility were noted during the pre-mating/mating period for 4 of 20 high dose group females and in the lactation period for 2 of 19 females for one or two days per animal.
Salivation was noted for 5 of 20 control females during the pre-mating/mating period for one or two test days per animal. In the low dose group, salivation was observed for all animals in the pre-mating/mating period and for 7 or 10 of 18 animals in the gestation and lactation period. Furthermore, all female animals of the intermediate and high dose group displayed salivation in all study periods. However, as also for the female animals the salivation was noted for not longer than 60 minutes post administration, the test item-related salivation was considered to be not adverse.
Detailed clinical observations: Males: No observations were noted during the detailed clinical observations for the male animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day). Females: No observations were noted during the detailed clinical observations for the female animals of the low dose groups (65 mg test item/kg b.w./day).
In the control group and in the intermediate dose group (200 mg test item/kg b.w./day) the female animals nos. 396 and 466 were noted with a loss of the tail tip from test week 1 or test week 9 onwards, respectively. In addition, the female animal no. 514 of the high dose group (600 mg test item/kg b.w./day) was noted with a wound in the neck region (approximately 4 mm in diameter) in test week 9. The single occurrences, of a missing tail tip and a wound in the neck region noted in the intermediate or high dose groups are considered to be spontaneous.

COHORT 1A: Males and females: No test item-related changes in behaviour, the external appearance or the faeces that were considered to be adverse were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Single occurrences of changes of behaviour or external appearance were noted for a few animals of different groups. In detail, piloerection was noted for the male animal no. 211 of the control group, for the male animal no. 298 of the intermediate dose group on TD 2 and 3 and for the female animal no. 231 of the control group on TD 2 to 7. Furthermore, reduced motility was noted in the high dose group for the male animal no. 339 and for the female animals nos. 341 and 352 on TD 15 or on TD 15 and 16. All changes were regarded to be spontaneous.
Moreover, salivation was noted for all male and female animals of all dose groups and additionally, for the female animal no 236 of the control group on TD 44. As salivation was noted with increasing severity and with an increasing number of days per animal, salivation was considered to be test item-related. However, as salivation was noted at maximum up to 60 minutes post administration, salivation was considered to be not adverse.
COHORT 2A: Males and females: No adverse changes in the behaviour, the external appearance or the faeces were noted for the male and female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day). Salivation was noted for all male and female animals of any dose group with a slight increase in severity and with an increasing number of days per animal. For one high dose male and one intermediate dose female, salivation was red discoloured on three days each. However, salivation was also noted for one male animal and for 6 of 10 female animals of the control group. In addition, reduced motility was noted for one high dose female on one test day. As salivation ended 60 minutes after administration at the latest and was also noted for male and female animals of the control group, salivation was considered to be test item related but not adverse. Body weight at autopsy: No test item-related differences for the body weight at autopsy were noted between the control group and male and female animals of low dose animals (65 mg test item/kg b.w./day) and for the intermediate dose females (200 mg test item/kg b.w./day).
Although not statistically significant, the decreased body weight at autopsy for the male animals dosed with 200 mg test item/kg b.w./day (9.5% below the value of the control group) was considered to be test item-related due to distinct difference and as the body weight on TD 56 was test item-related decreased.
In the high dose group (600 mg test item/kg b.w./day), statistically significantly decreased body weights at autopsy were observed for the male and the female animals (12.6% or 6.8% below the value of the value of the control group, p ≤ 0.05 or 0.01). These distinctly decreased body weights at autopsy were considered to be test item-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
COHORT 1B: Males and females: Males and females
No premature death was noted for the female animals of any group and for the male animals of the control group and the low and high dose group (65 or 600 mg test item/kg b.w./day).
In the intermediate dose group (200 mg test item/kg b.w./day), the male animal no. 457 was found dead in the morning of TD 74. Necropsy revealed a reddened thymus and dark-red discoloured lungs. No changes were noted for the behaviour or the external appearance of animal no. 457. Therefore, the death of the animal was considered to be not test item-related but most likely due to a misgavage.

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

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

Body weight at autopsy: Males and females: No test item-related differences were noted between the control group and the female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day) and the male low and intermediate dose animals (65 or 200 mg test item/kg b.w./day).
The male animals of the high dose group (600 mg test item/kg b.w./day) were noted with a decreased body weight at autopsy (14.9% below the value of the control group, statistically significant at p ≤ 0.01). As this in accordance with the test item-related decreased body weight from TD 8 onwards, also the decreased body weight at autopsy of the male high dose animals was considered to be test item-related and adverse.

COHORT 1A: Males: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In accordance with the reduced body weights of the F1 pups of the high dose males (600 mg test item/kg b.w./day), also after transfer into Cohort 1A, the body weight of the male high dose animals remains decreased (at maximum 11.7% below the value of the control group, statistically significant between TD 8 and 64 at p ≤ 0.05 or 0.01). Body weight gain: No differences in body weight gain were noted between the control group and the low and intermediate dose groups. Females: No test item-related changes in body weight and body weight gain were noted for the female rats between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day). In the high dose group, a reduced body weight was noted on TD 1, TD 8 and TD 15 (12.8%, 13.1% and 8.2% below the value of the control group, statistically significant on TD 8 at p ≤ 0.01 and on TD 15 at p ≤ 0.05). This was in accordance with the reduced body weight of the F1 pups throughout the lactation period. However, after TD 15 the body weight recovered and ranged between TD 22 and TD 64 between 4.7% and 0.8% of the control value. Therefore, the transiently reduced body weight between TD 1 and TD 15 was considered to be not test item-related but due to the already lower body weight before transfer into Cohort 1A. Body weight gain: The increased body weight gain of the high dose females in comparison to the control group was due to a lower body weight of the high dose group on TD 1 and the recovery until TD 64. Therefore, the increased body weight gain of the high dose group was considered to be not test item-related.
Body weight at autopsy: Males and females: No test item-related influence on the body weight at autopsy were noted for the female animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day) and for the male animals of the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day). A decreased body weight at autopsy was noted for the male animals dosed with 600 mg test item/kg b.w./day (11.9% below the value of the control group, statistically significant at p ≤ 0.01). The distinctly decreased body weight at autopsy was considered to be test item-related and adverse.




COHORT 2A: Males: No test item-related differences in comparison to the control group was noted for the body weight and the body weight gain of the male animals of the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), a reduced body weight compared to the control group was noted from TD 2 until necropsy on TD 56. In detail, the difference between the control group and the intermediate dose group ranged between 8.5% and 12.0% below the value of the control group (statistically significant from TD 36 until TD 56 at p ≤ 0.05). In the high dose group, the decrease in body weight ranged from 6.6% to 13.5% below the value of the control group and was statistically significant from TD 29 until TD 56 at p ≤ 0.05 or 0.01. Body weight gain: In accordance with the decreased body weight, also the body weight gain of the male high dose animals was decreased in comparison to the control group. The body weight gain of the intermediate dose group however, appeared to be only slightly decreased when compared to the control group. This was due to the low body weight of the intermediate dose group on TD 2. Females: No test item-related differences for the body weight of the female animals was noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), a decreased body weight was noted from TD 2 until necropsy on TD 56. On TD 2, the body weight of the high dose females was 20.5% below the value of the control group (statistically significant from TD 2 to TD 36 at p ≤ 0.01) and the difference decreased until TD 56 to 6.0% below the value of the control group. Due to the high difference in the beginning of the Cohort 2A and the constantly decreased values in comparison to the control group, the lower body weight of the high dose group was considered to be test item-related. Body weight gain:
The body weight gain of the female animals of the dose groups from TD 2 to TD 56 was higher in comparison to the control group. This was due to the fact that the difference between the control group and the dose groups on TD 2 was higher than the difference on TD 56. Therefore, the increased body weight gain of the females of the high dose group was considered to be not test item-related. Body weight at autopsy: Males and females: No test item-related differences for the body weight at autopsy were noted between the control group and male and female animals of low dose animals (65 mg test item/kg b.w./day) and for the intermediate dose females (200 mg test item/kg b.w./day).
Although not statistically significant, the decreased body weight at autopsy for the male animals dosed with 200 mg test item/kg b.w./day (9.5% below the value of the control group) was considered to be test item-related due to distinct difference and as the body weight on TD 56 was test item-related decreased.
In the high dose group (600 mg test item/kg b.w./day), statistically significantly decreased body weights at autopsy were observed for the male and the female animals (12.6% or 6.8% below the value of the value of the control group, p ≤ 0.05 or 0.01). These distinctly decreased body weights at autopsy were considered to be test item-related.

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
COHORT 1B: Males: No test item-related difference in the relative food consumption was noted between the male animals of the control group and all dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly decreased food consumption was noted between TD 92 and TD 99 for the low dose group (5.7% below the value of the control group, p ≤ 0.05) and a statistically significantly increased food consumption was noted for the intermediate dose group between TD 99 and TD 106 (6.8% above the value of the control group, p ≤ 0.01). Furthermore, a statistically significantly increased food consumption was noted for the high dose group between TD 50 and TD 113 (at maximum 14.0% above the value of the control group, statistically significantly at p ≤ 0.01). However, an increased relative food consumption was not considered to be test item-related but was most likely due to the decreased body weight of the male high dose animals.
Females: No test item-related difference in food consumption was noted between the female animals of the control group and allthe dose groups (65, 200 or 600 mg test item/kg b.w./day).
A statistically significantly increased food consumption was noted for the intermediate and high dose females between TD 64 and TD 71 (22.5% or 14.4% above the value of the control group, p ≤ 0.01). However this transiently increased food consumption was considered to be not test item-related.

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

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

COHORT 1A: Testosterone hormone level analysis: No test item-related differences for the serum testosterone were noted for the male Cohort 1A animals dosed with 65, 200 or 600 mg test item/kg b.w./day) compared to the males of the control group at PND 53.
Thyroid hormone levels: Males and females: No statistically significant differences for the levels of the examined thyroid hormones T4 and TSH were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).

COHORT 2A: Testosterone level analysis: No test item-related differences for the serum levels of testosterone of the male animals were noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1A: Males and females: No test item-related changes for the urine parameters were noted for the male and female animals of the low dose group (65 mg test item/kg b.w./day).
In the intermediate dose group (200 mg test item/kg b.w./day), statistically significant reductions were noted for the pH values of the male animals (5.3% below the value of the control group, p ≤ 0.05). Additionally, even more pronounced reductions were noted for the pH values of the male as well as the female animals (10.3% or 8.5% below the value of the control group, p ≤ 0.01) in the high dose group (600 mg test item/kg b.w./day).
The urine pH of the male and female high dose animals was outside the 5% to 95%-confidence interval of the Provivo background data, whereas the pH of the male intermediate dose animals was within this range. However, the decreased urine pH of the males of both the intermediate and the high dose group was considered to be test item-related as a dose-response relationship was present and the urine pH was also decreased for the male intermediate and high dose animals of the F0 generation. Also, the decreased urine pH of the female high dose animals was considered to be test item-related and adverse.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B: Organ weights (relative and absolute): Males: No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the male animals of the the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), statistically significantly increased relative organ weights were noted for the adrenal glands. The weights of the adrenals were within the 5% to 95% confidence interval of the Provivo background data. However, as the female intermediate and high dose animals of Cohort 1B, the high dose animals of Cohort 1A and the intermediate and high dose animals of the F0 Generation also displayed test item-related increased adrenal weights, the increased weights of the adrenal glands in the high dose males of Cohort 1B were considered to be test item-related. Further organ weights of the high dose males that were statistically significantly increased in comparison to the male animals of the control group: Epididymis (left & right; relative weight), Testis (left & right; relative weight), Pituitary (relative weight), Thyroid incl. parathyroid (relative weight). However, for most of these, no dose response-relationship was present and therefore, the increases were considered to be not test item-related but due to the reduced body weight at autopsy.
Females: No test item-related differences for the examined absolute and relative organ weights were noted between the female animals of the control group and the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), increased values were noted for the relative and absolute weights of the left and right adrenals. An increase in the adrenal weights were noted also for the Cohort 1B males and for the Cohort 1A males and females dosed with 600 mg test item/kg b.w./day as well as for the males and females in the intermediate and high dose group of the F0 Generation. Therefore, the increased weights of the adrenal glands for the intermediate and high dose Cohort 1B females were considered to be test item-related. Additionally, statistically significantly increased relative and absolute weights were noted for the thyroids of the intermediate dose females. However, as no dose response-relationship is present, the increased thyroid weights were considered to be not test item-related.

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

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

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

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

COHROT 2A: At termination of Cohort 2A animals (PND77 to 81) and Cohort 2B (PND 21 to 22) animals necropsy revealed no pathological changes for the male and female animals of the control group and of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

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

COHORT 2A: Histopathology on liver and seminal vesicles: Histopathological examinations were conducted on organs of male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day). Pathological changes were observed in form of centrilobular hepatocellular hypertrophy in the livers of 10 male and 10 female high dose animals compared to none of the male and female control animals.
During histopathological examination of the seminal vesicles and the coagulating glands of the male control and high dose Cohort 2A animals, no abnormalities were noted.

COHORT 2A and 2B: Neurohistopathology: The histopathological evaluation of the brains was performed for the male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day) from PND77 to 81 (Cohort 2A) or from PND21 to 22 (Cohort 2B).
The image analysis and the histomorphometry of the brains of the male and female Cohort 2A and 2B animals revealed no test item-related differences between the control animals and the animals of the high dose group.



Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
COHORT 1A: Lymphocyte typing in spleen: Males and females: No statistically significant differences were noted in the proportion of the examined lymphocyte subtypes in the male and female animals between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Bone marrow: Males and females: No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (600 mg test item/kg b.w./day) of both sexes.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
COHORT 1B: Stage of estrous cycle at necropsy: No test item-related differences were noted between the female animals of the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day) in the distribution of the stages of the estrous cycle.
A slightly decreased number of females in the metestrus stage and an increased number of females in the diestrus stage were noted for the females of the high dose group. However, these slightly differences were considered to be spontaneous.

COHORT 1A: The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 50 and 63 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day). Stage of the estrous cycle at necropsy: No test item-related differences were noted between the female animals of the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day) in the distribution of the stages of the estrous cycle.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
COHORT 1A: Examination of the sperm number, viability and morphology: No test item-related difference was noted between the rats of the control group and the rats treated with 65, 200 or 600 mg test item/kg b.w./day for the sperm number, the viability and morphology.
Reproductive performance:
no effects observed
Description (incidence and severity):
COHORT 1B: Female fertility: No test item-related influence on the fertility index was noted for the female rats of the treatment groups (65, 200 or 600 mg test item/kg b.w./day).
One female each of the control and the low and intermediate dose group (nos. 395, 423 and 470) did not mate with its male partner within the 14-day mating period and one female each of the low and intermediate dose group (nos. 428 and 462) did not become pregnant.
However, these numbers are within the range of normal biological variability.
Gestation index: No test item-related influence on the gestation index was noted for the female rats of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Dams nos. 382 and 511 of the control group or the high dose group lost all of their implantations. However, these numbers are within the range of normal biological variation.
Pre-coital time and gestation length: No test item-related influence was noted on the pre-coital time and for the gestation length for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly and not statistically significantly shorter pre-coital time was noted for the females of the high dose group (2.1 days in the high dose group compared to 3.0 days in the control group, not statistically significant). However, as no dose response-relationship was present, the slightly shorter pre-coital time was considered spontaneous.

Effect levels (P1)

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Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity (F1)
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity (F1)
Effect level:
65 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity (P1)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups: No test item-related changes of behaviour or external appearance were noted for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day) during the lactation period.
From LD 13 or 14 until necropsy of the pups on LD 22 or 23, changes in the external appearance of the pups in form of thin fur were noted for all remaining pups of one litter each of the control group (no. 39) and the high dose group (no. 192), and for all remaining pups of two litters of the low dose group (nos. 77 and 91). As also one litter of the control group was noted with thin fur and no dose-response relationship was present, the observations of thin fur were considered to be not test item-related.


Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 pups: Viability index - Pre-and post-cull period: No test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
Number of live pups: No test item-related differences were noted for the mean number of live pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) during the lactation period.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 pups: No test item-related influence on the body weight of pups was noted for the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
In the high dose group (600 mg test item/kg b.w./day), reduced body weights for the male and female pups were noted throughout the lactation period from LD 1 until LD 21 (at maximum 13.5% below the value of the control group for the female pups on LD 1, statistically significant at p ≤ 0.01). Furthermore, the means of the male and female litters per dam combined were only marginally above the 5% percentile of the Provivo background data for the LD 1, LD 4 and LD 7 and were below the 5% to 95% confidence interval on LD 14 and LD 21.
Hence, the distinctly and constantly reduced pup body weights that were partly outside the 5% to 95% confidence interval of the Provivo background data were considered to be test item-related.




Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
F1 pups: Thyroid hormone determination: T4 levels: No test item-related differences between the control group and the treatment groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the T4 serum level on LD 4 and LD 22 for the male and female pups.
Statistically significantly decreased T4 serum levels were noted for the male pups of the intermediate dose group and for the male and female pups of the high dose group. However, the mean values of the T4 levels on LD 4 for the male and female pups were within the 5% to 95% confidence interval of the Provivo background data and no statistically significant difference was observed for the T4 serum levels on LD 22. Hence, the decreased T4 levels on LD 4 were considered to be not test item-related. TSH level: No statisctically significant differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the TSH level on LD 22 for the male and female pups.

Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1A, 1B and 2B: Males: No test item-related differences between the control group and the test item-treated groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the time point of balano-prepuital separation and the body weight at the time point of balano-prepuital separation.
A statistically significantly lower body weight at the time point of balano-preputial separation was noted for the high dose animals. However, this was not due to an influence on the sexual maturation but due to the slightly earlier time point of balano-preputial separation and the lower body weight already in the lactation period of the high dose animals.
Additionally, the summarized comparison all F1 males of the appropriate age (Cohorts 1A, 1B and 2A) revealed the same result: No statistically significant difference was noted for the time point of the balano-preputial separation between the control group and the dose groups. For the body weight at the time point of the balano-preputial separation a statistically significant decrease was noted in the high dose group (9.9% below the value of the control group, p ≤ 0.01).

Females: No test statistically significant differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the body weight at the time point of vaginal opening. A slight but not statistically significant body weight loss was noted for the high dose females. However, this was due to the reduced body weight noted already during the gestation period and after transfer into Cohort 1A rather than an influence on the sexual maturation of the female animals. Furthermore, no difference was noted for the appearance of cornified cells, that marks the entry into the estrus stage of the estrous cycle for the first time in their life.
No statistically significant differences between the control group and the dose groups were observed for the day of vaginal opening for the Cohort 1A females only. However, a slight delay in the day of vaginal opening was noted for the high dose group. This was also noted for the Cohort 1B and for Cohort 2A, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when all F1 females (Cohorts 1A, 1B, and 2A) of appropriate age were included in the comparison. Additionally, the number of female animals with a day of vaginal opening above the 5% to 95% confidence interval of the Provivo background data increased with the dose level. However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not significant) at the high dose in Cohort 1A and when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the high dose treatment. Taking all findings together, the delayed day of vaginal opening for the F1 females of the high dose group (600 mg test item/kg b.w./day) was considered to be test item related but not adverse.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 pups: No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 pups: No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), a statistically significant increase was noted for the number of dams with nipple-bearing male pups and also for the number of nipples per pup. In detail, 0.9 male pups with nipples per dam each were noted in the intermediate or high dose group in comparison to 0.2 pups per dam in the control group (p ≤ 0.05). Furthermore, the number of nipples per pup was increased for the intermediate and high dose group (0.44 or 0.46 nipples per pup) when compared to the male pups of the control group (0.11 nipples per pup, statistically significant at p ≤ 0.05). The increase of these two parameters also led to an increase in the number of nipples per dam (2.2 for the intermediate or high dose group compared to 0.6 nipples on male pups per dam of the control group, statistically significant at p ≤ 0.05). However, the toxicological relevance of the increased number of pups with nipple retention for the intermediate and the high dose group is unclear but limited by the following facts:
1) the finding is isolated as no influence was noted on a related endpoint (e.g. ano-genital distance)
2) no dose response relationship was noted for the number of male pups with nipples per dam and for the number of nipples per dam
3) no influence was noted on the male pups of the F2 Generation of any dose group
4) for all calculated mean values, the standard deviation is higher than the mean value, indicating large variety
5) the investigation of this end point within this study type was only introduced in 2015, so that the data base of historical control data is limited as well as the interpretation of the described effects.
Based on the above mentioned considerations, the observation was considered to be spontaneous and no test item related.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F1 pups: No statistically significant differences for the examined absolute organ weights were noted for the F1 pups between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1 pups: Macroscopic internal and/or external examinations were performed on animals found dead during the lactation period, at litter adjustment on LD 4 or at termination of the lactation period between LD 22 and 24. During these macroscopic post mortem examinations, no gross abnormalities (e.g. malformations or variations) were noted for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
For the control pup no. 35-11 necropsy after terminal sacrifice on LD 22 revealed inflated stomach and intestines, a thymus being reduced in size and a reddened brain. These single occurrences for one control pup were considered to be spontaneous.

Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
F1 pups: Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Male to female ratio of the pups: No test item-related influence on the male to female ratio was noted for all dose groups (65, 200 or 600 mg test item/kg b.w./day).


Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurological screening - Cohort 2A: Auditory startle response: No test item-related difference was noted between the male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) for their auditory startle response.
When the last block of trials for a group was compared to the respective first one, the male and female control animals as well as the animals of the dose groups showed a slight habituation between the blocks.
Observational screening - Cohort 2A: No abnormalities were noted during the observational screening for any male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Functional tests - Cohort 2A: Grip strength:
No test item-related differences between the male and female animals of the control group and those of the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the grip strength of the fore- and hind-limbs.
A statistically significantly increased grip strength was noted for the fore-limbs of the male animals of the low and high dose groups (37.0% or 32.8% above the value of the control group, respectively; statistically significant at p ≤ 0.01). However, as no dose response-relationship was present and no differences were noted for the grip strength of the hind-limbs, the increased fore-limb grip strength for the male low and high dose animals was considered to be not test item-related.
Spontaneous motility: No statistically significant differences between the male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the spontaneous motility.

Neurohistopathology - Cohort 2A and Cohort 2B: The histopathological evaluation of the brains was performed for the male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day) from PND77 to 81 (Cohort 2A) or from PND21 to 22 (Cohort 2B).
The image analysis and the histomorphometry of the brains of the male and female Cohort 2A and 2B animals revealed no test item-related differences between the control animals and the animals of the high dose group.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

F1 pups: No test item-related effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post-implantation loss).
For the post-natal development, a decrease in body weight and accordingly a decrease in litter weight was noted for the pups of the high dose group (600 mg test item/kg b.w./day) during the lactation period.
No test item-related effect was noted on the postnatal development of the pups regarding the viability indices (pre- and post-cull) and the ano-genital distance.
No effect that was considered to be test item-related was noted for the nipple retention of the male pups. The increased number of pups with nipples in the intermediate and high dose were considered to be spontaneous.
The examination of the thyroid hormone levels on lactation days 4 and 22 revealed no test item related differences.
The gross inspection (internal and/or external) of the pups at necropsy revealed no changes for the pups for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
No test item-related influence was noted on the organ weights from pups sacrificed after weaning.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Prenatal development (conceptus to birth)
Generation:
F1
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Postnatal development (pup)
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Neurological development
Generation:
F1
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F2 pups: Pre- and post-cull period: No test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
Number of live pups: No test item-related differences were noted for the mean number of live pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) during the lactation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2 pups: No test item-related difference for the body weight of the male and female F2 pups was noted between the control group and the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose group (600 mg test item/kg b.w./day), a constant decrease of the pup body weights in comparison to the control was noted on the investigated days during the lactation period. In detail, slightly lower but not statistically significantly different pup body weights were noted on LD 1 for males as well as females (3.1% below the value of the control group for the male and female pups each). At later time points, the difference in body weight between the pups of the control group and the high dose group increased. Accordingly, a statistically significantly decreased body weight of the male and female high dose pups was noted on LD 14 (10.8% or 11.4% below the value of the control group, p ≤ 0.01) and on LD 21 (11.2% or 11.6% below the value of the control group, p ≤ 0.01). Therefore, the reduced body weights of the F2 pups were considered to be test item-related.
Litter weight: No test item-related difference for the litter weight was noted between the control group and the low and intermediate dose group (65 or 200 mg test item/kg b.w./day).
Due to the decreased body weight of the pups of the high dose group (600 mg test item/kg b.w./day), also the weight of the high dose litters was decreased before culling (12.1% or 13.6% below the value of the control group for the male and female litters combined on LD 1 or LD4, both statistically significant at p ≤ 0.05). Decreased litter weights in the high dose group were also noted after culling on LD 14 and LD 21 (11.6% or 11.8% below the value of the control group for the male and female litters combined, both statistically significant at p ≤ 0.01).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F2 pups: Thyroid hormone determination: T4 level: No test item-related differences were noted for the serum T4 levels of the pups at sacrifice between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Decreased T4 levels were noted for the pups of the intermediate dose group. However, as no dose response relationship was present, the decreased serum T4 levels for the intermediate dose group were considered to be not test item-related.
TSH level: No test item-related differences were noted for the serum TSH levels of the pups at sacrifice between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F2 pups: No test item-related differences for the ano-genital distance on LD 4 were noted between the pups of the control group and the pups of the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F2 pups: No test item-related influence was noted on the nipple retention on PND 13 for the male pups of the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F2 pups: No test item-related differences were noted for the examined organ weights of the pups in the F2 Generation between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Gross pathological findings:
no effects observed
Description (incidence and severity):
F2 pups: No pathological changes were noted for pups that were found dead during the lactation period and also no pathological changes were noted during necropsy of the pups in any dose group (65, 200 or 600 mg test item/kg b.w./day).
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
F2 pups: Pre-and postnatal development: Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly higher value was noted for the post-implantation loss in the high dose group (12.5% for the high dose group compared to 5.8% in the control group) leading to a lower birth index (87.5% in the high dose group in comparison to 94.9% in the control group). However, as no statistically significant difference and no dose response-relationship were present and also no influence on the post-implantation loss was noted for the F0 Generation, the slightly increased post-implantation loss was considered to be not test item-related.
Male to female ratio of the pups: No test item-related influence on the ratio of male to female pups was observed for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Prenatal development (conceptus to birth)
Generation:
F2
Effect level:
>= 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Postnatal development (pup)
Generation:
F2
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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

The aim of the study was to evaluate the effects of the test item at dose levels of 65, 200 or 600 mg/kg b.w./day on the reproductive toxicity of the F0 Parental Generation and the F1 Cohort 1B as offsprings as well as F1 Parental Generation. Further, general toxicity and developmental toxicity of the F1 and F2 Generation from weaning until adulthood (OECD 443) including effects on neurological development were evaluated.
Triggered by findings in a screening assay(Pubertal Development assay, Marty et.al, 2011), particular focus was put on the analysis of reproductive organs and tissues as well as the pubertal development of male rats, i.e. seminal vesicle weight, serum level of testosterone and preputial separation as marker for sexual maturation. Due to the extended treatment period, including the treatment between PND 23-53, this higher tier study especially aimed to investigate the concern in relation to reproductive/developmental toxicity in juvenile rats in more detail.


 


Maximum tolerated dose
Based on the findings in the OECD 421 study, doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death.
Taking all study results into account and considering the extended exposure time of the F0 and F1 Generation within the OECD 443 study (compared to the short-term exposure time in OECD 421), 600 mg/kg b.w. per day were considered the maximum tolerated dose for the F0 Generation of the OECD 443 study, with no systemic effects expected in the F1 Generation. Furthermore, 600 mg/kg b.w. per day have been selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).

General and reproductive toxicity
General toxicity (F0 Generation and Cohorts 1A, 1B and 2A)
No test item-related mortality was noted for any of the dose groups. Test item-related but non-adverse salivation was noted for all animals of all dose groups (65, 200 or 600 mg test item-related/kg b.w./day).
A test item related and adverse decreased body weight was noted for the males of the F0 Generation and the F1 Cohort 1A and 1B dosed with 600 mg test item/kg b.w./day) as well as the males of the F1 Cohort 2A dosed with 200 or 600 mg test item/kg b.w./day. No test item-related changes in body weight were noted for the female animals.
No influence was noted on food consumption, serum T4 and TSH levels, haematological parameters and the ratios of the lymphocyte sets in the spleen. Additionally, no test-item related influence was noted on the serum testosterone levels. Thus, the finding in the Pubertal Development assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.


The evaluation of the clinical biochemical parameters revealed increased serum levels of globulin, total cholesterol and total protein for the male and female high dose animals of the F0 Generation and for the male and female intermediate and high dose animals of the F1 Cohort 1A (200 or 600 mg test item/kg b.w./day).
During urinalysis, a lower pH was observed for the male animals of the intermediate and high dose group of the F0 Generation as well as the female and male animals of the F1 Cohort 1A.
For the intermediate and high dose females of the F0 Generation (200 or 600 mg test item/kg b.w./day), macroscopic findings were noted during necropsy, i.e. enlarged adrenals. This observation was in accordance with the increased absolute and relative adrenal glands weight of the female as well as male animals of the intermediate and/or high dose group of the F0 Generation and the F1 Cohorts 1A and 1B (200 or 600 mg test item/kg b.w./day).
Furthermore, increased weights were noted for the kidneys and livers of both sexes of the intermediate and/or high dose group of the F0 Generation and the F1 Cohorts 1A . Additionally, a decreased heart weight was noted for the high dose males of Cohort 1A. These effects were considered as test-item related. However, a treatment-related reduced weight of seminal vesicles in juvenile rats as observed in the Pubertal Development assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.
The histopathological evaluation revealed test-item related changes in the liver (at 65 and /or 200 and 600 mg test item/kg b.w./day), the adrenal glands and the thyroid glands (at 200 and 600 mg test item/kg b.w./day) of both sexes of the F0 Generation and the F1 Cohort 1A. Additionally, the treatment-related finding in the liver was also observed in both sexes (at 600 mg test item/kg b.w./day) from F1 Cohort 2A. Furthermore, histopathological changes were detected in the kidneys of males from F0 Generation and F1 Cohort 1A at 65 and/or 200 and 600 mg test item/kg b.w./day.
The microscopic finding in the liver tissue, i.e. centrilobular hepatocellular hypertrophy (observed with dose-dependent increases in incidence and/or severity) was considered to have contributed to the increased liver weights in each generation and cohort. Meanwhile, in any generations or cohorts, there were no further indicators of liver injury.
Therefore, for the duration of the dose administered under the circumstances of this study, this change was considered to be of metabolic nature and of adaptive character like an enzyme induction, and not adverse.
However, when liver responses exceed adaptive changes, e.g., by chronic exposure, toxicity and hepatocarcinogenicity occur (Hall et al., 2012) as observed in male mice after 2 years exposure with 500 mg test item/kg b.w./day in the carcinogenicity study (NTP, 1986).
Taking the observed effects of previous studies into account, i.e., in NTP, 1986 and Marty, et al., 2011, the findings in the liver are considered potentially adverse because it is assumed that longer or higher dosages would eventually lead to a failure of adaptive mechanism.
The microscopic finding in the kidneys, i.e. the enhanced hyaline droplet accumulation observed in the proximal tubular cells was treatment-related. The change was considered to be induced by the alpha2µ-globin associated mechanism. Thus, the noted effect was considered as adverse. However, this male-rat-specific process which is driven by an overload of synthetic protein derived from hyperfunction of the liver, is assumed to be not relevant to human.
In the adrenal glands, diffusely increased fine vacuolation in the zona fasciculata was noted in the histopathological examination. These changes were considered to have contributed to increased adrenal weights in both F0 and F1 Cohort 1A generations and grossly enlarged adrenal glands that was recorded in F0 generation. Diffuse fine (microvesicular) vacuolation is a normal pattern of the adrenal glands and enhanced vacuolation was considered to be an indicative of increased cholesterol utility. In the meantime, there were no further indicators of functional impairment (e.g. increase in coarse (macrovesicular) vacuolation) and tissue injury (e.g. necrosis, inflammation) in either generations. Thus, increase in fine vacuoles with no toxicologically relevant histomorphological abnormalities was considered to be of adaptive nature and hence, deemed not to be adverse.
The microscopic finding in the thyroid glands revealed a slight increase in incidence of follicular cell hypertrophy. This was thought to be the change elicited as a secondary consequence of hepatic enzyme induction (Gopinath C, et al., 2014) and hence, deemed not to be adverse.


No test item-related changes were noted for the reproductive organs of the male and female animals. Especially, no abnormalities were found in the seminal vesicles of male animals. Thus, the finding in Marty et al. 2011, was not established in this higher tier study with an extended treatment period.
Additionally, no lesions were noted during the neurohistomorphological examinations of the Cohorts 2A and 2B.


 


Reproductive toxicity (F0 Generation and Cohort 1B)
No test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).
The test item did not affect the prenatal development of the pups (number of resorptions, stillbirths and live born pups).
A reduction was noted for the body weight of the male and female high dose pups (600 mg test item/kg b.w./day) of F0 and Cohort 1B and consequently, the weights of the high dose litters. The distinctly and constantly reduced pup body weights were considered to be test item-relate because the means of the male and female litters per dam combined were partly (i.e. on LD 14 and LD 21) outside the 5% to 95% confidence interval of the Provivo background data.


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


 


Developmental toxicity (F1 - Cohorts 1A, 1B and 2A)
During their post-weaning development no test item-related prematurely deceased animals were noted in any dose group.
The adult F1 animals of all dose groups displayed non-adverse post-dosing salivation and the body weight was decreased for the high dose males of the Cohorts 1A and 1B, as well for the intermediate and high dose males and the high dose females of Cohort 2A. No influence was noted on the food consumption, on the haematological parameters investigated and on the serum levels of the thyroid hormones T4 and TSH of the male and female animals of the dose groups. Furthermore, he serum testosterone levels at PND 53 of the males was not changed. Regarding the biochemical parameters and the urinalysis, reductions of the serum levels of globulin, total cholesterol and total protein and a lower pH of the urine were observed for the male and female intermediate and high dose animals of Cohort 1A. Macroscopic post mortem examination revealed no test item-related pathological changes.
Increased organ weights were noted for the adrenal glands (high dose animals of Cohort 1A, high dose males and intermediate and high dose females of Cohort 1B), for the kidneys (intermediate and high dose animals of Cohort 1A) and for the liver (intermediate and high dose animals of Cohort 1A and high dose males and intermediate and high dose females of Cohort 2A). Additionally, a decreased absolute heart weight was noted for the male high dose animals of Cohort 1A. Histopathological examinations of the reproduction organs of the high dose animals of Cohort 1A revealed no pathological changes.
For the sexual maturation, the time point of balano-preputial separation of the male animals did not differ between the control group and the dose groups in the Cohorts 1A, 1B and 2A. Thus, the finding in the Pubertal Development Assay (Marty et al., 2011) was not established in this higher tier study with an extended treatment period.
A statistically significant delay for the time point of vaginal opening for the female animals was noted for the high dose group (600 mg test item/kg b.w./day) when comparing all F1 females of the Cohorts 1A, 1B and 2A combined with the control group. However, no statistically significant differences between the control group and the dose groups were observed for the day of vaginal opening for the Cohort 1A females and the Cohort 2A females, respectively when the Cohorts were considered separately.
Additionally, the mean body weight of the juvenile rats at the time point of vaginal opening was also reduced (not significant) in all Cohorts (except of Cohort 1B) as well as when comparing Cohort 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment indicating that there is no delay in sexual maturation. Consequently, this finding was considered as not adverse.


 


Neurotoxicity (F1 - Cohorts 2A and 2B)
The neurological screening of the animals of Cohort 2A between PND 58 and 67 revealed no test item-related differences between the dose groups and the control group. Also, the examination of the brain weights on PND21/22 (Cohort 2B) or between PND77 to 81 (Cohort 2A) showed no test item-related difference between the control group and the dose groups.


 


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


F0 Generation:


General toxicity


NOAEL                                             65 mg test item/kg b.w./day


Based on the reduced body weight, increased organ weights, histopathological changes as well as the biochemical and urine changes in the high and/or intermediate dose group of the F0 animals.


 


Reproductive toxicity


NOAEL                                            600 mg test item/kg b.w./day


 


F1 Generation:


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


NOAEL                              65 mg test item/kg b.w./day


Based on the reduced body weight, increased organ weights, histopathological changes, as well as the biochemical and urine changes in the high and/or intermediate dose group of F1 Cohort 1A , 1B and 2A animals.


 


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


Adverse effects on pre- and postnatal development



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


NOAEL                 600 mg test item/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                200 mg test item/kg b.w./day


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


Neurological developmental toxicity (Cohorts 2A and 2B)


NOAEL                      600 mg test item/kg b.w./day


 


 


Reproductive toxicity


NOAEL                        600 mg test item/kg b.w./day


F2 Generation


Developmental toxicity


Adverse effects on pre- and postnatal development



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


NOAEL                600 mg test item/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                200 mg test item/kg b.w./day


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