Tin sulphide

Toxicity to reproduction

Administrative data

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

Data source

Reference

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

Tin sulphide

EC Number:

215-248-7

EC Name:

Tin sulphide

Cas Number:

1314-95-0

Molecular formula:

SSn

IUPAC Name:

stannanethione

Test material form:

solid: particulate/powder

Test animals

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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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).

Details on results (P0)

-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).

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Details on results (P1)

-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.

Effect levels (P1)

1

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

2

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

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

-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).

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

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

2

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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