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Diss Factsheets

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:
26.02.2020 to 15.03.2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to the ECHA final decision on a compliance check from 2018-12-21 (Decision number CCH-D-2114453561-52-01/F) the study design of this EOGRTS according to OECD 443 is as follows:

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

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

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:
2022

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


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

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

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

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

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

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

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

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

DURATION OF STUDY:
- at least 5 adaptation days of the F0 generation
- F0 (parental generation):
2 weeks pre-exposure
2 weeks pre-mating
2 weeks mating
3 weeks pregnancy
3 weeks lactation
- F1 Cohort 1A: approx. 10 weeks
- F1 Cohort 1B: approx. 10 weeks
2 weeks mating for establishing of F2 3 weeks pregnancy
3 weeks lactation
- F2 Generation: lactation until PND 21
- F1 Cohort 2A: approx. 10 weeks
- F1 Cohort 2B: lactation until PND 21/22
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 that results in a greater metabolism rate of testosterone. The thus reduced testosterone level consequently leads to a subsequent delay in the maximum luteinizing hormone (LH) release and attainment of sufficient testosterone levels to drive puberty onset and accessory sex tissue size, e.g. seminal vesicles (Marty et al., 2018). It is assumed that prepubertal animals are particularly sensitive to this mode of action because opposite to adult males, they possess a positive feedback loop where higher levels of testosterone are stimulatory, potentiating the pituitary’s response to GnRH (Gonadotropin releasing-hormone), resulting in maximum LH release that further increases testosterone secretion (Marty et al., 2018, Stoker et al., 2000).
In the OECD 421 study, female animals treated with 1000 mg/kg b.w. had to be sacrificed after the first dosing and male animals after the third dosing due to animal welfare reasons. Two female animals dosed with 750 mg/kg b.w. were found dead on GD 20 and GD 21. Both male and female animals treated with 750 mg/kg b.w. showed slight to severe salivation, slightly to moderately reduced motility and yellow discoloured urine. Furthermore, a reduction in body weight was noted for the male animals during dosing period, while female animals showed a reduced body weight between GD 14 and GD 20. Accordingly, also the body weight gain was reduced for male and female animals during the respective periods. Reduced body weights were also noted at necropsy for both male and female animals. Therefore, the results of the above-mentioned historical studies could not completely be reproduced under the test conditions of the DRF OECD 421 study (LPT Study No. 37311, 2020).
For the pups of animals treated with 750 mg/kg b.w. reduced weights were noted for male and female animals on lactation days 1, 4 and 13.
Male animals treated with 300 mg/kg b.w. showed slight to moderate salivation, a slightly reduced motility and yellow discoloured urine, while slight salivation was observed in females treated with 300 mg/kg b.w.
Based on these findings, doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death. Taking all study results into account and considering the extended exposure time of the F0 and F1 Generation within the OECD 443 study, 600 mg/kg b.w. per day were considered the maximum tolerated dose for the F0 Generation of the OECD 443 study, with no systemic effects expected in the F1 Generation. Furthermore, 600 mg/kg b.w. per day were selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).

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

Examinations

Parental animals: Observations and examinations:
In order to derive the NOAELs the following results from the F0 generation + F1 generation Cohorts 1A, 1B and 2A were used. For an easier understanding these results are included under "parental animals" even though only the F0 and F1 Cohort 1B animals were used to establish a following generation.
CLINICAL SIGNS:
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

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

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

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

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

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

For male and female reproductive organs, no test item-related changes were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Bone marrow: Males and females: No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group of both sexes.

Reproductive function / performance (P0)

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

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

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

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

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

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

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

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

Details on results (P0)

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

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

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:
Males
No test item-related changes of behaviour, external appearance or the faeces that were considered to be adverse were noted for the male animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Transient changes in behaviour or external appearance were noted for the control group and for the high dose group. In detail, the male control animal no. 367 was observed with a hemorrhagic nose/snout on TD 108. Furthermore, in the high dose group, three male animals (nos. 483, 488 and 494) displayed reduced motility on TD 15 and animal no. 494 additionally, showed an increased water consumption on TD 105. The high dose animal no. 498 was noted with breathing sounds and gasping on TD 90 and with piloerection on TD 94 and 95. Due to the low number of observations, the above-mentioned changes were considered to be not test item-related.
In addition to these single occurrences, also in Cohort 1B salivation was noted in all male animals of any dose group with a dose-dependent increase in severity and number of days with salivation per animal. For two animals each of the intermediate dose group (nos. 446 and 456) and the high dose group (nos. 491 and 492) the saliva was occasionally red discoloured. However, as salivation for the male animals of any dose group was noted in almost all cases not longer than 60 minutes post administration, salivation was considered to be test item-related but not adverse.
Females:
No test item-related observations considered to be adverse were noted for the female animals of any dose group (65, 200 or 600 mg test item/kg b.w./day).
Single occurrences of reduced motility were noted during the pre-mating/mating period for 4 of 20 high dose group females and in the lactation period for 2 of 19 females for one or two days per animal.
Salivation was noted for 5 of 20 control females during the pre-mating/mating period for one or two test days per animal. In the low dose group, salivation was observed for all animals in the pre-mating/mating period and for 7 or 10 of 18 animals in the gestation and lactation period. Furthermore, all female animals of the intermediate and high dose group displayed salivation in all study periods (see Text Table 9 2 on the following page). However, as also for the female animals the salivation was noted for not longer than 60 minutes post administration, the test item-related salivation was considered to be not adverse.
Start and duration of observations
With the exception of two observations of salivation for male animals that were noted before start of application, all observations were noted immediately after application or within 5 minutes after application. The duration of salivation was for one male animal consistently during the day. For one male animal, salivation ended one to two hours and for another animal 6 to 24 hours after application. All other observations of salivation ended 5 to 20 minutes or 20 to 60 minutes after application for the male and female animals. The observation of gasping for one male animal appeared immediately to 5 minutes after administration and disappeared 6 to 24 hours after administration. Reduced motility was noted for the male and female animals from immediately to 5 minutes after application until 5 to 20 minutes or 20 to 60 minutes after application.
Detailed clinical observations
The detailed clinical observations were performed once weekly.
Males:
No observations were noted during the detailed clinical observations for the male animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Females:
No observations were noted during the detailed clinical observations for the female animals of the low dose group (65 mg test item/kg b.w./day).
In the control group and in the intermediate dose group (200 mg test item/kg b.w./day) the female animals nos. 396 and 466 were noted with a loss of the tail tip from test week 1 or test week 9 onwards, respectively. In addition, the female animal no. 514 of the high dose group (600 mg test item/kg b.w./day) was noted with a wound in the neck region (approximately 4 mm in diameter) in test week 9. The single occurrences of a missing tail tip and a wound in the neck region noted in the intermediate or high dose groups are considered to be spontaneous.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

COHORT 2A and 2B: Neurohistopathology: The histopathological evaluation of the brains was performed for the male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day) from PND77 to 81 (Cohort 2A) or from PND21 to 22 (Cohort 2B).
The image analysis and the histomorphometry of the brains of the male and female Cohort 2A and 2B animals revealed no test item-related differences between the control animals and the animals of the high dose group.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
COHORT 1A: Lymphocyte typing in spleen: Males and females: No statistically significant differences were noted in the proportion of the examined lymphocyte subtypes in the male and female animals between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Bone marrow: Males and females: No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (600 mg test item/kg b.w./day) of both sexes.

Reproductive function / 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. For females nos. 478 (group 3) and 520 (group 4), the presence of sperm was not noticed, hence these animals were pregnant and littered without a verified copulation.
However, these numbers are within the range of normal biological variability.

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

Pre-coital time and gestation length: No test item-related influence was noted on the pre-coital time and for the gestation length for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly and not statistically significantly shorter pre-coital time was noted for the females of the high dose group (2.1 days in the high dose group compared to 3.0 days in the control group, not statistically significant). However, as no dose response-relationship was present, the slightly shorter pre-coital time was considered spontaneous.

Details on results (P1)

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

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

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

COHORT 2A and COHORT 2B
This part of Study 37312 reports the influence of the test item 3,5,5-trimethylcyclohex-2-enone on the neurological development of the male and female animals in the adulthood (Cohort 2A) and in the early lifetime (Cohort 2B).
General toxicity
No test item-related premature deaths were noted for the male and female animals of Cohort 2A and Cohort 2B.
Test item-related but non-adverse salivation was noted for the male and female Cohort 2A animals of all dose groups.
A reduction in body weight, body weight gain and consequently in the body weight at autopsy was noted for the male animals of the intermediate and high dose groups and for the female animals of the high dose group of Cohort 2A.
In Cohort 2A, no test item-related differences were noted for the food consumption.
No test item-related pathological changes were observed in any dose group of Cohorts 2A and 2B.
Increased liver weights were noted for the male animals of the high dose group and the female animals of the intermediate and high dose groups of Cohort 2A.
No test item-related difference was noted for the brain weights of male and female animals of Cohort 2A and Cohort 2B.
The histopathological examinations revealed test item-related changes in the liver in form of centrilobular hepatocellular hypertrophy in the male and female animals of the high dose group (600 mg test item/kg b.w./day) of Cohort 2A. As this effect indicates towards changes previously reported in carcinogenicity studies, this finding was considered to be adverse.

Effect levels (P1)

open allclose all
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 (dam no. 39) and the high dose group (dam no. 192), and for all remaining pups of two litters of the low dose group (dams nos. 77 and 91). As also one litter of the control group was noted with thin fur and no dose-response relationship was present, the observations of thin fur were considered to be not test item-related.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 pups: Viability index - Pre-and post-cull period: No test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
In the high dose group, a slightly increased number of prematurely deceased pups in comparison to the control group was noted: In the pre-cull period, 12 prematurely deceased pups in 22 litters were noted for the high dose group compared to 5 prematurely deceased pups in 23 litters in the control group. In the post-cull period, 8 prematurely deceased pups in 22 litters were noted for the high dose group whereas in the control group, there were 3 deceased pups in 23 litters. However, the resulting values for the viability indices of the high dose group for the pre-cull period (96.6%) and the post-cull period (96.4%) were only slightly below the values of the control group (98.6% or 98.7%). Therefore, the slightly increased numbers of prematurely deceased pups in the pre- and post-cull period were considered to be not test item-related but to be within the normal variability.

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

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

COHORT 2A: Males:
No influence was noted on the time point of the balano-preputial separation of the male animals of the dose groups (65, 200 or 600 mg test item/kg b.w./day). Thus, the effect observed in Marty et al., 2011, i.e. a delayed balano-preputial separation, was not established.
A slightly lower body weight was noted for the body weight of the high dose males at balano-preputial separation. However, this was due to the test item-related reduction of the body weight already during lactation period.
Females:
No statistically significant differences were noted for the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear, and the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear for Cohort 2A females of all dose groups (65, 200 or 600 mg test item/kg b.w./day).
No influence was noted on the time point of vaginal opening of the female animals of the low and intermediate dose groups (65 or 200 mg test item/kg b.w./day).
For the high dose females (600 mg test item/kg b.w./day), a slight but statistically not significant delay was observed for the day of vaginal opening (8.1% above the value of the control group). This was also noted for the females of Cohort 1A and Cohort 1B, leading to a statistically significantly delayed day of vaginal opening for the high dose group (600 mg test item/kg b.w./day) when Cohort 1A, 1B and 2A females of appropriate age were included in the comparison. However, the mean body weights of the juvenile rats at the time point of vaginal opening were also reduced (not statistically significant) in Cohort 2A and when comparing Cohorts 1A, 1B and 2A combined with the control. Furthermore, the time between the day of vaginal opening and the day of the appearance of cornified cells was not affected by the treatment. Taking all findings together, the delay in vaginal opening for the F1 females of the high dose group was considered to be test item related but not adverse.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 pups: No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 pups: No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the low dose group (65 mg test item/kg b.w./day).
In the intermediate and high dose group (200 or 600 mg test item/kg b.w./day), a statistically significant increase was noted for the number of dams with nipple-bearing male pups and also for the number of nipples per pup. In detail, 0.9 male pups with nipples per dam each were noted in the intermediate or high dose group in comparison to 0.2 pups per dam in the control group (p ≤ 0.05). Furthermore, the number of nipples per pup was increased for the intermediate and high dose group (0.44 or 0.46 nipples per pup) when compared to the male pups of the control group (0.11 nipples per pup, statistically significant at p ≤ 0.05). The increase of these two parameters also led to an increase in the number of nipples per dam (2.2 for the intermediate or high dose group compared to 0.6 nipples on male pups per dam of the control group, statistically significant at p ≤ 0.05). However, the toxicological relevance of the increased number of pups with nipple retention for the intermediate and the high dose group is unclear but limited by the following facts:
1) the finding is isolated as no influence was noted on a related endpoint (e.g. ano-genital distance)
2) no dose response relationship was noted for the number of male pups with nipples per dam and for the number of nipples per dam
3) no influence was noted on the male pups of the F2 Generation of any dose group
4) for all calculated mean values, the standard deviation is higher than the mean value, indicating large variety
5) the investigation of this end point within this study type was only introduced in 2015, so that the data base of historical control data is limited as well as the interpretation of the described effects.
Based on the above mentioned considerations, the observation was considered to be spontaneous and no test item related.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F1 pups: No statistically significant differences for the examined absolute organ weights were noted for the F1 pups between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Gross pathological findings:
no effects observed
Description (incidence and severity):
F1 pups: Macroscopic internal and/or external examinations were performed on animals found dead during the lactation period, at litter adjustment on LD 4 or at termination of the lactation period between LD 22 and 24. During these macroscopic post mortem examinations, no gross abnormalities (e.g. malformations or variations) were noted for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
For the control pup no. 35-11 necropsy after terminal sacrifice on LD 22 revealed inflated stomach and intestines, a thymus being reduced in size and a reddened brain. These single occurrences for one control pup were considered to be spontaneous.
Histopathological findings:
no effects observed
Description (incidence and severity):
Neurohistopathology - Cohort 2A and Cohort 2B: The histopathological evaluation of the brains was performed for the male and female animals of the control group and the high dose group (600 mg test item/kg b.w./day) from PND77 to 81 (Cohort 2A) or from PND21 to 22 (Cohort 2B).
The image analysis and the histomorphometry of the brains of the male and female Cohort 2A and 2B animals revealed no test item-related differences between the control animals and the animals of the high dose group.
Other effects:
no effects observed
Description (incidence and severity):
F1 pups: Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Male to female ratio of the pups: No test item-related influence on the male to female ratio was noted for all dose groups (65, 200 or 600 mg test item/kg b.w./day).

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurological screening - Cohort 2A:
Auditory startle response: Males and females
No test item-related difference was noted between the male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) for their auditory startle response.
When the last block of trials for a group was compared to the respective first one, the male and female control animals as well as the animals of the dose groups showed a slight habituation between the blocks.
Observational screening - Cohort 2A: Males and females
No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faces were noted for the male and female animals of all treatment groups (65, 200 or 600 mg test item/kg b.w./day). Furthermore, no test item-related differences were noted with regards to the body temperature.
A statistically significantly increased hind-leg splay was noted for the male and female animals at the intermediate and/or high dose level. However, all individual values of the male animals were within the test institute´s background range, while for the female animals three values (animals no. 539 (control group) as well as nos. 551 and 556 (group 2)) were lower than the historical background data. Additionally, the mean values of the control group were rather low compared to the historical background data for both sexes. Hence, the changes in the hind-leg splay were considered to be spontaneous.
Functional tests - Cohort 2A: Males and females
Grip strength
No test item-related differences between the male and female animals of the control group and those of the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the grip strength of the fore- and hind-limbs.
A statistically significantly increased grip strength was noted for the fore-limbs of the male animals of the low and high dose groups (37.0% or 32.8% above the value of the control group, respectively; statistically significant at p ≤ 0.01). However, as no dose response-relationship was present and no differences were noted for the grip strength of the hind-limbs, the increased fore-limb grip strength for the male low and high dose animals was considered to be not test item-related.
Spontaneous motility: No statistically significant differences between the male and female animals of the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day) were noted for the spontaneous motility.

Developmental immunotoxicity (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 group were considered to be spontaneous.
The examination of the thyroid hormone levels on lactation days 4 and 22 revealed no test item-related differences.
The gross inspection (internal and/or external) of the pups at necropsy revealed no changes for the pups for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).
No test item-related influence was noted on the organ weights from pups sacrificed after weaning.

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

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

Neurological development (Cohort 2A and 2B)
During neurological screening no test item-related influences were noted on the male and female Cohort 2A animals of any dose group.
No test item-related differences between the control groups and the high dose group animals were noted during neurohistopathological examination of Cohorts 2A and 2B.

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.
Runts: One runt was noted for the low dose group (65 mg test item/kg b.w./day). The single occurrence of one runt was regarded to be spontaneous.
Litter weight: No test item-related difference for the litter weight was noted between the control group and the low and intermediate dose group (65 or 200 mg test item/kg b.w./day).
Due to the decreased body weight of the pups of the high dose group (600 mg test item/kg b.w./day), also the weight of the high dose litters was decreased before culling (12.1% or 13.6% below the value of the control group for the male and female litters combined on LD 1 or LD 4, both statistically significant at p ≤ 0.05). Decreased litter weights in the high dose group were also noted after culling on LD 14 and LD 21 (11.6% or 11.8% below the value of the control group for the male and female litters combined, both statistically significant at p ≤ 0.01).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F2 pups: Thyroid hormone determination: T4 level: No test item-related differences were noted for the serum T4 levels of the pups at sacrifice between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Decreased T4 levels were noted for the pups of the intermediate dose group. However, as no dose response relationship was present, the decreased serum T4 levels for the intermediate dose group were considered to be not test item-related.
TSH level: No test item-related differences were noted for the serum TSH levels of the pups at sacrifice between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F2 pups: No test item-related differences for the ano-genital distance on LD 4 were noted between the pups of the control group and the pups of the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F2 pups: No test item-related influence was noted on the nipple retention on PND 13 for the male pups of the dose groups (65, 200 or 600 mg test item/kg b.w./day ).
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
F2 pups: No test item-related differences were noted for the examined organ weights of the pups in the F2 Generation between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Decreased spleen weights were noted for the male and female pups of the high dose group (24.2 % or 24.7 % below the control, statistically not signiifcant or p ≤ 0.05).
The reduced spleen weight was due to the reduced pup body weight at autopsy and the reduced live body weight on PND 21 and not due to an adverse effect of the test item on the spleen.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F2 pups: No pathological changes were noted for pups that were found dead during the lactation period and also no pathological changes were noted during necropsy of the pups in any dose group (65, 200 or 600 mg test item/kg b.w./day).
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
F2 pups: Pre-and postnatal development: Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups (65, 200 or 600 mg test item/kg b.w./day).
A slightly higher value was noted for the post-implantation loss in the high dose group (12.5% for the high dose group compared to 5.8% in the control group) leading to a lower birth index (87.5% in the high dose group in comparison to 94.9% in the control group). However, as no statistically significant difference and no dose response-relationship were present and also no influence on the post-implantation loss was noted for the F0 Generation, the slightly increased post-implantation loss was considered to be not test item-related.
Male to female ratio of the pups: No test item-related influence on the ratio of male to female pups was observed for any of the dose groups (65, 200 or 600 mg test item/kg b.w./day).

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

F2 pups – pre- and postnatal development
Also, no test item-related difference was noted for the pre-natal development (numbers of implantation sites, resorptions, post-implantation loss, pups born).
For the post-natal pup development, a reduction was noted on the body weight of the F2 pups and consequently on the weight of the litters of the high dosed animals (600 mg test item/kg b.w./day). No test item-related differences between the control group and the dose groups were noted for the viability of the pups, the ano-genital distance, the numbers of nipples of the male pups and for the T4 and TSH levels.

F2 pups – examination at necropsy
Necropsy of the pups on LD 4 or LD 21 revealed no pathologic changes and no test item-related differences for the organ weights of the F2 pups.

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:

Conclusion


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


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


 


Maximum tolerated dose


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


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


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


 


General and reproductive toxicity


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


 


Reproductive toxicity (F0 Generation and Cohort 1B)


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


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


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


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


 


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


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


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


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


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


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


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


 


Neurotoxicity (F1 - Cohorts 2A and 2B)


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


 


Overall Conclusion


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


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


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


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


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


 


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


F0 Generation:


General toxicity


NOAEL                                       65 mg/kg b.w./day


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


 


Reproductive toxicity


NOAEL                                     600 mg/kg b.w./day


 


F1 Generation:


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


NOAEL                                       65 mg/kg b.w./day


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


 


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


Adverse effects on pre- and postnatal development



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


NOAEL                         600 mg/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                         200 mg/kg b.w./day


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


Neurological developmental toxicity (Cohorts 2A and 2B)


NOAEL                                     600 mg/kg b.w./day


 


Reproductive toxicity


NOAEL                                     600 mg/kg b.w./day


 


F2 Generation:


Developmental toxicity


Adverse effects on pre- and postnatal development



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


NOAEL                         600 mg/kg b.w./day



  1. Adverse effects on postnatal development (pup)


NOAEL                         200 mg/kg b.w./day


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