Decahydronaphthalene

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase from 08.07.2020 to 23.03.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In a final decision on a testing proposal dated 12 December 2018 (Decision number TPE-D-2114453345-50-01/F) ECHA requested to perform an extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten 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); and
-  Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.

Furthermore, as there is only limited relevant data to be used for dose-level setting, a dose range-finding study according to OECD 421 was performed.
In the OECD 421 study the test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.
The NOAEL for general toxicity was considered to be 300 mg/ kg b.w./day based on mortality, clinical signs, reduced body weight, reduced food consumption and macroscopic findings at 1000 mg /kg b.w./day. The NOAEL for reproductive toxicity is considered to be 100 mg/kg b.w./day based on reduced numbers of implantation sites and live body weight of pups at 300 mg/kg b.w./day, even though effects on the reproductive parameters of the parental females were only noted at 1000 mg/ kg b.w./day.
Based on available toxicological data and the results of an OECD 421 study in rats in the main OECD 443 study doses of 30, 100 and 300 mg/kg b.w./day by oral gavage were proposed.
However, dose levels were increased from 30 to 60 mg/kg b.w./day (low dose), from 100 to 200 mg/kg b.w./day (intermediate dose), and from 300 to 600 mg/kg b.w./day (high dose) as of test day 30 due to lack of any toxicity observed at the high dose.

The requested EOGRTS according to OECD 443 started as scheduled on 08 July 2020. An increased number of non-pregnant females in the F0 Generation and a similar finding in the previously finished OECD 421 dose range finding study were observed.
In order to clarify the possible effects on reproductive performance, the contributing study director recommended the extension of the study to include the mating of the F1 animals to yield a further F2 generation:

Cohort 1B animals were maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures were similar to those for the parental animals (F0 generation); sibling mating was avoided.
Reproductive performance of F1 animals will be assessed accordingly and animals of the F2 generation will be assessed in the same way as F1 animals. Furthermore, as the findings of the preliminary histopathological examination of the F0 females indicated issues in the vagina/uterus and ovaries, the LTH, FSH and LH hormone levels in F1 dams were be determined to clarify the effect of the test item on reproductive performance. In particular, the additional hormone determinations make it possible to show that the findings are only a rat-specific effect (change LTH (prolactin)) that has no relevance to humans.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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:

According to the ECHA final decision on a testing proposal from 2018-12-12 (Decision number TPE-D-2114453345-50-01/F) the study design of this EOGRTS according to OECD 443 is as follows:

Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity); and
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.

Alteration: Based on data generated with the F0 generation and to clarify possible effects on the reproduction, this study will be extended to include F2 generation pups and additional blood sampling for hormone level determinations.

The aim of the study is to evaluate the pre- and postnatal effects of Decahydronaphthalene on development as well as systemic toxicity in pregnant and lactating females and young and adult offspring.

Test material

Test material information

1

Reference substance name:

Decahydronaphthalene

EC Number:

202-046-9

EC Name:

Decahydronaphthalene

Cas Number:

91-17-8

Molecular formula:

C10H18

IUPAC Name:

decahydronaphthalene

Test material form:

liquid

Details on test material:

decahydronaphthalene from Evonik, Batch: 20040915

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 start of dosing: 56 days
- Number of parental animals:
Pre-exposure period: At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study: 192 animals (96 males and 96 females)
A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.
- Weight at study initiation: Males: 37.8 g to 446.1 g, Females: 192.1 g to 317.0 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 at a room temperature of 22°C ± 3 °C (maximum range) and a relative humidity of 55% ± 10% (maximum range).
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages are cleaned and changed once a week.
The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
- Diet: ssniff® R/M-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest), ad libitum with the exception of the night before the day of blood withdrawal for laboratory examinations
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C
- Humidity (%): 55% ± 10%
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle
IN-LIFE DATES: From: 08.07.2020 To: 23.03.2021

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

- Route of administration: Oral, via gavage
- Frequency of administration: Once daily
- Vehicle: Corn Oil
Administration volume: 2 mL/kg b.w./ day
Dosages: 0, 30, 100, 300, from test day 30 increased to 0, 60, 200, 600 mg/kg bw/day
Selection of route of administration: According to international guidelines.

The test item was administered orally at a constant administration volume per kilogram body weight once daily.
The amount of test item to be administered was daily adjusted to each animal's current body weight.
The control animals received the vehicle at the same administration volume daily in the same way.
The homogeneity and concentration of the test item formulations was monitored.

Details on mating procedure:

Sexually mature male and female rats of the F0 Generation were randomly paired for mating. Mating was monogamous: One male and one 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 its male partner after 2 weeks without further opportunity for mating.
No deaths of male animals were noted before pairing. Therefore, it was not necessary to pair a male animal that had already mated before with a second female to ensure that all females had been paired.

Establishment of F2 generation using Cohort 1B (investigation of potential reproductive toxicity):
Due to a high number of non-pregnant females in groups 3 and 4 of the F0 generation, a preliminary histopathological examination of the reproductive organs of the F0 animals was performed. As the findings of this examination indicated changes in the vagina/uterus and ovaries, the contributing pathologist recommended the extension of the study to include the mating of the F1 animals to yield a further generation and clarify the possible effects on reproductive performance.
Cohort 1B animals were maintained on treatment beyond PND 90 and bred to obtain the F2 generation. The mating procedures were similar to those for the parental animals. Mating of siblings was avoided as far as possible. The animals of the F2 generation were assessed in the same way as the F1 animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item formulations, two (2) aliquots per sample of approx. 3 mL each were taken as scheduled and stored at -20°C ±2°C until analysis.

Formulation samples of the F0 generation - Groups 2 to 4:
Sampling time/ Parameter/ Sampling per group/ Samples (aliquots)
- At start of the treatment period of the F0 animals (1st dosing day - test day 1)/ Concentration
and homogeneity / At start of administration, During (middle) administration, Before administration to the last animal/ 3 (6)
- At dose level change for groups 2, 3 and 4 (test day 30)/ Concentration and homogeneity/ At start of administration, During (middle) administration, Before administration to the last animal/ 3 (6)
- At a time when most F0 females have littered (test day 111)/ Concentration/ During treatment always before administration to the last animal/ 3 (6)
- Near the end of the F0 dosing period at a time when the majority of animals is dosed (test day 129)/ Concentration/ During treatment always before administration to the last animal/ 3 (6)
- Total F0 samples (aliquots): 24 (48)

Formulation samples of the F1 generation - Groups 2 to 4:
Sampling time/ Parameter/ Sampling per group/ Samples (aliquots)
- At start of the treatment period of the F1 animals (1st dosing day - test day 1)/ Concentration and homogeneity/At start of administration, During (middle) administration, Before administration to the last animal/ 3 (6)
- Test day 69 (approximately in the middle of the F1 dosing period)/ Concentration/ During treatment always before administration to the last animal, Near the end of the F1 dosing period at a time when the majority of animals is dosed (test day 123)/ Concentration/ During treatment always before administration to the last animal/ 3 (6)
- Total F1 samples (aliquots): 15 (30)

The samples were labelled with the study number, species, type of sample, aliquot number, concentration, generation and cohort number (if applicable), group number, sampling time, and date. Only one aliquot was analysed. The second aliquot served as back-up. The samples were analysed at the test institute using a validated method. The analytical method was partially re-validated to include the full concentration range of the formulation samples used in this study, i.e. 15 to 300 mg/mL for the dose levels ranging from 30 to 600 mg/kg b.w.
The following parameters were determined:
- Accuracy
- Precision
- Linearity
- Sensitivity
- Stability (only for the low dose of 15 mg/mL, the stability of the range of 50 to 500 mg/mL had been validated before)
Three months after the issuance of the Final Study Report, the remaining aliquots (back-up) will be destroyed unless the Sponsor requests otherwise.

Duration of treatment / exposure:

Treatment schedule
F0 ANIMALS:
MALES: 10 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: 10 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).
F1 ANIMALS: Until weaning, the F1 animals were indirectly exposed to the test item through the breast milk. After weaning, dosing was continued in the same way as for the parental generation.
COHORT 1A: Until a dosing period of 10 weeks had been completed (up to and including the day before sacrifice, PND 87 to PND 96).
COHORT 1B: Until a dosing period of at least 11 weeks had been completed (up to and including the day before sacrifice, PND 92 to PND 98).
F2 ANIMALS: Until sacrifice at PND 22, the F2 animals were indirectly exposed to the test item through the breast milk.

Frequency of treatment:

once daily

Details on study schedule:

PARENTAL ANIMALS, PRE-EXPOSURE:
At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
MAIN STUDY:
192 animals (96 males and 96 females), a sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.

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

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

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

DURATION OF STUDY:
- At least 5 adaptation days of the F0 generation
- F0 (parental generation):
- 2 weeks pre-exposure
- 10 weeks pre-mating
- 2 weeks mating
- 3 weeks gestation
- 3 weeks lactation
- F1 - Cohort 1A: approx. 10 weeks
- F1 - Cohort 1B:
- beyond PND 90
- 2 weeks mating for establishment of F2
- 3 weeks gestation
- 3 weeks lactation
- F2 Generation: lactation until PND 22

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

low dose group (from test day 30, before 30 mg/ kg bw/ day)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

intermediate dose group (from test day 30, before 100 mg/ kg bw/ day)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

high dose group (from test day 30, before 300 mg/ kg bw/ day)

No. of animals per sex per dose:

F0 generation: 20 m/f per group
F1 generation Cohorts 1A + 1B: 20 m/f per group
F2 generation: all

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
The dose levels had been selected in agreement with the study monitor and the study director based on available toxicological data and the results of an OECD 421 study in rats. In the OECD 421 study, the test item was administered orally to rats at dose levels of 100, 300 or 1000 mg/kg b.w./day.

General toxicity:
Parental male and female animals
Three female animals dosed with 1000 mg/kg b.w./day were prematurely sacrificed for animal welfare reasons on TD 22, GD 22 or LD 14.
In the high dose group (1000 mg/kg b.w./day), changes in the behaviour in form of salivation were noted for the male and female animals. Additionally, piloerection and pale faeces were noted for the female high dose animals. The female animals that were prematurely sacrificed displayed loss of hind-limb function.
In the high dose group (1000 mg/kg b.w./day), reductions were noted for the body weight at the end of the gestation period and the beginning of the lactation period and a reduction was noted for the body weight gain in the gestation period.
At 1000 mg/kg b.w./day, a test item-related decrease was noted for the food consumption of the female animals during the pre-mating period.
No influence on the T4 levels was noted for the male animals.
Macroscopic examination during necropsy of the males of the high dose group (1000 mg/kg b.w./day) revealed a reddened stomach for 3 of 10 animals.
Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related histomorphological changes.

Reproductive toxicity:
Parental females
In the high dose group (1000 mg/kg b.w./day), an increased number of animals was noted with an elongated period of days in the diestrus stage in the pre-mating period leading to a reduced number of females with complete oestrus cycles.
No influence was noted on the fertility of the male and female animals in any of the dose groups.
Pups
Test item-related influence on the pre-natal development was noted in the intermediate and high dose group (300 or 1000 mg/kg b.w./day) in form of reduced number of implantation sites and pups born. Furthermore, an increase was observed for the post-implantation loss of the high dose group.
For the post-natal development, changes in form of a reduced body weight and accordingly in form of a reduced litter weight was noted for the pups of the high dose group (1000 mg/kg b.w./day).
No influences on the T4 levels were noted for the male and female pups on LD 13.
Hence, doses of 30, 100 and 300 mg/kg b.w./day by oral gavage were proposed for the OECD 443 study.

RATIONALE FOR DOSE LEVEL INCREASE
Dose levels of the test item were increased from 30 to 60 mg/kg b.w./day (low dose), from 100 to 200 mg/kg b.w./day (intermediate dose), and from 300 to 600 mg/kg b.w./day (high dose) as of test day 30 due to lack of any toxicity observed at the high dose.


- Due to a high number of non-pregnant females in group 3 and 4 of the F0 generation, a preliminary examination of the reproductive organs of the F0 animals was performed. As the findings of the preliminary histopathological examination of the F0 females indicated issues in the vagina/uterus and ovaries, the contributing pathologist recommended the extension of the study to include the mating of the F1 animals to yield a further generation and clarify the possible effects on reproductive performance.

- Fasting period before blood sampling for clinical biochemistry: the night before the day of blood withdrawal for laboratory examinations

Positive control:

no

Examinations

Parental animals: Observations and examinations:

F0 generation + F1 generation Cohort 1B
CLINICAL SIGNS:
- All animals:
Throughout the test period, each animal was observed individually for clinical signs at least once daily. In case of changes, the animals were observed until the symptoms had disappeared.
Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. An increase of the frequency of observations was not necessary as no noteworthy signs of toxicity were observed.
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness.
The cageside observations included skin, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
Dated and signed records of appearance, change and disappearance of clinical signs observed for individual animals were maintained on clinical history sheets.
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.

Additionally, 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 exposure (to allow for within-subject comparisons) and weekly thereafter until termination. F1 Cohort 1B animals were examined weekly after weaning until termination.
The detailed clinical observations were made for all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

MORTALITY:
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. These provisions allowed for recording of premortal symptoms in detail and for performing post mortem examinations as soon as possible after exitus.

BODY WEIGHT:
F0: The animals were weighed and the weights recorded as scheduled.
Pre-mating period: Daily, starting on the first day of dosing*
Mating period: Daily*
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 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 F1 Cohort 1B animals
Mating period: Daily*
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:
F0: Food residue is weighed and recorded as follows:
Pre-mating period: weekly
Mating period: none
Gestation period (females): GD 0#2, 7, 14, 21#3
Lactation period (females): PND 1#2, 7, 14, 21#3
#1 starting on test day 15 of the F0 generation
#2 only the initial amount of food was weighed
#3 only the food residue was weighed (on the other days, the food residue, followed by the initial amount of food was weighed)

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#1 Females: See gestation and lactation period
#1 starting on test day 5 of the F1 generation
The drinking water consumption was monitored by visual appraisal daily throughout the study.

SEXUAL MATURATION:
Animals are evaluated daily for balano-preputial separation or vaginal patency commencing before expected achievement of these endpoints, i.e. completion of balano-preputial separation (approx. PND 40) or vaginal potency, to detect if sexual maturation occurs early. Any abnormalities of the genitals, such as persistent vaginal thread, hypospadia or cleft penis, will be recorded. Sexual maturity of the F1 animals is compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).

URINALYSIS:
Urine samples were collected from animals fasted overnight and analysed as given below.
At the end of the F0 dosing period: 10 males and 10 females randomly selected from each F0 group (animals also selected for laboratory examinations).
The urine was collected for 16 hours in a URIMAX funnel cage. The collection of urine was terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination. The parameters measured and the methods used are given in the text table below.
Parameters of urinalysis:
Parameter/ Unit/ Method:
- Volume/ mL/ Graduated vessel
- pH/ (n/a)/ Using a digital pH meter, type WTW InoLab pH 720
- Specific gravity/ g/mL/ Using Kern Refractometer, type ORA 2PA, Sample compared with water (nominal value of 1.000)
In addition, the tests given below were performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration.
Analytes in urinalysis:
Parameter/ Unit:
Protein/ g/L
Glucose/ mmol/L
Bilirubin/ (n/a)
Urobilinogen/ µmol/L
Ketones/ -
Haemoglobin (Hb, approx. values)/ ery/µL
Nitrite/ (n/a)
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 in the centrifugal deposit will be 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 Generation and F1 Cohort 1B animals
The reproductive parameters and reproductive indices listed in the sections following below were determined to evaluate the reproductive performance.
Reproductive parameters
General parameters
- stages of the estrous 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 and 13
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4 and 13
Number of stillbirths
- per group
- per dam
Number of pups with malformations
- per group
- per dam

s. also reproductive indices

Oestrous cyclicity (parental animals):

Vaginal smears were taken and the estrous cycle stages were determined as given below:
Animals/ Monitoring schedule
F0 animals/ 14 days pre-exposure period to select 96 main study animals with regular estrous cycles (4 or 5 days per cycle) and during 10 weeks of pre-mating until evidence of mating.
F1 animals, Cohort 1A/ Start after onset of vaginal patency until first appearance of cornified cells and two weeks starting around PND 75 (F1 study test days 49 to 63)
F1 animals, Cohort 1B/ Starting from the time of pairing until evidence of mating is detected.
All adult F0 and F1 animals/ On the day of sacrifice, shortly before necropsy.

Sperm parameters (parental animals):

All F0 and Cohort 1A males:
Sperm analysis (motility, morphology and counting) was performed from the cauda of the right epididymis. After weighing the cauda was incised and the emergent sperm sample was used for the examination of motility and morphology of the sperm cells.
After incision and receipt of the sperm sample for the examination of motility and morphology, the cauda of the epididymis was prepared, weighed and frozen at 80°C. The frozen cauda was homogenized and the suspension obtained was used for sperm counting (Kuriyama S.N. (2005), S. Plassmann and H. Urwyler (2001).

Litter observations:

LITTERING:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Based on these parameters, the reproductive performance of the dams was evaluated.
Any abnormal behaviour or changes in the external appearance of the pups noted during the daily cage side inspections were recorded.
The examinations and/or observations performed on the offspring are described in the sections following hereafter.

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 normalised to the cube root of body weight was calculated.

LITTER ADJUSTMENT
After counting on PND 4, the litters will be adjusted to 10 pups per litter (5 pups/sex per litter) by eliminating surplus pups following a randomisation scheme. Selective elimination of pups, e.g. based upon body weight is not appropriate. In case of unequal gender distribution, partial litter size adjustment is acceptable (e.g. 6 male and 4 female pups).

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

SEXUAL MATURATION:
All F1 pups (in Cohort 1A and Cohort 1B) were evaluated daily for balano-preputial separation or vaginal opening indicating 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.
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 the observation was determined as the time point of the onset of preputial gland function.

URINALYSIS:
Urine samples were collected from animals fasted overnight and analysed as given below.
At the end of the F1 cohort 1A dosing period: 10 males and 10 females randomly selected from each F1 cohort 1A group (animals also selected for laboratory examinations).
The urine was collected for 16 hours in a URIMAX funnel cage. The collection of urine was terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination. The parameters measured and the methods used are given in the text table below.
Parameters of urinalysis:
Parameter/ Unit/ Method:
- Volume/ mL/ Graduated vessel
- pH/ (n/a)/ Using a digital pH meter, type WTW InoLab pH 720
- Specific gravity/ g/mL/ Using Kern Refractometer, type ORA 2PA, Sample compared with water (nominal value of 1.000)
In addition, the tests given below were performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration.
Analytes in urinalysis:
Parameter/ Unit:
Protein/ g/L
Glucose/ mmol/L
Bilirubin/ (n/a)
Urobilinogen/ µmol/L
Ketones/ -
Haemoglobin (Hb, approx. values)/ ery/µL
Nitrite/ (n/a)
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 in the centrifugal deposit will be 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.

Postmortem examinations (parental animals):

LABOBRATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples were 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 F0 group.

HAEMATOLOGY:
The parameters listed in the text table below were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald Germany).
Parameter/ Unit
- Haemoglobin content (HGB)/ mmol/L
- Erythrocytes (RBC)/ 10⁶/µL
- Leucocytes (WBC)/ 10³/µL
- Reticulocytes (RET)/ %
- Platelets (PLT)/ 10³/µL
- Haematocrit value (HCT)/ %
- Differential blood count (relative)/ %
- Differential blood count (absolute)/ 10³/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 a possible histopathological examination in case of pathological findings. However, no histopathological examination was performed so far.

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

CLINICAL CHEMISTRY:
The parameters listed below were determined:
Parameter/ Unit
Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany
- Albumin/ g/L
- Globulin/ g/L
- Bile acids/ µmol/L
- Bilirubin (total)/ µmol/L
- Cholesterol (total)/ mmol/L
- Creatinine/ µmol/L
- Glucose/ mmol/L
- Protein (total)/ g/L
- Urea (in blood)/ 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 calculation:
- Albumin/globulin ratio/ non-dimensional
- BUN((blood urea nitrogen) = urea (in blood))/creatinine ratio/ non-dimensional
- Sodium/Potassium ratio/ non-dimensional

HORMONE DETERMINATIONS:
As the findings of the preliminary histopathological examination of the F0 females indicated issues in the vagina/uterus and ovaries, the contributing pathologist recommended the LTH, FSH and LH levels in F1 dams be investigated to clarify the effect of the test item on reproductive performance.
The blood samples were obtained always at the same time of day, as far as possible, as given below.
Blood sampling for hormone determination:
Samples stored at -20°C ±2°C:
F0: 10 males and 10 females randomly selected from each group (same animals selected for laboratory examinations), At sacrifice, 80, Fasted, T4 + TSH, 2 × 100 µL each
Samples stored at -80°C ±8°C:
F1 cohort 1B: 10 females randomly selected from each group#3, At sacrifice (weaning of F2 pups), 40, Non-fasted, LTH + LH + FSH, 2 × 150 µL each
The serum samples were divided into aliquots, as far as possible, and stored at -20°C ±2°C (T4, TSH) or at -80°C ±8°C (LTH, FSH, LH) until analysis within 6 months of collection. The hormone levels were determined at the test institute using commercial kits as stated below (Instrument: Tecan Sunrise
microplate reader, Tecan Deutschland GmbH, 74564 Crailsheim Germany) :
Hormone/ Test kit
- T4/ T4 ELISA Kit, cat. no. RE 55261, batch no. 304K021, IBL
- TSH/ Rat TSH ELISA Kit, cat. no. RE 45021, batch no. V052, IBL
- FSH/ Rat FSH ELISA Kit LS-F38636, batch no. 197836, LSBio
- LH/ Rat LH ELISA Kit LS-F20636, batch no. 197835, LSBio
- LTH/ Rat PRL / Prolactin ELISA Kit LS-F3900, batch no. 197649, LSBio
IBL: IBL International GmbH, Flughafenstraße 52a, 22335 Hamburg, Germany
LSBio: LifeSpan BioSciences, Inc., 2401 4th Ave Suite 900, Seattle, WA 98121, USA
The LODs (limit of detection) and LLOQs (lower limit of quantification) for each parameter are given below:
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
The samples for the determination of LTH, FSH and LH were analysed as scheduled below:
Sampling and sample analysis schedule for FSH, LH, and LTH
Cohort/ Sampling/ Samples analysed
- Cohort 1B/ All 40 females of groups 1 to 4/ Groups 1 and 4 (n = 20)

GROSS NECROPSY:
For adult F0 and F1 females, a vaginal smear taken on the day of sacrifice shortly before necropsy was examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The adult animals were euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled.
F0: Males, all, after weaning of the F1 animals, full necropsy
F0: Females, all, shortly after weaning of the F1 animals, full necropsy + vaginal smears
F1 Cohort 1B: Males, all, shortyl after weaning of the F2 animals, full necropsy
F1 Cohort 1B: Females, all, shortyl after weaning of the F2 animals, full necropsy + vaginal smears
In the case the time of dissection would fall on a weekend or bank holiday, necropsy will be postponed to the next working day and dosing will be continued up to and including the day before sacrifice.
Animals which are prematurely sacrificed or die during the study are necropsied as soon as possible after exitus.

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals were dissected and 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. 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 conditions of the thoracic viscera were 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, stomach and 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. The organ weights of selected organs were determined for each generation and cohort. Organ weights of animals which died or were sacrificed prematurely were recorded but not included in the mean value comparison

ORGAN WEIGHTS:
F0 generation:
The number of implantation sites was recorded and used to evaluate reproductive performance. Apparently non-pregnant uteri 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 weights of the organs listed below of all adult F0 animals were determined before fixation, where applicable. Paired organs were identified as left or right and weighed individually.
- Brain
- Pituitary
- Thymus
- Epididymis (2)
- Prostate (dorsolateral and ventral parts combined)
- Thyroid (1, including parathyroid, post-fixation)
- Heart
- Kidney (2)
- Seminal vesicles with coagulating glands
- Uterus including cervix
- Liver
- Identified target organs
- Ovary (2)
- Spleen
- Adrenal glands (2)
- Oviducts (2)
- Testis (2)

The organs or parts thereof listed below of all adult 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)
- Testicle (1)
The second epididymis and testicle were not preserved but used for the spermiogram
7% buffered formalin:
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Spleen
- Stomach
- Intestine, large (colon, rectum)
- Thyroid (2, including parathyroids)
- Kidney and ureter (2)
- Thymus
- Liver
- Trachea (including larynx)
- Lungs (with mainstem bronchi and bronchioles)
- Urinary bladder
- Mammary gland
- Uterus (including cervix)
- Muscle (skeletal)
- Vagina
- Nerve (sciatic)
- Vas deferens
- Oesophagus
- Identified target organs (if any)
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.

F1 generation Cohort 1B:
Determination of organ weight and organ preservation were restricted to the organs listed in the text table below:
Organ/ Weighing/ Fixative
Endocrine system:
- Adrenal gland (2)/ Yes/ 7% formalin
- Pituitary/ Yes/ 7% formalin
- Thyroid (2, including parathyroids)/ 1, post-fixation/ 7% formalin
Reproductive system:
- Epididymis (2)/ Yes/ Modified Davidson’s
- Ovary (2)/ Yes/ 7% formalin
- Prostate/ Yes/ 7% formalin
- Seminal vesicles with coagulating glands/ Yes/ 7% formalin
- Testicle (2)/ Yes/ Modified Davidson’s
- Uterus (including oviducts and cervix)/ Yes/ 7% formalin
- Vagina/ No/ 7% formalin
- Vas deferens/ No/ 7% formalin
- Identified target organs (if any)/ No/ As appropriate
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 histopathologically. However, histopathological examination of the F1 Cohort 1B animals was not deemed necessary.
Apparently non-pregnant uteri were placed in 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.

BONE MARROW:
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears per animal) of randomly selected animals as given in the text table below and stained according to PAPPENHEIM. The myeloid:erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the scheduled animals (10 males and 10 females randomly selected from each group ).

HISTOPATHOLOGY:
Blood smears were prepared from selected animals during the haematological examination to be available for a possible examination of pathological changes depending on necropsy findings. So far, no histopathological examination was requested by the Study Monitor.
In a first step (Step 1), to aid in the decision-making process regarding the F2 generation, paraffin sections of the organs listed below were prepared and evaluated. The slides were shipped to AnaPath Services GmbH in Liestal, Switzerland. The initial evaluation was performed non-GLP by Dr. K. Weber. In the first step only HE and PAS stained sections were evaluated (no ORO-stained sections were included).
Groups 1 and 4: all animals (n = 96)
Males:
- Adrenal glands (2)
- Epididymis (left)#
- Kidneys (right and left)
- Liver
- Pituitary
- Prostate
- Seminal duct
- Testicle (left)#
Females:
- Adrenal glands (2)
- Kidneys (right and left)
- Liver
- Ovaries (right and left)
- Oviducts (right and left)
- Pituitary
- Uterus with cervix
- Vagina
Group 3:
non-pregnant females (n = 6, animal nos. 122, 127, 133 136, 139, and 140); No group 3 males
- Adrenal glands (2)
- Kidneys (right and left)
- Liver
- Ovaries (right and left)
- Oviducts (right and left)
- Pituitary
- Uterus with cervix
- Vagina
# HE and PAS stained sections of one epididymis and one testicle.

In a second step, a full histopathological examination was performed on the preserved organs as follows:
• F0 animals: 20 animals per sex and group of groups 1 and 4
• All deceased or prematurely sacrificed animals

The organs were examined histopathologically after preparation of paraffin sections and HE staining. Parathyroids were examined microscopically if they could be identified macroscopically and were in the plane of section, and in all cases they were noted as grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined.
A detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of the interstitial testicular structure was performed on one testicle and one epididymis of the selected F0 males of groups 1 and 4 following HE and PAS staining.

The Sponsor was given sufficient notice to decide if organs of further animals were to be processed and examined histopathologically based on test item-related findings in the high dose group 4. As treatment-related microscopic findings observed under the conditions of this study were changes not to be adverse (liver), not relevant to humans (kidneys), to be stress-related (female reproductive organs, adrenal glands and thymus), or not due to systemic effects of the test item (stomach), the histopathological examination in the low and intermediate dose groups of the F0 and F1-C1A were not performed in the present study.

F1 Cohort 1B animals:
The Study Monitor was given sufficient notice to decide if the corresponding organs of the animals of Cohort 1B of the F1 generation were to be dissected and examined histopathologically based on test item-related findings in the organs of the animals of the F0 generation and of Cohort 1A of the F1 generation. As no adverse findings were observed in the animals of the F0 Generation and of Cohort 1A , no histopathological examination of the animals of Cohort 1B of the F1 generation was necessary.
Histopathological evaluation:
The histotechnique processing for all animals was performed by the test institute. All slides prepared at the test institute were labelled with the study number, species, animal number, block number and were dispatched to Test Site 1 (see Section 2.6) for the histopathological evaluation. The transport of the slides and tissues to Test Site 1 was arranged by the test institute, whereas the return transport to the Test Facility was arranged by Test Site 1.
The histopathological evaluation was performed by Test Site 1 according to all relevant Test Site SOPs.
All observations upon final assessment were reported per animal and the findings considered relevant for the treatment are listed in an incidence and occurrence table. All microscopic findings were recorded, reported and archived with the PathData system version 6.2e2.
The report of the histopathology phase of the study comprises a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments, if any, were addressed and the Draft Final Phase Report of the histopathology phase of the study was presented to the TSQAU for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by the Test Site.

Postmortem examinations (offspring):

LABORATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples were 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
F1 cohort 1A: 10 males and 10 females randomly selected from each group.

HAEMATOLOGY:
The parameters listed in the text table below were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald Germany).
Parameter/ Unit
- Haemoglobin content (HGB)/ mmol/L
- Erythrocytes (RBC)/ 10⁶/µL
- Leucocytes (WBC)/ 10³/µL
- Reticulocytes (RET)/ %
- Platelets (PLT)/ 10³/µL
- Haematocrit value (HCT)/ %
- Differential blood count (relative)/ %
- Differential blood count (absolute)/ 10³/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 a possible histopathological examination in case of pathological findings. However, no histopathological examination was performed so far.

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

CLINICAL CHEMISTRY:
The parameters listed below were determined:
Parameter/ Unit
Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany
- Albumin/ g/L
- Globulin/ g/L
- Bile acids/ µmol/L
- Bilirubin (total)/ µmol/L
- Cholesterol (total)/ mmol/L
- Creatinine/ µmol/L
- Glucose/ mmol/L
- Protein (total)/ g/L
- Urea (in blood)/ 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 calculation:
- Albumin/globulin ratio/ non-dimensional
- BUN((blood urea nitrogen) = urea (in blood))/creatinine ratio/ non-dimensional
- Sodium/Potassium ratio/ non-dimensional

HORMONE DETERMINATIONS:
Blood samples were taken from the scheduled animals always, processed for serum, and the T4 and TSH levels were analysed.
In addition, to clarify a test item effect on reproductive performance, the LTH, FSH and LH levels in F1 dams were investigated. This was recommended by the contributing pathologist as the findings of the preliminary histopathological examination of the F0 females indicated issues in the vagina/uterus and ovaries.
The animals scheduled for blood sampling were anaesthetised and euthanised as follows:
• PND 4 pups: euthanized by decapitation followed by blood collection
• PND 22 pups and adults: sampling from the retrobulbar venous plexus under isoflurane anaesthesia; euthanized by carbon dioxide (CO2) inhalation
• PND 4 pups not needed for blood sampling are euthanized by decapitation
The blood samples were obtained always at the same time of day, as far as possible, as given below. The blood samples of F2 pups (2x50 2x 50 μL, 2x 70 μL) are stored at - 20°c ±1 0 % until further notice.
Blood sampling for hormone determination:
Samples stored at -20°C ±2°C:
F1 pups#2:
2 surplus pups per litter,: all litters, if possible/ PND 4/ Non-fasted/ T4 only/ 1 × 75 µL
F1 pups: 2 surplus pups per litter, all litters/ PND 22/ Non-fasted/ T4 + TSH/ 2 × 50 µL + 2 × 70 µL
F1 cohort 1A: 10 males and 10 females randomly selected from each group (same animals selected for laboratory examinations)/ At sacrifice/ Fasted / T4 + TSH/ 2 × 100 µL each
F2 pups#3: One pup per sex of each dam selected for hormone sampling, 40 pups per sex/ At sacrifice/ PND 22/ Non-fasted/ T4 +TSH (not analysed so far)/ 2 × 50 µL
2 × 70 µL

Samples stored at -80°C ±8°C:
F1 cohort 1A: 10 females randomly selected from each group (same animals selected for laboratory examinations)/ At sacrifice/ Fasted/ LTH + LH+ FSH/ 2 × 150 µL each
The serum samples were divided into aliquots, as far as possible, and stored at -20°C ±2°C (T4, TSH) or at -80°C ±8°C (LTH, FSH, LH) until analysis within 6 months of collection. The hormone levels were determined at the test institute using commercial kits as stated below (Instrument: Tecan Sunrise
microplate reader, Tecan Deutschland GmbH, 74564 Crailsheim Germany) :
Hormone/ Test kit
- T4/ T4 ELISA Kit, cat. no. RE 55261, batch no. 304K021, IBL
- TSH/ Rat TSH ELISA Kit, cat. no. RE 45021, batch no. V052, IBL
- FSH/ Rat FSH ELISA Kit LS-F38636, batch no. 197836, LSBio
- LH/ Rat LH ELISA Kit LS-F20636, batch no. 197835, LSBio
- LTH/ Rat PRL / Prolactin ELISA Kit LS-F3900, batch no. 197649, LSBio
IBL: IBL International GmbH, Flughafenstraße 52a, 22335 Hamburg, Germany
LSBio: LifeSpan BioSciences, Inc., 2401 4th Ave Suite 900, Seattle, WA 98121, USA
The samples for the determination of LTH, FSH and LH were analysed as scheduled below:
Sampling and sample analysis schedule for FSH, LH, and LTH
Cohort/ Sampling/ Samples analysed
- Cohort 1A/ All 40 females of groups 1 to 4/ None

GROSS NECROPSY:
For adult F0 and F1 females, a vaginal smear taken on the day of sacrifice shortly before necropsy was examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The adult animals and the PND 22 pups were euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled.
PND 4 pups not needed for blood sampling were euthanized by decapitation.
F1 Generation:
- "Surplus" pups, all, on PND 4, gross necropsy
- "Surplus" pups, all#1, on PND 22, gross necropsy; partial organ preservation of only 10 pups/ sex/ group
- Cohort 1A, at the end of the dosing period (PND 91), full necropsy + vaginal smears
F2 Generation#2:
-all, on PND 22, Gross necropsy; partial organ preservation of 10 pups per sex and group
#1 All F1 “surplus” pups from PND 22 were subject to a gross necropsy, however only a limited number of pups was selected for tissue preservation.
#2 F2 pups were selected from the 10 F1 Cohort 1B females per group selected for blood sampling. The same pups (10 per sex and group) were subject to blood sampling for hormone analysis.

Examination of the "surplus" F1 pups and F2 pups:
Dead pups and pups sacrificed on day 4 post partum (PND 4) were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
Pups sacrificed on day 22 post partum (PND 22) were dissected and examined macroscopically for any abnormalities or pathological changes.
Of those F1 and F2 pups sacrificed on PND 22, organs of up to 10 pups per sex per group of each generation and from as many litters as possible were weighed and preserved in 7% buffered formalin or other appropriate fixative as listed in Text table 4-2 below. Paired organs were identified as left or right and weighed individually.
Organs to be preserved: surplus F1 pups (PND 22) and selected F2 pups
Organ/ Weighing/ Fixative
- Brain/ Yes 7% formalin
- Gross abnormalities/ No/ 7% formalin
- Mammary gland No/ 7%/ formalin
- Ovary (2)/ Yes/ 7% formalin
- Spleen/ Yes/ 7% formalin
- Thymus/ Yes/ 7% formalin
- Uterus including cervix/ Yes/ 7% formalin
- Identified target organs/ No/ As appropriate
A histopathological examination of the preserved organs of the "surplus" F1 pups and F2 pupswill be conducted only in agreement with the Study Monitor. So far, no examination was performed.

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals were dissected and 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. 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 conditions of the thoracic viscera were 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, stomach and 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.
The organ weights of selected organs were determined for each generation and cohort. Organ weights of animals which died or were sacrificed prematurely were recorded but not included in the mean value comparison.

F1 GENERATION - COHORT 1A:
The weights of the organs of all adult F1 animals of Cohort 1A were determined before fixation, where applicable.
However, lymph nodes were preserved and weighed for only 10 animals per sex and group (one animal per litter, all litters represented by at least one randomly selected pup).
Paired organs were identified as left or right and weighed individually.
Weighed organs:
- Adrenal gland (2)
- Ovary (2)
- Testicle (2)
- Brain
- Oviducts (2)
- Thymus
- Epididymis (2)
- Prostate (dorsolateral and ventral parts combined)
- Thyroid (1) (including para-thyroid, post-fixation)
- Heart
- Kidney (2)
- Pituitary
- Uterus including cervix
- Liver
- Seminal vesicles with coagulating glands
- Identified target organs
- Lymph node (1, cervical)#
- Lymph node (1, mesenteric)#
- Spleen
# For 10 animals per sex and group (one animal per litter, all litters represented by at least one randomly selected pup)

The organs or parts thereof listed below of all adult male and female animals of Cohort 1A of the F1 generation were preserved in an appropriate fixative. For the special handling of lymph nodes and spleen see the respective table footnotes.
Preserved organs:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: Modified Davidson’s solution
- Epididymis (1)#
- Testicle (1)#
Fixative: 7% buffered formalin
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Spleen#3
- Stomach
- Intestine, large (colon, rectum)
- Thyroid (2, (including parathyroids)
- Kidney and ureter (2)
- Thymus
- Liver
- Trachea (including larynx)
- Lungs (with mainstem bronchi and bronchioles)
- Urinary bladder
- Lymph node (1, cervical)#2
- Uterus (including cervix)
- Lymph node (1, mesenteric)#2
- Vagina
- Mammary gland
- Vas deferens
- Muscle (skeletal)
- Identified target organs (if any)
- Nerve (sciatic)
- Oesophagus
#1 The second epididymis and the second testicle were not preserved but used for the spermiogram
#2 For selected animals of Cohort 1A of the F1 generation only
#3 For 10 randomly selected animals per sex and group of all groups in Cohort 1A of the F1 generation (the same animals as selected for weighing of the lymph nodes): One half of the spleen was preserved for the histopathological evaluation, the second half was used for splenic lymphocyte subpopulation analysis.

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

BONE MARROW
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears per animal) of randomly selected animals as given in the text table below and stained according to PAPPENHEIM. The myeloid:erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the scheduled animals
Bone marrow smears
Generation/ Bone marrow smears prepared/ Subject to evaluation
F1 Cohort 1A/ 10 males and 10 females randomly selected from each group/ Groups 1 and 4

PHENOTYPIC ANALYSIS OF SPLEEN CELLS– Cohort 1A animals
After weighing, the spleens of selected 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 cell suspensions were used for FACS analysis using a flow cytometer (MACSQuant® Analyzer 10/16, Miltenyi Biotec B.V. & Co. KG, 51429 Bergisch Gladbach, Germany, and software MACSQuantify Version 2.11).
The following splenic lymphocyte subpopulations were determined:
- T-cells (CD4+ T-lymphocytes)
- Helper T-cells (Pan-T-lymphocytes, CD3+)
- Sup./cyt. T-cells (CD8+ T-lymphocytes)
- NK-cells (natural killer cells, CD161+)
- B-cells (B-lymphocytes, CD45RA+)
The evaluation was performed by the test institute.

HISTOPATHOLOGY
Blood smears:
Blood smears were prepared from selected animals during the haematological examination to be available for a possible examination of pathological changes depending on necropsy findings. So far, no histopathological examination was requested by the Study Monitor.

F1 Cohort 1A animals:
A full histopathological examination was performed on the preserved organs as follows:
• F1 animals: groups 1 and 4 of Cohort 1A
• All deceased or prematurely sacrificed animals

The organs were examined histopathologically after preparation of paraffin sections and HE staining. Parathyroids were examined microscopically if they could be identified macroscopically and were in the plane of section, and in all cases they were noted as grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined.
A detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of the interstitial testicular structure was performed on one testicle and one epididymis of all F1 Cohort 1A males of groups 1 and 4 following HE and PAS staining.
A detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea was performed on one ovary of the F1 Cohort 1A females of groups 1 and 4.
The ovary was processed as follows:
• Ten (10) 2 to 4 µm step sections with 50 µm steps in between.
• Each slide labelled with the slide number to follow the sequence.
The Sponsor was given sufficient notice to decide if organs of further animals were to be processed and examined histopathologically based on test item-related findings in the high dose group 4. As treatment-related microscopic findings observed under the conditions of this study were changes not to be adverse (liver), not relevant to humans (kidneys), to be stress-related (female reproductive organs, adrenal glands and thymus), or not due to systemic effects of the test item (stomach), histopathological examination in the low and intermediate dose groups of the F0 and F1-C1A were not performed in the present study.

Histopathological evaluation:
The histotechnique processing for all animals was performed by the test institute. All slides prepared at were labelled with the study number, species, animal number, block number and were dispatched to Test Site 1 for the histopathological evaluation. The transport of the slides and tissues to Test Site 1 was arranged bythe test institute, whereas the return transport to the Test Facility was arranged by Test Site 1.
The histopathological evaluation was performed by Test Site 1 according to all relevant Test Site SOPs.
All observations upon final assessment were reported per animal and the findings considered relevant for the treatment are listed in an incidence and occurrence table. All microscopic findings were recorded, reported and archived with the PathData system version 6.2e2.
The report of the histopathology phase of the study comprises a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments, if any, were addressed and the Draft Final Phase Report of the histopathology phase of the study was presented to the TSQAU for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by the Test Site.

Statistics:

Evaluation of data captured by Provantis®:
The following settings were used for the statistical evaluation of the parametrical values captured by Provantis® (see the flow chart of decision tree in Section 6.3 on page 86):
Homogeneity of variances and normality of distribution was tested using BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, a 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 DUNNETT's test (p ≤ 0.01 and p ≤ 0.05).

Reproductive data (not captured by Provantis®):
The statistical methods described in the following were used for the reproductive data that were not captured by the Provantis® software.
For the parametric values (number of pups etc.), homogeneity of variances was tested using BARTLETT's test. In case of homogeneity, intergroup comparison were performed by DUNNETT's test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group was performed using STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
For non-parametric values (for example the indices of reproduction or the survival rates), the statistical analysis was performed using FISHER's exact test (n < 100) or the Chi²-test with YATES' correction for continuity (n ≥ 100) at significance levels of p ≤ 0.05 and p ≤ 0.01.
All data were evaluated statistically according to the procedures described above. Individual results differing significantly from those of the control group are indicated in the tables of the report (marked with '*' if p ≤ 0.05 or with '**' if p ≤ 0.01).
Mean values and standard deviations were calculated to the highest possible degree of accuracy and afterwards rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ±1 may occur caused by rounding.

Reproductive indices:

For each F0 and F1 Cohort 1B group the gestation index is determined:
Gestation Index = (Number of litters with live pups/ Number pregnant) x 100
Fertility Index female [%] = (Number of pregnant rats/ Number of females used) x 100

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 Index post-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

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):

Daily cage side observations:
Males
A dose-related slight to moderate post-dosing salivation was observed at all dose levels. At the low dose of 30/60 mg/kg b.w./day (group 2), the effect was noted for only 50% of the animals (12 of 24) whereas all animals (24 of 24) treated with 100/200 mg (group 3) or 300/600 mg/kg b.w./day (group 4) were affected.
At all dose levels, the post dose salivation started within 5 minutes after administration and typically lasted up to 20 minutes, in a few cases up to 60 minutes.
The following observations for individual male animals are considered as coincidental findings being not test item-related:
- Animal no. 69 (group 2) revealed extreme salivation before administration on test day 67.
- Animal no. 98 (group 3) revealed a haemorrhagic right canthus on test days 104/105.
- Animal no. 99 (group 3) revealed piloerection on test days 110 and 111.
- Animal no. 166 (group 4) revealed a haemorrhagic right canthus on test days 104/105.

Females
A dose-related slight to extreme post-dosing salivation was observed at all dose levels throughout the periods of premating/mating, gestation, and lactation. The number of affected animals ranged from 19% (lactation period) to 41% (gestation period) of animals employed at the low dose of 30/60 mg/kg b.w./day (group 2), and from 67% (lactation period) to 82% (gestation period) of animals employed at the intermediate dose of 100/200 mg/kg b.w./day (group 3). At the high dose of 300/600 mg/kg b.w./day (group 4), all animals were affected throughout the periods of premating/mating, gestation and lactation.
The following other observations made for individual female animals are considered as coincidental findings being not test item-related:
- Animal no. 134 (group 3) revealed an increased drinking water consumption on test day 86 (last day of the premating/mating period).
- Animal no. 170 (group 4) revealed piloerection, a pale skin (whole body), an uterus prolapse, and its body felt cold to touch on gestation day 24 (test day 110, the littering day). The animal gave birth to only one male pup, which, however, was cannibalized on lactation day 2. Further, pale eyes were observed on lactation days 2 to 7 (test days 112 to 117), piloerection on lactation days 2 to 7 (test days 112 to 117), and a haemorrhagic vagina on lactation day 2. The animal always recovered from all findings noted and survived until terminal dissection on test day 133.
- Animal no. 182 (group 4) revealed an uterus prolapse on gestation day 23 (test day 109, the day before littering). Further, piloerection and a pale skin (whole body) were noted and its body felt cold to touch on gestation day 24 (test day 110, littering day). Piloerection was observed once more on lactation day 4 (test day 113). The animal recovered from all transient findings and survived until terminal dissection on test day 133.
The post-dosing salivation is regarded as a test item-related effect due to the palatability properties of the test item. However, as transient salivation is a common finding in oral rat studies with gavage administration, the effect is considered to have no toxicological relevance.

Males and females
The start and duration of salivation observed for male and female animals are summarized in Text table 7-3 which can be found in the attached document "Text Tables OECD443 DHN". The salivation observed is regarded as a short lasting post-dosing symptom in all cases.

Detailed clinical observations
The detailed clinical observations were performed once weekly.
Males and females
No noteworthy further observations were noted in addition to those made during the daily cage side observations for the animals of the control group and for the treatment with 30/60, 100/200 300/600 mg/kg b.w./day.
For one female animal (no. 170) treated with the high dose of 300/600 mg/kg b.w./day (group 4) piloerection and pale eyes were noted during the observations in test week 18. Another high-dosed female animal (no. 170) revealed an erected tail in test week 18. These findings are considered as spontaneous and not test item-related due to their isolated occurrences.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

MALES AND FEMALES
In total, 9 animals (one male and 8 females) of the F0 generation deceased prematurely or had to be prematurely sacrificed for animal welfare reasons. Deaths or humane sacrifice occurred at each dose level including the control, and, for the female animals, during the pre-mating period, at littering or shortly thereafter, or during the lactation period.
The unscheduled deaths or premature sacrifices of 8 of the 9 deceased animals occurred during the pre-mating period (animals nos. 48, 87, 125, 172), at littering (no. 121) or early during the lactation period (no. 124), or at the end of the study (nos. 119, 179) and are not considered to be test item-related.
For one female animal (no. 179) treated with the high dose of 300/600 mg/kg b.w./day (group 4) and found dead on test day 128 (lactation day 20) the actual cause of death could not be established and remains unclear. Therefore, a relation to the test item-treatment cannot be excluded .
Details on unscheduled deaths and premature sacrifices of animals are given following below.

Unscheduled deaths and premature sacrifices considered not related to treatment
FEMALE ANIMALS
The control animal no. 48 (group 1) had to be sacrificed prematurely on test day 49 because of persistent scratch wounds in the neck area. In addition, a slight body weight loss was observed as of test day 36. The body weight was reduced by approx. 24%14% on test day 43 (192.9 g) compared to test day 29 (222.9 g). The histopathological examination revealed no noteworthy pathological findings. Due to the lack of test item-treatment, the animal’s death is regarded as a coincicdental event.
Animal no. 87 (group 2) treated with the low dose of 30/60 mg/kg b.w./day had to be sacrificed prematurely on test day 49 because of persistent scratch wounds in the neck area. A slight body weight loss was noted as of test day 29. The body weight was reduced by approx. 27% on test day 43 (183.6 g) compared to test day 29 (252.2 g). No histopathological examination was performed. The necessity of the premature sacrifice of the animal was not related to the test item treatment.
Animal no. 125 (group 3) treated with the intermediate dose had to be sacrificed prematurely on test day 50 because of persistent scratch wounds in the neck area. A slight body weight loss was observed as of test day 36. The body weight was reduced by approx. 9% on test day 50 (211.6 g) compared to test day 27 (231.6 g). No histopathological examination was performed. The necessity of premature sacrifice of the animal was not related to the test item treatment.
Animal no. 172 (group 4) treated with the high dose of 300/600 mg/kg b.w./day had to be sacrificed prematurely on test day 44 due to an injury of the spine. A loss of hind limb function was noted during clinical observations. There were no other observations, except for slight salivation starting within 5 minutes after administration lasting up to 20 minutes on test days 32 to 33, 37, and 41 to 43. The animal's body weight and body weight gain were in the normal range throughout the study period until death. The external examination at necropsy revealed a haematoma in the back region with a diameter of approx. 8 mm. The histopathological examination revealed minimal haemorrhage in the parenchyma of the thoracic segment of spinal cord and a treatment-related hepatocellular hypertrophy (slight), stress-related changes (slight diffuse hypertrophy of the adrenal cortex, as well as minimal atrophy and increased tangible body macrophages in the thymus). Spontaneous lesions were noted in several organs but are not considered to be related to the animal’s morbidity. The vertebral fracture and the haemorrhage in the spinal cord were considered to be involved in deterioration of animal’s general condition. The primary cause of vertebral fracture is unknown, but it was most likely due to unexpected traumatic injury. Hence, the necessity of premature sacrifice of the animal was not related to the test item treatment.

Unscheduled deaths and premature sacrifices possibly related to dosing procedure but not considered test item-related
MALE ANIMALS
Animal no. 119 (group 3) treated with 100/200 mg/kg b.w./day (intermediate dose) was found dead during test day 123. The animal revealed slight salivation on test days 84 and 85. No other symptoms that might provide an indication for a cause of death were observed. The animal's body weight and body weight gain were in the normal range throughout the study period, both being slightly higher than the mean values of the control and the intermediate dose group 3 on test day 120. The macroscopic inspection at necropsy revealed reddened lungs, partly dark-red stained. No histopathological examination was performed. The animal's death is considered to be possibly associated with the dosing procedure (such as gavage error, regurgitation or incidental influx of the dosing solution into the respiratory tract), but not to be test item-related.
FEMALE ANIMALS
Animal no. 84 (group 2) treated with 30/60 mg/kg b.w./day (low dose) was found dead in the morning of test day 111. The animal had died during littering the night before. The animal revealed piloerection and a pale skin (whole body), and felt cold to touch on gestation days 23 and/or 24 (test days 109/110). The animal's body weight was slightly lower compared to the mean body weight in group 2 on gestation day 21 (424.5 g vs. 445.9 g). The body weight gain from gestation day 0 to gestation day 21 was slightly higher than the respective mean body weight gain of the group (53.6% vs. 49.9%). The macroscopic inspection at necropsy revealed an indurated and yellowish discoloured liver, yellow-brown discoloured kidneys, a thorax filled with clear liquid (approx. 3 mL), and oedematous lungs. No histopathological examination was performed. The animal's death is considered to be possibly associated with the dosing procedure (such as gavage error, regurgitation or incidental influx of the dosing solution into the respiratory tract), but not to be test item-related.
Animal no. 121 (group 3) treated with 100/200 mg/kg b.w./day (intermediate dose) had to be sacrificed prematurely during littering on test day 112 due to its deteriorated general conditions. The animal littered 14 stillborn pups before sacrifice. Previously, the animal revealed slight salivation starting within 5 minutes after administration lasting up to 20 minutes on test day 65 (premating/mating period) and on gestation days 3 and 17 (test days 91 and 105). Further, piloerection and slight laboured breathing were noted and the animal felt cold to touch on gestation day 23 (test day 111). On test day 112 (littering day), a haemorrhagic nose and snout, haemorrhagic canthi, piloerection, slightly laboured breathing, and lateral position were observed and the animal felt cold to touch. The animal's body weight was in the normal range throughout the study period up to gestation day 21. The body weight gain during the gestation period was slightly lower than the mean body weight in group 3 (28.3% vs. 45.2%). The external examination at necropsy revealed a haemorrhagic nose and a haemorrhagic vagina. Pale inner organs, a yellowish discoloured liver, and yellowish-brown discoloured kidneys were noted at macroscopic inspection at necropsy. Further, 16 implantation sites were noted (as 14 pups were littered, though stillborn, there were 2 resorptions). No histopathological examination was performed.
The animal's death is considered to be possibly associated with the dosing procedure (such as gavage error, regurgitation or incidental influx of the dosing solution into the respiratory tracts), but not to be test item-related.
Animal no. 124 (group 3) treated with the intermediate dose, was found dead on lactation day 2 (the day after littering, test day 111). The animal revealed a pale body and/or piloerection, and felt cold to touch on gestation day 23 (test day 109) and/or the littering day (test day 110). The animal's body weight and body weight gain were in the normal range throughout the study period until death. The macroscopic inspection at necropsy revealed enlarged kidneys and approx. 2.5 to 3.0 mL of a clear liquid in the thorax. No histopathological examination was performed. The animal's death is considered to be possibly associated with the dosing procedure (such as gavage error, regurgitation or incidental influx of the dosing solution into the respiratory tracts), but not to be test item-related.

Death with unclear cause
FEMALE ANIMAL
Animal no. 179 (group 4) treated with the high dose was found dead on test day 128 (lactation day 20). Before, the animal had revealed mostly slight, sporadically moderate or extreme salivation starting within 5 minutes after administration lasting up to 60 minutes on test days 22, 32, 43 to 44, 46, 53 to 56, 71 to 72, 79, and 83 to 84 during the premating/mating period, on gestation days 1, 7, 15, and 19 (test days 87, 93, 101, 105), and on lactation days 4,6 to 10,and 12 to 16 (test days 113, 115 to 119, and 121 to 125). Further, piloerection was observed on lactation days 8 to 10 (test days 117 to 119), a decreased drinking water consumption was noted on lactation days 8 and 9 (test days 117 and 118). In addition, the animal showed an abnormal tail posture on lactation days 11 to 19 (test days 120 to 128). The animal's body weight and body weight gain were in the normal range throughout the periods of premating/mating, gestation and lactation. The external examination at necropsy revealed a haemorrhagic nose and a thickened abdomen. A severely dilated and inflated intestine, oedematous and dark-red discoloured lungs, and an enlarged heart were noted at macroscopic inspection at necropsy. Slight inflammation with foreign material was observed in the laryngeal ventral pouch in the histological examination. There were also treatment-related hepatocellular hypertrophy (minimal) and atrophic mucosa of vagina, stress-related changes (minimal thymic atrophy and moderate diffuse hypertrophy of the adrenal cortex) and spontaneous lesions in several organs, however, these were unlikely to be related directly to the animal’s death. Thus, slight laryngeal inflammation might have been partially involved in this animal's morbidity, however, the actual cause of death could not be established.
The unscheduled deaths and premature sacrifices among the parental animals of the F0 generation are summarised in the text table 7-2 which can be found in the attached document "Text Tables OECD 443 DHN".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
No test item-related influence on the body weight and the body weight gain was noted for the male animals treated with 30/60 or 100/200 mg/kg b.w./day.
The body weight of the animals treated with the high dose of 300/600 mg/kg b.w./day was slightly reduced by up to 7.4% (on test day 138) compared to the control group from test day 50 onwards (statistically significant at p ≤ 0.05 on test days 92, 134, and 138). The body weight gain was reduced accordingly by up to approx. 10 percentage points (on test day 138) compared to the control.
The slightly reduced body weight is regarded as test item-related because it was noted starting after the dose increase from 300 to 600 mg/kg b.w./day on test day 30, and therefore is considered an adverse effect.

FEMALES
No test item-related influence on the body weight and the body weight gain was noted for the female animals treated with 30/60 or 100/200 mg/kg b.w./day throughout the periods of premating/mating, gestation, and lactation.
The body weight of the animals treated with the high dose of 300/600 mg/kg b.w./day was slightly reduced by 9.5% on gestation day 21 (statistically significant at p ≤ 0.01) and by 5.3% on lactation day 1 (statistically significant at p ≤ 0.05). The body weight gain was reduced accordingly by approx. 12 percentage points on gestation day 21. The body weight recovered to nearly the control group's value until the end of the lactation period. The body weight gain of the high dosed females from lactation day 1 to lactation day 7 was approx. 2 percentage points above that of the control group.
The transiently reduced body weight of the female animals at the high dose level is considered to be test item-related and adverse.

Body weight at autopsy
MALES
No test item-related differences were noted for the body weight at autopsy (test days 139 to 141) of the male animals between the control group and the groups treated with 30/60 or 100/200 mg/kg b.w./day.
The body weight at autopsy of the male animals treated with 300/600 mg/kg b.w./day was reduced by 7.9% compared to the control group (statistically significant at p ≤ 0.05). This change is a consequence of the generally lower body weights of the high-dosed male animals during the study period and is therefore also considered as a test item-related and adverse effect.

FEMALES
No test item-related differences were noted for the body weight at autopsy (test days 129 to 134) for the female animals between the control group and the groups treated with 30/60, 100/200, or 300/600 mg/kg b.w./day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MALES
No test item-related influence was noted on the food consumption of the animals treated with 30/60, 100/200 or 300/600 mg/kg b.w./day.
The food consumption was slightly but statistically significantly (at p ≤ 0.05 at all dose levels) reduced in comparison to the control group in all test item treated groups during the time interval from test day 15 to test day 22. However, the changes are regarded to be within the range of normal biological variation, and the slightly reduced food intake is not considered to be test item-related but to be a coincidental effect caused by a relative high food intake in the control group during the respective period.
Following test day 22, a slight but statistically significant (at p ≤ 0.01 or p ≤ 0.05) increase in the food consumption was observed in comparison to the control group for a few examination intervals for the animals of the intermediate dose group treated with 100/200 mg/kg b.w./day and for nearly all examination intervals for the animals of the high dose group treated with 300/600 mg/kg b.w./day. However, the apparently increased food consumption is regarded as a consequence of the slightly lower body weights observed for the respective animals during the examined time intervals and is not considered to be a test item-related effect.

FEMALES
No test item-related influence on the food consumption was noted for the animals treated with 30/60, 100/200 or 300/600 mg/kg b.w./day throughout the periods of premating/mating, gestation, and lactation.
The food consumption of the test item-treated animals was slightly increased by up to 16.2% in most of the time intervals examined between test days 22 and 71. All dose levels were affected, the changes in the intermediate and high dose groups being statistically significant (at p ≤ 0.01 or p ≤ 0.05) in many cases. However, all changes are regarded to be within the range of normal biological variation and are not considered to be test item-related.

No food intake was recorded for the male and the female animals during the mating period as the animals of both sexes were housed together.
Statistically significant differences noted for the food consumption of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 7-4 which can be found in the attached document "Text Tables OECD443 DHN".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual appraisal of the drinking water consumption did not reveal any differences between any of the test item treated groups and the control group.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related influence was noted.
All data are considered to be within the limits of normal biological variation and are completely covered by the range of the test institute's historical control data . Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
A statistically significant (at p ≤ 0.01) decrease by 4.7% in comparison to the control group was noted for the number of erythrocytes (RBC) of the male animals treated with the high dose level. However, the high dose group's value of 8.659×10³ cells/μL plasma is within the 5% and 95% percentiles of the historical control data range of 8.29 to 9.72×10³ cells/μL plasma. Hence, the slight change in the number of erythrocytes (RBC) is considered to be a coincidental effect being not test item related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the biochemical parameters of the male and female animals treated with 30/60, 100/200 or 300/600 mg/kg b.w./day.
All data are considered to be within the limits of normal biological variation and are completely covered by the range of the test institute's historical control data (see Text table 7-6). Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
A few statistically significant (at p ≤ 0.01 or p ≤ 0.05) changes were noted in comparison to the control group for the plasma levels of globulin, chloride, glucose, and urea (in blood), the albumin/globulin ratio, the ratio of urea in blood and creatinine, and the enzyme activity of ALAT for the male and/or female animals treated with 100/200 or 300/600 mg/kg b.w./day (see Text table 7-5). However, all data are well within the historical data ranges. Hence, all changes are considered to be coincidental effects being not test item related.
Statistically significant differences noted for biochemical parameters of the male and female animals of the F0 generation in comparison to the control group that are not considered to be test item-related are listed in the text table 7-5. Text tables can be found in the attached document "Text Tables OECD443 DHN".

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the serum levels of T4 and TSH at any dose level.
There were a few slight differences in T4 and TSH levels between the test item-treated groups and the control (never statistically significant at p ≤ 0.05 or p ≤ 0.01), and the values of individual animals, including control animals, were slightly outside the range of the test institute's historical control data. However, no dose dependency was observed in any case, all data are considered to be within the limits of normal biological variation. Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
Further, no test-item-related changes were noted for the thyroid weights in male and female animals at any dose level. All data are considered to be within the limits of normal biological variation.
A slight increase (+17.9% in comparison to the control group, not statistically significant at p ≤ 0.05 or p ≤ 0.01) noted for the relative thyroid weight of the high dosed male animals (group 4) is not considered as a test item-related effect but to be a consequence of the respective animals' slightly lower body weights relative to the control. The same effect was observed for the male high dosed animals of Cohort 1A of the F1 generation.
A small number of individual relative and absolute thyroid weights of animals treated with the low and intermediate dose level and of control animals were outside the limits of the test institute's historical control data, but all individual data of the female high dosed animals were entirely within this range.
Finally, no noteworthy changes were noted during the histopathological examination of the thyroid glands from the high dosed animals in the male and female animals. It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels . The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weight was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values. As observed in this study, nearly all T4 values are in the range of the historical control data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the urinary parameters of the male and female animals treated with 30/60, 100/200 or 300/600 mg/kg b.w./day.
All data are considered to be within the limits of normal biological variation and are completely covered by the range of the test institute's historical control data (see Text table 7-8). Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
A few slight but statistically significant (at p ≤ 0.01 or p ≤ 0.01) changes were noted in comparison to the control group for the specific gravity, the pH value and/or the relative urine volume for male and/or female animals at all dose levels. However, all data are well within the historical data ranges. Hence, all changes are considered to be coincidental effects being not test item related.
Statistically significant differences noted for urinary parameters of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 7-7. Text tables can be found in the attached document "Text Tables OECD443 DHN".

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):

The histopathological examination was performed on 20 animals per sex and group of the control group 1 and the high dose group 4 of the F0 generation.
Microscopic changes attributed to the test item-treatment were observed in the animals treated with the high dose of 300/600 mg/kg b.w./day as follows:
- Liver
Increased incidence/severity of centrilobular hepatocellular hypertrophy in both sexes. There were no further indicators of tissue injury.
This is considered to be of metabolic nature (like as enzyme induction) and hence, not adverse.
- Kidneys
Treatment-related changes were observed in males only:
Hyaline droplet deposit in the proximal tubular epithelia, accompanied by tubular cell necrosis with granular casts, tubular dilatation, as well as increased incidence/severity of tubular basophilia, peritubular fibrosis, hyaline casts and mononuclear cell infiltration (mononuclear cell focus/foci).
The renal lesions are most likely to be a male-rat specific alpha 2-microglobulin nephropathy, and not relevant to humans.
- Stomach
Hyperkeratosis in both sexes (8/20 males and 11/19 females); there was also focal/multifocal mucosal degeneration in 3 affected males.
These findings are likely due to local effect related to the irritating nature of the test item (not due to systemic effects).
Adrenal glands and thymus
- Adrenal glands: Increased incidence/severity of diffuse cortical hypertrophy in both sexes.
- Thymus: Group mean severity grade of thymic atrophy was increased in both sexes.
These changes are considered to be a stress-related secondary effect.
Female reproductive organs
- Ovaries: Persistent corpora lutea (4 of 18 females) and atrophy (1 of 18 female).
- Uterus: Granulomatous inflammation was observed in the implantation sites of 2 of 18 females.
- Vagina: Increase in the number of animals showing anestrus (acycling mucosa) or permanent diestrus.
These findings are considered secondary to maternal stress (s. also reproductive function: oestrus cycle).

Male reproductive organs
There were no toxicologically relevant microscopic changes in the male reproductive organs and tissues including testis, epididymis, vas deferens, prostate, coagulating glands and seminal vesicles.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles (exposure period)
After allocation of the animals to the 4 test groups and start of treatment on test day 15, the estrous cycles were monitored during the pre-mating and mating period until one day before copulation was verified.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per dam during the pre-mating period between the female animals of the control group and the female animals treated with 30/60 or 100/200 mg/kg b.w./day.
The elongated mean cycle length (5.86 days) noted at the intermediate dose level was caused by the non-pregnant female no. 133 which revealed only 2 complete estrous cycles, each lasting 33 test days. Comparison of the data of all other animals in the intermediate dose group to the data of the control group revealed that there was no noteworthy difference in the mean cycle length between the two groups.
The mean estrous cycle length of the animals treated with 300/600 mg/kg b.w./day appeared to be slightly extended to 5.26 days compared to 4.41 days in the control group (statistically significant at p ≤ 0.01) due to an increased number of females (13 out of 24) with elongated estrous cycles (1 to 3 oestrous cycles lasting for 10 or more days). Accordingly, the mean number of cycles during the premating period was slightly reduced to 12.4 compared to 14.4 observed for the control group (statistically significant at p ≤ 0.01).
However, the mean cycle length of the high-dosed dams was within the range of the individual data of the control group animals, nearly all females were successfully mated (with exception of nos. 180, 183, and 191), there were no changes in pregnancy duration, and females produced litters. Hence, there was no correlation between estrous cyclicity and pregnancy. Only 2 out of 24 animals (nos. 170 and 183) revealed a mean cycle length slightly exceeding the 95% percentile of the test institute's historical control data (7.4 and 6.6 days compared to 6.4 days). There was also one animal (no. 186) the mean cycle length of which was below the lower bound of the historical data range (3.7 days compared to 3.8 days).
No correlation was noted between the non-pregnant animals from each group 3 and group 4 and the length of the estrous stages.
No differences were noted for the pre-coital time during mating of the F0 generation at any dose level.
Further, no prolongation of the mean cycle length was observed for the females in Cohort 1A of the F1 generation (4.05 days at the high dose compared to 4.11 days in the control group).
Concluding the above, the changes in mean estrous cycle length at the high dose, though related to the test item treatment, are considered non-adverse. A similar effect was noted during a reproduction/developmental toxicity screening study (according to OECD guideline 421) performed in advance of the present study (refer to the IUCLID entry of the OECD 421 study from 2020).
The estrous cycle data of the F0 generation and the historical control data are summarised in the text tables 7.20/7.21/7.22/7.23 which can be found in the attached document "Text Tables OECD 443 DHN".

Estrous stage at necropsy Females
No test item-related influence was noted on the distribution of the stages of the estrous cycle at necropsy of the female animals treated with 30/60 or 100/200 mg/kg b.w./day. There were no noteworthy differences between the test item-treated groups and the control group.
A diestrus stage was noted for 11 (group 2) to 14 (group 4) animals per test item-treated group compared to 16 animals in the control group after examination of the vaginal lavages taken from 23 (groups 2 to 4) or 24 (group 1) animals per group at necropsy.
The histopathological examination of the vagina revealed that 4 animals treated with 300/600 mg/kg b.w./day were in the anestrus stage compared to only one animal in the control group. This effect is considered to be test item-related but not adverse due to following reasons:.
These findings in the ovaries and vagina are consistent with other findings encountered in the F0 and/or F1-C1A females during the in-life part, clinical biochemistry and pathology evaluation indicative for stress. They include:
- deceased body weight at 300/600 mg/kg bw of the F0 (9.5% on gestation day 21 and 5.3% on lactation day 1 with a subsequent recovery) and at 600 mg/kg bw of the F1-C1A (it was transient, but reduced up to 5.6% on PNDs 22 to 43);
- reduced glucose levels in the F0 (-12.6% at 100/200 mg/kg bw (MD group), and -15.6% at 300/600 mg/kg bw);
- increased blood urea nitrogen/creatinine ratio (+28.2% at 300/600 mg/kg bw);
- increased urea in blood in the F0 (+25.1% at 300/600 mg/kg bw);
- enlarged adrenal glands and reduced thymic size (small) at 300/600 mg/kg bw of the F0, which were recorded in the macroscopic observations;
- increased weight of adrenal glands and decreased weight of thymus at 300/600 mg/kg bw of the F0;
- increased incidence and/or severity of diffuse adreno-cortical hypertrophy and thymic atrophy, at 300/600 mg/kg bw of the F0, and increased incidence of diffuse adreno-cortical hypertrophy at 600 mg/kg bw of the F1-C1A.
Thus, the above mentioned findings are typical responses to stress including inactive ovaries and uterus, atrophy and mucification of vaginal mucosa, body weight reduction, reduced thymic and adrenal weights associated with thymus atrophy and adrenal cortex hypertrophy, decreased glucose levels (not very sensitive parameter), and increased urea.
A similar effect was observed for the female animals of F1 generation Cohort 1A.
The stages of estrous cycle at necropsy can be found in the text table 7.13, which can be found in the attached document "Text Tables OECD443 DHN".
There appeared to exist serious discrepancies between the findings of the estrous cycle obtained during the in-life part and the findings at histopathology. However, one has to keep in mind that different techniques were used to analyse the estrous cycle stages.
The estrous cycle monitoring of the F0 and F1 Generation animals during the in-life part was performed using vaginal smears at necropsy. The smears were examined without staining by means of a light microscope. The histopathological examination of the estrous cycle was performed by an expert pathologist using H+ E stained slides of the reproductive organs. The results from the histopathological examination have more weight than the examination of the vaginal smears.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the sperm count, the sperm motility, and the sperm morphology in the male animals at any dose level.
Morphology: In total, only 2 malformed sperms were noted across all groups examined. One sperm cell per 200 sperms examined each for 2 animals treated with the intermediate dose level (group 3) revealed banana-like sperm heads. These malformations are considered as coincidental changes and are not considered as test item-related.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-coital time: No test item-related influence was noted.
Gestation length: No test item-related influence was noted.
Gestation index: No test item-related influence was noted on the gestation index for the female animals treated with 30/60, 100/200 or 300/600 mg/kg b.w./day.
The control animal no. 35 did not litter live pups.
Animal no. 84 treated with the low dose died during littering. Only stillbirths were noted. The animal was excluded from the calculation of the gestation index.
At the intermediate dose level, animal no. 121 was prematurely sacrificed during littering due to poor health conditions with only stillbirths being noted. The animal was excluded from the calculation of the gestation index. Further, animal no. 124 was found dead on lactation day 2 with one stillbirth noted on lactation day 1 and 10 remaining live pups being prematurely sacrificed on lactation day 2.
At the high dose level of 300/600 mg/kg b.w./day, all pregnant females delivered live pups.
The gestation indices and the proportionate numbers of the dams with live pups of all pregnant animals are listed in Text table 7-24 which can be found in the attached document "Text Tables OECD443 DHN".
Fertility index: No test item-related influence on the fertility index was noted for the female animals treated with 30/60 mg/kg b.w./day.
For 6 females treated with 100/200 mg/kg b.w./day, and for 3 females dosed with 300/600 mg/kg b.w./day copulation was verified, but the animals were not pregnant. The fertility index was reduced to 74% at the intermediate dose level, and to 87% for the high dosed animals. Each one non-pregnant female with verified copulation was noted at the low dose level and in the control group. The higher number of non-pregnant animals at the intermediate and high dose level is considered to be test item-related. However, there is no clear dose-response relationship as the number of non-pregnant animals in the mid-dose group is higher than in the high dose group. Additionally, a reduced fertility index was not observed for the dams of the F1 generation (study cohort 1B). Therefore, the reduced fertility index of the dams of the F0 generation is considered to be test item-related but not an adverse effect.
Non-pregnant females without verified copulation (i.e. no sperm detection) were noted only in the control group (2 animals, nos. 31 and 38).
The non-pregnant animals are listed in the text table 7-25 which can be found in the attached document "Text Tables OECD443 DHN".
The fertility indices and the proportionate numbers of the pregnant females with verified copulation of females with verified copulation are listed in Text table 7-26 which can be found in the attached document "Text Tables OECD443 DHN".

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity (F0)

Effect level:

>= 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

findings are all secondary to maternal stress therefore not adverse

2

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity (F0)

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily cage side observations
COHORT 1A Males
A dose-related slight to moderate post-dosing salivation was observed at all dose levels. At the low dose of 60 mg/kg b.w./day, the effect was noted for only 25% of the animals (5 out of 20) whereas all animals (20 out of 20) treated with 200 mg/kg b.w./day and 95 % (19 out of 20) treated with the high dose of 600 mg/kg b.w./day were affected.
At the high dose, in total 331 observations were recorded whereas there were only 100 observations at the intermediate dose and just 5 at the low dose.
At all dose levels, the post dose salivation started within 5 minutes after administration and typically lasted up to 20 minutes, sometimes up to 60 minutes (see Text table 8-2 which can be found in the attached document "Text Tables OECD443 DHN"). In a very few cases at the intermediate and high dose, salivation already started before administration.
A haemorrhagic right canthus observed for the male control animal no. 211 (group 1) on post-natal days 77 to 79 was a spontaneous finding.
The following other observations made for individual male animals are considered as coincidental findings and being not test item-related (if observed for test item-treated animals):
- Animal no. 241 (group 2, 200 mg/kg) revealed haemorrhagic eyes on test day 59 and an increased drinking water consumption on post-natal days 79 to 87.
- Animal no. 329 (group 4, 600 mg/kg) revealed an increased drinking water consumption on post-natal days 81, 82, 85, 86, and 88.

COHORT 1A Females
A dose-related slight to moderate post-dosing salivation was observed starting at the intermediate dose of 200 mg/kg b.w./day (group 3). All animals (20 out of 20) treated with the intermediate dose were affected and 90% (18 out of 20) of the animals treated with the high dose of 600 mg/kg b.w./day (group 4). The total count of observations was 398 at the high dose compared to 128 at the intermediate dose.
The post-dose salivation started within 5 minutes after administration and typically lasted up to 20 minutes, sometimes up to 60 minutes. In very rare cases, start of salivation was already observed before administration of the intermediate and or high dose.
The following further observations made for individual female animals are considered as coincidental findings being not test item-related:
- Animal no. 263 (group 2) revealed a haemorrhagic left eye on test day 59.
- Animal no. 265 (group 3) revealed a haemorrhagic nose/snout on test days 38 and 39.
- Animal no. 342 (group 4) revealed gasping starting up to 5 min after administration lasting up to 20 min and a red discharge from the nose/snout on test day 50.
- Animal no. 347 (group 4) revealed convulsions, slight laboured breathing, and an increased respiratory rate starting 2 to 6 hours after administration ending within 2 to 6 hours after administration, and piloerection on test day 43.
- Animal no. 352 (group 4) revealed an increased drinking water (visual appraisal, not quantified) on test day 81.
- Animal no. 353 (group 4) revealed convulsions, slight laboured breathing, and an increased respiratory rate starting 2 to 6 hours after administration ending within 2 to 6 hours after administration on test day 43..
The post-dosing salivation is regarded as a test item-related effect due to the palatability properties of the test item. However, as transient salivation is a common finding in oral rat studies with gavage administration, the effect is considered to have no toxicological relevance

COHORT 1A MALES AND FEMALES
The start and duration of symptoms lasting for a certain period that were observed for male and female animals of Cohort 1A of the F1 generation are summarized in the text table 8-2 which can be found in the attached document "Text Tables OECD443 DHN". For the findings of gasping, laboured breathing and increased respiratory rate a possible reflux or regurgitation of the test item partly into the lungs cannot be excluded with certainty.

COHORT 1B Males
A dose-related, typically slight, only in rare cases up to extreme, post-dosing salivation, was observed at all dose levels. At the low dose of 60 mg/kg b.w./day, the effect was noted for only 10% of the animals (2 of 20) whereas nearly all animals (39 of 40) treated with 200 mg or 600 mg/kg b.w./day were affected.
At all dose levels, salivation started within 5 minutes after administration, sometimes even before start of administration, and typically lasted up to 20 minutes, in a few cases up to 60 minutes, and for one animal (no. 491) up to 2 hours.
Red discoloured urine observed for the male control animal no. 374 (group 1) on post-natal day 140 was a spontaneous finding.
The following observations made for individual male treated animals are considered as coincidental findings being not test item-related:
- Animal no. 374 (group 1, Control) revealed red discoloured urine on post-natal day 140.
- Animal no. 405 (group 2, 60 mg/kg) revealed haemorrhagic urine on post-natal day 63.
- Animal no. 458 (group 3, 200 mg/kg) revealed a haemorrhagic left canthus on post-natal days 93 and 94.
- Animal no. 491 (group 4, 600 mg/kg) revealed breathing sounds starting 0 to 5 min p.a. lasting up to 60 min on post-natal day 33.

COHORT 1B Females
A dose-related, typically slight, in rare cases up to extreme, post-dosing salivation was observed at all dose levels during the period of premating/mating, and starting at the intermediate dose of 200 mg/kg b.w./day (group 3) during the periods of gestation and lactation. The number of affected animals ranged from 15% of the animals employed at the low dose of 60 mg /kg b.w./day (group 2; only during the premating/mating period), and from 60% (gestation period) to 95% (premating/mating period) of the animals employed at the intermediate dose of 200 mg /kg b.w./day. At the high dose of 600 mg/kg b.w./day (group 4), all animals were affected during the periods of the periods of premating/mating and gestation, and 72% of the animals employed during the lactation period.
A decreased water consumption (visual appraisal, not quantified) observed for the female control animal no. 386 (group 1) on lactation day 13 was a spontaneous finding.
The following observations made for individual female treated animals are considered as coincidental findings being not test item-related:
- Animal no. 424 (group 2) revealed a decreased water consumption (visual appraisal, not quantified) on lactation day 13.
- Animal no. 462 (group 3) revealed a haemorrhagic right canthus on post-natal day 65.
- Animal no. 470 (group 3) revealed piloerection on post-natal days 92 and 93 (premating period), on the mating day (post-natal day 97), and on days 3,11 to 12, and 14 to 25 following mating. Further, the animal revealed a haemorrhagic nose/snout on days 1, 14, and 22 to 25, a red discharge from the vagina on days 11 and 12, a hunched posture on days 11 and 16 to 21, and 24 to 25, pale eyes on days 15 to 21, and a haemorrhagic throat on day 19 following mating.
- Animal no. 510 (group 4) revealed convulsions starting 2 to 6 h p.a. lasting 2 to 6 h on lactation days 7 and 12.
- Animal no. 516 (group 4) revealed a reduced amount of faeces on post-natal day 57.
The post-dosing salivation observed is regarded as a test item-related effect due to the palatability properties of the test item. However, as transient salivation is a common finding in oral rat studies with gavage administration, the effect is considered to have no toxicological relevance.

COHORT 1B MALES AND FEMALES
The start and duration of salivation observed for male and female animals are summarized in Text table 9-3 which can be found in the attached document "Text Tables OECD443 DHN". The salivation observed is regarded as a short lasting post-dosing symptom in all cases.

COHORT 1B Detailed clinical observations
The detailed clinical observations were performed once weekly.
MALES AND FEMALES
No noteworthy further observations were noted in addition to those made during the daily cage side observations for the animals of the control group and for the treatment with 60, 200 600 mg/kg b.w./day.
A scratch wound in the neck was observed for one male animal (no. 489) treated with the high dose of 600 mg/kg b.w./day (group 4) and one female animal (no. 489) treated with the low dose of 60 mg/kg b.w./day (group 2) in test week 8 or test weeks 8 and 9, respectively. One high dosed male animal revealed reddened eyelids in test weeks 16 and 17. For the female animal no. 470 treated with the high dose, a haemorrhagic snout, piloerection, pale eyes, and a hunched body posture were observed in test week 14. All findings described afore are considered as spontaneous and not test item-related due to their isolated occurrences.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

COHORT 1A
MALES AND FEMALES
In total, one male and 4 female animals of Cohort 1A of the F1 generation treated with the high dose of 600 mg/kg b.w./day (group 4) deceased prematurely or had to be prematurely sacrificed for animal welfare reasons. The cause of death for 2 of the 5 animals is unclear, a relation to the test item-treatment cannot be excluded.
Details on the death or premature sacrifice of animals are given following below.

Premature sacrifice due to morbidity considered not related to treatment
FEMALE ANIMAL
Animal no. 349 (group 4) was humanely sacrificed due to paralyzed hind limbs noted on PND 83. The animal had not revealed any clinical symptoms, except for slight salivation observed on several days. The animal's body weight was slightly below the group average as of test day 29. At macroscopic inspection at necropsy, the animal's vagina was smeared with urine. Further, a tightly filled urinary bladder containing brownish particles and a haematoma (diameter approx. 2 mm) near the ninth thoracic vertebra was noted. The histopathological examination revealed a moderate muscle fibre necrosis with moderate inflammation and slight haemorrhage in the dorsal paravertebral region. Further, minimal parenchymal necrosis and moderate haemorrhage in the spinal cord, slight sub- and epidural haemorrhage, fluid material retention mainly in epidural space and the related moderate cord compression in the thoracic segment, and minimal parenchymal haemorrhage in the cervical segment were observed. These lesions were considered to be the cause of hind limb paralysis and the animal’s death. The primary cause(s) that produced these lesions were unknown, however, incidental traumatic injury was speculated.

Unscheduled deaths possibly related to dosing procedure but not considered test item related
FEMALE ANIMALS
Animal no. 348 (group 4) was found dead in the morning of PND 38. No clinical symptoms were observed in advance of the death. The animal's body weight was above the group average up to test day 36. Dark red-stained oedematous lungs and a thorax filled with a clear liquid (approx. 1.0 to 1.5 mL) were noted at macroscopic inspection at necropsy. The histopathological examination revealed severe tracheal mucosal necrosis, as well as minimal to slight laryngeal mucosal necrosis, which are considered to be the cause of the animal's death. These lesions were considered to be associated with the technical procedure of dosing (such as gavage error, regurgitation or incidental influx of the dosing solution into the respiratory tracts), and deemed not to be test item-related.
Animal no. 359 (group 4) was found dead in the morning of PND 31. No clinical symptoms were observed for the animal. The animal's body weight was slightly above the group average on test days 22 and 29. A haemorrhagic snout and dark red-stained oedematous lungs were noted at macroscopic inspection at necropsy. The histopathological examination revealed severe tracheal mucosal necrosis, as well as minimal to slight laryngeal mucosal necrosis, which are considered to be the cause of the animal's death. These lesions were considered to be associated with the technical procedure of dosing (such as gavage error, regurgitation or incidental influx of the dosing solution into the respiratory tracts), and deemed not to be test item-related.

Deaths with unclear cause
MALE ANIMAL
Animal no. 328 (group 4) was found dead during the day on PND 28. No clinical symptoms were observed in advance of the death. The animal's body weight was above the group average on test day 22. Dark red-stained oedematous lungs were noted at macroscopic inspection at necropsy. The histopathological examination revealed minimal multifocal inflammatory cell infiltrates in terminal sacs, slight alveolar haemorrhage, slight alveolar enema and/or moderate congestion in the lungs. These findings are regarded as non-specific changes. There were also treatment-related hepatocellular hypertrophy and/or stress-related changes (increased tingible body macrophages in thymus, lymphoid atrophy in spleen and/or diffuse hypertrophy of the adrenal cortex). However, these were unlikely to be related directly to the animal’s death. Thus, the direct cause of the animal's death could not be specified histologically. and a test item related mortality cannot be excluded.
FEMALE ANIMAL
Animal no. 350 (group 4) was found dead in the morning of PND 49. The animal had not revealed any clinical symptoms, except for slight to moderate salivation observed on several days. No noteworthy difference was noted between the animal's body weight and the average body weight in group 4 up to test day 43. Dark red-stained oedematous lungs and a haemorrhagic snout were noted at macroscopic inspection at necropsy. The histopathological examination revealed minimal multifocal inflammatory cell infiltrates in terminal sacs, slight alveolar haemorrhage, slight alveolar enema and/or moderate congestion in the lungs. These findings are regarded as non-specific changes. There were also treatment-related hepatocellular hypertrophy and hyperkeratosis with mucosal degeneration, and/or stress-related changes (increased tingible body macrophages in thymus, lymphoid atrophy in spleen and/or diffuse hypertrophy of the adrenal cortex). However, these were unlikely to be related directly to the animal’s death. Thus, the direct cause of the animal's death could not be specified histologically and a test item related mortality cannot be excluded. (Details can be found in Text table 8-1, s. attached document "Text Tables OECD443 DHN").

COHORT 1 B
MALES AND FEMALES
In total, 3 male and 4 female animals of Cohort 1B of the F1 generation deceased prematurely or had to be prematurely sacrificed for animal welfare reasons due to their morbidity. Deaths or humane sacrifice occurred only at the intermediate and high dose level, and, in case of females, during the pre-mating period or the lactation period.
The cause of death for the 3 prematurely deceased female animals treated with the high dose of 600 mg/kg b.w./day (nos. 504, 509, and 520) is unclear, a relation to the test item-treatment cannot be excluded.
Details on deaths and premature sacrifices of animals are given following below.

Death and premature sacrifice considered unrelated to the test item:
MALE ANIMALS
Animal no. 454 treated with the intermediate dose of 200 mg/kg b.w./day (group 3) died immediately after administration on post-natal day 118. No premortal symptoms were observed. The animal did not reveal any clinical signs of toxicity, just slight salivation was noted on post-natal days 40, 44, 88, and 89, and a haemorrhagic right canthus on post-natal day 51. The animal's body weight and body weight gain appeared to be slightly lower than the respective group average of group 3 but was still in the normal range until its death. The macroscopic inspection at necropsy revealed no pathological changes. No histopathological examination was performed. Due to the lack of findings suggesting a relation to the test item-treatment, the animal's death is considered to be a coincidental event.
Animal no. 459 treated with the intermediate dose (group 3) was humanely sacrificed on post-natal day 37 due to its deteriorated health conditions. The animal revealed swollen fore and hind limbs and a swollen tail, a haemorrhagic canthus. In addition, multiple rashes in hairless skin areas were noted at macroscopic inspection at necropsy. The animal's body weight was approx. 33% lower than the group average. No histopathological examination was performed. Due to the lack of any similar findings in any male animal treated with the high dose level, the animal's morbidity is regarded as unrelated to the test-item-treatment.

Deaths with unclear cause (possibly test item related)
Deaths for which no clear explanation could be established and for which a relation to the test item treatment cannot be excluded were noted only at the high dose level of 600 mg /kg b.w./day (group 4) as described in the following.
MALE ANIMAL
Animal no. 486 treated with the high dose of 600 mg/kg b.w./day (group 4) was humanely sacrificed on post-natal day 74 due to paralysed hind legs. The animal did not reveal any clinical signs of toxicity, just slight to extreme salivation was noted on post-natal days 32, 43, 44, 57 to 58, 65, and 70 to 71, and breathing sounds on post-natal day 32. The animal's body weight and body weight gain were in the normal range until sacrifice. The macroscopic inspection at necropsy revealed a dilated urinary bladder (diameter 18 mm) being tightly filled with a clear liquid, a finding not regarded as test item-related. No histopathological examination was performed. It is assumed that the paralysis of the hindlegs resulted from incidental traumatic injury as described in the histopathology report for animal no. 349, which revealed similar hindleg paralysis.
FEMALE ANIMALS
Animal no. 504 was found dead in the morning of post-natal day 48. No premortal symptoms were observed. The animal did not reveal any clinical signs of toxicity, just slight salivation was noted on post-natal days 39 and 42. The animal's body weight and body weight gain were in the normal range until its death, being even slightly higher than the group average of group 4. A haemorrhagic nose and oedematous, dark-red stained lungs were noted at macroscopic inspection at necropsy. The animal proved to be not pregnant by a negative result of the Salewski staining. No histopathological examination was performed. The cause of death of the animal is unclear, a relation to the test item-treatment cannot be excluded.
Animal no. 509 was found dead in the morning of its lactation day 19 (= post-natal day 140). No premortal symptoms were observed. The animal did not reveal any clinical signs of toxicity, just slight salivation was noted on post-natal days 1 and 4 to 6. The animal's body weight and body weight gain were in the normal range until its death, the body weight being nearly equal to the group average of group 4 on lactation day 14. A haemorrhagic and bluish discoloured nose and dark-red stained lungs were noted at macroscopic inspection at necropsy. No histopathological examination was performed. The cause of death of the animal is unclear, a relation to the test item-treatment cannot be excluded.
Animal no. 514 treated with with the high dose of 600 mg/kg b.w./day (group 4) was humanely sacrificed on post-natal day 61 due to paralysed hind legs. The animal did not reveal any clinical signs of toxicity, only slight salivation was noted on post-natal days 42, 53, 58, and 61. A tightly filled urinary bladder was noted at macroscopic inspection at necropsy. The animal's body weight was approx. 6% above the group average on post-natal day 57. No histopathological examination was performed. It is assumed that the paralysis of the hindlegs resulted from incidental traumatic injury as described in the histopathology report for animal no. 349, which revealed similar hindleg paralysis.
Animal no. 520 was found dead in the morning of its lactation day 19 (= post-natal day 139). No premortal symptoms were observed. The animal did not reveal any clinical signs of toxicity. The animal's body weight was slightly lower than the group average of group 4 on lactation day 14 but well within in the range covered by the control animals on that day. A haemorrhagic nose, oedematous, dark-red stained lungs, and an enlarged left adrenal gland were noted at macroscopic inspection at necropsy. No histopathological examination was performed. The cause of death of the animal is unclear, a relation to the test item-treatment cannot be excluded.
The deaths and humane sacrifices among the parental animals of Cohort 1B of the F1 generation are summarised in the text table 9-2 which can be found in the attached document "Text Tables OECD443 DHN".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A MALES
No test item-related influence on the body weight and the body weight gain was noted for the male animals of Cohort 1A of the F1 generation treated with 60 or 200 mg/kg b.w./day.
The body weight of the animals treated with the high dose of 600 mg/kg b.w./day was reduced by up to 8.5% (on post-natal day 50) compared to the control group from post-natal 29 onwards (statistically significant at p ≤ 0.05 on post-natal days 43 and 50). The body weight gain was reduced accordingly by up to approx. 45 percentage points (on post-natal day 85) compared to the control.
A comparable effect was observed for the male animals of the F0 generation.

COHORT 1A FEMALES
No test item-related influence was noted on the body weight and the body weight gain of the female animals treated with 60, 200 or 600 mg/kg b.w./day throughout the study period.
The body weight of the female animals treated with the high dose of 600 mg/kg b.w./day appeared to be reduced by up to 5.6% (on post-natal day 29) on post-natal days 22 to 43 (not statistically significant at p ≤ 0.01 or p ≤ 0.05 in any case). However, the body weight gain of the female high dose animals was higher than in the control group as of post-natal day 29 and there was no noteworthy difference in body weight between the high dose group and the control group as of post-natal day 50. Therefore, the transient slight differences in body weight between control and high dose group are considered to be within the normal range of biological variation and not to be test item-related.

Body weight at autopsy
COHORT 1A MALES
No test item-related differences were noted for the body weight at autopsy (on test day 87 to 96 ) of the male animals between the control group and the groups treated with 60 or 200 mg/kg b.w./day.
The body weight at autopsy of the male animals treated with 600 mg/kg b.w./day was reduced by 10.9% compared to the control group (statistically significant at p ≤ 0.01). This change is a consequence of the generally lower body weights of the high-dosed male animals of Cohort 1A of the F1 generation during the study period and is considered as a test item-related and adverse effect.

COHORT 1A Females
No test item-related influence was noted.

COHORT 1B MALES
No test item-related influence on the body weight and the body weight gain was noted for the male animals treated with 60 or 200 mg/kg b.w./day.
The body weight of the animals treated with the high dose of 600 mg/kg b.w./day was reduced by up to 10.8% (on post-natal day 141) compared to the control group from post-natal day 22 onwards (statistically significant at p ≤ 0.05 on post-natal days 43, 50, 57, 71, 78, 85, 92, and 113, and at p ≤ 0.01 on post-natal days 99, 106, 120, 127, 134, and 141). The body weight gain was reduced accordingly and was approx. 65 percentage points below the value of the control on post-natal day 141.The reduced body weight is regarded as test item-related and to be an adverse effect.
A comparable effect on body weight was observed for the male animals of the F0 generation and the male animals of Cohort 1A of the F1 generation

COHORT 1B FEMALES
No test item-related influence on the body weight and the body weight gain was noted for the female animals treated with 60 or 200 mg/kg b.w./day throughout the periods of premating/mating, gestation, and lactation.
The body weight of the female animals treated with the high dose of 600 mg/kg b.w./day was reduced by 9.1% compared to the control on day 21 of the pre-mating period (statistically significant at p ≤ 0.05). The difference to the control group decreased during the course of the pre-mating period to be just -4.0% on post-natal day 92.
At the end of the gestation period (gestation day 21), the body weight of the high dosed female animals was reduced by 10.8% compared to the control (statistically significant at p ≤ 0.01). This difference is considered to be related to the smaller litter weight (see Section 9.11.5) which is a consequence of the lower number of pups born in the high dosed group (see Section 9.11.3).
On lactation day 1, the body weight of the high dosed female animals was still reduced by 6.6% compared to the control group (statistically significant at p ≤ 0.05). During the course of the lactation period, the divergence disappeared and no noteworthy difference in body weight was noted between the high dose group and the control group on lactation day 21.
The transiently reduced body weight of the female animals at the high dose level is considered to be test item-related and adverse.

Body weight at autopsy
COHORT 1B MALES AND FEMALES
No test item-related differences were noted between the control group and the groups treated with 60 or 200 mg/kg b.w./day for the body weight at autopsy (post-natal days 87 to 96 for the males, and test days 88 to 96 for the females).
The body weight at autopsy of the male animals treated with 600 mg/kg b.w./day was slightly reduced by 10.6% compared to the control group (statistically significant at p ≤ 0.01). This change is a consequence of the generally slightly lower body weights of the high-dosed male animals of Cohort 1B of the F1 generation during the study period and is considered as a test item-related and adverse effect.
No noteworthy difference was noted for the body weight at autopsy of the female animals between the control group and the high dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

COHORT 1A
Male and females
No test item-related influence was noted on the food consumption of the animals treated with 60, 200 or 600 mg/kg b.w./day.
For the male animals treated with the low dose of 60 mg/kg b.w./day, the food consumption was slightly but statistically significantly (at p ≤ 0.05) increased by 3.7% compared the control group in the time interval from PND 47 to PND 54. This change is considered to be a coincidental effect being not test item-related due to the single occurrence over time of such an effect within the group.
At the high dose level of 600 mg/kg b.w./day, a slight increase of the food consumption by up to 13.8% (time interval from PND 47 to PND 54) in comparison to the control group was observed for the male and the female animals in all time intervals from PND 5 to PND 68 (being statistically significant at p ≤ 0.01 or p ≤ 0.05 in most of the time intervals for both sexes). However, these changes are regarded as a consequence of the slightly lower body weights observed for the respective animals during the examined time intervals and are not considered as a test item-related effect.
Statistically significant differences noted for the food consumption of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 8-3 (attached document "Text Tables OECD443 DHN").

COHORT 1B
MALES
No test item-related influence was noted on the food consumption of the animals treated with 60, 200 or 600 mg/kg b.w./day.
The food consumption was slightly but statistically significantly (at p ≤ 0.05 at all dose levels) reduced in comparison to the control group in all test item treated groups during the time interval from test day 15 to test day 22. However, the changes are regarded to be within the range of normal biological variation, and the slightly reduced food intake is not considered to be test item-related but to be a coincidental effect caused by a relative high food intake in the control group during the respective period.
Following test day 22, a slight but statistically significant (at p ≤ 0.01 or p ≤ 0.05) increase in the food consumption was observed in comparison to the control group for a few examination intervals for the animals of the intermediate dose group treated with 200 mg/kg b.w./day and for nearly all examination intervals for the animals of the high dose group treated with 600 mg/kg b.w./day. However, the apparently increased food consumption is regarded as a consequence of the slightly lower body weights observed for the respective animals during the examined time intervals and is not considered to be a test item-related effect.
FEMALES
No test item-related influence on the food consumption was noted for the animals treated with 60, 200 or 600 mg/kg b.w./day throughout the periods of premating/mating, gestation, and lactation.
The food consumption of the test item-treated animals was slightly increased by up to 16.2% in most of the time intervals examined between test days 22 and 71. All dose levels were affected, the changes in the intermediate and high dose groups being statistically significant (at p ≤ 0.01 or p ≤ 0.05) in many cases. However, all changes are regarded to be within the range of normal biological variation and are not considered to be test item-related.

No food intake was recorded for the male and the female animals during the mating period as the animals of both sexes were housed together.
Statistically significant differences noted for the food consumption of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 9-4 (attached document "Text Tables OECD443 DHN".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

COHORT 1A and COHORT 1B
Daily visual appraisal of the drinking water consumption did not reveal any differences between any of the test item treated groups and the control group.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

COHORT 1A
MALES AND FEMALES
No test item-related influence was noted on the haematological parameters of the animals treated with 60, 200 or 600 mg/kg b.w./day.
A few statistically significant (at p ≤ 0.01 or p ≤ 0.05) changes were noted in comparison to the control group for the haemoglobin content (HGB), the mean corpuscular volume (MCV), and/or the mean corpuscular haemoglobin (MCH) for the male or female animals treated with 200 or 600 mg /kg b.w./day (see Text table 8-4). However, all data are well in the range of the test institute's historical control data (see text table Text table 8-5). Therefore, any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
Statistically significant differences noted for haematological parameters of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 8-4. Text tables can be found in the attached document "Text Tables OECD443 DHN".

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

COHORT 1A
MALES AND FEMALES
No test item-related influence was noted on the biochemical parameters of the male and female animals treated with 60, 200 or 600 mg/kg b.w./day.
All data are considered to be within the limits of normal biological variation and are completely covered by the range of the test institute's historical control data (see Text table 8-6). Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
A few statistically significant (at p ≤ 0.01 or p ≤ 0.05) changes were noted in comparison to the control group for the plasma levels of goblin, protein, and/or glucose, the albumin/globulin ratio, and the enzyme activity of ALAT and/or ASAT for the male and/or female animals treated with 60 or 600 mg/kg b.w./day (see Text table 8-6). However, all data are well within the historical data ranges. Hence, all changes are considered to be coincidental effects being not test item related.
Statistically significant differences noted for biochemical parameters of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 8-5. Text tables can be found in the attached document "Text Tables OECD443 DHN".

Endocrine findings:

no effects observed

Description (incidence and severity):

COHORT 1A
MALES AND FEMALES
No test item-related influence was noted on the serum levels of T4 and TSH for the male and female animals treated with 60, 200 or 600 mg/kg b.w./day.
The TSH levels of the test item-treated male animals appeared to be increased by up to 113.9% compared to the control group at all dose levels, being statistically significant (at p ≤ 0.01) only at the intermediate dose. No clear dose dependency was observed. The changes are regarded to be due to the relatively low value of only 1.8907 ±0.7173 ng TSH/mL serum noted for the control group. In the F0 generation, the control group revealed a value of 4.4781 ±3.1677 ng TSH/mL serum (see Section 7.9) which is even higher than the value observed for the intermediate dose group of Cohort 1A of the F1 generation.
With very few exceptions, all data were within the range of historical control data. All differences between the test item-treated groups of F1 generation Cohort 1A and the control group are regarded as coincidental and being not test item-related.
No test-item-related changes were noted for the thyroid weights in male and female animals.
A slight increase (+24.5% in comparison to the control group, statistically significant at p ≤ 0.05) noted for the relative thyroid weight of the high dosed male animals (group 4) is not considered as a test item-related effect but to be a consequence of the respective animals' slightly lower body weights relative to the control. The same effect was observed for the male high dosed animals of the F0 generation.
A small number of individual relative and/or absolute thyroid weights of male test item-treated and control animals were slightly outside the limits of the test institute's historical control data. However, all data are considered to be within the range of normal biological variation.
Finally, no changes were noted during the histopathological examination of the thyroid glands from the high dosed animals in the male and female animals. It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels . The validation report of OECD guideline 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weight was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values. As observed in this study, nearly all T4 values are in the range of the historical control data(see Text table 8-10, atatched document "Text Tables OECD443 DHN").

COHORT 1B
FEMALES (control and high dose group only)
Hormone levels - FSH, LH, LTH
No test item-related influence was noted on the serum levels of FSH, LH, and LTH for the female animals treated with 600 mg/kg b.w./day.
The LTH levels of the test item-treated female animals appeared to be increased by approx. 50% compared to the control group (not statistically significant at p ≤ 0.01 or p ≤ 0.05, see TABLE 7-1-Co1B). However, 8 of the 10 values in the high dose group are within the range covered by the individual data of the control group. Two values in the control group were at or just slightly above the limit of quantification for LTH (1.024 ng/mL serum). Only two values in the high dose group (animals nos. 501 and 516) were slightly above the maximum observed for the control group. Both animals had normal pregnancy and live litters. Therefore, the slight change in the high dose group is considered to be of spontaneous nature and not to be test item-related.

Urinalysis findings:

no effects observed

Description (incidence and severity):

COHORT 1A
MALES AND FEMALES
No test item-related influence was noted on the urinary parameters of the male and female animals treated with 60, 200 or 600 mg/kg b.w./day.
All data are considered to be within the limits of normal biological variation and are completely covered by the range of the test institute's historical control data. Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.
A few slight but statistically significant (at p ≤ 0.01 or p ≤ 0.01) changes were noted in comparison to the control group for the urine pH value of the male animals at the intermediate and high dose level. However, the data are well within the historical data ranges . Hence, all changes are considered to be coincidental effects being not test item related.
Statistically significant differences noted for urinary parameters of the male and female animals in comparison to the control group that are not considered to be test item-related are listed in the text table 8-8, historical control data in text table 8-9 (see attached document "Text Tables OECD443 DHN".)

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

COHORT 1A Males and females
No test item-related differences were noted for the relative proportions of the examined lymphocyte subtypes and the absolute counts of cell subpopulations between the male and female animals of the control group and the animals treated with 60, 200 or 600 mg /kg b.w./day.
The slight differences (not statistically significant at p ≤ 0.01 or p ≤ 0.05 in any case) noted for the relative count of various cell subpopulations of the female animals between the control group on the one hand and all test item treated-groupstreated groups on the other hand (independent of the dose level) are due to the relatively low or high values observed in the control group. The control group animals' data were close to the 95% percentile (in case of T-cells, helper T-cells, or suppressor/cytotoxic T-cells) or the 5% percentile (in case of B-cells) of the test institute's historical control data, whereas all data of the test item-treated groups were within the 25% and 75% percentiles, or at least very close to the respective boundary.
Hence, all data are considered to be within the limits of normal biological variation. The data of the relative proportions of the examined cell subpopulations are completely covered by the range of the historical control data. Any differences between the test item-treated groups and the control group are regarded as spontaneous changes being not test item-related.

COHORT 1A Males and females (control and high dose, groups 1 and 4 only)
No test item-related differences were noted for the myeloid/erythroid ratio of the bone marrow between the animals treated with 600 mg/kg b.w./day and the animals of the control group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A Males
A dose-related increase was noted for the kidney weights of the male animals starting at the low dose of 60 mg/kg b.w./day (statistically significant at p ≤ 0.05 or p ≤ 0.01 in nearly all cases at all dose levels) at terminal dissection between test day 87 and test day 96. These changes are considered to be test item-related.
Increased kidney weights, with a similar dose-response relationship, were also noted for the male animals of the F0 generation, likewise starting at the low dose level.
For the animals treated with the high dose level, the increased kidney weights correspond to microscopic changes in the kidneys noted at histopathological examination which are considered to be due to an alpha 2-microglobulin-mediated process being specific for male rats. Therefore, the increased kidney weights are regarded as not relevant for humans and, hence, non-adverse.
Further changes noted in relative and/or absolute weights of various organs of the male animals treated with 600 mg/kg b.w./days in comparison to the control group which are considered as test item-related are described following below.
Adrenal glands
The slightly increased group means of the relative weights of the left and right adrenal gland of the high dose male animals were within the range of the respective 5% and 95% percentiles of the test institute's historical control data (refer to Text table 8-23). However, the relative weights of the left adrenal gland of 8 of 19 animals (nos. 322, 332, 334, 335, 337, 339, 339, 340) were above the 95% percentile of the test institute's historical control data (refer to Text table 8-23), one value (for animal no. 338) was above the maximum of the historical control data range (not considering excluded values). Similar relative weights of the left adrenal gland above the 95% percentile of the test institute's historical control data were also noted for 3 of 20 male control animals (nos. 202, 217, and 220). For the relative weights of the right adrenal gland, the values of 8 animals (nos. 322, 325, 326, 328, 334, and 337) were slightly above the 95% percentile of the test institute's historical control data, one value (from animal no. 338) was above the upper limit of the range.
Histopathological findings corresponding to the weight increase were noted during the microscopic examination of the adrenal glands in form of a slightly increased incidence and/or severity of diffuse cortical hypertrophy in comparison to control group. However, these changes are considered to be a stress-related secondary effect.
A similar slight increase of the relative adrenal gland weights was also noted for the males of the F0 generation and, less pronounced, of Cohort 1B of the F1 generation.
Liver
The individual relative liver weights of 6 of 19 high dosed animals were above the 95% percentile of the test institute's historical control data, the group mean value (43.06 g/kg b.w.) was below the 95% percentile value (44.3 g/kg b.w., refer to Text table 8-23).
A similar slight increase of the relative liver weight was also noted for the males of the F0 generation.
Thymus
The mean absolute thymus weight was slightly reduced by 16.1% compared to the control group (statistically significant at p ≤ 0.05, refer to Text table 7-14). The data were within the range of the 5% and 95% percentiles of the test institute's historical control data (see Text table 7-18).

The minor changes in absolute and/or relative organ weights described in the following are not considered as an immediate effect of the test item but are regarded as spontaneous or stress-related changes or to be secondary effects due to the slightly lower body weights of the high dosed male animals.
Brain
The slightly increased group mean of the relative brain weight of the high dose male animals was within the range of the 5% and 95% percentiles of the test institute's historical control data (refer to Text table 8-23).The relative brain weights of 7 of 19 animals (nos. 322, 326, 330, 334, 335, 338, and 340) were above the 95% percentile of the test institute's historical control data (refer to Text table 8-23), but none of the values was above the maximum of the historical control data range (not considering values excluded from evaluation).
A similar slight increase of the relative brain weight was also noted for the males of the F0 generation.
Heart
The mean absolute heart weight of the male animals (1.381 g/kg b.w., refer to Text table 8-23) was slightly decreased (refer to Text table 8-22) compared to the control group (1.529 g/kg b.w.) but within the range of the 5% and 95% percentiles of the test institute's historical control data (see Text table 8-23).
A similar slight decrease of the absolute heart weight was also noted for the males of the F0 generation.
Prostate gland
The mean absolute prostate gland weight of the male animals (1.2464 g/kg b.w.) was slightly reduced compared to the control group (1.5276 g/kg b.w., refer to Text table 8-22) but was well within the range of the 5% and 95% percentiles of the test institute's historical control data (see Text table 8-23).
Spleen
The mean relative and the mean absolute spleen weights of the male high dosed animals were slightly reduced compared to the control group (refer to Text table 8-22) but were all within the ranges of the respective 5% and 95% percentiles of the test institutes historical control data (see Text table 8-23).
Testes
The mean absolute weights of the left and the right testis of the male high dosed animals were slightly reduced compared to the control group (refer to Text table 8-22) but were well within the range of the range of the 5% and 95% percentiles of the test institute's historical control data (see Text table 8-23).
Thyroid/Parathyroid
The mean relative weight of the left part of the thyroid/parathyroid was slightly reduced compared to the control group (refer to Text table 8-22) but was well within the range of the 5% and 95% percentiles of the test institute's historical control data (see Text table 8-23).
Concluding all above observations, the minor changes noted for the absolute and/or relative organ weights described afore are not considered as an immediate effect of the test item but are regarded as secondary effects due to the slightly lower body weights of the high dosed male animals or as stress-related.

COHORT 1A Females
No test item-related changes in relative and absolute organ weights were noted for the female animals treated with 60 or 200 mg/kg b.w./day.
The relative and absolute liver weights of the animals treated with 600 mg/kg b.w./day appeared to be slightly increased by 10.0% and 13.4%, respectively, compared to the control group (not statistically significant at p ≤ 0.05 or p ≤ 0.01 in any case). Similar increases were observed for the female animals of the high dosed group of the F0 generation (see Section 7.11.5 and Text table 7-15). The liver weight changes can be correlated with the histopathological findings of increased incidence and/or severity of centrilobular hepatocellular hypertrophy ( and are considered to be test item-related.
In contrast to the observations made for the female animals of the F0 generation and of Cohort 1B of the F1 generation, no increase was noted for the relative adrenal gland weights for the females of Cohort 1A of the F1 generation. This difference is considered to be due to the younger in age of these animals at the time of dissection.

COHORT 1B
MALES AND FEMALES
No test item-related influence was noted on the relative and absolute organ weights of the male and female animals treated with 60, 200 or 600 mg/kg b.w./day.
Slight but statistically significant differences (at p ≤ 0.01 or p ≤ 0.05) were noted for the relative weight of the left testis and adrenal gland, and the absolute weight of the left epididymidis for the male animals between the high dose group and the control group. Further, the female animals treated with the intermediate dose revealed a slightly increased absolute weight of the right ovary in comparison to the control group. However, most of the individual data are within the ranges of the test institute's historical control data, and if not, there is no noteworthy difference between the control group and the respective treatment group with regard to the values outside that range. Therefore, the slight changes observed are not considered to be test item-related but to be of spontaneous nature.
The kidney weights of the animals of Cohort 1B of the F1 generation were not examined. Details see Text tables 9-9, 9-10 an 9-11.

The mentioned text tables can be found in the attached document "Text Tables OECD443 DHN".

Gross pathological findings:

no effects observed

Description (incidence and severity):

COHORT 1A Males and females (surviving animals)
Macroscopic post-mortem findings
No toxicologically relevant macroscopic changes that could be associated with the test item were recorded for the surviving animals treated with 60, 200 or 600 mg/kg b.w./day at necropsy. All macroscopic findings recorded, with exception of enlarged kidneys (see further below), were lesions within the range of normal background changes or physiological alterations which may be observed in the present type of study or in animals of this strain and age, or are incidental gross appearances without corresponding histomorphological changes).
Three (3) male animals (nos. 288, 293, and 294) treated with the intermediate dose and one animal treated with the high dose (no. 329) revealed enlarged kidneys. This finding corresponds to increased kidney weights noted for the affected animals. and is considered to be test item-related. However, increased kidney weights are considered to be due to an alpha 2-microglobulin-mediated process being specific for male rats and are regarded as not relevant for humans and, hence, non-adverse.
All other findings noted at macroscopic inspection at necropsy are not considered to be test item-related and are summarized in Text table 8-19 which can be found in the attached document "Text Tables OECD 443 DHN".

COHORT 1 B Males and females (surviving animals)
No toxicologically relevant macroscopic changes associated with the test item were recorded at necropsy for the surviving male and female animals treated with 60, 200 or 600 mg /kg b.w./day. All macroscopic findings recorded, with exception of enlarged kidneys (see further below), were lesions within the range of normal background changes or physiological alterations which may be observed in the present type of study or in animals of this strain and age, or are incidental gross appearances.
Two male animals (nos. 488, 498) treated with 600 mg/kg b.w./day revealed enlarged kidneys. This finding was confirmed by increased kidney weights noted for the affected animals and is considered to be test item-related. However, increased kidney weights are considered to be due to an alpha 2-microglobulin-mediated process being specific for male rats and are regarded as not relevant for humans and, hence, non-adverse.
Comparable observations were made for the male animals of the F0 generation in form of increased kidney weights (see Section 7.11.5) and for the animals of Cohort 1A of the F1 generation in form of enlarged kidneys noted macroscopically and increased kidney weights. The findings noted at macroscopic inspection at necropsy are summarized in Text table 9-7 which can be found in the attached document "Text Tables OECD 443 DHN".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A:
The histopathological examination was performed on up to 20 animals per sex and group of the control group 1 and of the high dose group 4 of Cohort 1A of the F1 generation .
Microscopic changes attributed to the test item-treatment were observed in the animals treated with the high dose of 600 mg/kg b.w./day as follows:
Liver
- Increased incidence/severity of centrilobular hepatocellular hypertrophy in both sexes. As with the F0 generation, there were no further indicators of tissue injury.
This is considered to be of metabolic nature (like as enzyme induction) and hence, not adverse.
Kidneys
Treatment-related changes were observed in males only:
- Hyaline droplet deposit in the proximal tubular epithelia, accompanied by tubular cell necrosis with granular casts, as well as mononuclear cell infiltration (mononuclear cell focus/foci) and increased incidence/severity of tubular basophilia.
- The renal lesions are most likely to be a male-rat specific alpha 2-microglobulin nephropathy, and not relevant to humans.
Stomach
- Hyperkeratosis in both sexes (8 of 19 males and 5 of 16 females); there was also focal/multifocal mucosal degeneration in 2 males and 2 females.
These findings are likely due to local effect related to the irritating nature of the test item (not due to systemic effects).
Adrenal glands and thymus
- Adrenal glands: Increased incidence/severity of diffuse cortical hypertrophy in both sexes.
- Thymus: Group mean severity grade of thymic atrophy was increased in both sexes.
These changes are considered to be a stress-related secondary effect.
Female reproductive organs
- Ovaries: Decreased new corpora lutea, concomitant with increased atretic follicles, were noted in 5 of 16 females. Meanwhile, old corpora lutea were identified even in these affected females. These findings suggest that the regularity of the estrous cycle had been disrupted (i.e. there was an interruption of the ovulation and estrous cycling at least within the last 1 to 3 cycles). In the quantitative evaluation (see Text table 8-26 and Text table 8-27), a significant decrease in the values of corpora lutea and a significant increase in the values of growing follicles were detected in comparison to the control group. However, no differences were seen in the numbers of primordial follicles.
- Vagina: Increase in the number of animals showing anestrus (acycling mucosa).
- These findings are considered secondary to maternal stress

Male reproductive organs
As with the F0 generation, there were no toxicologically relevant microscopic changes in the male reproductive organs and tissues including testis, epididymis, vas deferens, prostate, coagulating glands and seminal vesicles of the males from Cohort 1A of the F1 generation.
The results of the histopathological examination of the animals of Cohort 1A of the F1 generation are summarised in the text table 8-25 which can be found in the attached document "Text Tables OECD443 DHN".

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous stage at necropsy COHORT 1 A Females
The stage of estrous cycle of the animals of Cohort 1A was monitored for 14 test days between test day 53 and test day 67 of the F1 generation part of the 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 animals treated with 60, 200 or 600 mg/kg b.w./day.
The estrous cycle data of the female animals of Cohort 1A are summarised in the text table 8-18 which can be found in the attached document "Text Tables OECD443 DHN".

Estrous stage at necropsy COHORT 1 B Females
No test item-related influence was noted on the distribution of the stages of the estrous cycle at necropsy of the female animals treated with 60, 200 or 600 mg/kg b.w./day.
The stages of estrous cycle at necropsy are summarised in the text table 9-8 which can be found in the attached document "Text Tables OECD443 DHN".

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

COHORT 1A
Sperm number
No test item-related influence was noted on the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue for the male animals treated with 60, 200 or 600 mg/kg b.w./day. There were no noteworthy differences between any of the test item-treated groups and the control group.
Sperm motility
No test item-related influence was noted on the total number of motile and non-motile spermatozoa and for the percentage of motile spermatozoa in the epididymal cauda of the male animals treated with 60, 200 or 600 mg/kg b.w./day. There were no noteworthy differences between any of the test item-treated groups and the control group.
Sperm morphology
No test item-related influence was noted on the sperm morphology for the male animals treated with 60, 200 or 600 mg/kg b.w./day. There were no noteworthy differences between any of the test item-treated groups and the control group.
The examination of up to 4000 spermatozoa (200 per animal) per test group did not reveal any malformation.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive parameters of the parental females (COHORT 1B)
Pre-coital time: No test item-related influence was noted.
Gestation length: No test item-related influence was noted.
Gestation index: No test item-related influence was noted. All pregnant females of all dose groups littered live pups. The gestation indices and the proportionate numbers of the dams with live pups of all pregnant animals of Cohort 1B of the F1 generation are listed in Text table 9-12 which can be found in the attached document "Text Tables OECD443 DHN".
Fertility index: No test item-related influence was noted. For one female treated with the low dose of 60 mg/kg b.w./day, and for 2 females treated with the intermediate dose level of 200 mg /kg b.w./day copulation was verified (by sperm detection), but the animals were not pregnant. Accordingly, the fertility index was reduced to 88% at the intermediate dose level, and to 95% for the low dosed animals.
At the intermediate dose level, 2 non-pregnant female animals with verified copulation were noted. Further, there were 3 non-pregnant females for which copulation could not be verified.
The non-pregnant animals are listed in the text table 9-13, the fertility indices and the proportionate numbers of the pregnant females with verified copulation of females with verified copulation are listed in Text table 9-14 which can be found in the attached document "Text Tables OECD443 DHN".

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity (Cohort 1B)

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

Remarks:

no adverse effects on reproductive parameters

2

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity (Cohort 1A and Cohort 1B)

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No test item-related differences were noted for the viability indices of the pre- and the post-cull period between the control group and any of the test item-treated groups. The viability index was highest at approx. 97% in both the low and the high dose group. At the intermediate dose, the viability index being at 92% was just below the control group's value of approx. 93% (see Text table 7-29, Text table 7-30, and Text table 7-31 which can be found in the attached document "Text Tables OECD443 DHN").

Number of live F1 pups
No test item-related influence was noted on the number of live pups for the animals treated with 30/60 mg/kg b.w./day (group 2).
At the intermediate dose of 100/200 mg/kg b.w./day (group 3), the number of live pups per dam was reduced by 20.4% (statistically significant at p ≤ 0.05) on lactation day 1 and by 21.6% (not statistically significant) on lactation day 4 (pre-culling) in comparison to the control group.
The dams treated with the high dose of 300/600 mg/kg b.w./day (group 4) revealed a decrease in the number of live pups by 25.3% (statistically significant at p ≤ 0.01) on lactation day 1 and by 22.0% (statistically significant at p ≤ 0.05) on lactation day 4 (pre-culling) compared to the control.
The reduced number of live pups per dam correlated with the reduced number of implantation sites noted at the intermediate and high dose level (see details on results) and is therefore considered to be test-item-related. However, these finding can be correlated to maternal stress as described in the histopathologically report and also seen in the biochemistry and heamatological parameters. Therefore, these findings are not adverse.
As a consequence of the smaller numbers of live pups at the intermediate and high dose, lesser pups were culled on lactation day 4 at these dose levels. As a result, there were no noteworthy differences in the numbers of live pups per dam in comparison to the control group on lactation days 7, 14, and 21.
The mean numbers of live pups per dam are summarised in the text table 7-32 which can be found in the attached document "Text Tables OECD443 DHN".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup litter weight: No test item-related influence was noted.
The changes in litter weight of F1 pups are considered as result of a smaller number of live pups in the affected group and not to be an adverse effect on the post-natal development of pups.
On lactation day (LD) 1, the mean litter weight combined for male and female F1 pups was reduced by up to 30.4% compared to the control group starting at the low dose level of 30/60 mg/kg b.w./day (statistically significant at p ≤ 0.05 at the intermediate dose and at p ≤ 0.05 at the high dose).
The slight litter weight decrease in comparison to the control group observed at the low dose level (group 2) was caused by changes noted only for female F1 pups. For the male F1 pups, no noteworthy litter weight changes at all compared to the control group were noted throughout the entire lactation period.
For the female F1 pups, the litter weight decrease at the intermediate dose level of 100/200 mg/kg b.w./day (group 3) had subsided on LD 7 and a slight increase in litter weight compared to the control was noted for LD 7, LD 14, and LD 21.
At the high dose of 300/600 mg/kg b.w./day, the change in litter weight ranged from -27.3% (LD 1) to -10.5% (LD 21) for the male F1 pups (not statistically significant at p ≤ 0.01 or p ≤ 0.05 in any case) and from -29.6% (LD 1) to -15.8% (LD 7) for the female F1 pups (statistically significant at p ≤ 0.01 or p ≤ 0.05 on LD 1, LD 14, and LD 21). The decreases in litter weight when assessed combined for male and female F1 pups ranging from -30.4 % (LD 1) to -11.3% (LD 7) were always statistically significant at p ≤ 0.01 on all lactation days of examination.
At all dose levels, the differences in litter weight in comparison to the control diminished with progress of the lactation period.
The slightly decreased litter weights correlate with the slightly reduced number of live pups observed at each respective dose level, and, at the high dose, with the slightly reduced pup body weights, which, however, are regarded as unrelated to the test item. Therefore, the lower litter weights in comparison to the control group are considered to be a result of the smaller number of live pups in the affected test item treated groups and not to be an adverse effect on the postnatal development of pups. The statistically significant changes noted for the litter weight during the lactation period that are not considered as adverse events are given in the text table 7-34 which can be found in the attached document "Text Tables OECD443 DHN".

Pup body weight: No test item-related influence was noted.
A slight decrease by up to 7.5% (females on lactation day 14) compared to the control group was noted for the mean body weight of male and female pups in the high dose group treated with 300/600 mg/kg b.w./day on lactation days 14 and 21. The female pups appeared to be slightly more affected than the male pups. However, none of the changes attained statistical significance (at p ≤ 0.01 or p ≤ 0.05) and the data were well within the 5% and 95% percentiles of the test institute's historical control data. In addition, no noteworthy difference was noted for the body weight at autopsy between the pups of the high dose group and the control group pups.
During the further progress of the study, the F1 pups from the high dose group assigned to cohorts 1A and 1B revealed opposite changes in body weight at the adult stage than at the pup stage: the high dosed male animals' body weight was slightly reduced compared to the control group whereas the females animals did not reveal any noteworthy body weight changes in comparison to the control.
Based on the considerations from above, the slight difference in body weight between the pups of the high dose group and of the control pups is regarded as a coincidental effect and to be not test item-related.
Runts
In total two runts were noted on lactation day 1: one female pup (no. 74-09) at the low dose level (group 2) and one female pup (no. 176-10) at the high dose level (group 4). The body weight of pup no. 74-09 recovered during the lactation period and the pup was selected for Cohort 1A. Pup no. 176-10 (high dose level, group 4) was cannibalised on lactation day 2. An incidence of one runt per group is not an uncommon finding in rats. Therefore, the occurrences of the runts are considered to be coincidental events and not 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):

Thyroid hormone levels: No test item-related influence was noted on the serum levels of T4 and/or TSH on post-natal day 4 and/or 22 at any dose level.
The T4 levels of the male pups in the group treated with 300/600 mg/kg b.w./day appeared to be increased by 35.2% on PND 4 (statistically significant at p ≤ 0.05). No noteworthy change was noted for the female pups. However, the change noted for the male pups is regarded to be due to the relatively low value observed in the control group which is just marginally above the 5% percentile of the test institute's historical control data. The T4 level of the pups of the high dose group was close to the 25% percentile of the historical control data. Further, no effect was observed on the TSH levels of the male pups of the high dose group. Therefore, the increase noted for the high dose group compared to the control group is not considered as test item-related but to be due to coincidental variation. Comparisons of the T4 levels of male F1 pups from the present study with the test institute's historical control data are given in the text table 7-39 which can be found in the attached document "Text Tables OECD443 DHN".
Thyroid-stimulating hormone (TSH - PND 22 only)
No test item-related influence was noted on the TSH serum levels of the male and female F1 pups of the control group and of the groups treated with 30/60, 100/200 or 300/600 mg/kg b.w./day on post-natal day 22.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Males - Cohort 1A alone
No test item-related differences were noted for the time point of balano-preputial separation and the body weight at the time point of balano-preputial separation between the males of the control group and the animals treated with 60, 200, or 600 mg/kg b.w./day.
The day of balano-preputial gland separation and the body weight on the day of balano-preputial gland separation appeared to be slightly increased at the low and intermediate dose level in comparison to the control (statistically significant at p ≤ 0.01 in each case). There was no clear dose-response-relationship, no noteworthy difference was noted between the high dose-treated animals and the control animals. Therefore, the changes at the low and intermediate dose level are considered as coincidental effects having no toxicological relevance.
The sexual maturation parameters of the male animals of Cohort 1A are summarised in the text table 8-12.

Males - Cohort 1A and Cohort 1B combined
The combined assessment of the day of the balano-preputial gland separation of Cohorts 1A and 1B did not result in any noteworthy difference compared to the assessment of Cohort 1A alone.
The day of balano-preputial gland separation and the body weight on the day of balano-preputial gland separation appeared to be slightly increased at the low and intermediate dose level in comparison to the control (statistically significant at p ≤ 0.01 or p ≤ 0.05) but there was no clear dose-response-relationship, and no noteworthy difference was noted between the high dose-treated animals and the control animals. The changes at the low and intermediate dose level are considered as coincidental effects having no toxicological relevance.
The sexual maturation parameters of the male animals of Cohorts 1A and 1B combined are summarised in the text table 8-13.

Females - Cohort 1A alone
No test item-related differences were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening between the females of the control group and the animals treated with 60, 200 or 600 mg/kg b.w./day
The day of vaginal opening appeared to be slightly decreased at the intermediate dose level in comparison to the control (statistically significant at p ≤ 0.01) but there was no dose-response-relationship and no noteworthy difference was noted between the high dose-treated animals and the control animals. All data were well within the range of the test institute's historical control data (obtained for combined Cohorts 1A and 1B, see Text table 8-16). Therefore, the change noted at the intermediate dose level is considered as a coincidental effect being not test item-related.
The sexual maturation parameters of the female animals of Cohort 1A are summarised in the text table 8-14.

Females - Cohort 1A and Cohort 1B combined
The combined assessment of the day of vaginal opening of Cohorts 1A and 1B did not result in any noteworthy difference compared to the assessment of Cohort 1A alone.
The day of vaginal opening appeared to be slightly decreased at the intermediate dose level in comparison to the control (statistically significant at p ≤ 0.01) but there was no dose-response-relationship and no noteworthy difference was noted between the high dose-treated animals and the control animals. All data were well within the range of the test institute's historical control data (see Text table 8-16). Therefore, the change noted at the intermediate dose level is considered as a coincidental effect being not toxicologically relevant.
The sexual maturation parameters of the female animals of Cohorts 1A and 1B of the F1 generation combined are summarised in the text table 8-15, historical ontrol data can be found in text table 8-16.

Appearance of cornified cells in the vaginal lavage
The day of the appearance of cornified cells in the vaginal lavage indicates the time point the rat cycle reaches the stage of estrus for the first time and is given as post-natal day (PND).
No test item-related differences were noted for the day of appearance of cornified cells in the vaginal lavage between the females of the control group and the animals treated with 60, 200 or 600 mg/kg b.w./day.
The day of appearance of cornified cells in the vaginal lavage appeared to be decreased in all test item-treated groups in comparison to the control. However, these differences are due to the relatively high mean value noted for the control group, which is influenced by the very high value of 15 days observed for animal no. 224 , and are not considered as test item-related (no statistical significance at p ≤ 0.05/p ≤ 0.01 was noted).
The appearance of cornified cells in the vaginal lavage of the female animals of Cohorts 1A and 1B combined are summarised in the text table 8-17.

Males - Cohort 1B
No test item-related differences were noted for the time point of balano-preputial separation and the body weight at the time point of balano-preputial separation between the males of the control group and the animals treated with 60, 200 or 600 mg/kg b.w./day.
The sexual maturation parameters of the male animals of Cohort 1B of the F1 generation are summarised in the text table 9-5.

Cohort 1A and Cohort 1B combined
The combined assessment of the day of the balano-preputial gland separation of Cohort 1A and Cohort 1B of the F1 generation did not result in any noteworthy difference compared to the assessment of Cohort 1B alone

Females - Cohort 1B
No test item-related differences were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening between the females of the control group and the animals treated with 60, 200 or 600 mg/kg b.w./day
The sexual maturation parameters of the female animals of Cohort 1B of the F1 generation are summarised in the text table 9-6.

Cohort 1A and Cohort 1B combined
The combined assessment of the day of vaginal opening of Cohort 1A and Cohort 1B of the F1 generation did not result in any noteworthy difference compared to the assessment of Cohort 1B alone.

The mentioned text tables can be found in the attached document "Text Tables OECD443 DHN".

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item-related influence was noted.
The absolute ano-genital distance (in ocular units) of the male F1 pups was slightly but statistically significantly (at p ≤ 0.05) increased by approx. 7% at the low and intermediate dose level. However, the marginal changes were well within the 5% and 95% percentiles of the test institute's historical data. No noteworthy changes were noted for the relative ano-genital distance (in ocular units per kilogram body weight) for the male F1 pups. Further, no dose dependency was observed because there was no noteworthy difference between the pups in the high dose group and the control group's pups. Therefore, none of the changes observed is considered to be test item-related.
The changes in the ano-genital distance of the male F1 pups considered not test item-related are listed in the text table 7-35 the historical control data can be found in text table 7-36 which can be found in the attached document "Text Tables OECD443 DHN".

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item-related influence was noted.
In total, 11 male pups with up to 3 nipples per pup were noted across all treatment groups and the control group. Of these, 7 pups revealed only 1 nipple per pup. For one pup treated with the intermediate dose, 3 nipples were observed. However, there were no noteworthy differences between the control group and any of the test item-treated groups. The highest number of affected pups and the highest total count of nipples per group were observed for the control group. Any differences between the groups are considered to be of spontaneous nature. Details on the affected pups and the nipple counts are given in the text tables 7-37 and 7-38 which can be found in the attached document "Text Tables OECD443 DHN".

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for the absolute weights of the organs examined between the control group and the groups treated with 30/60, 100/200, or 300/600 mg/kg b.w./day after terminal sacrifice on lactation day 22.
The mean thymus weight of the male F1 pups of the high dose group 4 appeared to be decreased by 19.6% (statistically significant at p ≤ 0.05) compared to the control group. However, this change is considered to be due to the relatively high thymus weights in the control group. The mean thymus weight of the control F1 pups was increased by approx. 15% compared to the control F2 pups. The mean thymus weight of the F1 pups of the high dose group was reduced by only 4% compared to the F2 pups of the control group.
Therefore, the reduced mean thymus weight noted for the F1 pups in the high dose group is regarded to be within the range of normal biological variation and considered as a coincidental effect that is not test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External and internal examination of the pups of F1 pups
An external examination was performed on all F1 pups that were culled on PND 4, were terminally sacrificed on PND 22/23/24, or were eventually found dead during the lactation period. An internal examination was performed only on the animals terminally sacrificed.
External examination (on PND 4 or 22/23/24, or when found dead)
No test item-related gross abnormalities, in particular no malformations and no variations, were noted at external macroscopic examination of the F1 pups from the groups treated with 30/60, 100/200 or 300/600 mg/kg b.w./day and the control group after culling on lactation day 4, after terminal sacrifice on lactation day 22/23/24, or when found dead earlier.
One control group pup (no. 42-12) revealed a thickened abdomen . This was a spontaneous change due to the lack of test item-treatment.
For one pup (no. 129-05) of a dam treated with the intermediate dose, piloerection was observed on the day of terminal sacrifice (LD 22). A stillborn pup (no. 129-05) of a dam treated with the high dose revealed a stubby tail (LD 1).
The findings noted for the two pups of the two dosed dams are regarded as coincidental changes due to the single occurrence and not to be test item-related.

Internal examination (PND 22/23/24)
No gross abnormalities, in particular no malformations and no variations, were noted at macroscopic examination of the inner organs and tissues of the F1 pups from the control group and the groups treated with 30/60, 100/200 or 300/600 mg/kg b.w./day after terminal sacrifice on lactation day 22/23/24.
One control group pup (no. 29-10) revealed a dilatation (approx. 3 cm) of the renal pelvis in the capsule of the right kidney. This was a spontaneous change due to the lack of test item-treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the male to female ratio of the pups for the groups treated with 30/60, 100/200 or 300/600 mg/kg b.w./day on lactation days 1 and 4.
The sex ratio at the high dose level was nearly the same ast in the control group. At the low dose level, the fractions of male and female pups were nearly identical (126 males, 128 females), whereas in the control group and at the intermediate and high dose level slightly more female pups than male pups were noted. There was no difference between lactation day 1 and lactation day 4. See table 7-33 The changes in the ano-genital distance of the male F1 pups considered not test item-related are listed in the text table 7-33- which can be found in the attached document "Text Tables OECD443 DHN".

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Birth indices and post-implantation loss - F1 pups
No test item-related differences were noted for the mean number of implantation sites per dam, the mean total number of pups (live and dead) born per dam, and the mean number of live born pups per dam between the control group and the group treated with 30/60 mg/kg b.w./day.
The dams treated with 100/200 or 300/600 mg/kg b.w./day revealed a slightly reduced number of implantation sites in comparison to the control group (12.4 and 13.0, respectively, compared to 15.5, statistically significant at p ≤ 0.05 at both dose levels).
The numbers of live born pups and the sum of pups born alive and dead were slightly decreased compared to the control group at the intermediate and high dose level (always statistically significant at p ≤ 0.01 or p ≤ 0.05).
The birth index per group of the dams treated with 300/600 mg/kg b.w./day (82.31%) was slightly reduced by approx. 6 percentage points compared to the control group (89.07%).
The post-implantation loss at group level in the high dose group (21.15%) was nearly 8 percentage points above the control group's value (12.22%) due to increased numbers of resorptions (46 compared to 34 in the control) and stillbirths (9 compared to 4 in the control) within the group. The increased post-implantation loss at the high dose was approx. 6 percentage points above the upper bound of the test institute's historical control data (14.86%). This post-implantation loss in the high dose group was not statistically significant and resulted majorly from increased numbers of resorptions. This is in line with increased levels of maternal stress as described histopathologically above (see histopathological report attached to this report).

The slightly reduced numbers of implantation sites and of live born pups, the slightly decreased sum of pups born alive and dead, the slightly reduced birth index and the slightly increased post-implantation loss described above are considered to be test item-related. However, these effects are most likely secondary to maternal stress related findings which can interfere with follicle maturation with decrease numbers of mature follicles, and a lower number of oocytes are expected and subsequently a lower number of implantation sites.
As not statistically significant, an increased post-implantation loss and reduced birth index is considered to be test item related but not adverse. But most importantly, no test item-related influence was observed on the live birth index at any dose level.

The reproductive data of the F0 generation are summarised in text tables 7-27 and 7-28 which can be found in the attached document "Text Tables OECD443 DHN".

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Postnatal 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

2

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

Remarks:

Effects can clearly be allocated to maternal stress affecting estrous cyclicity and follicle maturation.

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Viability index of F2 pups
Pre- and post-cull period
No test item-related differences were noted for the viability indices of the pre- and the post-cull period between the control group and the groups treated with 60, 200 or 600 mg/kg b.w./day. The pre-culling viability index was highest at 99.62% in the intermediate dose group and lowest at 78.84% in the control group. At the high dose, the viability index being at 83.49% was nearly 5% above the control group's value (see Text table 9-17, Text table 9-18, and Text table 9 19 in the attached document "Text Tables OECD443 DHN").

Number of live F2 pups
No test item-related influence was noted on the number of live pups for the animals treated with 60 or 200 mg/kg b.w./day (groups 2 and 3).
At the high dose of 600 mg/kg b.w./day (group 4), the number of live pups per dam was reduced by 17.3% on lactation day 1 and by 12.5% on lactation day 4 (pre culling) in comparison to the control group (not statistically significant at p ≤ 0.01 or p ≤ 0.05 in either case).
The reduced number of live pups per dam correlated with the reduced number of implantation sites noted at the high dose level and is therefore considered to be test-item-related but not adverse as the reduced number of implantation sites was based on disturbance of the estrous cyclicity, subsequently lower numbers of oocytes as discussed earlier as stress relating finding.
As a consequence of the smaller numbers of live pups at the high dose, lesser pups were culled on lactation day 4 at this dose level. As a result, there were no noteworthy differences in the numbers of live pups per dam in comparison to the control group on lactation days 7, 14, and 21.
The mean numbers of live pups per dam are summarised in the text table 9-20 which can be found in the attached document "Text Tables OECD443 DHN".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Litter weight
No test item-related influence was noted on the mean litter weight for the animals treated with 60, 200 or 600 mg/kg b.w./day.
At the high dose level of 600 mg/kg b.w./day, the mean litter weight combined for male and female F2 pups appeared to be reduced by up to 20.5% compared to the control group (statistically significant at p ≤ 0.05 on lactation day 1). The differences in litter weight diminished with progress of the lactation period, being at 12.5% compared to the control group on lactation day 21. The changes in litter weight were mainly due to differences in the female pups' weights whereas no noteworthy changes were noted for the male pups' weights as of lactation day 4.
The slightly decreased litter weight correlates with the slightly reduced total number of live pups observed at the high dose level. Further, there were 56% female pups in the litters of the control group (165 of in total 293 pups) compared to 50% in the high dose group (110 of in total 218 pups). Therefore, the lower litter weights in the high dose group in comparison to the control group are considered to be due to the lower number of female pups and not to be an adverse effect on the post-natal development of pups. The statistically significant changes noted for the litter weight during the lactation period that are not considered as adverse events are given in the text table 9-22 which can be found in the attached document "Text Tables OECD443 DHN".

Pup body weight
No test item-related influence was noted on the body weight of the male and female F2 pups for the groups treated with 60, 200 or 600 mg/kg b.w./day.
All data were well within the 5% and 95% percentiles of the test institute's historical control data (obtained from F1 pups).
The mean body weight at autopsy of the male F2 pups of the intermediate dose group appeared to be slightly increased by 16.5% compared to the control pups (statistically significant at p ≤ 0.05). A slight increase (by 13.8%, not statistically significant at p ≤ 0.01 or p ≤ 0.05) was also noted at the low dose level whereas no change was observed at the high dose. The increased mean body weight at autopsy correlates with the higher body weights noted for the male F2 pups at the low and intermediate dose level. Due to the lack of dose dependence, the changes at the low and intermediate dose are considered as coincidental effects that are not test item-related.

Runts
One runt was noted on lactation day 1 at the intermediate dose level (group 3, pup no. 466 09). This pup was culled on lactation day 4. The occurrences of the runt is considered to be a coincidental event and not 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:

not examined

Description (incidence and severity):

So far it was not considered necessary to analyse the hormones T4 and TSH of the F2 pups as the results of the hormone analyses of the F0 and F1 Generation animals revealed no test item-related findings and all results were within the normal range of variation.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the ano-genital distance of the male and female F2 pups for the groups treated with 60, 200 or 600 mg/kg b.w./day.
The absolute ano-genital distance of the female F2 pups (in ocular units) was slightly but statistically significantly (at p ≤ 0.05) increased by 13.2% at the intermediate dose level. However, the marginal change was well within the 5% and 95% percentiles of the test institute's historical data. No noteworthy change was noted for the relative ano-genital distance (in ocular units per kilogram body weight) for the female F2 pups. Further, no dose dependency was observed because there was no noteworthy difference between the pups in the high dose group and the control group's pups. Therefore, the change observed is not considered to be test item-related. Comparisons of the ano-genital distance from the present study with the test institute's historical control data are given in Text table 9-23 which can be found in the attached document "Text Tables OECD443 DHN".

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test-item-related differences were noted for the number of nipples between the male pups of the control group and the male pups of the groups treated with 60, 200 or 600 mg/kg b.w./day.
In total, 10 male F2 pups with one nipple per pup were noted across all treatment groups and the control group. Two nipples per pup were observed only for one pup in the high dose group. In the high dose group, the number of pups with nipples was slightly increased compared to the control group (4 dams with 5 affected pups and in total 6 nipples vs. 2 dams with 2 affected pups and in total 2 nipples). However, there is no noteworthy difference between the F2 pups of the high dose group and the F1 pups of the control group. Therefore, the slightly higher number of nipples noted for the high dose group in comparison to the control group in F2 pups is considered to be within the range of normal biological variation and not to be test item-related. Details on the affected F2 pups and the nipple counts are given in the text tables 9-24 and 9-25 which can be found in the attached document "Text Tables OECD443 DHN".

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for the absolute organ weights examined between the control group and the groups treated with 60, 200 or 600 mg/kg b.w./day after terminal sacrifice on lactation day 22.
A few slight but statistically significant (at p ≤ 0.05 or p ≤ 0.01) changes were noted for various organ weights of the F2 pups in the intermediate dose group in comparison to the control group. However, these slight changes are regarded as related to the slightly higher body weights of the pups.
Due to the lack of noteworthy changes in the pups of the high dose group and the absence of any dose dependency, all changes are considered be not test item-related but to be within the range of normal biological variation. The statistically significant changes in organ weights of F2 pups considered not test item-related are listed in the text table 9-26 which can be found in the attached document "Text Tables OECD443 DHN".

Gross pathological findings:

no effects observed

Description (incidence and severity):

The external examination was performed on all pups that were culled on PND 4, were terminally sacrificed on PND 22/23/24, or were eventually found dead earlier during the lactation period. The internal examination was performed only on the animals terminally sacrificed.
External examination (on PND 4 or 22/23/24, or when found dead)
No test item-related gross abnormalities, in particular no malformations and no variations, were noted at external macroscopic examination of the F2 pups from the groups treated with 60, 200 or 600 mg/kg b.w./day and the control group after culling on lactation day 4, after terminal sacrifice on lactation day 22, 23, or 24, or when found dead earlier.
One pup (no. 440-01) in the low dose group revealed a tail with annular restrictions at terminal sacrifice on lactation day 22. This finding is regarded as a coincidental change due to the single occurrence and not to be test item-related.
Internal examination (PND 22/23/24)
No gross abnormalities, in particular no malformations and no variations, were noted at macroscopic examination of the inner organs and tissues of the F2 pups from the control group and the groups treated with 60, 200 or 600 mg/kg b.w./day after terminal sacrifice on lactation day 22, 23, or 24.
One control group pup (no. 398-01) revealed yellow spotted kidneys. This was a spontaneous change due to the lack of test item-treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the male to female ratio of the F2 pups for the groups treated with 60, 200 or 600 mg/kg b.w./day on lactation days 1 and 4.
The sex ratio of pups at the high dose level was nearly the same in all test item-treated groups. In the control group, slightly more female pups were noted than male pups (165 females vs. 128 males). There was no noteworthy difference between lactation day 1 and lactation day 4 for all test groups including the control (s. also text table 9-21, which can be found in the attached document "Text Tables OECD443 DHN").

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

Birth indices and post-implantation loss - F2 pups
No test item-related differences were noted for the mean number of implantation sites per dam, the mean total number of pups (live and dead) born per dam, and the mean number of live born pups per dam between the control group and the group treated with 60 or 200 mg/kg b.w./day.
The dams treated with or 600 mg/kg b.w./day revealed a slight decrease by 11% for the mean number of implantation sites in comparison to the control group (13.9 compared to 15.6). This effect is similar to that observed for the F0 generation, but is less pronounced and not statistically significant (at p ≤ 0.01 or p ≤ 0.05).
The number of live born pups and the sum of pups born alive and dead were slightly reduced compared to the control group at the high dose level (though not statistically significant at p ≤ 0.01 or p ≤ 0.05, see Text table 9-15 for details).
The birth index per group of the dams treated with 600 mg/kg b.w./day was slightly reduced by approx. 6 percentage points compared to the control group (87.60% compared to 93.35%).
The post-implantation loss at the group level in the high dose group was 5.5 percentage points above the control group's value (12.80% compared to 7.28%) due to a slightly increased number of resorptions (31 compared to 21 in the control) within the group. The increased post-implantation loss at the high dose was approx. 6 percentage points above the upper bound of the test institute's historical control data (6.73%).
However, as none of these effects (reduced numbers of implantation sites and of live born pups, the slightly decreased sum of pups born alive and dead, and the slightly increased post-implantation loss described above) were statistically significant and similar effects were observed at F0/F1 Generation but with much higher severity, these findings are considered test item-related. As these effects are most likely secondary to maternal stress related findings which can interfere with follicle maturation with decrease numbers of mature follicles and a lower number of oocytes are expected and subsequently a lower number of implantation sites these effects were considered to be not adverse as the same effects were observed for the F0 generation but with higher severity in F0.
As for the F1 pups, no test item-related influence was observed on the live birth index at any dose level.
The reproductive data of the animals of Cohort 1B of the F1 generation are summarised in the text table 9-15, the historical control data can be found in text table 9-16 which can be found in the attached document "Text Tables OECD443 DHN".

Effect levels (F2)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Postnatal development

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

2

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:

other: findings can clearly be allocated to maternal stress affecting estrous cyclicity and follicle maturation.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

s. attached documents "Text Tables OECD443 DHN" and "Tables Study Nr 37571"

Applicant's summary and conclusion

Conclusions:

The aim of the study was to evaluate the effects of the test item with regard to general and reproductive toxicity in the F0 parental animals, and to developmental toxicity in the F1 generation from weaning until adulthood (according to OECD guideline 443). The initial dose levels of 30, 100, and 300 mg/kg b.w./day were increased to 60, 200, and 600 mg/kg b.w./day as of test day 30 as no relevant toxicological findings had been observed in the animals of the F0 generation up to test day 29.
The substance had no effect on reproductive parameters. NOAEL for general toxicity: 200 mg/kg bw/day.

Executive summary:

The aim of the study was to evaluate the effects of the test item with regard to general and reproductive toxicity in the F0 parental animals, and to developmental toxicity in the F1 generation from weaning until adulthood (according to OECD guideline 443). The initial dose levels of 30, 100, and 300 mg/kg b.w./day were increased to 60, 200, and 600 mg/kg b.w./day as of test day 30 as no relevant toxicological findings had been observed in the animals of the F0 generation up to test day 29.

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

F0 generation

General toxicity

NOAEL 200 mg/kg b.w./day.

Based on one premature death and reduced body weight observed for the animals treated with 300/600 mg/kg b.w./day.

Reproductive toxicity

NOAEL at least 600 mg/kg b.w./day.

Observed effects on reproductive performance in F0 generation are all originating form stress related findings (decreased body weights, reduced glucose levels, increased blood urea nitrogen/creatinine ratio, enlarged adrenal glands and increased weights as well as reduced thymic size and decreased thymus weights, together with increased incidence and/or severity of diffuse adreno-cortical hypertrophy and thymic atrophy). As general stress is known to affect estrous cyclicity and follicle maturation these findings are all secondary to maternal stress.

 

F1 generation

Developmental toxicity

Adverse effects on pre- and postnatal development:

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

NOAEL at least 600 mg/kg b.w./day

The slightly reduced number of implantation sites and subsequently  slight decrease of the numbers of live born pups and the sum of pups born alive and dead noted for the animals treated with 200 or 600 mg/kg b.w./day, and a slightly reduced birth index for the animals treated with 600 mg/kg b.w./day can clearly be allocated to maternal stress affecting estrous cyclicity and follicle maturation.

 

1. b) Adverse effects on postnatal development (pups):

NOAEL at least 600 mg/kg b.w./day.

 

General toxicity (Cohort 1A and Cohort 1B)

NOAEL 200 mg/kg b.w./day.

Based on premature deaths and reduced body weight observed for the animals treated with 600 mg/kg b.w./day.

Reproductive toxicity (Cohort 1B)

NOAEL at least 600 mg/kg b.w./day.

 

F2 generation

Developmental toxicity

Adverse effects on pre- and postnatal development:

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

NOAEL at least 600 mg/kg b.w./day.

Slight, but statistically not significant effects were seen on numbers of implantation sites and of live born pups as seen also in F0 Generation but with lower severity. In F0 females these findings can clearly be allocated to maternal stress affecting estrous cyclicity and follicle maturation.

No developmental toxicity was seen in F2 pups. Any effects (not statistically significant) were based on systemic maternal toxicity in F1B females.

 

1. b) Adverse effects on postnatal development (pups):

NOAEL at least  600 mg/kg b.w./day.