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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:
OECD 443 study started in May 2020. All available information is submitted in this updated dossier by 12 April 2021 as requested in a decision on a TP from ECHA (Decision number TPE-D-2114465819-31-01). A draft report is expected in April 2021.
Adequacy of study:
key study
Study period:
Start in May 2020. Draft report is expected in April 2021.
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

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
basic test design (Cohorts 1A, and 1B without extension)
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: Approximately 20 weeks** (young adults; sexually mature)
- Body weight (at 1st administration): **
- Number of parental animals: Pre-exposure period: At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study: 192 animals (96 males and 96 females) A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.
Identification of animals: Each rat receives a continuous number. For animals of the F1 generation a continuous number will be given starting at 201. Points are set on paws and/or tail either by tattoo or marker; additionally, the animal cages are labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception, and dates of administration.
- Housing: With the exception of the mating period, the animals are kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages are cleaned and changed once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL (see Section 9.2 Limitation for contaminants in the bedding material).
- Diet (ad libitum): Commercial diet ssniff® R/M-Z V1154 (ssniff Spezialdiäten GmbH, 59494 Soest, Composition of the diet will be stated in the report) serves as diet. The diet is offered ad libitum with the exception of the night before the day of blood withdrawal for laboratory examinations (see section 3.6).
Periodic analysis of the food for contaminants based on EPA/USA1 is conducted by LUFA-ITL2 (see Section 9.1 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.
- Water (ad libitum): Tap water is offered daily ad libitum.
Samples of the drinking water are taken periodically by Wasserwerk Wankendorf and are analysed according to the "Deutsche Trinkwasserverordnung 2001" [German Regulations on Drinking Water 2001] (see Section 9.3 Limitation for contaminants in the drinking water).
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL for means of bacteriological investigations according to the "Deutsche Trinkwasserver-ordnung 20013, Anlage 1" [German Regulations on Drinking Water 2001, Addendum 1].
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Air changes per hour: **
- 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, 250 mg/ kg b.w./ day
Selection of route of administration: According to international guidelines.

The test item formulations will be prepared daily. The test item formulations will be continously agiated by stirring throughout the entire administration procedure to ensure homogeneity.
The amount of the test item will be adjusted to the animal's current body weight daily.
The control animals receive the vehicle at the same administration volume daily in the same way.
The homogeneity and concentration of the test item formulations will be monitored
Details on mating procedure:
Sexually mature male and female rats of the same dose group are paired randomly monogamously, i.e. 1 male and 1 female animal are placed in one cage during the dark period. The female is placed with the same male until evidence of mating is observed or two weeks have elapsed. If mating has not occurred after 2 weeks, the animal will be separated without further opportunity for mating. The females are examined each morning for the presence of sperm. The day of conception (gestation day 0) is considered to be the day on which sperm is found. If there are insufficient males, for example due to male death before pairing, then male(s) which have already mated may be paired with a second female such that all females are paired.
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 are 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

- Sampling time 2: 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 3: 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): 15 (30)

F1-Generation (Groups 2 to 4):
- Sampling time 1: At start of dosing of the F1 animals
- Parameter: Concentration and homogeneity
- Sampling per group: at the start of administration/ during (middle) administration/ before administration to the last animal

- Sampling time 2: Near the end of the Cohort 1A 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

- Sampling time 3: Near the end of the Cohort 1B 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): 15 (30)
Duration of treatment / exposure:
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).
Frequency of treatment:
daily
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 will be 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 with 20, 60 or 180 mg/kg b.w. daily from test day 1 to test d ay 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 grou p (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 int ermediate and the high dose group. In the OECD 421 study, the test item was administered orally to rats at dose levels of 15, 50 or 150 mg/kg b.w./day.

GENERAL TOXICITY (Parental male and female animals)
None of the animals died prematurely. During the daily cage side observations a short post-dosing salivation was noted with an increasing incidence of affected 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.

REPRODUCTIVE TOXICITY
- Parental females: No influence was noted on the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.
- Pups: No adverse effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post implantation loss) and the 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 b.w.)

As no relevant toxicological findings were observed in this batch of animals up to test day 58, it has been agreed to increase the group 4 dose level on day 64 from 150 mg/ kg b.w., p.o. to 250 mg/ kg b.w., p.o. for F0 animals. Dose change for F1 group 4 to 250 mg/ kg b.w. as the F1 generation is dosed at the same levels as the respective F0 group.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CLINICAL SIGNS:
- All animals:
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded. Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness. Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed will be recorded. In addition, animals will be checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals will be checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Dated and signed records of appearance, change, and disappearance of clinical signs will be maintained on clinical history sheets for individual animals.
- F0 animals:
Additionally, a more detailed examination of all F0 and F1 Cohort 1B animals is conducted on a weekly basis. F0 animals will be examined once before the first exposure (to allow for within-subject comparisons) and weekly thereafter until termination. Detailed clinical observations will be made in all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self mutilation, walking backwards) will also be recorded.

MORTALITY:
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m. Premortal symptoms will be recorded in detail; as soon as possible after exitus, a post mortem examination will be performed . In the case of prematurely sacrificed animals, laboratory examinations will be performed, if possible.

BODY WEIGHT:
The animals will be weighed and the weights 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#
Gestation period Not applicable GD 0, 7, 14, 21
Lactation period Not applicable PND 1, 4, 7, 14, 21
Post-mating period Daily# See gestation and lactation period
#: Weekly values will be reported
GD: Gestation day
PND: Post-natal day

FOOD AND WATER CONSUMPTION:
Food residue is weighed and recorded as follows:
Study period F0 males F0 females
Pre-mating period Weekly Weekly
Mating period None None
Gestation period Not applicable GD 0 (#1), 7, 14, 21 (#2)
Lactation period Not applicable PND 1 (#1), 7, 14, 21 (#2)
Post-mating period Weekly# See gestation and lactation period
#: Starting on a suitable day after the mating period to consolidate all male animals
#1: Days on which only the amount of food at food start was weighed.
#2: Days on which only the amount of food at food residue was weighed. On the other days, the amount of food at food residue, followed by food start was weighed.
GD: Gestation day
PND: Post-natal day

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

REPRODUCTIV PERFORMANCE - F0 animals
Evaluation/parameters:
- Number of pregnant females
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups absolute
- at birth (alive and dead)
- after 4 days of life, after 21 days
Number of pups per dam
- at birth
- after 4 days of life, after 21 days
Number of male and female pups
- at birth
- after 4 days of life, after 21 days
Number of stillbirths
- absolute
- per dam
Number of pups with malformations
- absolute
- per dam
s. also reproductive indices
Oestrous cyclicity (parental animals):
Vaginal smears will be taken and the oestrus cycle stages will be determined at the following time points:
- F0 animals: 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days). + During 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):
Spermcount, motility and morphology (spermiogram:
- all F0 animals
One epididymis and one testicle will be used for the sperm count. The sperm motility is determined and the sperm morphology is examined according to the method described by S. Plassmann and H. Urwyler (2001).
- all F1 Cohort 1A males
The epididymis not preserved for histopathology is used for enumeration of cauda epididymis sperm reserves. In addition, sperm from the cauda epididymis (or vas deferens) is collected for evaluation of sperm motility and morphology.
Litter observations:
CLINICAL SIGNS:
- All animals:
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded. Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness. Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed will be recorded. In addition, animals will be checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals will be checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Dated and signed records of appearance, change, and disappearance of clinical signs will be maintained on clinical history sheets for individual animals.
- F1 Cohort 1B animals after weaning:
Additionally, a more detailed examination of all F0 and F1 Cohort 1B animals is conducted on a weekly basis. F1 Cohort 1B animals will be examined weekly after weaning until termination. Detailed clinical observations will be made in all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self mutilation, walking backwards) will also be recorded.

MORTALITY:
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m. Premortal symptoms will be recorded in detail; as soon as possible after exitus, a post mortem examination will be performed . In the case of prematurely sacrificed animals, laboratory examinations will be performed, if possible.

BODY WEIGHT:
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#
#: Weekly values will be reported
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:
Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (OECD guideline 443 defines runts as animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) and the presence of gross abnormalities. Based on these parameters, the reproductive performance of the dams will be evaluated. The pups will be examined as described below. Any abnormal behavior will be recorded.

COUNTING, SEXING AND WEIGHING:
Live pups will be counted and sexed. Live pups are weighed on PND 1 and regularly thereafter.

ANO-GENITAL DISTANCE:
On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups will be determined using a scale.

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

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

SEXUAL MATURATION:
Animals (all selected) are evaluated daily for balano-preputial separation or vaginal patency before expected achievement of these endpoints to detect if sexual maturation occurs early. Any abnormalities of the genitals are recorded. Sexual maturity of the F1 animals is compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).
Postmortem examinations (parental animals):
LABORATORY EXAMINATIONS:
Blood samples will be taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight as scheduled. The blood samples collected will be divided into tubes as follows:
EDTA anticoagulant (whole blood) ........................ for haematological investigations
Citrate anticoagulant (plasma) ............................................... for coagulation tests
LiHeparin anticoagulant (plasma) .................................... for clinical chemistry tests
Sampling time: at sacrifice
Animals: 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 will be taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, if possible, as scheduled below. The blood is processed for serum:
- Animals: F0: 10 males and 10 females randomly selected from each group (animals also selected for laboratory examinations)
- time of sampling: at sacrifice
- expected number of samples: 80
- feeding status: fasted
- analysed hormones: T4 + TSH
- sample volume: 2 x 100 µL each

The serum samples will be divided into aliquots, if possible, and stored at -20°C ± 10% until analysis using ELISA. The T4 and TSH ELISA (commercial kits) will be conducted at LPT.
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:
Urine samples are collected from animals fasted overnight at the following times and the parameters listed below are determined.
- 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 urine is collected for 16 hours in a URIMAX funnel cage. The collection of urine will be terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination.
The following parameters will be 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 will 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 will be carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit will be 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 will be examined visually.

PATHOLOGY AND HISTOPATHOLOGY:
GROSS NECROPSY:
For adult F0 and F1 females a vaginal smear taken on the day of sacrifice shortly before necropsy will be examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs. The animals will be euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled:
Animals No. of animals Necropsy date Examination Vaginal smears
Males All (96) After weaning of the F1 animals Full necropsy Not applicable
Dams All (96) Shortly after weaning of the F1 animals Full necropsy Yes
In the case the time of dissection would fall on a weekend or bank holiday, necropsy will be postponed to the next working day and dosing will be continued up to and including the day before sacrifice. Animals which are prematurely sacrificed or die during the study are necropsied as soon as possible after exitus.

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
All superficial tissues will be examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.
Organ weights of selected organs will be determined for each generation and cohort. Organ weights of the animals which die or are sacrificed prematurely will be recorded but not included in the mean value comparison.

F0 Generation:
The number of implantation sites will be recorded and used to evaluate reproductive performance. Apparently non-pregnant uteri will be placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI. Organ weights of all adult F0 animals will be determined before fixation, where applicable. Paired organs will be 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 will be preserved in an appropriate fixative:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: modified Davidson’s solution
- Epididymis (1)#
- Testicle (1)#
Fixative: 7% buffered formalin
- Adrenal gland (2)
- Ovary (2)
- Bone
- Oviducts
- Bone marrow (os femoris)
- Pituitary
- Brain (cerebrum, cerebellum, pons)
- Prostate
- Gross lesions observed
- Seminal vesicles with coagulating glands
- Heart (3 levels: right and left ventricle, septum)
- Spinal cord (3 sections)
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Spleen
- 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 will not be preserved but used for the spermiogram.

Any other organs displaying macroscopic changes will also be preserved. In addition, sperm viability and morphology will be evaluated for all male F0 animals, and bone marrow smears will be prepared for selected F0 animals.

BONE MARROW:
During dissection fresh bone marrow will be 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).
The myeloid:erythroid ratio will be determined by cell differentiation (counting of 200 nuclei-containing cells) for scheduled animals.

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

F0 animals:
Full histopathology will be 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 are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically; they are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged. In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined. Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of the selected F0 males of groups 1 and 4 following H-E and PAS staining.
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.

HISTOPATHOLOGICAL EVALUATION:
The histotechnique for all animals will be performed by LPT. All slides (labelled with study number, species, animal number, block number) prepared at LPT and will be dispatched to AnaPath GmbH for histopathological evaluation. The transport of slides and tissues to AnaPath GmbH will be arranged by LPT, whereas the return transport to the Test Facility will be arranged by AnaPath GmbH. Histopathological evaluation will be performed by the Test Site according to all relevant Test Site SOPs. All observations upon final assessment will be reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings are recorded, reported and archived with the PathData system version 6.2e2. The report of this phase of the study will comprise a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report will be 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 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 archivedby the Test Site. The Test Site Phase Report will be appended to the final LPT Study Report No. 37627.
Postmortem examinations (offspring):
LABORATORY EXAMINATIONS:
Blood samples will be taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight as scheduled. The blood samples collected will be divided into tubes as follows:
EDTA anticoagulant (whole blood) ........................ for haematological investigations
Citrate anticoagulant (plasma) ............................................... for coagulation tests
LiHeparin anticoagulant (plasma) .................................... for clinical chemistry tests
Sampling time: at sacrifice
Animals: 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 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 will be taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, if possible, as scheduled below. The blood is processed for serum:
- 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
- expected number of samples: 160
- 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 22
- expected number of samples: 160
- 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
- expected number of samples: 80
- feeding status: fasted
- analysed hormones: T4 + TSH
- sample volume: 2x 100 µL each

The serum samples will be divided into aliquots, if possible, and stored at -20°C ± 10% until analysis using ELISA. The T4 and TSH ELISA (commercial kits) will be conducted at LPT.
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:
Urine samples are collected from animals fasted overnight at the following times and the parameters listed below are determined.
- 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 is collected for 16 hours in a URIMAX funnel cage. The collection of urine will be terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination.
The following parameters will be 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 will 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 will be carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit will be 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 will be examined visually.

PATHOLOGY AND HISTOPATHOLOGY:
GROSS NECROPSY:
For adult F0 and F1 females a vaginal smear taken on the day of sacrifice shortly before necropsy will be examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs. The animals will be euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled:
- 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
- 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 91)
- 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 98)
- Examination: Full necropsy
- Vaginal smears: Yes

In the case the time of dissection would fall on a weekend or bank holiday, necropsy will be postponed to the next working day and dosing will be continued up to and including the day before sacrifice. Animals which are prematurely sacrificed or die during the study are necropsied as soon as possible after exitus.

EXAMINATION OF THE "SURPLUS" F1 PUPS:
Dead pups and pups sacrificed on day 4 post-partum will be carefully examined externally for gross abnormalities. The external reproductive genitals will be examined for signs of altered development.
Pups sacrificed on day 21 or 22 post-partum will be dissected and examined macroscopically for any abnormalities or pathological changes. Of those F1 pups sacrificed on PND 21 or 22, the following organs of up to 10 pups per sex per group of each generation, from as many litters as possible will be preserved in 7% buffered formalin:
- Brain#
- Gross abnormalities
- Mammary gland
- Ovary (2)#
- Spleen#
- Thymus#
- Uterus including cervix#
#: Organs will be weighed before fixation. Paired organs will be weighed individually and identified as left or right.
Histopathological examination of the preserved organs will be conducted only in agreement with the Study Monitor.

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
All superficial tissues will be examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.
Organ weights of selected organs will be determined for each generation and cohort. Organ weights of the animals which die or are sacrificed prematurely will be recorded but not included in the mean value comparison.

F1 generation- Cohort 1A:
The weights of the following organs of all adult F1 animals of Cohort 1A will be determined before fixation, where applicable. However, lymph nodes will be preserved and weighed for only 10 animals/sex/group (1 animal per litter, all litters represented by at least 1 pup; randomly selected). Paired organs will be 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 will be 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 will not be 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 will be preserved for histopathological evaluation, the second half will be used for splenic lymphocyte subpopulation analysis.
Any other organs displaying macroscopic changes will also be preserved. In addition, sperm viability and morphology will be evaluated for all male F1 Cohort 1A animals, and bone marrow smears will be prepared for selected F1 Cohort 1A animals.

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 will be 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 will be determined by cell differentiation (counting of 200 nuclei-containing cells) for scheduled animals.

PHENOTYPIC ANALYSIS OF SPLEEN CELLS - COHORT 1A ANIMALS:
After weighing, spleens of selected Cohort 1A animals will be split in two halves. The portion of the spleen not preserved for histopathology will be minced using a mechanic dissociator to prepare single cell suspensions. Samples will then be used for the following examinations:
- Splenic lymphocyte subpopulation analysis via FACS using the MACSQuant® Analyzer 10/168
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 will be available for possible examination of pathological changes, but examined and evaluated only depending on necropsy findings and upon agreement with the Study Monitor.

F1 Cohort 1A:
Full histopathology will be performed on the preserved organs of:
- F1 animals: groups 1 and 4 of Cohort 1A
- All deceased or prematurely sacrificed animals
The organs listed above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically; they are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged. In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined. Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of all F1 Cohort 1A males of groups 1 and 4 following H-E and PAS staining. Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea will be performed on one ovary of the F1 Cohort 1A females of groups 1 and 4. Therefore, the ovary will be processed as follows:
- Ten (10) 2-4 μm step sections with 50 μm steps in between;
- Each slide will be labelled with the slide number to follow the sequence.
In case of test item-related changes in group 4, the Sponsor will be given sufficient notice before the corresponding organs of 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 will be given sufficient notice before the corresponding organs of F1 Cohort 1B animals are sectioned and examined histopathologically.

HISTOPATHOLOGICAL EVALUATION:
The histotechnique for all animals will be performed by LPT. All slides (labelled with study number, species, animal number, block number) prepared at LPT and will be dispatched to AnaPath GmbH for histopathological evaluation. The transport of slides and tissues to AnaPath GmbH will be arranged by LPT, whereas the return transport to the Test Facility will be arranged by AnaPath GmbH. Histopathological evaluation will be performed by the Test Site according to all relevant Test Site SOPs. All observations upon final assessment will be reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings are recorded, reported and archived with the PathData system version 6.2e2. The report of this phase of the study will comprise a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report will be 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 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 archivedby the Test Site. The Test Site Phase Report will be appended to the final LPT Study Report No. 37627.
Statistics:
A) The following settings will be used for the statistical evaluation of the parametrical values captured by (Provantis®9 Integrated preclinical software, Instem LSS Ltd):
Homogeneity of variances and normality of distribution will be tested using the BARTLETT’s and SHAPIRO-WILK test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values will be performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group will be made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
B) The following statistical methods will be used for data not captured by Provantis software (reproductive data).
For the parametric values (number of pups etc.), homogeneity of variances will be tested using the BARTLETT test.
In case of homogeneity, intergroup comparison will be performed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01).
In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group will be performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
For the non parametric values (for example the indices of reproduction or the survival rates), the statistical analysis will be performed using the FISHER's exact test (n < 100) or the chi2-test with YATES' correction for continuity (n ≥ 100) at significance levels of p ≤ 0.05 and p ≤ 0.01.
All data are evaluated statistically in this manner. Individual results which differ significantly from those of the control group will be indicated in the tables of the report.
The mean values and standard deviations will be calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
For each F0 group the gestation index is determined:
Gestation Index = (Number of litters with live pups/ Number pregnant) x 100
Fertility Index female [%] = (Number of pregnant rats/ Number of females used) x 100
For each litter and group the following indices are determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars) x 100
Live Birth Index = (Number of pups born alive on day 0/1/ Total number born (live + dead)) x 100
Viability Index pre-select = (Number of pups alive on day 4 (pre-select)/ Number of pups live on day 0/1) x 100
Viability Indexpost-select = (Number of pups alive on day 21 (post-select)/ Number of pups live on day 0/1) x 100
Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100
Offspring viability indices:
Birth Index, Live Birth Index, Viability Birth Index, Post Implantation loss

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: in life-phase completed; draft repport not yet available, expected in April 2021

Results: F1 generation

Effect levels (F1)

Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: in-life phase completed; draft report not yet available, expected in April 2021

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The OECD 443 study started in May 2020. The first draft report of the study is expected to be available in April 2021.
Executive summary:

The OECD 443 study started in May 2020. The first draft report of the study is expected to be available in April 2021.