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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022
Reference Type:
publication
Title:
Characterization of Xenobiotic–Induced Hepatocellular Enzyme Induction in Rats: Anticipated Thyroid Effects and Unique Pituitary Gland Findings
Author:
Zabka, T.S.
Year:
2011
Bibliographic source:
ZABKA T. S.: Characterization of Xenobiotic–Induced Hepatocellular Enzyme Inductoion in Rats: Anticipated Thyroid Effects and Unique Pituitary Gland Findings. In: Toxicologic Pathology , 39:664-677, 2011)
Reference Type:
publication
Title:
Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights
Author:
Gallavan, RH
Year:
1999
Bibliographic source:
Gallavan R. H. et al.: Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights, Reproductive Toxicology (1999) 13: 383 - 390.
Reference Type:
publication
Title:
Deetman PE, Bakker SJ, Kwakernaak AJ, Navis G, Dullaart RP; PREVEND Study Group. The relationship of the anti-oxidant bilirubin with free thyroxine is modified by insulin resistance in euthyroid subjects. PLoS One. 2014;9(3):e90886. Published 2014 Mar 3.
Author:
Deetman, PE
Year:
2014
Bibliographic source:
Deetman PE, Bakker SJ, Kwakernaak AJ, Navis G, Dullaart RP; PREVEND Study Group. The relationship of the anti-oxidant bilirubin with free thyroxine is modified by insulin resistance in euthyroid subjects. PLoS One. 2014;9(3):e90886. Published 2014 Mar 3.
Reference Type:
publication
Title:
Thyroid hormones and the hepatic handling of bilirubin. I. effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the wistar rat
Author:
Van Steenbergen, W
Year:
1989
Bibliographic source:
Van Steenbergen W, Fevery J, De Vos R, Leyten R, Heirwegh KP, et al. (1989) Thyroid hormones and the hepatic handling of bilirubin. I. effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the wistar
Reference Type:
publication
Title:
Hormonal control of hepatic bilirubin transport and conjugation
Author:
Gartner, LM
Year:
1972
Bibliographic source:
Li F, Lu S, Zhu R, Zhou Z, Ma L, et al. (2011) Heme oxygenase-1 is induced by thyroid hormone and involved in thyroid hormone preconditioning-induced protection against renal warm ischemia in rat. Mol Cell Endocrinol 339: 54–62.
Reference Type:
publication
Title:
Heme oxygenase-1 is induced by thyroid hormone and involved in thyroid hormone preconditioning-induced protection against renal warm ischemia in rat
Author:
Li, F.
Year:
2011
Bibliographic source:
Li F, Lu S, Zhu R, Zhou Z, Ma L, et al. (2011) Heme oxygenase-1 is induced by thyroid hormone and involved in thyroid hormone preconditioning-induced protection against renal warm ischemia in rat. Mol Cell Endocrinol 339: 54–62.
Reference Type:
publication
Title:
Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats
Author:
Smith, TJ
Year:
1991
Bibliographic source:
Smith TJ, Drummond GS (1991) Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats. Biochim Biophys Acta 1075: 119–122
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2,3,4-tetrahydronaphthalene
EC Number:
204-340-2
EC Name:
1,2,3,4-tetrahydronaphthalene
Cas Number:
119-64-2
Molecular formula:
C10H12
IUPAC Name:
1,2,3,4-tetrahydronaphthalene
Test material form:
liquid
Details on test material:
1, 2, 3, 4-Tetrahydronaphthalene from Evonik, Batch: 20032012

Test animals

Species:
rat
Strain:
other: CD/ Crl:CD (SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies and required by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: Corn oil
Administration volume: 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).
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.





Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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.


Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.


Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Treatment related:
no

Any other information on results incl. tables

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.


Applicant's summary and conclusion

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.