Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

In a final decision on a testing proposal dated 5 April 2019 (Decision numberTPE-D-2114465819-31-01/F ) ECHA requested to perform an extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:

- Ten weeks premating exposure duration for the parental (P0) generation;

-  Dose level setting shall aim to induce some toxicity at the highest dose level;

-  Cohort 1A (Reproductive toxicity);

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

The OECD 443 study was initially scheduled to be completed in April 2021 before the REACH submission deadline. The first unaudited draft report was scheduled for the end of December 2020. However, due to the great extent of parameters to be evaluated and as the histopathological examination is not completed yet due to the Covid 19-pandemic situation, the completion of the study is delayed. The end of the In-life phase was 21 December 2020 and the first (unaudited) draft report is expected mid of April 2021. The dossier will be updated in a timely manner and submitted to ECHA.

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Remarks:
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.
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.
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
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
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
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
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
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
Critical effects observed:
not specified
Reproductive effects observed:
not specified
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.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08.01.2020 to 13.03.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This OECD 421 study is used as dose range finding study for the OECD 443 study (LPT, 2021).
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
This OECD 421 study is used as dose range finding study for the OECD 443 study (LPT, 2021).
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD / Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age: 71 days
- Body weight (at 1st administration): Males: 399.2 g - 477.1 g, Females 241.6 g - 286.3 g
- Identification of animals: After randomisation, each rat received a continuous number on the tail, either by tattoo or marker. Additionally, the animal cages were labelled with study number, animal ID number, sex, type of study, route of administration, and treatment group.
- Number of animals: Pre-exposure period: 58 female animals were evaluated pre-exposure for oestrous cyclicity to yield 40 females with regular oestrous cycle for the study. Prior to any study related activity, animals were allowed to adapt to the new environment for 7 days.
Main study: 80 animals (40 males and 40 females) A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 Generation.
- Housing: With the exception of the mating period the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx.18 cm Except during the mating period the animals were placed in the animal room as follows: Male animals: On one side of the room with each dose group separated by an empty row.
Female animals: On the other side of the room with each dose group separated by an empty row.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL. Certificates of analysis of the bedding material are included in the raw data.
- Diet (ad libitum): A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed. Samples of the food are analysed for contaminants based on EPA/USA3 by LUFA-ITL4 at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water (ad libitum): Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 20015]. In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the ‘Deutsche Trinkwasserverordnung 2001, Anlage 1’ [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.
- Acclimation period: 7 days

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

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

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

The measured concentrations of the test item in the test item-vehicle mixtures were between 93.7% and 99.5% of the nominal concentrations, indicating correctly prepared and homogenous test item-vehicle mixtures that were stable for at least 24 hours at room temperature.
Duration of treatment / exposure:
The study animals were treated during the following periods:
- Males + Females: Pre-mating day 15 to 29 (pairing was in the evening of test day 29) and Mating 30 to 44 (first mating day, confirmed by positive sperm detection, was in the morning of test day 30)
- Males: Post-mating until test day 44 (one day before necropsy on test day 45)
- Females: Gestation and lactation period until test days 65 to 79 (one day before necropsy on test days 66 to 80; corresponding to lactation days 14 to 16)
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control group
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study have been selected in agreement with the Sponsor based on results of a 2-week dose-range-finding study in rats (LPT, 2020, 14 day tolerance test). In this study, the rats were treated with 20, 60 or 180 mg/kg b.w. daily from test day 1 to test day 14. None of the animals died prematurely. No changes in behaviour, the external appearance or the appearance of the faeces were noted, with the exception of a severely reduced motility that was noted for all male and female animals of the high dose group (180 mg/kg b.w./day) on test day 1. The male animals showed a slightly reduced body weight at 60 and 180 mg/kg/ b.w./day and the female animals a marginally reduced body weight at 180 mg/kg b.w./day in comparison to the animals that were dosed with 20 mg/kg b.w./day. A slightly to moderately reduced food consumption was noted for the male animals that were dosed with 60 or 180 mg/kg b.w./day during the first test week. For the female animals a moderately reduced food consumption was noted during the first and the second test week at a dose level of 180 mg/kg b.w./day. No test item-related changes were noted during the macroscopic inspection at necropsy. The examination of the organ weights revealed no test item-related differences between the low, the intermediate and the high dose group. Hence, the dose levels of 15, 50 or 150 mg/kg b.w./day were selected for this study.

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

- Fasting period before blood sampling for clinical biochemistry: over night
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS:
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal. Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

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

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

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

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

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

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

REPRODUCTIVE PARAMETERS:
- stages of the ooestrous cycle
- pre-coital time
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4 and 13
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4 and 13
Number of stillbirths
- per group
- per dam
Number of pups with malformations
- per group
- per dam

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

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

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

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

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

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

DISSECTION OF ADULT ANIMALS:
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. During necropsy the number of implantation sites was recorded in the female animals. Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.

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

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

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

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

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

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

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

FEMALES: Pre-mating-, gestation- and lactation period
No test item-related differences in body weight or body weight gain were noted between the female rats of the control group and the female rats of the low and the intermediate dose group (15 or 50 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (150 mg test item/kg b.w./day) a slightly reduced body weight (statistically significant at p ≤ 0.05 or not statistically significant ) was noted during the gestation period and the lactation period. The difference between the high dose group and the control group was nearly constant, being around 5% to 6% below the control group during the gestation period and the lactation period.
In detail, on gestation days 0 and 20 the high dosed values were 4.7% or 6.1% below the control value. On lactation days 1 and 13 the high dosed values were 6.4% or 6.6% below the control values.
As these differences were soon noted at the begin of the gestation period and still noted during the lactation period they can be considered as a test item-related effect on maternal body weight, as an influence of the litter weight can be ruled out at the beginning of the gestation period and during the lactation period.
A marginally reduced body weight gain in comparison to the control group was noted at the high dose level during the gestation period (see text table 7-5 below). This fits to the slightly reduced body weight that was noted at the high dose level during the gestation period. During the lactation period the body weight gains of the control group and the high dose group were nearly identical.


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

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

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

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

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


Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A reduced fur at the head and the neck was noted for all pups from dam no. 59 of the intermediate dose group and a reduced fur on the whole body was noted for all pups of dam no. 71 of the high dose group.
As there were no further observations for the pups from these 2 dams, a test item-related effect was ruled out. Hence, the observation of reduced fur was considered to be spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pre- and post-cull period
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
A marginally increased number of prematurely deceased pups was noted at the high dose level for the pre-cull period (4 prematurely deceased pups in comparison to 1 or 2 prematurely deceased pups in the other test groups). This marginal difference was considered spontaneous as there was no difference in the number of prematurely deceased pups between the high dose group and the other test groups during the further course of the lactation period (post-cull period). Hence, the observed variability is considered as incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
PUP BODY WEIGHT:
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted during the lactation period.
Slightly reduced pup body weights (statistically not significant) were noted at the intermediate and the high dose level on lactation day 13 (4.2 % or 3.9 % below the control value). However, these slight differences were still in the range of LPT background data and considered to be spontaneous.

LITTER WEIGHT:
Slight reductions in litter weight (statistically significant or not statistically significant) in comparison to the control group were noted at the intermediate (LD 4: statistical significant p ≤ 0.05) and the high dose level (50 or 150 mg test item/kg b.w./day) on lactation days 1, 4 and 13.
The slight reductions in litter weight that were noted on lactation days 1 and 4 at the intermediate (11.1% or 10.3% below the control) and the high dose level (7.3% and 10.6% below the control) were due to a slightly reduced number of pups per dam on lactation days 1 and 4 (14.7 or 15.3 live pups per dam in comparison to 16.7 pups per dam in the control group on lactation day 1).
Thereafter, on lactation day 13, when the number of pups per dam was adjusted between the study groups due to the culling process, the differences in litter weight between the control group and the intermediate and the high dose group became much smaller (4.0% or 7.2% below the control). The remaining difference was due to the slightly reduced pup body weights that were noted at the intermediate and the high dose level on lactation day 13 (4.2% or 3.9% below the control).
Note that the number of pups per dam at the low dose level was also reduced in comparison to the control group on lactation days 1 and 4 (15.3 pups per dam in comparison to 16.7 pups per dam in the control group on lactation day 1). However, due to slightly increased pup body weights at the low dose level, no decreased litter weight was noted, in contrast to the intermediate and the high dose level. Hence, in conclusion, the effect was considered as not test item-related but incidental.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day) were noted for the T4 levels of the male and female pups on lactation day 13.
Increased T4 serum levels were noted at the high dose level for the male pups, the female pups and the male and female pups combined (18.6%, 12.1% and 15.3%, respectively, above the control values; statistically significant for the male pups at p ≤ 0.05). However, all values were in the range of LPT background data and the differences were considered to be spontaneous.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (15, 50 or 150 mg test item/kg b.w./day).
For most of the affected male pups only 1 or 2 nipple was noted per pup. The number of these pups is identical in control and intermediat dose group (7 pups).
Male pups with 2 nipples were noted in the control group, low and intermediat dose groups (2, 2 and 3, respictivly pups in each group) but not in the high dose group.
Two male pup with 3 nipples was noted in the control group and one pups with 3 nipples in the low dose group. Hence, the number of pups with nipples in the control group is above the values of the Provivo background data (see Appendix 6).
At the high dose level only 1 pup with 1 nipple was noted in comparison to 7 pups with altogether 13 nipples in the control group. This led to a statistically significantly reduced number of nipples / number of pups per dam at the high dose level (0.02 nipples / pup / dam in comparison to 0.29 nipples /pup / dam; p ≤ 0.05). As a reduced number of male nipples is not a toxicological effect, the reduced number of nipples / pup / dam was considered to be spontaneous.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 15, 50 or 150 mg test item/kg b.w./day after terminal sacrifice between lactation days 13 and 16.
Furthermore, no gross abnormalities were noted for the pups that were found dead (stillborns or pups that died during the lactation period).
A reduced fur was noted for the pups of dam no. 59 and dam no. 71 of the intermediate and the high dose group. This observation was considered to be spontaneous.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
MALE to FEMALE RATIO OF THE PUPS:
No test item-related influence on the male to female ratio was noted for all treatment groups (15, 50 or 150 mg test item/kg b.w./day).

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

Text Table 3‑3:     Groups and dose levels.

Group

THN dose levels

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

Number and sex of animals

Animal number

1

0

(vehicle control)

10

10

m

f

1 -

11 -

10

20

2

15

(low dose)

10

10

m

f

21 -

31 -

30

40

3

50

(intermediate dose)

 

10

10

m

f

41 -

51 -

50

60

4

150

(high dose)

10

10

m

f

61 -

71 -

70

80

m:

male

f:

female

 

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

THN

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Females started dosing

10

10

10

10

Females used for pairing

10

10

10

10

Females mated

10

10

10

10

Females not mated

0

0

0

0

Females pregnant

9

10

10

10

Females not pregnant

1 (no. 11)

0

0

0

Females with total resorption of implants

0

0

0

0

Females with live born pups

9

10

10

10

 

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

Observation (males)

Affected animals

First to last seen

(test days)

Range of days observed #

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

Haemorrhagic urine

1 of 10

36

1 (28)

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

Salivation (slight)

6 of 10

26 - 34

1 - 2

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

Salivation (slight to extreme)

8 of 10

25 - 44

1 - 15

Reduced motility

10 of 10

15

1

#:

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

 

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

 

Affected females per period

Range of days observed per period #

Observation

(females)

Pre-mating/

Mating

Gestation

Lactation

Pre-mating/

Mating

Gestation

Lactation

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

Salivation

(slight to moderate)

n.d.

n.d.

4 of 10

-

-

1 - 9

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

Salivation

(slight to extreme)

3 of 10

5 of 10

9 o 10

1 - 4

1 - 4

1 - 11

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

Salivation

(slight to extreme)

7 of 10

10 of 10

10 of 10

1 - 8

2 - 18

1 - 12

Reduced motility

10 of 10

n.d.

n.d.-

1

-

-

n.d.:

Not detected

#:

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

 

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

Males

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Body weight gain #

(test day 15 to test day 44)

+20.2 %

+18.3 %

+18.5 %

+14.3 %

#:

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

 

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

Females

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Body weight gain (%) #1

(pre-mating period)

(test days 15 to 29)

+4.1

+6.7

+6.4

+6.3

Body weight gain (%) #2

(gestation period)

+53.1

+53.5

+52.3

+50.2

Body weight gain (%) #3

(lactation period)

+12.5

+12.8

+11.6

+12.4

#1:

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

#2:

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

#3:

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

 

 

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

Stage of oestrous cycle at necropsy

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

Prooooestrus

3 of 9

-

-

1 of 10

Oooestrus

-

1 of 10

-

-

Metoooestrus

1 of 9

2 of 10

1 of 10

2 of 10

Dioooestrus

5 of 9

7 of 10

9 of 10

7 of 10

- :

not detected

 

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

Parameter

Values from this study

Group mean values ± SD

Range of individual animals #2

(nmol/L)

LPT Background Data #1

obtained from the control groups of 14 OECD 422 / 421 studies performed at LPT from 2016 - 2019

T4 serum level

(nmol/L)

(males)

 

 

Group 1

 

52.0794 ± 4.1264

46.486 - 58.113

Mean value from 140 control animals from 14 studies

64.7317 ± 14.4674

 

Range of 140 control animals

from 14 studies:

33.066 - 114.800

 

Group 2

 

53.6975 ± 5.6497

44.143 - 61.416

 

Group 3

 

61.4492 ± 8.0228*

50.984 - 79.258

 

Group 4

 

59.6764 ± 9.5186

48.174 - 80.485

*/**:

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

#1:

Data not audited by QAU

#2:

Values are taken from table 12-1 'Thyroid Hormone Level Analysis - Summary - Males'.

 

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

Parameter

Group 1

Control

Group 2

15 mg/kg

Group 3

50 mg/kg

Group 4

150 mg/kg

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

Mean cycle length (days)

4.37

4.18

4.00

4.17

Number of cycles

1.8

2.3

2.5

1.9

# :

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

 

Text Table 7-9:     Fertility indices per group.

Group / Dose level

Fertility index %

Pregnant rats /

Females used for pairing #

Group 1 (Control)

90

9 / 10

Group 2 (15 mg/kg)

100

10 / 10

Group 3 (50 mg/kg)

100

10 / 10

Group 4 (150 mg/kg)

100

10 / 10

#:

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

 

Text Table 7-10:   Gestation indices per group.

Group / Dose level

Gestation index %

Dams with live pups / pregnant rats #

Group 1 (Control)

100

9 / 9

Group 2 (15 mg/kg)

100

10 / 10

Group 3 (50 mg/kg)

100

10 / 10

Group 4 (150 mg/kg)

100

10 / 10

#:

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

 

Text Table 7-11:   Comparison of the birth index and post-implantation loss (group values) with the LPT background data.

Parameter

Values from this study #2

Group values

LPT Background Data #1

obtained from the control groups of 27 OECD 422 / 421 studies performed at LPT from 2012 - 2019

Live birth index

(%)

Group 1

99.34

Mean value from the group mean values

of 27 control groups:

99.42 ± 0.82

 

Range of group mean values from

27 control groups:

97.1 - 100.0

Group 2

99.35

Group 3

99.33

Group 4

97.45

#1:

Data not audited by QAU

#2:

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

 

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

 

Reproductive data

Group 1

(control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

Parametrical values

(see section 3.15.1 'Reproductive parameters')

Implantation sites (mean per dam) #1

17.8

16.1

16.0

16.4

Pups (born alive and dead)

(mean per dam) #1

16.8

15.4

14.9

15.7

Pups born alive (mean per dam) #1

16.7

15.3

14.8

15.3

Indices [%]

(see section 3.15.2 'Reproductive indices')

Birth Index

mean per dam #1

group             #2

94.34

94.38

95.18

95.06

93.14

93.13

95.73

95.73

Live birth Index

mean per dam #1

group             #2

99.38

99.34

99.47

99.35

99.41

99.33

96.61

97.45

Post-implantation loss

mean per dam #1

group             #2

6.24

6.25

5.34

5.56

7.45

7.50

7.36

6.71

Resorptions and stillbirths

Difference between

no. of implantation sites and no. of pups born alive

mean per dam #3

 

 

sum per group #3

1.1

 

 

10

0.9

 

 

9

1.2

 

 

12

1.1

 

 

11

Number of stillbirths #3

1

1

1

4

#1:

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

#2:

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

#3:

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

 

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

Pre-cull period

Parameter

Group 1

(control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

Prematurely deceased pups between lactation days 0/1 to 4

 /

Total number of live born pups #

2 / 150

1 / 153

2 / 147

4 / 153

Viability index (group values) #

98.67

99.35

98.64

97.39

#:

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

 

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

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

Group 1

(control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

dam no.

deceased pups

dam no.

deceased pups

dam no.

deceased pups

dam no.

deceased pups

17

2

38

1

53

1

73

1#2

 

 

55

1

79

1

 

80

2

#1:

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

#2:

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

                 

 

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

Post-cull period

Parameter

Group 1

(control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

Prematurely deceased pups between lactation days 5 and 13

/

Number of pups alive on lactation day 4 after culling #

1 / 90

0 / 100

1 / 100

1 / 97

Viability index (group values) #

98.89

100.00

99.00

98.97

#:

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

           

 

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

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

Group 1

(control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

dam no.

deceased pups

dam no.

deceased pups

dam no.

deceased pups

dam no.

deceased pups

16

1 #2

 

51

1

80

1

#1:

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

#2:

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

 

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

Male / Female ratio of the pups

Time point

Group 1

(control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

Lactation day 1 #

1.03

1.01

1.23

1.13

Lactation day 4 #

1.03

1.03

1.23

1.13

#:

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

 

 

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

Pup body weight

Changes in comparison to the control group

[%] #

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

 

Lactation day 1

males + females

combined

+5.2

+0.2

- 0.5

Lactation day 4

males + females

combined

+6.4

+1.5

0.0

Lactation day 13

males + females

combined

+ 2.4

- 4.2

- 3.9

#:

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

 

Text Table 7-19:   Comparison of the pup body weights (group mean values) on lactation day 13 with the LPT background data.

Parameter

Values from this study

Group mean values ± SD #2

(g)

LPT Background Data #1

obtained from the control groups of 15 OECD 422 / 421 studies performed at LPT from 2016 - 2019

Pup body weight

on lactation day 13

 

(males + females combined)

 

 

Group 1

 

29.591 ± 1.917

Mean value from the group mean values of 11 control groups:

28.8937 ± 1.1887

 

Range of group mean values from

11 control groups:

26.311 - 30.617

 

Group 2

 

30.296 ± 3.573

 

Group 3

 

28.359 ± 2.045

 

Group 4

 

28.441 ± 2.972

#1:

Data not audited by QAU

#2:

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

 

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

Litter weight #

Changes in comparison to the control group

[% / mean litter weight]

 

Group 1

(Control)

Group 2

(15 mg/kg)

Group 3

(50 mg/kg)

Group 4

(150 mg/kg)

Lactation day 1

males + females

combined

n.a.

112.26 g

- 3.8

108.00 g

- 11.1

99.80 g

- 7.8

103.52 g

Lactation day 4

males + females

combined

n.a.

153.09 g

- 2.5

149.30 g

- 10.3*

137.37 g

- 10.6

136.87 g

Lactation day 13

males + females

combined

n.a.

292.34 g

+ 3.6

302.96 g

- 4.0

280.69 g

- 7.3

271.14 g

*/**:

 

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

n.a.

 

Not applicable

#:

 

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

 

Text Table 7-21:   Mean number of live pups per dam during the lactation period.

Mean number of live pups per dam

(males + females combined)

Mean number of live pups per dam #

Group 1

(control)

Group 2

(3mg/kg)

Group 3

(10 mg/kg)

Group 4

(30 mg/kg)

 

Lactation

day 1

-

16.7

15.3

14.7

15.3

Lactation

day 4

before culling

16.4

15.2

14.5

14.9

after culling

10.0

10.0

10.0

9.7

Lactation

day 13

-

9.9

10.0

9.9

9.6

#:

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

 

Text Table 7-22:   Comparison of the number of live pups per dam (group mean values) on lactation day 1 with the LPT background data.

Parameter

Values from this study

Group mean values ± SD #2

LPT Background Data #1

obtained from the control groups of 27 OECD 422 / 421 studies performed at LPT from 2016 - 2019

Number of live pups per dam on lactation day 1

 

(males + females combined)

 

 

Group 1

 

16.7 ± 2.1

Mean value from the group mean values of 27 control groups:

13.90 ± 0.88

 

Range of group mean values from

27 control groups:

12.0 - 15.1

 

Group 2

 

15.3 ± 2.6

 

Group 3

 

14.7 ± 1.5

 

Group 4

 

15.3 ± 3.4

#1:

Data not audited by QAU

#2:

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

 

Text Table 7-23:   Comparison of the T4 serum levels of the male pups on lactation day 13 with the LPT background data.

Parameter

Values from this study

Group mean values ± SD #2

LPT Background Data #1

obtained from the control groups of 14 OECD 422 / 421 studies performed at LPT from 2016 - 2019

T4 level

on lactation day 13

(nmol/L)

 

(male pups)

 

 

Group 1

 

53.8282 ± 6.8546

Mean value from the group mean values of 14 control groups:

62.2943 ± 10.1984 nmol/L

 

Range of group mean values from

14 control groups:

42.0223 - 78.1673 nmol/L

 

Group 2

 

60.2185 ± 8.1706

 

Group 3

 

56.2724 ± 7.6752

 

Group 4

 

63.8528 ± 7.1342*

*/**:

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

#1:

Data not audited by QAU

#2:

Values are taken from table 25-1 'T4 Level of the Pups – Mean Values per Dam - Summary'.

 

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

Parameter

Sampling

Percent of nominal concentration [%] #

Concentration

Immediately after preparation on test day 15 (first day of administration)

and before administration to the last animal on test day 64.

93.3% - 99.5%

Stability

Left at room temperature after preparation for 8h or 24 h on test day 15.

94.2% - 95.8%

Homogeneity

On test day 15, at the start of administration, during administration and before administration to the last animal.

93.7% - 96.0%

#:

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

 

 

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

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and / or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 15, 50 or 150 mg/kg b.w./day.

 

General toxicity

Parental male and female animals

None of the animals died prematurely.

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

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

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

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

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

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

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

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

 

Reproductive toxicity

Parental females

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

 

 

Pups

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

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

 

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

General toxicity

 

 

NOAEL (for systemic toxicity)

 

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

 

Reproductive toxicity

 

 

 

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

 

NOAEL

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

 

 

b) adverse effects on pre- and postnatal development

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

 

NOAEL

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

 

 

- b2) adverse effects on postnatal development (pup)

 

NOAEL

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

 

 

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

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

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

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

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

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

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

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

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

Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

DRF study according to OECD 421

The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and / or development (LPT, 2020). The test item was administered orally to rats at dose levels of 15, 50 or 150 mg/kg b.w./day. Additionally, this study serves as dose range finding study for the ongoing OECD 443 (LPT Study No. 37627). The NOAEL for systemic toxicity was 50 mg/ kg bw/ day, based on clinical observations and reduced body weight. The NOAEL for reproductive toxicity (adverse effects on the reproductive parameters of the parental females/ pre- and postnatal development/ postnatal development) is above 150 mg/ kg bw/ day.None of the parental male and female animals died prematurely. During the daily cage side observations, a short post-dosing salivation was noted with an increasing incidence of affected male animals from 50 to 150 mg/kg b.w./day and from 15 to 150 mg/kg b.w./day for the females. Furthermore, a reduced motility was noted for all male and female animals after the first dosing with 150 mg/kg b.w./day on test day 15. The brief post-dosing salivation and the isolated observation of a reduced motility on test day 15, were both rated as adaptation effects are not of any toxicological relevance. A slightly reduced body weight and / or body weight gain was noted for the male and female animals at 150 mg/kg b.w./day. A transient and slightly reduced food consumption was noted for the male and female animals at 150 mg/kg b.w./day after the start of treatment.

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

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

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

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

NTP studies

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

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

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

EOGRTS according to OECD 443

The requested OECD 443 study in rats and with dose groups of 15, 50 or 150 mg/kg b.w./day started in May 2020. The in-life phase was completed in December 2020 and the first draft report of the study is expected to be available in April 2021. The dossier will then immediately be updated and submitted to ECHA without delay.


Effects on developmental toxicity

Description of key information

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

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

Link to relevant study records

Referenceopen allclose all

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Based on the results of the 28-day study in rats (Hüls AG, 1995 a), four groups of 24 mated female Sprague-Dawley rats received 1,2,3,4-tetrahydronaphthalene by daily oral administration (gavage) at 0 (sesame oil = control), 15, 45 and 135 mg/kg bw/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414 (2001) (Ehling, 2004). The dams showed no treatment-related death. Clinical signs were not observed. Mean absolute and relative food consumption was distinctly to slightly decreased in high dose animals as compared to the controls, attaining statistical significance on study days 6 - 18 (absolute 6 - 9: 33 %: 9 - 13: -12 %; 13 - 16: -10 %; 16 - 18: -12 %; relative 6 - 9: -32 %; 9 - 13: - 10 %; 13 - 16: -7 %; 16 - 18: -9 %). In mid dose females, mean absolute and relative food consumption was slightly to marginally lower (statistically significant) during study days 6 - 9 (absolute: -15 %; relative: -16 %) and days 13 - 16 (absolute and relative: -6 %) . The terminal body weight (gestation day 20) was decreased in a statistically significant way (-5 %) for high dose females as compared to controls. A significantly lower body weight gain was recorded for the whole treatment period (0 - 20: -15 %).

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

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

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

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

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

Justification for classification or non-classification

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

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