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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Public available NTP repeated dose toxicity studies from 2011 showed no indications of an adverse effect on vaginal cytology, sperm and on reproductive organs from the 13-week inhalation study on Fischer rats (NTP, 2011). In B6C3F1 mice, no effects on vaginal cytology and sperm were noted in the 13-week inhalation study, but uterus atrophy and atrophy of the ovary were found in the absence of systemic toxicity (NTP, 2011). In the 2-years inhalation study (NTP, 2011) with mice no uterus atrophy and no ovary atrophy were observed at the same doses that are used in the 13-week inhalation study. Therefore, it can be concluded that these may be mice specific and transient effects. However a 2 year inhalation study with rats (NTP, 2011) showed effects on the uterus (incidences of stromal polyp and endometrium hyperplasia in the high dose group 660 mg/m³ were significantly greater than those in the chamber controls; see Chapter 7.7 "Carcinogenicity" of IUCLID and Chapter 5.8 of this CSR).


In a final decision on a testing proposal dated 5 April 2019 (Decision numberTPE-D-2114465819-31-01/F ) ECHA requested to perform an extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./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);


-  Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation;


 


A reproduction screening study according to OECD guideline 421 (LPT, 2020) was used as dose range finding study for the OECD 443 study (LPT, 2021). In this DRF study, the test item was administered orally to Sprague Dawley rats at dose levels of 15, 50 or 150 mg/kg b.w./day. No adverse effect on mating performance, fertility, or gestation length was detected. The NOAEL for systemic toxicity was considered to be 50 mg/ kg bw/ day, based on clinical observations (salivation, reduced motility) and reduced body weight. The NOAEL for reproductive toxicity (adverse effects on the reproductive parameters of the parental females/ pre- and postnatal development/ postnatal development) is above 150 mg/ kg bw/ day. Histopathological examinations did not reveal any test-item related changes of the reproductive organs. Based on this study the dose levels for the OECD 443 study were set. Due to the reduced body weight of the female animals and the clinical symptoms the maximum tolerated dose was considered to be 150 mg/kg.


Based on the ECHA decision, the OECD 443 study with the basic study design with a 10-week pre-mating time (Cohorts 1A, and 1B) without extension to mate the Cohort 1 B animals to produce the F2 generation was conducted.


In this OECD 443 study (Provivo, 2022) no adverse effect on mating performance, fertility or gestation length was detected.


The oral exposure to 15, 50, and 150/250 mg test item/kg bw (male and female Sprague-Dawley rats) led to no detectable changes in fertility. In detail, no test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).


The prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) was also not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of oestrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).


Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive was considered to be above 250 mg test item/kg/day.


However, the parental animals as well as the animals of the F1 generation showed hematological and biochemical effects indicating hemolytic anemia. Also histopathological findings indicated anemia. Furthermore, the parental dams showed a decreased body weight during the lactation period. Based on these findings the systemic general NOAEL was determined to be 50 mg/kg bw d.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Remarks:
As requested in a decision on a TP by ECHA (Decision no. TPE-D-2114465819-31-01) we herewith submit the results of a OECD 443.
Adequacy of study:
key study
Study period:
2020-05-27 to 2021-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to the ECHA final decision on a testing proposal examination from 2019-04-05 (Decision number TPE-D-2114465819-31-01/F) the study design of this EOGRTS according to OECD 443 is as follows:

Based on Article 40 of Regulation ((EC) No 1907/2006) (the REACH Regulation), ECHA examined your testing proposal(s) and decided as follows.
Your testing proposal is accepted and you are requested to carry out: 1. 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);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
basic test design (Cohorts 1A, and 1B without extension); adopted June 25, 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
There is a decision from ECHA available regarding the implementation/study design of the extended one generation reproductive toxicity study (EOGRTS, OECD 443) with tetrahydronaphthalene. The decision is from 5th of April 2019 (Decision number: TPE-D-2114465819-31-01/F).
The testing proposal is accepted by ECHA:

Basic study design (Cohorts 1A, and 1B without extension):
10 weeks premating exposure duration for parental (P0) generation
- Dose level setting shall aim to induce some toxicity at the highest dose level
- Cohort 1A (Reproductive toxicity)
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation

- Route of administration: oral

- There is no trigger for Cohorts 2A/2B or Cohort 3.
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, Research Models and Services, Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age at first dosing: Males and females: 70 days
- Body weight (at 1st administration): Males: 338.9 g – 413.4 g, Females 188.3 - 263.2 g

- Pre-exposure period
120 female animals were evaluated pre-exposure for estrous cyclicity to yield 96 females (i.e. 24 per group) with a regular estrous cycle for the study.
Main study
192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation.

-Identification of the F0 animals
After randomisation, each rat received a continuous number. For animals of the F1 Generation the numbering started with 201. Points were set on paws and/or tail by tattoo or marker. Additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose levels and dates of administration.

HOUSING
With exception of the mating period, the male and female animals (F0 Generation) are kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 10% (maximum range). Deviations from the maximum range caused for example during cleaning procedures were dealt with in SOPs. No values exceeding the maximum range were noted during the course of the study.
Rooms were alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL (see Appendix 3: 'Limitation for Contaminants in the Bedding Material').

DIET
A certified commercial diet (ssniff® R/M-ZV1154, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL (see Appendix 3: 'Limitation for Contaminants in the Diet'). Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.

DRINKING WATER
Tap water was offered ad libitum.
Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001 ] (see Appendix 3 'Limitation for Contaminants in the Drinking Water'). In addition, drinking water samples taken at Provivo are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001, Anlage 1' [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.
- Adaption period: 7 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Air changes per hour: 15-20
- Photoperiod: 12 hours dark/12 hours light, 150 lux at approximately 1.5 m room height
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: Corn oil
Administration volume: 2 mL/kg b.w./day
Dosages: 0, 15, 50, 150/250 mg/ kg b.w./ day
Selection of route of administration: According to international guidelines.

The test item formulations were administered at a constant administration volume of 2 mL/kg b.w. once daily. The control animals received the vehicle at the same administration volume in the same way. The test item formulations were prepared daily.
The test item formulations were continuously agitated by stirring throughout the entire administration procedure to ensure homogeneity.The animals were treated with the test item during the following periods:
F0 Generation
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 and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).

F1 Generation
F1 Pups Until weaning, the pups were indirectly exposed to the test item through the breast milk. During the last week of lactation, the pups additionally received the test item directly when they commence eating for themselves.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage.
Cohort 1A The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 86 to 95).
Cohort 1B The male and female animals were dosed up to and including the day before sacrifice (males and females: PNDs 94 to 102).

Details on mating procedure:
Sexually mature male and female rats of the F0 Generation were randomly paired for mating . Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug.
The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from its their male partner after 2 weeks without further opportunity for mating.
If there would have been insufficient males, for example due to male death before pairing, then males which had already mated would have been paired with a second female such that all females would have been paired. However, as 3 males and 3 females prematurely deceased before the start of mating, sufficient male animals were always available in this study.
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 of approximately 2 mL each were taken as scheduled and stored at -20°C ± 10% until analysis.

F0-Generation (Groups 2 to 4):
- Sampling time 1: At start of the treatment period of the F0 animals (1st dosing day)
- Parameter: Concentration and homogeneity
- Sampling per group: at the start of administration/ during (middle) administration/ before administration to the last animal

-Sample time 2: At dose increase of group 4 on test day 64
- Parameter: Concentration and homogeneity
Sampling per group: At the start, during (middle) and before administration to the last animal of dose level group 4.

- Sampling time 3: At a time when most F0 females have littered
- Parameter: Concentration
- Sampling per group: During treatment always before administration to the last animal

- Sampling time 4: Near the end of the F0 dosing period at a time when the majority of animals is dosed
- Parameter: Concentration
- Sampling per group: During treatment always before administration to the last animal

Total F0 samples (aliquots): 18 (36)

F1-Generation (Groups 2 to 4):
- Sampling time 1: After all selected pups were transferred to the F1 cohorts (test day 5 of the F1 Study)
- Parameter: Concentration and homogeneity
- Sampling per group: at the start of administration/ during (middle) administration/ before administration to the last animal

- Sampling time 2: At termination of the Cohort 1 A treatment period (when the majority of animals was dosed; test day 68 of the F1 Study)
- Parameter: Concentration
- Sampling per group: During treatment always before administration to the last animal

- Sampling time 3: At termination of the Cohort 1 B treatment period (when the majority of animals was dosed; test day 76 of the F1 Study)
- Parameter: Concentration
- Sampling per group: During treatment always before administration to the last animal

Total F0 samples (aliquots): 15 (30)
Duration of treatment / exposure:

The animals were treated with the test item during the following periods:
F0 Generation
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 and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).

F1 Generation
F1 Pups Until weaning, the pups were indirectly exposed to the test item through the breast milk. During the last week of lactation, the pups additionally received the test item directly when they commence eating for themselves.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage.
Cohort 1A The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 86 to 95).
Cohort 1B The male and female animals were dosed up to and including the day before sacrifice (males and females: PNDs 94 to 102).
Frequency of treatment:
daily
Details on study schedule:
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 a dosing period of at least 11 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 98).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high dose group (F0 until test day 63)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
high dose group (F0 from test day 64 + F1 group 4)
No. of animals per sex per dose:
20 males and 20 females
Control animals:
yes, concurrent vehicle
Details on study design:
RATIONALE FOR DOSE SELECTION:
The dose levels were selected in agreement with the Sponsor based on available toxicological data and the results of a 2-week dose-range-finding study in rats (LPT Study No. 37750) and the subsequently performed OECD 421 study in rats (LPT Study No. 37626).
In the dose range finding study, the rats were treated daily with 20, 60 or 180 mg/kg b.w. test itemdaily from test day 1 to test day 14.
None of the animals died prematurely.
No changes in behaviour, the external appearance or the appearance of the faeces were noted, with the exception of a severely reduced motility that was noted for all male and female animals of the high dose group (180 mg/kg b.w./day) on test day 1.
The male animals showed a slightly reduced body weight at 60 and 180 mg/kg b.w./day and the female animals a marginally reduced body weight at 180 mg/kg b.w./day in comparison to the animals that were dosed with 20 mg/kg b.w./day.
A slightly to moderately reduced food consumption was noted for the male animals that were dosed with 60 or 180 mg/kg b.w./day during the first test week.
For the female animals a moderately reduced food consumption was noted during the first and the second test week at a dose level of 180 mg/kg b.w./day.
No test item-related changes were noted during the macroscopic inspection at necropsy.
The examination of the organ weights revealed no test item-related differences between the low, the intermediate and the high dose group.

In the OECD 421 study, the test item was administered orally in marginally reduced doses to rats at dose levels of 15, 50 or 150 mg/kg b.w./day to prevent malnutrition of the female animals. None of the parental animals of eigther sex died prematurely in the OECD 421 study.
During the daily cage side observations a short post-dosing salivation was noted with an increasing incidence of affected animals from 50 to 150 mg/kg b.w./day for the males and from 15, 50 to 150 mg/kg b.w./day for the females.
Furthermore, a reduced motility was noted for all male and female animals after the first dosing with 150 mg/kg b.w./day on test day 15.
The short post-dosing salivation and the isolated observation of a reduced motility that are rated as an adaptation effect are not of any toxicological relevance.
A slightly reduced body weight was noted for the male and female animals at 150 mg/kg b.w./day.
A transient and slightly reduced food consumption was noted for the male and female animals at 150 mg/kg b.w./day after the start of treatment.
No test item-related influence was noted on the T4 serum levels of the male animals.
The macroscopic inspection at necropsy and the microscopic examination of the reproductive organs of the male and female animals of the high dose group revealed no test item-related changes.
No test item-related differences were noted for the organ weights of the reproductive organs and the thyroid of the male and female animals. 
No influence was noted on the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period. Therefore, no changes in the reproduction was noted.

Pups
No adverse effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post implantation loss) and the post-natal development of the pus (viability indices, pup body weight, ano-genital distance, the number of nipples per male pup, T4 serum levels on lactation day 13).
The macroscopic external examination of the pups at necropsy or after premature death revealed no abnormalities.

Hence, doses of 15, 50, 150 mg/kg b.w./day by oral gavage are proposed for the OECD 443 study because moderately reduced food consumption in females in the high dose group (=180 mg/kg).
However, the animals adapted well to the test substance and no relevant toxicological effects up to test day 64 occurred. As a consequence, the highest dose level was increased to 250 mg/kg b.w./day.

Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS:
- Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal.
-F0 and F1 Generation:
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.
In case signs of toxicity occurred the frequency of observations was increased.
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded.
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.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
-F0 and F1 Generation (Cohort 1B) after weaning:
A more detailed examination of all F0 and F1 Cohort 1B animals was conducted on a weekly basis. F0 animals were examined once before the first test item treatment on test day 14 to allow for within-subject comparisons. Thereafter, the examination was performed weekly until termination. The F1 animals of Cohort 1B were examined weekly after weaning until termination.
Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included 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:
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.
If necessary, these provisions allowed to record any premortal symptoms in detail and a post mortem examination to be carried out during the working period of a day.


BODY WEIGHT:
The body weight the animals was recorded as follows:
F0 animals:
Study period F0 males F0 females
Pre-mating period Daily, starting on the first day of dosing#
Mating period Daily, report of weekly values
Gestation period Not applicable GD 0, 7, 14, 21
Lactation period Not applicable PND 1, 4, 7, 14, 21
Post-mating period Daily report of weekly values

GD: Gestation day
PND: Post-natal day

FOOD AND WATER CONSUMPTION:
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days that are listed below.
Study period F0 males F0 females
Pre-mating period Weekly Weekly
Mating period None None
Gestation period Not applicable GD 0, 7, 14, 21
Lactation period Not applicable PND 1, 7, 14, 21
Post-mating period Weekly values for males#
#: Starting at the end of the 14 day mating period on test day 99.
GD: Gestation day
PND: Post-natal day

Water consumption was monitored by visual appraisal daily throughout the study.

REPRODUCTIV PERFORMANCE - F0 animals
Evaluation/parameters:
- Number of pregnant females
-Stages of estrous cycle
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4, 7, 14 and 21.
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4, 7, 14 and 21.
Number of stillbirths
- per group
- per dam
Number of pups with malformations
- per group
- per dam
Oestrous cyclicity (parental animals):
Vaginal lavages were taken and the oestrous cycle stages were determined at the following time points:
- F0 animals: 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days). + During 10 weeks of premating until evidence of mating.
- F1 animals, cohort 1A: Start after onset of vaginal patency until first appearance of cornified cells. + Two weeks starting around PND 75.
- 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. Sperm count (spermatids) was additionally performed on one testicle for the males of the F0 Generation and Cohort 1A but not reported as the guideline requests a sperm count from the cauda 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 minus 80°C. The frozen cauda was homogenized homogenised and the obtained suspension was used for sperm counting (Kuriyama S.N. (2005), S. Plassmann and H. Urwyler (2001)).
Litter observations:
CLINICAL SIGNS:

Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.
In case signs of toxicity occurred the frequency of observations was increased.
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded.
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.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.



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


BODY WEIGHT:
F1 animals (F1 Cohort 1A):
Study period F1 males and F1 females
Lactation period PND 1, 4, 7, 14, 21
After weaning Daily, starting on PND 22 report of weekly values

PND: Post-natal day
In addition, all animals will be weighed at sacrifice.

FOOD AND WATER CONSUMPTION:
Study period Cohorts 1A + 1B
Starting after weaning Weekly

Water consumption is monitored by visual appraisal daily throughout the study.

LITTERING:
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (i.e. body weight less than 70% of mean litter weight) and the presence of gross abnormalities.
Abnormal behaviour or changes in the external appearance of the pups noted during the daily cage side inspections were recorded.


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

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

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


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

SEXUAL MATURATION:
All F1 Pups (Cohorts 1A and 1B) were evaluated daily for balano-preputial separation or vaginal opening which indicate sexual maturity of the animals. The genitals were examined for any abnormalities. The body weight was recorded at the time point of balano-preputial separation or vaginal opening.

Preputial separation:
During this examination the time point of the onset of the function of the preputial glands is determined. A soft pressure is exerted against the root of the penis. If this leads to the observation of small drops of secretion from the preputial glands on both sides of the foreskin, the postnatal day of this observation is determined as the time point of the onset of preputial gland function.
Postmortem examinations (parental animals):
LABORATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
EDTA anticoagulant (whole blood) ........................ for haematological investigations
Citrate anticoagulant (plasma) ............................................... for coagulation tests
LiHeparin anticoagulant (plasma) .................................... for clinical chemistry tests
Sampling time: at sacrifice
Animals: F0: 10 males and 10 females randomly selected from each group.

HAEMATOLOGY:
Parameter Units
Differential blood count^5 (relative) %
Differential blood count (absolute) 10^3/μL
Erythrocytes (RBC) 10^6/μL
Leucocytes (WBC) 10^3/μL
Haematocrit value (HCT) %
Haemoglobin content (HGB) mmol/L
Platelets (PLT) 10^3/μL
Reticulocytes (RET) %
Mean corpuscular volume (MCV) fL
Mean corpuscular haemoglobin (MCH) fmol
Mean corpuscular haemoglobin concentration (MCHC) mmol/L
Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany
^5 Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells will be simultaneously quantified during measurement of the differential blood count.
Following the haematological examinations using the ADVIA system, blood smears will be prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
The parameters listed below are determined:
Parameter Units
Prothrombin time (PT) sec
Activated partial thromboplastin time (aPTT) sec
Instrument: Amax Destiny PlusTM, Tcoag Deutschland GmbH, 32657 Lemgo, Germany

CLINICAL CHEMISTRY:
The parameters listed below are determined:
Parameter Units
Sodium mmol/L
Potassium mmol/L
Calcium mmol/L
Chloride mmol/L
Albumin g/L
Total bilirubin μmol/L
Total cholesterol mmol/L
Glucose mmol/L
Total protein g/L
Blood urea (BUN) mmol/L
Creatinine μmol/L
Alanine amino-transferase (ALAT/GPT) U/L
Alkaline phosphatase (aP) U/L
Aspartate aminotransferase (ASAT/GOT) U/L
Bile acids μmol/L
Lactate dehydrogenase (LDH) U/L
Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany
Sodium/Potassium ratio non-dimensional (by calculation)
Globulin g/L (by subtraction)
Albumin/globulin ratio non-dimensional (by calculation)
BUN/creatinine ratio non-dimensional (by calculation)

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
- Animals: F0: 10 males and 10 females randomly selected from each group (animals also selected for laboratory examinations)

- time of sampling: at sacrifice (Test days 129 - 136)
- expected number of samples: 80 (10 per Group/sex)
- feeding status: fasted
- analysed hormones: T4 + TSH
- sample volume: 2 x 100 µL each

Blood samples were processed for serum, divided into aliquots and stored
-20°C ± 10% at Provivo until analyses using commercial ELISA kits as follows:

Parameter Method
Total thyroxine T4 ELISA Kit, cat. no. RE 55261, IBL, Batch no. 304K090
Tyroid stimulating hormone (TSH) Rat TSH ELISA Kit, cat. no. RE 45021, IBL, Batch no.
V051
Instrument: Tecan Sunrise

URINANALYSIS
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:

- Sampling time: At the end of the F0 dosing period
- Animals: F0: 10 males and 10 females randomly selected from each group (animals also selected for laboratory examinations).

The following parameters were determined by using the instruments given below:
Parameter Units Method
Volume mL Graduated vessel
pH - 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)

The following tests were also be performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration:
- protein
- glucose
- bilirubin
- urobilinogen
- ketones
- haemoglobin (Hb) (approx. values)
- nitrite
reporting convention: s. Any other information on materials and methods incl. tables

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 was recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.

PATHOLOGY AND HISTOPATHOLOGY:
GROSS NECROPSY:
On the day of necropsy, vaginal lavages of the adult animals (F0 and F1 Generation) were obtained and examined to determine the stage of estrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis. A gross or full necropsy of the animals of the F0 and F1 animals was carried out. At gross necropsy the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighting of selected organs. The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:

Animals No. of animals Time Examination Vaginal smears
Males All (96) TD 134- 136 Full necropsy Not applicable
Dams All (96) TD 129 - 143 Full necropsy Yes

In the case the time of dissection was fallen on a weekend or bank holiday, necropsy would have been postponed to the next working day and dosing would have been continued up to and including the day before sacrifice.
Animals which were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.


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

During necropsy the number of implantation sites in the uteri was recorded in the female animals and used to evaluate reproductive performance.
Apparently non-pregnant uteri of the F0 animals were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI
The following organs of the male and female F0 animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.

Organs to be weighed:
- Adrenal gland (2)
- Oviducts (2)
- Testicle (2)
- Brain
- Pituitary
- Thymus
- Epididymis (2)
- Prostate (dorsolateral and ventral parts combined)
- Thyroid (1) (including para-thyroid, post-fixation)
- Heart
- Kidney (2)
- Seminal vesicles with coagulating glands
- Uterus including cervix
- Liver
- Identified target organs
- Ovary (2)
- Spleen

The following organs or parts thereof of all adult male and female animals of the F0 generation were preserved in an appropriate fixative:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: modified Davidson’s solution
- Epididymis (1)#
- Testicle (1)#
Fixative: 7% buffered formalin
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Spleen
- 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
#: The second epididymis and testicle were not preserved but used for the spermiogram.

Any other organs displaying macroscopic changes were also preserved.

Sperm analysis (motility, morphology and counting) was performed from the cauda of the right epididymis. Sperm count (spermatids) was additionally performed on one testicle for the males of the F0 Generation and Cohort 1A but not reported as the guideline requests a sperm count from the cauda 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 minus 80°C. The frozen cauda was homogenized homogenised and the obtained suspension was used for sperm counting (Kuriyama S.N. (2005), S. Plassmann and H. Urwyler (2001)).


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

HISTOPATHOLOGY:
BLOOD SMEARS:
The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.

F0 animals:
Full histopathology was performed on the preserved organs of:
- F0 animals: 20 animals/sex/group of groups 1 and 4
- All deceased or prematurely sacrificed animals

The organs listed above were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids could not always be identified macroscopically; they were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged. In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined. Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure were performed on one testicle and one epididymis of the selected F0 males of groups 1 and 4 following H-E and PAS staining.
In case of test item-related changes in group 4, the Sponsor was given sufficient notice before the corresponding organs of further animals are processed and examined histopathologically.

HISTOPATHOLOGICAL EVALUATION:
Histotechnique was performed by Provivo.
The slides were labelled with study number, test species, animal number and block number and were dispatched to the Test Site (AnaPath Services GmbH, Switzerland, see Section 2.6 'Test Site for histopathological evaluation' for details) for histopathological evaluation on the following dates:
- 10 February 2021 (F0 and Cohort 1A animals: groups 1 and 4),
- 01 June 2021 (F0 and Cohort 1A animals: additional organs from group 2 and 3),
- 17 June 2021 (Cohort 1A: spleens not included in the shipment on 10 February 2021).
The transport of slides to the histopathology Test Site (TS) was arranged by Provivo, whereas the return transport to the Test Facility will be arranged by the TS.
The study phase was recorded under the TS reference number 12848C.
All observations upon final assessment were reported per animal and the findings considered to be relevant for the treatment were recorded in incidence and occurrence tables. All microscopic findings are recorded, reported and archived with the with the PathData system .
The report of this study phase comprises a description of objective, materials, methods and results (macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TS Quality Assurance Unit (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 as scheduled. The blood samples collected were divided into tubes as follows:
EDTA anticoagulant (whole blood) ........................ for haematological investigations
Citrate anticoagulant (plasma) ............................................... for coagulation tests
LiHeparin anticoagulant (plasma) .................................... for clinical chemistry tests
Sampling time: at sacrifice
Animals: F1 cohort 1A: 10 males and 10 females randomly selected from each group.

HAEMATOLOGY:
Parameter Units
Differential blood count5 (relative) %
Differential blood count (absolute) 10^3/μL
Erythrocytes (RBC) 10^6/μL
Leucocytes (WBC) 10^3/μL
Haematocrit value (HCT) %
Haemoglobin content (HGB) mmol/L
Platelets (PLT) 10^3/μL
Reticulocytes (RET) %
Mean corpuscular volume (MCV) fL
Mean corpuscular haemoglobin (MCH) fmol
Mean corpuscular haemoglobin concentration (MCHC) mmol/L
Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany
^5 Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood count.
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
The parameters listed below were determined:
Parameter Units
Prothrombin time (PT) sec
Activated partial thromboplastin time (aPTT) sec
Instrument: Amax Destiny PlusTM, Tcoag Deutschland GmbH, 32657 Lemgo, Germany


CLINICAL CHEMISTRY:
The parameters listed below were determined:
Parameter Units
Sodium mmol/L
Potassium mmol/L
Calcium mmol/L
Chloride mmol/L
Albumin g/L
Total bilirubin μmol/L
Total cholesterol mmol/L
Glucose mmol/L
Total protein g/L
Blood urea (BUN) mmol/L
Creatinine μmol/L
Alanine amino-transferase (ALAT/GPT) U/L
Alkaline phosphatase (aP) U/L
Aspartate aminotransferase (ASAT/GOT) U/L
Bile acids μmol/L
Lactate dehydrogenase (LDH) U/L
Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany
Sodium/Potassium ratio non-dimensional (by calculation)
Globulin g/L (by subtraction)
Albumin/globulin ratio non-dimensional (by calculation)
BUN/creatinine ratio non-dimensional (by calculation)

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
- Animals: Pups (If the individual volume obtained from the pups is insufficient for analysis, the samples may be pooled by litter.), 2 surplus pups per litter, all litters, if possible
- time of sampling: PND 4
- number of samples: 103
- feeding status: non-fasted
- analysed hormones: T4 only
- sample volume: 1x 75 µL

- Animals: Pups, 2 surplus pups per litter, all litters
- time of sampling: PND 21/22
- number of samples: 162
- feeding status: non-fasted
- analysed hormones: T4 + TSH
- sample volume: 2x 50 µL (T4) + 2x 70 µL (TSH)

- Animals: F1 cohort 1A: 10 males and 10 females randomly selected from each group (animals also selected for laboratory examinations)
- time of sampling: at sacrifice
- number of samples: 80
- feeding status: fasted
- analysed hormones: T4 + TSH
- sample volume: 2x 100 µL each

The serum samples were divided into aliquots, if possible, and stored at -20°C ± 10% until analysis using ELISA. The T4 and TSH ELISA (commercial kits) were conducted at Provivo.
Parameter Method
T4 T4 ELISA Kit, cat. no. RE 55261, IBL
TSH Rat TSH ELISA Kit, cat. no. RE 45021, IBL
Instrument: Tecan Sunrise

URINALYSIS:
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
- Sampling time: At the end of the F1 cohort 1A dosing period
- Animals: F1 cohort 1A: 10 males and 10 females randomly selected from each group (animals also selected for laboratory examinations).
The urine wascollected 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 following parameters were measured using the methods given below:
Parameter Units Method
Volume mL Graduated vessel
pH - 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)

The following tests were also performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration:
- protein
- glucose
- bilirubin
- urobilinogen
- ketones
- haemoglobin (Hb) (approx. values)
- nitrite
reporting convention: s. Any other information on materials and methods incl. tables

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.

PATHOLOGY AND HISTOPATHOLOGY:
GROSS NECROPSY:
On the day of necropsy, vaginal lavages of the adult animals (F1 Generation) were obtained and examined to determine the stage of estrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis.
The pups were euthanized by decapitation (PND4) or by carbon dioxide (CO2) inhalation (PND 22 to 24).
A gross or full necropsy of the animals of the F1 animals was carried out. At gross necropsy the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighting of selected organs.
Blood samples for determination of haematological and biochemical parameter as well as for thyroid hormone determination were taken.
The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:

- Animals: “Surplus” pups
- No. of Animals: All (up to 192)
- Necropsy date: On PND 4
- Examination: Gross necropsy
- Vaginal smears: No

- Animals: “Surplus” pups
- No. of Animals: All (up to 640) (All F1 “surplus” pups from PND 22 will be subject to a gross necropsy, however only a limited number of pups will be selected for tissue preservation)
- Necropsy date: On PND 22-24
- Examination: Gross necropsy; partial organ preservation of only 10 pups/sex/group
- Vaginal smears: No

- Animals: Cohort 1A
- No. of Animals: All (160)
- Necropsy date: At the end of the dosing period (PND 86-95)
- Examination: Full necropsy
- Vaginal smears: Yes

- Animals: Cohort 1B
- No. of Animals: All (160)
- Necropsy date: At the end of the dosing period (approx. PND 94- 102)
- Examination: Full necropsy
- Vaginal smears: Yes

In the case the time of dissection was fallen on a weekend or bank holiday, necropsy would have been postponed to the next working day and dosing would have been continued up to and including the day before sacrifice.
Animals which were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.

EXAMINATION OF THE "SURPLUS" F1 PUPS:
External inspection for gross abnormalities
Dead pups and culled F1 Pups on PND 4 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
External and/or internal inspection for gross abnormalities
Pups not selected for the F1 Cohorts were sacrificed on PND 22 to 24 and examined macroscopically for any abnormalities or pathological changes.
Ten ‘surplus’ pups per sex and group were used for organ weighing and preservation.
The following organs/tissues of the F1 Pups were weighed and/or preserved in 7% formalin:

- Brain#
- Gross abnormalities
- Mammary gland
- Ovary (2)#
- Spleen#
- Thymus#
- Uterus including cervix#
#: Organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.
Histopathological examination of the preserved organs was conducted only in agreement with the Study Monitor.

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

F1 generation- Cohort 1A:
The following organs of all adult male and female F1 Cohort 1A animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.
Organs to be weighed:
- 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/sex/group (1 animal per litter, all litters represented by at least 1 pup; randomly selected)

The following organs or parts thereof of all adult male and female animals of the F1 generation Cohort 1A were preserved in an appropriate fixative. For special handling of lymph nodes and spleen see footnotes ## and ###:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: modified Davidson’s solution
- Epididymis (1)#
- Testicle (1)#
Fixative: 7% buffered formalin
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Spleen###
- Intestine, large (colon, rectum)
- Stomach
- Kidney and ureter (2)
- Thyroid (2) (including parathyroids)
- Liver
- Thymus
- Lungs (with mainstem bronchi and bronchioles)
- Trachea (including larynx)
- Lymph node (1, cervical)##
- Urinary bladder
- Lymph node (1, mesenteric)##
- Uterus (including cervix)
- Mammary gland
- Vagina
- Muscle (skeletal)
- Vas deferens
- Nerve (sciatic)
- Identified target organs
- Oesophagus
#: The second epididymis and testicle were not preserved but used for the spermiogram.
##: For selected cohort 1A animals only.
###: For 10 animals/sex/group of all cohort 1A groups, randomly selected (same animals as selected for weighing of the lymph nodes): One half of the spleen was preserved for histopathological evaluation, the second half was used for splenic lymphocyte subpopulation analysis.
Any other organs displaying macroscopic changes were also preserved. In addition, sperm viability and morphology were evaluated for all male F1 Cohort 1A animals, and bone marrow smears were prepared.
F1 Generation - Cohort 1 B
Determination of organ weight and organ preservation is restricted to the following organs:
Organ Weigh 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 No As appropriate

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

PHENOTYPIC ANALYSIS OF SPLEEN CELLS - COHORT 1A ANIMALS:
The spleens of the male and female F1 Cohort 1A animals were split in two halves. The portion of the spleen not preserved for histopathology was minced using a mechanic dissociator to prepare single cell suspensions.
The following parameters were determined in the samples by using the instruments given below:

CD4+ T-Lymphocytes
CD8+ T-Lymphocytes
Pan-T-lymphocytes (CD3+)
B-lymphocytes (CD45RA+)
Natural killer cells (CD161+)
Evaluation will be performed by LPT.

HISTOPATHOLOGY:
BLOOD SMEARS:
The blood smears prepared from the selected animals during the haematological examination were available for possible examination of pathological changes, but examined and evaluated only depending on necropsy findings and upon agreement with the Study Monitor.

F1 Cohort 1A:
Full histopathology was performed on the preserved organs of:
- F1 animals: groups 1 and 4 of Cohort 1A
- All deceased or prematurely sacrificed animals
The organs listed in Text table 4-Text table 4-4, 4-7, 4-9 and 4-10 were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section.
In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the control and high dosed animals of groups 1 and 4 following H&E and PS staining:
- F1 Generation (Cohort 1A): 20 male animals per group
-Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea was performed on one ovary of the following control and high dosed animals of groups 1 and 4:
In case of test item-related changes in group 4, the Sponsor will be given sufficient notice before the corresponding organs of further animals are processed and examined histopathologically.

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

HISTOPATHOLOGICAL EVALUATION:
Histotechnique was performed by Provivo.
The slides were labelled with study number, test species, animal number and block number and were dispatched to the Test Site (AnaPath Services GmbH, Switzerland, see Section 2.6 'Test Site for histopathological evaluation' for details) for histopathological evaluation on the following dates:
- 10 February 2021 (F0 and Cohort 1A animals: groups 1 and 4),
- 01 June 2021 (F0 and Cohort 1A animals: additional organs from group 2 and 3),
- 17 June 2021 (Cohort 1A: spleens not included in the shipment on 10 February 2021).
The transport of slides to the histopathology Test Site (TS) was arranged by Provivo, whereas the return transport to the Test Facility will be arranged by the TS.
The study phase was recorded under the TS reference number 12848C.
All observations upon final assessment were reported per animal and the findings considered to be relevant for the treatment were recorded in incidence and occurrence tables. All microscopic findings are recorded, reported and archived with the with the PathData system .
The report of this study phase comprises a description of objective, materials, methods and results (macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TS Quality Assurance Unit (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:
The statistical evaluation of the parametrical values was done by Provantis® using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
For the statistical evaluation of the histopathological data FISHER's exact test was used.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to 1 may occur caused by rounding.
Significantly different data are indicated in the summary tables.
Reproductive indices:
For each F0 group the gestation index was determined:


Female Fertility Index [%] = (Number of pregnant females with verified copulation /Number of females with a verified copulation) x 100

Gestation Index = (Number of litters with live pups/ Number pregnant) 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-cull= (Number of pups alive on day 4 (pre-cull)/ Number of pups live on day 0/1) x 100

Viability Index post-cull = (Number of pups alive on day 13 (post-select)/ Number of pups live on day 4) x 100

Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100
Offspring viability indices:
Birth Index, Live Birth Index, Viability Birth Index, Post Implantation loss
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male (surviving males)
No test item-related changes were noted in the control and low dose group (15 mg test item/kg b.w./day) for the male animals.
However, in the control group an increased water consumption was noted for one male animal on test day 133 which was considered to be spontaneous.
Additionally, a post-dosing salivation was noted for 3 animals on one test day each and an increased water consumption for one male (no. 57) on one test day at the low dose animals.
Due to their low incidences the observations of salivation and increased water consumption were considered to be spontaneous in both groups.
Test item-related changes in behaviour were noted in the form of post-dosing salivation for nearly all and all male animals of the intermediate and the high dose group (50 or 150/250 mg test item/kg b.w./day), respectively.
The observation of a post dosing salivation was considered to be test item-related but not adverse (or of toxicological relevance), as in all cases it did not last longer than 60 min.
The observations of a decreased water consumption and piloerection that were noted for 2 male animals of the intermediate dose group on 2 consecutive test days each, were considered to be spontaneous due to their low incidence.

Females
No test item-related changes were noted in the control and low dose group (15 mg test item/kg b.w./day) for the female animals.
However, at the low dose group (15 mg test item/kg b.w./day) a post dosing salivation was noted during the pre-mating / mating period and the gestation period for one female on one test day each. Due to the low incidence, this observation was considered to be spontaneous.
At the intermediate dose level (50 mg test item/kg b.w./day) a post-dosing salivation was noted for nearly all females during the pre-mating / mating period and for approx. half of the females during the gestation and the lactation period.
At the high dose level (150/250 mg test item/kg b.w./day) a post-dosing salivation was noted for all or nearly all females during the pre-mating, the gestation and the lactation period, respectively. The observation of a post dosing salivation was considered to be test item-related but not adverse (or of toxicological relevance), as in all cases it did not last longer than 60 min

Detailed clinical observations
Males and females
No further observations in addition to those made during the daily cage side observations were noted for the surviving and the prematurely deceased male and female animals of the control group and the treatment groups during the once weekly performed detailed clinical observation.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males
No test item-related death was noted in the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
Male No. 67 (Group 2) and male No. 116 (Group 3) were found dead on Test Day 82 and 83, respectively. During the macroscopic observation, hemorrhage in the external nose/snout area and esophageal injury were recorded in both animals, and the lungs in one male appeared reddened. These findings indicate that the cause of these animals’ deaths was misgavage during the dosing procedure. This was supported by inflammatory changes which were observed microscopically.

Females
No premature death was noted for the female animals at 15, 50 or 150/250 mg test item/kg b.w./day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES
Pre-mating-, mating- and post-mating period
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
However, a marginally and statistically not significantly reduced body weight was noted for the males of the high dose group during the post-mating period from test day 92 onwards (2.6 % below the control) until the end of the study (3.1% below the control on test day 133).
This marginal difference could be considered as spontaneous and non adverse.

Body weight gain
Corresponding to the marginally reduced body weight during the post-mating period, a marginally reduced body weight gain was noted for the male animals of the high dose group (150/250 mg test item/kg b.w./day) from test day 15 until the end of the study on test day 133 (49.5 % in comparison to 52.9 % in the control group). They were not considered to be adverse.

FEMALES
Pre-mating-, gestation- and lactation period
No test item-related changes in body weight and body weight gain were noted for the female rats between the control group and the low and the intermediate dose group (15, 50 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (150/250 mg test item/kg b.w./day), a marginal reduction in body weight was noted at the end of the pre-mating period on test day 85 (2.8 % below the control, statistically not significant).
During the gestation period the difference between the high dose group and the control group increased and became statistically significant on gestation days 14 and 21 (5.6 % or 10.0 % below the control, p ≤ 0.01) (non-pregnant animals are not considered in this calculation). Nearly the same difference in body weight was still noted after the process of littering was completed (on lactation day 1).
Further on , a statistically significantly (p ≤ 0.01) reduced body weight was noted at the high dose level on lactation days 1, 4, 7 and 14. The biggest difference between the high dose group and the control group was noted on lactation day 1 (11.3 % below the control). Thereafter the difference between the control group and the high dose group declined. On lactation day 14 the difference between the high dose group and the control group has declined to 8.1 %, but was still statistically significant at p ≤ 0.01. On lactation day 21 the difference between the high dose group and the control group has decreased further and was no longer statistically significant (4.1 % below the control).
The high dose group decline in body weight from the end of the pre-mating period until the end of the lactation period, with maximum differences on gestation day 21 and lactation day 1 were considered to be test item-related.



Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
No influence of toxicological relevance on food consumption was noted between the control group and in the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
Statistically significantly reduced amounts of food consumed were noted on a few test weeks at the intermediate and the high dose level. The maximal reduced food consumption was noted after the start of treatment between test days 15 and 22 (8.5 % below the control at the intermediate dose level and 8.9 % below the control at the high dose level, p ≤ 0.01). These differences had disappeared in the following week (between test days 22 and 29) and can be considered as a non-adverse process of adaptation, which was without toxicological relevance and has also been noted for the female animals (see section below).
Further statistically significantly reduced food consumption were noted at the intermediate and the high dose level inindividual test weeks during the further course of the study. As these were only slight and temporary they were considered spontaneous

Females: Pre-mating, gestation and lactation period
No influence of toxicological relevance on food consumption was noted between the control group and in the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
However, a statistically significantly reduced food consumption was noted during the first 2 weeks after the start of treatment for the female animals of the high dose group between test days 15 and 22 and test days 22 to 29 (14.3 % or 5.9 % below the control, p ≤ 0.01). This was considered to be a non-adverse process of adaptation.
A further slight but statistically significant reduction in food consumption was observed for 2 test weeks between test days 64 to 78. As this reduction in food consumption was only slight (at maximum 6.9 % below the control, p ≤ 0.01) and temporary, it was considered to be spontaneous.
No statistically significant differences were noted during the gestation and the lactation period.
No food intake of female animals was recorded during the mating period as both sexes were housed together.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption for males and females was performed by visual appraisal during the daily cage side observations. No decreased or increased water consumption was noted for nearly all animals.
A decreased water consumption was noted for one male animal of the low and the intermediate dose group each, on one or 2 test days. On the other hand, one animal of the control group showed an increased water consumption on one test day.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
No test item-related differences for the examined haematological parameters were noted between the control group, low and intermediate dose level (15 and 50 mg test item/kg b.w./day).
A test item-related effect on the haematological parameter was noted test item for the male animals at the high dose level (150/250 mg test item/kg b.w./day).
For the number of red blood cells only a slight reduced number of red blood cells was noted at the high dose level (6.0 % below the control (statistically not significant). Nevertheless, the erythrocytes values are still in the range of the Provivo background data.
However, a significant increase in the reticulocytes, which are the precursors of the erythrocytes, was noted at the high dose level (55.9 % above the control, p ≤ 0.01). A comparison with the Provivo background data revealed that for the high dosed males 8 individual values were above the Provivo background range. Yet, at the intermediate dose level the percentages of reticulocytes from all individual males were within the Provivo background data.
Also other hematological parameter which are related to the number of red blood cells are shown to be significantly affected such as the MCHC concentration (2.6 % below the control, p ≤ 0.01).
All these haematological effects and the changes in the clinical biochemistry (bilirubin increase) and the histopathological observations (in spleen, liver) are indicative for an enhanced erythrocyte degradation and a subsequent extra medullary hematopoiesis.
As a reactive change to the enhanced erythrocyte degradation a hypercellularity (increased cellularity) of erythroid elements was noted in the bone marrow. These histopathological changes were noted for the males and females of the F0 Generation in group 4, and for the males and females of the F1 Generation (Cohort 1A) in groups 3 and 4.

Females
No test item-related differences for the examined haematological parameters were noted between the control group and females of the treatment groups (15 and 50 mg test item/kg b.w./day).
A test item-related effect on the haematological parameter was noted for the female animals at the high dose level (150/250 mg test item/kg b.w./day).
For the number of red blood cells a dose-related significant decrease was noted for the female animals at the intermediate and the high dose level (7.6 % or 16.1 % below the control, p ≤ 0.01). A comparison with the Provivo background data revealed that for the high dosed females 4 individual values were above the Provivo background range. Yet, at the intermediate dose level the percentages of erythrocytes from only 4 female animals were slightly below the Provivo background data.
The mostly affected parameter was the percentage of reticulocytes for the females (+168.8%) of the high dose group. This was in line with the observations from 10 animals of the high dose group, an increased erythroid cellularity in the bone marrow was noted for 7 female animals during the histopathological examination.
A comparison with the Provivo background data for the high dosed females 4 individual values were above the Provivo background range. At the intermediate dose level the percentages of reticulocytes from all individual female animals were within the Provivo background data.
Further statistically significant changes that were noted for the red blood cell parameters of the females of the high dose group were a decrease in HGB content (minus 9.4 %, p ≤ 0.01), a decreased HCT value (minus 8.1 %, p ≤ 0.01), an increased MCV value (plus 9.5 %, p ≤ 0.01) and an increased MCH value (plus 8.0 %, p ≤ 0.01).
At the intermediate dose level a statistically significant difference in comparison to the control group for the female animals was only noted in form of an increased MCH value (plus 4.3 %, p ≤ 0.05).
Therefore, only the changes in red blood cells, reticulocytes and red blood cells related parameters of the high dose level are considered to be test-item related changes in the females.
In line with the clinical biochemistry and histopathological detection of a haemolytic anemia, changes in different parameters that belong to the red blood cells (e.g. increased percentage of reticulocytes, decreased number of red blood cells) were noted for the female animals of the high dose group. The histopathological examination of the spleen revealed changes that are generally found in conditions with enhanced erythrocyte degradation (e.g. haemolytic anemia). As a reactive change to the enhanced erythrocyte degradation a hypercellularity (increased cellularity) of erythroid elements was noted in the bone marrow. These histopathological changes were noted for the males and females of the F0 Generation in group 4, and for the males and females of the F1 Generation (Cohort 1A) in groups 3 and 4.

Non-test item-related changes (Male and Female animals):
An increased number of neutrophilic granulocytes were noted for the male animals of the intermediate and the high dose group (52.5 % or 82.9 % above the control, p ≤ 0.05 or 0.01). However, only one individual value each of the intermediate and the high dose group was above the Provivo background range, whereas one individual value of the control group was below the Provivo background range. Hence, as nearly all individual values were within the Provivo background range, the changes were considered to be spontaneous.
The number of monocytic granulocytes was statistically significantly increased for the male and the female animals (48.3 % or 47.7 % above the control, p ≤ 0.01). Whereas in case of the male animals only one individual value was above the Provivo background range, in case of the female animals 4 individual values were above the Provivo background range. However, no statistically significant changes were noted for the number of monocytic granulocytes of the male and female animals of Cohort 1A,The noted changes at the high dose level for the F0 Generation were considered to be spontaneous,
An increased number of large unstained cells (LUCs) was noted for the female animals of the high dose group (74.4 % above the control, p ≤ 0.01). As only one individual value was above the Provivo background range and no statistically significant changes were noted for the male animals of the F0 Generation and the male and female animals of Cohort 1A, the observed increased number of LUCs that was noted for the female animals of the F0 Generation was considered to be spontaneous.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males
No test item-related differences for the examined biochemical parameters were noted between the control group and the low and intermediate dose group (15 mg and 50 test item/kg b.w./day) for the male animals.
A test item-related significant increase of the bilirubin concentration (32.4 % above the control, p ≤ 0.01) was only noted test itemfor the male animals at the high dose level (150/250 mg test item/kg b.w./day. For the male animals 5 individual values of the high dose group were above the Provivo background range. An increased bilirubin concentration is correlated with the decreased number of red blood cells, as bilirubin is one of the metabolic product of heme which is released by degrading erythrocyte. Due to this correlation, the increased bilirubin concentration that was noted for the male animals at the high dose level was considered to be test item-related.
Additionally, also other liver produced enzymes are increased in the highest treated dose like globulin, total protein and alkaline phosphatase.
In detail, a statistically significantly increased globulin concentration was noted at the high dose level for the male animals (9.4 % above the control, p ≤0.05).
The concentration of total protein was slightly, but statistically significantly increased at the high dose level (5.2 % above the control, p ≤ 0.05). However, nearly all individual values of the high dosed males were within the Provivo background range. The protein concentration of 3 high dosed males was marginally (68 g protein (total)/L) above the Provivo background range (57 to 67 g protein (total)/L). This slight increase above the background range has no scientific relevance. Furthermore, as no statistically significant increase was noted for the female animals, the slight increase that was noted for the high dosed male animals was considered to be secondary.
An increased activity of the enzyme alkaline phosphatase was noted the high dosed male animals (36.4 % above the control).
All this additional increased liver related parameters facilitate the histopathology observed centrilobular hepatocellular hypertrophy in the high dose. This histopathological change is a sign for an increased hepatic enzyme induction. Which is supported by subsequent changes in the biochemical parameters e.g. slightly increased T4 values, the total protein values, the globulin, alkaline phosphatase and bilirubin values. Secondly, no other indicator for a hepatic injury were observed histopathological then the centrilobular hepatocellular hypertrophy.

Females
No test item-related differences for the examined biochemical parameters were noted between the control group and the low dose group (15 mg test item/kg b.w./day) for the female animals.
A slightly but already statistically significant increased bilirubin concentration was noted for the females of the intermediate dose group (50 mg test item/kg b.w./day)
A test item-related significant increase of the bilirubin concentration was noted for the female animals at the high dose level (150/250 mg test item/kg b.w./day).
In detail, increased concentrations of bilirubin were noted for the female animals at the intermediate and the high dose level (32.4 % or 82.7 % above the control, p ≤ 0.05 or 0.01).
For the female animals 6 individual values of the high dose group were above the Provivo background range. From the animals of the intermediate dose group the bilirubin concentrations from two females were above the Provivo background range.
The increased bilirubin concentration could be correlated with the decreased number of red blood cells that was noted at the high dose level for the female animals. Due to this correlation, the increased bilirubin concentration that was noted for the female at the high dose level was considered to be test item-related.
Compared to the males of the F0 generation only one liver related functional marker is increased in the highest dose, alkaline phosphatase. Nevertheless, this additional increased liver related parameter further confirms observed centrilobular hepatocellular hypertrophy in the high dos. This histopathological change is a sign for an increased hepatic enzyme induction. Which is also the case for the slightly increased T4 values, alkaline phosphatase and bilirubin. Secondly, no other indicators for a hepatic injury are given then the increased hepatic enzyme induction.

Non-test item-related changes (Female animals):
An increased BUN / Creatinine ratio was noted for the female animals of the high dose group (23.2 % above the control, p ≤ 0.05). As all values were within the Provivo background range and no increase was noted for the male animals, the increased BUN / Creatinine ratio that was noted for the female animals of the high dose group was considered to be spontaneous.
The increased concentration of urea in blood that was noted for the female animals of the intermediate and the high dose group (23.6 % or 26.1 % above the control, p ≤ 0.05 / 0.01) was also considered to be spontaneous. All individual values were within the Provivo background range and no statistically significantly increased values were noted for the male animals.
Although both values are common markers for reduced kidney function, they are still in the range of the historical control data. Furthermore, no changes have been observed in the histopathological examination of the kidney in females , as a consequence, the biochemical effects were considered spontaneous.
An increased glucose concentration was noted for the female animals of the high dose group (15.3 % above the control, p ≤ 0.05). This was considered to be spontaneous as all individual values were within the Provivo background range and no changes were noted for the male animals.
Endocrine findings:
no effects observed
Description (incidence and severity):
THYROID HORMONE LEVELS
Males
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
However, distinct changes were observed for the T4 and the TSH concentrations of the male and female animals. These changes are of secondary nature as they are results of the increased bilirubin production (Van Steenbergen et al., 1988; Deetman at al., 2014). The observations are discussed in detail below.
For the male animals a slightly increased T4 concentration was noted between the control group and the high dose group (5.9 % above the control, statistically not significant).
A comparison of the T4 concentration of the male animals with the Provivo background data revealed that nearly all T4 concentrations of the individual male animals were within the background range (see Text Table 7-23). These changes are of secondary nature as they are results of the increased bilirubin production. As the thyroid hormones (T4) stimulate the heme oxygenase-1 activity (HO-1), which is the main enzyme responsible for heme destruction and therefore the bilirubin genesis (Smith and Drummond, 1991; Li et al., 2011). Secondly, thyroid hormones downregulate the enzymatic activity of uridine 5′-diphospho-glucuronosyltransferase (UDP-GT), which stimulates bilirubin conjugation, thereby facilitating bilirubin excretion (Gartner & Arias, 1972; Van Steenbergen et al., 1989). Hence, the increased T4 values are only a feedback loop to increase degradation of heme and the excretion of conjugated bilirubin.

Distinctly increased TSH concentrations were noted at the low, the intermediate and the high dose level (49.4 %, 26.9 % or 53.4 % above the control, statistically not significant, see Text Table 7-22). Nevertheless, the TSH values are still in the range of the historical control and therefore considered to be not test item relevant.

Females
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
However, distinct changes were observed for the T4 and the TSH concentrations of the female animals. These changes are of secondary nature as they are results of the increased bilirubin production.

For the female animals an increased T4 concentration was noted at the low dose level and the high dose level (26.3 % or 43.2 % above the control, statistically not significant). No difference in the T4 concentration was noted at the intermediate dose level (3.1 % above the control).
A comparison of the T4 concentration of the high dosed females with the Provivo background data revealed that the T4 concentrations of 5 individual female animals were above the background range. These changes are of secondary nature as they are a result of the increased bilirubin production. As the thyroid hormones (T4) stimulate the heme oxygenase-1 activity (HO-1), which is the main enzyme responsible for heme destruction and therefore the bilirubin genesis (Smith and Drummond, 1991; Li et al., 2011). Secondly, thyroid hormones downregulate the enzymatic activity of uridine 5′-diphospho-glucuronosyltransferase (UDP-GT), which stimulates bilirubin conjugation, thereby facilitating bilirubin excretion (Gartner & Arias, 1972; Van Steenbergen et al., 1989). Hence, the increased T4 values are only a feedback loop to increase degradation of heme and the excretion of conjugated bilirubin.

For the TSH concentration increased values were noted at the intermediate and the high dose level (23.4 % or 22.2 % above the control, statistically not significant).

However, a possible influence on the T4 / TSH homeostasis by the observed pathological changes in the liver as suspected for the male animals of the F1 Generation could also be possible for the male and female animals of the F0 Generation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Males
No test item-related differences for the examined urinalysis parameters were noted between the control group and the lowest dose group (15mg test item/kg b.w./day).
Test item related influences on the examined urinalysis parameters were noted for the intermediate and high treatment groups (50 or 150/250 mg /kg b.w./day).
A slightly but statistically significantly increased specific gravity was noted at the high dose level for the male animals (1.3 % above the control, p ≤ 0.05). Yet, all individual values of the high dosed animals were within the Provivo background range. However, as also similar not significantly increases were observed in the intermediate dose group and histopathological kidney changes have been observed, this effect was considered to be test item related but not human relevant.
The relative urine volume of the male animals was statistically significantly decreased at the intermediate dose level (31.4 % below the control, p ≤ 0.05). Also in the highest dose a decrease was noted which was with -16% not significantly reduced compared to the control. However, no dose response relationship was noted and only one individual value of the male animals of the intermediate dose group (7.2 mL/kg b.w./24 h) was below the Provivo background range (9.1 to 33.3 mL/kg b.w./24 h). Secondly, also histopathological changes in the kidney of the males were observed and a decrease in the urine volume is a sign for kidney function alternations this was considered to be test item related yet not human relevant. As the histopathology revealed an overload of synthetic proteins like alpha-2-u microglubulin.
Additionally, a statistically significant changes were noted for the pH value, which were not considered to be test item-related:
Statistically significantly decreased pH values were noted for the male animals of the intermediate and the high dose level (pH = 6.35 or pH = 6.29 in comparison to pH = 6.85 in the control group, p ≤ 0.01). However, nearly all values of the intermediate and the high dose level were within the Provivo background range. Only the pH value of one individual male animal from the intermediate dose group (pH = 5.9) was marginally below the Provivo background range for the pH values of the male animals (pH = 6.0 to pH = 7.3).As nearly all individual pH values from the males of the intermediate and the high dose group were within the Provivo background range, the decreased pH values that were noted at the intermediate and the high dose level were considered to be spontaneous.
However, a statistically significantly decreased pH value was also noted for the male animals of Cohort 1A, which was also considered as spontaneous.

Females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (15, 50 or 150/ 250mg test item/kg b.w./day).
Nevertheless, for the female animals a statistically significantly increased pH value (pH = 7.37 in comparison to pH = 6.71 in the control group, p ≤ 0.01) was noted at the intermediate dose level. The pH values from 5 individual females of the intermediate dose group were above the Provivo background range. However, as no dose response-relationship was noted (no statistically significantly increased pH value was noted for the female animals of the high dose group), the increased pH value that was noted for the female animals of the intermediate dose group was considered to be spontaneous.

Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females (survivors)
No test item-related differences were noted between the control group and the low dose groups (15mg /kg b.w./day) in the histopathological observations.
Test item-related differences were noted between the control group and the intermediate dose group (50 mg /kg b.w./day) for the kidney histopathology. Test item-related differences were noted between the control group and the high dose group (150/250 mg /kg b.w./day) for the kidney, spleen, liver, bone marrow, thyroid gland, adrenal glands and thymus.

In detail, histopathological, treatment-related findings were observed in the spleen, bone marrow, liver, thyroid glands, thymus and adrenal glands of both sexes from F0, in the kidneys of males from F0.

SPLEEN
In the spleen, the histologic severity of hemosiderin deposits and extramedullary hematopoiesis (EMH) increased in both sexes of Group 4, and the mean severity grade in Group 4 was higher than that in Group 1. There was also increased incidence and severity of congestion in both sexes of Group 4.

FEMUR
In the femur bone marrow, the number of animals showing hypercellularity (increased cellularity) of erythroid elements were large in both sexes of Group 4 compared to that of Group 1.
These findings were consistent with the changes that are generally found in hemolytic anemia, and the increased splenic weights were thought to reflect these histologic changes. Hypercellularity (increased cellularity) of erythroid elements in the bone marrow was deemed to be reactive change to hemolytic anemia and not to be due to direct effects of the test item to bone marrow.

LIVER
In the liver, centrilobular hepatocellular hypertrophy was observed at minimal to moderate severity in both sexes of Group 4. There were no further indicators of liver injury in any animals examined.
Hemopoietic cell foci (extramedullary hematopoiesis in the liver) were observed in a few animals of Group 4, however, there were no differences to be toxicologically concerned in the incidence and severity in the liver between Groups 1 and 4.
In the liver, there were increase in or increased tendency of absolute and/or relative weights of both sexes of Group 4 of F0. These were considered to correlate microscopically with centrilobular hepatocellular hypertrophy. Thus, this was considered to be of metabolic nature and of adaptive character, and hence, deemed not to be adverse.

THYROID
In the thyroid glands, there were increased incidence and/or severity of follicular cell hypertrophy in both sexes of Group 4.
Increased incidence and/or severity of thyroid follicular cell hypertrophy was considered to be the changes associated with hepatic enzyme induction that was indicated by increased liver weights and hepatocellular hypertrophy, and hence, deemed not to be adverse as well.

KIDNEY
The incidence and group mean severity grade of hyaline droplets in the proximal tubular cells increased in males of Group 4 in comparison with that of the control males (Group 1). Such droplets were not observed in renal tubules of any females.
Increases in or increased tendency of absolute and/or relative kidney weights that were detected in F0 males of Groups 3 and 4 were thought to reflect the enhanced accumulation of hyaline droplets.
Increase in hyaline droplets was considered to be induced by overload of synthetic protein, which is specific in male rat like as alpha-2 microglobulin, derived from hyperfunction of the liver.
In some high dose males of F0, degenerating and/or regenerated renal tubules were found at slightly higher severity compared to the corresponding control males. This was considered to be secondary to enhanced accumulation of hyaline droplets in the tubular cell, but not due to direct effects of the test item. In addition, enhanced hyaline droplet accumulation associated with hyperfunction of the liver is believed to be a male rat specific phenomenon and not relevant to human. Thus, renal lesions recorded only in male rats were deemed not to be adverse.

ThymusIn the thymus, there was a slight increase in the severity of atrophy in both sexes of Group 4. In males, the group mean severity grade was not so much different between Groups 1 and 4, however, the number of animals showing grade 3 and higher was larger in Group 4 compared to the control group; that is, the number of animals showing thymic atrophy at severity grade 3 or higher was 9 males (8 at grade 3; 1 at grade 4) and 14 males (all at grade 3) in Group 4.

ADRENAL GLAND
In the adrenal glands, diffuse cortical hypertrophy was observed in both sexes of Group 4 and the incidence of animals showing increased lipid vacuoles in zona fasciculata was increased in males of Group 4. These findings were considered to be stress-related changes, however, they were found only in F0 generation, but not observed in F1-C1A animals. As with the histological findings, significant changes in the thymus and adrenal weights were detected only in F0 animals.
The remainders of microscopic findings recorded in F0 animals were within the range of normal background lesions or physiological alterations which may be observed in this study type or animals of this strain and age.

MALE REPRODUCTICE ORGANS – including detailed qualitative examination for testes
No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs including the testis, epididymis, vas deferens, prostate glands, seminal vesicles and coagulating glands from the F0 high dose (Group 4) animals.
All findings recorded in the male reproductive organs and tissues were within the range of normal background changes or physiological alterations that may be observed in this study type and animals of this strain and age, and hence, deemed not to be treatment-related.
For the testis, the stages were checked on completeness of cell populations and stages while taking also into consideration any degenerative changes and the interstitial cell structure, for both generations. As a result, all testes examined showed completeness of stages and cell population, and there were no alterations that could be attributed to treatment with the test item as well.

FEMALE REPRODUCTIVE ORGANS
No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the female reproductive organs including ovaries, oviducts, uterus, uterine cervix and vagina from the F0 high dose (Group 4) animals.
All findings recorded in the female reproductive organs and tissues were within the range of normal background changes or physiological alterations that may be observed in this study type and animals of this strain and age.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BONE MARROW
Males and females
At the high dose level (150/250 mg test item/kg b.w./day) a slight shift in the ratio of myeloid to erythroid cells (statistically not significant) was noted, which was more pronounced for the female as for the male animals.
In detail, for the male animals the myeloid to erythroid ratio was 2.098 to 1 in the control group in comparison to 1.836 to 1 at the high dose level.
For the female animals the myeloid to erythroid ratio was 1.756 to 1 in the control group in comparison to 1.424 to 1 at the high dose level.
This shift correlated with the histopathological finding of an enhanced erythropoiesis in the bone marrow in form of hypercellularity of erythroid elements which is the result of the counteract of the body to the treatment related enhanced erythrocyte degradation.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
P Animals
After the allocation of the animals to the test groups and the start of treatment on test day 15, the oestrous cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign (verification of copulation) was noted.
No test item-related differences were noted for the mean length and the mean number of oestrous cycles per dam during the pre-mating period between the female animals of the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).

ADULT F1 OESTRUS CYCLE EVALUATIONS
Cohort 1A Animals
The stages of the oestrous cycle of the cohort 1A females were monitored on 14 test days between test days 50 and 63 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of oestrous cycles per female animal between the females of the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day.

Cohort 1B Animals
No test item-related differences were noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day) in the distribution of the stages of the oestrous cycle at necropsy for the adult Cohort 1B females .
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
P Generation
Sperm number
No test item-related difference was noted between the rats of the control group and the rats treated with 15, 50 or 150/250 mg test item/kg b.w./day for the number of sperm cells per gram cauda epidymides.
Sperm motility
No test item-related differences were noted between the rats of the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day) for the percentage of motile sperm cells in the cauda epididymis. Motile sperm cells were noted for all examined male animals.
Sperm morphology
The examination of the sperm cells from the cauda epidymides revealed no increased numbers of sperm cells with a malformation in the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day) in comparison to the control group.
In detail, 4 sperm cells with a malformation were noted in the control group, one in the low dose group and no malformed sperm cells were noted in the intermediate and the high dose group.
Males that did not inseminate their female partner
No verified copulation (no sperm detection in the vaginal smear) during the mating period of 14 days was noted for one pair of the control group and one pair of the high dose group.
The investigated sperm parameter revealed motile sperm cells for both male animals (male no. 17 with 71.5 % and male no. 161 with 63.0 % motile sperm cells).
The sperm count revealed 252.2 x10E6 cells / g cauda epididymis for male no. 17 of the control group, which was below the mean value of the control group (432.04 ± 236.79 x10E6 g cells). However, due to the high variance that was noted for the sperm count, it can be concluded that the reduced number of sperm cells that was noted for male no. 17 was without an adverse effect on the fertility of male no. 17. No sperm count was performed for male no. 161 due to a mistake in the lab.
Hence, the missing verified copulation cannot be attributed to the sperm properties of male nos. 17 and 161.

COHORT 1A Animals
No test item-related difference was noted between the rats of the control group and the rats treated with 15, 50 or 250 mg test item/kg b.w./day for the sperm number, the viability and morphology.
Reproductive performance:
no effects observed
Description (incidence and severity):
FEMALE FERTILITY
No test item-related influence on the fertility index was noted for the female rats of the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
Females with verified copulation:
An increased number of non-pregnant animals (all with a verified copulation) was noted at the low dose level (3 of 24; nos. 79, 81 and 92) in comparison to one non-pregnant animal with a verified copulation in the high dose group (no. 174). No non-pregnant animals with a verified copulation were noted in the control group and in the intermediate dose group. This resulted in reduced fertility indices of 88 % and 96 % at the low and the high dose level, respectively, in comparison to fertility indices of 100 % in the control group and the intermediate dose group.
The fertility index of 96 % that was noted at the high dose level was still in the range of the Provivo background data (95 % to 100 %). The fertility index of 88 % that was noted in the low dose group was below the Provivo background range.
However, as no dose-response relationship was noted, the reduced fertility index at the low dose level of 88 % was considered to be spontaneous.

GESTATION INDEX
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
Two pregnant females with a resorption of all implants were noted at the low and the intermediate dose group each (nos. 86, 95 of the low dose group and nos. 133, 140 of the intermediate dose group). This lead to reduced gestation indices at the low and the intermediate dose group of 90 % and 92 %, respectively. Consequently, both indices were below the range of the Provivo background data of 95 % to 100 %.
However, as no female with a resorption of all implants was noted at the high dose level, the observations of 2 females each at the low and the intermediate dose group and no dose response-relationship was noted. . Furthermore, one pregnant female with a resorption of all implants was also noted in the control group (no. 48). Therefore, these observations were considered to be spontaneous.

PRE-COITAL TIME
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
An elongated pre-coital time was noted at the high dose level in comparison to the control group (4.1 ± 4.1 days in comparison to 2.8 ± 2.7 days in the control group; statistically not significant). This was due to 3 pregnant high dosed females (nos. 171, 179 and 190) with elongated pre-coital intervals between 11 and 14 days, whereas in the control group no pregnant animal was noted with such an elongated pre-coital time. Furthermore, one non-pregnant female was noted without a verified copulation (no. 186) during the mating period of 14 days. This female was considered in the mean length of pre-coital time with 14 days. However, such a non-pregnant female without a verified copulation was also noted in the control group (no. 42).
During their elongated pre-coital time the 3 pregnant high dosed females (nos. 171, 179 and 190) remained in a dioestrous stage until or one day before a copulation was verified by sperm detection. Such an elongated pre-coital time with a persistent dioestrous stage was also noted for the pregnant female no. 76 of the low dose group.
The occurrence of 3 pregnant females with such elongated dioestrous stages during the mating period is still in the Provivo background range. In detail, in the control group from 6 OECD 443 studies performed at Provivo, one control group showed 2 females with elongated dioestrous stages between 8 and 12 days and another control group 3 females with elongated dioestrous stages between 9 and 11 days during the mating period were noted.
Additionally, no dose response-relationship was noted, as the low and intermediate dose groups even showed a shortened pre-coital time compared to the control groupe. Hence, the observed elongated pre-coital time that was noted at the high dose level can be considered as spontaneous.

GESTATION LENGTH
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day). The gestation length was observed between 22.4 and 22.6 days for all four groups.





The following test item-related changes were considered adverse and for setting the NOAEL of the parental animals:
• A slightly reduced body weight that was noted for the females of the high dose group (150/250 mg/kg b.w./day) from the end of the pre-mating until the end of the lactation period with a max. difference on gestation day 21 and lactation day 1 (statistically significant).
• Changes in the red blood cell parameter and an increased bilirubin concentration that were noted for the female animals at (50 mg/kg b.w./day) and for the male and female animals at (150/250 mg/kg b.w./day).
• An increased spleen weight that was noted for the male and female animals of the high dose group (150/250 mg/kg b.w./day).
• Changes in the spleen and the bone marrow that were noted during the histopathological examination and related to conditions of an haemolytic anemia at the high dose level for the male and female animals (150/250 mg/kg b.w./day).
• Enlarged spleens that were noted for one male and one female animal of the high dose group (150/250 mg/kg b.w./day) at necropsy.
An enlarged spleen was also noted in one control animal. However, due to the adverse effects on the spleens at the high dose level in general (histopathological spleen findings for several animals, a generally increased spleen weight), the enlarged spleens that were noted at the high dose level at necropsy were considered adverse and test item-related.
• A slight shift that was noted during the bone marrow examination for the myeloid / erythroid ratio of the male and female animals of the high dose group (150/250 mg/kg b.w./day). This correlated with the histopathological finding of an enhanced erythropoiesis in the bone marrow in form of hypercellularity of erythroid elements.
Hence, with the exception of a decreased body weight that was noted for the high dosed females, all adverse effects that were noted for the parental animals were correlated to a haemolytic anemia of the high dosed animals.

The following observations were considered to be test item-related but not adverse and, hence, not considered for the NOAEL of the parental animals:
• A short lasting post dosing salivation that was noted for the male and female animals at the intermediate and the high dose level.
• A marginally reduced body weight that was noted for the male animals at the high dose level.
• A reduced food intake that was noted after the start of dosing for the male (intermediate and high dose level) and female (high dose level) animals This was considered to be a process of adaptation.
• Observations that were noted during the histopathological examination for the male and female animals of the intermediate and the high dose group in the liver, and the kidneys (males only). Furthermore, observations in the thyroid, the thymus and the adrenal glands that were noted for the male and female animals of the high dose group only.
• Changes in the organ weights of the liver, the kidneys (males only), the thymus and the adrenal glands, as they were related to the non-adverse changes of these organs that were noted during the histopathological examination.
Key result
Dose descriptor:
NOAEL
Remarks:
for reproductive performance
Effect level:
> 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Hemolytic anemia
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
other: hematopoetic system (hemolytic anemia)
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1 A Animals
Males
A post-dosing salivation was noted for one male of the control group on one test day. No further observations were noted for the male animals of the control group.
At the low dose level (15 mg test item/kg b.w./day) a post-dosing salivation was noted for 2 males (nos. 248, 257) on one test day each. Due to the low incidence, the observed post-dosing salivation was considered to be spontaneous. In addition, an increased water consumption was noted for male no. 247 on 10 test days between postnatal days 76 and 89. As only one male animal was affected, the observation of an increased water consumption was considered to be spontaneous.
At the intermediate dose group (50 mg test item/kg b.w./day) a post-dosing salivation was noted for all male animals. In addition, piloerection was noted for one male animal (no. 281) on one test day. Due to the low incidence, the observation of piloerction was considered to be spontaneous.
At the high dose level (250 mg test item/kg b.w./day) a post-dosing salivation was noted for all male animals as in the intermediate dose group. No further observation as the mentioned post-dosing salivation was noted for the male animals of the high dose group.
The observation of a post dosing salivation was considered to be test item-related but not adverse (or of toxicological relevance), as in all cases it did not last longer than 60 min.

Females
No observations were noted for the female animals of the control group.
At the low dose level (15 mg test item/kg b.w./day) a post dosing salivation was noted for one female on one test day. This low incidence was considered to be spontaneous. No further observations were noted at the low dose level.
At the intermediate dose level and the high dose level (50 or 250 mg test item/kg b.w./day) a post-dosing salivation was noted for all females.
No further observation was noted for the female animals of the of the intermediate and the high dose level.
The observation of a post dosing salivation was considered to be test item-related but not adverse (or of toxicological relevance), as in all cases it did not last longer than 60 min.

Males and females
The observation of salivation was a short lasting post-dosing symptom that disappeared between 20 and 60 min after dosing at the latest.

COHORT 1 B Animals
Males and females
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male and female animals of the control group.
Test item related but non adverse changes (or toxicological relevant) were noted in the treatment groups (15, 50 or 250 mg test item/kg b.w./day) in form of post dosing salvation.

A post-dosing salivation was noted for 2 male animals of the low dose group (15 test item/kg b.w./day) on one test day each. This observation was considered to be spontaneous due to its low incidenceand no observations were noted for the females.
As for the parental generation and for the animals of Cohort 1A, a post-dosing salivation was noted for all male and female animals of the intermediate and the high dose group (50 or 250 mg test item/kg b.w./day).
In addition, one male animal (no. 498) with an decreased water consumption on 3 consecutive test days was noted at the high dose level. Due to its low incidence, the observed observation of an decreased water consumption was considered to be spontaneous.
With the exception of the above mentioned post-dosing salivation, no further observations were noted for the female animals of the low and the intermediate dose group.

Males and females
The observation of salivation was a short lasting post-dosing symptom in all cases and in all test groups forboth sex. In nearly all cases salivation started immediately to 5 min after dosing and disappeared between 5 min and 60 min after dosing.
Only for 2 male animals of the high dose group (nos. 483, 488) salivation was noted before the start of dosing on one test day each. For 4 female animals of the high dose group (nos. 504, 507, 516, 519) salivation was noted before the start of dosing on one test day each. This is most likely related to conditioning.


Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
PUPS
VIABILITY INDEX OF F1 PUPS
Pre- and post-cull period
No test item-related differences between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
During the pre-cull period 6 prematurely deceased pups were noted in the high dose group in comparison to 2 prematurely deceased pups in the control group, leading to viability indices of 97.74 % (group value) at the high dose level in comparison to 99.32 % (group value) in the control group. This was in the range of the Provivo background data for the pre-cull period (96.71% to 99.36 %). Hence, the marginally reduced viability index of the pre-cull period that was noted at the high dose level was considered to be spontaneous.

ADULTS
MORTALITY OF COHORT 1A
Males and females
No test item-related death was noted in the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day) of both sexes.
However, 2 male animals of the control group were prematurely sacrificed.
Male no. 212 was prematurely sacrificed for ethical reasons due to scratch wounds in the neck and back region on postnatal day 66. Microscopically, the lesions were scab and ulceration in the skin of neck and back areas, accompanied by inflammation and fibrosis in the dermis and hypodermis, loss and atrophy of skin appendages and epidermal squamous hyperplasia and hyperkeratosis (see section 3.1 ‘Mortality and Cause of Animals’ Death/Morbidity’ of the Histopathology Phase Report in Appendix 5).
With the exception of haemorrhagic scratch wounds on the neck and the back region of male no. 212 no further observations were noted during necropsy.
During the daily cage side observations only an isolated observation of piloerection was noted for no. 212 on postnatal day 56 (10 days before premature sacrifice). No other observation was noted during the daily cage side observations.
Male no. 220 was prematurely sacrificed due to moribund conditions on postnatal day 89.
No observations were noted for animal no. 220 during the daily cage side observations on the days before sacrifice. Only on the day of sacrifice a haemorrhagic nose/snout and an extremely reduced motility were noted. The following necropsy revealed emphysematous lungs for animal no. 220, which corresponded to the histopathological finding of an alveolar emphysema. All these findings, however, were found at minimal to slight severity, unlikely to be the direct causes of the moribundity. No microscopic lesions that could be causes of animal’s morbidity were also not found in other organs and tissues, and the cause of animal’s morbidity was not established in this animal.


MORTALITY OF COHORT 1 B
Males and females
No test item-related death was noted in the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
Male no. 494 of the high dose group was prematurely sacrificed on postnatal day 494 for ethical reasons due to scratch wounds in the neck and back region.
With the exception of post-dosing salivation no further observations were noted for animal no. 494 during the daily cage side observations. Necropsy revealed a haemorrhagic scratch wound on the neck for male no. 494. No further observation was noted at necropsy for male no. 494. A histopathological examination of animal no. 494 was not performed, as this was not requested by the Study Plan.
As such a case was also noted for one male animal from the control group of Cohort 1A (no. 212), the reason for the premature sacrifice of no. 494, was considered to be spontaneous.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT OF F1 PUPS
No test item-related influence on the body weight of the pups was noted in all treatment groups (15 ,50 or 150/250 mg test item/kg b.w./day).
However, a statistically significantly reduced pup body weight was noted for the male and female pups (alone or combined) of the high dose group (150/250 mg test item/kg b.w./day) on lactation days 7, 14 and 21.
In detail, the body weight from the combined high dose male and female pups was 7.5 %, 10.7 % or 8.1 % below the control group on lactation days 7, 14 and 21 (p ≤ 0.05 or 0.01).
The reduced pup body weight at the high dose level was considered to be a secondary effect on the post-natal development of the pups due to the following reasons:
At the high dose level, the mean pup body weight from 3 individual dams was below the Provivo background range on lactation day 7. On lactation day 14 even the mean pup body weight from 9 individual dams was below the Provivo background range.
On lactation day 21 the mean pup body weight from 4 individual dams (nos. 175, 180, 182 and 188; mean pup body weight between 33.62 g (no. 175) and 40.37 g (no. 182)) was below the Provivo background range (43.65 to 60.24 g) and also below the lowest value of the control group (43.90 g). Due to the culling process on lactation day 4, a maximum of 10 pups had to be fed per dam. Hence, the reduced mean pup body weights that were noted for several dams could not be explained by a large number of pups. Yet, it can be driven by the reduced body weight of the dams. The comparison of the individual dam and the pup body weight match. Of the dams which exhibit a low body weight during the lactation period consequently those pups showed a lower body weight as well. For some of the dams ( e.g. Nr. 175 and 180) already on LD1 the pups exhibited a lower body weight to begin with compared to the pups of other dams of the high dose group.
Furthermore, the difference in pup body weight between the high dose group and the control group was reduced in a non objective manner due to the high dosed female no. 184 which delivered only 3 pups. Due to the low number of pups, the pups from female no. 184 had an increased pup body weight (for example: 71.73 g on post-natal day 21). Without female no. 184 and her few, but therefore heavier pups, the body weight difference between the control group and the high dose group would have been even bigger.
The difference in pup body weight between the pups of the high dose group and the pups of the control group that was noted on post-natal day 21 was also noted for the male and female animals of Cohort 1A and Cohort 1B at start of the F1 Study on post-natal day 22. After start of the F1 Study the difference in body weight between the F1 animals of the control group and the high dose group further increased, before the difference declined during the further course of the F1 Study.

ADULTS
BODY WEIGHT OF COHORT 1A ANIMALS
Males and females
No test item-related differences in body weight and body weight gain were noted for the male and female animals between the control group and the low dose group (15 mg test item/kg b.w./day) during the post-weaning development.
A marginally reduced body weight was noted at the intermediate dose level (50 mg test item/kg b.w./day) for the male animals on post-natal days 43, 50 and 57 (5.1 %, 6.3 % or 5.6 % below the control, statistically significant on post-natal day 43 at p ≤ 0.05). However, the difference in body weight between the control group and the intermediate dose group declined to the end of the study. Furthermore, only a marginally reduced body weight gain was noted at the intermediate dose level in comparison to the control group (663.4 % in comparison to 696.7 % in the control group). Hence, the observed reduction in body weight that was noted at the intermediate dose level was not considered adverse.
At the high dose level (250 mg test item/kg b.w./day) a slight but statistically significantly reduced body weight was noted for the male and female animals at the start of Cohort 1A on postnatal day 22. The body weight of the male and female animals from the high dose group was 11.5 % (p ≤ 0.01) or 9.8 % (p ≤ 0.5) below the control on post-natal day 22, respectivly. The differences in body weight that were noted on post-natal day 22 corresponded to the differences in pup body weight that were noted before weaning on post-natal day 21.
After post-natal day 22 the differences in body weight between the control group and the high dose group increased for the male and the female animals. The maximal difference in body weight between the control group and the high dose group was reached for the male animals on post-natal day 50 (17.3 % below the control, p ≤ 0.01) and for the female animals on post-natal day 29 (16.3 % below the control, p ≤ 0.01). Thereafter the difference in body weight between the high dosed animals and the control animals decreased. To the end of the study, on post-natal day 85 the body weight of the high dosed males was 9.9 % below the control (p ≤ 0.05) and the body weight of the high dosed females was 6.5 % below the control (statistically not significant).
In conclusion, there is a test item-related effect on the male and female animals of the high dose group during their post-weaning development in form of an increasing reduction in body weight between the control group and the high dose group during the first weeks (males) or week (females) after weaning. However, this effect was not considered to be an adverse effect on post-weaning development due to the following reasons:
• The increasing difference in body weight that was noted between the high dose group and the control group from post-natal day 22 onwards was caused by a reduced pup body weight that was noted at the high dose level during the lactation period and which can be considered as secondary to maternal toxicity.
• The difference in body weight between the male and female animals of the high dose group and the control group declined during the further course of the study but failed to reach the level of the control group before termination.
• Body weight gain of the male and female animals of group 4 was not affected during the course of the F1 Study and was slightly higher in group 4 as in the control group.


Body weight gain
As the differences in body weight that were noted between the control group and the high dose group were established at the end of the lactation period, no test item-related differences in body weight gain were noted between the control group and the treatment groups for the male and female animals.

Body weight at autopsy of Cohort 1 A animals
Males and females
No test item-related differences between the control group and the treatment groups (15 or 50 mg test item/kg b.w./day) were noted for the body weight at autopsy.
At the high dose level (250 mg test item/kg b.w./day) the body weight at autopsy was 8.6 % below the control for the male animals (p ≤ 0.05) and 8.2 % for the female animals (statistically not significant). These differences between the high dose group and the control group corresponds well with the differences between the high dose group and the control group that were noted for the live body weight at the end of the study.

BODY WEIGHT and BODY Weight GAIN OF COHORT 1B ANIMALS
Males
No test item-related differences in body weight were noted for the male animals between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day).
Test-item related differences in body weight were noted for the male animals between the control group and high dose group (250 mg test item/kg b.w./day).
At the high dose level a reduced body weight was noted at the start of the study (11.1 % below the control, p ≤ 0.01). This was correlated with the reduced body weight of the high dosed pups at weaning on post-natal day 21 (10.2 % below the control, p ≤ 0.01).
Thereafter the difference in body weight between the control group and the high dose group further increased. The maximum difference between the control group and the high dose group was noted on post-natal day 36 (18.2 % below the control). From post-natal day 43 onwards, the difference between the control group and the high dose group slightly declined to 13.3 % (p ≤ 0.01) at the end of the study on post-natal day 93.

Females
No test item-related differences in body weight were noted for the female animals between the control group, the low and intermediate dose group (15 or 50 mg test item/kg b.w./day).
However, a marginally reduced body weight was noted at the intermediate dose level (50 mg test item/kg b.w./day) on post-natal day 29 (9.3 % below the control, p ≤ 0.01) for the female animals. Thereafter the differences between the control group and the intermediate dose group slightly declined and no further statistically significant difference was noted. At the end of the study on post-natal day 93 the body weight at the intermediate dose level was 5.9 % below the control (statistically not significant).
As the difference in body weight between the intermediate dose group and the control group declined to the end of the study and only a marginally reduced body weight gain was noted at the intermediate dose level (399.3 % in comparison to 412.0 in the control), the decreased body weight at the intermediate dose level was not considered to be adverse.
A test item-related differences in body weight were noted for the female animals between the control group and the high dose group (250 mg test item/kg b.w./day).
At the high dose level (250 mg test item/kg b.w./day) the body weight of the female animals on post-natal day 29 (one week after the start of the F1 Study on post-natal day 22) was 16.8 % below the control (p ≤ 0.01). Thereafter the difference between the control group and the high dose group slightly declined. At the end of the study on post-natal day 93 the body weight of the high dosed females was 10.2 % below the control (p ≤ 0.01). Therefore, these changes in the body weight of the high dose females were considered to be test-item related.
Note:
The only slightly reduced body weight of the high dosed females of Cohort 1B on post-natal day 22 (-3.4%) did not match neither with the differences in body weight between the control group and the high dose group for the female pups on post-natal day 21 (8.0 % below the control, p ≤ 0.01) nor the females of Cohort 1A on post-natal day 22 (9.8 % below the control, p ≤ 0.01),
This was due to the high dosed female nos. 506 and 507 of Cohort 1B. This showed an increased body weight in comparison to the other high dosed females of Cohort 1B. Both females were descended from dam no. 184 that delivered only 3 pups, which consequently revealed an increased body weight in comparison to the pups from the other dams with more pups per litter. Without female nos. 506 and 507 the difference in body weight between the high dosed females of Cohort 1B and the control group on post-natal day 22 would have been in the range of those from cohort 1A on post-natal day 22.

Body weight gain (males and females)
No test item-related differences in body weight gain were noted between the control group and the treatment groups for the male animals. This was due to the fact that a reduced body weight was already noted for the male animals on post-natal day 22.
In the case of the female animals a slightly reduced body weight gain was noted at the high dose level (382.6 % in comparison to 412.0 % between post-natal days 22 and 93). This was due to the reduced difference in body weight (3.4 % below the control) that was noted for the high dosed females on post-natal day 22.

However, these effects were considered to be adverse effects on post-weaning development due to the following reasons:
• The increasing difference in body weight that was noted between the high dose group and the control group from post-natal day 22 onwards was not only caused by a reduced pup body weight. This further increased and then reached a steady state for the males and again increased again form PND 50 onwards
• Body weight gain of the female animals of group 4 was affected during the course of the F1 Study.

Body weight at autopsy of Cohort 1 B animals
Males and females
No test item-related differences between the control group and the treatment groups (15 or 50 mg test item/kg b.w./day) were noted for the body weight at autopsy.
Test item-related differences between the control group and the highest treatment group ( 250 mg test item/kg b.w./day) were noted for the body weight at autopsy
At the high dose level (250 mg test item/kg b.w./day) the body weight at autopsy was 14.7 % below the control for the male animals (p ≤ 0.01) and 9.8 % below the control for the female animals (p ≤ 0.01). These differences between the high dose group and the control group corresponds well with the differences between the high dose group and the control group that were noted for the live body weight at the end of the study.


Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
ADULT COHORT 1A ANIMALS
Males
No test item-related difference in food consumption was noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
Nevertheless, a slight increase in food consumption was noted for the high dose males compared to the control group. This increase was noted nearly during the whole treatment period, yet it is not toxicological relevant.

Females
No test item-related difference in food consumption was noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).

COHORT 1B ANIMALS
Males and females
No test item-related difference in food consumption was noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day) for the male and female animals.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
ADULT
COHORT 1A ANIMALS
Males and females
No test item-related differences for the examined haematological parameters were noted between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day).
Test item-related changes were noted at the high dose level (150/250 mg test item/kg b.w./day) for the red blood cell parameter, due to a haemolytic anemia.
These changes were more pronounced in animals in the F1 CoA then in when they were already observed in the high dose animals of the F0 generation. As for the F0 animals, the most pronounced changes were noted for the percentage of reticulocytes and the number of red blood cells.

Statistically significantly reduced numbers of red blood cells were noted at the high dose level for the male and female animals (8.4 % or 11.8 % below the control, respectively, p ≤ 0.01). The numbers of red blood cells from 3 individual high dosed males and from 7 individual high dosed females were below the Provivo background range.
However, a significant increase in the reticulocytes, which are the precursors of the erythrocytes, was noted noted for the male and female animals of the high dose group was 50.0 % or 84.3 % above the control (p ≤ 0.01). A comparison with the Provivo background data revealed that the percentage of reticulocytes from 8 individual females of the high dose group was above the background range. For the male animals this was only the case for two animals.
Further statistically significant changes of the red blood cell parameters at the high dose level for the male and / or female animals were noted for the haemoglobin concentration, the haematocrit value, the MCV content, the MCH content and the MCHC concentration.

All these haematological effects and the changes in the clinical biochemistry (bilirubin increase) and the histopathological observations (in spleen, liver) are indicative for an enhanced erythrocyte degradation and the subsequent extra medullary hematopoiesis. The corresponding histopathological changes in the spleen and the bone marrow were noted for the male and female animals of Cohort 1A at the intermediate and the high dose level. However, statistically significant changes for the red blood cell parameter were only noted at the high dose level.


LYMPHOCYTE TYPING IN SPLEEN 1A ANIMALS
Males and females
No test item-related influence was noted in the proportion of the examined lymphocyte subtypes to each other between the male and female animals of the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
Though test item-related effects were noted on the spleen in the form of an increased spleen weight and findings during the histopathological examination (extramedullary haematopoiesis), nearly all individual values of the examined lymphocytes were within the Provivo background range.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
ADULT CLINICAL BIOCHEMISTRY
Males and females Cohort 1A
No test item-related differences for the examined biochemical parameters were noted between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day).
A test item-related significant increase in the concentration of bilirubin was noted at the high dose level (250 mg test item/kg b.w./day) for the male and female animals.
As for the male and female animals of the parental F0 Generation a statistically significantly increased concentration of bilirubin was noted for the male and female animals of the high dose group (19.4 % or 35.8 % above the control, respectively, p ≤0.05 / 0.01). In contrast to the parental F0 Generation, nearly all individual values of the Cohort 1A animals were within the Provivo background range (only 1 value of the female animals was above the Provivo background range.
However, the increased bilirubin concentrations that were noted at the high dose level for the male and female animals of Cohort 1A were considered to be test item-related due to the following reasons:
• An increased bilirubin concentration was also noted for the male and female animals of the F0 Generation.
An increased bilirubin concentration is correlated with the decreased number of red blood cells, as bilirubin is one of the metabolic products of heme which is released by degrading erythrocyte.
• An increased bilirubin concentration could be correlated with histopathological findings (extramedullary haematopoiesis) that could be correlated with enhanced erythrocyte degradation (e.g. haemolytic anemia).

Non-test item-related changes (F1 Cohort 1A – Male and Female animals):
A statistically significantly increased concentration of cholesterol was noted for the male animals of the high dose group (29.9 % above the control, p ≤ 0.01). As all high dosed values were within the Provivo background range, these observation was considered to be spontaneous.
The statistically significantly decreased glucose concentration that was noted for the male animals of the high dose group (11.5 % below the control, p ≤ 0.01) was also considered to be spontaneous. Since, only one individual value from the high dosed males (6.02 mmol/L) was marginally below the Provivo background range (6.03 to 11.80 mm0l/L).


THYROID HORMONE (T4 and TSH)
DETERMINATION F1 PUPS ON PND 4 AND PND 21/22
No test item-related differences between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day) were noted for the T4 level and the TSH level on lactation days 4 and 22 for the male and female pups.
Increased T4 levels were noted for the male pups on PND 22 at the low dose level (11.7 % above the control, statistically not significant) and the high dose level (19.4 % above the control, p ≤ 0.05). Whereas the T4 level of the intermediate dose group was only 6.7 % above the control, statistically not significant).
In detail, the T4 levels of the male pups from 4 individual dams of the low dose group (59.252 to 65.309 nmol/L) and of the high dose group (59.700 to 63.296 nmol/L) were marginally to slightly above the Provivo background range (28.244 to 57.824 nmol/L).
Hence, as only a few individual values were marginally or slightly above the background range, no dose-response relationship was noted and no statistically significant changes were noted for the female pups. Lastly, a corresponding slight mean decrease in the THS values of the high dose male pups were noted, which indicates a working feedback loop of the thyroid hormones. Therefore, the increased T4 levels that were noted for the male pups at the low and the high dose level were considered to be spontaneous.

ADULT COHORT 1A Animals Thyroid hormone levels
Males
No test item-related differences were noted for the T4 concentration between the control group and the low dose group (15, 50 or 250 mg test item/kg b.w./day).
However, in contrast to the high dosed males of the F0 Generation, statistically significantly increased T4 concentrations were noted for the F1 male animals of the intermediate and the high dose group (50 or 250 mg test item/kg b.w./day) (28.4 % or 46.2 % above the control, respectively, p ≤ 0.05 / 0.01). Five individual values of the male intermediate dose group and 8 individual values of the high dose group were above the Provivo background range. These changes are of secondary nature as they are results of the increased bilirubin production.

The increased T4 concentrations can be related to the observed adaptive changes in the thyroid glands and the liver which were noted during the histopathological examination of the male and female rats of group 3 and 4 as stated in the histopathology report:
In the liver, there were increases in or increased tendency of absolute and/or relative weights of F0 and F1-C1A Group. These were considered to correlate microscopically with centrilobular hepatocellular hypertrophy. There were no further indicators of liver injury in either generation. Thus, this was considered to be of metabolic nature and of adaptive character, and hence, deemed not to be adverse.
Increased incidence and/or severity of thyroid follicular cell hypertrophy was considered to be the changes associated with hepatic enzyme induction that was indicated by increased liver weights and hepatocellular hypertrophy, and hence, deemed not to be adverse as well
These changes are of secondary nature as they are results of the increased bilirubin production. As the thyroid hormones (T4) stimulate the heme oxygenase-1 activity (HO-1), which is the main enzyme responsible for heme destruction and therefore the bilirubin genesis (Smith and Drummond, 1991; Li et al., 2011). Secondly, thyroid hormones downregulate the enzymatic activity of uridine 5′-diphospho-glucuronosyltransferase (UDP-GT), which stimulates bilirubin conjugation, thereby facilitating bilirubin excretion (Gartner & Arias, 1972; Van Steenbergen et al., 1989). Hence, the increased T4 values are only a feedback loop to increase degradation of heme and the excretion of conjugated bilirubin.

In repeat-dose studies in rodents, the induction of hepatic enzymes can have an impact on other organ systems beyond the liver. In particular, rats are uniquely sensitive to the induction of hepatic enzymes that ultimately overstimulate the thyroid gland as a result of increased TSH secretion (Zabika et al., 2011).
Hence, the observed increased T4 concentrations that were noted for the male animals at the intermediate and the high dose level can be considered as not adverse, as they were most probably caused by non-adverse changes in the liver.

Males – TSH
No test item-related differences were noted for the TSH concentrations between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
However, high differences were noted for the TSH concentration between the control group and the treatment groups (at maximum 91.2 % above the control value for the males of the intermediate dose group; 4.32 to 2.26 ng/ml in the control group). As no dose-response relationship and no statistical significance were noted, the observed changes were considered to be spontaneous.

Females
No test item-related differences were noted for the T4 and the TSH concentrations between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
Increased T4 concentrations were also noted for the female animals (25.1 %, 21.4 % or 24.3 % above the control for the females of the low, the intermediate and the high dose level, respectively). However, no dose response-relationship and no statistical significance was noted for the female animals. A comparison with the Provivo background range showed that the T4 concentration from one female animal of the control group, 3 females of the low dose group and 2 females each of the intermediate and the high dose group each were above the background range.
As no dose response and no statistical significance were noted, the changes were considered to be spontaneous. Furthermore, due to the uniformly increased T4 concentrations in all treatment groups, and unusually low control value this is also a possible explanation for the increased T4 values in the treatment groups.

Females – TSH
As for the male animals, high differences, between the control group and the treatment groups were noted for the TSH concentrations of the female animals (at maximum 75.6 % above the control value at the intermediate dose level, 1.35 to 0.77 ng/ml in the control group). These changes were considered to be spontaneous, as no statistical significance and no dose response relationship was noted.




Urinalysis findings:
no effects observed
Description (incidence and severity):
ADULT COHORT 1A ANIMALS
Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
A slight but statistically significantly increased specific gravity was noted at the high dose level for the male and female animals (1.6 % or 1.8 % above the control, respectively, p ≤ 0.01). However, as nearly all individual values at the high dose level were within the Provivo background range, these changes were considered to be spontaneous.
The pH value was slightly reduced at the high dose level of the males (5.2 % below the control, p ≤ 0.05). Two individual values from the high dose level (pH = 5.9) were marginally below the Provivo background range (pH = 6.0 to 7.3). As nearly all individual values were within the Provivo background range the reduced pH value that was observed at the high dose level was considered to be spontaneous.
Sexual maturation:
no effects observed
Description (incidence and severity):
Males (Cohort 1A and 1B combined)
No test item-related differences between the control group and the test item-treated groups (15, 50 or 250 mg test item/kg b.w./day) were noted for the time point of balano-prepuital separation.
The reduced body weight of around 10.8% for the highest dose group that was noted on post-natal day 22 was also noted at the time point of preputial separation. Nevertheless, no adverse effect on the time point of preputial separation was noted for any of the dose groups. The time point of preputial separation ranged between post-natal days 22.1 and 22.3.

Females (Cohort 1A & 1B)
No test item-related differences between the control group and the low dose group (15 or 50 mg test item/kg b.w./day) was noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.
Yet, for the combined females of Cohort 1A and Cohort 1B a statistically significant (p ≤ 0.01) delay was noted for the time point of vaginal opening at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day).
In detail, the time point of vaginal opening for the female animals of the control group was on post-natal day 32.3 ± 1.9, at the low dose at 33.2 ± 2.1, at the intermediate dose level on 33.6 ± 2.0 and at the high dose level on post-natal day 36.8 ± 3.5. See Text Table 8.25 and Figure 8-9.
A comparison with the Provivo background data revealed that the day of vaginal opening from 13 individual high dosed dams (Co1A/1B combined) (time points of vaginal opening between post-natal days 38 to 47) was above the background range (post-natal days 30 to 37). This detailed analysis shows that the time point of vaginal opening was significantly and even further delayed in the high dose groups then for other to treatment groups.
However, it has to be considered that the time point of vaginal opening is correlated with the body weight of the animals as demonstrated in Figure 8-10.
Due the correlation between the time point of vaginal opening and the body weight of the females, the observed delays in the time-point of vaginal opening were due to the decreased body weight of the female animals of the high dose level around the time point of vaginal opening. The maximal difference in body weight was noted for the females in the high does at PND 29, aka one day before normally vaginal opening is observed in the control groups. Looking further on the days at whom the vaginal opening was observed at 250 mg test item/kg b.w./day all animals still exhibited a body weight that was more than 10% lower than the body weight of the control animals at the same time point. Therefore, the observed delay in vaginal opening at the high dose was indeed a secondary consequence of decreased female body weight in the high dose group and only exhibit a developmental delay.
Hence, the observed delays for the F1 females were secondary to the decreased body weight of the F1 females and not considered as an adverse effect on sexual maturation.
As the timepoint of vaginal opening was observed in older animals in the high dose compared to the control, no statistically significant difference was noted for the body weight at the time point of vaginal opening. Instead of a decreased body weight as noted for the weekly body weights, slightly increased body weights were noted at the time point of vaginal opening at the high dose level (6.9 % above the control), as this was later in time and the animals have grown in the mean time. This indicates a correlation between the time point of vaginal opening and the body weight of the animals.


Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
PUPS No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
The ano-genital distance is dependent on pup body weight and increases with pup body weight. Hence, apparent alterations in the ano-genital distance might occur after treatment with agents that affect overall pup body size. In such cases, hormonal activity might be associated incorrectly with the test item treatment.

The correlation between the ano-genital distance and the pup body weight can be decreased by normalizing the ano-genital distance with the cube root of the pup body weight (see Gallavan et al., 1999 ). Normalizing ano-genital distance with the cube root of body weight yielded a normalized variable that was constant over the range of weights observed in this analysis with no significant relationship between the AGD/cube root of body.
The correlations between the ano-genital distance (normalized to the cube root of pup body weight or not) are given in Figure 7-15. The flattened regression line demonstrated that the results for the ano-genital distance that were obtained in this study were influenced by the pup body weight.

Nipple retention in male pups:
no effects observed
Description (incidence and severity):
PUPS
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
In detail, 7 pups with nipple retention from 5 different dams were noted in the control group and 3 pups with nipple retention from 3 different dams were noted in the high dose group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
PUP
Pup organ weights (absolute) - F1 Pups
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).


ADULT organ weights
COHORT 1A ANIMALS
Males and females
No test item-related differences between the control group and the low dose animals (15 mg test item/kg b.w./day) and intermediate dose females were noted for the organ weight at autopsy.
At the intermediate dose level (50 mg test item/kg b.w./day) test item related changes were noted for the kidney weights of the male animals.
Test item-related changes were noted for the male and female animals of the high dose group (250 mg test item/kg b.w./day) for the organ weights of the spleen and the liver and for the male animals for the kidneys.
The observed changes for the weights of the spleen and the liver (males and femaleswere considered to be test item-related due to correlated findings during the histopathological examination (extramedullary haematopoiesis). Additionally, the increased kidney weights of the males were also considered to be test-item related, enhanced accumulations of hyaline droplets, yet not human relevant However, in case of the liver and the kidneys, most of the observed increase in the relative organ weight was due to the reduced body weight at autopsy that was noted for the male and female animals.
Spleen
Statistically significantly increased absolute and relative spleen weights were noted for the male and the female animals at the high dose level. In detail, the relative and the absolute spleens weights for the high dosed females were 36.7 % and 25.4 % above the control, respectively (p ≤ 0.01). For the males of the high dose group the relative and absolute spleen weights were 28.6 % and 17.4 % above the control, respectively (p ≤ 0.01).
The more pronounced changes that were noted for the relative organ weights could be due to the reduced body weight at autopsy that was noted for the high dosed male and female animals . However, the increased spleen weight was mostly due to the correlating histopathological findings (extramedullary haematopoiesis).
For the male animals increased relative and absolute spleen weights were also noted at the intermediate dose level (53.4 % or 56.8 % above the control, statistically not significant). This was due to one male. Male no. 293 of the intermediate dose group showed an extreme spleen weight of 9.59 g due to a malignant lymphomatous infiltration. As the observations for male no. 293 were considered to be incidental,, the increased spleen weights that were noted for the intermediate dosed male were considered to be spontaneous.

Liver
A statistically significant increase was noted for the relative liver weight from the male and female animals of the high dose group (13.9 % & 12.6 % above the control, respectively, p ≤ 0.05 / 0.01).
In contrast, only a marginal increase was noted for the absolute liver weight of the male and female animals (4.7 % and 3.8 % above the control, respectively, statistically not significant). The only marginally increased absolute liver weight indicated that the observed effect was mostly due to the reduced body weight at autopsy that was noted for the male and female animals of the high dose group of Cohort 1A. However, there were similar histopathological changes in the liver observed (centrilobular hepatocellular hypertrophy) as in the F0 animals. Therefore, the slight increase in absolute liver weight can be attribute to the increased hepatic enzyme induction following the erythrocyte degradation.

As for the spleen weight the male animal no 293 of the intermediate dose groups showed massiv increase in the liver weight (143.2 g) compared to control groupe mean of 36.43 g. Subsequently, an increased relative and absolute liver weight was noted for the male animals of the intermediate dose group (20.1 % and 21.0 % above the control, respectively, statistically not significant). As the observations for male no. 293 were considered to be incidental , the increased liver weights for the male animals at the intermediate dose level were considered to be spontaneous.

Kidney
For the male animals of the high dose group statistically significantly increased relative organ weights were noted for the left and the right kidneys (12.5 % and 16.6 % above the control, respectively, p ≤ 0.01).
As for the absolute liver weight, the absolute kidney weights of the high dose group were only marginally increased (3.4 % and 7.3 % above the control, respectively, statistically not significant). The only marginally increased absolute kidney weight indicated that the observed effect was mostly due to the reduced body weight at autopsy that was noted for the male animals of the high dose group of Cohort 1A. However, there were similar histopathological changes in the kidney observed (hyaline droplets) as in the F0 animals. Therefore, the slight increase in absolute kidney weight can be attribute to the increased deposition of hyaline droplet in the kidney.
Changes in organ weights that were not considered to be test item-related:
Brain
Due to the decreased body weights at autopsy for the male and female animals of the high dose group, a slight but statistically significantly decreased absolute brain weight was noted for the male and female animals of the high dose group (4.9 % and 4.8 % below the control, respectively, p ≤ 0.01). No decrease was noted for the relative brain weight of the male and female animals of the high dose group.
Hence, as the decreased absolute brain weight was due to a reduced body weight at autopsy and no corresponding changes were noted during the histopathological examination, the observed decrease in the absolute brain weight at the high dose level was considered to be not of toxicological relevance.
Heart
Also the decreased absolute heart weight that was noted for the male animals of the high dose group (11.4 % below the control, p ≤ 0.01) was due to the decreased body weight at autopsy. For the relative heart weight of the male animals from the high dose group only a marginal reduction was noted (3.1 % below the control, statistically not significant).
As for the decreased absolute brain weight, the observed decreased absolute heart weight was not considered to be of toxicological relevance, as it was due to a decreased body weight at autopsy and no corresponding changes were noted during the histopathological examination.
Cervical lymph node
Increased relative and absolute organ weights were noted at the high dose level for the cervical lymph nodes (52.2 % and 38.4 % above the control, respectively p ≤ 0.05 or statistically not significant). However, no corresponding changes or abnormalities were observed during the histopathological examination in the lymph nodes of any animals from this study.
Mesenteric lymph node
Increased relative and absolute organ weights were noted for the mesenteric lymph nodes form the male animals of all treatment groups. The increased weights were statistically significant for the absolute weight of the mesenteric lymph nodes from the male animals at the intermediate (37.1 % above the control, p ≤ 0.05) and the high dose level (62.1 % above the control, p ≤ 0.01).
For the absolute weight of the mesenteric lymph nodes from the male animals a statistically significant increase was only noted at the high dose level (47.4 % above the control, p ≤ 0.05).
Kidneys
In contrast, the observed weight changes that were noted for the kidneys of the female animals were not correlated to histopathological findings but due to a decreased body weight at autopsy. Therefore, they were considered not toxicological relevant
In detail, slightly increased relative kidney weights were noted for both kidneys at the intermediate and the high dose level (between 5.6 and 8.1 % above the control, statistically not significant and p ≤ 0.05 / 0.01, respectively).
No statistically significant increase was noted for the absolute weights of the left and right kidneys. Hence, the observed increase at the high dose level for the relative weights was due to the decreased body weights at autopsy and considered not as toxicological relevant.

COHORT 1B ANIMALS
Males
No differences of toxicological relevance were noted for the examined absolute and relative organ weights between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
However, at the high dose level a reduced absolute organ weight was noted for the right epididymis (13.2 % below the control, p ≤ 0.05) and prostate gland (19.3 % below the control, p ≤ 0.01).
Increased relative organ weights were noted for the right testis (8.4 % above the control, p ≤ 0.01) and the pituitary gland (13.5 % above the control, p ≤. 0.01).
As the changes for the above mentioned organs were due to the reduced body weight at autopsy of the male animals, they were considered to be not of toxicological relevance.
Females
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
However, statistically significantly increased relative organ weights were noted for the pituitary gland (18.5 % above the control, p ≤ 0.01) and the left oviduct (27.0 % above the control, p ≤ 0.01).
As the changes for the above mentioned organs were due to the reduced body weight at autopsy of the female animals, they were considered to be not of toxicological relevance.
At the intermediate dose level an increased relative organ weight was noted for the thyroid (including parathyroid) (17.6 % above the control, p ≤ 0.05). As no dose response-relationship was noted and no increase was noted for the females of Cohort 1A, the observation was considered to be spontaneous.




Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
PUPS
External and internal examination of the pups – F1 Pups
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external and internal examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).

ADULT COHORT 1A Animals
Males and females (survivors)
No test item-related macroscopic changes were recorded for the surviving male and female animals of the low, the intermediate and the high dose group (15, 50 or 250 mg test item/kg b.w./day).
All macroscopic findings recorded in F1-C1A animals were within the range of normal background lesions or physiological alterations which may be observed in this type of study or animals of this strain and age, or the lesions without dose-response relationship in the incidence. There were no macroscopic findings that could be attributed to treatment with the test item in either sex.

However, scratch or eschar sites were noted on shoulder, neck or ear from one male of the control group, one male of the low dose group and 2 males of the intermediate and the high dose group each. A histopathological examination of the scratch sites was performed for the affected male of the control group (no. 202) and the 2 affected males of the high dose group (nos. 325 and 337). The histopathological examination of the scratch sites revealed ulceration, inflammation, fibrosis and squamous hyperplasia/hyperkeratosis in the skin. However, these and other findings were not considered to be test item-related.

Enlargement of the spleen, liver and lymph nodes (tracheal, bronchial and iliac) was recorded in one male (No. 293) of group 3. In this animal, dark-red discoloration of the lymph node, as well as enlargement and light-brown discoloration of the adrenal glands, were also recorded. These gross changes indicate malignant lymphoma, and this diagnosis was supported by the histological examination of the liver and spleen of this animal.
Under the condition of this study, there were no neoplastic lymph proliferative lesions in the high dose group of either generation observed. Therefore, gross lesions recorded in this intermediate dosed male animal were considered to be of incidental nature and not treatment-related.

ADULT COHORT 1B Animals
Males (surviving) and females
No test item-related observations were noted for the male (surviving) and the female animals of the treatment groups (15, 50 or 250 mg test item/kg b.w./day).
However, scratch wounds or eschar on neck or shoulder were noted for 3 male animals of the low and the high dose group each and for one male animal of the intermediate dose group. A similar distribution and incidence of scratch sites or eschar sites was noted for the males of Cohort 1A, which were considered as not test item-related. Hence, the observed scratch wounds or the observed eschar that was noted for the male animals of Cohort 1B can also be considered as not test item-related.
A small testis (left side only) was noted for male no. 451 of the intermediate dose group and for male no 498 (left and right testis affected) of the high dose group. For the high dosed male no. 498 a small epididymis (left and right side) was additionally noted. These observations were correlated with the weights of the testes and epididymides of the affected animals.
Small testes and small epididymides were also noted for the low dose male no. 260 of Cohort 1A.
None of the affected testes and epididymides were histopathologically examined, as this was not requested by the Study Plan. However, the histopathological examination of the testes and epididymides from all high dosed animals of Cohort 1A revealed no test item-related changes. Additionally, no dose response can best established. Hence, the observations at necropsy can be considered as spontaneous.
The macroscopic examination at necropsy for the females of Cohort 1B only revealed one high dose female with a dilated uterus, which was considered as spontaneous.



Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
ADULT COHORT 1A ANIMALS
Males and females (Survivors)
No test item-related differences were noted between the control group and the low dose groups (15mg /kg b.w./day) in the histopathological observations.
Test item-related differences were noted between the control group and the intermediate dose group (50 mg /kg b.w./day) for the kidney histopathology.
Test item-related differences were noted between the control group and the high dose group (250 mg /kg b.w./day) for the kidney, spleen, liver and bone marrow.

In detail, histopathological, treatment-related findings were observed in the spleen, bone marrow, liver, thyroid glands, glands of both sexes from F1 Co1A, in the kidneys of males from F1 Co1A.

The histomorphologic changes in the spleen and bone marrow same as the findings observed in F0 animals were also found in both sexes of F1-C1A animals of Group 4. These included increased severity of hemosiderin deposits and EMH in the spleen and increased incidence and/or severity of congestion in the spleen and erythroid elements hypercellularity in the bone marrow.
These findings were consistent with the changes that are generally found in hemolytic anemia, and the increased splenic weights were thought to reflect these histologic changes. Hypercellularity (increased cellularity) of erythroid elements in the bone marrow was deemed to be reactive change to hemolytic anemia and not to be due to direct effects of the test item to bone marrow.


The histomorphologic liver changes qualitatively same as the findings observed in F0 animals were also found in both sexes of F1-C1A animals of Group 4, which included centrilobular hepatocellular hypertrophy (minimal to slight severity) in the liver and increased incidence and/or severity of thyroid follicular cell hypertrophy. In the liver, there were increase in or increased tendency of absolute and/or relative weights of both sexes of Group 4 of F1 C1A. These were considered to correlate microscopically with centrilobular hepatocellular hypertrophy. Thus, this was considered to be of metabolic nature and of adaptive character, and hence, deemed not to be adverse.
As with F0 generation, there were no further indicators of liver injury in any animals examined and there were no differences to be toxicologically noted in the incidence and severity of hemopoietic cell foci in the liver between Groups 1 and 4.

The histomorphologic kidney changes qualitatively same as the findings observed in F0 animals were also found in F1-C1A males of Group 4, which included increased incidence and group mean severity grade of hyaline droplets in the proximal tubular cells, as well as increased number of males showing higher severity of tubular degeneration/regeneration in comparison with the control males.
As with F0 generation, hyaline droplets were not found in renal tubules of any females.
Increases in or increased tendency of absolute and/or relative kidney weights that were detected in F1 C1A males of Groups 3 and 4 were thought to reflect the enhanced accumulation of hyaline droplets.
Increase in hyaline droplets was considered to be induced by overload of synthetic protein, which is specific in male rat like as alpha-2 microglobulin, derived from hyperfunction of the liver.
In some high dose males of F1 C1A, degenerating and/or regenerated renal tubules were found at slightly higher severity compared to the corresponding control males. This was considered to be secondary to enhanced accumulation of hyaline droplets in the tubular cell, but not due to direct effects of the test item. In addition, enhanced hyaline droplet accumulation associated with hyperfunction of the liver is believed to be a male rat specific phenomenon and not relevant to human. Thus, renal lesions recorded only in male rats were deemed not to be adverse.


There were no differences to be toxicologically concerned in the incidence and severity of the findings observed in the thymus and adrenal glands between Groups 1 and 4.

Diffuse adreno-cortical hypertrophy was found in one female of Group 4 and increased lipid vacuoles in zona fasciculata was observed in five males of Group 4. However, these were within the range of normal background changes or physiological alterations which may be observed in this study type or animals of this strain and age, and therefore, deemed to be of incidental nature and not treatment-related.

Male Reproductive Organs - including Detailed Qualitative Examination for Testes
The remainders of microscopic findings recorded in F1-C1A animals were within the range of normal background lesions or physiological alterations which may be observed in this study type or animals of this strain and age.

No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs including the testis, epididymis, vas deferens, prostate glands, seminal vesicles and coagulating glands from the F1-C1A high dose (Group 4) animals.

All findings recorded in the male reproductive organs and tissues were within the range of normal background changes or physiological alterations that may be observed in this study type and animals of this strain and age, and hence, deemed not to be treatment-related.

For the testis, the stages were checked on completeness of cell populations and stages while taking also into consideration any degenerative changes and the interstitial cell structure, for both generations. As a result, all testes examined showed completeness of stages and cell population, and there were no alterations that could be attributed to treatment with the test item as well.


Female Reproductive Organs - including Quantitative Examination of Ovaries in F1 Generation Cohort 1A Females
No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the female reproductive organs including ovaries, oviducts, uterus, uterine cervix and vagina from the F1-C1A high dose (Group 4) animals.

All findings recorded in the female reproductive organs and tissues were within the range of normal background changes or physiological alterations that may be observed in this study type and animals of this strain and age, and hence, deemed not to be treatment-related.

Further, in F1-C1A females, there was also no direction that was biased to specific status of estrus cycles.

For ovaries, the quantitative evaluation was performed in F1 generation cohort 1A, in addition to qualitative histopathology examination.
In the quantitative evaluation of F1-C1A ovaries, statistically significant increases in the values were detected in the parameters of primordial follicles, growing follicles, summed up primordial and growing follicles and antral follicles of the HD group, compared with that of the corresponding control group.
However, when comparing the values in the parameters from the control group of the present study with that from other studies in which the quantitative evaluation was performed in the same strain (Sprague-Dawley rats) of animals, the control group of the present study showed overall lower values in relative to the reference studies (Table 13). Thus, significant differences between Groups 1 and 4 in the present study were deemed to be of incidental nature detected due to lower values in the parameters of the control group, and it was considered that there was no toxicological significance in the higher values detected in Group 4 in comparison with that in Group 1.
Other effects:
no effects observed
Description (incidence and severity):
BIRTH INDICES AND POST IMPLANTATION LOSS OF F1 PUPS
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
Also the reproductive indices birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).
The percentage of post-implantation loss (considering the number of resorptions and stillbirths) at the high dose level was nearly at the same level as in control group. In detail, the mean percentage of post-implantation loss per dam was 15.04 ± 20.50 % in the control group and 15.28 ± 11.29 % in the high dose group. The post-implantation loss per group was 14.79 % in the high dose group in comparison to 13.99 % in the control group. As already mentioned under gestation index two dames in the low and intermediate dose group showed total resorptions of all implantations. Consequently, the post-implantation loss for these both groups was higher compared to the control and the high dose group.
The mean number of stillbirths and resorptions per dam was 2.1 ± 2.2 in the control group and 2.1 ± 1.5 in the high dose group.
An increased number of 7 stillbirths was noted in the control group (from 5 different dams). In comparison to one stillbirth in the low dose group and the high dose group each were noted. No stillbirths were noted in the intermediate dose group. As the increased number of stillbirths was noted in the control group, this was considered to be spontaneous.

MALE TO FEMALE RATIO OF THE F1 PUPS
No test item-related influence on the male to female ratio was noted for all treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).

LITTER WEIGHT OF F1 PUPS
No test item-related influence on the litter weight was noted at the low and the intermediate dose levels (15, 50 or 150/250 mg test item/kg b.w./day).
Nevertheless, at the high dose level (150/250 mg test item/kg b.w./day) the litter weight was nearly constantly reduced during the whole lactation period from post-natal day 1 (15.3 % below the control; p ≤ 0.01) to post-natal day 21 (12.6 % below the control; p ≤ 0.01). Which is a consequence of the reduced individual pup body weight at the high dose. Therefore, this was a secondary outcome of the reduced dam body weight in the high dose group.
On post-natal days 1 and 4 the reduced litter weight at the high dose level was due to a slightly reduced number of live pups and a slightly reduced pup body weight. After the adjustment of the pup number on post-natal day 4, the further reduced litter weight at the high dose level was caused by a more pronounced reduction of the body weight of the high dose pups.
As the reduced pup body weight was considered to be secondary maternal effect, the reduced litter weight at the high dose level, which was mostly caused by a reduced pup body weight, must also be considered as secondary maternal effect

NUMBER OF LIVE F1 PUPS
No test item-related differences were noted for the mean number of live pups per dam between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day) during the lactation period.
A slightly reduced number of live born pups was noted on post-natal day 1 for the females of the intermediate and the high dose group (12.0 ± 4.7 or 12.0 ± 2.8 in comparison to 12.8 ± 3.6 in the control group, statistically not significant) (see Text Table 7-45). However, as the difference was only slight and not statistically significant, it was considered to be spontaneous.
After the culling process on post-natal day 4, the mean number of live pups per dam was nearly identical between the control group and the high dose group (post-natal day 21: 9.5 ± 2.1 pups per dam in the control group and 9.5 ± 1.5 pups per dam in the high dose group).

EXTERNAL AND INTERNAL EXAMINATION OF THE F1 PUPS
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external and internal examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).

PUP ORGAN WEIGHT OF F1 PUPS (ABSOLUTE)
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).

ADULT
BONE MARROW OF COHORT 1A ANIMALS
Males and females
As for the animals of the F0 Generation, a slight shift in the ratio of myeloid to erythroid cells (statistically not significant) was noted at the high dose level (250 mg test item/kg b.w./day). The shift was more pronounced for the female as for the male animals.
In detail, for the male animals the myeloid to erythroid ratio was 2.043 to 1 in the control group in comparison to 1.758 to 1 at the high dose level.
For the female animals the myeloid to erythroid ratio was 1.864 to 1 in the control group in comparison to 1.475 to 1 at the high dose level.
This shift correlated with the histopathological finding of an enhanced erythropoiesis in the bone marrow in form of hypercellularity of erythroid elements. Which is the result of the counteract of the body to the treatment related enhanced erythrocyte degradation.


Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
PUPS
For the pots-natal development a reduced pup body weight that was noted at the high dose level (150/250 mg/kg b.w./day) on lactation days 7, 14 and 21. This was a secondary consequence of the reduced maternal weight during gestation and lactation period. However, no changes in birth indices, pup viability, number of live pups, anao-genital distance, nipple retention or in the thyroid horman levels were observed after the treatment with thes test-item.

ADULTS
Summarised results - Cohort 1A
All test item-related and adverse observations that were noted for the male and female animals of Cohort 1A were related to a haemolytic anemia. This was also the case for the male and female animals of the F0 Generation. All these observations were considered for the setting of the NOAEL:
• Changes in the red blood cell parameter and an increased bilirubin concentration that were noted for the male and female animals of the high dose group (250 mg/kg b.w./day).
• An increased spleen weight that was noted for the male and female animals of the high dose group (250 mg/kg b.w./day).
• Histopathological changes in the spleen and the bone marrow of the male and female animals of the intermediate and the high dose level (50 or 250 mg/kg b.w./day).
• A slight shift that was noted during the bone marrow examination for the myeloid / erythroid ratio of the male and female animals of the high dose group (250 mg/kg b.w./day).
• Slightly to moderately reduced body weight that was noted at the high dose level for the male and female animals.

The following test item-related changes that were noted for the animals of Cohort 1A were not considered to be adverse and, hence, not considered for the NOAEL:
• A short lasting post dosing salivation that was noted for the male and female animals of the intermediate and the high dose level.
• A marginally to slightly reduced body weight at the intermediate dose level that was noted for the male animals.
The observed reduction in body weight that was noted at the intermediate dose level for the male animals was not considered as adverse, as the difference declined to the end of the study and no difference was noted in body weight gain.
• The delay in vaginal opening that was noted at the high dose level, was due to the reduced body weight of the female animals (Cohort 1A and 1B combined) and the high dose level Therefore, this observation is a secondary effect.
• An increased T4 concentration at the intermediate and the high dose level, were related to the liver changes observed during the histopathological examinations at the intermediate and the high dose level.
• Observations that were noted during the histopathological examination for the male and female animals of the intermediate and the high dose group in the liver and the kidneys (males only) and for the male and female animals of the high dose group in the thyroid gland.
• Changes of the absolute organ weights that were noted for the male and the female animals of the high dose group for the liver and the kidneys (males only). The changes were mainly due to the reduced body weight at autopsy.

Summarised results - Cohort 1B
All test item-related and adverse observations that were noted for the male and female animals of Cohort 1B which are considered for the NOAEL:
• A slightly to moderately decreased body weight that was noted for the male and female animals of the high dose group.
In Cohort 1B no parameters were examined that could be affected by the haemolytic anemia that was noted for the animals of the F0 Generation and of Cohort 1A. Hence, no further test item-related adverse changes, then the reduced body weight, were noted for the male and female animals of Cohort 1B.

However, the following test item-related changes that were not considered to be adverse were noted for the male and female animals of Cohort 1B
• A short lasting post dosing salivation that was noted for the male and female animals.
• A marginally to slightly decreased body weight that was noted for the females at the intermediate dose level one week after the start of dosing (PND 29). As the difference declined thereafter and no difference in body weight gain between the control group and the females of the intermediate dose group were noted, this was considered as not adverse.


Key result
Dose descriptor:
NOAEL
Remarks:
for developmental toxicity of F1 pups
Generation:
F1
Effect level:
> 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
for general toxicity for adult F1 animals
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Key result
Reproductive effects observed:
no
Treatment related:
no

Text Table 7‑1:     Reproductive outcome of the female animals.


















































































































Test item



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



Females started dosing



24



24



24



24



Females used for pairing


(paired together with a male at the beginning of the mating period)



24



24



24



24



Females with no verified copulation during the mating period of 14 days



1 (42) #



-



-



1 (186)



 



- thereof non-pregnant



1 (42)



-



-



1 (186)



 



- thereof pregnant



-



-



-



-



Females with verified copulation during the mating period of 14 days



23



24



24



23



 



- thereof non-pregnant



-



3


(79, 81, 92)



-



1


(174)



 



- thereof pregnant



23



21



24



22



Fertility index


(Pregnant females with verified copulation related to females with verified copulation)



100 %


(23 of 23)



88 %


(21 of 24)



100 %


(24 of 24)



96 %


(22 of 23)



Females with resorption of all implants



1


(48)



2


(86, 95)



2


(133, 140)



0



Females with live born pups



22



19



22



22



Females that delivered only stillbirth



0



0



0



0



#:



Number of the animals is given in brackets.


       

Text Table 7‑11:    Statistically significant changes that were noted for the male or female animals for the parameters of the red blood cells.






































































































































 



 



Changes in comparison to control


[%]



Reason



Parameter #


F0



Sex



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



 



 



HGB


[mmol/L]



Males



+0.3



+0.3



- 4.4



A



HGB


[mmol/L]



Females



+1.0



- 3.3



- 9.4**



RBC


[x10E6/µL]



Males



+2.0



- 3.9



- 6.0



RBC


[x10E6/µL]



Females



- 0.3



- 7.6**



- 16.1**



Reticulocytes


[%]



Males



- 8.1



- 5.6



+55.9**



Reticulocytes


[%]



Females



+1.0



+59.9**



+168.8**



HCT


[%]



Males



+1.2



- 1.1



- 2.0



HCT


[%]



Females



+1.2



- 3.9



- 8.1**



MCV


[fL]



Males



- 0.7



+3.0



+4.4



MCV


[fL]



Females



+1.5



+3.9



+9.5**



MCH


[fmoL]



Males



- 1.6



+4.7



+1.6



MCH


[fmoL]



Females



+1.2



+4.3*



+8.0**



MCHC


[mmol/L]



Males



- 0.7



+1.7



- 2.6**



MCHC


[mmol/L]



Females



- 0.3



+0.5



-1.4



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#:



Values taken from table 8-1 'Haematological Parameters - Summary - Males' and table 8-2 'Haematological Parameters - Summary - Females'.



A:



A haemolytic anemia was noted for the male and female animals of the high dose group during the histopathological examination. As the listed parameters belongs to the red blood cell parameters, noticeable differences in comparison to the control (statistically significant or not) that were noted at the intermediate and the high dose level were considered to be test item-related.



Text Table 7‑12:    Statistically significant changes of the haematological parameters that were not considered to be test item-related.



































































 



 



Changes in comparison to control


[%]



Reason



Parameter #


F0



Sex



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



 



 



Neutrophilic granulocytes


[x10E3/µL]



Males



+8.7



+52.5*



+82.9**



A



Monocytic granulocytes


[x10E3/µL]



Males



+6.7



+17.2



+48.3**



A



Monocytic granulocytes i


[x10E3/µL]



Females



+1.6



0.0



+47.7**



A



LUC


[x10E3/µL]



Females



- 6.4



+14.1



+74.4**



A



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#:



Values taken from table 8-1 'Haematological Parameters - Summary - Males' and table 8-2 'Haematological Parameters - Summary - Females'.



A:



The changes were not considered to be test item-related. The individual values were all or nearly all in the range of the background data and / or no changes were noted for the animals of Cohort 1A.



 


Text Table 7-13:    Male animals - F0 Generation: comparison of statistically significantly different parameters with the Provivo background data.



































































































Parameter


(males)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Reticulocytes


(%)



Group 1



2.70 ± 0.73


(2.0 – 4.4)  [n=1]



5% to 95% Percentile


 


1.5 – 3.4



Group 2



2.48 ± 0.41


(1.7 – 3.1)



Group 3



2.55 ± 0.63


(1.0 – 3.2)



Group 4



4.21 ± 1.00**


(3.4 – 6.4)  [n=8]



Neutrophilic granulocytes


(x10E3/µL)



Group 1



1.228 ± 0.516


[n=1]  (0.54 – 2.23)



5% to 95% Percentile


 


0.72 – 2.70



Group 2



1.335 ± 0.385


(0.99 – 2.23)



Group 3



1.873 ± 0.676*


(0.84 – 2.88)  [n=1]



Group 4



2.246 ± 0.657**


(1.52 – 3.86)  [n=1]



Monocytic granulocytes


(x10E3/µL)



Group 1



0.238 ± 0.066


(0.16 – 0.39)



5% to 95% Percentile


 


0.12 – 0.46



Group 2



0.254 ± 0.068


(0.16 – 0.38)



Group 3



0.279 ± 0.122


[n=1]  (0.08 – 0.52)  [n=1]



Group 4



0.353 ± 0.090**


(0.29 – 0.58)  [n=1]



MCHC


(mmol/L)



Group 1



21.105 ± 0.371


(20.60 – 21.78)  [n=1]



5% to 95% Percentile


 


19.65 – 21.60



Group 2



20.948 ± 0.248


(20.48 – 21.21)



Group 3



21.473 ± 0.919


(20.54 – 23.95)  [n=1]



Group 4



20.555 ± 0.262**


(20.23 – 21.01)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 8-1 and 8-3 'Haematological Parameters - Summary and Individual Data - Males'.



 



 



 


Text Table 7-14:    Female animals - F0 Generation: Comparison of statistically significantly different parameters with the Provivo background data.



















































































Parameter


(females)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



HGB


(mol/L)



Group 1



10.19 ± 0.36


(9.6 – 10.8)  [n=1]



5% to 95% Percentile


 


9.2 – 10.7



Group 2



10.29 ± 0.20


(10.0 – 10.5)



Group 3



9.85 ± 0.42


[n=1]  (9.1 – 10.6)



Group 4



9.23 ± 0.35**


[n=5]  (8.6 – 9.8)



RBC


(x10E6/µL)



Group 1



8.437 ± 0.391


(7.74 – 9.08)



5% to 95% Percentile


 


7.74 – 9.10



Group 2



8.410 ± 0.333


(7.89 – 8.87)



Group 3



7.799 ± 0.262**


[n=4] (7.35 – 8.14)



Group 4



7.077 ± 0.430**


[n=10]  (6.18 – 7.72)



Reticulocytes


(%)



Group 1



1.92 ± 0.49


(1.1 – 2.6)



5% to 95% Percentile


 


0.6 – 4.9



Group 2



1.94 ± 0.40


(1.6 – 2.8)



Group 3



3.07 ± 0.84**


(2.0 – 4.4)



Group 4



5.16 ± 1.26**


(3.6 – 7.2)  [n=4]



HCT


(%)



Group 1



47.50 ± 1.43


(45.3 – 49.9)



5% to 95% Percentile


 


44.0 – 53.3



Group 2



48.05 ± 1.22


(46.4 – 49.8)



Group 3



45.66 ± 2.16


[n=3]  (42.9 – 49.8)



Group 4



43.65 ± 2.26**


[n=6] (39.1 – 46.9)







































































































Parameter


(females)


F0



Values from this study


Mean value per group ± SD


(range of the individual


females) #2



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 - 2021



Monocytes


[x10E3/µL]



Group 1



0.365 ± 0.109


(0.23 – 0.55)



5% to 95% Percentile


 


0.06 – 0.55



Group 2



0.371 ± 0.087


(0.27 – 0.56)  [n=1]



Group 3



0.365 ± 0.130


(0.08 – 0.50)



Group 4



0.539 ± 0.142**


(0.35 – 0.78)  [n=4]



LUC


[x10E3/µL]



Group 1



0.078 ± 0.027


(0.05 – 0.14)



5% to 95% Percentile


 


0.02 – 0.22



Group 2



0.073 ± 0.022


(0.05 – 0.11)



Group 3



0.089 ± 0.039


(0.02 – 0.17)



Group 4



0.136 ± 0.058**


(0.06 – 0.27)  [n=1]



MCV


[fL]



Group 1



56.38 ± 2.52


[n=1]  (52.9 – 62.5)



5% to 95% Percentile


 


53.1 – 64.0



Group 2



57.21 ± 1.76


(54.1 – 59.8)



Group 3



58.56 ± 2.28


(55.2 – 62.0)



Group 4



61.72 ± 1.59**


(58.6 – 64.5)  [n=1]



MCH


[fmoL]



Group 1



1.210 ± 0.056


[n=1]  (1.10 – 1.31)  [n=1]



5% to 95% Percentile


 


1.12 – 1.30



Group 2



1.224 ± 0.035


(1.16 – 1.28)



Group 3



1.262 ± 0.037*


(1.21 – 1.32)  [n=1]



Group 4



1.307 ± 0.045**


(1.24 – 1.39)  [n=5]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 8-2 and 8-4 'Haematological Parameters - Summary and Individual Data - Females'.


     

 


Text Table 7‑15:    Statistically significant changes of the biochemical parameters that were considered to be test item-related.

















































Parameter #


(F0)



 



Changes in comparison to control


[%]



Reason



Sex



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



 



 



Bilirubin (total)


[µmol/L]



Males



0.0



+9.3



+32.4**



A



Bilirubin (total)


[µmol/L]



Females



+8.5



+32.4*



+82.7**



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:



Considered to be test item-related as it was correlated to the test item-related decrease in the number of red blood cells.



#:



Values taken from table 9-1 'Biochemical Parameters - Summary - Males' and from table 9-2 'Biochemical Parameters - Summary - Females'.



 


Text Table 7‑16:    Statistically significant changes of the biochemical parameters that were not considered to be test item-related.

















































































































 



 



Changes in comparison to control


[%]



Reason



Parameter #


(F0)



Sex



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



 



 



Globulin


[g/L]



Males



+1.5



- 1.2



+9.4*



A



Protein (total)


(g/L)



Males



+0.5



- 1.8



+5.2*



BUN / Creatinine


ratio



Females



+6.5



+23.5*



+23.2*



Glucose


(mmol/L)



Females



+0.4



- 1.6



+15.3*



Urea (in blood)


(mmol/L)



Females



+6.3



+23.6*



+26.1**



aP


(U/L)



Females



+9.1



- 3.4



+61.6**



B



aP


(U/L)



Males



- 7.8



- 9.3



+36.4



C



aP


(U/L)



Males


(Co1A)



0.0



- 3.6



- 12.6



aP


(U/L)



Females


(Co1A)



+1.0



- 8.6



+2.8



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:


 



Considered to be spontaneous, as all or nearly all individual values were within the background range and no changes were noted for the other sex.



B:



Considered to be spontaneous, as no statistically significant increase was noted for the male animals and the male and female animals of Cohort 1A.



C:



Only given for reason of comparison.



#:


 



Values taken from table 9-1 'Biochemical Parameters - Summary - Males' and from table 9-2 'Biochemical Parameters - Summary - Females'.



 



 



 


Text Table 7-17:    Comparison of the biochemical parameters of the male animals with statistically significant changes with the Provivo background data.













































































Parameter


(males)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Globulin


[g/L]



Group 1



29.02 ± 1.81


[n=1]  (24.7 – 30.6)



5% to 95% Percentile


 


26.6 – 35.9



Group 2



29.46 ± 1.97


[n=1]  (25.4 – 31.7)



Group 3



28.68 ± 1.87


[n=1]  (25.0 – 30.9)



Group 4



31.76 ± 2.29*


(28.5 – 34.6)



Bilirubin (total)


[µmol/L]



Group 1



3.21 ± 0.53


(2.5 – 3.9)



5% to 95% Percentile


 


2.5 – 4.3



Group 2



3.21 ± 0.43


(2.6 – 3.9)



Group 3



3.51 ± 0.31


(2.9 – 4.0)



Group 4



4.25 ± 0.53**


(3.3 – 5.1)  [n=5]



Protein (total)


(g/L)



Group 1



61.5 ± 2.6


[n=1]  (55 – 65)



5% to 95% Percentile


 


57 – 67



Group 2



61.8 ± 1.8


(58 – 64)



Group 3



60.4 ± 1.9


(57 – 63)



Group 4



64.7 ± 2.8*


(61 – 68)  [n=3]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 9-1 and 9-3 'Biochemical Parameters - Summary and Individual Data - Males'.



 


Table 7-18:           Comparison of the biochemical parameters of the female animals with statistically significant changes with the Provivo background data.



















































































Parameter


(females)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Bilirubin (total)


[µmol/L]



Group 1



2.84 ± 0.47


[n=1)  (2.2 – 3.7)



5% to 95% Percentile


 


2.4 – 4.5



Group 2



3.08 ± 0.57


[n=1]  (2.3 – 4.0)



Group 3



3.76 ± 0.68*


(2.7 – 4.9)  [n=2]



Group 4



5.19 ± 1.01**


(3.7 – 6.6)  [n=6]



BUN / Creatinine


ratio



Group 1



142.266 ± 20.389


(113.54 – 188.00)



5% to 95% Percentile


 


107.17 – 225.58



Group 2



151.508 ± 12.549


(125.49 – 165.80)



Group 3



175.765 ± 33.161*


(122.45 – 229.18)  [n=1]



Group 4



175.292 ± 28.090*


(177.33 – 222.50)



Glucose


(mmol/L)



Group 1



7.410 ± 1.147


[n=1]  (5.56 – 8.96)



5% to 95% Percentile


 


5.88 – 9.72



Group 2



7.442 ± 0.755


(6.82 – 9.26)



Group 3



7.289 ± 0.881


(6.12 – 8.47)



Group 4



8.546 ± 0.713*


(7.52 – 9.98)  [n=1]



Urea (in blood)


(mmol/L)



Group 1



6.761 ± 0.903


(5.45 – 8.46)



5% to 95% Percentile


 


5.32 – 11.94



Group 2



7.188 ± 0.673


(6.09 – 8.29)



Group 3



8.356 ± 1.527*


(6.00 – 11.23)



Group 4



8.525 ± 1.550**


(5.52 – 10.35)



 









































Parameter


(females)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



aP


(U/L)



Group 1



96.6 ± 27.0


(67 – 143)



5% to 95% Percentile


 


48 – 164



Group 2



105.4 ± 31.4


(70 – 150)



Group 3



93.3 ± 30.0


(54 – 151)



Group 4



156.1 ± 59.0**


(62 – 244)  [n=5]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 9-2 and 9-4 'Biochemical Parameters - Summary and Individual Data - Females'.



 


Text Table 7‑19:    Statistically significant changes of the urinary parameters that were not considered to be test item-related.









































































Parameter #


(F0)



Absolut values


(Changes in comparison to control %)



Reason



Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



 



 



Specific gravity


[g/mL]



Male



1.0495


-



1.0447


(- 0.5)



1.0665


(+1.6)



1.0628*


(+1.3)



A



pH



Male



6.85


-



6.73


(- 1.8)



6.35**


(- 7.3)



6.29**


(- 8.2)



A



pH



Female



6.71


-



6.70


(- 0.1)



7.37**


(+ 9.8)



6.95


(+3.6)



B



Urine volume (relative)


(mL/kg b.w./24 h)



Male



22.11


-



23.31


(+5.4)



15.17*


(- 31.4)



18.57


(- 16.0)



A



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:



Considered to be spontaneous, as nearly all individual values were within the background range.



B:



Considered to be spontaneous, as no dose-response relationship.



#:



Values taken from table 10-1 'Urinalysis - Summary - Males' and from table 10-2 'Urinalysis - Summary - Females'.



 


Text Table 7-20:    Comparison of urinalysis parameter with the Provivo background data.




















































































Parameter


(males)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Specific gravity


[g/mL]



Group 1



1.0495 ± 0.0139


(1.035 – 1.080)



5% to 95% Percentile


 


1.028 – 1.096



Group 2



1.0447 ± 0.0128


(1.030 – 1.070)



Group 3



1.0665 ± 0.0215


(1.045 – 1.120)  [n=1]



Group 4



1.0628 ± 0.0122*


(1.050 – 1.090)



pH



Group 1



6.85 ± 0.23


(6.5 – 7.2)



5% to 95% Percentile


 


6.0 – 7.3



Group 2



6.73 ± 0.24


(6.4 – 7.1)



Group 3



6.35 ± 0.29**


[n=1]  (5.9 – 7.0)



Group 4



6.29 ± 0.21**


(6.0 – 6.7)



Volume


(mL/kg b.w./


24 h)



Group 1



22.11 ± 6.77


(10.2 – 33.3)



5% to 95% Percentile


 


9.1 – 33.3



Group 2



23.31 ± 7.63


(11.0 – 31.8)



Group 3



15.17 ± 3.61*


[n=1]  (7.2 – 19.6)



Group 4



18.57 ± 6.04


[n=1]  (7.8 – 26.7)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from table 10-3 'Urinalysis - Individual Data - Males'.


     

Text Table 7-21:    Comparison of urinalysis parameter with the Provivo background data.
















































Parameter


(females)


F0



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



pH



Group 1



6.71 ± 0.26


(6.3 – 7.1)



5% to 95% Percentile


 


5.9 – 7.5



Group 2



6.70 ± 0.39


(5.9 – 7.3)



Group 3



7.37 ± 0.56**


(6.2 – 8.1)  [n=5]



Group 4



6.95 ± 0.58


(6.2 – 8.1)  [n=1]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from table 10-4 'Urinalysis - Individual Data - Females'.


     

 


 


Text Table 7-22:    Changes of the T4 and TSH hormones, not considered to be test item-related.






























































Parameter #1


(F0)



Sex



Changes in comparison to control [%]



Reason



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



 



 



T4


[nmol/L]



males



+1.6



- 2.9



+5.9



A



T4


[nmol/L]



females



+26.3



+3.1



+43.2



TSH


[ng/mL]



males



+49.4



+26.9



+53.4



TSH


[ng/mL]



females



+0.0



+23.4



+22.0



#1:



Values taken from table 11-1 'Thyroid Hormone Level Analysis - Summary - Males' and from table 11-2 ‘Thyroid Hormone Level Analysis - Summary - Females'.



A:



The observed changes were considered to be spontaneous, as no statistical significance and no dose-response relationship was noted..



 



 



 


Text Table 7-23:    Comparison of the T4 concentrations with the Provivo background range.































































Parameter


(F0)



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



T4


[nmol/L]


(males)



Group 1



53.5879 ± 7.7345


(37.141 – 64.639)



5% to 95% Percentile


 


33.618 – 66.057



Group 2



54.4205 ± 11.9293


[n=1]  (30.889 – 71.826)  [n=1]



Group 3



52.0175 ± 13.6619


[n=1]  (29.863 – 72.820)  [n=2]



Group 4



56.7236 ± 12.4255


(34.047 – 74.743)  [n=3])



T4


[nmol/L]


(females)



Group 1



33.6680 ± 12.9124


[n=2]  (13.777 – 47.395)



5% to 95% Percentile


 


18.464 – 49.518



Group 2



42.5252 ± 6.2503


(33.051 – 52.011) )  [n=2]



Group 3



34.7108 ± 15.8118


[n=2]  (13.465 – 57.574)  [n=2]



Group 4



48.2003 ± 18.4321


[n=1]  (16.691 – 78.771)  [n=5]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 11-1 and 11-3 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males' and from tables 11-2 and 11-4 'Thyroid Hormone Level Analysis – Summary and Individual Data – Females’.



 



 



 


Text Table 7-27:        Stage of the oestrous cycle at necropsy. The stage of the oestrous cycle was evaluated from vaginal lavages that were taken at necropsy and during the microscopic examination of the vagina (only group 1 and 4).









































































Stage of oestrous cycle


at necropsy


(F0 Generation) #1



Group 1


Control #2



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



N



H



N



N



N



H



Prooestrus



1 of 24



10 of 21



2 of 24



1 of 24



1 of 24



2 of 20



Oestrus



1 of 24



1 of 21



3 of 24



5 of 24



5 of 24



4 of 20



Metoestrus



5 of 24



1 of 21



5 of 24



3 of 24



2 of 24



2 of 20



Dioestrus



17 of 24



9 of 21



14 of 24



15 of 24



16 of 24



12 of 20



-:



not detected.



#1:



The values are taken from table 15 ‘Stage of Oestrous Cycle at Necropsy - Individual Data - F0 Generation’.



N:



Stage of the oestrous cycle was determined from the vaginal lavage at necropsy.



H:



Stage of the oestrous cycle was determined during the microscopic examination of the vagina (see Appendix 5 'Histopathological Phase Report').



 


Text Table 7-28:        Test item-related changes in organ weights






































































































































F0 Generation


Parameter



Sex



Changes in comparison to control


[%]



Reason



Group 2



Group 3



Group 4



 



 



Spleen


(relative) #1



Male



- 11.4



- 6.8



+11.5**



A



Spleen


(absolute) #2



Male



- 8.4



- 5.5



+ 5.7**



Spleen


(relative) #3



Female



- 5.3



+2.7



+44.0**



Spleen


(absolute) #4



Female



- 2.8



+2.2



+42.9**



Liver


(relative) #1



Male



- 2.3



- 0.4



+10.8*



Liver


(absolute) #2



Male



+1.1



+1.0



+5.3



Liver


(relative) #3



Female



- 1.9



- 2.5



+8.4**



Liver


(absolute) #4



Female



+2.0



- 2.5



+8.9



Kidney


(left, relative) #1



Male



+2.7



+7.1*



+20.8**



Kidney


(left, absolute) #2



Male



+6.1



+8.9*



+15.3**



Kidney


(right, relative) #1



Male



+2.1



+7.7*



+22.1**



Kidney


(right, absolute) #2



Male



+5.4



+9.7**



+16.4**



Thymus


(relative) #1



Male



0.0



- 3.2



- 9.3



Thymus


(absolute) #2



Male



+2.7



- 1.1



- 13.9



Thymus


(relative) #3



Female



- 6.5



+13.0



- 23.2**



Thymus


(absolute) #4



Female



- 5.0



+12.6



- 23.6**



- to be continued -


 


 











































































































F0 Generation


Parameter



Sex



Changes in comparison to control


[%]



Reason



Group 2



Group 3



Group 4



Adrenal gland


(left, relative) #3



Female



- 2.2



+3.0



+13.5*



A



Adrenal gland


(left, absolute) #4



Female



+1.1



+3.1



+13.8*



Adrenal gland


(right, relative) #3



Female



- 1.8



- 0.7



+7.5



Adrenal gland


(right, absolute) #4



Female



+1.5



-0.2



+7.7



Adrenal gland


(left, relative) #1



Male



- 2.2



- 1.5



+10.3



Adrenal gland


(left, absolute) #2



Male



+1.2



0.0



+4.3



Adrenal gland


(right, relative) #1



Male



- 1.8



- 3.4



+12.3



Adrenal gland


(right, absolute) #2



Male



+1.5



- 2.1



+6.3



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Taken from table 17-1 ‘Relative Organ Weights - Summary - Males - F0’.



#2:



Taken from table 18-1 ‘Absolute Organ Weights - Summary - Males - F0’.



#3:



Taken from table 17-2 ‘Relative Organ Weights - Summary - Females - F0’.



#4:



Taken from table 18-2 ‘Absolute Organ Weights - Summary - Females - F0’.



A:



The observed changes could be correlated with histopathological findings.


       

Text Table 7-29:        Statistically significant changes in organ weights, not considered to be test item-related.































































































































F0 Generation


Parameter



 



Changes in comparison to control


[%]



Reason



Sex



Group 2



Group 3



Group 4



 



 



Brain


(relative) #1



Male



- 5.5



- 3.4



+1.4



A



Brain


(absolute) #2



Male



- 2.0



- 1.6



- 3.4**



Heart


(relative) #1



Male



- 3.4



- 5.8*



+0.9



B



Heart


(absolute) #2



Male



- 0.2



- 4.1



- 4.0



Ovary


(left, relative) #3



Female



- 5.6



- 18.6*



- 13.3



C



Ovary


(left, absolute) #4



Female



- 2.9



- 19.3*



- 13.0



Ovary


(right, relative) #3



Female



- 1.7



- 1.7



- 6.0



D



Ovary


(right, absolute) #4



Female



+ 0.1



- 2.2



- 5.9



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Taken from table 17-1 ‘Relative Organ Weights - Summary - Males - F0’.



#2:



Taken from table 18-1 ‘Absolute Organ Weights - Summary - Males - F0’.



#3:



Taken from table 17-2 ‘Relative Organ Weights - Summary - Females - F0’.



#4:



Taken from table 18-2 ‘Absolute Organ Weights - Summary - Females - F0’.



A:



Considered to be spontaneous as no changes were noted fort eh relative brain weight and no changes were noted during the histopathological examination.



B:



Considered to be spontaneous, as no dose-response relationship was noted.



C:



Considered to be spontaneous as no dose response relationship was noted and no changes were noted for the right ovary.



D:



Only given for comparison.


       

 


Text Table 7-30:    Comparison of organ weights with the Provivo background data.



























































Parameter


(F0)



Values from this study #2


Mean value per group ± SD


(range of the individual values (n= up to 24))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021



Spleen


[g]


(male)



Group 1



0.953 ± 0.421


(n=2]  (0.57 – 2.79)  [n=2]



 


5 to 95 % Percentile


0.66 – 1.13



Group 2



0.873 ± 0.104


(0.66 – 1.06)



Group 3



0.900 ± 0.131


(0.70 – 1.18)  [n=1]



Group 4



1.008 ± 0.101**


(0.84 – 1.27)  [n=3]



Spleen


[g]


(female)



Group 1



0.607 ± 0.088


(0.45 – 0.73)



5 to 95 % Percentile


0.45 – 0.77



Group 2



0.590 ± 0.064


(0.47 – 0.72)



Group 3



0.620 ± 0.074


(0.47 – 0.72)



Group 4



0.868 ± 0.180**


(0.62 – 1.34)  [n=14]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 18-1 and 18-3 ‘Absolute Organ Weights – Summary and Individual Data - Males - F0’ and from tables 18-2 and 18-4 ‘Absolute Organ Weights - Summary and Individual Data - Females - F0’.



 


Text Table 7-32:    Mean length and mean number of oestrous cycles.























































Parameter



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150/250 mg/kg



Pre-mating: Test day 15 (start of treatment) until pairing (test day 85) #1



Mean cycle length (days)



4.30 ± 0.47



4.29 ± 0.73



4.34 ± 0.48



4.30 ± 0.58



Mean number of cycles



15.4 ± 1.4



15.6 ± 2.1



15.0 ± 1.6



15.6 ± 1.9



Pre-mating: Test day 85 (start of pairing) until verification of copulation #1



Cycle length (days)



2 #2



 



 



 



Number of cycles



1



 



 



 



#1:



Values taken from table 19-1 'Oestrous Cycle Data – Start of Dosing until Mating - Summary - F0 Generation'.



#2:



Female no. 42 revealed an incomplete oestrous cycle between test days 88 and 89, consisting of an oestrous stage and a metoestrous stage. Thereafter female no. 42 remained in an oestrous stage for the remaining 10 days of the mating period. No evidence of copulation was noted for female no. 42 during the mating period and the non-pregnancy status of female no. 42 was confirmed by Salewski Staining at the end of its pseudo-gestation period.






Text Table 7-36:    Overview of the reproductive data.













































































































Parental females


F0 Generation



Reproductive data



Group 1


(Control)


(23 / 22) #4



Group 2


15 mg/kg


(21 / 19)



Group 3


50 mg/kg


(24 / 22)



Group 4


150/250 mg/kg


(22 / 22)



Number of implantation sites and pups



Implantation sites #1



14.9 ± 2.5



14.3 ± 4.9



13.8 ± 4.1



14.1 ± 2.8



Pups #1


(born alive and dead)



13.1 ± 3.7



13.0 ± 5.2



12.0 ± 4.7



12.1 ± 2.9



Pups born alive #1



12.8 ± 3.6



13.0± 5.1



12.0 ± 4.7



12.0 ± 2.8



Reproductive indices [%]



Birth index



per dam (group mean) #1


per group #2



86.85 ± 20.52


88.05



84.11 ± 28.74


90.70



79.97 ± 28.70


86.79



85.03 ± 11.62


85.53



Live birth index



per dam (group mean) #1


per group #2



97.83 ± 4.84


97.68



99.65 ± 1.53


99.63



100.00 ± 0.00


100.00



99.70 ± 1.42


99.62



Post-implantation loss



 


per dam (group mean) #1


per group #2



 


15.04 ± 20.50


13.99



 


16.21 ± 28.59


9.63 #3



 


20.03 ± 28.70


13.21 #3



 


15.28 ± 11.29


14.79



Resorptions and stillbirths



Sum of resorptions and stillbirths


(difference between number of implantation sites and stillbirths)



per dam (group mean) per group



2.1 ± 2.2


48



1.4 ± 1.2


29



1.8 ± 1.8


44



2.1 ± 1.5


46



Number of stillbirths



7



1



-



1



#1:



Statistical calculation was performed by ANOVA / DUNNETT


(*/**: p ≤ 0.05 / p ≤ 0.01).



#2:



No statistical evaluation was performed for the group values.



#3:



The differences between the mean and the group values for the birth index and the post-implantation loss that were noted at the low and the intermediate dose level were due to 2 females each with a resorption of all implants. This resulted in a birth index of 0 % and a post-implantation loss of 100 % for the individual females with a resorption of all implants. This led to an increased group mean value, though the total number of resorption from the animals with total resorption of all implants was in the range of the other females from the low or the intermediate dose group.



#4:



(number of pregnant animals / number of animals with pups). More pregnant animals in comparison to animals with pups means that females with a total resorption of all implants (NVP = no viable pups) occurred. These NVP animals are considered in the mean number of implantation sites and the resulting parameter (e.g. number of resorptions).



 


Text Table 7-42:    Changes of pup body weight in comparison to the control group.





















































































































Pup body weight



Changes in comparison to the control group


[%] #



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150/250 mg/kg)



 



Lactation day 1



males



+1.2



+3.6



- 5.3



females



+3.3



+4.1



- 2.7



males + females


combined



+1.8



+3.3



- 4.0



Lactation day 4



males



+0.1



+3.4



- 5.0



females



+0.9



+3.2



- 1.8



males + females


combined



+0.2



+3.1



- 3.1



Lactation day 7



males



+1.2



+2.6



- 9.6**



females



+0.6



+2.6



- 6.8*



males + females


combined



+1.1



+2.7



- 7.4*



Lactation day 14



males



+2.5



+1.9



- 12.8**



females



+1.8



+2.3



- 10.2**



males + females


combined



+2.3



+2.2



- 10.7**



Lactation day 21



males



+2.8



+1.5



- 10.2**



females



+2.6



+1.5



- 7.6**



males + females


combined



+2.7



+1.5



- 8.1**



#:



Values are taken from table 26-1 'Mean Body Weight of the Pups per Dam - Summary'.



 


Text Table 7-43:    Comparison of pup body weight with the Provivo background data.













































































Parameter


(g)



Values from this study #2


Mean value per group ± SD


(Range of the individual values)


[number of individual values below the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



PND 7


Male and female


combined



Group 1



15.521 ± 0.953


(13.21 – 16.80)



5% to 95% Percentile


 


13.05 – 17.90



Group 2



15.690 ± 1.810


(13.52 – 21.03)



Group 3



15.938 ± 2.262


(13.36 – 23.33)



Group 4



14.359 ± 2.161*


[n=3]  (10.10 – 20.50)



PND 14


Male and female


combined



Group 1



31.983 ± 1.677


(28.54 – 35.24)



5% to 95% Percentile


 


27.66 – 35.98



Group 2



32.730 ± 2.750


(29.43 – 41.23)



Group 3



32.664 ± 4.626


[n=1]  (26.50 – 50.20)



Group 4



28.571 ± 4.108**


[n=9]  (20.83 – 40.67)



PND 21


Male and female


combined



Group 1



52.443 ± 2.956


(43.90 – 58.39)



5% to 95% Percentile


 


43.65 – 60.24



Group 2



53.874 ± 5.826


(45.48 – 70.88)



Group 3



53.270 ± 7.554


(45.90 – 80.80)



Group 4



48.184 ± 7.667**


[n=4]  (33.62 – 71.73)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 26-1 ’Mean Body Weight of the Pups per Group – Summary – F1 Pups’.



 


Text Table 7-44:    Changes of litter weight in comparison to the control group for the male and female pups combined. Not considered to be test item-related.

























































Litter weight



Changes in comparison to the control group


[%] #



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150/250 mg/kg)



 



Lactation day 1



males + females


combined



+7.3



- 0.8



- 15.3**



Lactation day 4



males + females


combined



+4.5



- 1.3



- 15.7**



Lactation day 7



males + females


combined



- 1.8



- 2.3



- 12.6**



Lactation day 14



males + females


combined



- 0.1



- 2.5



- 14.8**



Lactation day 21



males + females


combined



+0.1



- 3.1



- 12.6**



#:



Values are taken from table 27-1 'Litter Weight - Summary'.



 


Text Table 8‑10:    Test item-related changes for the haematological parameters that were considered to be test item-related.





































































































































Parameter #


F1



 



Changes in comparison to control


[%]



Reason



Sex



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



 



 



HGB


[mmol/L]



Males



- 0.7



- 1.6



- 5.2*



A


 



HGB


[mmol/L]



Females



+0.1



- 0.1



- 6.7**



RBC


[x10E6/µL]



Males



- 1.2



- 3.9



- 8.4**



RBC


[x10E6/µL]



Females



+1.6



- 0.3



- 11.8**



Reticulocytes


[%]



Males



+8.3



+18.9



+50.0**



Reticulocytes


[%]



Females



- 0.3



+8.3



+84.3**



HCT


[%]



Males



- 0.9



- 1.5



- 1.7



HCT


[%]



Females



+0.5



+0.9



- 4.6**



MCV


[fL]



Males



+0.6



+2.6



+7.3**



MCV


[fL]



Females



- 1.0



+1.2



+8.3**



MCH


[fmoL]



Males



0.9



2.4



3.5



MCH


[fmoL]



Females



- 1.5



0.0



+5.9**



MCHC


[mmol/L]



Males



+0.1



- 0.2



-3.6**



MCHC


[mmol/L]



Females



- 0.4



- 1.1



- 2.1*



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#:



Values taken from table 6-1-Co1A 'Haematological Parameters - Summary - Males' and from table 6-2-Co1A 'Haematological Parameters - Summary - Females'



A:



A haemolytic anemia was noted for the male and female animals of the high dose group during the histopathological examination. As the listed parameters belongs to the red blood cell parameters, noticeable differences in comparison to the control (statistically significant or not) that were noted at the high dose level were considered to be test item-related.



 


Text Table 8-11:    Test item-related changes on the haematological parameters of the male animals that were not considered to be test item-related.





























































































Parameter #


(Co1A)



Sex



Changes in comparison to control [%]



Reason



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



 



 



Neutrophilic granulocytes


[x103/µL]



Males



- 1.8



+47.1



+68.8*



A



Neutrophilic granulocytes


[x103/µL]



Females



+23.2



+10.9



+62.7**



WBC


[x103/µL]



Males



- 9.5



+1.6



+37.6*



B



WBC


[x103/µL]



Females



+7.0



0.0



+35.5*



A



Lymphocytes


[x103/µL]



Females



+3.7



- 1.7



+32.4*



Basophilic granulocytes


[x103/µL]



Females



+50.0



+12.5



+62.5*



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Values taken from table 6-1-Co1A 'Haematological Parameters - Summary - Males' and from table 6-2-Co1A 'Haematological Parameters - Summary - Females'



A:



Considered to be spontaneous as all or nearly all individual values were within the background range.



B:



Though a few individual values were above the background range for the male animals, the increase was considered to be spontaneous, as all of the also increased individual female values were still in the background range.


         

 


Text Table 8-12:    Male animals – F1 Generation: comparison of statistically significantly different parameters with the Provivo background data.



















































































Parameter


(males)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



HGB


[mmol/L]



Group 1



9.55 ± 0.51


(8.7 – 10.2)



5% to 95% Percentile


 


8.6 – 10.2



Group 2



9.48 ± 0.38


(8.6 – 9.9)



Group 3



9.40 ± 0.21


(9.0 – 9.7)



Group 4



9.05 ± 0.34*


[n=1]  (8.4 – 9.5)



RBC


[x106/µL]



Group 1



8.695 ± 0.336


(8.27 – 9.21)



5% to 95% Percentile


 


7.74 – 9.21



Group 2



8.594 ± 0.603


[n=1]  (7.29 – 9.24)  [n=1]



Group 3



8.356 ± 0.183


(8.14 – 8.76)



Group 4



7.964 ± 0.262**


[n=3]  (7.53 – 8.24)



WBC


[x103/µL]



Group 1



10.041 ± 1.332


(7.82 – 12.26)



5% to 95% Percentile


 


6.25 – 13.41



Group 2



9.091 ± 2.858


[n=2]  (5.25 – 13.61)  [n=1]



Group 3



10.204 ± 2.099


(6.88 – 12.93)



Group 4



13.815 ± 4.051*


(8.63 – 21.07)  [n=5]



Reticulocytes


[%]



Group 1



2.64 ± 0.68


[n=2]  (1.7 – 3.7)



5% to 95% Percentile


 


1.9 – 4.4



Group 2



2.86 ± 0.66


(2.0 – 4.1)



Group 3



3.14 ± 0.42


(2.6 – 3.8)



Group 4



3.96 ± 0.91**


[n=1]  (1.7 – 5.0)  [n=2]



- to be continued -


 


 


 




















































































Parameter


(males)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Neutrophilic granulocytes


[x103/µL]



Group 1



1.130 ± 0.275


(0.81 – 1.51)



5% to 95% Percentile


 


0.81 – 2.49



Group 2



1.110 ± 0.695


[n=5]  (0.48 – 2.42)



Group 3



1.662 ± 0.541


(0.94 – 2.69)  [n=1]



Group 4



1.907 ± 0.537*


(1.09 – 2.95)  [n=1]



MCV


[fL]



Group 1



52.31 ± 1.47


[n=1]  (49.5 – 54.7)



5% to 95% Percentile


 


49.9 – 55.6



Group 2



52.62 ± 3.33


[n=1]  (48.6 – 60.5)  [n=1]



Group 3



53.68 ± 2.29


[n=1]  (49.4 – 57.3)  [n=2]



Group 4



56.14 ± 1.35**


(53.5 – 57.6)  [n=7]



MCHC


[mmol/L]



Group 1



21.001 ± 0.373


[n=1]  (20.14 – 21.60)  [n=1]



5% to 95% Percentile


 


20.18 – 21.28



Group 2



21.024 ± 0.450


(20.30 – 21.76)  [n=3]



Group 3



20.969 ± 0.398


(20.43 – 21.45)  [n=2]



Group 4



20.250 ± 0.179**


[n=4]  (19.97 – 20.59)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 6-1-Co1A and 6-3-Co1A 'Haematological Parameters - Summary and Individual Data - Males'.


     

 


Text Table 8-13:    Female animals – F1 Generation: comparison of statistically significantly different parameters with the Provivo background data.



















































































Parameter


(females)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



HGB


[mmol/L]



Group 1



9.21 ± 0.21


(8.8 – 9.6)



5% to 95% Percentile


 


8.4 – 9.7



Group 2



9.22 ± 0.32


(8.8 – 9.7)



Group 3



9.20 ± 0.29


(8.6 – 9.6)



Group 4



8.59 ± 0.41**


[n=1]  (8.1 – 9.0)



RBC


[x106/µL]



Group 1



8.035 ± 0.295


(7.55 – 8.46)



5% to 95% Percentile


 


7.33 – 8.55



Group 2



8.161 ± 0.297


(7.84 – 8.73)  [n=2]



Group 3



8.007 ± 0.256


(7.72 – 8.48)



Group 4



7.084 ± 0.488**


[n=7]  (6.51 – 7.84)



WBC


[x103/µL]



Group 1



6.545 ± 1.547


(4.49 – 9.41)



5% to 95% Percentile


 


4.33 – 11.52



Group 2



7.004 ± 2.066


(4.77 – 11.14)



Group 3



6.544 ± 1.569


(4.40 – 9.77)



Group 4



8.868 ± 2.113*


(4.94 – 11.42)



Reticulocytes


[%]



Group 1



3.12 ± 0.65


(2.3 – 4.4)  [n=1]



5% to 95% Percentile


 


1.9 – 3.9



Group 2



3.11 ± 0.64


(2.1 – 3.9)



Group 3



3.38 ± 0.75


[n=1]  (1.8 – 4.4)  [n=2]



Group 4



5.75 ± 1.42**


(3.6 – 8.1)  [n=8]



- to be continued -


 


 


 



















































































Parameter


(females)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



HCT


[%]



Group 1



43.05 ± 1.42


(40.8 – 44.7)



5% to 95% Percentile


 


39.0 – 45.8



Group 2



43.28 ± 1.25


(41.6 – 45.0)



Group 3



43.44 ± 1.01


(41.9 – 45.0)



Group 4



41.08 ± 1.84**


[n=1]  (38.7 – 45.0)



Neutrophilic granulocytes


[x103/µL]



Group 1



0.829 ± 0.241


(0.56 – 1.25)



5% to 95% Percentile


 


0.47 – 2.27



Group 2



1.021 ± 0.428


(0.52 – 1.72)



Group 3



0.919 ± 0.293


[n=1]  (0.40 – 1.54)



Group 4



1.349 ± 0.430**


(0.59 – 2.14)



Lymphocytes


[x103/µL]



Group 1



5.354 ± 1.322


(3.56 – 8.01)



5% to 95% Percentile


 


2.98 – 9.82



Group 2



5.554 ± 1.750


(3.34 – 9.00)



Group 3



5.261 ± 1.264


(3.56 – 7.66)



Group 4



7.087 ± 1.832*


(4.03 – 9.23)



Basophilic granulocytes


[x103/µL]



Group 1



0.016 ± 0.007


(0.01 – 0.03)



5% to 95% Percentile


 


0.00 – 0.04



Group 2



0.024 ± 0.014


(0.01 – 0.06)  [n=1]



Group 3



0.018 ± 0.006


(0.01 – 0.03)



Group 4



0.026 ± 0.007*


(0.01 – 0.03)














































































Parameter


(females)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



MCV


[fL]



Group 1



53.60 ± 1.28


(52.1 – 55.9)  [n=1]



5% to 95% Percentile


 


50.7 – 55.8



Group 2



53.05 ± 1.06


(51.5 – 54.5)



Group 3



54.25 ± 1.67


(50.9 – 57.0)  [n=1]



Group 4



58.07 ± 1.91**


(55.3 – 61.6)  [n=8]



MCH


[fmol]



Group 1



1.148 ± 0.026


(1.12 – 1.21)  [n=1]



5% to 95% Percentile


 


1.08 – 1.19



Group 2



1.131 ± 0.026


(1.10 – 1.16)



Group 3



1.148 ± 0.046


[n=1]  (1.06 – 1.22)  [n=1]



Group 4



1.216 ± 0.049**


(1.17 – 1.29)  [n=7]



MCHC


[mmol/L]



Group 1



21.398 ± 0.395


(20.79 – 22.12)  [n=1]



5% to 95% Percentile


 


20.45 – 21.91



Group 2



21.321 ± 0.338


(20.65 – 21.83)



Group 3



21.170 ± 0.318


(20.65 – 21.65)



Group 4



20.953 ± 0.393*


[n=1]  (20.30 – 21.66)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 6-2-Co1A and 6-4-Co1A 'Haematological Parameters - Summary and Individual Data - Females'.



 


 


Text Table 8-16:    Comparison of the biochemical parameters of the male animals with statistically significant changes with the Provivo background data.




























































































Parameter


(males)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Bilirubin


(µmol/L)



Group 1



2.84 ± 0.28


(2.4 – 3.3)



5% to 95% Percentile


 


2.4 – 5.2



Group 2



2.71 ± 0.47


[n=3]  (2.1 – 3.4)



Group 3



2.93 ± 0.52


[n=2]  (2.0 – 3.7)



Group 4



3.39 ± 0.33*


(2.8 – 3.9)



Cholesterol


(mmol/L)



Group 1



1.294 ± 0.343


[n=1]  (0.86 – 1.89)



5% to 95% Percentile


 


0.94 – 2.24



Group 2



1.373 ± 0.297


[n=1]  (0.88 – 1.73)



Group 3



1.498 ± 0.283


(1.06 – 2.02)



Group 4



1.681 ± 0.294*


(1.21 – 2.12)



Glucose


(mmol/L)



Group 1



8.159 ± 0.685


(7.15 – 9.12)



5% to 95% Percentile


 


6.03 – 11.80



Group 2



7.357 ± 1.020*


(6.61 – 9.54)



Group 3



7.410 ± 0.746


[n=1]  (5.93 – 8.61)



Group 4



7.219 ± 1.144**


[n=1]  (6.02 – 9.83)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 7-1-Co1A and 7-3-Co1A 'Biochemical Parameters - Summary and Individual Data - Males'.



 



 



 



 


     

 


Text Table 8-17:    Comparison of the biochemical parameters of the female animals with statistically significant changes with the Provivo background data.













































Parameter


(females)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Bilirubin


(µmol/L)



Group 1



2.88 ± 0.30


[n=1]  (2.2 – 3.3)



5% to 95% Percentile


 


2.4 – 4.6



Group 2



2.90 ± 0.43


[n=1]  (2.3 – 3.5)



Group 3



3.09 ± 0.51


(2.4 – 4.0)



Group 4



3.91 ± 0.61**


(3.0 – 5.1)  [n=1]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 7-2-Co1A and 7-4-Co1A 'Biochemical Parameters - Summary and Individual Data - Females'.



 



 



 


 


Text Table 8-18A: Male animals – F1 Generation: comparison with Provivo background data.













































































































Parameter


(Males)


Co1A



Values from this study


Mean value per group ± SD


(range of the individual


males) #2



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 - 2021



T-cells


[%]



Group 1



45.73 ± 5.86


[n=1]  (33.5 – 51.9)



5% to 95% Percentile


 


37.8 – 67.2



Group 2



44.59 ± 5.55


[n=2] (36.0 – 53.8)



Group 3



44.16 ± 2.52


(40.9 – 48.5)



Group 4



46.03 ± 6.54


[n=1]  (36.7 – 59.6)



Helper T-cells


[%]



Group 1



25.23 ± 3.62


[n=1]  (18.1 – 29.5)



5% to 95% Percentile


 


21.1 – 40.2



Group 2



26.22 ± 2.56


(23.0 – 29.4)



Group 3



25.00 ± 3.62


[n=1]  (19.2 – 32.2)



Group 4



25.21 ± 4.74


[n=2]  (17.9 – 34.7)



Suppressor and cytotoxic T-cells


[%]



Group 1



18.74 ± 2.68


(13.3 – 22.0)



5% to 95% Percentile


 


12.2 – 24.3



Group 2



16.15 ± 4.40


[n=2]  (8.1 – 22.3)



Group 3



17.28 ± 3.76


[n=1]  (8.4 – 22.4)



Group 4



18.69 ± 3.51


[n=1]  (12.1 – 23.1)



NK-cells


[%]



Group 1



5.97 ± 1.71


[n=1]  (2.7 – 8.9)



5% to 95% Percentile


 


3.3 – 9.4



Group 2



6.09 ± 1.79


(3.9 – 8.5)



Group 3



5.90 ± 1.49


[n=1]  (3.1 – 8.2)



Group 4



4.80 ± 1.07


(3.4 – 7.3)



B-cells


[%]



Group 1



49.77 ± 5.63


(43.0 – 59.3)  [n=1]



5% to 95% Percentile


 


31.0 – 59.0



Group 2



51.08 ± 6.61


(42.1 – 62.6)  [n=2]



Group 3



51.44 ± 2.73


(45.2 – 54.8)]



Group 4



50.84 ± 6.72


(38.5 – 60.7)  [n=1]



#1:



Data not audited by QAU



#2:



Taken from tables 8-3-Co1A 'Lymphocyte Typing in Spleen - Individual Data'.



 


 


Text Table 8-18B: Female animals – F1 Generation: comparison with Provivo background data.













































































































Parameter


(Females)


Co1A



Values from this study


Mean value per group ± SD


(range of the individual


females) #2



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 - 2021



T-cells


[%]



Group 1



46.30 ± 5.00


(37.5 – 51.9)



5% to 95% Percentile


 


37.5 – 69.4



Group 2



46.58 ± 5.29


[n=1] (37.3 – 53.7)



Group 3



47.95 ± 7.02


(39.3 – 57.1)



Group 4



43.77 ± 7.82


[n=2]  (32.8 – 60.4)



Helper T-cells


[%]



Group 1



24.59 ± 4.32


(19.7 – 31.8)



5% to 95% Percentile


 


19.7 – 42.5



Group 2



25.77 ± 3.62


(20.4 – 31.2)



Group 3



25.98 ± 5.05


[n=1]  (19.3 – 33.9)



Group 4



23.77 ± 4.24


[n=1]  (14.7 – 29.0)



Suppressor and cytotoxic T-cells


[%]



Group 1



19.59 ± 3.64


(15.5 – 25.9)  [n=1]



5% to 95% Percentile


 


10.9 – 25.4



Group 2



18.90 ± 2.29


(14.4 – 22.2)



Group 3



19.32 ± 3.06


(14.0 – 24.8)



Group 4



18.05 ± 5.06


(11.4 – 29.0)  [n=1]



NK-cells


[%]



Group 1



4.88 ± 1.10


(3.3 – 7.4)



5% to 95% Percentile


 


2.5 – 11.1



Group 2



5.43 ± 1.60


(3.7 – 8.4)



Group 3



5.50 ± 1.73


(3.5 – 8.0)



Group 4



5.10 ± 1.53


(3.3 – 8.5)



B-cells


[%]



Group 1



50.23 ± 5.59


(43.2 – 59.5)  [n=1]



5% to 95% Percentile


 


24.5 – 58.0



Group 2



49.46 ± 5.05


(43.7 – 59.9)  [n=1]



Group 3



47.90 ± 6.52


(39.2 – 56.0)



Group 4



52.44 ± 7.06


(38.6 – 62.7)  [n=3]



#1:



Data not audited by QAU



#2:



Taken from tables 8-4-Co1A 'Lymphocyte Typing in Spleen - Individual Data'.



 


Text Table 8‑19:    Statistically significant changes of the urinary parameters that were not considered to be test item-related.






































































Parameter #


(Co1A)



Absolut values


(Changes in comparison to control %)



Reason



Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



 



 



Specific gravity


[g/mL]



Male



1.0491


-



1.0466


(- 0.2)



1.0530


(+0.4)



1.0663*


(+1.6)



A



Specific gravity


[g/mL]



Female



1.0357


-



1.0506


(+1.4)



1.0501


(+1.4)



1.0543*


(+1.8)



A



pH



Male



6.59


-



6.58


(- 0.2)



6.37


(- 3.3)



6.25*


(- 5.2)



A



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



A:



Considered to be spontaneous as all or nearly all values were within the Provivo background range.



#:



Values taken from table 9-1-Co1A 'Urinalysis - Summary - Males' and from table 9-2-Co1A 'Urinalysis - Summary - Females'


        

 


 


Text Table 8-20:    Comparison of the urinary parameter with the Provivo background data.



























































Parameter


(males)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Specific gravity


[g/mL]



Group 1



1.0491 ± 0.0122


(1.040 – 1.072)



5% to 95% Percentile


 


1.030 – 1.090



Group 2



1.0466 ± 0.0075


(1.034 – 1.062)



Group 3



1.0530 ± 0.0133


[n=1]  (1.028 – 1.070)



Group 4



1.0663 ± 0.0183*


(1.031 – 1.093)  [n=1]



pH



Group 1



6.59 ± 0.23


(6.4 – 7.1)



5% to 95% Percentile


 


6.0 – 7.3



Group 2



6.58 ± 0.18


(6.3 – 7.0)



Group 3



6.37 ± 0.33


(6.1 – 7.2)



Group 4



6.25 ± 0.35*


[n=2]  (5.9 – 7.0)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 9-1-Co1A and 9-3-Co1A 'Urinalysis - Summary and Individual Data - Males'.



 


 


 


Text Table 8-21:    Comparison of the urinary parameter with the Provivo background data.









































Parameter


(Females)


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Specific gravity


[g/mL]



Group 1



1.0357 ± 0.0119


(1.025 – 1.066)



5% to 95% Percentile


 


1.024 – 1.090



Group 2



1.0506 ± 0.0182


(1.025 – 1.080)



Group 3



1.0501 ± 0.0148


(1.025 – 1.073)



Group 4



1.0543 ± 0.0131*


(1.034 – 1.078)



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 9-2-Co1a and 9-4-Co1A 'Urinalysis - Summary and Individual Data - Females'.



 


Text Table 8-22:    Statistically significant changes of the thyroid hormones T4 and TSH.































































Parameter #1


(Cohort 1A)



Sex



Changes in comparison to control [%]



Assessment



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



 



 



T4


[nmol/L]



males



+13.9



+28.4*



+46.2**



A



T4


[nmol/L]



females



+25.1



+21.4



+24.3



B



TSH


[ng/mL]



males



+48.2



+91.2



- 1.3



TSH


[ng/mL]



females



- 4.2



+75.6



+38.7



#1:



Values taken from table 10-1-Co1A 'Thyroid Hormone Level Analysis - Summary - Males' and from table 10-2-Co1A ‘Thyroid Hormone Level Analysis - Summary - Females'.



A:



The increased values were not considered to be adverse, as they were probably due to changes in the liver which were observed during the histopathological examination of the liver.



B:



The observed changes were considered to be spontaneous, as no statistical significance and no dose response relationship were noted.



 


Text Table 8-23:    Comparison of the thyroid hormone T4 of the male and female animals with the Provivo background data.































































Parameter


Co1A



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 10))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



T4


[nmol/L]


(males)



Group 1



60.1688 ± 12.1206


(50.228 – 89.927)  [n=1]



5% to 95% Percentile


 


42.528 – 76.219



Group 2



68.5087 ± 13.7150


(46.327 – 90.619) )  [n=4]



Group 3



77.2314 ± 21.3415*


(45.140 – 107.260)  [n=5]



Group 4



87.9498 ± 13.1864**


(64.671 – 113.175)  [n=8]



T4


[nmol/L]


(females)



Group 1



39.4754 ± 13.7384


(22.759 – 66.513)  [n=1]



5% to 95% Percentile


 


21.861 – 59.502



Group 2



49.3690 ± 17.0393


(32.493 – 81.838)  [n=3]



Group 3



47.9274 ± 11.1714


(33.703 – 66.932)  [n=2]



Group 4



49.0770 ± 17.7528


(23.635 – 89.429)  [n=2]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 10-2-Co1A and 10-4-Co1A 'Thyroid Hormone Level Analysis - Individual Data – Males and Females'.



 



 



 


Text Table 8-25:    Time points (postnatal day) of vaginal opening.






































Parameter



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



Day of vaginal opening (PND)



Cohort 1A and 1B combined #1



32.3 ± 1.9



33.2 ± 2.1



33.6 ± 2.0**



36.8 ± 3.5**



Body weight on the day of vaginal opening (% changes in comparison to control)


Cohort 1A and 1B combined #1



not


evaluable



+ 5.6 %



+ 1.7 %



+ 6.9 %



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Values taken from table 11-3-Co1A/1B ‘Vaginal Opening - Summary'.



 


 


Text Table 8-26:    Comparison of the day of vaginal opening of the female animals with the Provivo background data.









































Parameter


(females)


Co1A/1B


combined



Values from this study #2


Mean value per group ± SD


(Range of the individual values (n = 40))


[number of individual values below or above the stated range]



Provivo Background Data #1


obtained from the control groups of 6 OECD 443 studies performed at Provivo


from 2019 – 2021



Day of vaginal opening



Group 1



32.3 ± 1.9


(30 - 39)  [n=2]



5% to 95% Percentile


 


30 – 37



Group 2



33.2 ± 2.1


(30 - 39)  [n=1]



Group 3



33.6 ± 2.0**


(31 - 38)  [n=2]



Group 4



36.8 ± 3.5**


(30 - 47)  [n=13]



#1:



Data not audited by QAU



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test)



#2:



Taken from tables 11-3-Co1A/1B 'Vaginal Opening - Summary – F1 Generation – Cohort 1A/1B combined. The individual values were taken from table 11-4-Co1A/1B 'Vaginal Opening - Individual Data’.



 


Text Table 8-27:    Sexual maturation of female animals.




















































Parameters #


Co1A females alone



Parameters of sexual maturation


Mean values per group



Group 1


(Control)



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



-



day of vaginal opening (PND)



32.3 ± 1.9



33.2 ± 2.1



33.6 ± 2.0**



36.8 ± 3.5**



-



day of appearance of cornified cells (PND)



33.6 ± 2.1



34.1 ± 2.3



34.2 ± 2.3



38.9 ± 3.9**



-



period between day of vaginal opening and day of appearance of cornified cells (test days)



1.0 ± 1.3



1.0 ± 1.7



1.0 ± 1.4



2.1 ± 2.4



**:



p ≤ 0.01 (ANOVA / DUNNETT test)



PND:



Postnatal day



#:



Values taken from table 11-3-Co1A ‘Vaginal Opening - Summary'.



 


Text Table 8-32:        Test item-related changes in organ weights







































































































































Parameter


F1 Generation, Cohort 1A



Sex



Changes in comparison to control


[%]



Reason



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



 



 



Spleen


(relative) #1



Male



- 5.0



+53.4



+28.6**



A



Spleen


(absolute) #2



Male



- 6.7



+56.8



+17.4**



Spleen


(relative) #3



Female



+2.0



+6.8



+36.7**



Spleen


(absolute) #4



Female



+1.2



+3.8



+25.4**



Liver


(relative) #1



Male



- 1.6



+20.1



+13.9*



B



Liver


(absolute) #2



Male



- 2.4



+21.0



+4.7



Liver


(relative) #3



Female



- 1.1



+6.1



+12.6**



Liver


(absolute) #4



Female



- 1.9



+3.4



+3.8



Kidney


(left, relative) #1



Male



+0.9



+4.4



+12.5**



Kidney


(left, absolute) #2



Male



- 0.4



+3.5



+3.4



Kidney


(right, relative) #1



Male



+4.6



+8.7*



+16.6**



Kidney


(right, absolute) #2



Male



+3.5



+7.9



+7.3



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Taken from table 18-1-Co1A ‘Relative Organ Weights - Summary - Males’.



#2:



Taken from table 19-1-Co1A ‘Absolute Organ Weights - Summary - Males’.



#3:



Taken from table 18-2-Co1A ‘Relative Organ Weights - Summary - Females’.



#4:



Taken from table 19-2-Co1A ‘Absolute Organ Weights - Summary - Females’.



A:



The observed increased organ weights for the spleen were mostly due to the correlating histopathological findings and only secondly due to the decreased body weight at autopsy.



B:



The increased relative organ weights that were noted for the liver and the kidneys were mostly due to the decreased body weight at autopsy. However, a participation of histopathological findings is likely.



 


Text Table 8-33:        Changes in organ weights of animals of Cohort 1A, unrelated to the test item.



























































































































































Parameter


F1 Generation, Cohort 1A



 



Changes in comparison to control


[%]



Reason



Sex



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


250 mg/kg



 



 



Brain (relative) #1



Male



+2.3



+2.5



+4.7



A



Brain (absolute) #2



Male



+0.1



+1.1



- 4.9**



Brain (relative) #3



Female



+3.3



+5.6



+4.1



Brain (absolute) #4



Female



+1.9



+1.9



- 4.8**



Heart


(relative) #1



Male



- 2.6



- 4.0



- 3.1



Heart


(absolute) #2



Male



- 4.1



- 4.9



- 11.4**



Lymph node


(cervical, relative) #1



Male



+15.3



- 1.1



+52.2*



B



Lymph node


(cervical, absolute) #2



Male



+13.5



- 1.5



+38.4



Lymph node


(mesenteric, relative) #1



Male



+40.7



+37.1*



+62.1**



Lymph node


(mesenteric, absolute) #2



Male



+37.7



+36.6



+47.4*



Kidney, left


(relative) #3



Female



+4.1



+7.2*



+8.1**



C



Kidney, left


(absolute) #4



Female



+2.9



+4.4



- 0.6



Kidney, right


(relative) #3



Female



+2.3



+5.6



+7.5*



Kidney, right


(absolute) #4



Female



+1.2



+2.7



- 1.1



*:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Values taken from table 18-1-Co1A ' Relative Organ Weights - Summary - Males'



#2:



Values taken from table 19-1-Co1A ' Absolute Organ Weights - Summary - Males'



#3:



Values taken from table 18-2-Co1A ' Relative Organ Weights - Summary - Females'



#4:



Values taken from table 19-2-Co1A ' Absolute Organ Weights - Summary - Females'



A:



The statistically significantly reduced absolute organ weights of the brain and the heart were not considered to be of toxicological relevance, as they were due to a decreased body weight at autopsy.



B:



Considered to be spontaneous as no changes were noted during the histopathological examination.



C: 



The increased relative organ weights that were noted at the high dose level were not considered to be of toxicological relevance, as they were due to a decreased body weight at autopsy. The increased relative organ weight of the left kidney that was noted at the intermediate dose group was considered to be spontaneous as it was not caused by a decreased body weight at autopsy.


Conclusions:
The aim of the study was to evaluate the effects of the test item 1,2,3,4-Tetrahydronapthalene (test item) at dose levels of 15, 50 or 150/250 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood (OECD 443). The high dose level was increased from 150 to 250 mg/kg b.w./day on test day 64 due to no relevant external observed toxicological findings in group 4. For this reason, the high dose animals of the F1 Generation have received a dose level of 250 mg/kg b.w./day.

Examination of the haematological and biochemical parameters revealed test item-related changes for parameters of the red blood cells (e.g. increased percentage of reticulocytes, decreased number of red blood cells) resulting in an increased bilirubin concentration for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day).   This observations show an enhanced erythrocyte degradation.

The test item-related changes that were noted for the haematological and the biochemical parameters correlated with test item-related findings during the histopathological examination in the spleen and the bone marrow for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day), which can be found in conditions with haemolytic anemia. No such findings were not noted anymore during the additional histopathological examination of the spleen and the bone marrow from the male and female animals of the low and the intermediate dose group.

Correlating findings were also noted at necropsy in the form of enlarged spleens and increased spleen weights for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day).

No reproductive or developmental effects could be observed. The decreased pup body weight was  considered as secondary effect due to the decreased body weight of the dams during lactation period.

In the Cohort 1 A and 1 B animals also toxicological effects in hematology and biochenical parameters as well as in histopathology could be observed indicating hemolytic anemia. 





Executive summary:

The aim of the study was to evaluate the effects of the test item 1,2,3,4-Tetrahydronapthalene (test item) at dose levels of 15, 50 or 150/250 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood (OECD 443). The high dose level was increased from 150 to 250 mg/kg b.w./day on test day 64 due to no relevant external observed toxicological findings in group 4. For this reason, the high dose animals of the F1 Generation have received a dose level of 250 mg/kg b.w./day.


GENERAL AND REPRODUCTIVE TOXICITY (F0 GENERATION AND F1 PUPS)
General toxicity
No test item-related premature deaths were noted. The daily cage side observations for changes of behavior and the external appearance revealed a post dosing salivation for the male and female animals of the intermediate and the high dose group which was not considered to be adverse and, hence, not considered for the NOAEL.
For the body weight of the male high dose animals a marginally reduction was noted which was not considered as adverse.
However, a test item-related reduction in body weight that was considered to be adverse was noted at the high dose level for the female animals (150/250 mg test item/kg b.w./day).
Food consumption revealed reductions at start of dosing at the intermediate and / or the high dose level for the male and female animals. However, these changes were considered to be an adaptation to the start of dosing and not considered to be adverse.
Examination of the haematological and biochemical parameters revealed test item-related changes for parameters of the red blood cells (e.g. increased percentage of reticulocytes, decreased number of red blood cells) resulting in an increased bilirubin concentration for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day). This observations show an enhanced erythrocyte degradation (hemolytic anemia).
The test item-related changes that were noted for the haematological and the biochemical parameters correlated with test item-related findings during the histopathological examination in the spleen and the bone marrow for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day), which can be found in conditions with haemolytic anemia. No such findings were not noted anymore during the additional histopathological examination of the spleen and the bone marrow from the male and female animals of the low and the intermediate dose group.
Correlating findings were also noted at necropsy in the form of enlarged spleens and increased spleen weights for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day).


REPRODUCTIVE TOXICITY AND DEVELOPMENTAL TOXICITY
No test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).
No test item-related effect was noted on the prenatal development of the pups (number of resorptions, stillbirths and live born pups).
During the postnatal development of the pups during the lactation period a decrease in the pup body weight was noted at the high dose level (150/250 mg test item/kg b.w./day) on lactation days 7, 14 and 21. The observed reduced body weight at the high dose level was driven by reduced maternal body weight in the high dosed females. Therefore, the reduced pup body weight is secondary effect to the maternal toxicity.
Other parameter of the pups as the viability indices, the ano-genital distance, nipple retention, the thyroid hormone levels and the pup organ weights were not affected by the test item as well. Furthermore, no malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


GENERAL TOXICITY (F1 COHORTS 1 A and 1 B)
No premature death was noted during the post-weaning development of the male and female animals. A post-dosing salivation was noted during the daily cage side observations which was not considered to be adverse.
A marginally reduced body weight was noted at the intermediate dose level (50 mg test item/kg b.w./day) for the male animals of Cohort 1A and the female animals of Cohort 1B. A more pronounced reduction in body weight was noted at the high dose level (250 mg test item/kg b.w./day) post-weaning until necropsy for the male and female animals of Cohort 1B. However, no changes in food consumption were noted.
The changes that were noted in the context of a haemolytic anemia for the male and female animals of the F0 Generation were also noted for the male and female animals of the F1 Generation. The respective changes in the haematological and the biochemical parameter and the increased spleen weight were noted for the male and female animals of the high dose group (250 mg test item/kg b.w./day. The respective histopathological findings in the spleen and the bone marrow were noted at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day). Yet, only consider severe in the highest dose group. All these haematological effects and the changes in the clinical biochemistry (bilirubin increase) and the histopathological observations (in spleen, liver) are indicative for an enhanced erythrocyte degradation and a subsequent extra medullary hematopoiesis.


Furthermore, a delay in sexual maturation was noted in form of a delayed vaginal opening at the high dose level for the female animals of Cohort 1A and 1B combined. However, this delay was considered to be a secondary effect due to the decreased body weights that were noted for the female animals of the and the high dose group.


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


F0 Generation:
General toxicity (for systemic toxicity) NOAEL 50 mg test item/kg b.w./day
Due to changes in the haematological and biochemical parameters at the high dose level for the male and female animals together. This findings could be correlated for the male and female animals with histopathological observations in the spleen and the bone marrow, as well as increased spleen weights and enlarged spleens that were noted at necropsy. All these findings together showed the disease pattern of a haemolytic anemia. Furthermore, a reduced body weight was noted for the female animals of the high dose group.


Reproductive toxicity
a) adverse effects on the reproductive parameters of the parental females:
NOAEL above 150/250 mg test item/kg b.w./day


Developmental toxicity
b) adverse effects on the pre-natal development of the pups:
NOAEL above 150/250 mg test item/kg b.w./day
c) adverse effects on the post-natal development of the pups:
NOAEL above 150/250 mg test item/kg b.w./day


F1 Generation:
General toxicity (for systemic toxicity)(Cohorts 1A and 1B)
NOAEL 50 mg test item/kg b.w./day
Due to histopathological findings in the spleen and the bone marrow at the high dose level for the male and females. Further changes at the high dose level that were considered for the NOAEL were in the form of changes in the haematological and biochemical parameters and an increased spleen weight, both noted for the male and female animals. A slightly to moderately decreased body weight that was noted for the male and female animals of the high dose group.


 



Findings - F0 Generation and F1 Pups












































































































Mortality


 



Males


No test item-related premature death was noted for the male animals at 15, 50 or 150/250 mg test item/kg b.w./day.


One male of the low dose group and one male of the intermediate dose group died by misgavage.


Females


No premature death was noted for the female animals at 15, 50 or 150/250 mg test item/kg b.w./day.


  


Clinical signs



Males


A post-dosing salivation was noted for nearly all or all surviving male animals at the intermediate and the high dose level (50 or 150/250 mg test item/kg b.w./day), respectively (duration: <= 60 min)..


Females


A post-dosing salivation was noted for nearly all female animals at the intermediate and the high dose level (50 or 150/250 mg test item/kg b.w./day).


No further observations were noted for the female animals.


Start and duration


In all cases salivation was a short lasting post-dosing symptom (duration: <= 60 min).


Detailed clinical observations


No further observations in addition to those made during the daily cage side observations were noted for the male and female animals of the control group and the treatment group.


  

Body weight and


body weight gain



Males


No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).


At the high dose level (150/250 mg test item/kg b.w./day), marginally (statistically not significant) reduced body weights were noted from the begin of the post-mating period on test day 92 (2.6 % below the control) until the end of the study on test day 133 (3.1 % below the control). This reduction in body weight can be considered as spontaneous and non adverse.


 


Body weight gain


Accordingly, a marginally reduced body weight gain was noted at the high dose level (49.5 % in comparison to 52.9 % in the control group). This was not considered to be adverse.


 


Females



 



At the high dose level (150/250 mg test item/kg b.w./day), a test-item related reduced body weight was noted from the end of the pre-mating period until the end of the lactation period


Statistically significant reductions (p ≤ 0.01; with maximum reduced weight -11.3%) of) body weight of the females were noted on GD 14 & 21, as well as at LD 1,4,7, &14.


 


Body weight gain


At the high dose level a marginally reduced body weight gain was noted during the pre-mating period. A moderately reduced body weight gain was noted during the gestation period. The difference in body weight declined amongst the control group and the high dose group between lactation day 1 and 21. An increased body weight gain in comparison to the control group was noted at the high dose level during the lactation period.


 



Food consumption



Males and females


No influence of toxicological relevance on food consumption was noted for both sexes between the control group and in the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day).


A significantly reduced food consumption was noted at start of the treatment period for the male animals at the intermediate and the high dose level (8.5 % and 8.9 % below the control, respectively, p ≤ 0.01).


Also for the female animals a significantly reduced food consumption was noted at the high dose level during the first and the second week of treatment (14.3 % and 5.9 % below the control, respectively, p ≤ 0.01).


These reductions in food consumption during the first (males) or the first and the second treatment week (females) were considered to be caused by the adaptation and without toxicological relevance.


 



Haematology


(test days 129 to 136,


at necropsy)



Males


At the high dose level (150/250 mg test item/kg b.w./day) test-item related statistically significantly increased percentage of reticulocytes (plus 55.9 %, p ≤ 0.01), decrease in erythrocytes and changes in other hematological parameter (e.g. MCHC) were noted in comparison to the control group.


All these haematological effects are indicative for an enhanced erythrocyte degradation.


 


Females


A test item-related effect on the hematological parameter was noted for the female animals at the high dose level (150/250 mg test item/kg b.w./day).


At the high dose level (150/250 mg test item/kg b.w./day) statistically significantly increased values in comparison to the control group were noted for the percentage of reticulocytes (plus 168.8 %, p ≤ 0.01), the MCV value (plus 9.5 %, p ≤ 0.01) and the MCH value (plus 8.0 %, p ≤ 0.01).


Statistically significantly decreased values in comparison to the control group were noted at the high dose level for the number of red blood cells (minus 16.1 %, p ≤ 0.01), the HGB concentration (minus 9.4 % , p ≤ 0.01) and the HCT value (minus 8.1 %, p ≤ 0.01).


 




Clinical biochemistry


(test days 129 to 136,


at necropsy)


 



Males


At the high dose level (150/250 mg test item/kg b.w./day) a test-item related statistically significantly increased bilirubin concentration (plus 32.4 %, p ≤ 0.01) was noted in comparison to the control group.



 


 


 



Females


A slightly but already statistically significant increased bilirubin concentration was noted for the females of the intermediate dose group (50 mg test item/kg b.w./day) (plus 32.4 %, p ≤ 0.05).


A test item-related significant increase of the bilirubin concentration was noted for the female animals at the high dose level (150/250 mg test item/kg b.w./day).) (plus 82.7 %, p ≤ 0.01).



Urinalysis


(test days 129 to 136,


at necropsy)


 



Males


A slightly but statistically significantly increased specific gravity was noted at the high dose level for the male animals (1.3 % above the control, p ≤ 0.05). Yet, all individual values of the high dosed animals were within the Provivo background range. However, as also some similar but not significant increases were observed in the intermediate dose group and also histopathological kidney changes have been observed, this effect was considered to be test item related but not human relevant.


Females


No test item-related changes were noted.


  

Thyroid hormone levels


(test days 129 to 136,


at necropsy)


 


 


 



Males and females


No test item-related changes were noted. However, distinct changes were observed for the T4 and the TSH concentrations of the male and female animals. These changes are of secondary nature as they are results of the increased bilirubin production.


  

Sperm parameter


(test days 134 to 136,


at necropsy)


 


 



Males


No test item-related changes were noted.




Necropsy


(test days 134 to 136 for the males,


test days 129 to 143 for the females)



Macroscopic post-mortem


findings



 


Males and females


An enlarged spleen was noted for one male and one female each of the high dose group (150/250 mg test item/kg b.w./day), but also for one animal of the control group.


However, as there was an adverse effect on the spleen at the high dose level in general (microscopic spleen findings for several animals and a generally increased spleen weight) the observation of enlarged spleens at the high dose level was considered to be test item-related.


  

Body weight at autopsy



Males and females


No test item-related differences between the control group and the treatment groups (15, 50 or 150/250 mg test item/kg b.w./day) were noted for the body weight at autopsy for the male and female animals.


However, a marginally reduced body weight at autopsy was noted at the high dose level (4.7 % below the control, statistically not significant).


 


 


  

Oestrous stage at necropsy



No test item-related differences were noted.


  

Organ weights



Males and females


A the high dose level (150/250 mg test item/kg b.w./day) statistically significantly increased organ weights (p ≤ 0.05 / 0.01) that were considered to be test item-related were noted for the spleen, the liver, the kidneys (males only) and the adrenal glands.


Test-item related decreased organ weights were noted for the thymus.


The changes in the organ weights were considered to be test item-related, as they correlated with  changes that were noted during the histopathological examination.



 



 



Bone marrow examination



Males and females


At the high dose level (150/250 mg test item/kg b.w./day) a slight shift was noted for the myeloid : erythroid ratio towards the erythroid cells. Which is the result of the counteract of the body to the treatment related enhanced erythrocyte degradation











Histopathological examinations


(Groups 1 and 4, plus additional examinations of organs from animals of groups 2 and 3):



 


 


 


Males and females


Test item-related observations were noted for the examined male and female animals of group 4 (150/250 mg test item/kg b.w./day) in the following organs:


Spleen: increased severities of hemosiderin deposits, increased severities of extramedullary hematopoiesis in erythroid elements (erythropoiesis in spleen) and increased incidence and severity of congestion.


Bone marrow: increased incidence of hypercellularity (increased cellularity) of erythroid elements.  Hypercellularity (increased cellularity) of erythroid elements in the bone marrow was deemed to be reactive change to hemolytic anemia and not to be due to direct effects of the test item to bone marrow


Liver: increased incidence and severity of centrilobular hepatocellular hypertrophy. This was considered to be of metabolic nature and of adaptive character, and hence, deemed not to be adverse.


Thyroid glands: increased incidence and / or severity of follicular cell hypertrophy. These changes were associated with hepatic enzyme induction that was indicated by increased liver weights and hepatocellular hypertrophy, and hence, deemed not to be adverse.


Kidneys (males only): increased incidence and / or severity grade of hyaline droplets in the proximal tubular epithelia. Increase in hyaline droplets was considered to be induced by overload of synthetic protein, which is specific in male rat like as alpha-2 microglobulin, derived from hyperfunction of the liver.


 


Thymus: slight increase in the severity of atrophy. These findings were considered to be stress-related.


Adrenal glands: increased incidence of diffuse cortical hypertrophy (both sexes) lipid vacuoles (males only). These findings were considered to be stress-related.


Only the findings in the spleen were considered as adverse and considered for the NOAEL.


 


Additional examinations in groups 2 and 3:


The additional examination of the organs with test item-related findings from the male and female animals of group 2 and group 3 revealed no test item-related changes.


 


Examination of reproductive organs


No test item-related observations were noted for the examined reproductive organs of the male and female animals of group 4.


All testes examined showed completeness of stages and cell population.




 



















































































































Reproductive toxicity


Reproductive parameters of the parental females



Oestrous cycle data


(test day15 until evidence of


copulation)



 


No test item-related influence was noted on the mean number and the mean length of the oestrous cycles.



Fertility index



No test item-related influence was noted.


A reduced fertility index of 88 % that was noted at the low dose group was considered to be spontaneous, as no dose-response relationship was noted.



Gestation index



No test item-related influence was noted.



Pre-coital time



No test item-related influence was noted.



Gestation length



No test item-related influence was noted.



 



 



F1 Pups - Pre- and postnatal development



- Prenatal development (from conceptus to birth)



Reproductive parameters



No test item-related influence was noted on the number of implantation sites, the number of live born pups, the birth index, the live birth index, and the percentage of post-implantation loss.


No increased number of resorptions or stillborns were noted in the treatment groups in comparison to the control group.


 



 



 



- Postnatal development (pup)



Mortality (Viability index)



Pre- and post-cull period


No test item-related influence was noted.


  

Pup body weight



No test item-related influence was noted.


Yet, at the high dose level (150/250 mg test item/kg b.w./day) slight but statistically significantly reduced pup body weights were noted on lactation days 7, 14 and 21 (7.5 %, 10.7 % and 8.1 % below the control for the male and female pups combined; respectively,  p ≤ 0.05 / 0.01).


The reduced pup body weight at the high dose level was considered to be a secondary adverse effect on the post-natal development of the pups due to the decreased maternal weight during gestation & lactation.


 


  

Ano-genital distance



No test item-related influence was noted.


  

Count of male nipples


(nipple retention)



 


No test item-related influence was noted.


  

 


 


F1 Pups - Examination at necropsy (surplus pups; not used for F1 generation)



External and


internal examination



 


No malformations or variations were noted.



 



 



T4 determination


(lactation day 4; culled pups)



 


No test item-related difference was noted.



 



 



T4, TSH determination


(lactation day 21/22)



 


No test item-related difference was noted.



 



 



Pup organ weights



No test item-related difference was noted.



 




Findings - Cohort 1A































































































































Mortality (Co1A)



Males


No test item-related premature death was noted for the male animals at 15, 50 or 250 mg test item/kg b.w./day.


Two male animals of the control group were prematurely sacrificed.


Females


No premature death was noted for the female animals at 15, 50 or 250 mg test item/kg b.w./day.


  

Clinical signs (Co1A)


 



Males and females


A post-dosing salivation was noted for all surviving male animals at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day).


 


Females


A post-dosing salivation was noted for nearly all female animals at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day).


 



Body weight and


body weight gain (Co1A)



 


Males


At the high dose level (250 mg THNtest item/kg b.w./day) a  decreased body weight was noted at the start of the F1 Study on post-natal day 22 (11.5 % below the control, p ≤ 0.01).


After the start of the F1 Study the difference between the control group and the high dose group increased until a maximum difference was reached on post-natal day 50 (17.3 % below the control, p ≤ 0.01). Thereafter the difference between the control group and the high dose group slightly decreased until the end of the study on post-natal day 85 (9.9 % below the control, p ≤ 0.05). Therefore, the changes in body weight were consider test item related.


Females


At the high dose level (250 mg THNtest item/kg b.w./day) a reduced body weight was noted at the start of the study on post-natal day 22 (9.8 % below the control, p ≤ 0.05).


During the following week, the difference between the control group and the high group increased and on postnatal day 29 the body weight of the high dosed females was 16.3 % (p ≤ 0.01) below the control.


Thereafter the difference between the control group and the high dose group decreased. At the end of the study on post-natal day 85 the body weight of the high dosed females was 6.5 % (statistically not significant) below the control value. Therefore, the changes in body weight were consider test item related.



 



Body weight gain


A marginally reduced body weight gain was noted for the male animals of the intermediate dose group (663.4 % in comparison to 696.7 % in the control).


As the difference in body weight between the control group and the high dose group was already present at the start of the F1 Study on post-natal day 22, no difference in body weight gain between the control group and the treatment groups was noted at the high dose level for the course of the study



Food consumption (Co1A)



Males and females


No test-item related changes were noted.


  

Haematology (Co1A)


(postnatal days 86 to 95,


at necropsy)



 


Males


At the high dose level (250 mg test item/kg b.w./day) test-item related increased values were noted for the percentage of reticulocytes (50.0 % above the control, p ≤ 0.01)  and the MCV value (7.3 % above the control, p ≤ 0.01).


Decreased values were noted for the number of red blood cells (8.4 % below the control, p ≤ 0.01), the HGB concentration (5.2 % below the control, p ≤ 0.05) and the MCHC concentration (3.6 % below the control, p ≤ 0.01). All the changes in the red blood cell parameter, were due to a haemolytic anemia.


 


Females


At the high dose level (250 mg test item/kg b.w./day) test-item related increased values were noted for the percentage of reticulocytes (84.3 % above the control, p ≤ 0.01), the MCV value (8.3 % above the control, p ≤ 0.01) and the MCH value (5.9 % above the control, p ≤ 0.01).


Decreased values were noted for the number of red blood cells (11.8 % below the control, p ≤ 0.01), the HGB concentration (6.7 % below the control, p ≤ 0.01), the HCT value (4.6 % below the control, p ≤ 0.01) and the MCHC concentration (2.1 % below the control, p ≤ 0.05). All the changes in the red blood cell parameter, were due to a haemolytic anemia.


  


Clinical biochemistry (Co1A)


(postnatal days 86 to 95,


at necropsy)



Males and females


An test-item related increased bilirubin concentration (males: 19.4 % above the control, p ≤ 0.05; females 35.8 % above the control, p ≤ 0.01) was noted at the high dose level (250 mg test item/kg b.w./day). An increased bilirubin concentration is correlated with the decreased number of red blood cells, as bilirubin is one of the metabolic products of heme which is released by degrading erythrocyte.


 


  

Lymphocyte typing (spleen)


(postnatal days 86 to 95,


at necropsy)



Males and females


No test-item related changes were noted.


  

Urinalysis (Co1A)


(postnatal days 86 to 95,


at necropsy)


 



Males and females


No test-item related changes were noted.


  

Thyroid hormone levels


(postnatal days 86 to 95,


at necropsy)


 



Males and females


No test item-related changes were noted for the TSH concentration.


Increased T4 concentrations were noted at the intermediate and the high dose level (28.4 % or 46.2 % above the control, p ≤ 0.05 / 0.01). These changes were caused by the observed changes in the liver and the increased bilirubin production. Therefore, they were  considered to be non adverse.


  

Sexual Maturation



Males (Cohort 1A and 1B combined)


No test item-related influence was noted on the day of preputial separation and even that the highest doses males show a body weight reduction of ~10 % on the day of preputial separation.


 


Females (Cohort 1A and 1B combined)


A delayed vaginal opening was noted at the the high dose level (250 mg test item/kg b.w./day) (post-natal days 36.8 in comparison to 32.3 in the control group, p ≤ 0.01). Due the correlation between the time point of vaginal opening and the body weight of the females, the observed delays in the time-point of vaginal opening were due to the decreased body weight of the female animals of the high dose level around the time point of vaginal opening


 


  

Oestrous cycle data (Co1A)



Females


No test item-related influence on the number and length of the oestrous cycles was noted during the examination of a 2-week period between test days 50 and 63.



Sperm parameter


(PND 86 to 95; at necropsy)


 



Males


No test-item related changes were noted.


  


Necropsy Co1A)


(PND 86 to 99 for the males and females)



Macroscopic post-mortem


Findings (Co1A)



 


Males and females


No test item-related observations were noted.


  

Body weight at autopsy (Co1A)



Males and females


A reduced body weight at autopsy was noted at the high dose level (250 mg test item/kg b.w./day) for the male and female animals (8.6 % or 8.2 % below the control, p ≤ 0.05 or not statistically significant).


  

Oestrous stage at necropsy



No test item-related influence was noted on the number of the different stages between the control group and the treatment groups.


  
   

 


 






















Organ weights (Co1A)



Males and females


Test item-related changes were noted for the male and female animals of the high dose group (250 mg test item/kg b.w./day) for the organ weights of the spleen and the liver and for the male animals for the kidneys.


  

Bone marrow examination



Males and females


At the high dose level (250 mg test item/kg b.w./day) a slight shift was noted for the myeloid : erythroid ratio towards the percentage of erythroid cells. Which is the result of the counteract of the body to the treatment related enhanced erythrocyte degradation.


 


 


  

 














Histopathology (Co1A)


(groups 1 and 4 plus additional examinations of organs from animals of groups 2 and 3)



 


 


 


Males and females


Test item-related observations were noted for the examined male and female animals of group 4 (250 mg test item/kg b.w./day) in the following organs:


Spleen: increased severities of hemosiderin deposits, increased severities of extramedullary hematopoiesis in erythroid elements (erythropoiesis in spleen) and increased incidence and severity of congestion.


Bone marrow: increased incidence of hypercellularity (increased cellularity) of erythroid elements. Hypercellularity (increased cellularity) of erythroid elements in the bone marrow was deemed to be reactive change to hemolytic anemia and not to be due to direct effects of the test item to bone marrow.


Liver: increased incidence and severity of centrilobular hepatocellular hypertrophy. This was considered to be of metabolic nature and of adaptive character, and hence, deemed not to be adverse.


Thyroid (including parathyroid): increased incidence and / or severity of follicular cell hypertrophy. Increased incidence and/or severity of thyroid follicular cell hypertrophy was considered to be the changes associated with hepatic enzyme induction that was indicated by increased liver weights and hepatocellular hypertrophy, and hence, deemed not to be adverse.


 


Kidneys (males only): increased incidence and / or severity grade of hyaline droplets in the proximal tubular epithelia. Increase in hyaline droplets was considered to be induced by overload of synthetic protein, which is specific in male rat like as alpha-2 microglobulin, derived from hyperfunction of the liver.


 


Additional examinations in group 2 and 3:


An additional examination of the above mentioned organs was performed for the male and female animals of group 2 and 3.


This additional examination confirmed the above mentioned test item-related findings for the male and female animals of group 3 (50 mg test item/kg b.w./day) for the spleen, the bone marrow, the liver and the kidneys (males only)


However, only the findings in the spleen were considered adverse and considered for the NOAEL.


 


Examination of reproductive organs


No test item-related observation were noted for the examined reproductive organs of the male and female animals of group 4.


All testes examined showed completeness of stages and cell population.


Quantitative evaluation of primordial and small growing follicles and corpora lutea


No test item-related differences were noted between the females of the control group and the females of the high dose group in the number of follicles and corpora lutea.


  


1.1.1        Findings - Cohort 1B



















































Mortality (Co1B)


 



Males


No test item-related premature death was noted for the male animals at 15, 50 or 250 mg test item/kg b.w./day.


One male of the high dose group was prematurely sacrificed due to scratch wounds..


Females


No premature death was noted for the female animals at 15, 50 or 250 mg test item/kg b.w./day.


  

Clinical signs (Co1B)



Males


A post-dosing salivation was noted for all surviving male animals at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day).


 


Females


A post-dosing salivation was noted for all female animals at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day.


 



Body weight and


body weight gain (Co1B)



Males


At the high dose level (250 mg test item/kg b.w./day) a test-item related decreased body weight was noted at the start of the F1 Study on post-natal day 22 (11.1 % below the control, p ≤ 0.05).


During the next two weeks the difference between the control group and the high dose group increased until a maximum difference was reached on post-natal day 36 (18.2 % below the control, p ≤ 0.01). Thereafter the difference between the control group and the high dose group only slightly decreased and at the end of the study on post-natal day 93 the body weight of the high dosed males was still 13.3 % below the control (p ≤ 0.01).



Females


A test-item related reduced body weight was noted at the high dose level (250 mg test item/kg b.w./day).


The difference between the control group and the high group increased and on postnatal day 29 the body weight of the high dosed females was 16.8 % (p ≤ 0.01) below the control.


Thereafter the difference between the control group and the high dose group declindedd. At the end of the study on post-natal day 93 the body weight of the high dosed females was 10.2 % (p ≤ 0.01) below the control value.



 



Body weight gain:


No difference in body weight gain was noted for the male animals of the high dose group, as the reduced body weight that was noted for the male animals of the high dose group was already present at the start of the study.


A marginally reduced body weight was noted for the female animals at the intermediate dose level (399.3 % in comparison to 412.0 % in the control).


At the high dose level the body weight of the female animals was 382.6 % in comparison to 412.0 % for the females of the control group. The slightly reduced body weight gain was due to an increased difference in body weight between the control group and the high dose group from post-natal day 22 to post-natal day 93 (3.4 % or 10.2 % below the control).


 



Food consumption (Co1B)



Males and females


No test-item related changes were noted.


  


Necropsy (Co1B) (PND 94 to 102)



Macroscopic post-mortem


Findings (Co1B)



 


Males and females


No test item-related observations were noted.


  
   


 


 


























Body weight at autopsy (Co1B)



Males and females


A test-item related statistically significantly reduced body weight at autopsy was noted for the male and female animals at the high dose level (250 mg test item/kg b.w./day) (14.7 % or 9.8 % below the control, p ≤ 0.01).



 



 



Oestrous stage at necropsy



No test item-related influence was noted.



 



 



Organ weights (Co1B)



Males and females


No test item-related differences were noted.


Statistically significant changes that were noted at the high dose level for the relative or the absolute organ weights of the right epididymis, the right testis, the pituitary gland and the prostate gland were due to the decreased body weight at autopsy and not considered to be of toxicological relevance.



 


 


 


 


 

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08.01.2020 to 25.08.2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This OECD 421 study is used as dose range finding study for the OECD 443 study (Provivo, 2021).
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
This OECD 421 study is used as dose range finding study for the OECD 443 study (Provivo, 2021).
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD / Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age: 71 days
- Body weight (at 1st administration): Males: 399.2 g - 477.1 g, Females 241.6 g - 286.3 g
- Identification of animals: After randomisation, each rat received a continuous number on the tail, either by tattoo or marker. Additionally, the animal cages were labelled with study number, animal ID number, sex, type of study, route of administration, and treatment group.
- Number of animals: Pre-exposure period: 58 female animals were evaluated pre-exposure for oestrous cyclicity to yield 40 females with regular oestrous cycle for the study. Prior to any study related activity, animals were allowed to adapt to the new environment for 7 days.
Main study: 80 animals (40 males and 40 females) A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 Generation.
- Housing: With the exception of the mating period the males and females (F0-Generation) were 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 Except during the mating period the animals were placed in the animal room as follows: Male animals: On one side of the room with each dose group separated by an empty row.
Female animals: On the other side of the room with each dose group separated by an empty row.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL. Certificates of analysis of the bedding material are included in the raw data.
- Diet (ad libitum): A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed. Samples of the food are analysed for contaminants based on EPA/USA3 by LUFA-ITL4 at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water (ad libitum): Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 20015]. In addition, drinking water samples taken at Provivo are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the ‘Deutsche Trinkwasserverordnung 2001, Anlage 1’ [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 15%
- Air changes per hour: 15 to 20
- Photoperiod: 12 hours dark/12 hours light, 150 lux at approximately 1.5 m room height
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- Route of administration: Oral by gavage
- Frequency of administration: Once daily
- Vehicle / Control: Corn oil
- Administration volume: 2 mL/kg b.w./day
- Dosages: 0, 15, 50, 150 mg/ kg b.w./day
- Selection of administration route: According to OECD guideline 421

The test item formulations were freshly prepared every day and volumes administered were adjusted to the animal's actual body weight daily. The test item was suspended in the vehicle and was administered orally at a constant volume once daily. The test item formulations were continuously agitated by stirring throughout the entire administration procedure. The control animals received the vehicle at the same administration volume daily in the same way.
Details on mating procedure:
Sexually mature male and female rats were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found. In case pairing would have been unsuccessful during the 2 week period, re-mating of females with proven males of the same group could have been performed. However, as all males successfully inseminated their female partners during the 2-week period, re-mating was not required.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, two (2) aliquots of approximately 2 mL each were taken at the following times and stored at -20°C ± 10% until analysis.
AT START OF THE TREATMENT PERIOD (first dosing day):
- Analysis of concentration: Immediately after preparation of the test itemvehicle formulation as well as after 8 and 24 hours storage at room temperature. (3 samples / dose level group; groups 2 - 4).
- Number of samples (aliquots): 3 x 3 = 9 (18)
- Analysis of homogeneity:
At the start of administration, during (middle) administration and before administration to the last animal of the dose level group. (3 samples / dose level group; groups 2 - 4).
- Number of samples (aliquots): 3 x 3 = 9 (18)
TOWARDS THE END OF THE TREATMENT PERIOD (when the majority of animals was dosed):
- Analysis of concentration: During treatment always before administration to the last animal/dose level group (1 sample / dose level group; groups 2 - 4)
- Number of samples (aliquots): 3 (6)
- Sum of all samples (aliquots): 21 (42)

The samples were labelled with the study number, species, type of sample, aliquot, concentration, test day, sampling time and date. Only one aliquot was analysed; the second aliquot served as back-up. The samples were analysed at Provivo using a validated method (LPT Study No. 37850).
Three months after the issuance of the Final Study Report, the remaining aliquots (backups) will be destroyed until the Sponsor requests otherwise.

The measured concentrations of the test item in the test item-vehicle mixtures were between 93.3% and 99.9% of the nominal concentrations, indicating correctly prepared and homogenous test item-vehicle mixtures that were stable for at least 24 hours at room temperature.
Duration of treatment / exposure:
The study animals were treated during the following periods:
- Males + Females: Pre-mating day 15 to 29 (pairing was in the evening of test day 29) and Mating 30 to 44 (first mating day, confirmed by positive sperm detection, was in the morning of test day 30)
- Males: Post-mating until test day 44 (one day before necropsy on test day 45)
- Females: Gestation and lactation period until test days 65 to 79 (one day before necropsy on test days 66 to 80; corresponding to lactation days 14 to 16)
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control group
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study have been selected in agreement with the Sponsor based on results of a 2-week dose-range-finding study in rats (Provivo, 2020, 14 day tolerance test). In this study, the rats were treated with 20, 60 or 180 mg/kg b.w. daily from test day 1 to test day 14. None of the animals died prematurely. No changes in behaviour, the external appearance or the appearance of the faeces were noted, with the exception of a severely reduced motility that was noted for all male and female animals of the high dose group (180 mg/kg b.w./day) on test day 1. The male animals showed a slightly reduced body weight at 60 and 180 mg/kg/ b.w./day and the female animals a marginally reduced body weight at 180 mg/kg b.w./day in comparison to the animals that were dosed with 20 mg/kg b.w./day. A slightly to moderately reduced food consumption was noted for the male animals that were dosed with 60 or 180 mg/kg b.w./day during the first test week. For the female animals a moderately reduced food consumption was noted during the first and the second test week at a dose level of 180 mg/kg b.w./day. No test item-related changes were noted during the macroscopic inspection at necropsy. The examination of the organ weights revealed no test item-related differences between the low, the intermediate and the high dose group. Hence, the dose levels of 15, 50 or 150 mg/kg b.w./day were selected for this study.

- Rationale for animal assignment (if not random):
The animals were allocated to the test groups at the last day of the pre-dosing period on test day 14 by using a Provantis2-generated randomization based on the body weights of the animals. Only those female animals were used for randomization that passed the oestrous cycle test.

- Fasting period before blood sampling for clinical biochemistry: over night
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS:
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal. Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays 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. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

MORTALITY:
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. If an animal would have died prematurely, this would have been allowed a post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. However, none of the animals died prematurely.

BODY WEIGHT:
The adult animals were weighed on each day of dosing for dose adjustment and at sacrifice (body weight at autopsy on test day 45); the individual body weights were recorded. The report included weekly values for the male animals (starting on test day 15) and the body weight one day before sacrifice (on test day 44). For the female animals the days on which body weights were reported during the different study periods are listed below:
- Pre-mating period Test days 15, 22, 28
- Gestation period Gestation days 0, 7, 14, 20
- Lactation period Lactation days 1, 4, 8, 13

FOOD AND DRINKING WATER CONSUMPTION:
Food intake per rat (g) was calculated using the total amount of food given to and left
by each rat in each group on those days:
Study period Males Females
Pre-mating period TD1(#1), TD8 and TD15 (#2) TD1 (#1), TD8 and TD15 (#2)
Mating period None None
Gestation period Not applicable GD0 (#1), GD7, GD14 and GD20 (#2)
Lactation period Not applicable LD1 (#1), LD8 and LD13 (#2)
#1: Days on which only the amount of food at food start was weighed.
#2: Days on which only the amount of food at food residue was weighed. On the other days, the amount of food at food residue, followed by food start was weighed.

From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula: Relative food consumption= (Total food given (g) - Total food left (g)
(g/kg b.w./day)) / (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

Drinking water consumption was monitored daily by visual appraisal throughout the study.

REPRODUCTIVE PERFORMANCE:
The reproductive parameters listed below and reproductive indices (s. "Reproductive indices") were determined to evaluate the reproductive performance.

REPRODUCTIVE PARAMETERS:
- stages of the ooestrous cycle
- pre-coital time
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- 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

THYROID HORMONE (T4) DETERMINATION
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 7.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below. Blood withdrawal was performed by randomization of the parental male and female animals. The male animals of all test groups were completely randomized in a one- step process, the female animals in different staggers according to their litter day.
Oestrous cyclicity (parental animals):
During the 14-day pre-exposure period (TD 1 to TD 14), the animals were not yet allocated to the test groups and were kept in the study pool. During these 14 days the estrous cycle of the animals was monitored. Only those animals that exhibit typical 4 to 5 day estrous cycles were considered in the randomization process for the allocation of the animals to the test groups 1 to 4. This randomisation procedure was based on the body weight of the animal. Most of the female animals that were used for the main study revealed 2 or 3 typical 4 to 5 day cycles during the 14-day pre-exposure period.
After the allocation of the animals to the study groups and the start of treatment, the estrous cycle was further monitored during the pre-mating period and the mating period until one day before a positive mating sign was noted. Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy.
Sperm parameters (parental animals):
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of all males of groups 1 and 4.
Litter observations:
EXAMINATION OF THE PUPS:
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Abnormal behaviour or changes in the external appearance of the pups that were noted during the daily cage side inspections were recorded.
The following examinations/observations were done for the offspring:

COUNTING, SEXING AND WEIGHING:
Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.

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

LITTER ADJUSTMENT ON PND 4:
After counting on PND 4 (lactation day 4), the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.

BLOOD SAMPLING FOR THYROID HORMONE (T4) DETERMINATION:
On PND 4 and on PND 13 blood samples for T4 hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection. On PND 13 the same pups that were used for T4 analysis were also used for the preservation of the thyroids.
Determination of the pups used for blood withdrawal: On lactation day 4 (PND4) and lactation day 13 (PND13) the litter sequence of pup blood withdrawal was determined by randomization of the dams. The collection of the pups for blood withdrawal from the individual litters occurred in an ascending order (the male and female pups per dam with the lowest number were used, if possible).

MALE NIPPLES COUNTING:
Nipples were counted in all male pups on PND 13.
Postmortem examinations (parental animals):
GROSS NECROPSY:
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle at necropsy. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males: On test day 45
Dams: On lactation days 14 to 16

DISSECTION OF ADULT ANIMALS:
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. During necropsy the number of implantation sites was recorded in the female animals. 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.

ORGAN WEIGHTS:
The weight of the following organs of all adult male and female animals was determined. Paired organs were weighed individually and identified as left or right.
- Epididymis (2)
- Thyroid (1) (including parathyroids)
- Ovary (2)
- As a whole: Prostate, seminal vesicles with coagulating glands
- Testicle (2)
- Uterus including cervix
The weights of the organs were determined before fixation. Only the weight of the thyroid glands was determined after fixation.

ORGANS FIXED FOR PRESERVATION:
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin:
Fixative: modified Davidson'solution
- Epididymis (2)
- Testicle (2)
Fixative: 7 % buffered formalin
- Gross lesions observed
- Thyroid (including parathyroids)
- Ovary and oviduct (2)
- Uterus (including cervix)
- Prostate
- Seminal vesicles with coagulating glands
Any other organs displaying macroscopic changes were also preserved.

HISTOPATHOLOGY:
Histopathological examination was performed on the organs listed below from all male and female animals of groups 1 and 4.
Organ HE-stained sections PAS-stained sections
Epididymis 2 (left and right) 1
Ovary 2 (left and right) None
Testicle 2 (left and right) 1
The following organs or parts thereof from the above selected animals and from every deceased or prematurely sacrificed animal were fixed for histopathology examination. Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of all males of groups 1 and 4. The other preserved organs will be examined when necessary.

HISTOPATHOLOGICAL EVALUATION:
Histotechnique was performed by Provivo. The slides (labelled with study number, species, animal number, block number) were dispatched to AnaPath Services GmbH on 31 March 2020.
The transport of the slides for the histopathology work to AnaPath Services GmbH was arranged by Provivo, whereas the return transport to the Test Facility will be arranged by AnaPath Services GmbH.
Postmortem examinations (offspring):
GROSS NECROPSY:
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Pups: On lactation days 13 to 16

EXAMINATION OF THE PUPS:
Dead pups and pups sacrificed between lactation days 13 to 16 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development. Thyroid preparation and blood withdrawal for T4 analyses were performed from 1 male and 1 female pup (same pups) from each litter. These pups were always sacrificed on
lactation day 13.
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd) using the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05). Significantly different data are indicated in the summary tables of the result sections and the result tables in the tables section (section 8) of this report.
Reproductive indices:
The following indices were calculated for each group:
- Female Fertility Index[%] = (Number of pregnant rats/ Number of rats paired with a male) x 100
The female fertility index reflects the total number of dams that had achieved
pregnancy, including dams which delivered at term, aborted or had fully resorbed
litters.
- Gestation Index [%] = (Number of dams with live pups/ Number of pregnant rats) x 100

For each litter and group the following indices were determined:
- Birth Index [%] #1 = (Total number of pups born (alive + dead)/ Number of implantation sites) x 100
- Live Birth Index [%] =( Number of pups alive on day 0/1 of lactation/ Total number of pups (alive + dead)) x 100
- Viability Index [%] pre-cull = (Number of pups alive on day 4 (pre cull)/ Number of pups alive on day 0/1) x 100
- Viability Index [%] post cull = (Number of pups alive on day 13/ Number of pups alive on day 4 (post cull)) x 100
- Post-implantation loss [%]#1 = (Implantations - number of pups born alive/ Implantation sites) x 100
#1: Twins (shared placentae) are considered as additional implantation sites in the
calculation of the birth index and the post-implantation loss, to avoid a birth index above
100% or a negative post-implantation loss.
Offspring viability indices:
Birth and live birth index, post-impantation loss and viability indices of the pups
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES AN FEMALES:
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male and female animals of the control group.
At the low dose level (15 mg test item/kg b.w./day) haemorrhagic urine was noted for one male animal on one test day. Due to the single occurrence, the observation of haemorrhagic urine was considered to be spontaneous.
At the low, intermediate and the high dose level (15, 50 or 150 mg test item/kg b.w./day) a post-dosing salivation was noted for several to all male and / or female animals in a dose-related manner.
Furthermore, a reduced motility was noted after the first dosing on test day 15 for all male and female animals of the high dose group (150 mg test item/kg b.w./day).
The observations of salivation and a reduced motility were considered to be test item-related. However, a short post-dosing salivation is not considered to be of toxicological relevance. The observation of a reduced motility after the first dosing (and no more thereafter) can be considered as an effect of adaptation and is also not considered to be of toxicological relevance.

MALES:
At 50 mg/kg b.w./day, a post-dosing salivation was noted for 6 of 10 male animals on 1 or 2 test days between test days 26 to 34.
At 150 mg/kg b.w./day, a post-dosing salivation was noted for all 10 male animals on 1 up to 15 test days between test days 25 and 44 (last day of dosing).
Additionally, a unique observation of reduced motility was noted for all high dosed males after the start of treatment on test day 15.
FEMALES:
At 15 mg/kg b.w./day, a post-dosing salivation was noted for 4 of 10 female animals during the lactation period on 1 up to 9 test days.
At 50 mg/kg b.w./day, a post-dosing salivation was noted for several females during the pre-mating/mating (on 1 to 4 test days), the gestation (on 1 to 4 test days) and the lactation period (on 1 to 11 test days).
At 150 mg/kg b.w./day, a post-dosing salivation was noted for all 10 females during the pre-mating/mating (on 1 to 8 test days), the gestation (on 2 to 18 test days) and the lactation period (on 1 to 12 test days).
Additionally, as with the male animals, a unique observation of reduced motility was noted for all high dosed females after the start of treatment on test day 15.
START AND DURATION:
On most test days, salivation started immediately to 5 min after administration and disappeared again between 20 to 60 min after administration.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the animals treated with 15, 50 or 150 mg/kg b.w./day died prematurely.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES: Pre-mating-, mating- and post-mating period
No test item-related changes in body weight or body weight gain were noted for rats in the low and the intermediate dose groups (15 or 50 mg test item/kg b.w./day).
At the high dose level (150 mg test item/kg b.w./day) a slightly (statistically not significant) reduced body weight was noted from test day 29 onwards until test day 44 (one day before necropsy).
In detail, the body weight from the male animals of the high dose group was 5.1% below the control group on lactation day 29 and 5.6% below the control group on test day 44.
Body weight gain was accordingly reduced in the high dose group. The slight reductions in body weight and body weight gain that were noted at the high dose level were considered to be test item-related.

FEMALES: Pre-mating-, gestation- and lactation period
No test item-related differences in body weight or body weight gain were noted between the female rats of the control group and the female rats of the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (150 mg test item/kg b.w./day) a slightly reduced body weight (statistically significant at p ≤ 0.05 on GD14, LD4 and LD13) was noted during the gestation period and the lactation period. The difference between the high dose group and the control group was nearly constant, being around 5% to 6% below the control group during the gestation period and the lactation period.
In detail, on gestation days 0 and 20 the high dosed values were 4.7% or 6.1% below the control value. On lactation days 1 and 13 the high dosed values were 6.4% or 6.6% below the control values.
As these differences were soon noted at the begin of the gestation period and still noted during the lactation period they can be considered as a test item-related effect on maternal body weight, as an influence of the litter weight can be ruled out at the beginning of the gestation period and during the lactation period.
A marginally reduced body weight gain in comparison to the control group was noted at the high dose level during the gestation period (see text table 7-5 below). This fits to the slightly reduced body weight that was noted at the high dose level during the gestation period. During the lactation period the body weight gains of the control group and the high dose group were nearly identical.


MALES AND FEMALES (at autopsy):
No test item-related differences were noted between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day) for the male and female animals.
At the high dose level (150 mg test item/kg b.w./day) the body weight at autopsy was 4.9% below the control value for the male animals (statistically not significant). For the high dose female animals the body weight at autopsy was 7.2% below the control value (p ≤ 0.05). These differences correspond to those values that were noted for the last live weighing.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
MALES: Pre-mating period
No test item-related difference in food consumption was noted between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day) after the start of treatment in test weeks 3 and 4 of the pre-mating period.
At 150 mg test item/kg b.w./day, a slightly reduced food consumption was noted for test week 3 and test week 4 (15.5% and 6.3% below the control; p ≤ 0.05 or p ≤ 0.01). This slight reduction that was noted at the high dose level is considered to be test item-related.
FEMALES: Pre-mating, gestation and lactation period
No test item-related differences in food consumption were noted between the control group and the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (150 mg test item/kg b.w./day), a slightly reduced food consumption (6.5% below the control value, statistically not significant) was noted for the first test week after the start of treatment (test week 3). This can be considered as test item-related. No test item-related difference in food consumption was noted between the high dose group and the control group during the gestation and the lactation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 15, 50 or 150 mg test item/kg b.w./day by visual assessment.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES:
No test item-related changes were noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) for the T4 levels of the parental males.
Increased serum levels were noted at the intermediate dose level (18.0% above the control value; statistically significant at p ≤ 0.05) and the high dose level (14.6% above the control value; not statistically significant). However, all individual values were within the Provivo background range (see Text Table 7-7). Hence, the increased values that were noted at the intermediate and the high dose level were considered not to be test item-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Salivation:
Salivation was recorded 284 times for the male and female animals out of altogether the whole study duration. Salivation started 267 times immediately to 5 min after administration and disappeared between 20 to 60 min after administration.
Reduced motility:
A reduced motility was noted for all male and female animals of the high dose group only after the first dosing. It was noted between 5 to 20 min after dosing and disappeared again during the night after the first dosing.
The observations of salivation and a reduced motility were considered to be test item-related.
However, a short post-dosing salivation is not considered to be of toxicological relevance. The observation of a reduced motility after the first dosing (and no more thereafter) can be considered as an effect of adaptation and is also not considered to be of toxicological relevance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
MALES:
Histological evaluation took place on testes and epididymides from all male animals of the control and high dose group 150 mg test item/kg b.w./day).
One testicle of the control group and the high dose group was checked for the completeness of cell populations and stages while taking into account the interstitial cell structure and the presence/absence of any degenerative changes. One epididymis from animals of the control and the high dose group was also carefully examined. As a result, no treatment-related effects on the testicular and epididymal histomorphology were observed.
As a result, there were no treatment-related changes in the reproductive organs examined in this study. The histopathology revealed that the test item did not produce any treatment-related histomorphological changes in testes, epididymides in animals receiving up to 150 mg/kg b.w./day of the test item.
The following findings were observed at a minimal severity in testes and epididymides of males from control and/or high dose group. The incidence and severity were within the range of normal background changes that may be recorded in this strain of rats:
- focal/multifocal Sertoli cell vacuolation in 4 males of control and 6 males of high dose;
- focal/single hypoplastic seminiferous tubule in one male of high dose;
- mononuclear cell focus/foci in the interstitium of epididymides in 9 males of control
and 9 males of high dose;
- focal epithelial vacuolation of epididymides in one male of high dose.

FEMALES:
Histological evaluation took place on ovaries from all female animals of the control and high dose group 150 mg test item/kg b.w./day).
The histopathology revealed that the test item did not produce any treatment-related histomorphological changes in ovaries in animals receiving up to 150 mg/kg b.w./day of the test item. As a result, there were no treatment-related changes nor abnormalities in ovaries of any animals examined.
The ovaries from one female (animal no. 11) that was not pregnant showed a normal histological appearance of diestrus/proestrus phases of the oestrous cycle, however, there were no morphological abnormalities that could be related to non-pregnant.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
After the allocation of the animals to the test groups and the start of treatment on test day 15, the oestrous cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign was noted (see section 3.3 'Oestrous cycle monitoring').
No test item-related differences were noted for the mean length and the mean number of oestrous cycles per dam during the pre-mating period between the female animals of the control group and the female animals of the treatment groups (15, 50 or 150 mg test item/kg b.w./day). None of the females showed a complete oestrous cycle during the mating period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
FEMALE FERTILITY:
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Only one female animal (no.11) of the control group did not became pregnant after insemination by its male partner (evidenced by sperm determination).

GESTATION INDEX:
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
All pregnant females delivered live pups.

PRE-COITAL TIME:
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
One control female (no. 13) was noted with an elongated pre-coital time of 15 test days. The occurrence of one female with an elongated pre-coital time was in the range of normal variability.

GESTATION LENGTH:
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).


Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A reduced fur at the head and the neck was noted for all pups from dam no. 59 of the intermediate dose group and a reduced fur on the whole body was noted for all pups of dam no. 71 of the high dose group.
As there were no further observations for the pups from these 2 dams, a test item-related effect was ruled out. Hence, the observation of reduced fur was considered to be spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Stillborns:
One dam with one stillborn pup each was noted in the control group (dam no. 13; excluded from the evaluation of reproductive parameter), the low dose group (dam no. 37) and in the intermediate dose group (dam no. 51). At the high dose level 2 dams were noted with altogether 4 stillborn pups (dam no. 72 with one stillborn and dam no. 73 with 3 stillborns).
The observation of 3 stillborns for dam no. 73 resulted in a reduced live birth index for dam no. 73 (72.7 %). This led to a slightly reduced live birth index at the high dose level (group values: 97.45% in comparison to 100.00% in the control group).
However, the occurrence of one dam with 3 stillborns at the high dose level was considered to be spontaneous. Due to the reasons discussed below:
With the exception of post dosing salivation (which was noted for all animals of the high dose group), no further signs of toxicity were noted for dam no. 73, body weight and food consumption were in the normal range. For pre-natal development one resorption was noted, which was also in the normal range. In addition to the 3 stillborns, 8 live born pups were noted for dam no. 73 of whom one further pup died on lactation day 3 (one prematurely deceased pup during the lactation period was in the normal range). The surviving 7 pups of dam no. 73 showed a normal development. The body weights of the pups from dam no. 73 were in the range of the pups from the other high dosed females.
Hence, no signs of maternal toxicity and no further signs of prenatal toxicity (increased number of resorptions) or postnatal toxicity (increased number of prematurely deceased pups or reduced pup body weights) were noted for dam no. 73. Furthermore, no further dam with more than one stillborn was noted at the high dose level and the live birth index was still in the range of the Provivo background data.

Viability index (Pre- and post-cull period):
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
A marginally increased number of prematurely deceased pups was noted at the high dose level for the pre-cull period (4 prematurely deceased pups in comparison to 1 or 2 prematurely deceased pups in the other test groups). This marginal difference was considered spontaneous as there was no difference in the number of prematurely deceased pups between the high dose group and the other test groups during the further course of the lactation period (post-cull period). Hence, the observed variability is considered as incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
PUP BODY WEIGHT:
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted during the lactation period.
Slightly reduced pup body weights (statistically not significant) were noted at the intermediate and the high dose level on lactation day 13 (3.6 % or 3.3 % below the control value, see Text table 7-18 below). However, all individual values of the control group and nearly all individual values of the treatment groups were within the range of Provivo background data. Only one value of the high dose group (24.28 g) was marginally below the background range (24.34 g – 32.75 g), whereas two values of the low dose group (34.52 g and 38.06 g) and one value of the high dose group (35.36 g) were above the background range. Hence, the slightly decreased pup body weights that were noted at the intermediate and the high dose level were considered to be spontaneous (see Text table 7-19 on the following page).
One runt (no. 17-12) was noted in the control group.

LITTER WEIGHT:
Slight reductions in litter weight (statistically significant or not statistically significant) in comparison to the control group were noted at the intermediate (LD 4: statistical significant p ≤ 0.05) and the high dose level (50 or 150 mg test item/kg b.w./day) on lactation days 1, 4 and 13.
The slight reductions in litter weight that were noted on lactation days 1 and 4 at the intermediate (11.1% or 10.3% below the control) and the high dose level (7.3% and 10.6% below the control) were due to a slightly reduced number of pups per dam on lactation days 1 and 4 (14.7 or 15.3 live pups per dam in comparison to 16.7 pups per dam in the control group on lactation day 1).
Thereafter, on lactation day 13, when the number of pups per dam was adjusted between the study groups due to the culling process, the differences in litter weight between the control group and the intermediate and the high dose group became much smaller (4.0% or 7.2% below the control). The remaining difference was due to the slightly reduced pup body weights that were noted at the intermediate and the high dose level on lactation day 13 (4.2% or 3.9% below the control).
Note that the number of pups per dam at the low dose level was also reduced in comparison to the control group on lactation days 1 and 4 (15.3 pups per dam in comparison to 16.7 pups per dam in the control group on lactation day 1). However, due to slightly increased pup body weights at the low dose level, no decreased litter weight was noted, in contrast to the intermediate and the high dose level. Hence, in conclusion, the effect was considered as not test item-related but incidental.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the T4 levels of the male and female pups on lactation day 13.
Increased T4 serum levels were noted at the high dose level for the male pups, the female pups and the male and female pups combined (17.9%, 16.0% and 17.0%, respectively, above the control values; statistically significant for the male pups and the male and female pups combined at p ≤ 0.05). However, all individual values for the male and female pups as well as for the male and female pups combined were in the range of Provivo background data and the differences were considered to be spontaneous.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
For most of the affected male pups only 1 or 2 nipple was noted per pup. The number of these pups is identical in control and intermediate dose group (7 pups).
Male pups with 2 nipples were noted in the control group, low and intermediate dose groups (3, 1 and 3, respectively pups in each group) but not in the high dose group.
One male pup with 4 nipples was noted in the control group and one pup with 3 nipples in the low dose group. Hence, the number of pups with nipples in the control group is above the values of the Provivo background data.
At the high dose level only 1 pup with 1 nipple was noted in comparison to 7 pups with altogether 13 nipples in the control group. This led to a statistically significantly reduced number of nipples / number of pups per dam at the high dose level (0.02 nipples / pup / dam in comparison to 0.33 nipples / pup / dam; p ≤ 0.05). As a reduced number of male nipples is not a toxicological effect, the reduced number of nipples / pup / dam was considered to be spontaneous.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 15, 50 or 150 mg test item/kg b.w./day after terminal sacrifice between lactation days 13 and 16.
Furthermore, no gross abnormalities were noted for the pups that were found dead (stillborns or pups that died during the lactation period).
A reduced fur was noted for the pups of dam no. 59 and dam no. 71 of the intermediate and the high dose group. This observation was considered to be spontaneous.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
MALE to FEMALE RATIO OF THE PUPS:
No test item-related influence on the male to female ratio was noted for all treatment groups (15, 50 or 150 mg test item/kg b.w./day).

NUMBER OF LIVE PUPS
No test item-related differences were noted for the mean number of live pups per dam between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) during the lactation period.
A slightly reduced mean number of pups per dam was noted on lactation days 1 and 4 in all treatment groups (between 7.2% and 11.6% below the control, statistically not significant).
The slightly reduced mean numbers per dam in the treatment groups in comparison to the control group that were noted on lactation days 1 and 4 were considered to be spontaneous. Nevertheless, the mean number of pups in the control group and in the treatment groups on lactation days 1 and 4 are in the range of the Provivo background data .
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
per- & postnatal development
Generation:
F1
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
Key result
Reproductive effects observed:
no
Treatment related:
no

Text Table 3‑3:     Groups and dose levels.




















































Group



dose levels


[mg/kg b.w./day, p.o.]



Number and sex of animals



Animal number



1



0


(vehicle control)



10


10



m


f



1 -


11 -



10


20



2



15


(low dose)



10


10



m


f



21 -


31 -



30


40



3



50


(intermediate dose)


 



10


10



m


f



41 -


51 -



50


60



4



150


(high dose)



10


10



m


f



61 -


71 -



70


80



m:



male



f:



female



 


Text Table 7-1:     Reproductive Outcome of the female animals
















































































test item



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Females started dosing



10



10



10



10



Females excluded from evaluation of reproductive parameters



1 (no. 13 #)



-



-



-



Females used for pairing



9



10



10



10



Females with


a positive mating sign



9



10



10



10



Females without


a positive mating sign



0



0



0



0



Females pregnant



8



10



10



10



Females not pregnant



1 (no. 11)



0



0



0



Females with total resorption of implants



0



0



0



0



Females with live born pups



8



10



10



10



#:



Female no. 13 was excluded from the evaluation of the reproductive parameters, as a verified copulation was noted on day 15 of the mating period, which was one day after the mating period should have been finished (scheduled length of the mating period was 14 days).






Text Table 7‑2:     Observations that were noted during the daily cage-side observations for the male animals.

















































Observation (males)



Affected animals



First to last seen


(test days)



Range of days observed #



Observations in group 2 (15 mg test item/kg b.w./day)



Haemorrhagic urine



1 of 10



36



1 (28)



Observations in group 3 (50 mg test item/kg b.w./day)



Salivation (slight)



6 of 10



26 - 34



1 - 2



Observations in group 4 (150 mg test item/kg b.w./day)



Salivation (slight to extreme)



8 of 10



25 - 44



1 - 15



Reduced motility



10 of 10



15



1



#:



If only 1 or 2 animals were affected, the animal numbers are listed in brackets.



 


Text Table 7‑3:     Observations that were noted during the daily cage-side observations for the female animals.









































































 



Affected females per period



Range of days observed per period #



Observation


(females)



Pre-mating/


Mating



Gestation



Lactation



Pre-mating/


Mating



Gestation



Lactation



Observations in group 2 (15 mg test item/kg b.w./day)



Salivation


(slight to moderate)



n.d.



n.d.



4 of 10



-



-



1 - 9



Observations in group 3 (50 mg test item/kg b.w./day)



Salivation


(slight to extreme)



3 of 10



5 of 10



9 o 10



1 - 4



1 - 4



1 - 11



Observations in group 4 (150 mg test item/kg b.w./day)



Salivation


(slight to extreme)



7 of 10



10 of 10



10 of 10



1 - 8



2 - 18



1 - 12



Reduced motility



10 of 10



n.d.



n.d.-



1



-



-



n.d.:



Not detected



#:



If only 1 or 2 animals were affected, the animal numbers are listed in brackets.



 


Text Table 7-4:     Body weight gain of the male animals during the treatment period from test day 15 to 44).
























Males



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150 mg/kg



Body weight gain #


(test day 15 to test day 44)



+20.2 %



+18.3 %



+18.5 %



+14.3 %



#:



Values taken from table 5-1 'Body Weight Gain - Summary - Males'



 


Text Table 7-5:     Body weight gain of the female animals during the pre-mating period, the gestation period and the lactation period.














































Females



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150 mg/kg



Body weight gain (%) #1


(pre-mating period)


(test days 15 to 29)



+4.1



+6.7



+6.4



+6.3



Body weight gain (%) #2


(gestation period)



+53.1



+53.5



+52.3



+50.2



Body weight gain (%) #3


(lactation period)



+12.5



+12.8



+11.6



+12.4



#1:



Values taken from table 5-2 'Body Weight Gain - Sum. - Females - Pre-mating Period'



#2:



Values taken from table 5-3 'Body Weight Gain - Sum. - Females - Gestation Period'



#3:



Values taken from table 5-4 'Body Weight Gain - Sum. - Females - Lactation Period'



 


Text Table 7-6:   Stage of the oestrous cycle at necropsy. The stage of the oestrous cycle was evaluated from vaginal smears that were taken at necropsy between lactation days 14 to 16.













































Stage of oestrous cycle at necropsy



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150 mg/kg



Proestrus



3 of 9



-



-



1 of 10



Estrus



-



1 of 10



-



-



Metestrus



1 of 9



2 of 10



1 of 10



2 of 10



Diestrus



5 of 9



7 of 10



9 of 10



7 of 10



- :



not detected



 


Text Table 7-7:     Comparison of the T4 levels of the male animals with the Provivo background data.









































Parameter



Values from this study #2


 


Mean value per group ± SD


(range of the individual values (n = 10))



Provivo Background Data #1


obtained from the control groups of 19 OECD 421/422 studies performed at Provivo from


2016 – 2020



T4 serum level


(nmol/L)


(males)


 



 


Group 1


 



52.0794 ± 4.1264


(46.486 - 58.113)



5% to 95% Percentile


42.611 – 90.506


 



 


Group 2


 



53.6975 ± 5.6497


(44.153 - 61.416)



 


Group 3


 



61.4492 ± 8.0228*


(50.598 - 79.258)



 


Group 4


 



59.6764 ± 9.5186


(48.174 - 80.485)



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Data not audited by QAU



#2:



Values are taken from table 12-2 'Thyroid Hormone Level Analysis – Individual Data - Males'.






Text Table 7-8:     Mean length and mean number of oestrous cycles.


































Parameter



Group 1


Control



Group 2


15 mg/kg



Group 3


50 mg/kg



Group 4


150 mg/kg



Pre-mating: Test day 15 (start of treatment) until pairing (test day 29)



Mean cycle length (days)



4.37



4.18



4.00



4.17



Number of cycles



1.8



2.3



2.5



1.9



# :



Values taken from table 13-1 'Oestrous Cycle Data – Start of Dosing until Mating - Summary'



 


Text Table 7-9:     Fertility indices per group.







































Group / Dose level



Fertility index %



Pregnant rats /


Females used for pairing #1



Group 1 (Control)



89



8 / 9 #2



Group 2 (15 mg/kg)



100



10 / 10



Group 3 (50 mg/kg)



100



10 / 10



Group 4 (150 mg/kg)



100



10 / 10



#1:



Values taken from table 15-1 'Reproductive Outcomes and Indices per Group'



#2:



Female no. 13 was excluded from the evaluation of the fertility index. Hence, only 9 females were left for the calculation of the fertility index.



 


 


Text Table 7-10:   Gestation indices per group.



































Group / Dose level



Gestation index %



Dams with live pups / pregnant rats #



Group 1 (Control)



100



9 / 9



Group 2 (15 mg/kg)



100



10 / 10



Group 3 (50 mg/kg)



100



10 / 10



Group 4 (150 mg/kg)



100



10 / 10



#:



Values taken from table 15-1 'Reproductive Outcomes and Indices per Group'



 


Text Table 7-11:   Comparison of the live birth index (group values) with the Provivo background data.





































Parameter



Values from this study #2


Group values



Provivo Background Data #1


obtained from the control groups of 20 OECD 421 / 422 studies performed between 2016 - 2020



Live birth index


(%)



Group 1



100.00



 


Range of  the group values from


20 control groups:


97.14 - 100.00



Group 2



99.35



Group 3



99.32



Group 4



97.45



#1:



Data not audited by QAU



#2:



Values were taken from table 16 'Number of Pups and Indices - Overview per Group'.



 


Text Table 7-12:   Overview of the reproductive parameters.
























































































































Parental females


F0 Generation



Reproductive data



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Parametrical values


(mean number per dam) #1



Implantation sites #3



17.6 ± 2.1



16.1 ± 2.6



16.0 ± 1.4



16.4 ± 2.7



Pups #3


(born alive and dead) (mean per dam)



16.6 ± 2.2



15.4 ± 2.7



14.8 ± 1.6



15.7 ± 2.7



Pups born alive #3



16.6 ± 2.2



15.3 ± 2.6



14.7 ± 1.5



15.3 ± 3.4



Indices [%] #2



Birth index



mean per dam #3


sum per group  #4



94.30 ± 4.28


94.33



95.18 ± 6.92


95.06



92.48 ± 5.50


92.50



95.73 ± 5.09


95.73



Live birth index



mean per dam #3


sum per group #4



100.00 ± 0.00


100.00



99.47 ± 1.66


99.35



99.41 ± 1.86


99.32



96.61 ± 8.65


97.45



Post-implantation loss



mean per dam #3


sum per group #4



5.70 ± 4.28


5.67



5.34 ± 6.71


5.56



8.11 ± 4.89


8.13



7.36 ± 11.00


6.71



Resorptions and stillbirths



Sum of resorptions and stillbirths


(difference between number of implantation sites and live born pups) #5



mean per dam


sum per group



1.0 ± 0.8


8



0.9 ± 1.1


9



1.3 ± 0.8


13



1.1 ± 1.4


11



Number of stillbirths



sum per group



0 #6



1



1



4



#1:



Statistical calculation was performed by ANOVA / DUNNETT.



#2:



Statistical calculation was performed by ANOVA / DUNNETT (mean values).



#3:



Values taken from table 17-1 'Birth Indices and Post-implantation loss - Values per Dam - Summary'



#4:



Taken from table 16 'Number of Pups and Indices - Overview per Group'.



#5:



Values taken from table 17-2 'Birth Indices and Post-implantation loss - Values per Dam - Individual Data'; columns ‘Stillborn’ and 'No. Resorptions + Stillborns'.



#6:



One stillborn was noted for the excluded female no. 13 (13-18).



 


Text Table 7-13:   Viability indices and prematurely deceased pups during the pre-cull period.


































Pre-cull period



Parameter



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Prematurely deceased pups between lactation days 0/1 to 4


 /


Total number of live born pups #



2 / 133



1 / 153



2 / 147



4 / 153



Viability index (group values) #



98.50



99.35



98.64



97.39



#:



Taken from table 16 'Number of Pups and Indices - Overview per Group'.



 


 


Text table 7-14:    Dams with prematurely deceased pups during the pre-cull period.



































































Number of prematurely deceased pups per dam (pre-cull period) #1



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



dam no.



deceased pups



dam no.



deceased pups



dam no.



deceased pups



dam no.



deceased pups



17



2



38



1



53



1



73



1#2



 



 



55



1



79



1



 



80



2



#1:



Taken from table 19-2 'Dead Pups per Dam and Viability Index - Individual Data'.



#2:



Dam no. 73 was also noted with 3 stillborn pups.


         

 


Text table 7-15:    Viability indices and prematurely deceased pups during the post-cull period.


































Post-cull period



Parameter



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Prematurely deceased pups between lactation days 5 and 13


/


Number of pups alive on lactation day 4 after culling #



1 / 80



0 / 100



1 / 100



1 / 97



Viability index (group values) #



98.75



100.00



99.00



98.97



#:



Taken from table 16 'Number of Pups and Indices - Overview per Group'.






Text table 7-16:    Dams with prematurely deceased pups during the post-cull period.










































Number of prematurely deceased pups per dam (post-cull period) #1



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



dam no.



deceased pups



dam no.



deceased pups



dam no.



deceased pups



dam no.



deceased pups



16



1 #2



 



51



1



80



1



#1:



Taken from table 19-2 'Dead Pups per Dam and Viability Index - Individual Data'.



#2:



Pup no. 16-12 died on lactation day 4 after the culling process was completed. Hence, pup no. 16-12 was considered in the Viability Index of the post-cull period.



 


Text table 7-17:    Male to female ratios in the test groups on lactation day 1 and lactation day 4.


































Male / Female ratio of the pups



Time point



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Lactation day 1 #



1.08



1.01



1.23



1.13



Lactation day 4 #



1.08



1.03



1.23



1.13



#:



Taken from table 16 'Number of Pups and Indices - Overview per Group'.



 


Text Table 7-18:   Changes of pup body weight in comparison to the control group for the male and female pups combined.











































Pup body weight



Changes in comparison to the control group


[%] #



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



 



Lactation day 1



males + females


combined



+5.4



+0.4



- 0.3



Lactation day 4



males + females


combined



+6.0



+1.1



- 0.4



Lactation day 13



males + females


combined



+ 3.0



- 3.6



- 3.3



#:



Values are taken from table 20-1 'Mean Body Weight of the Pups per Dam - Summary'.



 


Text Table 7-19:   Comparison of the pup body weights on lactation day 13 with the Provivo background data.





































Parameter



Values from this study #2


 


Mean value per group ± SD


(range of the individual values (n = 10))


[number of the individual values below or above the background range]



Provivo Background Data #1


obtained from the control groups of 20 OECD 421/422 studies performed at Provivo from


2016 – 2020



Pup body weight


on lactation day 13 (g)


(males + females combined)


 



 


Group 1


 



29.407 ± 1.962


(26.26 – 32.13)



5% to 95% Percentile


 


24.34 – 32.75



 


Group 2


 



30.296 ± 3.573


(26.52 – 38.06)  [n=2]



 


Group 3


 



28.359 ± 2.045


(25.09 – 31.84)



 


Group 4


 



28.441 ± 2.972


[n=1]  (24.28 – 35.36)  [n=1]



#1:



Data not audited by QAU



#2:



Values are taken from table 20-1 'Mean Body Weight of the Pups per Dam - Summary'.



 


Text Table 7-20:   Changes of litter weight in comparison to the control group for the male and female pups combined.




















































Litter weight #



Changes in comparison to the control group


[%]



 



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Lactation day 1



males + females


combined



- 3.2



- 10.6



- 7.2



Lactation day 4



males + females


combined



- 2.3



- 10.1



- 10.4



Lactation day 13



males + females


combined



+ 4.5



- 3.2



- 6.5



*/**:



 



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



n.a.



 



Not applicable



#:



 



Values are taken from table 21-1 'Litter Weight per Dam - Summary'.



 


Text Table 7-21:   Comparison of the litter weights on lactation day 1 with the Provivo background data.









































































Parameter



Values from this study #2


 


Mean value per group ± SD


(range of the individual values (n = 10))


[number of the individual values below or above the background range]



Provivo Background Data #1


obtained from the control groups of 20 OECD 421/422 studies performed at Provivo from


2016 – 2020



Litter weight


on lactation day 1 [g]


(males + females combined)


 



 


Group 1


 



111.60 ± 8.38


(99.0 – 127.1)  [n=1]



5% to 95% Percentile


 


70.3 – 122.6



 


Group 2


 



108.00 ± 12.19


(94.3 – 134.4)  [n=1]



 


Group 3


 



99.80 ± 9.26


(84.6 – 114.5)



 


Group 4


 



103.52 ± 22.42


[n=1]  (51.5 – 135.3)  [n=1]



Litter weight


on lactation day 4 [g]


(males + females combined)


 



 


Group 1


 



152.74 ± 9.40


(141.8 – 165.3)  [n=1]



5% to 95% Percentile


 


104.4 – 164.0



 


Group 2


 



149.30 ± 14.02


(132.9 – 179.3)  [n=2]



 


Group 3


 



137.37 ± 11.16


(119.7 – 149.7)



 


Group 4


 



136.87 ± 21.00


[n=1]  (84.6 – 156.6)



Litter weight


on lactation day 13 [g]


(males + females combined)


 



 


Group 1


 



290.05 ± 16.24


(282.0 – 318.9)



5% to 95% Percentile


 


205.9 – 322.8



 


Group 2


 



302.96 ± 35.73


(265.2 – 380.6)  [n=2]



 


Group 3


 



280.69 ± 21.43


(250.9 – 318.4)



 


Group 4


 



271.14 ± 22.21


(239.7 – 305.3)



#1:



Data not audited by QAU



#2:



Values are taken from table 21-2 'Litter Weight per Dam – Individual Data'.



 


Text Table 7-22:   Comparison of the number of live pups per dam (group mean values) on lactation days 1 and 4 before culling with the Provivo background data.























































Parameter



Values from this study


Group mean values ± SD #2


(range of the live born pups per dam in the test groups)



Provivo Background Data #1


obtained from the control groups of 20 OECD 422 / 421 studies performed at Provivo from 2016 - 2020



Number of live pups per dam on lactation day 1


 


(males + females combined)


 



 


Group 1


 



16.6 ± 2.2



Mean value from all individual values:


14.5 ± 2.8


 


Range of group mean values from


20 control groups


12.6 ± 4.1 to 16.7 ± 1.9



 


Group 2


 



15.3 ± 2.6



 


Group 3


 



14.7 ± 1.5



 


Group 4


 



15.3 ± 3.4



Number of live pups per dam on lactation day 4


 


(males + females combined)


 



 


Group 1


 



16.4 ± 1.8



Mean value from all individual values:


14.3 ± 2.8


 


Range of group mean values from


20 control groups:


12.4 ± 3.9 to 16.4 ± 1.8



 


Group 2


 



15.2 ± 2.5



 


Group 3


 



14.5 ± 1.5



 


Group 4


 



14.9 ± 3.3



#1:



Data not audited by QAU



#2:



Values are taken from table 18-1 'Number of Live Pups per Dam - Summary'.



 


Text Table 7‑23: Overview of the pups with nipple retention.































































































































































Pups with nipples #1



Group 1


(control)



Group 2


(15 mg/kg)



Group 3


(50 mg/kg)



Group 4


(150 mg/kg)



Dam no.



Pup no.


(number of nipples) #2



dam no.



Pup no.


(number of nipples)



dam no.



Pup no.


(number of nipples)



dam no.



Pup no.


(number of nipples)



14



14-02 (1)



32



32-01 (2)



51



51-06 (1)



75



75-03 (1)



15



15-04 (2)



 



32-05 (3)



54



54-04 (2)



 



 



17



17-03 (1)



38



38-07 (1)



55



55-03 (1)



 



 



 



17-09 (1)



 



 



56



56-04 (1)



 



 



19



19-07 (2)



 



 



57



57-07 (1)



 



 



20



20-01 (2)



 



 



 



57-08 (2)



 



 



 



20-03 (4)



 



 



58



58-08 (2)



 



 



Summary



5



7 Pups



2



3 Pups



6



7 Pups



1



1 Pup



Number of nipples per pup



1 Nip



3 Pups



1 Nip



1 Pup



1 Nip



4 Pups



1 Nip



1 pup



2 Nip



3 Pups



2 Nip



1 Pup



2 Nip



3 Pups



2 Nip



-



3 Nip



-



3 Nip



1 Pup



3 Nip



-



3 Nip



-



4 Nip



1 Pup



4 Nip



-



4 Nip



-



4 Nip



-



#1:



See table A ‘Individual Pup Data’ from the listed dams.



#2:



The number of nipples that were noted for the listed pup are given in brackets.



 


Text Table 7-24:   Comparison of the T4 serum levels of the pups on lactation day 13 with the Provivo background data.













































































Parameter



Values from this study #2


 


Mean value per group ± SD


(range of the individual values (n = 10))



Provivo Background Data #1


obtained from the control groups of 15 OECD 421/422 studies performed at Provivo from


2016 – 2020



T4 level


on lactation day 13


[nmol/L]


 


(male pups)


 



 


Group 1


 



54.1574 ± 7.2514


(43.682 – 62.964)



5% to 95% Percentile


 


36.709 – 82.736



 


Group 2


 



60.2185 ± 8.1706


(49.534 – 69.865)



 


Group 3


 



56.2724 ± 7.6752


(39.795 – 64.845)



 


Group 4


 



63.8528 ± 7.1342*


(57.557 – 81.989)



T4 level


on lactation day 13


[nmol/L]


 


(female pups)


 



 


Group 1


 



54.5993 ± 9.7180


(41.123 – 71.850)



5% to 95% Percentile


 


38.334 – 79.324



 


Group 2


 



61.3589 ± 6.4421


(52.405 – 71.453)



 


Group 3


 



59.5843 ± 8.4965


(48.109 – 74.841)



 


Group 4


 



63.3477 ± 6.8959


(47.566 – 73.741)



T4 level


on lactation day 13


[nmol/L]


 


(male and female pups combined)


 



 


Group 1


 



54.3783 ± 7.7049


(44.658 – 67.407)



5% to 95% Percentile


 


36.776 – 81.853



 


Group 2


 



60.7887 ± 6.1915


(50.970 – 70.659)



 


Group 3


 



57.9284 ± 7.6583


(44.316 – 68.994)



 


Group 4


 



63.6003 ± 5.5617*


(53.083 – 75.395)



*/**:



Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test)



#1:



Data not audited by QAU



#2:



Values are taken from table 25-2 'T4 Level of the Pups – Mean Values per Dam – Individual Data'.



 


Text Table 7-24:   Results of the test item-formulation analyses.






























Parameter



Sampling



Percent of nominal concentration [%] #



Concentration



Immediately after preparation on test day 15


(first day of administration) and


before administration to the last animal


on test day 44.



93.3% - 99.9%



Stability



Left at room temperature after preparation for


8 h or 24 h on test day 15.



94.2% - 95.8%



Homogeneity



On test day 15, at the start of administration, during administration and before administration


to the last animal.



93.7% - 96.0%



#:



Values are taken from table 26-1 'Concentration and Stability of the Test Item vehicle Mixtures' or table 26-2 'Homogeneity of the Test Item vehicle Mixtures'.



 

Conclusions:
The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and / or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 15, 50 or 150 mg/kg b.w./day. Additionally, this OECD 421 study serves as dose range finding study for the OECD 443 study with the test item (Provivo Study No. 37627). The dose levels of 15, 50 or 150/250 mg/kg b.w./day were selected for the Provivo Study No. 37627. The NOAEL for systemic toxicity was considered to be 50 mg/ kg bw/ day, based on clinical observations and reduced body weight. The NOAEL for reproductive toxicity (adverse effects on the reproductive parameters of the parental females/ pre- and postnatal development/ postnatal development) is above 150 mg/ kg bw/ day.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and / or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 15, 50 or 150 mg/kg b.w./day. Additionally, this OECD 421 study serves as dose range finding study for the OECD 443 study with the test item (Provivo Study No. 37627). The dose levels of 15, 50 or 150/250 mg/kg b.w./day were selected for the Provivo Study No. 37627.


 


General toxicity


Parental male and female animals


None of the animals died prematurely.


During the daily cage side observations a short post-dosing salivation was noted with an increasing incidence of affected male animals from 50 to 150 mg/kg b.w./day and from 15 to 150 mg/kg b.w./day for the females.


Furthermore, a reduced motility was noted for all male and female animals after the first dosing with 150 mg/kg b.w./day on test day 15.


The short post-dosing salivation and the isolated observation of a reduced motility that are rated as an adaptation effect are not of any toxicological relevance.


A slightly reduced body weight and / or body weight gain was noted for the male and female animals at 150 mg/kg b.w./day.


A transient and slightly reduced food consumption was noted for the male and female animals at 150 mg/kg b.w./day after the start of treatment.


No test item-related influence was noted on the T4 serum levels of the male animals.


The macroscopic inspection at necropsy and the microscopic examination of the reproductive organs of the male and female animals of the high dose group revealed no test item-related changes.


No test item-related differences were noted for the organ weights of the reproductive organs and the thyroid of the male and female animals.


 


Reproductive toxicity


Parental females


No influence was noted on the length and number of the oestrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.


 


 


Pups


No adverse effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post implantation loss) and the postnatal development of the pus (viability indices, pup body weight, ano-genital distance, the number of nipples per male pup, T4 serum levels on lactation day 13).


The macroscopic external examination of the pups at necropsy or after premature death revealed no abnormalities.


 


The following no-observed-adverse-effect levels (NOAEL) were established:































































General toxicity



 



 



NOAEL (for systemic toxicity)



 



                             50 mg test item/kg b.w./day, p.o.


Based on clinical signs observed in animals with 150 mg/kg b.w./day, and reductions in the body weight and food consumption at 150 mg/kg b.w./day.



 



Reproductive toxicity



 



 



 



a) adverse effects on the reproductive parameters of the parental females



 



NOAEL


            above 150 mg test item/kg b.w./day, p.o.



 



 



b) adverse effects on pre- and postnatal development



- b1) adverse effects on prenatal development (conceptus to birth)



 



NOAEL


            above 150 mg test item/kg b.w./day, p.o,



 



 



- b2) adverse effects on postnatal development (pup)



 



NOAEL


            above 150 mg test item/kg b.w./day, p.o.



 

Endpoint:
toxicity to reproduction
Remarks:
other: Subchronic toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-08-19 to 1996-11-22
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions: Limited documentation
Principles of method if other than guideline:
NTP Test Protocol, see Test Conditions
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Taconic, Germantown (New York, USA)
- Age: approximately 6 weeks at first exposure
- Number of animals: 10 per dose and sex
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
ADMINISTRATION / EXPOSURE 
- Type of exposure: whole-body inhalation
Details on mating procedure:
no mating
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Tetrahydronaphthalene concentrations in the exposure chambers were monitored by an online gas chromatograph. Samples were drawn from each exposure chamber approximately every 24 minutes during each 6‑hour exposure period.
Chamber concentration uniformity was maintained throughout the studies.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 h/day, 5 days/week; additionally: last sunday before terminal sacrifice
Dose / conc.:
0 ppm
Remarks:
0 mg/m3 (control group, exposure to air)
Dose / conc.:
7.5 ppm
Remarks:
41.2 mg/m3
Dose / conc.:
15 ppm
Remarks:
82.4 mg/m3
Dose / conc.:
30 ppm
Remarks:
165 mg/m3
Dose / conc.:
60 ppm
Remarks:
330 mg/m3
Dose / conc.:
120 ppm
Remarks:
660 mg/m3
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
SATELLITE GROUPS AND REASONS THEY WERE ADDED: As part of the disease  control program, five male and five female mice were submitted for a 
pre-exposure health examination. Sera were collected from five mice of  each sex from extra animals 20 days after arrival and from the control  
group at the end of the study. Sera were tested for viral and mycoplasmal  antibodies.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: twice daily including weekends
- Mortality: twice daily including weekends
- Body weight: weekly
- "Observations": weekly
- Hematology: All animals were bled at terminal necropsy. Hematologic  evaluations included: red blood cell count, volume of packed cells & spun  
hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular  hemoglobin, mean corpuscular hemoglobin concentration, white blood 
cell  count, differential count (absolute), absolute reticulocyte count,  platelet count & morphologic assessment, erythrocyte morphologic  
assessment.
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: Complete necropsy; weights of liver, thymus, right kidney,  right testis, heart, lungs.
- Microscopic: Complete histopathology on all 0- and 120-ppm mice  included the following tissues: adrenal glands, brain, clitoral glands,  
esophagus, femur (including bone marrow & joint surfaces), gallbladder,  gross lesions, tissue masses, regional lymph nodes, heart, aorta, large  
intestine (cecum, colon, rectum), small intestine (duodenum, jejunum,  ileum), kidneys (left only for males), larynx, liver, lungs & mainstem  
bronchi, lymph nodes (mandibular, mesenteric, bronchial, mediastinal),  mammary glands & adjacent skin, nasal cavity & nasal turbinates (three  
sections), ovaries, pancreas, parathyroid glands, pituitary glands,  preputial glands, prostate, salivary glands, spleen, stomach (forestomach  & 
glandular), testes, epididymis, seminal vesicles, thymus, thyroid  glands, trachea, urinary bladder, uterus.   
Target tissues identified at 120 ppm were examined at lower  concentrations to no-effect level or lowest exposure concentration. 
Gross  lesions were examined in all groups.
OTHER EXAMINATIONS: 
- Micronuclei in erythrocytes: Two blood smears were taken from all core  study animals at necropsy. One of these slides was subject to 
micronuclei  determination.
- Sperm morphology & vaginal cytology (SMVCE): Vaginal cytology was  evaluated for 12 days during the last 2 weeks of the study on all  remaining 
females in the 0-, 30-, 60-, and 120-ppm groups. Epididymal  sperm concentration, spermatid heads/testis, and left caudal, epididymal  & 
testicular weights were evaluated in surviving males from the same  groups.
Oestrous cyclicity (parental animals):
Vaginal cytology was evaluated for 12 days during the last 2 weeks of the study on all  remaining 
females in the 0-, 30-, 60-, and 120-ppm groups.
Sperm parameters (parental animals):
Epididymal  sperm concentration, spermatid heads/testis, and left caudal, epididymal  & 
testicular weights were evaluated in surviving males from the same  groups.
Statistics:
STATISTICAL METHODS: A modified Dunnett's t-test (Xybion Path / Tox  System; Cedar Knolls, New Jersey) was used to compare the treated groups  
to the control group with respect to body and organ weights, and  organ:body weight ratios. Corresponding statistics for hematology, and  clinical 
chemistry were calculated using the Statistical Analysis System  (SAS Institute; Berkeley, California).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain at the highest dose level
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Total erythrocytes and packed cell volumes were decreased, accompanied by increased mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration measurements and reticulocyte concentrations in both sexes from 30 ppm on (females more sensitive). Effects indicative for anemia.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Dark-colored urine at 30 ppm (7/10 each for males and females) and higher (all animals), effect obseved in the first weeks of treatment, not adverse
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Atrophy of the uterus and ovaries in females; Metaplasia and hyaline droplets in the nose
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
NOAEL (NOEL), LOAEL (LOEL): ignoring transitional epithelial eosinophilic  granules of urinary bladder:
- females: NOAEL = 7.5 ppm (uterus atrophy)
- males: NOAEL = 15 ppm (dark-colored urine); decreased kidney weights at  this dose, but not at 30 ppm
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death: no mortalities in any group
- Clinical signs: no gross observations in any group
- Body weight gain: lower by 8.9 % (males, significant) and 7.0 %  (females, insignificant), respectively, in highest dose groups
- Haematology: Total erythrocytes and packed cell volumes were decreased,  accompanied by increased mean corpuscular hemoglobin, mean 
corpuscular  volume, and mean corpuscular hemoglobin concentration measurements and  reticulocyte concentrations in both sexes at 60 or 
120 ppm. Platelet  concentrations were increased in these same groups.
- Urinalysis:    Dark-colored urine at 30 ppm (7/10 each for males and females) and  higher (all animals)
- Organ weights:   
Kidney: relative and absolute weights of right kidneys reduced in males  of 15, 60, and 120 ppm groups   
Liver: relative liver weights increased for males (120 ppm) and females (60 and 120 ppm), may be primarily attributed to lower  body weight gain in 
these groups   Heart: relative (120 ppm) and absolute (60 and 120 ppm) decrease in  males
- Gross pathology: no gross observations in any group
- Histopathology:   No lesions were observed in the liver, kidney, heart, or testes that  correlated with any of the weight changes observed.    
Atrophy of olfactory epithelium correlated very well with observations  in the previous 14-day study.    
Ovary and uterus atrophy was observed in high dose females. Incidences  of ovary atrophy at minimal doses of observation and above were 
4/10 (330  mg/m3), and 8/10 (660 mg/m3). Incidences of uterus atrophy at minimal  doses of observation and above were 2/10 (82.4 mg/m3), 
2/10 (165 mg/m3),  6/10 (330 mg/m3), and 8/10 (660 mg/m3). Information on severity is not  reported.    Transitional epithelial eosinophilic 
granules were observed in the  urinary bladder of all animals exposed (dose-related), the significance  of this finding is unclear.
Reproductive effects observed:
not specified
Executive summary:

Groups of 10 male and 10 female mice were exposed to tetrahydronaphthalene at concentrations of 0, 7.5, 15, 30, 60, or 120 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All mice survived to the end of the study. Mean body weights of 60 and 120 ppm males were significantly less than those of the chamber controls. Dark stained urine was observed in the catch pans of mice exposed to 30, 60, or 120 ppm during the first month of the study.

A minimal decrease in the erythron in both sexes that resulted in a hematopoietic response was observed. The relative liver weights of 120 ppm males and 30 ppm or greater females were significantly greater than those of the chamber controls. This was considered to be an adaptive response as no further histopathological adverse liver effects could be observed. Incidences of olfactory epithelium metaplasia in 60 and 120 ppm males and females, respiratory epithelium hyaline droplet accumulation in 120 ppm males and 60 and 120 ppm females, cytoplasmic eosinophilic granules within the transitional epithelium lining the urinary bladder in all exposed groups of males and females, and ovarian atrophy and uterine atrophy in 60 and 120 ppm females were significantly increased.

No effects on reproductive organs could be observed. Also no toxic effects on fertility (sperm parameters, oestrus cycle...) occured.

Endpoint:
toxicity to reproduction
Remarks:
other: Subchronic toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-08-19 to 1996-11-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions: Limited documentation
Principles of method if other than guideline:
NTP Test Protocol, see Test Conditions
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
common rodent species
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Taconic, Germantown (New York, USA)
- Age: approximately 6 weeks at first exposure
- Number of animals: Total of 25 males and 20 females per dose = 5 male  renal toxicity rats + 10 male and 
10 female core study rats + 10 male and  10 female clinical pathology rats
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
ADMINISTRATION / EXPOSURE 
- Type of exposure: whole-body inhalation
Details on mating procedure:
no mating
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The tetralin concentrations in the exposure chambers were monitored by an online gas chromatograph. Samples were drawn from each exposure chamber approximately every 24 minutes during each 6‑hour exposure period.
Chamber concentration uniformity was maintained throughout the studies.
Duration of treatment / exposure:
Exposure period: 14 weeks
Frequency of treatment:
6 h/day, 5 days/week; additionally: last sunday before terminal sacrifice
Dose / conc.:
0 ppm
Remarks:
0 mg/m3 (control group exposed to air)
Dose / conc.:
7.5 ppm
Remarks:
41.2 mg/m3
Dose / conc.:
15 ppm
Remarks:
82.4 mg/m3
Dose / conc.:
30 ppm
Remarks:
165 mg/m3
Dose / conc.:
60 ppm
Remarks:
330 mg/m3
Dose / conc.:
120 ppm
Remarks:
660 mg/m3
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post exposure period: sacrifice on day after last exposure
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: twice daily including weekends
- Mortality: twice daily including weekends
- Body weight: weekly (core study rats)
- "Observations": weekly
- Hematology: Sampling from 10 animals per dose and sex on days 3 and 23  (after exposures, clinical pathology rats) and at terminal sacrifice  
(core study rats). Evaluations included: red blood cell count, volume of  packed cells and spun hematocrit, hemoglobin, mean corpuscular volume,   mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,  white blood cell count, differential count (absolute), absolute  
reticulocyte count, platelet count, Morphological assessment. - Biochemistry: Blood urea nitrogen, sorbitol dehydrogenase, alanine  
aminotransferase, total protein, albumin, alkaline phosphatase, total  bile acids, creatine kinase, creatinine.
- Urinalysis: 16-hour collection during week 12 on all surviving core  study animals, with access to water but not food. Measurements included:  
volume, specific gravity, appearance (visual inspection), microscopic  examination of sediment from centrifuged sample, glucose, protein, 
 N-acetyl-beta-glucosaminidase, creatinine (to be used to normalize other  values), alkaline phosphatase, aspartate aminotransferase, lactate  
dehydrogenase, gamma glutamyl transaminase
Postmortem examinations (parental animals):
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: Complete necropsy; weights of liver, thymus, right kidney,  right testis, heart, lungs.
- Microscopic: Complete histopathology on all 0- and 120-ppm-rats  included the following tissues: adrenal glands, brain, clitoral glands,  
esophagus, femur (including bone marrow & joint surfaces), gross lesions,  tissue masses, regional lymph nodes, heart, aorta, large intestine  
(cecum, colon, rectum), small intestine (duodenum, jejunum, ileum),  kidneys (left only for males), larynx, liver, lungs, mainstem bronchi,  lymph 
nodes (mandibular, mesenteric, bronchial, mediastinal), mammary  glands & adjacent skin, nasal cavity & nasal turbinates (three sections),  ovaries, pancreas, parathyroid glands, pituitary glands, preputial  glands, prostate, salivary glands, spleen, stomach (forestomach &  glandular), testes / 
epididymis / seminal vesicles, thymus, thyroid  glands, trachea, urinary bladder, uterus.   
Target tissues identified at 120 ppm were examined at lower  concentrations to no-effect level or lowest exposure concentrations.  
Gross lesions were examined in all groups.
OTHER EXAMINATIONS: 
- Assessment of kidneys after 2 weeks (5 renal toxicity rats), at 6 weeks  (5 male clinical pathology rats), and at terminal sacrifice (5 male core  
study rats): Histopathology and evaluation of cell proliferation  (positive control: cross section of small intestine) in left kidney,  measurement 
of a2u-globulin in right kidney
- Sperm morphology & vaginal cytology (SMVCE): Vaginal cytology was  evaluated for 12 days during the last 2 weeks of the study in all  remaining 
females in the 0-, 30-, 60-, and 120-ppm groups. Epididymal  sperm concentration, spermatid heads/testis, and left caudal, epididymal  & 
testicular weights were evaluated in surviving males from the same  groups.
Statistics:
STATISTICAL METHODS: A modified Dunnett's t-test (Xybion Path / Tox  System; Cedar Knolls, New Jersey) was used to compare the treated groups  
to the control group with respect to body and organ weights, and organ:body weight ratios. Corresponding statistics for hematology, and clinical 
chemistry were calculated using the Statistical Analysis System  (SAS Institute; Berkeley, California).
Reproductive indices:
not available
Offspring viability indices:
not available
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight at 120 ppm males
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A modest regenerative anemia was observed in both sexes,  primarily in groups exposed to 60 and 120 ppm.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum alanine aminotransferase decreased at 60 and 120 ppm (both sexes)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Dark-stained urine at 30, 60, and 120 ppm.    Urine aspartate aminotransferase values significantly higher in males  (ca. 2.5) and 
females (ca. 17 times control values) at the 120 ppm level.   Urine lactic dehydrogenase (LDH):creatinine ratio significantly, but  modestly 
increased  in the two highest dose levels, LDH activity increased  in 120 ppm females group.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Olfactory necrosis and regeneration at 30 ppm, confirming the irritation potential of 1,2,3,4-tetrahydronaphthalene. 
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death: no mortalities in any group
- Clinical signs: no clinical abnormalities in any group
- Body weight gain: lower by 6.1 % (males) and 5.7 % (females),  respectively, in highest dose groups
- Clinical chemistry: Minimal nephropathy was observed in males in the  higher exposure groups. Clinical chemistry data were consistent with  
nephropathy.
- Haematology: A modest regenerative anemia was observed in both sexes,  primarily in groups exposed to 60 and 120 ppm.
- Urinalysis: Dark-stained urine at 30, 60, and 120 ppm.  Urine aspartate aminotransferase values significantly higher in males  (ca. 2.5) and females 
(ca. 17 times control values) at the 120 ppm level.   Urine lactic dehydrogenase (LDH):creatinine ratio significantly, but  modestly increased in the 
two highest dose levels, LDH activity increased  in 120 ppm females group.
- Organ weights: 
Kidney: Increased right kidney:body weight ratio in males (15, 60, and  120 ppm) and females (15 ppm and higher); mean absolute right kidney 
weight slightly increased in all treated groups;   
Liver:body weight ratios increased in males (15 and 120 ppm) and  females (60 and 120 ppm); mean absolute liver weight slightly increased  in all 
groups exposed;
- Gross pathology: no gross observations in any dose group - Histopathology:   Olfactory necrosis and regeneration, confirming the irritation  
potential of 1,2,3,4-tetrahydronaphthalene. 
The NOAEL for nasal lesions  was 15 ppm in males and 7.5 ppm in females.    
Hyaline droplet accumulation in kidneys of males increased slightly  with increased exposure; a NOAEL was not clear.   
Minimal nephropathy in males in the higher exposure groups
- Other: Concentrations of a2u-globulin generally increased with exposure  concentration and time on study.
Reproductive effects observed:
not specified
Executive summary:

Groups of 10 male and 10 female rats were exposed to tetrahydronaphthalene at concentrations of 0, 7.5, 15, 30, 60, or 120 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. The same exposure concentrations were given to additional groups of 10 male and 10 female clinical pathology study rats for up to 6 weeks and five male renal toxicity rats for 2 weeks.

All rats survived to the end of the study. Mean body weights of 120 ppm male rats were significantly less than those of the chamber controls. During the first 4 weeks of exposure, dark-stained urine was observed in the catch pans of rats exposed to 30, 60, or 120 ppm. Tetrahydronaphthalene induced a minimal decrease in the erythron in both sexes that resulted in a hematopoietic response.

Increased urine aspartate aminotransferase and urine lactate dehydrogenase activities (males and females) and glucose/creatinine ratio (males), suggestive of renal injury were observed. The absolute kidney weights of 60 and 120 ppm females and the relative kidney weights of males and females exposed to 15 ppm or greater were significantly greater than those of the chamber controls. Concentrations of [alpha]2u‑globulin in the kidney of exposed male rats were generally greater than those of the chamber controls at all time points and greater at 6 and 14 weeks than at 2 weeks.

No effects on reproductive organs could be observed. Also no toxic effects on fertility (sperm parameters, oestrus cycle...) occured.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

NTP studies


Partly cited from SIAR to SIAM 19 (Berlin, 19-22 October 2004):


In a subchronic inhalation study, 25 male and 20 female Fischer 344 rats per dose level were exposed (whole body) to nominal test item concentrations of 7.5; 15; 30; 60; 120 ppm = 41.2; 82.4; 165; 330; 660 mg/m3 = exposure levels 1; 2; 3; 4; 5 for 13 weeks on 6 h/day, 5 days/week. Rats were subdivided into groups of 5 male renal toxicity rats + 10 male and 10 female core study rats + 10 male and 10 female clinical pathology rats. General test conditions and observations are reported above in chapter 3.1.5 of SIAR (annotation: and chapter 7.5.2 of IUCLID 5). In order to determine whether 1,2,3,4-tetrahydronaphthalene may be a reproductive toxicant, vaginal cytology was evaluated for 12 days during the last 2 weeks of the study in all females in the 0-, 30-, 60-, and 120-ppm groups. Epididymal sperm concentration, spermatid heads/testis, and left caudal, epididymal and testicular weights were evaluated in all males from the same groups. No indications of reproductive toxicity were reported (NTP, 1997).


A similar subchronic inhalation study was performed by NTP (1997) in B6C3F1 mice, which were exposed whole-body in groups of 10 per dose and sex to nominal test item concentrations of 7.5; 15; 30; 60; 120 ppm = 41.2; 82.4; 165; 330; 660 mg/m3 = levels 1; 2; 3; 4; 5 for 13 weeks on 6 h/day, 5 days/week. General test conditions and observations are reported above in chapter 3.1.5 of SIAR (annotation: and chapter 7.5.2 of IUCLID 5). In order to determine whether 1,2,3,4-tetrahydronaphthalene may be a reproductive toxicant, vaginal cytology was evaluated for 12 days during the last 2 weeks of the study on all females in the 0-, 30-, 60-, and 120-ppm groups. Epididymal sperm concentration, spermatid heads/testis, and left caudal, epididymal & testicular weights were evaluated in all males from the same groups. In histopathology, ovary and uterus atrophy was observed in high dose females. Incidences of ovary atrophy at minimal doses of observation and above were 4/10 (330 mg/m3), and 8/10 (660 mg/m3). Incidences of uterus atrophy at minimal doses of observation and above were 2/10 (82.4 mg/m3), 2/10 (165 mg/m3), 6/10 (330 mg/m3), and 8/10 (660 mg/m3). Information on severity is not reported. No other indications of reproductive toxicity were reported. Uterus atrophy and atrophy of the ovary were found in the absence of systemic toxicity in mice, but no such effect was evident in equal studies on rats. Furthermore, in the 2-years inhalation study with mice no uterus atrophy and no ovary atrophy were observed at the same doses that are used in the 13-week inhalation study. Therefore, it can be concluded that these may be mice specific and transient effects. However a 2 year inhalation study with rats showed effects on the uterus (incidences of stromal polyp and endometrium hyperplasia in the high dose group 660 mg/m³ were significantly greater than those in the chamber controls; see Chapter 7.7 "Carcinogenicity" of IUCLID and Chapter 5.8 of this CSR).


 


DRF study according to OECD 421


The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and / or development (LPT, 2020) and to set suitable dose levels for the subsequently performed OECD 443 study.


The test item was administered orally to rats at dose levels of 15, 50 or 150 mg/kg b.w./day. Additionally, this study serves as dose range finding study for the ongoing OECD 443 (LPT Study No. 37627). The NOAEL for systemic toxicity was 50 mg/ kg bw/ day, based on clinical observations and reduced body weight. The NOAEL for reproductive toxicity (adverse effects on the reproductive parameters of the parental females/ pre- and postnatal development/ postnatal development) is above 150 mg/ kg bw/ day. None of the parental male and female animals died prematurely. During the daily cage side observations, a short post-dosing salivation was noted with an increasing incidence of affected male animals from 50 to 150 mg/kg b.w./day and from 15 to 150 mg/kg b.w./day for the females. Furthermore, a reduced motility was noted for all male and female animals after the first dosing with 150 mg/kg b.w./day on test day 15. The brief post-dosing salivation and the isolated observation of a reduced motility on test day 15, were both rated as adaptation effects are not of any toxicological relevance. A slightly reduced body weight and / or body weight gain was noted for the male and female animals at 150 mg/kg b.w./day. A transient and slightly reduced food consumption was noted for the male and female animals at 150 mg/kg b.w./day after the start of treatment.


No test item-related influence was noted on the T4 serum levels of the adult male animals, the observed values were in the range of the historic controls. The macroscopic inspection at necropsy and the microscopic examination of the reproductive organs of the male and female animals of the high dose group revealed no test item-related changes. No test item-related differences were noted either for the organ weights of the reproductive organs or the thyroid of the male and female animals.


No influence was noted on the length and number of the oestrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.


No adverse effect was noted on the prenatal development (birth and live birth index, percentage of post implantation loss) and the postnatal development of the pups (viability indices, pup body weight, ano-genital distance, the number of nipples per male pup, T4 serum levels on lactation day 13).


The macroscopic external examination of the all pups revealed no abnormalities.


 


 


EOGRTS according to OECD 443


The requested OECD 443 study in rats and with dose groups of 15, 50 or 150 mg/kg b.w./day started in May 2020. The in-life phase was completed in December 2020 and the first draft report of the study was available end of August 2021. 


The aim of the study was to evaluate the effects of the test item at dose levels of 15, 50 or 150/250 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood (OECD 443). The high dose level was increased from 150 to 250 mg/kg b.w./day on test day 64 due to no relevant external observed toxicological findings in group 4. For this reason, the high dose animals of the F1 Generation have received a dose level of 250 mg/kg b.w./day.


GENERAL AND REPRODUCTIVE TOXICITY (F0 GENERATION AND F1 PUPS)
No test item-related premature deaths were noted. The daily cage side observations for changes of behavior and the external appearance revealed a post dosing salivation for the male and female animals of the intermediate and the high dose group which was not considered to be adverse and, hence, not considered for the NOAEL.
For the body weight of the male high dose animals a marginally reduction was noted which was not considered as adverse.
However, a test item-related reduction in body weight that was considered to be adverse was noted at the high dose level for the female animals (150/250 mg test item/kg b.w./day).
Food consumption revealed reductions at start of dosing at the intermediate and / or the high dose level for the male and female animals. However, these changes were considered to be an adaptation to the start of dosing and not considered to be adverse.
Examination of the haematological and biochemical parameters revealed test item-related changes for parameters of the red blood cells (e.g. increased percentage of reticulocytes, decreased number of red blood cells) resulting in an increased bilirubin concentration for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day). This observations show an enhanced erythrocyte degradation (hemolytic anemia).
The test item-related changes that were noted for the haematological and the biochemical parameters correlated with test item-related findings during the histopathological examination in the spleen and the bone marrow for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day), which can be found in conditions with haemolytic anemia. No such findings were not noted anymore during the additional histopathological examination of the spleen and the bone marrow from the male and female animals of the low and the intermediate dose group.
Correlating findings were also noted at necropsy in the form of enlarged spleens and increased spleen weights for the male and female animals of the high dose group (150/250 mg test item/kg b.w./day).


REPRODUCTIVE TOXICITY AND DEVELOPMENTAL TOXICITY
No test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).
No test item-related effect was noted on the prenatal development of the pups (number of resorptions, stillbirths and live born pups).
During the postnatal development of the pups during the lactation period a decrease in the pup body weight was noted at the high dose level (150/250 mg test item/kg b.w./day) on lactation days 7, 14 and 21. The observed reduced body weight at the high dose level was driven by reduced maternal body weight in the high dosed females. Therefore, the reduced pup body weight is secondary effect to the maternal toxicity.
Other parameter of the pups as the viability indices, the ano-genital distance, nipple retention, the thyroid hormone levels and the pup organ weights were not affected by the test item as well. Furthermore, no malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


GENERAL TOXICITY (F1 COHORTS 1 A and 1 B)
No premature death was noted during the post-weaning development of the male and female animals. A post-dosing salivation was noted during the daily cage side observations which was not considered to be adverse.
A marginally reduced body weight was noted at the intermediate dose level (50 mg test item/kg b.w./day) for the male animals of Cohort 1A and the female animals of Cohort 1B. A more pronounced reduction in body weight was noted at the high dose level (250 mg test item/kg b.w./day) post-weaning until necropsy for the male and female animals of Cohort 1B. However, no changes in food consumption were noted.
The changes that were noted in the context of a haemolytic anemia for the male and female animals of the F0 Generation were also noted for the male and female animals of the F1 Generation. The respective changes in the haematological and the biochemical parameter and the increased spleen weight were noted for the male and female animals of the high dose group (250 mg test item/kg b.w./day. The respective histopathological findings in the spleen and the bone marrow were noted at the intermediate and the high dose level (50 or 250 mg test item/kg b.w./day). Yet, only consider severe in the highest dose group. All these haematological effects and the changes in the clinical biochemistry (bilirubin increase) and the histopathological observations (in spleen, liver) are indicative for an enhanced erythrocyte degradation and a subsequent extra medullary hematopoiesis.


Furthermore, a delay in sexual maturation was noted in form of a delayed vaginal opening at the high dose level for the female animals of Cohort 1A and 1B combined. However, this delay was considered to be a secondary effect due to the decreased body weights that were noted for the female animals of the and the high dose group.


 

Effects on developmental toxicity

Description of key information

The test substance did not show any developmental effects in a gavage study with rats performed in accordance with OECD TG 414 up to and including the highest tested dose level of 135 mg/kg bw/day. The NOAEL for maternal toxicity was 45 mg/kg bw/day, effects at 135 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is above 135 mg/kg bw/day (Ehling, 2004).


There is no study with regard to developmental toxicity in a second species (e.g. rabbit) available. A data waiver is claimed.


In the OECD 443 study (Provivo, 2021) no adverse effects in the prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were detected after the treatment with the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.


No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of estrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).A delay in sexual maturation was noted in form of a delayed vaginal opening at the high dose level for the female animals of Cohort 1A and 1B combined. However, this delay was considered to be a secondary effect due to the statistically significant decreased body weights that were noted for the female animals of the and the high dose group.


Summarising, the NOAEL for pre- and postnatal development was above 250 mg test item/kg b.w./day.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-08-12 to 2004-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
92/32/EEC, Appendiy 5
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
SD
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Harlan Winkelmann, D-33178 Borchen
- Strain: Hsd: Sprague Dawley SD
- Age: 9-11 weeks
- Weight at study initiation: 208 g (mean)
- Number of animals: 24 per group
- Controls: vehicle
Route of administration:
oral: gavage
Vehicle:
other:  sesame oil
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: sesame oil
- Concentration in vehicle: 0; 3.75; 11.25; 33.75 mg/ml
- Total volume applied: 4 ml/kg bw/day, adjusted to most recently  recorded body weight
Details on mating procedure:
MATING PROCEDURES: Virgin females in the pre-oestrus or oestrus phase  were mated overnight with sexually mature males (ratio 1 male : 1 female)  and were caged individually after the detection of sperm in vaginal  smears. The day of sperm detection was defined as day 0 of gestation.  Pregnancy was confirmed at necropsy.
Duration of treatment / exposure:
days 6 through 19 of pregnancy (day 0 = sperm detection)
Frequency of treatment:
once daily
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: sacrifice on day 20
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weights: days 0, 3, 6, 9, 13, 16, 18, 20
- Food consumption: for intervals between body weight determinations
- Clinical observations: survival, health condition and behavior twice  daily (once daily on weekends and public holidays); individual clinical 
observations once daily
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: P external and internal (thoracic and abdominal contents)  for macroscopically visible changes, with emphasis on the uterus; F1 for  
visible abnormalities
- Organ weights: gravid uterus weight with at least one fetus
Ovaries and uterine content:
- Examination of uterine content: Live and dead fetuses as well as  conceptuses undergoing resorption, placentas and corpora lutea were  counted, 
identified in numerical sequence from cervix to ovary and  examined macroscopically for visible abnormalities. The implantation  sites in the 
uterus were counted after staining with ammonium sulfide.
Fetal examinations:
- Examination of fetuses: Determination of weight, crown-rump length,  examination for gross external abnormalities.  Approximately half of the 
live fetuses of each litter were skinned and  fixed in alcohol, necropsied, sexed and checked for anomalies of the  internal organs (including 
particular attention to the reproductive tract  for signs of altered development). The carcasses were placed in a  solution of potassium hydroxide 
for clearing and stained with Alizarin  red S and Alcian blue (double-staining). The skeletons (bones and  cartilage) were examined and checked 
for stage of development and  abnormalities with the aid of a stereomicroscope. The remaining fetuses were transferred in Bouin's solution, 
necropsied,  sexed and examined for organ  anomalies (including particular attention  to the reproductive tract for signs of altered development) 
referring to  Wilson's slicing technique. Visceral and skeletal changes were subdivided into four categories (major  defects, minor defects, 
variations, and retardations) based on the  severity and / or the spontaneous incidence of the finding.
Statistics:
The statistical evaluation was based on the assumption of a monotonic dose-response relationship. A step-down trend test procedure was therefore applied, using all doses to determine the dose level at which there was no statistical significance of trend for the parameter. Statistical evaluations
over the low dose group(s) were only carried out if significant effects were detected in the higher dose group(s) (Hothorn & Lehmacher, 1991).
Maternal body weight was analyzed for statistical significance (p ≤ 0.05) using a 1-way ANOVAbased ordinal step-down trend test (see Tukey et al.,
1985). Food consumption was analyzed for statistical significance (p ≤ 0.05) using a Jonckheere test (Jonckheere, 1954; Lin & Haseman, 1975)
associated to the step-down ordinal test procedure.
Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
> 135 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
> 135 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Result: not teratogenic
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality: There was no treatment-related death.
- Description, severity, time of onset and duration of clinical signs:  Clinical signs did not occur.
- Body weight: Slightly decreased in high dose group, statistically  significant (p<=0.05) on days 9 (-3%), 18 (-5%), and 20 (-5%).

A  significantly lower body weight gain was recorded for the whole treatment  period (0-20: -15%). Body weights slightly 

decreased also in the mid dose group with  statistical significance on days 9 (-11%) and 13 (-6%) and subsequent  adaptation to 

normal, thus considered to be of questionable biological  relevance. Normal in low dose group.
- Food/water consumption: Food consumption distinctly to slightly  decreased in high dose group, statistically significant on study days 
6-9: -33%; 9-13: -12%; 13-16: -10%; 16-18: -12% (absolute); 
6-9: -32%; 9-13: -10%; 13-16: -7%; 16-18: -9% (relative). 
Food consumption slightly to marginally lower in mid dose females,  attaining statistical significance on days 6-9 (-15% abs., 

-16% rel.) and  13-16 (-6% abs. and rel.) only. Normal in low dose group.
- Number pregnant per dose level: 22/24; 22/24; 23/24; 23/24 (control /  low / mid / high dose) = not affected. One control female 

was pregnant  with only corpora lutea and empty implantation sites
- Number aborting: no abortions
- Number of resorptions: no total resorptions
- Number of implantations: 13.6; 14.5; 13.6; 13.7 (control / low / mid /  high dose) = not affected. 
- Pre and post implantation loss: 4.9; 6.2; 8.3; 10.9% pre-implantation  loss (%corpora lutea) (means of control / low / mid / high dose). One  total post implantation loss in control group.
- Number of corpora lutea: 14.3; 15.5; 14.6; 15.4 (means of control / low  / mid / high dose).
- Gross pathology incidence and severity: No compound-related gross  lesions were observed at necropsy.
- Organ weight changes: Insignificantly lower uterus weight for high dose  females (-7%).

FETAL DATA: 
There were no findings at caesarian section in any group of fetuses which  could be related to the test substance administration. 
There was no statistically significant difference  in the mean crown-rump  length for either male or female fetuses in any group. 

However,  evaluation of both genders together revealed a slight but statistically  significant (p<=0.05) decrease of the mean 

crown-rump length for all high  dose fetuses against the control (-2.3%). 
The mean placenta weight was slightly but statistically significantly  (p<=0.05) decreased in the high dose group 

(0.52; 0.51; 0.51; 0.46 g in  control, low, mid, and high dose groups, respectively).
Though considered not biologically significant, a treatment-related  influence on these endpoints could not be excluded.
- Litter size and weights: mean weights 3.64; 3.61; 3.64; 3.56 g = not  affected
- Number viable: Total number of live fetuses 12.4; 13.7; 13.0; 12.6  (control / low / mid / high dose) = not affected. No dead fetuses.
- Sex ratio: 44.4; 46.3; 44.6; 52.4% males (control / low / mid / high  dose) = not affected
- Grossly visible abnormalities: no substance-related findings
- External abnormalities: no substance-related findings
- Soft tissue abnormalities: no substance-related findings
- Skeletal abnormalities: Isolated findings of statistical significance  for high-dosed fetuses at the thoracic vertebra centra and in the 

rib  (here also for low-dosed fetuses): There was 1 fetus (out of 152, i.e.  0.7%) in the high dose group with a tail aplasia and 

spina bifida occulta  as a major defect, associated with several skeletal minor defects on the  vertebra and skeletal retardations. 

This complex finding was associated  secondary to insufficient oxygen supply of this fetus, which is known to  occur incidentally 

during embryonal development within relatively large  litters (14 fetuses in total in this litter). In the absence of  correlating findings 

either in other fetuses or other litters of this  group, these findings were considered to be incidental.
Minor skeletal defects of statistical significance included  aplasia/fused/fragmented thoracic vertrebra centra in 0% (control),  0.6%  (low dose), 0% (mid dose), and 2.0% (high dose, p<=0.05) animals. As the  incidences were only slightly above inhouse 

control data (0-1.5%) and did  not follow a dose response relationship, they were considered to be  incidental. Another minor defect 

of statistical significance was uni- or  bilateral knoddy ribs in 0% (control), 3.2% (low dose, p<=0.05), 0% (mid  dose), and 7.9% 

(high dose, p<=0.05) animals. As historical control data  were not yet available for this endpoint and the occurrence of this  effect 

did not follow a dose response relationship, it was considered to  be incidental.

Conclusions:
Regarding to the outcome of this developmental toxicity test there is no evidence of developmental toxicity in rats for Tetrahydronaphthalene, according to CLP regulation 1272/2008
Executive summary:

Daily oral (gavage) administration of Tetrahydronaphthalene at a dose level of 135 mg/kg bw/day during the sensitive phase of organogenesis and intrauterine development (days 6 – 20) of the fetuses induced decreased body weight gains and food intake during the treatment phase, which were interpreted as a first sign of maternal toxicity, without significantly altering pregnancy of the dams and intrauterine development of the conceptuses.

There was no maternal or embryofetal/developmental toxicity observed after administration of Tetrahydronaphthalene at either 45 or 15 mg/kg body weight/day. Tetrahydronaphthalene was not teratogenic in the rat.

The 'No Observed Adverse Effect Level' (NOAEL) was 45 and > 135 mg/ kg body weight/day for maternal and developmental effects, respectively.

Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Species:
rabbit
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
135 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1 (reliable without restriction)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Partly cited from SIAR to SIAM 19 (Berlin, 19-22 October 2004:


Based on the results of the 28-day study in rats (Hüls AG, 1995 a), four groups of 24 mated female Sprague-Dawley rats receivedthe test item by daily oral administration (gavage) at 0 (sesame oil = control), 15, 45 and 135 mg/kg bw/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414 (2001) (Ehling, 2004). The dams showed no treatment-related death. Clinical signs were not observed. Mean absolute and relative food consumption was distinctly to slightly decreased in high dose animals as compared to the controls, attaining statistical significance on study days 6 - 18 (absolute 6 - 9: 33 %: 9 - 13: -12 %; 13 - 16: -10 %; 16 - 18: -12 %; relative 6 - 9: -32 %; 9 - 13: - 10 %; 13 - 16: -7 %; 16 - 18: -9 %). In mid dose females, mean absolute and relative food consumption was slightly to marginally lower (statistically significant) during study days 6 - 9 (absolute: -15 %; relative: -16 %) and days 13 - 16 (absolute and relative: -6 %) . The terminal body weight (gestation day 20) was decreased in a statistically significant way (-5 %) for high dose females as compared to controls. A significantly lower body weight gain was recorded for the whole treatment period (0 - 20: -15 %).


Abortions, premature delivery or total resorptions were not observed in any of the test groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. There was no statistically significant difference in the mean crown-rump length for either male or female fetuses in any group. However, evaluation of both genders together revealed a slight but statistically significant decrease of the mean crown-rump length for all high dose fetuses against the control. In addition, the mean placenta weight was slightly but statistically significant decreased in the high dose group. These findings were marginal and considered to be within the physiological range for this rat strain and age. With respect to the fetuses, no test item related external or soft tissue malformations or variations were detected. Evaluation of skeletal defects revealed isolated findings of statistical significance for high-dose fetuses at the thoracic vertebra centra and in the rib (here also for low-dose fetuses): There was one tail aplasia in a fetus of a high dose female (1 fetus out of 152 examined, i.e. 0.7 %). This fetus also showed a large variety of other skeletal minor defects on the vertebra and skeletal retardations which were all associated with spina bifida occulta as the major defect diagnosis for this fetus. This complex finding was associated with insufficient oxygen supply of this fetus, which is known to occur incidentally during embryonal development within relatively large litters (14 fetuses in total in this litter). In the absence of correlating findings either in other fetuses or other litters of this group, these findings were considered to be incidental.


Minor skeletal defects of statistical significance included aplasia/fused/fragmented thoracic vertrebra centra in 0 % (control), 0.6 % (low dose), 0 % (mid dose), and 2.0 % (high dose) animals.


As the incidences were only slightly above inhouse control data (0 - 1.5 %) and did not follow a dose response relationship, they were considered to be incidental. Another minor defect of statistical significance was uni- or bilateral knobby ribs in 0 % (control), 3.2 % (low dose), 0 % (mid dose), and 7.9 % (high dose) animals. As historical control data were not yet available for this


endpoint and the occurrence of this effect did not follow a dose response relationship, it was considered to be incidental.


The NOAEL for maternal toxicity was 45 mg/kg bw/day and above 135 mg/kg bw/day for embryonic development (Ehling, 2004).

Justification for classification or non-classification

Based on the result of all available studies the test substance is not classified with regard to reproduction (fertility / development) according to the criteria of CLP Regulation 1272/2008.

Additional information