Tin sulphide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2020 to 23 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 June 2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

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

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

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

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


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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Details on mating procedure:

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

Duration of treatment / exposure:

Day 6 to 28 of gestation

Frequency of treatment:

Once daily

Duration of test:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

based on test material (at least 98% purity)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

based on test material (at least 98% purity)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

based on test material (at least 98% purity)

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

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

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: In addition to the detailed clinical observations, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Further checks were made early in the morning of each working day and again in the afternoon to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible.

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

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

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

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

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

Ovaries and uterine content:

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

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

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

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

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

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

Statistics:

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

Indices:

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

Historical control data:

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item-related changes in the consumption of drinking water were noted for the dams treated with 100, 300 or 1000 mg Tin(II)-sulfide/kg bw/day by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No changes in behavior were noted for the treatment groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

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

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

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

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

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

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

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

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

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

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

other: highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable

External malformations:

no effects observed

Description (incidence and severity):

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

Skeletal malformations:

no effects observed

Description (incidence and severity):

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

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

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

Description (incidence and severity):

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

Details on embryotoxic / teratogenic effects:

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

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: highest dose tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

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

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

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

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

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

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

 

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

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

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

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

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

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