Tin sulphide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Both a reproductive screening toxicity study and a 90-day repeated dose toxicity study with Tin sulfide did not demonstrate any obvious adverse effects on the reproductive organs and function at doses of 100, 300 and 1000 mg/kg bw/day by oral gavage. Therefore Tin sulfide was considered safe and well tolerated up to the dose of 1000 mg/kg bw/day. The reproductive NOAEL was defined at 1000 mg/kg. Referring to the results of OECD 421 test, there is no evidence that the test item is causing any developmental toxicity.

 

An extended one-generation reproductive toxicity study (EOGRTS) (OECD TG 443) was available, in which the effects of Tin sulfide were evaluated at dose levels of 100, 300 or 1000 mg/kg bw/day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood. In addition, the reproductive toxicity of the F1 Generation was evaluated until weaning of the F2 Pups. For the F0 Generation, all NOAELs were above 1000 mg/kg bw/day (i.e. general toxicity and reproductive toxicity (adverse effects on reproductive parameters of parental females, adverse effects on the prenatal development of the pups, and adverse effects on the post-natal development of the pups)). For the F1 generation, all NOAELs were above 1000 mg/kg bw/day (i.e. developmental toxicity Cohort 1A and 1B and reproductive developmental toxicity Cohort 1B (adverse effects on reproductive parameters of the parental females, adverse effects on the prenatal development of the pups, and adverse effects on the post-natal development of the pups)).

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2021 (study plan) – 22 April 2022 (2nd draft )

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: Males and females: 10 weeks prior to mating
- Basis for dose level selection: The dose levels have been selected in agreement with the Sponsor based on the results of an OECD 421 study in rats dosed at 100, 300 and 1000 mg/kg b.w./day (CETA study No. 111/09/18), a 90-day toxicity study according to OECD 408 in rats dosed at 100, 300 and 1000 mg/kg b.w./day (CETA study No. 32/09/C), and an embryotoxicity study according to OECD 414 conducted in rats dosed at 100, 300 and 1000 mg/kg b.w./day (LPT Study No. 37282).
In the OECD 421 study, NOAEL for the parents was >1000 mg/kg b.w./day; NOAEL for the reproduction was 1000 mg/kg b.w./day, whereas NOEL for the toxic effect on reproduction organs of males was 300 mg/kg b.w./day, and NOEL for the toxic effect on reproduction organs of females was 1000 mg/kg b.w./day; NOAEL for the development of pups was 1000 mg/kg b.w./day. The irrelevant negative influence of the test substance treatment expressed in males of the highest dose level (limit dose) consisted of an increase in absolute weight of pituitary gland (without histopathological changes) and an effect on the microscopical structure of the testes (sporadic occurrence of degeneration and/or atrophy of germ epithelium, residual bodies in germ epithelium and vacuolation of cytoplasm of spermiogonia) without effect on the spermiogenesis. The average number of pups and accompanied weight of the litters varied among groups, but fell withing historical ranges and these differences were not considered to be toxicologically relevant.
In the 90-day repeated dose toxicity study in rats, the test item caused a slight increase in food consumption, associated with slight increases in body weight and serum glucose concentrations in females dosed at 1000 mg/kg b.w./day. These were however not considered as adverse effects. Slight dose dependent decreases in serum Na and Cl, varying within physiological range, were observed. Except for the above mentioned changes, the test item did not cause any negative effect on body weight, food consumption, ophthalmoscopy, haematology and clinical chemistry parameters and organ weights. The test item further did not cause any organ weight changes nor gross or histopathological changes in the liver, kidneys, gastrointestinal tract or other organs of surviving animals indicating a toxic effect. Under the test conditions used, 90-day administration of the test item to rats was safe and well tolerated up to the dose of 1000 mg/kg b.w./day. The NOAEL was defined at 1000 mg/kg b.w./day and the NOEL at 300 mg/kg b.w./day.
In the rat embryotoxicity study, no premature death was noted for any dose group. No test item-related teratogenic activity or test item-related toxicity on the fetal organisms was noted. The NOAEL for the dams was at 1000 mg/kg b.w. For all dams dosed with 300 or 1000 mg/kg b.w./day, dark discoloured faeces were noted. However, the dark discoloured faeces were considered to be due to the grey colour of the administered test item and therefore, of no toxicological relevance. No test item-related changes were noted in the macroscopic examination during laparotomy.
Hence, doses of 100, 300, 1000 mg/kg b.w./day are proposed for the OECD 443 study.
- Inclusion/exclusion of extension of Cohort 1B: Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 Generation.
- Termination time for F2: sacrifice between PND 22 to 24
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not included
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not included
- Route of administration: Oral by gavage
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals:
Strain/species: CD® rat (The rat is a commonly used rodent species for such studies)
Vehicle: 0.5 % aqueous hydroxypropylmethylcellulose gel
Number of animals:
Pre-exposure period: 120 female animals were evaluated pre-exposure for oestrous cyclicity to yield 96 females (i.e. 24 per group) with a regular oestrous cycle for the study.
Main study: 192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation

Species:

rat

Strain:

other: 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 Germany GmbH Sandhofer Weg 7 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation:
(P) Males and females: 69 days (at first dosing); (F1) When all selected pups had reached postnatal day 21, all the selected pups were transferred to their respective Cohort of the F1 Study and the F1 Study started with test day 1.
- Weight at study initiation: (P) Males: 350.6 g – 444.0 g ; Females: 195.5 g – 272.6 g (at first dosing; i.e. test day 15);
- Fasting period before study:
- Housing: With exception of the mating period, the male and female animals (F0 Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm at a room temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55 % ± 10 % (maximum range). Deviations from the maximum range caused for example during cleaning procedures were dealt with in SOPs. No values exceeding the maximum range were noted during the course of the study.
After weaning and with the exception of Cohort 1B during its mating period, the animals of Cohort 1A and Cohort 1B were kept in pairs of the same sex, cohort and dose group in MAKROLON cages (type III plus or type IV, as appropriate.
After mating, F0 females and Cohort 1B females were housed individually. Cohort 1B males were returned to group housing in pairs.
Pups were housed with their dam until weaning.
Rooms were alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt / Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL
Environmental enrichment: The animals received one piece of aspen softwood (certified for animal use) to gnaw on once weekly at change of the cages.
Octagon-shaped red-tinted huts (polycarbonate) and cardboard tunnels (Plexx® play tunnels) of appropriate size were placed in the cages to offer the animals a resting and hiding place. The cardboard tunnels (certified for animal use) were also suitable for gnawing.
- Diet:
A certified commercial diet (ssniff® R/M-Z V1154, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. 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 (e.g. 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, 2001]. In addition, drinking water samples taken at Provivo are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001, Anlage 1' [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range) ; No values exceeding the maximum range were noted during the course of the study.
- Humidity (%): 55 % ± 10 % (maximum range); No values exceeding the maximum range were noted during the course of the study.
- Air changes (per hr): fifteen to twenty air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle
IN-LIFE DATES: From: 24 march 2021 (start of pre-estrus) To: 09 December 2021 (sacrifice of last animal)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous hydroxypropylmethylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared daily. The test item was suspended in the vehicle to the appropriate concentrations. The test item formulations were administered at a constant administration volume of 10 mL/kg b.w. once daily. The control animals received the vehicle at the same administration volume in the same way.
The test item formulations were continuously agitated by stirring throughout the entire administration procedure to ensure homogeneity. The amount of the test item was adjusted to the animal’s current body weight daily.

VEHICLE:
- Justification for use and choice of vehicle (if other than water ): Tin sulfdie was not water soluble, therefore suspension was made in 0.5 % aqueous hydroxypropylmethylcellulose gel.
- Concentration in vehicle: 10 mg/mL, 30 mg/mL, and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg b.w.
- Lot/batch no. (if required): Batch nos. 19G17-B04-194737 Fagron GmbH&Co. KG; 21509 Glinde, Germany

Details on mating procedure:

- M/F ratio per cage: Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period
- Length of cohabitation: The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 2 weeks of unsuccessful pairing: females without a positive mating sign were separated from its male partner without further opportunity for mating.
- After successful mating each pregnant female was caged (how): After mating, F0 females and Cohort 1B females were housed individually.
- Any other deviations from standard protocol: Cohort 1B animals were paired between their post-natal days 92 and 97 to obtain the F2 Generation. The mating procedure was similar to those of the parental animals of the F0 Generation. The pairing of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item formulations were prepared daily. The test item was suspended in the vehicle to the appropriate concentrations. The test item formulations were administered at a constant administration volume of 10 mL/kg b.w. once daily. The control animals received the vehicle at the same administration volume in the same way. The test item formulations were continuously agitated by stirring throughout the entire administration procedure to ensure homogeneity. The amount of the test item was adjusted to the animal’s current body weight daily. The homogeneity and concentration of the test item formulations were monitored.
For the analysis of the test item-vehicle formulations, 2 aliquots of at least 5 mL were taken at the following times and stored at -20°C ± 10% until shipment. One set of aliquots was shipped for analysis. The second set of aliquots is stored at the Test Facility.

F0 generation:
-At start of treatment period on test day 15 (07.04.2021) (first administration day): Analysis of concentration and homogeneity (At the start, during (middle) and before administration to the last animal of each dose level group. (3 samples / dose level group; groups 2 - 4)).
-At a time when most F0 females had littered (test day 112) (13.07.2021): Analysis of concentration (During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4)).
-At termination of the F0 treatment period (when the majority of animals was dosed; test day 132) (02.08.2021): Analysis of concentration (During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4)).

F1 generation:
-After all selected pups were transferred to the F1 cohorts (test day 2 of the F1 Study) (31.07.2021): Analysis of concentration and homogeneity (At the start, during (middle) and before administration to the last animal of each dose level group. (3 samples / dose level group; groups 2 - 4)).
-At termination of the Cohort 1 A treatment period (when the majority of animals was dosed; test day 75 of the F1 Study) (12.10.2021): Analysis of concentration (During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4)).
-At termination of the Cohort 1 B treatment period (when the majority of animals was dosed; test day 120 of the F1 Study) (26.11.2021): Analysis of concentration: (During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4)).

The samples were labelled with study number, species, type of sample, aliquot number, concentration, generation and cohort number, group number, sampling time, and date.
The samples were shipped on dry ice to Weyl Chem, Germany for analysis on 14 December 2021. The analytical method was validated for the OECD 414 study (LPT Study No. 37282) by Test Site 1 under Phase no. VP 033/2019.

Duration of treatment / exposure:

F0 generation:
Males were dosed 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 were dosed 10 weeks prior to mating, during the mating, gestation and lactation periods and until termination of weaning of their litters (up to and including the day before sacrifice).

F1 animals:
F1 Pups: Until weaning the pups would have been indirectly exposed to the test item through the breast milk. After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage in the same way as for the parental generation.
Cohort 1A: The male and female animals were dosed for 10 weeks up to and including the day before sacrifice.
Cohort 1B: The male and female animals were dosed until sacrifice of their F2 Pups up to and including the day before sacrifice.

F2 pups:
Until sacrifice between PND 22 to 24 the F2 pups were not directly exposed. The only exposure would have been indirectly through the breast milk

Frequency of treatment:

Once daily

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation: A few days before the pups from those dams with the earliest litter date had reached postnatal day 21 (PND 21) pups from all available litters were randomly selected for the F1 Generation using Provantis. When all selected pups had reached postnatal day 21, all the selected pups were transferred to their respective Cohort of the F1 Study and the F1 Study started with test day 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24 animals/sex/dose (F0 generation)
20 animals/sex/dose (F1 generation)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels have been selected in agreement with the Sponsor based on the results of an OECD 421 study in rats dosed at 100, 300 and 1000 mg/kg b.w./day (CETA study No. 111/09/18), a 90-day toxicity study according to OECD 408 in rats dosed at 100, 300 and 1000 mg/kg b.w./day (CETA study No. 32/09/C), and an embryotoxicity study according to OECD 414 conducted in rats dosed at 100, 300 and 1000 mg/kg b.w./day (LPT Study No. 37282).
In the OECD 421 study, NOAEL for the parents was >1000 mg/kg b.w./day; NOAEL for the reproduction was 1000 mg/kg b.w./day, whereas NOEL for the toxic effect on reproduction organs of males was 300 mg/kg b.w./day, and NOEL for the toxic effect on reproduction organs of females was 1000 mg/kg b.w./day; NOAEL for the development of pups was 1000 mg/kg b.w./day. The irrelevant negative influence of the test substance treatment expressed in males of the highest dose level (limit dose) consisted of an increase in absolute weight of pituitary gland (without histopathological changes) and an effect on the microscopical structure of the testes (sporadic occurrence of degeneration and/or atrophy of germ epithelium, residual bodies in germ epithelium and vacuolation of cytoplasm of spermiogonia) without effect on the spermiogenesis. The average number of pups and accompanied weight of the litters varied among groups, but fell withing historical ranges and these differences were not considered to be toxicologically relevant.
In the 90-day repeated dose toxicity study in rats, the test item caused a slight increase in food consumption, associated with slight increases in body weight and serum glucose concentrations in females dosed at 1000 mg/kg b.w./day. These were however not considered as adverse effects. Slight dose dependent decreases in serum Na and Cl, varying within physiological range, were observed. Except for the above mentioned changes, the test item did not cause any negative effect on body weight, food consumption, ophthalmoscopy, haematology and clinical chemistry parameters and organ weights. The test item further did not cause any organ weight changes nor gross or histopathological changes in the liver, kidneys, gastrointestinal tract or other organs of surviving animals indicating a toxic effect. Under the test conditions used, 90-day administration of the test item to rats was safe and well tolerated up to the dose of 1000 mg/kg b.w./day. The NOAEL was defined at 1000 mg/kg b.w./day and the NOEL at 300 mg/kg b.w./day.
In the rat embryotoxicity study, no premature death was noted for any dose group. No test item-related teratogenic activity or test item-related toxicity on the fetal organisms was noted. The NOAEL for the dams was at 1000 mg/kg b.w. For all dams dosed with 300 or 1000 mg/kg b.w./day, dark discoloured faeces were noted. However, the dark discoloured faeces were considered to be due to the grey colour of the administered test item and therefore, of no toxicological relevance. No test item-related changes were noted in the macroscopic examination during laparotomy.
Hence, doses of 100, 300, 1000 mg/kg b.w./day are proposed for the OECD 443 study.
- Rationale for animal assignment (if not random):
F0 generation: The animals were allocated to the test groups on the last day of the pre-dosing period on test day 14 by using a Provantis®-generated randomization based on the body weights of the animals. Only those female animals were used for randomization that passed the oestrous cycle test
F1 generation: The animals were allocated to the cohorts of their group by using a Provantis®-generated randomization.
- Fasting period before blood sampling for clinical biochemistry: yes overnight

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals:
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.
In case signs of toxicity occurred the frequency of observations was increased.
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded.
In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:00 p.m.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed 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.
Premortal symptoms were recorded in detail. A post mortem examination was performed as soon as possible after exitus.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A more detailed examination of all F0 and F1 Cohort 1B animals was conducted on a weekly basis. F0 animals were examined once before the first test item treatment on test day 14 to allow for within-subject comparisons. Thereafter, the examination was performed weekly until termination. The F1 animals of Cohort 1B were examined weekly after weaning until termination.
Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed daily and the body weight was recorded.

FOOD CONSUMPTION (no feeding study): Food residue (food removal) was weighed and recorded as follows:
-pre-mating period: weekly
-mating period: none
-post-mating period: weekly values for the males
-gestation period: GD 0, 7, 14, 21
-lactation period: PND 1, 7, 14, 21

WATER CONSUMPTION (no drinking water study): Yes
- Time schedule for examinations: Drinking water consumption was monitored daily by visual appraisal throughout the study
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

OTHER:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
EDTA anticoagulant (whole blood): for haematological examinations
Citrate anticoagulant (plasma): for coagulation tests
Serum: for clinical chemistry
The following sampling times and animals were employed:
At necropsy
F0 Generation: 10 males and 10 females randomly selected from each group
F1 Generation - Cohort 1A: 10 males and 10 females randomly selected from each group
-Haematology:
The following parameters were determined:
Differential blood count*: - relative (%)
Differential blood count*: - absolute (10³/µL)
Haemoglobin content (HGB) (mmol/L)
Erythrocytes (RBC) (10^6/µL)
Leucocytes (WBC) (10³/µL)
Reticulocytes (Reti) (%)
Haematocrit value (HCT) (%)
Platelets (PLT) (10³/µL)
Mean corpuscular volume (MCV) (fL)
Mean corpuscular haemoglobin (MCH) (fmol)
Mean corpuscular haemoglobin concentration (MCHC) (mmol/L)
* Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood count.
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings
-coagulation parameters:
The following parameters were determined:
Prothrombin time (PT) (sec)
Activated partial thromboplastin time (aPTT) (sec)
-biochemical parameters:
The following parameters were determined:
Albumin (g/L)
Bile acids (µmol/L)
Bilirubin (total) (µmol/L)
Cholesterol (total) (mmol/L)
Creatinine (µmol/L)
Glucose (mmol/L)
Protein (total) (g/L)
Blood urea nitrogen (BUN) (mmol/L)
Calcium (mmol/L)
Chloride (mmol/L)
Potassium (mmol/L)
Sodium (mmol/L)
Alanine aminotransferase (ALAT) (U/L)
Alkaline phosphatase (aP) (U/L)
Aspartate aminotransferase (ASAT) (U/L)
Lactate dehydrogenase (LDH) (U/L)
Globulin (g/L)
Albumin/globulin ratio
Sodium/potassium ratio
BUN/creatinine ratio

-Determination of thyroid hormone (T4 and TSH):
Blood samples were taken from animals fasted overnight always at the same time of day, as scheduled below:
F0 generation:
10 males and 10 females per group (animals also selected for laboratory examinations). Test days 129-134 (at sacrifice). Feeding status: fasted. Analyte: T4 (Sample volume 2x100 µL) and TSH (Sample volume 2x100 µL);
F1 generation:
10 males and 10 females per Cohort 1A group (animals also selected for laboratory examinations). PND 86 to 97 (at sacrifice). Feeding status: fasted. Analyte: T4 (Sample volume 2x100 µL) and TSH (Sample volume 2x100 µL);
The animals scheduled for blood sampling were anaesthetised and euthanized as follows:
adults: sampling from the retrobulbar venous plexus under isoflurane anaesthesia; euthanized by carbon dioxide (CO2) inhalation.

-Urinalysis
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
At the end of dosing period (prior to blood withdrawal)
F0 Generation:
10 males and 10 females randomly selected from each group (same animals as selected for laboratory examinations)
F1 Generation - Cohort 1A:
10 males and 10 females randomly selected from each group (same animals as selected for laboratory examinations)
The following parameters were determined:
Volume (mL)
pH (non-dimensional)
Specific gravity (g/mL)
The following examinations were also performed using Combur 9® Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine:
Proteins (g/L)
Glucose (mmol/L)
Bilirubin (-)
Urobilinogen (µmol/L)
Ketones (-)
Haemoglobin (Hb) (approximate values) (ery/µL)
Nitrite (-)
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
E Epithelial cells
L Leucocytes
R Erythrocytes
B Organisms
C Further constituents (i.e. sperm, casts)
A Crystalluria
The frequency of the above parameters were recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.

Oestrous cyclicity (parental animals):

The oestrous cycle stages were determined at the following time points:
F0 main study animals:
-14 days pre-exposure period to select 96 main study animals with regular oestrous cycles (4 to 5 days per cycle).
-During 10 weeks of pre-mating until evidence of mating.
F1 animals, cohort 1A:
- Start after onset of vaginal patency until first appearance of cornified cells.
- Two weeks starting around PND 75 (F1 study test days 53 to 66)
F1 animals, cohort 1B:
- Starting from the time of pairing until evidence of mating was detected
F0 and F1 animals – Cohort 1A,B:
- On the day of sacrifice, shortly before necropsy.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Following organs were weighed before fixation: F0 generation; F1 generation Cohort 1A; F1 generation Cohort 1B:
Epididymis (2)
Testis (2)
Prostrate (dorsolateral and ventral parts combined)
Seminal vesicles with coagulating gland

All F0 and Cohort 1A males:
Spermiogram: Sperm analysis (motility, morphology and counting) was performed from the cauda of the right epididymis for all male animals per group.
Time of sperm analysis: at necropsy: F0 (all surviving males from each group); F1 generation Cohort 1A (all males from each group).
Sperm motility and morphology were examined immediately after sacrifice. After weighing the cauda was incised and the emergent sperm sample was used for the examination of motility and morphology of the sperm cells. The percentage of progressively motile sperm was determined microscopically. For the evaluation of sperm morphology 200 sperm cells were examined from the sperm sample of the cauda epididymis. The sperm cells were classified as either normal (both head and midpiece / tail appeared normal) or abnormal. Examples of morphologic sperm abnormalities would include fusion, isolated heads, and misshapen heads and / or tails. Misshapen or large sperm heads may indicate defects in spermiation.
Sperm count: After incision and receipt of the sperm sample for the examination of motility and morphology the cauda of the epididymis was prepared, weighed and frozen at minus 80°C. The frozen cauda was homogenized and the obtained suspension was used for sperm counting.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.
After counting on PND 4 (lactation day 4), the litters were adjusted to 10 pups per litter (5 pups/sex/litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.
Selective elimination of pups, e.g. based upon body weight was not appropriate. In case of unequal gender distribution, a partial litter size adjustment was performed (e.g. 6 male and 4 female pups).

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (i.e. body weight less than 70% of mean litter weight) and the presence of gross abnormalities.
Abnormal behaviour or changes in the external appearance of the pups noted during the daily cage side inspections were recorded.
The following examinations/observations were done for the offspring:
-Live pups were counted, sexed and weighed on post-natal days (PND) 1, 4, 7, 14 and 21.
-On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalised to the cube root of body weight.
-Nipples/areolae were counted in all male pups on PND 13.
-All animals of Cohorts 1A and 1B were evaluated daily for balano-preputial separation or vaginal opening to detect, if sexual maturation occurred early. The evaluation started before the expected achievement of these endpoints, i.e. completion of balano-preputial separation or vaginal opening. Any abnormalities of the genitals, such as persistent vaginal thread, hypospadias or cleft penis, would have been reported. However, no abnormalities of the genitals were noted.
Sexual maturity was compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).
- Blood samples were taken from animals fasted overnight always at the same time of day, as scheduled below.
F1 Pups: 2 surplus pups per litter (If the individual volume obtained from the pups was insufficient for analysis, the samples may be pooled by litter). Sampling time: PND4. Feeding status: non-fasted. Analyte: T4 only (sample volume: 1x75 µL).
F1 Pups: 2 surplus pups per litter (If the individual volume obtained from the pups was insufficient for analysis, the samples may be pooled by litter). Sampling time: PND22. Feeding status: non-fasted. Analyte: T4 (sample volume: 2 x 50 µL) and TSH (sample volume: 2 x 70 µL).
F2 Pups: 2 surplus pups per litter (If the individual volume obtained from the pups was insufficient for analysis, the samples may be pooled by litter). Sampling time: PND4. Feeding status: non-fasted. Analyted: T4 (not to be analysed; sample volume: 1 x 75 µL).
F2 Pups: 1 male and 1 female pup from 10 different dams (If the individual volume obtained from the pups was insufficient for analysis, the samples may be pooled by litter). Dams selected in the sequence of randomised dam necropsy. Sampling time: PND22. Feeding status: non-fasted. Analyte: T4 (TSH not to be analysed; sample volume: 2x50 µL, 2x70 µL).
The animals scheduled for blood sampling were anaesthetised and euthanized as follows:
PND 4 pups: euthanized by decapitation followed by blood collection.
PND 22 pups: sampling from the retrobulbar venous plexus under isoflurane anaesthesia; euthanized by carbon dioxide (CO2) inhalation.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
-External inspection for gross abnormalities:
Dead pups and culled F1 Pups on PND 4 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not applicable
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not applicable

Postmortem examinations (parental animals):

SACRIFICE
F0:
- Male animals: All surviving animals (test days: 133-136)
- Maternal animals: All surviving animals (test days: 129-142 for females with litter; test days 111 or 125 for females without litter)
F1:
- Cohort 1A: test days 86-97
- Cohort 1B: test days 140-147 (males) & 121-153 (females) after weaning of F2 pups.

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

-F0 generation:
During necropsy the number of implantation sites in the uteri was recorded in the female animals and used to evaluate reproductive performance.
Apparently non-pregnant uteri of the F0 animals were placed in a 10 % aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.

-F1 Generation Cohort 1B:
During necropsy the number of implantation sites in the uteri was recorded in the female animals and used to evaluate reproductive performance.
Apparently non-pregnant uteri of the Cohort 1B animals were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.

HISTOPATHOLOGY / ORGAN WEIGHTS
Bone marrow examination:
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of male and female F0 and F1 Generation Cohort 1 A animals (10 males and 10 females randomly selected from group 1 and group 4; same animals as selected for laboratory examinations) and stained according to PAPPENHEIM. The myeloid / erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.
The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.

Phenotypic analysis of spleen cells:
The same male and female animals from that were selected for the laboratory examinations were used for the phenotypic analysis of the spleen cells. The spleens of the male and female F1 Cohort 1A animals were split in two halves. The portion of the spleen not preserved for histopathology was minced using a mechanic dissociator to prepare single cell suspensions.
The following parameters were determined in the samples by using the instruments given below:
CD4+ T-Lymphocytes
CD8+ T-Lymphocytes
Pan-T-lymphocytes (CD3+)
B-lymphocytes (CD45RA+)
Natural killer cells (CD161+)

Organ weights:
-F0 generation:
The following organs of the male and female F0 animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Ovary (2)
Oviducts (2)
Testicle (2)
Thyroid (1; left) (including parathyroid, post-fixation)
Thymus
Prostrate (dorsolateral and ventral parts combined)
Seminal vesicles with coagulating glands
Pituitary
Uterus including cervix
Spleen
The following organs or parts thereof obtained from the male and female animals of the F0 Generation were preserved in an appropriate fixative:
Fixative: Davidson’s solution: eye with optic nerve (2)
Fixative: Modified Davidson’s solution: Epididymis (1) (The right epididymis was not preserved but used for the spermiogram), Testis (1) (The right testis was frozen and stored for a possible spermatid count)
Fixative: 7% buffered formalin: Adrenal gland (2); Bone; Bone marrow (os femoris); Brain (cerebrum, cerebellum, pons); Gross lesions observed; Heart (3 levels: right and left ventricle, septum); Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method); Intestine large (colon, rectum); Kidney and ureter (2); Liver; Lungs (with mainstem bronchi and bronchioles); Mammary gland; Muscle (skeletal); Nerve (sciatic); Oesophagus; Ovary (2); Oviducts; Pituitary; Prostrate; Seminal vesicles with coagulating glands; Spinal cord (3 sections); Spleen; Stomach; Thyroid (2, incl. parathyroids); Thymus; Trachea (incl. larynx); Urinary bladder; Uterus (incl. cervix); Vagina; Vas deferens
Any other organs displaying macroscopic changes were also preserved.
Sperm viability and morphology were evaluated for all male F0 animals
In addition, bone marrow smears were prepared for selected F0 animals

-F1 generation cohort 1A:
The following organs of all adult male and female F1 Cohort 1A animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Ovary (2)
Oviducts (2)
Prostrate (dorsolateral and ventral parts combined)
Pituitary
Seminal vesicles with coagulating glands
Spleen
Testis (2)
Thymus
Thyroid (1; left) (including parathyroid, post-fixation)
Uterus including cervix
Lymph node (1, cervical)
Lymph node (1, mesenteric)
The following organs or parts thereof obtained from the male and female animals of F1 Generation Cohort 1 A were preserved in an appropriate fixative:
Fixative: Davidson’s solution: eye with optic nerve (2)
Fixative: Modified Davidson’s solution: Epididymis (1) (The right epididymis was not preserved but used for the spermiogram), Testis (1) (The right testis was frozen and stored for a possible spermatid count)
Fixative: 7% buffered formalin: Adrenal gland (2); Bone; Bone marrow (os femoris); Brain (cerebrum, cerebellum, pons); Gross lesions observed; Heart (3 levels: right and left ventricle, septum); Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method); intestine, large (colon, rectum); Kidney and ureter (2); Liver; Lungs (with mainstem bronchi and bronchioles); lymph node (1, cervical); lymph node (1; mesenteric) (Lymph nodes (mesenteric and cervical): Lymph nodes were preserved for all Cohort 1A animals, however, histopathology was only carried out for 10 animals/sex/group (1 animal per litter, all litters represented by at least 1 pup; randomly selected)) Mammary gland; Muscle (skeletal); Nerve (sciatic); Oesophagus; Ovary (2) (Ovary: One ovary of the F1 Cohort 1A females of groups 1 and 4 (20 females per group) was processed for detailed histopathological examination as follows: - Ten (10) 2-4-μm step sections with 50 μm steps in between; - Each slide was labelled with the slide number to follow the sequence); Oviducts; Pituitary; Prostrate; Seminal vesicles with coagulating glands; Spinal cord (3 sections); Spleen (For 10 animals/sex/group of Cohort 1A, randomly selected (same as selected for laboratory examination): One half of the spleen was preserved for histopathogical evaluation, the second half was used for splenic lymphocyte subpopulation analysis); Stomach; Thyroid (2, incl. parathyroids); Thymus; Trachea (incl. larynx); Urinary bladder; Uterus (incl. cervix); Vagina; Vas deferens
Any other organs displaying macroscopic changes were also preserved.
Sperm viability and morphology were evaluated for all male animals of F1 Cohort 1A.
In addition, bone marrow smears were prepared for selected animals of F1 Cohort 1A.

-F1 generation Cohort 1B:
Determination of organ weight and preservation was restricted to the organs listed below. Paired organs were weighed individually and identified as left or right. No target organs were identified.
Endocrine system:
Organ – weight – fixative – block
Adrenal gland (2) – yes – 7% formalin – no (The adrenals, thyroids, oviducts and vas deferens are not processed to block stage as this is not required by the current OECD TG 443)
Pituitary – yes – 7% formalin – yes
Thyroid (2) (including parathyroid) – 1, post-fixation (In the same manner as for Cohort 1 A, both thyroids with parathyroids were preserved, but only one thyroid with parathyroid (left) was weighed after fixation) – 7% formalin – no (The adrenals, thyroids, oviducts and vas deferens are not processed to block stage as this is not required by the current OECD TG 443)
Reproductive system:
Organ – weight – fixative – block
Epididymis (2) – yes – Modified Davidson’s – yes
Ovary (2) – yes – 7% formalin – yes
Oviducts – no – 7 % formalin – no (The adrenals, thyroids, oviducts and vas deferens are not processed to block stage as this is not required by the current OECD TG 443)
Prostrate – yes – 7% formalin – yes
Seminal vesicles with coagulating glands – yes – 7% formalin – yes
Testicle (2) – yes – Modified Davidson’s – yes
Uterus (including and cervix) – yes – 7% formalin – yes
Vagina – no – 7% formalin – yes
Vas deferens – no – 7% formalin – no (The adrenals, thyroids, oviducts and vas deferens are not processed to block stage as this is not required by the current OECD TG 443)

Histopathology F0 Generation and F1 Generation Cohort 1A:
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F0 Generation (parents): All males and females of group 1 and 4
- F1 Generation (Cohort 1A): All males and females of group 1 and 4 of Cohort 1A
- All deceased or prematurely sacrificed animals
A full histopathology was also performed for all deceased or prematurely sacrificed animals, from the F0 Generation and from Cohort 1A.
The organs listed above were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section. The parathyroids would also have been examined microscopically if they would have been noted as grossly enlarged. However, no grossly enlarged parathyroids were noted.
In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the control and high dosed animals of groups 1 and 4 following H&E and PS staining:
Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea was performed on one ovary of the F1 Cohort 1A females of group 1 and group 4. Therefore, the ovary was processed as follows:
- Ten (10) 2 - 4 µm step sections with 50 µm steps in between;
- Each slide was labelled with the slide number to follow the sequence.
In case of test item-related changes in the high dose level (group 4), the Sponsor would have given sufficient notice before the corresponding organs of the animals of the F0 Generation and F1 Generation Cohort 1A of the intermediate and low dose level groups (group 2 and 3) are sectioned and examined histopathologically.

Histopathology F1 Generation Cohort 1B:
Stained sections of the embedded organs of Cohort 1B group 1 and 4 male and female animals listed below were prepared in the same manner as for Cohort 1A.
Organ – HE stain – PAS stain
Pituitary – Yes – No
Epididymis, right – Yes – No
Epididymis, left – Yes – Yes
Ovary (left) – Yes – No
Ovary (right) – Yes – No
Prostrate – Yes – No
Seminal vesicles with coagulating glands – Yes – No
Testicle, left – Yes – Yes
Uterus (including cervix) – Yes – No
Vagina – Yes – No

Additional examination of the reproductive organs of the Cohort 1B females:
Due to statistically significant differences in the distribution of the oestrous cycle stages between the control and the high dosed F0 Generation females, the oestrous cycle stages of the F1 Cohort 1B groups 1 and 4 were additionally examined.
For this additional examination the prepared slides below were sent to Test Site 2 for the determination of the oestrous cycle stages:
Ovary (left), Ovary right (ten step sections)*, Uterus (including cervix), Vagina
* The organs listed above had been processed to block stage before they were requested for an additional examination. Thereby the right ovary was processed in ‘ten step sections’ for a possible quantification of the follicles and corpora lutea, as it was performed for the Cohort 1A females. Though quantification of the follicles and the corpora lutea was not necessary for the evaluation of the oestrous cycle stages, the ‘ten step sections’ were used for the evaluation of the oestrous cycle stages, as no other slides were available.

Histopathology evaluation:
Histotechnique was performed by Provivo.
The slides were labelled with study number, test species, animal number and block number and were dispatched to Test Site 2 for histopathological evaluation on the following dates:
16 September 2021: Group 1 and group 4: males and females of the F0 Generation.
26 November 2021: Group 1 and group 4: males and females of Cohort 1A.
03 February 2022: Group 1 and group 4: reproductive organs of Cohort 1B females.
The transport of slides to the histopathology Test Site (TS) was arranged by Provivo, whereas the return transport to the Test Facility will be arranged by the TS.
The study phase was recorded under the TS reference number 580.
All microscopic findings were recorded, reported and archived with the Provantis System of Test Site 2.

Postmortem examinations (offspring):

SACRIFICE
- Dead pups and culled F1 Pups on PND 4 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed between PND22 and 24. - These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of: External and internal inspection for gross abnormalities:
F1 Pups that were not selected for the F1 Cohorts were sacrificed between PND 22 and PND 24. They were examined macroscopically for any abnormalities or pathological changes.
The macroscopic examination included an external examination that was combined with a dissection for a gross internal inspection.
A more detailed examination of the inner organs that also included the preservation and weighing of different organs was performed for selected pups and is defined as ‘internal inspection’.
For this detailed examination, including the preservation and the weighing of different organs, 10 F1 and 10 F2 pups per sex and group should be used if possible (one male and one female pup per litter from altogether 10 litters per group).


HISTOPATHOLOGY / ORGAN WEIGTHS
Organs of selected F1 and F2 surplus pups (PND 22) to be weighed and / or preserved:
Organ – weight – fixative:
-brain – yes – 7% formalin
-gross abnormalities – no – 7% formalin
-mammary gland – no – 7% formalin
-spleen – yes – 7% formalin
-thymus – yes – 7% formalin

Histopathological examination of the preserved organs will be conducted only in agreement with the Study Monitor and Sponsor, if needed. There was no need.

Statistics:

see under “any information on materials and methods incl. tables”

Reproductive indices:

The reproductive parameters and reproductive indices listed below were determined to evaluate the reproductive performance for the F0 Generation and Cohort 1B.
Reproductive parameters
- stages of the oestrous cycle
- number of pregnant females
- pre-coital time
- gestation length calculated from day 0 of gestation
Implantation sites
- number per dam
- distribution in the uterine horns (implantation sites left and right)
- absolute number per group
- mean per group
Number of total pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4, 7, 14 and 21
Number of male pups and number of female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4, 7, 14 and 21
Number of stillbirths
- absolute
- per group
- per dam
Number of pups with malformations (see Appendix 4)
- absolute
- per dam

The following indices were calculated for each group:
-Female Fertility Index [%] = (Number of pregnant females with verified copulation)/(Number of females with a verified copulation) 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

Offspring viability indices:

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 21)/(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.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related observations were noted in the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). No further relevant observations in addition to those made during the daily cage side observations were noted for the surviving and the prematurely deceased male and female animals of the control group and the treatment groups during the once weekly performed detailed clinical observation.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related premature death or sacrifice were noted for the male and female animals of the F0 Generation. Four male animals were prematurely sacrificed. However, the reasons for sacrifice were not considered to be test item-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences between the control group and the treatment groups were noted for body weight and body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences between the control group and the treatment groups were noted for food consumption.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No differences between the control group and the treatment groups were noted for water consumption (visually).

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the haematological parameters revealed no test item-related differences between the control group and the treatment groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the biochemical parameters revealed no test item-related differences between the control group and the treatment groups.

Endocrine findings:

no effects observed

Description (incidence and severity):

The examination of the T4 and TSH levels revealed no test item-related differences between the control group and the treatment groups.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the urinary parameters revealed no test item-related differences between the control group and the treatment groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related changes in behaviour were noted for the male and female animals of the F0 Generation

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related findings were noted during the histopathological examination.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No test item-related findings were noted during the histopathological examination.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) in the distribution of the stages of the oestrous cycle .

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The examination of the sperm parameters revealed no test item-related differences between the control group and the treatment groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the reproductive performance of the parental animals (number and length of oestrous cycles, fertility and gestation index, pre-coital time and gestation length).

-mortality:
Males:
No test item-related premature death or premature sacrifice was noted in the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Four male animals were prematurely sacrificed due to animal welfare reasons. However, the reasons for the premature sacrifice of the animals were considered to be spontaneous or due to a misgavage.
Females:
No test item-related death was noted in the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). Three female animals were prematurely sacrificed due to animal welfare reasons. However, the reasons for the premature sacrifice of the animals were not considered to be test item-related.
-clinical signs – daily cage side observations:
Male (surviving males):
No test item-related observations were noted in the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). All observations were considered to be spontaneous as mostly only 1 or 2 animals per group were affected and / or no dose-response relationship was noted.
Female (surviving females):
No test item-related observations were noted in the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). All observations (see discussion below) were considered to be spontaneous as mostly only 1 or 2 animals per group were affected and / or no dose-response relationship was noted.
-detailed clinical observations:
Males and females
No further relevant observations in addition to those made during the daily cage side observations were noted for the surviving and the prematurely deceased male and female animals of the control group and the treatment groups during the once weekly performed detailed clinical observation.
-Body weight and body weight gain
Males: Pre-mating-, mating- and post-mating period
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
However, a marginally reduced body weight was noted at the intermediate dose level from test day 85 (2.3 % below the control, statistically not significant) until test day 132 (4.6 % below the control, statistically not significant). The maximum difference was noted on test day 113 (6.5 % below the control, p < 0.05). However, as the difference was only marginal and no dose-response relationship was noted, the marginally reduced body weight that was noted at the intermediate dose level was considered to be spontaneous.
Body weight gain
Corresponding to the marginally reduced body weight at the intermediate dose level, a marginally reduced body weight gain was noted for the male animals of the intermediate dose group (300 mg Tin(II)-sulfide/kg b.w./day) from test day 15 until the end of the study on test day 132 (47.3 % in comparison to 54.4 % in the control group). As no dose-response relationship was noted, this small difference was considered to be spontaneous.
Females: Pre-mating-, gestation- and lactation period
No test item-related changes in body weight and body weight gain were noted for the female rats between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the pre-mating, the gestation and the lactation period.
Body weight gain:
At the high dose level a marginally reduced body weight gain was noted during the gestation period (45.0 % in comparison to 50.2 % in the control group). This was due to a marginally increased body weight at the high dose level on gestation day 0 (2.2 % above the control, statistically not significant). Hence, the marginally reduced body weight gain that was noted for the high dosed females during the gestation period was considered to be spontaneous.
-Food and water consumption:
Food consumption:
Males:
No test item related differences in food consumption were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). No food intake of male animals was recorded during the mating period as both sexes were housed together.
Females: Pre-mating, gestation and lactation period
No test item-related differences in food consumption were noted between the control group and in the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the pre-mating / mating, the gestation and the lactation period.
No food intake of female animals was recorded during the mating period as both sexes were housed together.
Water consumption:
Water consumption was performed by visual appraisal during the daily cage-side observations. No decreased water consumption was noted for any of the animals.
An increased water consumption was noted for 5 males of the low dose group, 2 males of the high dose group and one female of the intermediate dose group on a few to several test days.
This was considered to be spontaneous, as no dose response relationship was noted and no increased water consumption was noted for the animals of the F1 Generation.
-Haematology:
Males
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). Reduced percentages of reticulocytes were noted at the low, the intermediate and the high dose level (23.2 %, 17.9 % or 11.6 % below the control, statistically significant at the low dose level (p ≤ 0.01). The percentage of reticulocytes from the individual males of all treatment groups was within the Provivo background range. As all individual values from the males of the treatment groups were within the background range and no statistically significant changes were noted for the males of Cohort 1A, the observed reduced percentages of reticulocytes that were noted for the F0 males of the treatment groups were considered to be spontaneous.
Females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
The following statistically significant changes that were noted for the haematological parameters of the F0 females were considered to be spontaneous.
Number of white blood cells and lymphocytes:
Statistically significantly decreased numbers of white blood cells and lymphocytes (absolute numbers) were noted at the low dose level (31.9 % or 28.6 % below the control, p ≤ 0.01). Reduced numbers of white blood cells and lymphocytes were also noted at the intermediate and the high dose level, but the reduction was less pronounced than at the low dose level and not statistically significant. A comparison with the Provivo background data revealed that with the exception of one or 2 individual values per group, all individual values were within the background range.
Number of large unstained cells (LUCs):
A statistically significantly reduced number of large unstained cells was noted at the low dose level (34.1 % below the control, p ≤ 0.05). The number of LUCs that was noted at the high dose level was less reduced than at the low dose level and the reduction was not statistically significant (19.8 % below the control, statistically not significant). No reduction in the number of LUCs in comparison to the control group was noted at the intermediate dose level (2.2 % above the control group, statistically not significant).
Nearly all individual values were within the Provivo background range, only at the intermediate dose level the number of large unstained cells of one female was marginally above the background range.
Number of basophilic granulocytes:
Statistically significantly reduced numbers of basophilic granulocytes were noted at the low and the intermediate dose level (42.9 % below the control at the low and the intermediate dose level, p ≤ 0.05). The reduction that was noted at the high dose level was in the same range, but not statistically significant (40.0 % below the control, statistically not significant). The individual values from the control group and all dose groups were within the Provivo background range.
As all values were within the background range and no reduced numbers of basophilic granulocytes in comparison to the control were noted for the Cohort 1A females, the reduced number of basophilic granulocytes that was noted for the F0 females in all dose groups in comparison to the control group was considered to be spontaneous.
Activated partial thromboplastin time (aPTT):
A slightly increased aPTT time was noted at the high dose level (8.6 % above the control, p ≤ 0.01). A slightly increased aPTT time was also noted for the high dosed Cohort 1A females (7.2 % above the control, statistically not significant). However, all individual values of the F0 Generation females of the high dose group were within the Provivo background range.
As all values at the high dose level were within the background range, the observed statistically significantly increased aPTT time that was noted for the F0 high dosed females was considered to be spontaneous.
-Clinical biochemistry:
Males
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the male animals.
Statistically significant changes which were considered to be spontaneous are discussed below.
Urea in blood and urea in blood / creatinine ratio:
A statistically significantly increased urea concentration in the blood was noted at the intermediate dose level (22.6 % above the control, p ≤ 0.05). An increased concentration of urea in the blood was also noted at the high dose level. However, the increase at the high dose level was less pronounced than at the intermediate dose level and not statistically significant (16.8 % above the control). A comparison with the Provivo background data revealed that in the control group only one individual value was above the background range, whereas at the low, the intermediate and the high dose level 3, 6 or 5 individual values were above the background range.
As there were no changes in the creatinine concentration, the urea / creatinine ratio corresponded to the course of the urea concentration. In detail, a statistically significantly increased urea / creatinine ratio was noted at the intermediate dose level (26.6 % above the control, p ≤ 0.01). At the high dose level the increase in the urea / creatinine ratio was less pronounced and not statistically significant (17.2 % above the control). In the control group and the low dose group only one individual value each was above the background range, whereas this was the case for 5 individual values at the intermediate and for 4 individual values at the high dose level.
However, for the Cohort 1A males a slightly increased urea in blood concentration was only noted at the high dose level (10.2 % above the control, statistically not significant). At the intermediate dose level the urea in blood concentration was in the range of the control group for the Cohort 1A males. A similar course was noted for the urea / creatinine concentration of the Cohort 1A animals. These slight and statistically not significant changes that were noted for the Cohort 1A males can be considered as spontaneous.
Hence, as a pronounced and statistically significantly increased urea in blood concentration was only noted for the F0 Generation males and not for the Cohort 1A males, the increase in the urea in blood concentration that was noted for the F0 Generation males was considered to be spontaneous. This was also true for the resulting increase of the urea / creatinine ratio.
Females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the female animals.
-Urinalysis
Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
A decreased relative urine volume was noted for the male animals of all treatment groups, statistically significant at the intermediate and the high dose level (35.9 % or 39.5 % below the control, p ≤ 0.05). In detail, the relative urine volume of 2 males of the low (nos. 57, 70), 3 males of the intermediate (nos. 103, 105, 119) and 2 males of the high dose group (no. 145, 157) was below the Provivo background range. With the exception of male no. 70, the reduced urine volume below the background range could be explained by a reduced duration of urine sampling for a few animals (urine collection erroneously started in the morning of scheduled sacrifice and not in the evening before scheduled sacrifice). Hence, as the decreased relative urine volume that was noted for the male animals in all treatment groups, could be explained by the reduced duration of urine sampling from a few male animals of the treatment groups, it was not considered to be test item-related. Furthermore, no statistically significantly decreased relative urine volume was noted for the Cohort 1A male animals nor it was observed in the F0 and Cohort 1A females.
-Thyroid hormone levels
Males and females
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-Examination of the sperm number, viability and morphology
Sperm number:
No test item-related difference was noted between the rats of the control group and the rats treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day for the number of sperm cells per gram cauda epididymis.
Sperm motility:
No test item-related differences were noted between the rats of the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the percentage of motile sperm cells in the cauda epididymis. Motile sperm cells were noted for all examined male animals.
Sperm morphology:
The examination of the sperm cells from the cauda epididymis revealed no malformed sperm cells in the control group and in the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Males that did not inseminate their female partner:
No verified copulation (no sperm detection in the vaginal smear) during the mating period of 14 days was noted for one pair of the low dose group (male no. 58 and female no. 81) and one pair of the intermediate dose group (male no. 115 and female no. 140).
Male no. 58 revealed a markedly reduced number of sperm cells. Furthermore, no motility was noted for the few sperm cells found. Hence, the failed mating could be assigned to the male animal.
Male no. 115 showed a normal number of total sperm cells and a normal number of motile sperm cells. Hence, the reason for the failed mating could not be explained.
-Final examinations:
1. Body weight at autopsy
Males and females:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the body weight at autopsy for the male and female animals.
However, at the high dose level a marginally reduced body weight at autopsy was noted for the male animals at the intermediate and the high dose level (3.3 % or 2.0 % below the control, statistically not significant), whereas no consistent tendency was noted for the female animals (1.3 % below or 1.9 % above the control, statistically not significant). These marginal differences that were noted for the male and female animals were considered to be spontaneous.
2. Macroscopic post-mortem findings
Males (terminally sacrificed)
No test item-related macroscopic changes were recorded for the surviving male animals of all treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
The observed changes were considered to be spontaneous, as only one male per group was affected or the observations were also noted in the control group (thin fur).
Thin fur or loss of fur was noted at the forelimb(s) from one male of the control group (no. 11), one male of the intermediate dose group (no. 107) and 3 males of the high dose group (nos. 147, 161, 165). The male of the intermediate dose group and the males of the high dose group additionally showed thin fur at the hindlimb(s).
The thin fur was combined with a wound on the forelimb(s) in case of the control male no. 19 and with partly reddened fore- and hindlimb(s) in case of the high dose male no. 165.
A small testis and epididymis (left and / or right) were noted for one male of the control group (no. 5, right), 2 males of the low dose group (nos. 58, 60, left and right), one male of the intermediate dose group (no. 100, left and right) and one male of the high dose group (no. 159, right). In addition, dark-brown or pink discoloured testis of soft consistency were observed in the test item-treated males nos. 60, 100 and 159. Furthermore, dark-brown or pink discoloured epididymis of soft consistency were noted for the males nos. 60 and 159.
As no dose-response relationship was noted (2 affected males in the low dose group and one each in the intermediate and the high dose group) the observations that were noted for the testis and the epididymis were considered to be spontaneous. Furthermore, one male with a small testis and a small epididymis was also noted in the control group.
During the evaluation of the sperm parameter using the right cauda epididymis (the right epididymis from all 5 males was macroscopically affected) of the 5 affected males (nos. 5, 58, 60, 100, 159) only a very low number of sperm cells was noted. Due to the observed macroscopic changes, the 5 males were excluded from the statistical evaluation of the sperm count and the number of motile sperm cells.
The histopathological evaluation of the left testis and the left epididymis (the right testis was stored for a possible sperm count and the right epididymis was used for the evaluation of sperm parameter) from control male no. 5 and from high dosed male no. 159 revealed no observations. However, for both males (no. 5 and no. 159) macroscopic observations were only noted for the right testis and the right epididymis, whereas no macroscopic observations were noted for the left testis and the left epididymis of no. 5 and no. 159.
A red or dark-red discoloured (spotted or marbled) thymus was noted for 2 males of the control group (nos. 11, 12) and 2 males of the intermediate dose group (nos. 106, 107). In the intermediate dosed male no. 107 the thymus was marbled and with a few red-foci.
Observations that were only noted for one male each of the control group and the low dose group were in the form of reddish secretion from the left canthus (no. 17) or a jejunum with a haemorrhagic mucosa (no. 60).
Females (terminally sacrificed)
No test item-related macroscopic changes were recorded for the surviving female animals of all treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
All observations were considered to be spontaneous as discussed below.
Thin fur on the forelimbs(s), the neck region, the mammary gland(s), the abdomen, the chest, the shoulders and / or partly on the whole body was noted for 3 females of the control group (nos. 33, 43, 44), 3 females of the low dose group (nos. 76, 90, 91), one female of the intermediate dose group (no. 143) and 4 females of the high dose group (nos. 176, 179, 180, 189). As females from the control group were affected with a similar incidence as females of the low and the high dose group, the observation of thin fur was considered to be spontaneous.
A jejunum with a haemorrhagic or reddened mucosa was noted for one female of the low dose group (no. 79), 2 females of the intermediate dose group (nos. 136, 143) and 2 females of the high dose group (nos. 173, 183). The jejunum of the high dosed females was additionally filled with liquid. The microscopic examination of the jejunum from the 2 high dosed females nos. 173 and 183 revealed no abnormalities.
The observation of a jejunum with a haemorrhagic or reddened mucosa was considered to be spontaneous due to the low incidence of affected animals. Furthermore, no jejunum with a haemorrhagic or reddened mucosa was noted for the male and female animals of Cohort 1A and Cohort 1B.
Black foci in the stomach were noted for the control female no. 45 and the intermediate dosed female no. 143. Due to the low incidence, and as this observation was also noted in the control group, the observation was considered to be spontaneous.
An uterus that was filled with clear liquid was noted for 2 females of the intermediate dose group (no. 123, 131). In the case of no. 131 the uterus was additionally dilated. As no dose-response relationship was noted and due to the low incidence, this observation was considered to be spontaneous.
Other observations as a canthus with a reddish secretion (no. 172), an enlarged spleen (no. 143), a thickened and enlarged pituitary gland (no. 36) and enlarged lymph nodes (no. 87) were only noted for one female each and considered to be spontaneous due to their low incidence or as they were noted in the control group.
3. Stage of oestrus cycle at necropsy:
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) in the distribution of the stages of the oestrous cycle.
However, during the histopathological examination of the vagina a statistically significantly (p ≤ 0.05) increased number of females with an oestrous or pro-oestrous stage were noted at the high dose level in comparison to the control group. In detail, 5 females each of 24 females in total were noted with an oestrous or a pro-oestrous stage, in comparison to none females with an oestrous or a pro-oestrous stage in the control group.
Due to this observed statistically significant difference, an additional histopathological examination was performed for the oestrous stages at necropsy of the Cohort 1B females, which were also sacrificed after weaning, as the F0 Generation females.
As the examination of the oestrous stages of the Cohort 1B females did not reveal a statistically significant difference between the control and the high dosed females, the observed difference that was noted for the females of the F0 Generation was considered to be spontaneous.
4. Bone marrow:
Males and females
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (1000 mg Tin(II)-sulfide/kg b.w./day).
5. Organ weights (relative and absolute)
Males and females
No test item-related differences were noted for the absolute and relative organ weights of the male and female animals between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
6. Histopathological examinations
Males and females (terminal sacrifice animals)
No test item-related observation was noted during the histopathological examination of the male and female animals of the control group and the high dose group (1000 mg Tin(II)-sulfide/kg b.w./day).
Histopathological examination performed on one testicle and one epididymis each (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) of all males of groups 1 and 4 (F0 Generation), did not reveal any test item-related effects.
A statistically significant difference was noted in the distribution of the stages of the oestrous cycle between the F0 Generation females of the high dose group and the control group.
In detail, in group 4, five of 24 females showed oestrous cycle stage versus 0/24 in the controls. Moreover, this is also the case for proestrus where proestrus stage was observed in the vagina in 5/24 versus 0/24 in controls. Both attained statistical significance and was subscribed to the test item in group 4 animals.
However, an additional examination of the stages of the oestrous cycle from the females of Cohort 1B revealed no statistically significant differences between the females of the control group and the high dose group.
Hence, the observed statistically significant difference between the control group and the high dose group for the F0 Generation females was considered to be spontaneous.
All other microscopic changes seen in all other organs in all animals were either incidental, or were considered to lie within the normal range of background alterations, which may be seen in untreated rats of this age and strain.
-Reproductive parameters of the F0 females:
1. Monotoring of oestrous cycles – exposure period
After the allocation of the animals to the test groups and the start of treatment on test day 15, the oestrous cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign (verification of copulation) was noted.
No test item-related differences were noted for the mean length and the mean number of oestrous cycles per dam during the pre-mating period between the female animals of the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). The pre-mating period was from test day 15 until test day 85 (pairing was in the evening of test day 85).
No female with a complete oestrous cycle was noted during the mating period from test day 86 (first morning after the start of pairing in the evening of test day 85) until verification of mating (sperm detection).
2. Female fertility:
No test item-related influence on the fertility index was noted for the female rats of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Females with verified copulation:
A verified copulation was noted for all paired 23 females of the control group (no. 25 deceased during pre-mating) and all paired 24 females of the high dose group. At the low and the intermediate dose group one female each without a verified copulation was noted (nos. 81, 140), leading to 22 females with a verified copulation at the low dose level (no. 94 deceased during pre-mating) and 23 females with a verified copulation at the intermediate dose level.
From the females with a verified copulation one non-pregnant female each was noted in the control group (no. 31), the low dose group (no. 83) and the intermediate dose group (no. 123), leading to fertility indices of 96 % in the control group, 95 % at the low dose level and 96 % at the intermediate dose level.
Two non-pregnant females were noted at the high dose level (nos. 177, 178), leading to a fertility index of 92 %, which was slightly below the Provivo Background range (95 % to 100 %). In total, the number of non-pregnant females at the high dose level was one non-pregnant female higher than the background number (2 non-pregnant females at the high dose level for the current study, instead of one non-pregnant female in the background range). However, historical control groups with 2 non-pregnant females are available from OECD 422 studies carried out by Provivo. Hence, this slight increase in the total number can be considered as spontaneous, moreover, as the fertility index at the high dose level in Cohort 1B was 100 %.
3. gestation index:
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
All pregnant females of the control group and the intermediate dose group delivered live pups, leading to gestation indices of 100 %.
Two pregnant females that did not deliver live pups due to a total resorption of all implants were noted at the low dose level (nos. 92, 96) and one pregnant female with a resorption of all implants was noted at the high dose level (no. 189). The resulting gestation indices were 90 % at the low dose level and 95 % at the high dose level. However, whereas the gestation index at the high dose level was in the range of the Provivo background data (95 % to 100 %), the gestation index at the low dose level was below the Provivo background range. However, as no dose response relationship was noted, the reduced gestation index at the low dose level was considered to be spontaneous, moreover, as in Cohort 1B a gestation index of 100 % was noted for all groups.
4. 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 (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
However, elongated periods of pre-coital time (statistically not significant) in comparison to the control group were noted for all treatment groups. In detail, the pre-coital time interval was 2.0 ± 1.1 days in the control group, 2.3 ± 2.8 days at the low dose level, 2.5 ± 2.7 days at the intermediate dose level and 3.0 ± 3.5 days at the high dose level.
At the low and the intermediate dose level this was mainly due to one non-pregnant female each without a verified copulation after 14 days of mating (nos. 81, 140). At the high dose level the 2 pregnant female nos. 172 and 180 with pre-coital time intervals of 14 or 13 days were mainly responsible for the elongated mean value of pre-coital time.
However, a pregnant female with an elongated pre-coital time interval was also noted in the control group of Cohort 1B (no. 392 with a pre-coital time of 14 days). Moreover, in Cohort 1B the mean pre-coital time intervals at the control group and the high dose group were nearly in the same range. Hence, the elongated mean pre-coital time interval that was noted for the F0 high dosed females can be considered as spontaneous.
5. 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 (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive parameters of the parental females

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No premature death or sacrifice was noted for the Cohort 1A and 1B animals during their post-weaning development until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were noted for the Cohort 1A and 1B animals between the control group and the treatment groups for body weight and body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No differences were noted for the Cohort 1A and 1B animals between the control group and the treatment groups for food consumption.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No test item-related influence on water consumption was noted for Cohort 1A and 1B animals.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the haematological parameters for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The examination of the biochemical parameters for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the T4 and TSH levels for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The examination of the urinary parameters for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes in behaviour were noted for the Cohort 1A and 1B animals.

Immunological findings:

no effects observed

Description (incidence and severity):

The examination of the spleen cell population (lymphocyte typing) for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The weights of the evaluated organs from the Cohort 1A and Cohort 1B animals revealed no test item-related differences between the control group and the treatment groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related findings were noted during the macroscopic examination at necropsy for the cohort 1A and 1B animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histopathological examination, including a quantitative evaluation of the number of corpora lutea and follicles of the ovaries, which was performed for the Cohort 1A animals revealed no test item-related findings.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The histopathological examination, including a quantitative evaluation of the number of corpora lutea and follicles of the ovaries, which was performed for the Cohort 1A animals revealed no test item-related findings.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The examination of the monitoring of the oestrous cycle during a 2 week period for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the sperm parameter for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

Description (incidence and severity):

No test item-related influence was noted on the reproductive performance of the parental Cohort 1B animals, regarding the number and length of the oestrous cycles, the fertility index, the gestation index, the pre-coital time and the gestation length.

-mortality:
Males and females:
No premature death or premature sacrifice was noted in the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-clinical signs – daily cage side observations:
Males:
No observations were noted for the male animals of the control group and the male animals of the intermediate and the high dose group (300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
At the low dose level (100 mg Tin(II)-sulfide/kg b.w./day) male no. 241 showed cuts / wounds on the head on post-natal day 38 and hair loss on the head on post-natal days 39 to 42. In addition, cuts / wounds on the neck were noted for no. 241 on several test days between post-natal days 61 and 84.
Furthermore, male no. 259 of the low dose group showed hair loss on the neck on 9 consecutive days between post-natal days 69 and 77.
The observations at the low dose level were considered to be spontaneous, as only 2 animals were affected and no dose-response relationship was noted.
Females:
No observations were noted for the female animals of the control group and the females animals of the intermediate and the high dose group (300 or 1000 mg Tin-II sulfide/kg b.w./day).
At the low dose level (100 mg Tin(II)- sulfide/kg b.w./day) a reddened canthus (right eye) was noted for the female animal no. 263 on 5 consecutive between post-natal days 37 and 41.
As only one female was affected the observation of a reddened canthus, noted at the low dose level, was considered to be spontaneous.
-Detailed clinical observations:
No further observations in addition to those made during the daily cage side observations were noted for the male and female animals of the control group and the treatment groups during the once weekly performed detailed clinical observation.
-body weight and body weight gain:
Males:
No test item-related differences in body weight and body weight gain were noted for the male animals between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the post-weaning development.
Body weight gain: No test item-related differences in body weight gain were noted between the control group and the treatment groups for the male animals.
Females:
No test item-related differences in body weight and body weight gain were noted for the male animals between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the post-weaning development.
Body weight gain: No test item-related differences in body weight gain were noted between the control group and the treatment groups for the female animals.
-food and water consumption:
-Food consumption:
Males:
No test item-related difference in food consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Females:
No test item-related difference in food consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-water consumption (male and female):
Water consumption was performed by visual appraisal during the daily cage side observations. No decreased or increased water consumption was noted for the male and female animals.
-haematology:
Males:
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
A slightly, but statistically significantly reduced number of red blood cells was noted at the high dose level (4.6 % below the control, p ≤ 0.05). The number of red blood cells from all individual high dosed males was in the range of the Provivo background data.
As all individual high dosed values were within the background range , the observed reduced number of red blood cells in comparison to the control group that was noted at the high dose level, was considered to be spontaneous.
Females:
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the female animals.
-clinical biochemistry:
Males and females:
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the male and female animals.
-lymphocytes typing in spleen:
Males and females:
No test item-related influence was noted in the proportion of the examined lymphocyte subtypes to each other between the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-urinalysis:
Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/day).
-thyroid hormone levels:
Males and females:
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the male and female animals.
Statistically significantly increased T4 values were noted for the male animals at the high dose level (27.2 % above the control, p ≤ 0.05) and for the female animals at the intermediate and the high dose level (140.0 % or 121.6 % above the control, p ≤ 0.01).
A comparison with the Provivo background data revealed that most of the individual values from the male and female animals of the control group and the low dose group were below the background range.
In detail, for the male animals 8 individual values each of the control and the low dose group were below the background range. On the other hand, at the intermediate dose level only 4 individual values from the males were below the background range and at the high dose level all 10 individual male values were within the background range.
For the female animals nearly all individual values from the control group and the low dose group were below the background range. At the intermediate dose level 5 individual values were below the background range and at the high dose level 6 individual values were below the background range.
In conclusion, as nearly all individual values from the male and female animals of the control group were below the background range, the statistically significantly increased T4 values that were noted for the male animals at the high and for the female animals at the intermediate and the high dose level were considered to be spontaneous.
-sexual maturation – cohort 1A and 1B combined:
Males:
No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the time point of balano-prepuital separation and the body weight at the time point of balano-prepuital separation.
Females:
No test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the time point of vaginal opening, the body weight at the time point of vaginal opening and the time point of the first appearance of cornified cells after vaginal opening. For an overview see Text Table 8-8 and the detailed discussion below.
Time point (postnatal day) of vaginal opening (Cohort 1A and Cohort 1B combined):
Vaginal opening was on post-natal day 32.6 for the females of the control group, on post-natal days 32.5, 32.6 and 32.5 for the females of the low, the intermediate and the high dose group. The marginal difference between the control group and the treatment groups (at maximum 0.1 days at the low and the high dose level; statistically not significant) was considered to be spontaneous.
Body weight at the time point (postnatal day) of vaginal opening (Cohort 1A and Cohort 1B combined):
The body weight at the time point of vaginal opening was in the range of the control group for all dose groups. In detail, the body weight at the time point of vaginal opening was 0.8 % below the control at the low dose level and 1.1 % or 2.8 % above the control at the intermediate and the high dose level. These marginal and statistically not significant differences were considered to be spontaneous.
First appearance of cornified cells after vaginal opening (only examined for Cohort 1A):
Shortly earlier time points (statistically not significant) for the first appearance of cornified cells in comparison to the control group were noted at the low and the high dose level, whereas at the intermediate dose level the time point was in the range of the control group.
In detail, the first appearance of cornified cells was on post-natal day 34.4 for the females of the control group and on post-natal days 33.4, 34.6 and 33.5 for the females of the low, the intermediate and the high dose group. As the differences were not statistically significant and no dose-response relationship was noted, the observed differences for the time points of the first appearance of cornified cells after vaginal opening were considered to be spontaneous.
-monitoring of oestrus cycles – 2 week period:
The stages of the oestrous cycle of the Cohort 1A animals were monitored on 14 test days between test days 53 and 66 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of oestrous cycles per female animal between the females of the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-examination of the sperm number, viability and morphology:
No test item-related difference was noted between the rats of the control group and the rats treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day for the sperm number, the viability and morphology.
Two malformed sperm cells (banana-like each were noted in the control group, the low dose group and the high dose group. One malformed sperm cell was noted in the intermediate dose group. All observed malformations were in the form of banana-like sperm heads.
As the number of malformed sperm cells was very low and malformed sperm cells were also detected in the control group, the observation of malformed sperm cells in the low, the intermediate and the high dose group was considered to be spontaneous.
-final examinations:
-body weight at autopsy:
Males and females
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the body weight at autopsy of the male and female animals.
-macroscopic post-mortem findings:
Males
No test item-related observations were noted for the male animals of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) at necropsy.
The following observations were only noted for a few individual animals and considered to be spontaneous:
Control:
• A reddened testicle (right) and an epididymis (right) that was reduced in size (or small) and transparent were noted for male no. 202 of the control group.
During the evaluation of the sperm parameter using the cauda of the right epididymis no sperm cells were noted. Due to the observed macroscopic changes for the right epididymis male no. 202 was excluded from the evaluation of the sperm parameter.
Group 2:
• A urinary bladder that was filled with a white-yellow mass was noted for no. 244.
• A testicle (left) that was enlarged and of soft consistency was noted for no. 249.
No observations were noted for the males of the intermediate and the high dose group.
Females
No test item-related observations were noted for the female animals of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) at necropsy.
Observations were noted for the uterus and the spleen which were considered to be spontaneous as discussed below.
A dilated uterus (mostly filled with clear liquid) was noted for 2 females of the control group, one female of the low dose group, 3 females of the intermediate dose group and 4 females of the high dose group. A dilated uterus was also noted for one F0 Generation and one Cohort 1B female of the intermediate dose group.
The macroscopic finding of a dilated uterus was verified during the histopathological examination of the affected control and high dosed animals.
The finding of a dilated uterus and / or an uterus that was filled with clear liquid can be associated with normal reproductive cyclicity and is not related to the treatment with the test item.
An enlarged spleen was noted for the intermediate dosed female no. 311. As an enlarged spleen was only noted for one animal, it was considered to be spontaneous.
-stage of the oestrus cycle at necropsy:
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) in the distribution of the stages of the oestrous cycle determined from lavages taken at necropsy and during the histopathological examination of the vagina.
-bone marrow:
Males and females:
No test item-related differences for the myeloid / erythroid ratio of the bone marrow were noted between the control group and the high dose group (1000 mg Tin(II)-sulfide/kg b.w./day).
-organ weights (relative and absolute):
Males and females:
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the absolute and relative organ weights of the male and female animals.
Slightly decreased values were noted in the male animals for the absolute and relative organ weights of the left and right adrenal glands at the intermediate dose level (between 10.0 % and 12.9 % below the control value, p ≤ 0.05 or not statistically significant). As no dose-response relationship was noted, the observed decreased values that were noted at the intermediate dose level were considered to be spontaneous.
-histopathological examinations:
Males and females (terminal sacrifice animals):
Histopathological examination performed on one testicle and one epididymis each (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) of all males of groups 1 and 4 (F1 Generation Cohort 1A), did not reveal any test item-related effects.
All microscopic changes seen in the organs of the Cohort 1A male and female animals were either incidental, or were considered to lie within the normal range of background alterations, which may be seen in untreated rats of this age and strain.
Quantitative assessment of Primordial and Small growing Follicles (PSF) and Corpora Lutea (CL) - Females F1 Generation Cohort 1A:
The quantitative assessment (including statistical analyses) of the right oavary for the presence of primordial and small growing follicles (PSF) and corpora lutea did not show any significant and/or toxicologically relevant differences between females of groups 1 and 4.

Results – F1 generation – Cohort 1B:
-mortality:
Males and females:
No premature death or premature sacrifice was noted in the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide /kg b.w./day).
-clinical sings:
Males:
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male animals of the control group and the high dose group (1000 mg Tin(II)- sulfide /kg b.w./day).
No test item-related changes were noted at the low and the intermediate dose group (100 or 300 mg Tin(II)-sulfide /kg b.w./day).
Salivation was noted for one male animal of the low (no. 409) and the intermediate (no. 449) dose group on one test day each. Additionally, animal no. 417 of the low dose group showed scratch wounds (cuts/wounds) on 4 consecutive test days between post-natal days 86 and 89, which healed afterwards. Due to the low number of affected animals and the low incidence (salivation was only noted on one test day) these observations were considered to be spontaneous.
Females:
No changes in behaviour, the external appearance and the consistency of the faces were noted in the control group and the low and the intermediate dose group (100 or 300 mg Tin(II)-sulfide /kg b.w./day).
No test item-related changes were noted in the high dose group (1000 mg Tin(II)-sulfide /kg b.w./day).
To the end of the lactation period hair loss on the forelimb was noted on 6 consecutive test days for female no. 520 of the high dose group. Hair loss was first noted on lactation day 17 and last noted on the day of necropsy on lactation day 22.
As only one animal was affected, this observation was considered to be spontaneous.
-detailed clinical observations:
Males and females
No further relevant observations in addition to those made during the daily cage side observations were noted for the male and female animals of the control group and the treatment groups during the once weekly performed detailed clinical observation.
-body weight and body weight gain:
Males:
No test item-related differences in body weight and body weight gain were noted for the male animals between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
A marginally reduced body weight (statistically not significant) was noted at the high dose level from PND 50 onwards until PND 139 (last day before necropsy on which all males were available) with a maximum difference on PND 99 (3.5 % below the control, statistically not significant. This marginal difference was considered to be spontaneous.
Body weight gain: No test item-related differences in body weight gain were noted between the control group and the treatment groups for the male animals.
Females:
No test item-related differences in body weight and body weight gain were noted for the female animals between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the pre-mating / mating period, the gestation period and the lactation period.
Body weight gain: No test item-related differences in body weight gain were noted between the control group and the treatment groups for the female animals.
-food and water consumption:
-Food consumption
Males:
No test item-related difference in food consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the male animals.
Females
No test item-related difference in food consumption was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the female animals during the premating period, the gestation period and the lactation period.
-Water consumption
Water consumption was performed by visual appraisal during the daily cage side observations. No decreased or increased water consumption was noted for nearly all animals.
-final examinations:
-body weight at autopsy:
Males and females
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-macroscopic post-mortem findings:
Males
No observations were noted for the male animals of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) at necropsy.
The only observation was noted in the control group in the form of enlarged and spotted caudal and medial liver lobes from male no. 371.
Females
No test item-related observations were noted for the female animals of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) at necropsy.
The following observations were only noted for single animals and considered to be spontaneous:
Control:
• The right kidney from female no. 397 was enlarged and of soft consistency.
Group 2:
• The uterus horns from female no. 426 were cystic.
• The right kidney from female no. 432 was swollen with a vascular congestion on the surface.
Group 3:
• The uterus from female no. 473 was dilated.
Group 4:
• Lungs with multiple grey foci and a thymus with a reddish focus were noted for female no. 501.
• Thin fur on the left and right forelimb was noted for female no. 520.
-stage of oestrus cycle at necropsy:
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) in the distribution of the stages of the oestrous cycle determined from lavages taken at necropsy or determined during the histopathological examination of the vagina.
-organ weights:
Males:
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Statistically significant differences in comparison to the control group were noted for the weight of the pituitary gland and the weight of the prostate gland (see Text Table 9-7 below). However, these differences were considered to be spontaneous as discussed below.
Increased organ weights (statistically significant or not) were noted for the relative and absolute weights of the pituitary gland at the low and the intermediate dose level.
In detail, at the low and the intermediate dose level the relative organ weights of the pituitary gland were 8.4 % or 7.9 % above the control (p ≤ 0.05) and the absolute organ weights of the pituitary gland were 8.4 % or 9.1 % above the control (p ≤ 0.05 or not statistically significant). However, at the high dose level the relative organ weight of the pituitary gland was in the range of the control group (0.7 % above the control) and the absolute organ weight of the pituitary gland was slightly to marginally decreased versus the control group (4.0 % below the control, not statistically significant).
Hence, as no dose-response relationship was noted, the increased relative and absolute organ weights of the pituitary gland that were noted at the low and the intermediate dose level were considered to be spontaneous.
Slightly decreased relative and absolute organ weights (statistically significant or not) were noted at the high dose level for the prostate gland.
In detail, at the high dose level, the relative organ weight of the prostate gland was 11.6 % below the control (not statistically significant) and the absolute organ weight was 14.8 % below the control (p ≤ 0.05).
However, the relative and the absolute organ weights of the prostate gland from the Cohort 1A males were in the range of the control group.
Hence, as the decreased organ weights of the prostate gland that were noted for the high dosed Cohort 1B males were only slight and not noted for the high dosed Cohort 1A males, they were considered to be spontaneous.
Females:
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) for the female animals.
-reproductive parameters of F1 females:
-monitoring of oestrus cycles mating period:
The stage of the oestrous cycle was monitored for the Cohort 1B females during the mating period. The monitoring started on test day 77, which was the first day after pairing in the evening of test day 76, and ended one day before a positive sperm detection was noted or on the last morning of the 14 day mating period on test day 90.
Elongated mating periods (pre-coital time intervals) were noted for no. 392 (control), nos. 427 and 433 (low dose group) and no. 520 (high dose group). The elongated mating periods consisted of consecutive test days with a dioestrous stage (between 10 and 13 days) until a positive sperm detection was noted. No oestrous stage was noted within these consecutive periods of dioestrous stages, hence no mating opportunity was passed.
The two non-pregnant females without a positive sperm detection, no. 423 of the low dose group and no. 512 of the high dose group, revealed consecutive 13 days (no. 512) or 14 days (no. 423) of dioestrous stage during the14-day mating period.
-female fertility:
No test item-related influence on the fertility index was noted at any dose level (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
In detail, a verified copulation was noted for all 20 females of the control group and the intermediate dose group. Females without a verified copulation were noted at the low (no. 423) and the high dose level (no. 512) (both females without a verified copulation did not became pregnant). The 2 females without a verified copulation were excluded from the calculation of the fertility index.
From the females with a verified copulation one female each of the control (no. 398), the low (no. 426) and the intermediate dose group (no. 464) did not became pregnant, leading to a fertility index of 95 % for the control group and the low and the intermediate dose group.
At the high dose level all 19 females with a verified copulation became pregnant, leading to a fertility index of 100 % at the high dose level.
-gestation index:
No test item-related influence on the gestation index was noted for the female rats of all treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
All pregnant females with a verified copulation that were noted in the control group and the treatment groups delivered live pups. Hence, a gestation index of 100 % was noted for the control group and all treatment groups.
-pre-coital time:
No test item-related differences were noted in the length of the pre-coital time interval between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
The pre-coital time interval was 3.3 ± 2.8 days in the control group, 3.6 ± 4.5 days at the low dose level, 2.2 ± 1.0 days at the intermediate dose level and 3.5 ± 3.3 days at the high dose level.
-gestation index:
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 (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
The observed gestation lengths were 22.2 ± 0.4 in the control group and in the low dose group, 22.1 ± 0.2 in the intermediate dose group and 22.3 ± 0.5 in the high dose group.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity (cohort 1A + Cohort 1B)

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dosse tested

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive parameters of the parental females

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest tested dose

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related observations were noted.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related influence on the body weight of the pups was noted in any of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the sexual maturation of the male and female animals during the combined evaluation of the Cohort 1A and Cohort 1B animals.

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 (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No increased number of pups with nipple retention was noted for the male pups of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) in comparison to the male pups of the control group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external and internal examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

-pre- and postnatal development – F1 pups
-birth indices and post-implantation loss – F1 pups:
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
The percentage of post-implantation loss (considering the number of resorptions and stillbirths) at the high dose level was nearly at the same level as in control group. In detail, the mean percentage of post-implantation loss per dam was 16.06 ± 13.57 % in the control group and 18.40 ± 22.48 % in the high dose group. The post-implantation loss per group was 16.40 % in the high dose group in comparison to 16.12 % in the control group.
The mean number of stillbirths and resorptions per dam was 2.5 ± 2.0 in the control group and 2.4 ± 2.2 in the high dose group. The total number of resorptions in the control group was 54 and in the high dose group 52. Four stillbirths were noted in the control group and 3 in the high dose group.
-viability index – F1 pups:
Pre- and post-cull period:
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.
Viability indices – pre-cull:
The viability index of the pre-cull period at the control group (98.22 %) and the high dose group (97.74 %) were in the range of the Provivo background data (96.71 % to 99.36 %).
However, viability indices below the background range were noted for the low (93.50 %) and the intermediate dose group (93.51 %). As no dose-response relationship was noted, this was considered to be spontaneous.
Viability indices - post-cull:
A reduced viability index for the post-cull period was noted at the high dose level (94.03 %) that was below the Provivo background range for the post-cull period (99.36 % to 100 %).
This was due to the pups from dam no. 180. All the 10 remaining pups of dam no. 180 deceased on lactation day 20. In detail, one pup was cannibalized, one was found dead and 8 pups were prematurely sacrificed due to poor health conditions. The reason for the poor health condition of the pups from dam no. 180 is not clear yet. With the exception of a slight body weight loss 2 days before lactation day 20 no further signs that could be related to a systemic toxicity were noted for dam no. 180. Furthermore, the body weight of dam no. 180 recovered after the removal of their pups. Examination of the stomach from the sacrificed pups revealed milk in all stomachs, indicating an ongoing nursing of the pups by the dam. Hence, the poor health conditions of the pups are probably not a secondary effect of a neglected breeding care of dam no. 180. A full necropsy was performed for one of the prematurely deceased pups (no. 180-9) and revealed a severely intertwined jejunum.
As the pups from only one litter were affected, the poor health conditions of the pups from this litter can be considered as spontaneous. Moreover, as no pup with poor health conditions was noted at the high dose level of Cohort 1B during the post-cull period.
-male to female ratio of the pups – F1 pups:
No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
A slightly reduced male to female ratio in comparison to the control group was noted at the low and the high dose level (0.94 or 0.89 in comparison to 1.11 in the control group on lactation day 1). However, as no dose-response relationship was noted and no decreased male to female ratio was noted for the high dosed F2 pups of Cohort 1B, the slightly reduced male to female ratio at the high dose level that was noted for the F1 pups was considered to be spontaneous.
-male to female ratio of the pups – F1 pups:
No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
A slightly reduced male to female ratio in comparison to the control group was noted at the low and the high dose level (0.94 or 0.89 in comparison to 1.11 in the control group on lactation day 1). However, as no dose-response relationship was noted and no decreased male to female ratio was noted for the high dosed F2 pups of Cohort 1B, the slightly reduced male to female ratio at the high dose level that was noted for the F1 pups was considered to be spontaneous.
-Body weight of pups – F1:
No test item-related influence on the body weight of the pups was noted in any of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Runts:
On lactation day 1 two runts each were noted in the control group (nos. 47-09, 48-03), the low dose group (nos. 84-05, 91-11) and in the intermediate dose group (nos. 134-15, 135-14). One runt (no. 172-11) was noted at the high dose level.
-litter weight – F1 pups:
No test item-related influence on the litter weight was noted in any of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-number of live pups – F1 pups:
No test item-related differences were noted for the mean number of live pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the lactation period.
A marginal and statistically not significantly reduced number of live pups (male and female pups combined) in comparison to the control group was noted on lactation day 1 at the low and the high dose level.
In detail, 12.8 ± 3.1 live pups were noted in the control group, 12.3 ± 5.1 at the low dose level, 14.0 ± 3.0 at the intermediate dose level and 12.0 ± 4.2 at the high dose level. The differences between the control group and the low and the high dose group were considered to be spontaneous, as they were only marginal and no dose-response relationship was noted. Furthermore, no reduced number of live pups in comparison to the control group was noted for the F2 high dosed level pups of Cohort 1B.
However, combined with the reduced male to female ratios at the low and the high dose level, the marginally reduced number of live pups at the low and the high dose level led to a reduced number of male pups at the low and the high dose level during the whole lactation period. These differences were statistically significant on lactation day 21 (21.4 % or 20.5 % below the control, p ≤ 0.05). As no dose-response relationship was noted (the number of male pups at the intermediate dose level was in the range of the control group or even marginally above during the whole lactation period) the observed statistically significant changes were considered to be spontaneous. Moreover, as the differences in the number of male pups were the result of 2 other parameters which were both not considered to be test item-related (slight differences in the male to female ratio and marginal differences in the whole number of pups).
-Ano-genital distance – F1 pups:
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-Nipple retation – F1 pups:
No increased number of pups with nipple retention was noted for the male pups of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) in comparison to the male pups of the control group.
In detail, 13 pups with nipple retention from 7 different dams were noted in the control group in comparison to only 5 pups with nipple retention from 3 different dams in the high dose group.
-Thyroid hormone determination – F1 pups:
T4 and TSH levels (PND 4 and / or PND 22)
No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the T4 level and the TSH level on PND 4 and / or PND 22 for the male and female pups.
Statistically significantly decreased TSH values were noted for the male and female pups of the low dose group on PND 22.
In detail, the TSH concentration at the low dose level was 52.5 % below the control for the male pups (p ≤ 0.05), 70.3 % below the control for the female pups (p ≤ 0.01) and 61.7 % below the control for the male and female pups combined (p ≤ 0.01). However, at the high dose level the TSH concentrations of the male and female pups were increased in comparison to the control (25.1 %, 22,5 % or 25.4 % above the control for the male, the female and the male and female pups combined; statistically not significant).
Hence, as no dose-response relationship was noted, the observed statistically significantly decreased TSH concentrations that were noted at the low dose level were considered to be spontaneous.
-External and internal examinations of the pups – F1 pups:
No test item-related gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external and internal examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
The following observations were considered to be spontaneous due to their singular occurrence:
Group 2: A malformation in the form of a hydrocephalus was noted pup no. 76-01, which was found dead on lactation day 20.
A slightly enlarged spleen was noted for pup no. 82-05.
Group 3: A malformation in the form of a hyperflexion of the left forepaw was noted for pup no. 137-01 at scheduled sacrifice on lactation day 22.
Group 4: A malformation in the form of an absent left eye was noted for pup no. 179-02 at scheduled sacrifice on lactation day 22.
Group 4: An unclassified observation in the form of an extremely intertwined jejunum was noted for pup no. 180-09 during the gross inspection of the inner organs after premature sacrifice on lactation day 20.
A further unclassified observation was noted for no. 180-11 (found dead on lactation day 20) in the form of yellow discharge from the right eye.
-pup organ weight (absolute) – F1 pups:
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Key result

Dose descriptor:

NOAEL

Remarks:

prenatal development pups

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

post-natal development of pups

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Description (incidence and severity):

No test item-related observations were noted.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related influence on the body weight of the pups was noted at the low, the intermediate and the high dose level (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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 (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Nipple retention in male pups:

no effects observed

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 (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external and internal examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

prenatal development of pups

Generation:

F2

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

post-natal development of pups

Generation:

F2

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Reproductive effects observed:

no

Conclusions:

The following no-observed-adverse-effect levels (NOAEL) were established for the parental animals of the F0 Generation and the F1 Generation:
F0 Generation:
General toxicity
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Reproductive toxicity
a) adverse effects on the reproductive parameters of the parental females:
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day
b) adverse effects on the prenatal development of the pups:
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day
c) adverse effects on the post-natal development of the pups:
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

F1 Generation:
Developmental toxicity (Cohorts 1A + Cohort 1B)
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Reproductive developmental toxicity (Cohort 1B)
a) adverse effects on the reproductive parameters of the parental females:
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day
b) adverse effects on the prenatal development of the pups:
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day
c) adverse effects on the post-natal development of the pups:
NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Executive summary:

The aim of the study was to evaluate the effects of the test item Tin(II)-sulfide at dose levels of 100, 300 or 1000 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood. In addition, the reproductive toxicity of the F1 Generation was evaluated until weaning of the F2 Pups (OECD 443).

 

General and reproductive toxicity (F0 Generation and F1 Pups)

General toxicity

No test item-related premature death or sacrifice and no test item-related changes in behaviour, the external appearance or the faeces were noted for the male and female animals of the F0 Generation.

No differences between the control group and the treatment groups were noted for body weight, body weight gain, and for food and water consumption.

The examination of the haematological parameters, the biochemical parameters, the urinary parameters, the T4 and TSH levels and the sperm parameters revealed no test item-related differences between the control group and the treatment groups.

No test item-related findings were noted during the macroscopic examination at necropsy and the histopathological examination. The determination of the organ weights revealed no test item-related differences between the control group and the treatment groups.

Reproductive toxicity

No test item-related influence was noted on the reproductive performance of the parental animals (number and length of oestrous cycles, fertility and gestation index, pre-coital time and gestation length).

No test item-related influence was noted on the prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights). No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.

 

Developmental toxicity (F1 - Cohorts 1A and 1B)

No premature death or sacrifice was noted for the Cohort 1A and 1B animals during their post-weaning development until scheduled necropsy.

Furthermore, no changes in behaviour, the external appearance or the faeces were noted for the Cohort 1A and 1B animals.

No differences were noted for the Cohort 1A and 1B animals between the control group and the treatment groups for body weight, body weight gain and food consumption.

No test item-related influence was noted on the sexual maturation of the male and female animals during the combined evaluation of the Cohort 1A and Cohort 1B animals.

The examination of the haematological parameters, the biochemical parameters, the spleen cell population (lymphocyte typing), the urinary parameters, the T4 and TSH levels, the monitoring of the oestrous cycle during a 2 week period, and the sperm parameter for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

No test item-related findings were noted during the macroscopic examination at necropsy for the cohort 1A and 1B animals.

The weights of the evaluated organs from the Cohort 1A and Cohort 1B animals revealed no test item-related differences between the control group and the treatment groups.

The histopathological examination, including a quantitative evaluation of the number of corpora lutea and follicles of the ovaries, which was performed for the Cohort 1A animals revealed no test item-related findings.

 

Reproductive toxicity (F1 - Cohort 1B)

No test item-related influence was noted on the reproductive performance of the parental Cohort 1B animals, regarding the number and length of the oestrous cycles, the fertility index, the gestation index, the pre-coital time and the gestation length.

The prenatal development of the F2 Pups (number of resorptions, stillbirths and live born pups) and their postnatal development until weaning (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were not affected by the test item. Furthermore, no malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.

 

 

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

F0 Generation:

General toxicity

NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Reproductive toxicity

a) adverse effects on the reproductive parameters of the parental females: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

b) adverse effects on the prenatal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

c) adverse effects on the post-natal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

 

F1 Generation:

Developmental toxicity (Cohorts 1A + Cohort 1B)

NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Reproductive developmental toxicity (Cohort 1B)

a) adverse effects on the reproductive parameters of the parental females: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

b) adverse effects on the prenatal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

c) adverse effects on the post-natal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-10 to 2010-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted by the Council on July 27th 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Albino laboratory rat Wistar Han ( outbred SPF quality - guaranteed)
- Supplier: SPF breeding, VELAZ s.r.o., Kolec u Kladna Czech Republic, RCH CZ 21760152
- Sex: males and virgin females
- Age at study initiation: 10 weeks
- total numer of animals: 48 males and 48 females (12 females and 12 males per group)
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage ( 40x25x20 cm) containing sterilised clean shavings of soft wood. During mating period - one male and one female in one cage, pregnant females -individually, offspring- with mother.
- Diet: Complete pelleted diet for rats and mice in SPF breeding- ST 1 BERGMAN was used, manufacturer: Mlyn Kocanda, V)'roba krmnych smesf, Kocanda No.19, 252 42 Jesenice u Prahy. Diet was sterilised before using .
- Water: Free access to drinking water (ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coil. of Law, Health Ministry. Water was sterilised before using.
- Acclimatization time: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.9 to 23.2°C
- Humidity: 35.8 to 66.6 %
- Air changes: approximately 15 air exchanges/hour
- Photoperiod: 12 hours daily

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose in water for injection

Details on exposure:

The concentrations of the suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by 0.5% methylcellulose in water for injection in the same volume. The application form (test substance suspension in 0.5% methylcellulose in water for injection) was prepared daily just before administration. This suspension was mixed for 10 minutes by magnetic stirrer (ca 1000 rpm).
The test substance was administered to the stomach by gavage as suspension in 0.5% methylcellulose in water for injection.Oral administration of rats was made daily.

Details on mating procedure:

Animals were mated from the 15th day of study. Mating 1: 1 ( one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical report includes the results of testing a homogeneity of Tin sulfide application form.
The application form for "Tin sulfide- Reproduction/Developmental Toxicity Screening Test" is the suspension of test substance in 0.5% methylcellulose. The test purpose was found out whether the application form prepared is homogenous.
Homogeneity of the application form was checked by determination of a concentration of the test substance in three levels of solution (at the bottom, in the middle and at the surface ).
The measurement was performed on two concentration levels (50 mg / 10 mL and 1000 mg / 10 mL). The application form was prepared in a way that is conformable with "Tin sulfide – Reproduction/Developmental Toxicity Screening Test" (the similar equipment: container, mixing stirrer). The analyses were performed by ICP - OE spectrophotometric method.
From the results of analyses follows that the Tin sulfide application form in vehicle at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogeneous. The differences in concentrations detected on the single levels of measurement to average are less then uncertainty of measurement.

Duration of treatment / exposure:

The treated groups were administered daily for the following periods:
males and females - 2 weeks prior to the mating period and during the mating period, pregnant females - during pregnancy and till the 3rd day of lactation, males - after mating period- totally for 42 days, non-pregnant females (mated females without parturition) - for 25 days after the confirmed mating.

Frequency of treatment:

once per day, 7 days per week

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks (10 weeks at start of study; mating from the 15th day of study)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for study- 100, 300 and 1000 mg/kg/day -were chosen with respect to the results of the dose-range finding experiment and on request of the sponsor.
The DRF experiment with 14-day application period was performed with 4 groups of treated rats. The doses for the DRF experiment were chosen with respect to the literature data : 125, 250, 500 and 1000 mg/kg/day. In the DRF experiment with Tin sulfide, the body weight increments and haematological parameters were relatively well-balanced at all dose levels during the 14-day application period. The pathological examination did not reveal any pathological changes. No animal died during the 14-day application period.
- Rationale for animal assignment (if not random): random

Positive control:

no

Parental animals: Observations and examinations:

Health condition control: daily - during the acclimatization and the experimental part;
Body weight: males - the first day of administration and then weekly,females - the first day of administration and then weekly in prematingand mating period; during pregnancy: 0., 7th, 14th, 20th day; during lactation: 0. or 1st and 4th day;
Food consumption: males- weekly (except the mating period), females - weekly during premating period and after mating period, during pregnancy and lactation on the same days as body weight;
Clinical observations: males and females - daily during the administration period;
Mortality control: daily.

Oestrous cyclicity (parental animals):

The oestrous cycle length and pattern was not evaluated before mating but the phases of oestrous cycle were recorded during histopathological examination.

Sperm parameters (parental animals):

In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to the SOP No. M/45.
Sperm motility:
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension (mixture of one epididymis cutted into pieces and 5 ml of 0.9% NaCl). The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 -no progressive motility, 4- nonmotile sperm.
Sperm morphology:
Sperm samples (taken from one epididymis) and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations - e.g. broken tail, abnormal form of tail, double head, amorphous head and abnormal form of neck - were recorded.

Litter observations:

All pups were observed in natural conditions in their cages daily during the lactation period. Changes in behavioural abnom1alities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Postmortem examinations (parental animals):

Parental males were killed at the end of the administration period after 42 days of administration. Parental females were killed on the 4th day of lactation. Mated females without delivery was killed 26th day after confirmed mating. Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded. The absolute weights of testes, one epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries; uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the relative weight of the organ was computed according to the following formula: weight of organ x 100/ body weight.

Postmortem examinations (offspring):

Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight - necropsy weight in males, body weight of females on the 20th day of pregnancy, necropsy weight of females; biometry of organs of males and females; number of live pups on the 0/1 st day and 4th day of lactation period and average weight of pup on the 0/1 st day and 4th day of lactation period. Control group with vehicle was compared with the three treated groups. The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables (no findings at probability level 0.01 and 0.001).

Reproductive indices:

Reproduction parameters: number of females achieving pregnancy, number of females bearing live pups and number of females with live pups at day 4 after parturition in treated groups were similar to the control or higher than the control groups. Duration of mating and pregnancy were similar in the control and treated females. Pre-implantation, post-implantation and postnatal losses were relatively well balanced at the treated groups and control group.

Offspring viability indices:

Pre-implantation loss, post-implantation loss, post-natal loss, mating index, fertility index, conception index, gestation index, percentage of postnatal loss days 0-4 post partum and viability index were calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
No treatment-related effects were found in treated males during the health condition control.
No relevant clinical changes were observed in males at all dose levels after application of the test substance.
Slight fall of weight ( one male at the dose level of 100 mg/kg/day in the 6th week) and thinner excrements ( one male at the dose of 1000 mg/kg/day in the 1 st week) were observed sporadically in treated males.
Females:
Slight decreases in body weights were sporadically (in 1 or 2 females from the group) recorded in the 3rd week at all groups including control.
In treated females of all dose levels, no signs of disease were found during the check-in, acclimatisation and application period. Treatment-related effects were not detected during health condition control and clinical observation of females ( except of vocalisation in one female at the dose level of 1000 mg/kg/day in the 5th week).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The average animal body weights at all dose levels were relatively balanced with the control group during the whole application period. No statistically significant differences were detected.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: Average food consumption values of males at all treated close levels were slightly increased against the control group during the whole study. These differences were not dependent on the dose level and therefore not considered relevant.
Fermales:
Females:
Pre-mating period
Average food consumption of treated females was analogous to food consumption of control females.
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy. Average food consumption of treated females was well-balanced with control females.
Lactation
Only mothers (females with live pups) were included in evaluation of food consumption during lactation period. Average food consumption of treated females was similar to consumption of control females.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The effect on the microscopical structure of the testes (sporadic degeneration and/or atrophy of germ epithelium, residual bodies in germ epithelium and vacuolation of cytoplasm of spermiogonia) had no effect on spermiogenesis, thus, were not relevant.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Body weight, food consumption, clinical status and macroscopic structure of reproductive organs of treated parental males were unaffected by treatment of the test substance. A statistically significant difference was detected in the weight of the pituitary gland. An absolute weight of pituitary gland was statistically significantly incrased in males at the dose level of 1000 mg/kg/day. In absence of any changes of microscopic structure of the pituitary gland, the effect could be considered not to be of toxicological importance. The test substance had effect on the microscopical structure of the tests without damage of spermiogenesis. Histopathological examination of testes of parental males showed increased incidence of irrelevant changes: degenerations and/or atrophies of germ epithelium and vacuolations of cytoplasm of spermiogonia in males of the dose level 1000 mg/kg/ day. However, there were no histopathological findings in testes in a 90-day study on tin sulfide. Food consumption, clinical observations, weight and structure of reproductive organs of treated parental females were not significantly affected by treatment of the test substance. Growth of parental females was slightly but insignificantly influenced by the test substance administration: the body weight of females at the dose levels of 300 and 1000 mg/kg/day were slightly decreased during the pregnancy and lactation period.
Reproduction parameters - number of females achieving pregnancy, number of females bearing live pups and number of females with live pups at day 4 after parturition in treated groups were similar to the control or higher than the control groups. Duration of mating and pregnancy were similar in the control and treated females. Pre-implantation, post-implantation and postnatal losses were relatively well balanced at the treated groups and control group.

Key result

Dose descriptor:

NOAEL

Remarks:

for the REPRODUCTION

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Key result

Dose descriptor:

NOEL

Remarks:

toxic effect on reproduction organs

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Remarks:

toxic effect on reproduction organs

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

One pup of control group and one pup of the dose level 300 mg/kg/day died after birth – in the period 0/1st day - 4th day.
No differences in development of pups were observed at the control group and at all treated groups.
Statistical evaluation was performed on the number of live pups.
The total numbers of live pups (on the day of parturition/1st day after parturition and the 4th day after parturition) were similar at the control and the dose level 1000 mg/kg. At the dose level 100 mg/kg/day the number was higher, and vice versa at the dose level 300 mg/kg/day, it was slightly lower.
Average numbers of pups per litter were well-balanced at the control and the dose level 100 mg/kg/day. At the dose levels 300 and 1000 mg/kg/day, the average number of pups per litter was decreased compared to the control, however they fell within the normal range of historical control data.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The average weights of the litters and pups at the dose level 100 mg/kg/day were slightly increased against control. At the dose levels of 300 and 1000 mg/kg/day, the average weight of the litter was lower compared to control, but the average body weights of pups were slightly higher compared to control group. No statistically significant changes were found.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopical examination was performed in all pups. Sporadic pathologic findings were recorded in pups of the treated and control groups. The number of pups with macroscopic changes was comparable in the control and 100 and 300 mg/kg dosed groups; an increase in livers with marked structure was noted in the 1000 mg/kg dose group.

Histopathological findings:

no effects observed

Description (incidence and severity):

The microscopical examination was performed in the pups with macroscopic changes: susp. hydrocephalus (litter No. 142 at the dose level 300) - no histopathological changes in cranial cavity; dark spot on testis (litter No. 167 at the dose level 1000) - no histopathological changes on testis and epididymis; oversized teeth (litter No. 127 at the dose level 100) – no histopathological changes and marked structure of liver (litter No. 164 and 171 at the dose level 1000) – extramedullary haemopoiesis was recorded (it is a physiological finding in pups).

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The number of corpora lutea, implantations, number of pups and accompanied weight of litter were slightly but insignificantly influenced by the administration of the test substance (decrease esp. at dose levels 300 and 1000 mg/kg/day). Sex ratio, average weight of pups and postnatal development of pups were unaffected. Macroscopic abnormalities were described sporadically in pups of all treated and the control groups.

Key result

Dose descriptor:

NOAEL

Remarks:

for the development

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Reproductive effects observed:

not specified

Conclusions:

A reproduction and development toxicity screening study according to OECD guideline 421 in rats with tin sulfide revealed the following values:
NOAEL for the Reproduction: 1000 mg/kg bw/day,
NOEL for the toxic effect on reproduction organs of males: 300 mg/kg bw/day,
NOEL for the toxic effect on reproduction organs of females: 1000 mg/kg bw/day,
NOAEL for the Development of pups: 1000 mg/kg bw/day.

Executive summary:

A reproduction and development toxicity screening study according to OECD guideline 421 in rats with tin sulfide revealed the following values:

NOAEL for the Reproduction: 1000 mg/kg bw/day,

NOEL for the toxic effect on reproduction organs of males: 300 mg/kg bw/day,

NOEL for the toxic effect on reproduction organs of females: 1000 mg/kg bw/day,

NOAEL for the Development of pups: 1000 mg/kg bw/day.

The irrelevant negative influence of the test substance treatment expressed in males of the highest dose level (limit dose) consisted of an increase in absolute weight of pituitary gland (without histopathological changes) and an effect on the microscopical structure of the testes (sporadic occurrence of degeneration and/or atrophy of germ epithelium, residual bodies in germ epithelium and vacuolation of cytoplasm of spermiogonia) without effect on the spermiogenesis. Moreover, there were no histopathological findings in testes in a 90-day study on tin sulfide. The average number of pups and accompanied weight of the litters varied among groups, but fell withing historical ranges and these differences were not considered to be toxicologically relevant.

Body weight, food consumption, clinical observations, weight of reproductive organs of parental males were not markedly affected by treatment of the test substance. Body weight, food consumption, clinical observations, organ weights and structure of reproductive organs of parental females were also not adversely affected by treatment of the test substance. Reproductive performance - ability of male and female animals to successfully mate and produce viable offspring was unaffected by the test substance treatment. Also sex ratio and development of pups were not changed in treated groups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study on tin sulfide

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key reproduction and development toxicity screening study according to OECD guideline 421 in rats with tin sulfide (Procházková, 2010) revealed the following values:

NOAEL for the Reproduction: 1000 mg/kg bw/day,

NOEL for the toxic effect on reproduction organs of males: 300 mg/kg bw/day,

NOEL for the toxic effect on reproduction organs of females: 1000 mg/kg bw/day,

NOAEL for the Development of pups: 1000 mg/kg bw/day.

The irrelevant negative influence of the test substance treatment expressed in males of the highest dose level (limit dose) consisted of an increase in absolute weight of pituitary gland (without histopathological changes) and an effect on the microscopical structure of the testes (sporadic occurrence of degeneration and/or atrophy of germ epithelium, residual bodies in germ epithelium and vacuolation of cytoplasm of spermiogonia) without effect on the spermiogenesis. The average number of pups and accompanied weight of the litters varied among groups, but fell withing historical ranges and these differences were not considered to be toxicologically relevant.

Body weight, food consumption, clinical observations, weight of reproductive organs of parental males were not markedly affected by treatment of the test substance. Body weight, food consumption, clinical observations, organ weights and structure of reproductive organs of parental females were also not adversely affected by treatment of the test substance. Reproductive performance - ability of male and female animals to successfully mate and produce viable offspring was unaffected by the test substance treatment. Also sex ratio and development of pups were not changed in treated groups.

 

A key 90-day repeated dose toxicity study in rats daily dosed with Tin sulfide was also available (Slais, 2010) - See endpoint 7.5.1. Tin sulfide to rats was safe and well tolerated up to the dose of 1000 mg/kg bw/day, with NOAEL defined at 1000mg/kg. Histological examination did not elicit any changes on the reproductive organs, therefore fertility was not considered to be adversely affected.

 

In an extended one-generation reproductive toxicity study (EOGRTS) (OECD TG 443), the effects of Tin(II)-sulfide were evaluated at dose levels of 100, 300 or 1000 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood. In addition, the reproductive toxicity of the F1 Generation was evaluated until weaning of the F2 Pups.

-General and reproductive toxicity (F0 Generation and F1 Pups):

-General toxicity:

No test item-related premature death or sacrifice and no test item-related changes in behaviour, the external appearance or the faeces were noted for the male and female animals of the F0 Generation.

No differences between the control group and the treatment groups were noted for body weight, body weight gain, and for food and water consumption.

The examination of the haematological parameters, the biochemical parameters, the urinary parameters, the T4 and TSH levels and the sperm parameters revealed no test item-related differences between the control group and the treatment groups.

No test item-related findings were noted during the macroscopic examination at necropsy and the histopathological examination. The determination of the organ weights revealed no test item-related differences between the control group and the treatment groups.

-Reproductive toxicity:

No test item-related influence was noted on the reproductive performance of the parental animals (number and length of oestrous cycles, fertility and gestation index, pre-coital time and gestation length).

No test item-related influence was noted on the prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights). No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.

-Developmental toxicity (F1 - Cohorts 1A and 1B):

No premature death or sacrifice was noted for the Cohort 1A and 1B animals during their post-weaning development until scheduled necropsy.

Furthermore, no changes in behaviour, the external appearance or the faeces were noted for the Cohort 1A and 1B animals.

No differences were noted for the Cohort 1A and 1B animals between the control group and the treatment groups for body weight, body weight gain and food consumption.

No test item-related influence was noted on the sexual maturation of the male and female animals during the combined evaluation of the Cohort 1A and Cohort 1B animals.

The examination of the haematological parameters, the biochemical parameters, the spleen cell population (lymphocyte typing), the urinary parameters, the T4 and TSH levels, the monitoring of the oestrous cycle during a 2 week period, and the sperm parameter for the Cohort 1A animals revealed no test item-related differences between the control group and the treatment groups.

No test item-related findings were noted during the macroscopic examination at necropsy for the cohort 1A and 1B animals.

The weights of the evaluated organs from the Cohort 1A and Cohort 1B animals revealed no test item-related differences between the control group and the treatment groups.

The histopathological examination, including a quantitative evaluation of the number of corpora lutea and follicles of the ovaries, which was performed for the Cohort 1A animals revealed no test item-related findings.

-Reproductive toxicity (F1 - Cohort 1B):

No test item-related influence was noted on the reproductive performance of the parental Cohort 1B animals, regarding the number and length of the oestrous cycles, the fertility index, the gestation index, the pre-coital time and the gestation length.

The prenatal development of the F2 Pups (number of resorptions, stillbirths and live born pups) and their postnatal development until weaning (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were not affected by the test item. Furthermore, no malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.

 

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

F0 Generation:

-General toxicity

NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

-Reproductive toxicity

1. a) adverse effects on the reproductive parameters of the parental females: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day


2. b) adverse effects on the prenatal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day


3. c) adverse effects on the post-natal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

F1 Generation:

-Developmental toxicity (Cohorts 1A + Cohort 1B)

NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

-Reproductive developmental toxicity (Cohort 1B)

1. a) adverse effects on the reproductive parameters of the parental females: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day


2. b) adverse effects on the prenatal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day


3. c) adverse effects on the post-natal development of the pups: NOAEL above 1000 mg Tin(II)-sulfide/kg b.w./day

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study was available in rats (OECD TG No. 414). Tin(II)-sulfide was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy. The NOAEL was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams. The NOAEL for the fetal organism was above 1000 mg Tin(II)-sulfide/kg bw/day. Under the conditions of the study, Tin(II)-sulfide did not show any embryotoxic potential in rats.

 

A prenatal developmental toxicity study was available in rabbits (OECD TG No. 414). Tin(II)-sulfide was administered orally to female rabbits at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 28th day of pregnancy. Under the present test conditions, the test item-related NOAEL was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams. The NOAEL for the fetal organism was also above 1000 mg Tin(II)-sulfide/kg bw/day. Under the conditions of the study, Tin(II)-sulfide did not show any teratogenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2020 to 23 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Commission Regulation (EU) No. 260/2014 adopted 24 January 2014, published in the Official Journal of the European Union L81, dated 19 March 2014

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Thomas Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Age at study initiation: on day 0 of gestation: 4 to 5 months
- Weight at study initiation: on day 6 of gestation: 3.84 kg to 5.12 kg
- Fasting period before study: not specified
- Housing: The dams were kept separately in cages with plastic floors (with an area of approx. 0.54 m2)
- Diet (e.g. ad libitum): Commercial ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany served as food. This food was offered daily ad libitum. Food residue was removed and weighed. Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL twice a year. Certificates of analysis of the content and for contaminants were provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water (e.g. ad libitum): Tap water (in drinking bottles) was offered ad libitum.
Samples of drinking water are taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany) and periodic analyses are performed according to the 'Deutsche Trinkwasserverordnung 2001 [German Regulations on drinking water 2001] by LUFA-ITL.
In addition, drinking water samples taken at the Test Facility 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: The animals arrived with the status of GD 2 to 4, start of dosing was on GD 6.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 19.5°C and 23.0°C
- Humidity (%): between 45% and 65%. Temporarily increased relative humidity of up to 80% were noted. These were caused for example during the cleaning procedure and are dealt with in SOPs.
- Air changes (per hr): The ventilation rate of the animal room was between fifteen to twenty air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 1st date of conception: 04 August 2020; First administration: 10 August 2020
To: Termination of the in-life part: 15 October 2020

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous hydroxypropylmethylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared on every administration day.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume (5 mL/kg bw) once daily from the 6th to the 28th day of gestation. The administration formulations were continuously agitated by stirring throughout the entire administration procedure to ensure homogeneity.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the stability, homogeneity, and concentration of the test item-vehicle formulations were monitored


VEHICLE: 0.5 % aqueous hydroxypropylmethylcellulose gel
- Justification for use and choice of vehicle (if other than water): test item is not soluble in water; therefore suspension was made.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day administration volume
- Lot/batch no. (if required): Lot no.: 18D04-B03, Supplier: Fagron Services B.V, 1911 DB UITGEEST, The Netherlands

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of approximately 10 mL each were taken at the following times and stored at 20°C ± 10% until dispatch:
-At start of dosing (GD 6):
• Concentration and stability: Immediately after preparation 3 samples; 8 hours storage of formulation at room temperature: 3 samples; 24 hours storage of formulation at room temperature: 3 samples.
• Homogeneity: At the start of administration: 3 samples; During (middle) administration: 3 samples; Before administration to the last animal: 3 samples
- At the end of the dosing period - at a time when the majority of animals was dosed:
• Concentration: During treatment always before administration to the last animal: 3 samples
Sum of all samples: 21
The samples as well as 1 x 10 mL of the vehicle and 2 x 1 g of the test item were labelled with the study number, species, type of sample, concentration, sampling time and date and stored at -20°C ± 10% until dispatch to WeylChem InnoTec GmbH.
The 21 formulation samples were dispatched on dry ice on 21 October 2020 to Test Site 1 (WeylChem InnoTec GmbH, Alt-Fechenheim 34, 60386 Frankfurt am Main, Germany) for analysis.
The results of the test item-formulation analyses were compiled by Provivo:
-Concentration:
• Immediately after preparation: 92% - 95% nominal concentration
• before administration to the last animal at the end of the dosing period (test day 43): 87% - 101% nominal concentration
-Stability:
• 8h after preparation: 88% - 94% nominal concentration
• 24h after preparation: 91% - 99% nominal concentration
-Homogeneity:
• before administration to the first animal: 91% - 98% nominal concentration
• during administration to the animals: 91% - 99% nominal concentration
• before administration to the last animal: 92% - 97% nominal concentration
The measured actual concentrations of the test item in the test item vehicle-mixtures were between 87% and 101% of the nominal concentrations, indicating correctly prepared, stable and homogeneous formulations.

Details on mating procedure:

For this experiment, sexually mature, purebred female artificially inseminated rabbits (New Zealand White) were used. The state of health of the animals was checked by the breeder prior to the start of the study. The animals arrived with the status of GD 2 to 4, start of dosing was on GD 6.
(day of conception is day 0 of pregnancy)

Duration of treatment / exposure:

Day 6 to 28 of gestation

Frequency of treatment:

Once daily

Duration of test:

23 administration days from gestation day 6 to 28. Laparotomy on gestation day 29

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

based on test material (at least 98% purity)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

based on test material (at least 98% purity)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

based on test material (at least 98% purity)

No. of animals per sex per dose:

102 females in groups 1 to 4:
Treated animals
Group 1 : 26 females
Groups 2 and 3: 24 females/group
Group 4: 28 females
Evaluated litters with viable fetuses
Group 1: 20 litters
Group 2: 18 litters
Group 3: 19 litters
Group 4: 20 litters

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in agreement with the Sponsor based on the results of a dose-range-finding for a prenatal developmental toxicity study which was conducted in pregnant rabbits with Tin(II)-sulfide dosed at 100, 300 and 1000 mg/kg bw/day (LPT Study No. 37283), as well as a prenatal developmental toxicity study according to OECD guideline 414 conducted in rats with Tin(II)-sulfide at 100, 300 and 1000 mg/kg bw/day (LPT Study No. 37282).
During the dose-range-finding study in rabbits (LPT Study No. 37283), one control animal was excluded from the study after misgavage and premature death. No changes in behavior, the external appearance and the appearance of the faeces were noted for the surviving animals. No test item-related changes were noted for body weight and food consumption. A slightly reduced body weight (approx. 9% below the mean of the 2 evaluated control animals, statistically not significant) was noted at 1000 mg/kg. However, this reduction was already noted before the start of dosing (pre-dose on gestation day 6). This is in the range of normal variability and due to the small number of animals in each group. There was no indication on teratogenic properties of the test item. The incidence of one malpositioned kidney is considered to be coincidental. The same applies to the incidence of one malrotated fore paw, a variation which is known to occur spontaneously in rabbits. Dark liquid contents in the gastro-intestinal region were noted in three fetuses of the same litter and are considered to be an artefact due to the process of laparotomy.
In the rat prenatal developmental toxicity study (LPT Study No. 37282), no premature death was noted for any dose group. No test item related teratogenic activity or test item related toxicity on the fetal organisms was noted. The NOAEL for the dams was at 1000 mg/kg bw. For all dams dosed with 300 or 1000 mg/kg bw/day, dark discolored faeces were noted. However, the dark discolored faeces were considered to be due to the grey color of the administered test item and therefore, of no toxicological relevance. No test item-related changes were noted in the macroscopic examination during laparotomy.
- Rationale for animal assignment (if not random): The animals were assigned to the test groups by body weight using a Provantis® -generated randomization scheme.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: In addition to the detailed clinical observations, 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.
Further checks were made early in the morning of each working day and again in the afternoon to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were individually observed before and after dosing at each time of dosing for any signs of behavioral changes, reaction to treatment or all signs of overt toxicity.
Immediately after administration any signs of illness, or reaction to treatment (including changes in fecal consistency, fecal output) were recorded. In case of changes the animals were observed until the symptoms disappeared.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rabbit was recorded on the day of delivery (used for randomization), followed by daily weighing starting on gestation day 6 - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29). Furthermore, the carcass weight and the net weight gain from day 6 are given.
Carcass weight = Terminal body weight minus uterine weight
Net weight change from GD 6 = Carcass weight minus body weight on GD 6
These values are stated in the report.
The body weight values of pregnant and non-pregnant females were not combined.
These measurements were also used for calculating the daily amount of test item to be administered.

FOOD CONSUMPTION AND COMPOUND INTAKE ( no feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each rabbit was recorded. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.
The relative food consumption (g/kg bw/day) was calculated using the following formula:
Daily food consumption [g/kg bw/day] = Total food intake in g/day / Body weight in kg

WATER CONSUMPTION AND COMPOUND INTAKE (no drinking water study):
Daily monitoring by visual appraisal of the drinking water consumption was maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug effects, a macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopic findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: all per litter. All fetuses (dead and alive) were inspected externally for damages, especially for malformations
- Soft tissue examinations: Yes: all per litter. Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discoloration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages.
- Skeletal examinations: Yes: all per litter. The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Head examinations: Yes: half per litter. The head was removed from 50% of the fetuses and fixed in BOUIN's solution. An examination according to WILSON was carried out, inspecting the internal head structures (e.g. eyes). The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.
- Anogenital distance of all live rodent pups: No

Fetal examinations:
The fetuses were removed and the following examinations performed:
a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations or abnormal appearance (e.g. size, colour, shape).
b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
d) Number and size of resorptions were determined.
e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) The gravid uterus weight was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations
(i) The fetuses were sacrificed with oral administration of approx. 50 μL/fetus pentobarbital.

Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages
The head was removed from 50% of the fetuses and fixed in BOUIN's solution. An examination according to WILSON was carried out, inspecting the internal head
structures (e.g. eyes).
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red
(according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).

Evaluation / Parameters
Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distributions in the uterine horns
- absolute number
Resorptions
- Number of early and late resorptions are differentiated
and counted per dam
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- number of early resorptions (< 2 g)
- number of late resorptions (> 2 g)
Weight of placentae
- individual data per fetus
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group
Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group
Fetuses
- number and % per dam (alive)
- number and % per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- sex ratio per litter
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- mean % per sex and group
Runts
- number per dam
- mean per group
Dead fetuses
- number per dam
- mean per group
Malformed fetuses
- individual data per fetus
- type of malformation: number and incidence (%) per group and litter
- number of affected fetuses per group
Total malformation rate [%] = malformed fetuses per group x 100 / fetuses per group
Fetuses with variations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- Number of affected fetuses per group
Total variation rate [%] = fetuses per group with variations x 100 / fetuses per group
Fetuses with retardations
- individual data per fetus
- type of retardation: number and incidence (%) per group and litter
- Number of affected fetuses per group
Total variation rate [%] = fetuses per group with retardations x 100 / fetuses per group

Statistics:

-Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis® using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
-Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis® (maternal macroscopic findings at necropsy or findings during the external macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).

Indices:

Indices of pre-implantation loss and post-implantation loss:
Pre-implantation loss [%] per group = [(corpora lutea – implantations) / corpora lutea] x 100
Post-implantation loss [%] per group = [(implantations – living fetuses) / implantations] x 100
Pre-implantation loss [%] mean per litter = Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group
Post-implantation loss [%] mean per litter = Sum of post-implantation losses per litter in a group [%] / Number of litters in a group

Historical control data:

Provivo Background Data: Summarized results of the 14 last embryotoxicity studies in rabbits (New Zealand White) performed at Provivo in the years 2013 to June 2017.
-General reproductive indices
-Skeletal retardations
-Skeletal variations
-External/internal variations
-Soft tissue variations of the fetal head
-External/internal malformations
-Visceral malformations of the fetal head

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in behavior, external appearance or faeces were noted for the treatment groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related death was noted for any dose group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted for the body weight and body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted for the food consumption.

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 were noted for the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes in behavior were noted for the treatment groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in the gravid uterus weight and the carcass weight (terminal body weight minus gravid uterine weight) in comparison to the control group were noted for the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day). The slight but not statistically significant decrease for the gravid uterus weight of the intermediate dose group was due to a slightly lower fetal weight together with a slightly lower number of fetuses per dam. This is regarded to be spontaneous.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related pathological findings were observed at necropsy in any of the dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

-Clinical signs:
No changes in behavior, external appearance or faeces were noted for the control dams or for the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day which survived until laparotomy.
Animal no. 73 displayed a haemorrhagic vagina on GD 26 one day before it aborted.
-Mortality:
No test item-related premature deaths were noted in the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
Premature death and/or humane sacrifice after misgavage occurred in individual animals in all dose groups and in the control group: In total, four dams were affected in the low dose group, two dams in the intermediate dose group and one dam each at the high dose level and in the control group.
The misgavages were most probably caused by stress during the administration procedure. As pregnant rabbits are known to be stress-prone and restless during handling, misgavages can occur even with experienced staff.
No changes were noted for the behavior, external appearance or the faeces. However, some of the prematurely deceased/sacrificed animals revealed distinctly reduced food consumption or refusal of food intake and a slightly reduced body weight on the days prior to their death/sacrifice.
-Body weight and weight changes:
No test item-related changes in body weight were noted for the dams after oral treatment with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day.
The difference for the body weight between the control group and the dose group ranged between +1.1% and -3.4% of the control value.
In accordance with the body weight, no test item-related differences between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day) were noted for the body weight gain.
The slight but constantly lower body weight in the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day), led to a not statistically significantly lower body weight gain during the study period from GD 6 to GD 29, that was considered to be not test item-related.
The body weight of the examined dams was not distinctly influenced by differences in the gravid uterus weight. The differences of -2.7%, -3.3% or -2.0% for the low, intermediate and high dose group noted for the live body weight on GD 29 were in good agreement with the differences in the carcass weights (-3.6%, -2.1% or 3.2%).
No test item-related changes between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day) were noted for the net body weight gain (without gravid uterus) between gestation day 6 and gestation day 29.
-Food consumption (no feeding study):
No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day.
From GD 13 onwards, a constantly lower body weight was noted for the dose groups in comparison to the control group that ranged between -1.1% and 27.8% of the value of the control group. The constantly lower food consumption for the dose groups could be due to a palatability effect. However, no dose-response relationship was present and no statistically significant differences were noted. Also, the food consumption during the whole dosing period from GD 6 to GD 29 did not show any relevant differences. Therefore, the constantly lower food consumption was considered to be not test item-related.
-Water consumption (no drinking water study):
No test item-related changes in the consumption of drinking water were noted for the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day by visual appraisal.
-Behaviour (functional findings):
No changes in behavior were noted for the control dams or for the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day which survived until laparotomy.
-Organ weight findings including organ/body weight ratios:
No test item-related changes in the gravid uterus weight and the carcass weight (terminal body weight minus gravid uterine weight) in comparison to the control group were noted for the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day). The slight but not statistically significant decrease for the gravid uterus weight of the intermediate dose group was due to a slightly lower fetal weight together with a slightly lower number of fetuses per dam. This is regarded to be spontaneous.
-Gross pathological findings:
No test item-related pathological findings were noted during macroscopic inspection of the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day at necropsy.
Discolorations of the liver (pale or yellow/brown discolored) were noted for 6 low dose animals (2 of those with laparotomy on GD 29 and 4 were found dead or were prematurely sacrificed) and for one dam of the high dose dam (abortion). However, as no dose-response relationship was present the discolorations of the livers were considered to be not test item-related.
The discolorations of the lungs, the thoracic or abdominal cavity filled with liquid that were noted for the prematurely deceased/sacrificed animals across all groups including the control group were considered to be due to the misgavage. Discolorations noted for animals that were found dead may be due to autolysis.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

An increased number of abortions was noted in the high dose group (1000 mg Tin(II)-sulfide/kg bw/day), however, this finding is considered to be stress-related due a high gastro intestinal volume load caused by a compound with low gastro-intestinal absorption.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related changes were noted for the pre- and post-implantation loss.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was observed on the number of resorptions or the number of live fetuses.
Slight but statistically significant increases compared to the control group (p ≤ 0.05) were noted for late resorptions at 100 mg Tin(II)-sulfide/kg bw/day. However, these incidences are not considered as adverse effects as no corresponding findings were noted at the high dose level (1000 mg Tin(II)-sulfide/kg bw/day) and the incidences were within the range of the Provivo background data.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Slight but statistically significant increases compared to the control group (p ≤ 0.05) were noted for the number of dead fetuses at 300 mg Tin(II)-sulfide/kg bw/day. However, these incidences are not considered as adverse effects as no corresponding findings were noted at the high dose level (1000 mg Tin(II)-sulfide/kg bw/day) and the incidences were within the range of the Provivo background data.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

-Number of abortions:
No abortion was noted in the control group or in the low dose group (100 mg Tin(II)-sulfide/kg bw/day). One intermediate dose dam (no. 64 at 300 mg Tin(II)-sulfide/kg bw/day) aborted on gestation day 27. This is regarded to be within the normal range of variation.
However, three high dose dams (nos. 73, 83 and 84 at 1000 mg Tin(II)-sulfide/kg bw/day) aborted between GD 27 and GD 29. The occurrence of 3 abortions out of 23 pregnant animals was outside the range of the Provivo background data. However, the animals nos. 73 and 83 were noted with very low food consumption or even complete refusal of food intake three days prior to the abortions. Animal no. 84 displayed a strongly decreased food consumption one day before abortion. The reduced food consumption was considered to be due to a high absolute test item application volume causing a high gastro-intestinal volume load caused by a compound with low gastro-intestinal absorption and due to a possible palatability effect leading to stress-related abortions. Therefore, the abortions of these animals were considered to be not directly related to the test item.
-Pre- and post-implantation loss:
No test item-related changes were noted for the pre- and post-implantation loss.
-Total litter losses by resorption:
-Early or late resorptions:
No test item-related differences for the numbers of corpora lutea, implantation sites or fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day). Furthermore, no test item-related influence was observed on the number of resorptions or the number of live fetuses.
Slight but statistically significant increases compared to the control group (p ≤ 0.05) were noted for late resorptions at 100 mg Tin(II)-sulfide/kg bw/day. However, these incidences are not considered as adverse effects as no corresponding findings were noted at the high dose level (1000 mg Tin(II)-sulfide/kg bw/day) and the incidences were within the range of the Provivo background data.
-Dead fetuses:
Slight but statistically significant increases compared to the control group (p ≤ 0.05) were noted for the number of dead fetuses at 300 mg Tin(II)-sulfide/kg bw/day. However, these incidences are not considered as adverse effects as no corresponding findings were noted at the high dose level (1000 mg Tin(II)-sulfide/kg bw/day) and the incidences were within the range of the Provivo background data.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No test item-related differences were observed for the fetal weights.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related deaths of fetuses were noted for of the any dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day). Three dead fetuses (nos. 49-3f, 59-1f and 71-11m) were noted in the intermediate dose
group (300 mg Tin(II)-sulfide/kg b.w./day). This incidence was considered to be
spontaneous as no corresponding findings were noted at the high dose level and the
incidence of three dead fetuses per group was within the range of the dose groups of the
Provivo background data.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related changes were noted for the sex distribution.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the mean fetal weights after administration of 100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day. No test item-related differences for the numbers of corpora lutea, implantation sites or fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable

External malformations:

no effects observed

Description (incidence and severity):

External gross inspection revealed no test item-related differences between the control group and the dose groups.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal skeletal examination according to DAWSON revealed no test item-related differences between the control group and the dose groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal examination of the internal organs according to WILSON revealed no test item-related differences between the control group and the dose groups. Multiple malformations for two fetuses each were noted for the control group and for the
low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg b.w./day) in form of a
shortened mandible, a cleft lip and a cleft palate. However, as also two control animals
were noted with these malformations, shortened mandible, cleft lip and cleft palate were
considered to be not test item-related.

Description (incidence and severity):

No statistically significant differences for the placental weights were noted between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Details on embryotoxic / teratogenic effects:

-Fetal body weight changes:
No test item-related influence was noted on the mean fetal weights after administration of 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day.
No test item-related effect was noted for the number of runts. In total, 24 runts were noted at laparotomy: 11 runts were noted for the control group, 7 runts were noted for the low dose group (100 mg Tin(II)-sulfide/kg bw/day) and 3 runts each for the intermediate and high dose groups (300 or 1000 mg Tin(II)-sulfide/kg bw/day).
-Reduction in number of live offspring:
No test item-related deaths of fetuses were noted for of the any dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
Three dead fetuses (nos. 49-3f, 59-1f and 71-11m) were noted in the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day). This incidence was considered to be spontaneous as no corresponding findings were noted at the high dose level and the incidence of three dead fetuses per group was within the range of the dose groups of the Provivo background data.
-Changes in sex ratio:
The sex distribution of the fetuses in the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day) was comparable to the control fetuses. Slight differences observed were within the biological variability.
-Changes in litter size and weights:
-Anogenital distance of all rodent fetuses:
Not examined
-Changes in postnatal survival:
Not applicable
-External malformations:
No test item-related pathological changes were noted for any of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
External examination revealed malformations and variations for the fetuses of the control group and the low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg bw/day).
One fetus of a control dam was found only rudimentary developed: The head was absent and only the spinal cord, liver, intestines and the tail were identified.
Furthermore, 6 fetuses of 2 control dams displayed malformations in form of a shortened mandible and a cleft of the lip and palate. These observations were also noted for four fetuses of two low dose dams (100 mg Tin(II)-sulfide/kg bw/day) and three fetuses of one high dose dam (1000 mg Tin(II)-sulfide/kg bw/day). All fetuses noted with malformations had a weight that was below the group mean weight of the respective sex.
The fetuses that were noted with a shortened mandible and the cleft of the lip and palate displayed also a uni- or bilateral flexure of the forepaws. Flexures of forepaws were noted also for a few fetuses as solitary findings. In total, flexure of forepaws was noted for 9 fetuses of 4 different control litters, for 5 fetuses of 3 low dose litters and for 4 fetuses of 2 high dose litters.
No gross alterations were noted for the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day).
The mean percentages per group of the malformations and variations for the dose groups were within the range of the Provivo background data. As also no dose-response relationship was present and no fetal alterations were noted for the intermediate dose group, the observed malformations and variations were considered to be spontaneous.
-Skeletal malformations:
No alterations in the form of malformations were noted during skeletal examinations of the fetuses according to DAWSON for the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
In the control group, the male fetus no. 1-6 that was observed to be only rudimentary developed was noted with multiple malformations in form of the absence of the skull, all left ribs, the thoracic vertebral arches, the caudal vertebral bodies and the left pectoral girdle. Furthermore, the right ribs were partly wavy and fused, the thoracic vertebral bodies were reduced in size, the lumbar and sacral arches were misaligned, the pelvic girdle as well as the right pectoral girdle were rudimentary and a spina bifida was noted. In addition, four further control fetuses (nos. 6-1, 6-13, 23-4 and 23-9) were noted with a small skull, the os nasale being beak-shaped, and a reduction in size of the maxilla as well as for nos. 23-4 and 23-9 also of the mandible. However, as no skeletal malformations were noted at any dose level, the observed malformations were considered to be spontaneous.
No test item-related difference between control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day) was noted for the incidence of the skeletal variations.
The skeletal variations observed during examination according to DAWSON were related to the skull (fontanelle enlarged or distinct areas not ossified), the sternum (sternebra(e) bipartite, misaligned, misshapen or fused to a slight degree), to the limbs (femur fused to tibia or humerus fused to ulna) or the rib(s) (accessory 13th ribs (uni- or bilateral), fused to a slight degree or short).
In the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day), a statistically significant increase was noted for the incidence of the sternebra(e) being misshapen (p ≤ 0.05). As the incidence of the misshaped sternebra(e) was within the Provivo background data range the observation was considered to be not test item-related.
The retardations observed during skeletal examination (according to DAWSON) were related to the skull (hyoid unossified), the sternum (sternebra(e) incompletely ossified, unossified or reduced in size), the thoracic vertebral bodies (bipartite), the limbs (absence of ossification in metacarpalia/metatarsalia 2 to 5, metacarpalia/metatarsalia not ossified, phalanges not ossified, femur not ossified, femur reduced in size, fibula not ossified, humerus not ossified, ulna not ossified or radius not ossified), the caudal vertebral bodies (bipartite), the os ischii (unossified), the os pubis (unossified) and the talus (reduced in size or unossified).
Statistically significant differences for the incidences of skeletal retardations were noted for the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). However, as nearly all of these incidences were within or marginally above the range of the Provivo background data or were decreased or no dose-response relationship was present, they were considered to be not test item-related.
-Visceral malformations:
A macroscopic internal examination was performed to detect gross alterations of the internal organs. No malformations or variations were noted during the internal examination of the fetuses of the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
Also the external inspection of the brain in 50% of the fetuses revealed no changes for any of the fetuses after opening of the cranium and removal of the brain.
No test item-related alterations in the form of malformations were noted during soft tissue examinations of the fetal head according to WILSON at any tested dose level (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
Multiple malformations for two fetuses each were noted for the control group and for the low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg bw/day) in form of a shortened mandible, a cleft lip and a cleft palate. However, as also two control animals were noted with these malformations, shortened mandible, cleft lip and cleft palate were considered to be not test item-related.
Soft tissue variations that were observed during the examination of the fetal head according to WILSON were in the form of dilatations of the 4th cerebral ventricle, subdural haemorrhages in the meninges, distinct or semicircular cystic areas or haemorrhages in the cerebrum or cystic areas in the cerebral hemisphere. No test item-related differences in the incidences of the observed variations of the fetal head were noted between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
In the low and intermediate dose groups (100 or 300 mg Tin(II)-sulfide/kg bw/day), a statistically significantly decreased number of cystic areas in the cerebral hemisphere was noted. However, decreased numbers of cystic areas in the cerebral hemispheres were considered to be not test item-related and also no dose-response relationship was present.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: highest dose tested

Key result

Developmental effects observed:

no

Conclusions:

Under the present test conditions, the test item-related no-observed-adverse-effect level (NOAEL) was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams.
The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 1000 mg Tin(II)-sulfide/kg bw/day.
Under the conditions of the study, Tin(II)-sulfide did not show any teratogenic potential.

Executive summary:

In this prenatal developmental toxicity study, the test item Tin(II)-sulfide was administered orally to female rabbits at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 28th day of pregnancy.

Under the present test conditions, the test item-related no-observed-adverse-effect level (NOAEL) was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams.

No test item-related death was noted for any dose group.

No changes in behavior, external appearance or faeces were noted for the treatment groups.

No test item-related differences were noted for the body weight, body weight gain and the food consumption.

No test item-related pathological findings were observed at necropsy in any of the dose groups.

An increased number of abortions was noted in the high dose group (1000 mg Tin(II)-sulfide/kg bw/day), however, this finding is considered to be stress-related due a high gastro intestinal volume load caused by a compound with low gastro-intestinal absorption.

 

The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 1000 mg Tin(II)-sulfide/kg bw/day.

No test item-related differences were noted for the number of corpora lutea, implantation sites or fetuses.

No test item-related changes were noted for the pre- and post-implantation loss or the sex distribution.

Also no test item-related differences were observed for the fetal and placental weights.

External and internal gross inspection as well as fetal skeletal examination according to DAWSON and also the examination of the internal organs according to WILSON revealed no test item-related differences between the control group and the dose groups.

Under the conditions of the study, Tin(II)-sulfide did not show any teratogenic potential.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 October 2019 to 13 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 24 January 2014, published in the Official Journal of the European Union L81, dated 19 March 2014

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Tribotecc, batch S903028
- Expiration date of the lot/batch: 31 October 2020
- Purity test date: 21 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C, stored in a dry place in tightly closed container.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The measured actual concentrations of the test item in the test item vehicle-mixtures were between 83% and 94% of the nominal concentrations, indicating correctly prepared, stable and homogeneous formulations.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were freshly prepared on every administration day.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation. The application formulation was continuously agitated by stirring throughout the entire administration procedure.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the homogeneity and concentration of the test item mixture were monitored.
The male rats for mating remained untreated.

Species:

rat

Strain:

other: CD®/ Crl:CD (SD)

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
- Age on day 0 of pregnancy: 58 - 67 days
- Weight on day 0 of pregnancy: 206.2 g - 281.7 g
- Fasting period before study: not specified
- Housing: The animals 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.
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 cleaned and changed 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.
The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered daily ad libitum.
Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
Samples of drinking water are taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany), and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on drinking water 2001].
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: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range)
- Air changes (per hr): between fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
-From:
Start of mating: 07 October 2019
First administration: 14 October 2019
-To: Termination of the in-life part: 07 November 2019

Vehicle: 0.5 % aqueous hydroxypropylmethylcullulose gel (Lot no.: 18D04-B03, Supplier: Fagron Services B.V, Uitgest, The Netherlands)
Administration volume 10 mL/kg bw/day
Day 6 to 20 of gestation

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous hydroxypropylmethylcullulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared on every administration day.
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation. The application formulation was continuously agitated by stirring throughout the entire administration procedure.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the homogeneity and concentration of the test item mixture were monitored.
The male rats for mating remained untreated.


VEHICLE 0.5 % aqueous hydroxypropylmethylcullulose gel
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: The amount of the test item was adjusted to the animal's current body weight daily.
- Amount of vehicle (if gavage): 10 mL/kg bw/day (vehicle or vehicle with test item at appropriate concentration)
- Lot/batch no. (if required): Lot no.: 18D04-B03, Supplier: Fagron Services B.V, Uitgest, The Netherlands

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C ± 10% until dispatch:
*At start of dosing:
-Analysis of stability and concentration: Immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature. (3 sample/test item group).
Number of samples: 3 x 3 = 9 (Sampling date: 15 October 2019)
-Homogeneity: At the start of treatment, during (middle) administration and before administration to the last animal of the test item group. (3 samples/test item group).
Number of samples: 3 x 3 = 9 (Sampling date: 15 October 2019)
* At the end of the dosing period, at a time when the majority of the animals was dosed:
-Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 1 x 3 = 3 (Sampling date: 29 October 2019)
* Sum of all samples: 21
The samples were labelled with the study number, species, type of sample, concentration, sampling time, date and test day.
The samples were dispatched on dry ice on 11 November 2019 to: WeylChem InnoTec GmbH, Alt-Fechenheim 34, 60386 Frankfurt am Main, Germany.
The measured actual concentrations of the test item in the test item vehicle-mixtures were between 83% and 94% of the nominal concentrations, indicating correctly prepared, stable and homogeneous formulations.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner.
- Further matings after two unsuccessful attempts: This procedure was repeated until 25 mated dams were available for each groups.
- Verification of same strain and source of both sexes: Sexually mature ('proved') male rats of the same breed served as partners.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
The non-pregnant rats were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:

Day 6 to 20 of gestation

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Treated animals: Groups 1-4: 25 females per group
Evaluated litters: Groups 1-4: 20 litters per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Tin(II)-sulfide has been tested for reproductive toxicity using OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test in Wistar rats dosed by oral gavage at 0, 100, 300, 1000 mg/kg bw/day in 0.5% methylcellulose in water (CETA study, 2010). Males and females were dosed from 2 weeks prior to mating, during the mating period and for pregnant females during pregnancy and till 3rd day of lactation, whereas males were dosed after mating period for a total of 42 days. The NOAEL for the REPRODUCTION was established at 1000 mg/kg bw/day, whereas a NOEL (No Observed Effect Level) for the toxic effect on reproduction was established at 300 mg/kg bw/day in males and 1000 mg/kg bw/day in females. The NOAEL for the DEVELOPMENT of pups was established at 1000 mg/kg bw/day. In males dosed at 1000 mg/kg bw/day (limit dose), an increase in absolute weight of the pituitary gland (without histopathological changes) and an effect on the microscopical structure of the testes (sporadic occurrence of degeneration and/or atrophy of germ epithelium, residual bodies in germ epithelium and vacuolation of cytoplasm of spermiogonia) were observed.
The average number of pups and accompanied weight of the litters varied among groups, but fell within historical ranges and these differences were not considered to be toxicologically relevant.
Body weight, food consumption, clinical observations, weight of reproductive organs of parental males were not markedly affected by treatment with the test substance. Body weight, food consumption, clinical observations, organ weights and structure of reproductive organs of the parental females were also not adversely affected by treatment with the test substance. Reproductive performance - ability of male and female animals to successfully mate and produce viable offspring - was unaffected by the test substance treatment. Also sex ratio and development of pups were not change in treated groups.
The dose levels were further selected in agreement with the Sponsor based on the results of a dose-range finding study in 3 pregnant CD® / Crl:CD(SD) rats per dose group dosed with Tin(II)-sulfide by oral gavage from days 6 to 20 of gestation at 0, 600 and 1000 mg/kg b.w. in 0.5% aqueous hydroxyprpoylmethylcellulose gel (LPT Study No. 37281). In this study, 3 females with live fetuses were obtained during laparotomy from groups 1 to 3 (0, 100 or 600 mg/kg bw) and 2 females with live fetuses and one non-pregnant female were noted in group 4 (1000 mg/kg bw) at laparotomy. For evaluation, 3 females with live fetuses were considered in groups 1 to 3 and 2 females with live fetuses in group 4. No changes in behaviour and the external appearance were noted. No differences were noted in body weight and food consumption. No influence on the development of the fetuses was noted (number of resorptions, fetal and placental weight). The external macroscopic examination of the fetuses revealed no malformations or variations.
Based on the existing reproductive testing and information from the dose-range finding study, doses of 100, 300 and 1000 mg/kg bw/day by oral gavage were proposed for the main OECD 414 study.
- Rationale for animal assignment (if not random): Four (4) groups of pregnant rats were established, each obtained from matings which were carried out on a daily basis. Vaginal lavages were taken each morning. Day 0 of pregnancy was the day on which sperm was found in the vaginal lavage. When positive, the animals were assigned to the test groups by mating day using a Provantis® -generated randomization based on the body weight of the animals (Provantis® Integrated preclinical software, version 10.2.1, Instem LSS Ltd.).

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting 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.
Special attention was paid to ascertain of there were any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. gestation day 0-3, 3 6, 6-9, 9-12, 12 15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.
These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

FOOD CONSUMPTION (no feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg bw/day) was calculated using the following formula: Daily food consumption [g/kg bw/day]= Total food intake in g / Body weight in kg

WATER CONSUMPTION (no drinking water study): Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
The ovaries, thyroid and the uteri were removed. Following organswere weighed: Thyroid (1) (including parathyroids), Gravid uterus including cervix
In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. The thyroid and any organs with macroscopic findings of all dams (including deceased or prematurely deceased or prematurely sacrificed animals) were fixed in 7% neutral buffered formalin. Organs to be preserved were: Thyroid (2) including parathyroids, Gross lesions.

OTHER:
*Thyroid hormone (T3, T4, TSH) determination: In order to obtain approximately 2 aliquots of 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight following a randomisation scheme. Blood samples were taken always at the same time of day (approximately from 7:00 a.m. to 10:00 a.m.).The samples were divided into aliquots, if possible, and stored at -20°C ± 10% at LPT until analysis using ELISA.The T3, T4 and TSH ELISA (commercial kits: IBL International GmbH, instrument: Tecan Sunrise) was conducted at LPT.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes (number per dam, absolute number per group, mean per group)
- Number of implantations: Yes (number per dam, distributions in the uterine horns, absolute number per group, mean per group)
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter: All fetuses (dead and alive) were inspected externally for damages, especially for malformations
- Skeletal examinations: Yes:half per litter: 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and
sternum) were opened and the location, size and condition of the internal organs were determined.
Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations)
- Soft tissue examinations: Yes: half per litter : The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined
according to WILSON. The fetuses were allocated to the evaluation of DAWSON or WILSON on
an alternating basis.
- Head examinations: Not applicable for rat study

Fetal examinations:
The fetuses were removed and the following examinations performed:
a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations or abnormal appearance (e.g. size, colour, shape).
b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
d) Number and size of resorptions were determined.
e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) The ano-genital distance (AGD) of all live fetuses was determined using a scale.
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations.
(i) The fetuses were sacrificed by CO2 inhalation.
(j) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and
sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined
(determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined
according to WILSON. The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
(k) External fetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations).
(l) Indication of incomplete testicular descent/cryptorchidism was noted in male fetuses.

Evaluation / Parameters
Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Resorptions
- number of early and late resorptions per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
- early resorptions < 2 mm
- late resorptions > 2 mm
Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and litter
Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- mean per sex and litter
Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + dead) per sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- male/female ratio (alive + dead)
- % of fetuses alive per litter
- mean % of fetuses alive per litter
Runts
- number per dam
- number per group
Dead fetuses
- number per group
- mean per dam
Malformed fetuses
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter
malformed fetuses per group
Total malformation rate [%] = malformed fetuses per group x 100 / fetuses per group
Fetuses with variations
- type of variation
- individual data per fetus
- number and incidence (%) per group8 and litter
Total variation rate [%] = fetuses per group with variations x 100 / fetuses per group
Fetuses with retardations
- type of retardation
- individual data per fetus
- number and incidence (%) per group11 and litter
Total retardation rate [%] = fetuses per group with retardations x 100 / fetuses per group

Statistics:

*Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or nonnormality 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).
*Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Significantly different data are indicated in the summary tables of the result sections of the report.

Indices:

*Calculation of group indices
Pre-implantation loss [%] = [(Corpora lutea (per group) - implantations (per group)) / Corpora lutea (per group)] x 100

Post-implantation loss [%] = [(Implantations (per group) - living fetuses (per group)) / Implantations (per group)] x 100

*Calculation of mean indices per litter
Pre-implantation loss [%] = Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group

Post-implantation loss [%] = Sum of post-implantation losses per litter in a group [%] / Number of litters in a group

Historical control data:

Summarized results of the 52 last embryotoxicity studies in Sprague-Dawley rats were available in Appendix of the study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No changes in behaviour or the external appearance were noted in the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
Dark discoloured faeces were noted for all dams dosed with 300 or 1000 mg Tin(II)-sulfide/kg b.w./day starting between GD 8 and GD 14 until study termination on GD 21. However, as macroscopic examination revealed no findings for the gastrointestinal tract, the dark discoloured faeces were considered to be due to the oral administration of the grey test item in high dose levels. Therefore, the dark discoloured faeces were considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No premature deaths were noted in the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences in body weight were noted between the dams of the control group and dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
The difference in the body weight between the control group and the dose groups ranged from -2.1% to +0.7% during the whole study period.
In accordance with the body weight, also no test item-related difference between the control group and the animals treated with 100, 300 or 1000 mg Tin(II) sulfide/kg bw/day was noted for the body weight gain from GD 0 to 21 and for the period after the start of dosing (GD 6 to GD 21). The slight difference for the body weight gain of approximately minus 6% in the high dose group compared to the control group is regarded to be still within the normal range of variability.
There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related difference was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
Between GD 10 and GD 11, statistically significant increases were noted for the groups dosed with 100 or 300 mg Tin(II)-sulfide/kg bw/day (6.3% or 7.8% above the value of the control group, statistically significant at p ≤ 0.05 or 0.01). However, as no dose-response relationship was present and as the food consumption was only increased for one day, this transiently higher food consumption was considered to be spontaneous and not test item-related.

Food efficiency:

not examined

Description (incidence and severity):

No test item-related differences in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for thyroid weights. Thyroid hormone concentration No test item-related differences were noted for the serum levels of T3, T4 and TSH. Histopathologic examination of the thyroids revealed no test item-related changes

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related differences to the control group were noted for the relative and absolute thyroid weights of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
No test item-related differences were noted between the gravid uterus weight of the control dams and the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related observations were noted for the dams of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day) during the macroscopic inspection of the organs and tissues.
In the intermediate dose group and the high dose groups, dilatations of the renal pelvis were noted for the right kidney (no. 70, intermediate dose group) or for the left and right kidney (no. 95, high dose group). However, as only one animal per group was affected, the dilatations of the renal pelvis were considered to be not test item-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathologic examination of the thyroids revealed no test item-related changes

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for the serum levels of T3, T4 and TSH in all dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
In the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day), a statistically significantly increased level of T3 (10.8% above the value of the control group, p ≤ 0.05) was noted. However, no difference in the T3 serum levels was noted for the high dose group (1000 mg Tin(II)-sulfide/kg bw/day). Therefore, the increased T3 serum level of the intermediate dose group was considered to be not test item-related.
No test item-related morphological lesions were noted during histopathological examination of the thyroids of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
The thyroid in 80 female control and test item-treated rats did not show any abnormalities. The follicles showed normal follicular cuboidal and flattened epithelial cells and homogeneous eosinophilic colloid. The epithelium showed no mitosis or cell activation. A small number of squamous cell cysts (also known as ultimobrancial remnants) was noted in the control animals and the animals treated with the test item Tin(II)-sulfide.
There was no morphological difference between the controls and the rats treated with the test item.

Details on results:

-Mortality:
No premature deaths were noted in the control group and the dose groups (100,
300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-Behaviour, external appearance, faeces:
No changes in behaviour or the external appearance were noted in the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Dark discoloured faeces were noted for all dams dosed with 300 or 1000 mg Tin(II)-sulfide/kg b.w./day starting between GD 8 and GD 14 until study termination on GD 21. However, as macroscopic examination revealed no findings for the gastrointestinal tract, the dark discoloured faeces were considered to be due to the oral administration of the grey test item in high dose levels. Therefore, the dark discoloured faeces were considered to be of no toxicological relevance.
-Body weight and body weight gain:
Body weight:
No test item-related differences in body weight were noted between the dams of the control group and dose groups (100, 300 or 1000 mg Tin(II)- sulfide/kg b.w./day).
The difference in the body weight between the control group and the dose groups ranged from -2.1% to +0.7% during the whole study period.
Body weight gain from GD 0 or GD 6 to GD 21:
In accordance with the body weight, also no test item-related difference between the control group and the animals treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day was noted for the body weight gain from GD 0 to 21 and for the period after the start of dosing (GD 6 to GD 21).
The slight difference for the body weight gain of approximately minus 6% in the high dose group compared to the control group is regarded to be still within the normal range of variability.
There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups.
-Food and drinking water consumption:
No test item-related difference was noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Between GD 10 and GD 11, statistically significant increases were noted for the groups dosed with 100 or 300 mg Tin(II)-sulfide/kg b.w./day (6.3% or 7.8% above the value of the control group, statistically significant at p ≤ 0.05 or 0.01). However, as no dose-response relationship was present and as the food consumption was only increased for one day, this transiently higher food consumption was considered to be spontaneous and not test item-related.
Drinking water consumption:
No test item-related differences in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
-Thyroid hormone concentrations:
No test item-related differences were noted for the serum levels of T3, T4 and TSH in all dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
In the intermediate dose group (300 mg Tin(II)-sulfide/kg b.w./day), a statistically significantly increased level of T3 (10.8% above the value of the control group, p ≤ 0.05) was noted. However, no difference in the T3 serum levels was noted for the high dose group (1000 mg Tin(II)-sulfide/kg b.w./day). Therefore, the increased T3 serum level of the intermediate dose group was considered to be not test item-related.
-necropsy findings:
No test item-related observations were noted for the dams of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the macroscopic inspection of the organs and tissues.
In the intermediate dose group and the high dose groups, dilatations of the renal pelvis were noted for the right kidney (no. 70, intermediate dose group) or for the left and right kidney (no. 95, high dose group). However, as only one animal per group was affected, the dilatations of the renal pelvis were considered to be not test item-related.
-Histopathology of the thyroids:
No test item-related morphological lesions were noted during histopathological examination of the thyroids of the dose groups (100, 300 or 1000 mg Tin(II)- sulfide/kg b.w./day).
The thyroid in 80 female control and test item-treated rats did not show any abnormalities. The follicles showed normal follicular cuboidal and flattened epithelial cells and homogeneous eosinophilic colloid. The epithelium showed no mitosis or cell activation. A small number of squamous cell cysts (also known as ultimobrancial remnants) was noted in the control animals and the animals treated with the test item Tin(II)-sulfide.
There was no morphological difference between the controls and the rats treated with the test item.
-Thyroid weights:
No test item-related differences to the control group were noted for the relative and absolute thyroid weights of the dose groups (100, 300 or 1000 mg Tin(II)- sulfide/kg b.w./day).
-Gravid uterus weight, carcass weight and body weight gain from day 6:
Gravid uterus weight and Carcass weight:
No test item-related differences were noted between the gravid uterus weight of the control dams and the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Net body weight gain from GD 6 to 21:
Also, no test item-related differences were noted for the net body weight gain from GD 6 to 21 between the dams of the control group and the dams of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
However, a reduced net body weight gain was noted at the high dose level (23.5 % below the control, statistically not significant). This reduction was mainly due to dam no. 83 with a negative net body weight gain of 41.1 g. Small or even negative body weight gains were noted for animal no. 83 on several of the investigated 3-day periods of body weight gain. As such a negative net body weight gain was only noted for 1 of 20 high dosed animals, this was considered to be spontaneous. Moreover, an obviously reduced body weight gain in comparison to the other dams of the high dose group was already noted for animal no. 83 before the start of treatment between gestation days 0 and 3.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortion occurred in the study.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Dead fetuses:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

at least 98% purity

Remarks on result:

other: highest dose tested

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
No runts were noted for the control group and the low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg bw/day).
In the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day), one runt (no. 51-8) was noted. The occurrence of one runt in the intermediate dose group is within the normal range of biological variability and therefore, was considered to be not test item-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead fetus was noted in the control group and in the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of
implantation sites, fetuses, resorptions and the index of pre- and post-implantation
loss) were noted between the dams of the control group and the dams of the
treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day). The d fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-
sulfide/kg b.w./day).

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No test item-related differences to the control group were noted for the fetal anogenital
distance of the dose groups (100, 300 or 1000 mg Tin(II)-
sulfide/kg b.w./day).

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopically visible external malformations were noted for the fetuses of the low and high dose group (100 or 1000 mg Tin(II)-sulfide/kg bw/day) during the external inspection at laparotomy.
In the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day), one fetus (no. 70 10) was noted with multiple malformations of the head in form of a short lower jaw, a short tongue and a small left eye. However, the occurrence of one fetus with malformations was considered to be spontaneous and not test item-related.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day) during the skeletal examination according to DAWSON.
No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day). Skeletal variations were noted for the ribs (less than 13 ribs ossified, ribs short or ribs wavy) and the sternum (bipartite or misaligned to a slight degree).
No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day was noted during skeletal examination according to DAWSON.
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossified or all bodies unossified), the lumbar vertebral bodies (unossified), the sacral vertebral bodies (unossified), the os ischii (incompletely ossified), the os pubis (incompletely ossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5).
An increased incidence of bipartite thoracic vertebral bodies (21 fetuses affected of 8 litters) was noted for the low dose group (100 mg Tin(II)-sulfide/kg bw/day) that was outside the range of the LPT background data. However, as no dose-response relationship was noted (in the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day) even a decreased incidence of bipartite thoracic vertebral bodies in comparison to the control group was present), the observation was considered to be spontaneous. Moreover, as also the number of litters with affected fetuses in the low dose group was not increased in comparison to the control group or the high dose group (for both 7 litters with affected fetuses).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
No malformations were noted for the fetuses of the control group and the fetuses of the low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg bw/day) during the soft tissue examination according to WILSON. In the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day), one fetus (no. 70-10) was noted with multiple malformations in form of a short lower jaw, a short tongue and a small left eye. However, as no malformations were noted in the high dose group, the occurrence of one fetus with malformations in the intermediate dose group was considered to be spontaneous and not test item-related.
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day).
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (hemorrhagic focus/foci).
No unclassified observations were noted for the the low and high dose group (100 or 1000 mg Tin(II)-sulfide/kg b.w./day).
An unclassified observation in form of a thoracic cavity filled with blood was noted for one fetus (no. 18-2) of the control and for three fetuses (nos. 52-11, 54-7 and 73-10) of the intermediate dose group (300 mg Tin(II)-sulfide/kg bw/day). This observation was considered to be a preparation-induced artefact.

Other effects:

no effects observed

Description (incidence and severity):

No cryptorchidism and no testicular malposition was noted during assessment of
the testicular development of the male fetuses of the control group and the dose
groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).

Details on embryotoxic / teratogenic effects:

-Mortality:
No dead fetus was noted in the control group and in the dose groups (100, 300 or
1000 mg Tin(II)-sulfide/kg b.w./day).
-Sex distribution:
No test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-Weight of placentae and fetuses:
The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)- sulfide/kg b.w./day).
-Number of runts:
No runts were noted for the control group and the low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg b.w./day).
In the intermediate dose group (300 mg Tin(II)-sulfide/kg b.w./day), one runt (no. 51-8) was noted. The occurrence of one runt in the intermediate dose group is within the normal range of biological variability and therefore, was considered to be not test item-related.
-Ano-genital distance:
No test item-related differences to the control group were noted for the fetal ano- genital distance of the dose groups (100, 300 or 1000 mg Tin(II)- sulfide/kg b.w./day).
-Macroscopic inspection of the fetuses at laparotomy:
1. External inspection at laparotomy:
No macroscopically visible external malformations were noted for the fetuses of the low and high dose group (100 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the external inspection at laparotomy.
In the intermediate dose group (300 mg Tin(II)-sulfide/kg b.w./day), one fetus (no. 70-10) was noted with multiple malformations of the head in form of a short lower jaw, a short tongue and a small left eye. However, the occurrence of one fetus with malformations was considered to be spontaneous and not test item- related.
2. Gross inspection of the organs and tissues as laparotomy:
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the dose groups (100, 300 or 1000 mg Tin(II)- sulfide/kg b.w./day).
-Testicular development:
No cryptorchidism and no testicular malposition was noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
-Skeletal examination according to DAWSON
1. Skeletal malformations:
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the skeletal examination according to DAWSON.
2. Skeletal variations:
No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
Skeletal variations were noted for the ribs (less than 13 ribs ossified, ribs short or ribs wavy) and the sternum (bipartite or misaligned to a slight degree).
3. Skeletal retardations:
No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day was noted during skeletal examination according to DAWSON.
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossified or all bodies unossified), the lumbar vertebral bodies (unossified), the sacral vertebral bodies (unossified), the os ischii (incompletely ossified), the os pubis (incompletely ossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5).
An increased incidence of bipartite thoracic vertebral bodies (21 fetuses affected of 8 litters) was noted for the low dose group (100 mg Tin(II)-sulfide/kg b.w./day) that was outside the range of the LPT background data. However, as no dose- response relationship was noted (in the intermediate dose group (300 mg Tin(II)- sulfide/kg b.w./day) even a decreased incidence of bipartite thoracic vertebral bodies in comparison to the control group was present), the observation was considered to be spontaneous. Moreover, as also the number of litters with affected fetuses in the low dose group was not increased in comparison to the control group or the high dose group (for both 7 litters with affected fetuses).
-Soft tissue examination according to WILSON
1. Malformations:
No malformations were noted for the fetuses of the control group and the fetuses of the low and high dose groups (100 or 1000 mg Tin(II)-sulfide/kg b.w./day) during the soft tissue examination according to WILSON.
In the intermediate dose group (300 mg Tin(II)-sulfide/kg b.w./day), one fetus (no. 70-10) was noted with multiple malformations in form of a short lower jaw, a short tongue and a small left eye.
However, as no malformations were noted in the high dose group, the occurrence of one fetus with malformations in the intermediate dose group was considered to be spontaneous and not test item-related.
2. Variations:
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 or 1000 mg Tin(II)-sulfide/kg b.w./day).
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (hemorrhagic focus/foci).
3. Unclassified observations:
No unclassified observations were noted for the the low and high dose group (100 or 1000 mg Tin(II)-sulfide/kg b.w./day).
An unclassified observation in form of a thoracic cavity filled with blood was noted for one fetus (no. 18-2) of the control and for three fetuses (nos. 52-11, 54-7 and 73-10) of the intermediate dose group (300 mg Tin(II)-sulfide/kg b.w./day). This observation was considered to be a preparation-induced artefact.
-Abstract and Assessment of fetal alterations:
No test item-related malformations or variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON).
Furthermore, no test item-related retardations (delay in ossification) were noted in any of the treatment groups (100, 300 and 1000 mg Tin(II)-sulfide/kg b.w./day).

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

at least 98% purity

Sex:

male/female

Remarks on result:

other: highst dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1. Overview on reproduction parameters

Parameter		Group 1 Control (n=20)	Group 2 100 mg/kg (n=20)	Group 3 300 mg/kg (n=20)	Group 4 1000 mg/kg (n=20)
Corpora lutea	total mean per dam	323 16.2	306 15.3	327 16.4	314 15.7
Implantation sites	total mean per dam	317 15.9	304 15.2	320 16.0	308 15.4
Resorptions	total mean per dam	12 0.6	10 0.5	12 0.6	14 0.7
Early resorptions	total mean per dam	12 0.6	6 0.3	10 0.5	13 0.7
Late resorptions	total mean per dam	0 0.0	4 0.2	2 0.1	1 0.1
Live fetuses	total mean per dam	305 15.3	294 14.7	308 15.4	294 14.7
Dead fetuses	total	0	0	0	0
Pre-implantation loss [%]	per group mean per dam	1.9 1.8	0.7 0.6	2.1 2.2	1.9 2.3
Post-implantation loss [%]	per group mean per dam	3.8 3.7	3.3 3.1	3.8 3.6	4.5 4.9

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

 

Conclusions:

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg Tin(II) sulfide/kg bw/day.
Under the conditions of the study, Tin(II)-sulfide did not show any embryotoxic potential in rats.

Executive summary:

In this prenatal developmental toxicity study, the test item Tin(II)-sulfide was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams.

No premature death was noted for any dose group.

No test item-related changes in behaviour or external appearance were noted for the treatment groups.

For all dams dosed with 300 or 1000 mg Tin(II)-sulfide/kg bw/day, dark discoloured faeces were noted. However, the dark discoloured faeces were considered to be due to the grey colour of the administered test item and therefore, of no toxicological relevance.

No test item-related changes of the body weight, the body weight gain, the carcass weight and the food consumption was noted for the females of any dose group.

No test item-related pathologic changes were noted for the animals of the dose groups.

No differences for the thyroid weights were noted and histopathologic examination of the thyroids revealed no test item-related changes.

No test item-related increased values were noted for the serum levels of T3, T4 and TSH.

 

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg Tin(II)-sulfide/kg bw/day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No influence was noted on fetal development (ano-genital distance or testicular development of the male fetuses).

No deaths of fetuses and no test item-related malformations, variations or retardations were noted.

 

Under the conditions of the study, Tin(II)-sulfide did not show any embryotoxic potential in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable

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

In a supporting dose-range-finding for a prenatal developmental toxicity study, Tin(II)-sulfide was administered orally to female rats at dose levels of 300, 600 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy (Hansen, 2020). Under the present test conditions, the NOAEL was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams. No prematurely deceased dams were noted during the study. No changes in behaviour or external appearance were noted. No test item-related changes were noted for the body weight, the body weight gain or the food consumption. No pathological changes were noted in the macroscopic examination during laparotomy. The NOAEL for the fetal organism was also above 1000 mg Tin(II)-sulfide/kg bw/day. The reproductive parameters (number of implantation sites, number of resorptions, number of fetuses and sex distribution) were not influenced by the test item. No test item-related influence was noted on the weights of the placentae or the fetuses. No runts were noted in any of the dose groups. No dead fetuses, no malformations and no variations were noted. Based on the data obtained in this dose-range-finding study, the following dose levels are suggested for the main Prenatal developmental toxicity study in rats:

Group 1: Control

Group 2: 100 mg Tin(II)-sulfide / kg bw/day

Group 3: 300 mg Tin(II)-sulfide / kg bw/day

Group 4: 1000 mg Tin(II)-sulfide / kg bw/day

 

In a key prenatal developmental toxicity study, Tin(II)-sulfide was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy. Under the present test conditions, the NOAEL was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams. No premature death was noted for any dose group. No test item-related changes in behaviour or external appearance were noted for the treatment groups. For all dams dosed with 300 or 1000 mg Tin(II)-sulfide/kg bw/day, dark discoloured faeces were noted. However, the dark discoloured faeces were considered to be due to the grey colour of the administered test item and therefore, of no toxicological relevance. No test item-related changes of the body weight, the body weight gain, the carcass weight and the food consumption was noted for the females of any dose group. No test item-related pathologic changes were noted for the animals of the dose groups. No differences for the thyroid weights were noted and histopathologic examination of the thyroids revealed no test item-related changes. No test item-related increased values were noted for the serum levels of T3, T4 and TSH. The NOAEL for the fetal organism was above 1000 mg Tin(II)-sulfide/kg bw/day. The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item. No influence was noted on fetal development (ano-genital distance or testicular development of the male fetuses). No deaths of fetuses and no test item-related malformations, variations or retardations were noted. Under the conditions of the study, Tin(II)-sulfide did not show any embryotoxic potential in rats.

 

In a supporting dose-range-finding for a prenatal developmental toxicity study, Tin(II)-sulfide was administered orally (per gavage) to female rabbits at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 28th day of pregnancy. Under the present test conditions, the NOAEL was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams. No test item-related premature death was noted for any dose group. No changes in behaviour, external appearance or faeces were noted for the treatment groups. No test item-related changes of the body weight, the body weight gain, the carcass weight and the food consumption was noted for the females of any dose group. In the high dose group (1000 mg Tin(II)-sulfide/kg bw/day) a reduction was noted for the gravid uterus weight that was considered to be a secondary finding due to the reduced number of fetuses per dam, in particular caused by one of three dams. No test item-related pathological changes were noted for the animals of the dose groups during necropsy. The NOAEL for the fetal organism was 300 mg Tin(II)-sulfide/kg bw/day. An increased number of resorptions and accordingly a lower number of fetuses were noted for the dams dosed with 1000 mg Tin(II)-sulfide/kg bw/day, again, in particular caused by one of three dams. No dead fetuses, no test item-related malformations or variations and no influence on the fetal body weight were noted.

Based on the data obtained in this dose-range-finding study, the following dose levels were selected for LPT Study No. 37284 (Prenatal developmental toxicity study in rabbits):

Group 1: Control

Group 2: 100 mg Tin(II)-sulfide/kg bw/day, p.o.

Group 3: 300 mg Tin(II)-sulfide/kg bw/day, p.o.

Group 4:1000 mg Tin(II)-sulfide/kg bw/day, p.o.

 

In a key prenatal developmental toxicity study, Tin(II)-sulfide was administered orally to female rabbits at dose levels of 100, 300 or 1000 mg/kg bw/day from the 6th to 28th day of pregnancy. Under the present test conditions, the test item-related NOAEL was above 1000 mg Tin(II)-sulfide/kg bw/day for the dams. No test item-related death was noted for any dose group. No changes in behavior, external appearance or faeces were noted for the treatment groups. No test item-related differences were noted for the body weight, body weight gain and the food consumption. No test item-related pathological findings were observed at necropsy in any of the dose groups. An increased number of abortions was noted in the high dose group (1000 mg Tin(II)-sulfide/kg bw/day), however, this finding is considered to be stress-related due a high gastro intestinal volume load caused by a compound with low gastro-intestinal absorption. The NOAEL for the fetal organism was also above 1000 mg Tin(II)-sulfide/kg bw/day. No test item-related differences were noted for the number of corpora lutea, implantation sites or fetuses. No test item-related changes were noted for the pre- and post-implantation loss or the sex distribution. Also no test item-related differences were observed for the fetal and placental weights. External and internal gross inspection as well as fetal skeletal examination according to DAWSON and also the examination of the internal organs according to WILSON revealed no test item-related differences between the control group and the dose groups. Under the conditions of the study, Tin(II)-sulfide did not show any teratogenic potential.

Justification for classification or non-classification

Based on the results of the available studies, tin sulfide is not subject to classification as reproductive toxic according to the criteria of  EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.