Tin sulphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 AUG 2020 to 15 FEB 2022 (final report amendment outstanding)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Range finder for OECD TG 408 study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 MAY 2020 to 2021 (final report outstanding)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Principles of method if other than guideline:

The objective of this study was to determine the toxicity of the test article, following daily oral (dietary) administration to the rat for 14 days and to provide the basis for the selection of dose levels for subsequent 90-day toxicity study. Male and female Crl:WI(Han) rats were assigned to four groups receiving 830, 2500 or 6000 ppm in diet. The control article (vehicle) was 5KB3 EU Rodent Diet (Expanded, Ground Fine). Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, test article consumption, organ weights and macroscopic observations. Blood samples were collected for toxicokinetic evaluation.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Rat: Crl:WI(Han)

Details on species / strain selection:

The rat was selected because it is a rodent species well known and accepted for repeated dose testing

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Carles River Laboratories, Margate, United Kingdom
- Age at study initiation: Animals were obtained of approx. 29 to 35 days of age upon arrival and were between approx. 6 and 7 weeks old at the start of dosing
- Weight at study initiation: Males weighed between 160.0 and 229.5 g, and females weighed between 127.1 and 170.3 g
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of three.
- Diet (e.g. ad libitum): Animals had ad libitum access to 5KB3 EU Rodent Diet (Expanded, Ground Fine) (International Product Supplies Ltd., London, United Kingdom).
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles
- Acclimation period: Animals were acclimated for 18 days

DETAILS OF FOOD AND WATER QUALITY:
The water is periodically analyzed for specific contaminants and each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the water or diet at levels that might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 40 to 70%
- Air changes (per hr): minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark, except when otherwise dictated by experimental procedures

IN-LIFE DATES: From: 08 June 2020 To: 22 June 2020

Route of administration:

oral: feed

Details on route of administration:

The study is performed by oral route due to the considerations on the test method and testing strategy given in the ECHA Final Decision.

Vehicle:

other: in diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): formulations were prepared weekly
- Mixing appropriate amounts with (Type of food): The test article was formulated as a diet mix in 5K3 EU Rodent Diet (Expanded, Ground Fine) following dispensary SOPs and the formulation method (Method 8420250_D_02D)
- Storage temperature of food: Formulations were stored at room temperature (15 to 25°C) in a sealed container

VEHICLE
- Justification for use and choice of vehicle: The control article (vehicle) was 5KB3 EU Rodent Diet (Expanded, Ground Fine).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

continuous

Dose / conc.:

0 mg/kg diet

Dose / conc.:

830 mg/kg diet

Remarks:

male: 84.2 mg/kg/day
female: 85.1 mg/kg/day

Dose / conc.:

2 500 mg/kg diet

Remarks:

males: 268.4 mg/kg/day
female: 238.7 mg/kg/day

Dose / conc.:

6 000 mg/kg diet

Remarks:

male: 692.1 mg/kg/day
female: 680.5 mg/kg/day

No. of animals per sex per dose:

6 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Doses were selected based on available repeated dose toxicity studies on similar tin(II) compounds, investigating a period of 28 and 90-day.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed at the beginning and end of each working day for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was given a detailed physical examination twice weekly during the predose phase, once daily during the dosing phase, and on the day of terminal necropsy. An individual record of the clinical condition of each animal was maintained.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of all animals were recorded on Day 1 of the predose phase, once weekly from Day 1 of the dosing phase (predose), and before each necropsy. Body weights taken on Days 1 and 8 of the dosing phase were taken on the same day of the week, and the body weight taken on Day 14 of the dosing phase was taken the day prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (g/animal/day)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

CLINICAL CHEMISTRY/PLASMA ANALYSIS:
- Time schedule for collection of blood: blood samples (0.3 mL nominal) were taken from all animals at 1, 4, 8, and 24 hours postdose after the animals were fed with test article formulated diet on Days 1 and 13 of the dosing phase.

Sacrifice and pathology:

All animals were subjected to necropsy.
The scheduled necropsies were performed on Day 15 of the dosing phase, after an overnight period without food. Necropsies were carried out by controlled randomization (i.e., one cage of animals from Group 1, 4, 2, then 3 and repeated in the same sequence until all animals were sacrificed). Each animal was administered isoflurane anesthesia. Once a suitable deep plane of anesthesia was established, the animal was exsanguinated by severing its major blood vessels.
Animals were weighed before necropsy. Organs denoted by W in the following tissue list from all animals were dissected free from fat and other contiguous tissues and weighed before fixation. Left and right organs were weighed together. A full macroscopic examination was performed under the general supervision of a Pathologist, and all lesions were recorded. The following tissues from each animal were preserved in 10% neutral-buffered formalin, unless otherwise indicated: Adrenal (a W); animal identification; aorta; brain (W); cecum; colon; duodenum; epididymis (b W); esophagus; eye (c); femur with bone marrow including femorotibial joint; gut-associated lymphoid tissue (GALT)/Peyer’s patch; gross lesions sternum with bone marrow; heart (W); ileum; jejunum; kidney (W); liver (W); lungs with main stem bronchi and bronchioles; lymph node, mandibular; lymph node, mesenteric; muscle, biceps femoris; nerve, optic; nerve, sciatic; ovary (Wd); oviduct; pituitary (W); prostate (W); rectum; seminal vesicle including coagulating glands; spinal cord, cervical; spinal cord, lumbar; spinal cord, thoracic; spleen (W); stomach; testis (b W); thymus (W); thyroid with parathyroid (W); trachea; urinary bladder; uterus with cervix (W); vagina
* W = Weighed.
Note: Bone designated for microscopic examination was decalcified using Kristenson’s fluid.
a Tissues weighed as a pair; b Modified Davidson’s fixative and processed to at least block stage. Tissue weighed as a pair; c Davidson’s fluid fixative; d Tissue weighed with oviduct.

Statistics:

Various models of calculators, computers, and computer programs were used to analyze data in this study. Data from test article-treated animals were compared with control data. Statistical analyses were performed when appropriate. Please refer to 'Any other information on materials and methods incl. tables' for further details on Statistical Analysis.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical observations were observed

Mortality:

no mortality observed

Description (incidence):

No mortality occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreased body weight gain was observed on Days 1 through 8 and 8 through 14 of the dosing phase for males given 2500 or 6000 ppm; the body weight gain for animals given 6000 ppm was statistically significant. This resulted in a statistically significantly decreased mean cumulative body weight gain (40.3%) for males given 6000 ppm; compared with control, and due to the magnitude of decrease this observation was considered adverse, although animals were clinically healthy and were not observed with clinical observations or effects on food consumption. Decreased absolute body weights in males of groups given 2500 or 6000 ppm were not statistically different.
A slight decreased in body weight gain (approx. 12%) was also observed for females given 6000 ppm. However, this decrease in body weight gain as well as the absolute body weight in all dose groups was not statistically different compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effects on food consumption were observed.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment-related, marginally decreased absolute and relative (to brain weight) adrenal gland (up to 17%), heart (up to 14%), spleen (up to 12%), thymus (up to 15%), and thyroid gland weights (up to 12%) were observed for males administered 6000 ppm. These observations were considered not adverse; as such, the differences were not statistically significant, compared with controls. Moreover, no effects on organ weights were observed for females.
Statistically significant differences, when observed, were noted without a dose response and considered incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic observations were noted. All macroscopic observations were considered incidental, as they were also observed in controls, were noted infrequently, and/or had no relationship to dose (compared with controls).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Critical effects observed:

no

Conclusions:

In conclusion, daily dietary administrations of Stannous Chloride Anhydrous to Crl:WI(Han) rats at 830, 2500, and 6000 ppm for 14 days did not cause mortality. Body weight gain was decreased for males given 6000 ppm and was considered adverse due to magnitude of decrease (40.3%) compared to control. Non adverse decrease in adrenal, heart, spleen, thymus, and thyroid weights were observed in males administered 6000 ppm. Besides a slight decrease in body weight gain (approx. 12%) in the group given 6000 ppm, no effects were observed in females. Based on the results, the dose levels 830, 2500 or 6000 ppm are considered for the subsequent 90 days repeated dose toxicity study.

Executive summary:

The objective of this study was to determine the toxicity of the test article, Stannous Chloride Anhydrous, following daily oral (dietary) administration to the rat for 14 days and to provide the basis for the selection of dose levels for subsequent 90-day toxicity study. The toxicokinetic profile of the test article was also assessed.

The purpose of this study was to determine suitable dose levels for use in subsequent toxicity studies.

Male and female Crl:WI(Han) rats were assigned to four groups (6 animals/sex/dose), receiving 0, 830, 2500 or 6000 ppm Stannous chloride in diet. Animals were dosed via oral (dietary) administration. The control article (vehicle) was 5KB3 EU Rodent Diet (Expanded, Ground Fine).

Assessment of toxicity was based on mortality, clinical observations, body weights, food consumption, test article consumption, organ weights and macroscopic observations. Blood samples were collected for toxicokinetic evaluation.

No mortality occurred. No Stannous Chloride Anhydrous-related clinical observations were observed. Stannous Chloride Anhydrous-related decreased body weight gain was observed for males given 2500 or 6000 ppm. The magnitude of decrease was higher (40.3%) in males given 6000 ppm and was considered adverse although no relevant clinical observation was recorded. Besides a slight decrease in body weight gain (approx. 12%) in the group given 6000 ppm, no effects were observed in females. No Stannous Chloride Anhydrous-related effects on food consumption were observed. The achieved Stannous Chloride Anhydrous consumption by males and females was as expected. Stannous Chloride Anhydrous consumption was 84.2, 268.4, or 692.1 mg/kg/day for males in Groups 2, 3, and 4, respectively, and 85.1, 238.7, or 680.5 mg/kg/day for females in Groups 2, 3, and 4, respectively.

Stannous Chloride Anhydrous-related, non-adverse, marginally decreased absolute and relative (to brain weight) adrenal gland (up to 17%), heart (up to 14%), spleen (up to 12%), thymus (up to 15%), and thyroid weights (up to 12%) were observed for males. No effects on organ weights were observed for females. No Stannous Chloride Anhydrous-related macroscopic observations were recorded.

In conclusion, daily dietary administrations of Stannous Chloride Anhydrous to Crl:WI(Han) rats at 830, 2500, and 6000 ppm for 14 days did not cause mortality. Body weight gain was decreased for males given 6000 ppm and was considered adverse due to magnitude of decrease (40.3%) compared to control. Non adverse decrease in adrenal, heart, spleen, thymus, and thyroid weights were observed in males administered 6000 ppm. Besides a slight decrease in body weight gain (approx. 12%) in the group given 6000 ppm, no effects were observed in females. Based on the results, the dose levels 830, 2500 or 6000 ppm are considered for the subsequent 90 days repeated dose toxicity study.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

immunotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 AUG 2020 to 15 FEB 2022 (final report amendment outstanding)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD TG 408

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Carles River Laboratories, Margate, United Kingdom
- Age at study initiation: Animals were obtained of approx. 29 to 35 days of age upon arrival and were between approx. 6 and 7 weeks old at the start of dosing
- Weight at study initiation: Males weighed between 174.8 and 258.8 g, and females weighed between 107.6 and 172.5 g
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of two separated by sex.
- Diet (e.g. ad libitum): Animals had ad libitum access to 5KB3 EU Rodent Diet (Expanded, Ground Fine) (International Product Supplies Ltd., London, United Kingdom).
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles
- Acclimation period: Animals were acclimated for 17 days

DETAILS OF FOOD AND WATER QUALITY:
The water is periodically analyzed for specific contaminants and each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the water or diet at levels that might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 40 to 70%
- Air changes (per hr): minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark, except when otherwise dictated by experimental procedures

IN-LIFE DATES: From: 09 September 2020 (Toxicity); 11 September 2020 (Immunotoxicity) To: 08 and 09 December 2020 (Toxicity); 10 December 2020 (Immunotoxicity)

Route of administration:

oral: feed

Vehicle:

other: in diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): formulations were prepared weekly
- Mixing appropriate amounts with (Type of food): The test article was formulated as a diet mix in 5K3 EU Rodent Diet (expanded, ground fine) following dispensary SOPs and the formulation method (Method 8398349_D_01D)
- Storage temperature of food: Formulations were stored at room temperature (15 to 25°C) in a sealed container

VEHICLE
- Justification for use and choice of vehicle: The control article (vehicle) was 5KB3 EU Rodent Diet (Expanded, Ground Fine).

IMMUNOTOXICITY
Keyhole Limpet Hemocyanin
The test article, a white lyophilized powder, was identified as KLH (batch number 029M4885V) and supplied by Sigma-Aldrich.

Keyhole Limpet Hemocyanin Control Vehicle
The KLH control vehicle was water for injection and NaCl supplied by Baxter.

Keyhole Limpet Hemocyanin Formulation
Formulations were prepared on days of dosing.
Stock of Keyhole limpet hemocyanin (KLH) was reconstituted with water for injection following dispensary SOPs and the formulation method (Method 8398349_KLH_0_01D), as maintained in the study data.
Keyhole limpet hemocyanin was prepared at concentrations of 0.15 mg/mL. The stock vials containing lyophilized KLH/buffer salts (20 mg KLH), were reconstituted in 2 mL of water for injection to achieve a concentration of 10 mg/mL. A further dilution was made using 0.9% NaCl to achieve the final concentration of 0.15 mg/mL.
Formulations were stored refrigerated (2 to 8°C) in Dispensary and at 19 to 25°C in the animal room.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared for use in Weeks 1, 3 and 13 of the dosing phase were taken for analysis of achieved concentration. Two samples of 20 g were collected from each formulation including the control group. Samples were collected at random from formulations. Samples were dispatched at room temperature (15 to 25°C) to the Principal Investigator for analysis.
Thereby, data were acquired and integrated using MassHunter (version C.01.05, Agilent). Peak area results were exported into Watson LIMS (7.5 SP1, Thermo Scientific) in text file format. Watson LIMS was used to calculate standard curve parameters and concentration data for tin.
Formulations were found stable and homogenous for 83 days at an ambient temperature for Stannous Chloride Anhydrous in tin dichloride rodent diet formulations over a concentration range of 830 to 6000 ppm tin dichloride.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Dose / conc.:

0 mg/kg diet

Dose / conc.:

830 mg/kg diet

Remarks:

male: 57.3 mg/kg/day
female: 73.3 mg/kg/day

Dose / conc.:

2 500 mg/kg diet

Remarks:

male: 175.7 mg/kg/day
female: 234.1 mg/kg/day

Dose / conc.:

6 000 mg/kg diet

Remarks:

male: 449.0 mg/kg/day
female: 669.5 mg/kg/day

No. of animals per sex per dose:

6 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Based on the background information and toxicokinetic exposure (available repeated dose toxicity studies on similar tin(II) compounds and the 14-d DRF study), dose levels of 830, 2500, and 6000 ppm were selected as the low-, intermediate-, and high-dose levels, respectively. The high-dose level of 6000 ppm was expected to induce adequate extent toxicity in 90-day toxicity study, but no severe suffering. The low- and intermediate-dose levels were expected to produce little or no toxicity.

Observations and clinical examinations performed and frequency:

Please refer to information given in section 7.5.1 (see cross-reference).

Sacrifice and pathology:

Please refer to information given in section 7.5.1 (see cross-reference).

Humoral immunity examinations:

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): Yes
- Method: Keyhole limpet hemocyanin was administered to immunotoxicity animals via intravenous injection to the lateral caudal vein at a dose volume of 2 mL/kg and an approximate administration rate of 4 mL/min on Days 70 and 78 of the dosing phase. Blood samples were collected from the jugular vein of immunotoxicity animals before administration of KLH (on Days 68 and 78 of the dosing phase) and on Days 72, 82, and 89 of the dosing phase; terminal samples were collected from the abdominal aorta of immunotoxicity animals on Day 91 of the dosing phase. Analysis of anti-KLH antibodies (IgM and IgG) in the resultant serum was performed using an enzyme linked immunosorbent assay method following Analytical Procedure (AP) 11-010.
- Dose groups: all dose groups
- No. of animals: 6 animals/sex/group

Positive control:

none

Statistics:

Data from test article-treated animals were compared with control data. Statistical analyses were not performed.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreases in body weight and/ or body weight gain was observed for animals given 2500 or 6000 ppm; compared with control.
Mean cumulative body weight gain (Days 1 through 90) was statistically significantly decreased by 13 and 31% for males given 2500 or 6000 ppm, respectively. This was caused by statistically significant decreases in body weight gain on Days 1 through 15 for males given 2500 ppm and on Days 1 through 15, 22 through 29, 36 through 43, 50 through 57, and 64 through 71 for males given 6000 ppm. For males at 6000 ppm, the magnitude of decrease in cumulative body weight gain was higher (31%), compared with the control and resulted in statistically significant decrease of absolute body weight from Day 8 until the end of dosing (up to 18% compared to control) and at termination. Therefore, the observation was considered adverse, although, animals had gained weight from Day 1 through 90 in all treatment groups and moreover had no relevant clinical observation. For males at 2500 ppm, the reduction in cumulative body weight gain (13%) was considered to be due to statistically significantly decreased body weight gain in the first two weeks of treatment (days 1 through 15). From day 15 onwards the body weight gain was comparable to the control. No effects on absolute body weight and no relevant clinical observations were recorded; as such the reduced body weight gain was considered not adverse.
For females given 2500 or 6000 ppm, mean body weight gain was statistically significantly decreased from Day 57 through 64. Dose related decrease in mean cumulative body weight gain from Day 1 through 90 was not statistically different; compared with control and moreover, the absolute body weight was not affected; as such the effect on body weight gain was considered not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No noteworthy difference was identified in the anti-KLH immunoglobulin G (IgG) or immunoglobulin M (IgM) titer kinetics of controls and test article-treated animals; any evidence of marked variation was attributed to high inter-animal variability and was not a dose-dependent effect. Therefore, the provision of test item in the rat diet did not result in any notable KLH TDAR changes considered immunotoxic in nature, which was supported by the lack of adverse clinical pathology findings.
See 'Specific immunotoxic examinations' for further details.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to information given in section 7.5.1 (see cross-reference).

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings that suggested treatment-related effects were recorded. Most tissues were macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Cell viabilities:

not examined

Humoral immunity examinations:

effects observed, non-treatment-related

Description (incidence and severity):

Immunoglobulin G
In males, evaluation of the anti-KLH IgG kinetics demonstrated that, following KLH administration, the average IgG titre levels increased markedly between Days 78 and 91 in all controls (average IgG titre range: 2599 to 37989) and test article-treated males (average IgG titre range: 1160 to 102,771), compared to pre immunization on Day 68 (average IgG titre range: 100 to 127) or post immunization on Day 72 (average IgG titre range: 100 to 133). Peak titres were noted between Days 82 and 91. The anti-KLH IgG titres of males given 2500 or 6000 ppm were comparable to those of controls throughout the dosing phase.
Compared to pre-immunization levels on Day 68, average anti-KLH IgG titres were higher on Days 78 in males given 830 ppm (average IgG titre: 5960 [60-fold increase]), compared to controls (average IgG titre: 2499 [25-fold increase]). These differences in IgG titres were more pronounced on Days 82 to 91 in males given 830 ppm (average IgG titre range: 100,649 to 102,771 [1006- to 10,028 fold increase]), compared to controls (average IgG titre range: 19,161 to 37,989 [184- to 365-fold increase). Assessment of individual animal IgG responses indicated that the aforementioned differences were mainly attributed to particularly enhanced IgG responses noted for two males given 830 ppm (Animals R0116 and R0113).
In females, anti-KLH IgG kinetics were similar to that observed in males. Following KLH immunization, the anti-KLH titres on Days 78 to 91 were generally comparable between controls (average IgG titre range: 10,098 to 141,040) and females given the test article (average IgG titre range: 7212 to 190,247). Where higher IgG titers were noted in females given 2500 or 6000 ppm (average IgG titer: 187,698 and 161,228, respectively), compared to controls (average IgG titer: 101,852), this effect was mostly restricted to Day 89; effects were not dose-dependent and were associated with high inter-animal variability. Thus, this was considered not test article related.

Immunoglobulin M
The anti-KLH IgM titres in males increased markedly, with IgM titres on Days 78 to 91 comparable between controls (average IgM titre range: 1399 to 32,984) and males given 830 ppm (average IgM titre range: 5713 to 36,074), compared pre-immunisation levels of control/test-article-treated males on Day 68 (average IgM titre range: 100 to 227) and similarly following immunization on Day 72 (average IgM titre range: 100 to 208).
The IgM titers were lower in males given 2500 or 6000 ppm (average IgM titers: 19,865 and 24, 885, respectively [125- and 110 fold increase, respectively]), compared to controls (average IgM titer: 32,984 [330-fold increase]) on Day 82 -. This trend was also observed on Days 78 and 89 in males given 6000 ppm.
A similar pattern of anti-KLH IgM response following KLH immunization was noted in control and Stannous Chloride Anhydrous-administered females, with markedly increased IgM titres between Days 78 and 91. Peak IgM antibody responses were observed on Day 82 in controls (average IgM titre: 34,458 [341-fold increase]) and were comparable in females given 830, 2500, or 6000 ppm (average IgM titres: 40,553, 50,052, and 44,480, respectively [269-, 501-, and 445-fold increases, respectively]), compared to pre immunization IgM levels on Day 68. No noteworthy differences were observed in the IgM titre kinetics of controls and test article-treated animals; and any marked differences were inconsistent between sexes and associated with high inter-animal variability.

Specific cell-mediated immunity:

not examined

Non-specific cell-mediated immunity:

not examined

Other functional activity assays:

not examined

Other findings:

not examined

Dose descriptor:

NOAEL

Effect level:

6 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

immunology

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Conclusions:

In conclusion, the provision of Stannous Chloride Anhydrous in the rat diet did not result in any notable KLH TDAR changes considered immunotoxic in nature, supported by the lack of adverse clinical pathology findings.

Executive summary:

The objective of the study was to determine the toxicity of the test article, Stannous Chloride Anhydrous, following daily oral (dietary) administration to the rat for at least 90 days. All groups were dosed via ad libitum access to diet for 90 days, excluding the day of necropsy.

This report details the immunological response to the test article,Stannous Chloride Anhydrous, as determinedby keyhole limpet hemocyanin (KLH) T cell dependent antibody response (TDAR) analysis.

No noteworthy difference was identified in the anti-KLH immunoglobulin G (IgG) or immunoglobulin M (IgM) titer kinetics of controls and test article-treated animals; any evidence of marked variation was attributed to high inter-animal variability and was not a dose-dependent effect.

In conclusion, the provision of Stannous Chloride Anhydrous in the rat diet did not result in any notable KLH TDAR changes considered immunotoxic in nature,supported by the lack of adverse clinical pathology findings.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

stannous chloride

IUPAC Name:

stannous chloride

Test animals

Species:

rat

Strain:

other: Crl:WI (Han)

Details on species / strain selection:

The rat was selected because it is a rodent species well known and accepted for repeated dose testing and due to the availability of adequate background data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Carles River Laboratories, Margate, United Kingdom
- Age at study initiation: Animals were obtained of approx. 29 to 35 days of age upon arrival and were between approx. 6 and 7 weeks old at the start of dosing
- Weight at study initiation: Males weighed between 174.8 and 258.8 g, and females weighed between 107.6 and 172.5 g
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of two separated by sex.
- Diet (e.g. ad libitum): Animals had ad libitum access to 5KB3 EU Rodent Diet (Expanded, Ground Fine) (International Product Supplies Ltd., London, United Kingdom).
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles
- Acclimation period: Animals were acclimated for 17 days

DETAILS OF FOOD AND WATER QUALITY:
The water is periodically analyzed for specific contaminants and each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the water or diet at levels that might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 40 to 70%
- Air changes (per hr): minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark, except when otherwise dictated by experimental procedures

IN-LIFE DATES: From: 09 September 2020 (Toxicity); 11 September 2020 (Immunotoxicity) To: 08 and 09 December 2020 (Toxicity); 10 December 2020 (Immunotoxicity)

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The study is performed by oral route due to the considerations on the test method and testing strategy given in the ECHA Final Decision.

Vehicle:

other: in diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
formulations were prepared weekly
- Mixing appropriate amounts with (Type of food): The test article was formulated as a diet mix in 5K3 EU Rodent Diet (expanded, ground fine) following dispensary SOPs and the formulation method (Method 8398349_D_01D)
- Storage temperature of food: Formulations were stored at room temperature (15 to 25°C) in a sealed container

VEHICLE
- Justification for use and choice of vehicle: The control article (vehicle) was 5KB3 EU Rodent Diet (Expanded, Ground Fine).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared for use in Weeks 1, 3 and 13 of the dosing phase were taken for analysis of achieved concentration. Two samples of 20 g were collected from each formulation including the control group. Samples were collected at random from formulations. Samples were dispatched at room temperature (15 to 25°C) to the Principal Investigator for analysis.
Thereby, data were acquired and integrated using MassHunter (version C.01.05, Agilent). Peak area results were exported into Watson LIMS (7.5 SP1, Thermo Scientific) in text file format. Watson LIMS was used to calculate standard curve parameters and concentration data for tin.
Formulations were found stable and homogenous for 83 days at an ambient temperature for Stannous Chloride Anhydrous in tin dichloride rodent diet formulations over a concentration range of 830 to 6000 ppm tin dichloride.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg diet

Doses / concentrations 2

Dose / conc.:

830 mg/kg diet

Remarks:

male: 57.3 mg/kg/day
female: 73.3 mg/kg/day

Doses / concentrations 3

Dose / conc.:

2 500 mg/kg diet

Remarks:

male: 175.7 mg/kg/day
female: 234.1 mg/kg/day

Doses / concentrations 4

Dose / conc.:

6 000 mg/kg diet

Remarks:

male: 449.0 mg/kg/day
female: 669.5 mg/kg/day

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on the background information and toxicokinetic exposure (available repeated dose toxicity studies on similar tin(II) compounds and the 14-d DRF study), dose levels of 830, 2500, and 6000 ppm were selected as the low-, intermediate-, and high-dose levels, respectively. The high-dose level of 6000 ppm was expected to induce adequate extent toxicity in 90-day toxicity study, but no severe suffering. The low- and intermediate-dose levels were expected to produce little or no toxicity.
- Fasting period before blood sampling for clinical biochemistry: Samples were collected after animals were fasted overnight

Positive control:

n.a.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and end of each working day for signs of ill health or overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination once daily including the day of terminal necropsy. An individual record of the clinical condition of each animal was maintained. Additional observations were recorded when deemed necessary

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of all animals were recorded on Day 1 of the predose phase, once weekly from Day 1 of the dosing phase (predose), and before each necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (The amount of food consumed by each cage of animals was determined twice weekly from Day 1 of the dosing phase. Consumption was calculated as g/animal/day)

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmic examinations were conducted on all animals during the predose phase and on animals in Groups 1 and 4 during Week 12 of the dosing phase. A mydriatic agent was instilled into eyes prior to indirect ophthalmic examinations.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all toxicity animals for hematology (1 x 0.5 mL [EDTA], nominal) and coagulation (1 x 0.5 mL [trisodium citrate], nominal), and were withdrawn from the jugular vein on Day 84 (Week 12) of the dosing phase. Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood travelled all the way to the top and bottom of the tube each time, followed by at least 5 minutes on an automatic mixer. Blood smears were prepared from each hematology specimen and used when necessary to confirm results produced by the analyzer. No observational comments were recorded as a result of blood smear review.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: hemoglobin. red blood cell count(a), packed cell volume(b), mean cell volume(a), mean cell hemoglobin(b), mean cell hemoglobin concentration(b), reticulocyte count(c), red cell distribution width(a), hemoglobin distribution width
total and differential white cell count, platelet count(d), platelet crit, mean platelet volume, platelet distribution width
* (a) Reported from the gated reticulocyte population, (b) Derived from the gated reticulocyte population, (c) Absolute reticulocyte count derived from the gated reticulocyte population, (d) Includes platelet clump assessment. Clump counts below 100 were considered none detected; clump counts over 100 were considered platelet clumps present and were confirmed by review of Advia cytogram or blood film examination

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical chemistry (1 x 0.8 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy. Samples were collected after animals were fasted overnight via controlled randomization i.e., one cage of animals from Groups 1, 4, 2, and then 3 and repeated in the same sequence until all animals were sampled. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring that the blood travelled all the way to the top and bottom of the tube each time to mix with the clot activator.
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, alkaline phosphatase, total cholesterol, low density lipoprotein cholesterol, triglycerides, total bilirubin, total protein, albumin, globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, creatine kinase, glucose, high density lipoprotein cholesterol, bile acid

PLASMA/SERUM HORMONES/LIPIDS: Yes
See 'Any other information on materials and methods incl. tables'

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples from all toxicity animals were collected overnight on Day 84 (Week 12) of the dosing phase. Food was removed during collection; water remained available.
- Parameters were examined.: volume, color, turbidity, specific gravity, pH(a), protein(a), glucose(a), ketones(a), urobilinogen(a), bilirubin(a), blood(a), microscopy of sediment
*(a) Determined semi-quantitatively

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Observations were performed at approximately the same time every day. At the time of recording, the observer was unaware of each animal’s dose level. All toxicity animals were observed within the home cage, in the hand, and in the arena (2-minute duration) before the initiation of treatment and once a week thereafter. Animals were observed for potential effects on: Behavior, Gait, Posture, Respiration, Secretion, Excretion, Involuntary movements, Skin, Tail, Eyes, Pelage, Activity
In addition, animals were subjected to elicited responses in order to assess their reaction to a stimulus/manipulation. Quantitative measures of latency to first step, number of rears, fecal boli, and urine pools were recorded during the arena observation.
Toxicity animals were observed for approach response, corneal tactile reflex test, touch response, auditory startle response, tail pinch, hindlimb foot splay, air righting ability, quantitative forelimb and hindlimb grip strength, and pupillary response
during Week 12 of the dosing phase. Locomotor activity was recorded for all toxicity animals at 60 minutes and reported in 10 minute bins during Week 12 of the dosing phase. Room lights were turned of and white noise turned on; animals were acclimatized to the white noise (between 60 and 80 decibels) for at least 10 minutes prior to any procedure.
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: Keyhole limpet hemocyanin was administered to immunotoxicity animals via intravenous injection to the lateral caudal vein at a dose volume of 2 mL/kg and an approximate administration rate of 4 mL/min on Days 70 and 78 of the dosing phase. Blood samples were collected from the jugular vein of immunotoxicity animals before administration of KLH (on Days 68 and 78 of the dosing phase) and on Days 72, 82, and 89 of the dosing phase; terminal samples were collected from the abdominal aorta of immunotoxicity animals on Day 91 of the dosing phase
- How many animals: 6 animals per sex
- Dose groups that were examined: all dose groups
- Parameters were examined: Analysis of anti-KLH antibodies (IgM and IgG) in the resultant serum was performed using an enzyme-linked immunosorbent assay method following Analytical Procedure (AP) 11-010.

OTHER:
Estrous Cycles
The stage of the estrous cycle was recorded for all females within 1 hour of blood sample collection for thyroid hormone at necropsy.

Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy for toxicity animals. Tissues were air-dried and fixed in methanol, but no examination was required

Thyroid Hormones
Blood samples were collected from all toxicity animals for thyroid hormones (2 x 0.8 mL [serum separator tubes], nominal) and were withdrawn from the abdominal aorta at necropsy. Samples were collected between 09:00 and 13:00 hours at necropsy to allow for accurate comparisons between groups. Samples were collected after animals were fasted overnight. For further details please refer to 'any other information on materials and methods incl. tables'.
Thyroid Hormone Tests
- Triiodothyronine (T3) measured using Advia Centaur CP.
- Thyroid stimulating hormone (TSH) and Thyroxine (T4) measured using Siemens Immulite.

Keyhole Limpet Hemocyanin Antibody (Immunoglobulin M)
For further details please refer to 'any other information on materials and methods incl. tables'.

Sacrifice and pathology:

Necropsy, Organ Weights, and Macroscopic Observations
With the exception of fasting, these procedures were also followed for the sacrifice at an unscheduled interval.
All animals, including the animal sacrificed at an unscheduled interval, were subjected to necropsy.
The scheduled necropsies were performed on Day 91 or 92 of the dosing phase, after an overnight period without food. Necropsies were carried out in controlled randomization (i.e., one cage of animals from Groups 1, 4, 2, and then 3 and repeated in the same sequence until all animals were sacrificed). Each animal was given isoflurane anesthesia. Once a suitable deep plane of anesthesia was established, the animal was exsanguinated by severing its major blood vessels.
Animals were weighed before necropsy. Organs denoted by W in the following tissue list from all toxicity animals, were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. The spleen only was weighed for immunotoxicity animals. A full macroscopic examination was performed under the general supervision of a Pathologist, and all lesions were recorded.
The following tissues from each toxicity animal were preserved in 10% neutral-buffered formalin, unless otherwise indicated:
adrenal (W E); animal identification; aorta (E); bone marrow smear (femur) (a,b); brain (c W E); cecum (E); colon (E); duodenum (E); epididymis (d W E); esophagus (E); eye (e E); femur with bone marrow and femorotibial joint (E); gut-associated lymphoid tissue (GALT)/ Peyer’s patch (E); gross lesions (E); Harderian gland (f ); head; heart (W E); ileum (E); jejunum (E); kidney (W E); liver (W E); lungs with main stem bronchi and bronchioles (E); lymph node, mandibular (E); lymph node, mesenteric (E); mammary gland (E); mandibular salivary gland (E); muscle, biceps femoris (E); nasopharynx (E); nerve, optic (E); nerve, sciatic (E); nose/nares (E); ovary (g W E); oviduct (E); pancreas (E); pituitary (W E); prostate h W E; rectum (E); seminal vesicle with coagulating glands (h W E); skin and subcutis (E); spinal cord, cervical (E); spinal cord, lumbar (E); spinal cord, thoracic (E); spleen (W E); sternum with bone marrow (E); stomach (E); testis (d W E); thymus (W E); thyroid with parathyroid (i W E); tongue (E); trachea (E); urinary bladder (E); uterus with cervix (W E); vagina (E)

* E = Processed and examined microscopically; W = Weighed.
Note: Bone designated for microscopic examination was decalcified using Kristenson’s fluid.
(a) Methanol fixative; (b) tissues were air-dried and fixed in methanol, but no examination was required; (c) Cerebrum, cerebellum, medulla/pons included in evaluation; (d) Modified Davidson’s fixative; tissues weighed as a pair; (e) Davidson’s fluid fixative; (f) Preserved with head in situ; (g) Weighed with oviducts; (h) Prostate, seminal vesicle and coagulating gland weighed as a whole, then prostate weighed separately; (i) The thyroid and parathyroid were weighed pre-fixation in error. The raw organ weight data were retained with study date. The thyroid and parathyroid weight was removed from Pristima and amended to the post fixation weight. Corrective action was taken, therefore no impact to study outcome occurred.

The left testes and epididymides were examined in all toxicity males for the assessment of spermatogenesis, with CASA sperm counts, motility, and morphology assessed. The following tissues from each immunotoxicity animal were preserved in 10% neutral-buffered formalin.
animal identification; gross lesions (variable); spleen (W E)
* E = Processed and examined microscopically; W = Weighed.

Histology
The following tissues were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
Toxicity Animals
Group 1 (Control): All tissues denoted by E in the previous tissue list
Group 2 (Low): Gross lesions and the kidney, liver, and spleen only
Group 3 (Intermediate): Gross lesions and the kidney, liver, and spleen only
Group 4 (High): All tissues denoted by E in the previous tissue list
Unscheduled Sacrifice All tissues denoted by E in the previous tissue list

Immunotoxicity Animals
Groups 1, 2, 3, and 4 Spleen only
Additional sections of the testes and epididymides from toxicity animals were stained with periodic acid Schiff and Eosin Y for the assessment of spermatogenesis.

Microscopic Observations
The following tissues were examined microscopically by the Study Pathologist.
Toxicity Animals
Group 1 (Control): All tissues denoted by E in the previous tissue list
Group 2 (Intermediate): Gross lesions only
Group 3 (Low): Gross lesions only
Group 4 (High): All tissues denoted by E in the previous tissue list
Unscheduled Sacrifice All tissues denoted by E in the previous tissue list

Immunotoxicity Animals
Groups 1, 2, 3, and 4 Spleen only
Additional sections of the testes and epididymides from toxicity animals were stained with periodic acid Schiff and Eosin Y for the assessment of spermatogenesis.

Statistics:

Various models of calculators, computers, and computer programs were used to analyze data in this study. Data from test article-treated animals were compared with control data. Statistical analyses were performed when appropriate. Please refer to 'Any other information on materials and methods incl. tables' for further details on Statistical Analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment–related clinical observation was recorded. However, mild or moderate vocalization was observed from dosing day 10 in eight females (Animals R0705, R0706, R0708, R0710, R0712, R0713, R0714 and R0715) given 6000 ppm. In consideration that vocalization was also observed in eight females during predose and across the groups including control during the dosing phase [four control females (Animal R0403, R0411, R0412 and R0414), four females (Animals R0509, R0510, R0511 and R0516) given 830 ppm, and one female (Animal R0610) given 2500 ppm], this observation was considered not test article-related.
All other clinical observations were considered incidental as they were observed in controls, infrequent, exhibited no relationship to dose, and/or were comparable with observations routinely noted at this laboratory for rats.

Mortality:

no mortality observed

Description (incidence):

One toxicity male (Animal R0110) given 830 ppm test item was sacrificed in a moribund condition on Day 65 due to the severity of clinical observations, which included broken claws, thinning pelage, and red discoloration of the eye. Although the cause of demise could not be determined, as no microscopic finding considered the primary cause of death was noted, it was considered not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreases in body weight and/ or body weight gain was observed for animals given 2500 or 6000 ppm; compared with control.
Mean cumulative body weight gain (Days 1 through 90) was statistically significantly decreased by 13 and 31% for males given 2500 or 6000 ppm, respectively. This was caused by statistically significant decreases in body weight gain on Days 1 through 15 for males given 2500 ppm and on Days 1 through 15, 22 through 29, 36 through 43, 50 through 57, and 64 through 71 for males given 6000 ppm. For males at 6000 ppm, the magnitude of decrease in cumulative body weight gain was higher (31%), compared with the control and resulted in statistically significant decrease of absolute body weight from Day 8 until the end of dosing (up to 18% compared to control) and at termination. Therefore, the observation was considered adverse, although, animals had gained weight from Day 1 through 90 in all treatment groups and moreover had no relevant clinical observation. For males at 2500 ppm, the reduction in cumulative body weight gain (13%) was considered to be due to statistically significantly decreased body weight gain in the first two weeks of treatment (days 1 through 15). From day 15 onwards the body weight gain was comparable to the control. No effects on absolute body weight and no relevant clinical observations were recorded; as such the reduced body weight gain was considered not adverse.
For females given 2500 or 6000 ppm, mean body weight gain was statistically significantly decreased from Day 57 through 64. Dose related decrease in mean cumulative body weight gain from Day 1 through 90 was not statistically different; compared with control and moreover, the absolute body weight was not affected; as such the effect on body weight gain was considered not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect on food consumption was observed.
Food consumption was transiently and statistically significantly decreased on Day 1 through 8 for males given 6000 ppm; compared with control, but the mean cumulative food consumption from Day 1 through 90 were not affected.
Food consumption for females was generally increased during the dosing phase and attained statistical significance on Days 8 through 36, 43 through 57, 64 through 71, and 78 through 90 and resulted in statistically significantly increased mean cumulative food consumption from Day 1 through 90 for females given 6000 ppm (28% compared to control); compared with control. This increased food consumption did not adversely effect the absolute body weight, and no relevant clinical observations were noted.

Food efficiency:

no effects observed

Description (incidence and severity):

Mean test article consumption was 57.3, 175.7 and 449.0 mg/kg/day and 73.3, 234.1, and 669.5 mg/kg/day for males and females given 830, 2500, or 6000 ppm, respectively.

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related ophthalmic observations were recorded.
One female (Animal R0707) given 6000 ppm had a slightly hemorrhagic right eye. This observation was considered incidental as this slight observation was noted in only one female and was not noted in males.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effect on hematology parameters was observed. However, mean red cell distribution width percentage was statistically significantly decreased for males given 6000 ppm (11.3%, compared to 11.9% for control) and mean platelet volume was statistically significantly increased for males given 2500 or 6000 ppm (8.6 fL, compared to 8.2 fL of control) and females given 6000 ppm (8.7 fL, compared to 8.3fL of control). These observations were considered not test item-related as the individual and mean values were within or near the range of concurrent control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related, statistically significant increase in HALP was observed for males given 6000 ppm (83 IU/L compared to 60 IU/L for control) and females given 2500 or 6000 ppm (50 and 58 IU/L, respectively, compared to 31 IU/L for control). In the absence of relevant microscopic changes noted in liver, muscle, kidney, or heart, this observation was considered not adverse effect of test article. Where other statistically significant differences were observed, they were only observed in a single sex and were not associated with a dose response or were in the wrong direction for biological relevance.
All plasma samples were analyzed in accordance with the validated method and within the documented frozen storage period. There were no samples with an irreconcilable discrepancy. No evidence of tin was noted in the control group samples.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed for the measured urine parameters. The urine parameters values in test article groups were comparable to concurrent control.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No treatment -related effects on detailed clinical observations or quantitative assessments (latency, number of fecal boli, number of rears, number of urine pools, hindlimb foot splay, or limb grip strengths) were observed. Mild vocalization observed in females given control, 830, 2500 or 6000 ppm were in similar number and were also recorded during the predose.
A treatment–related, nonadverse, effect on motor activity was observed.
Statistically significantly increased basic movements, fine movement, total ambulations, total rears and total distance travelled were observed during the first (1 to 10 minutes) and second 10-minute (11 to 20 minutes) intervals for males given 6000 ppm and statistically significantly increased fine movements was observed at second 10-minute (11 to 20 minutes) interval for males given 2500 ppm; compared with control. Basic movements, fine movements, and total distance travelled were also statistically significantly decreased during the sixth 10-minutes interval (51 to 60 minutes) for animals given 2500 or 6000 ppm. These observations were considered nonadverse because of the varying consistency (increased initially and then decreased at a later interval).
Statistically significantly increased basic movements, fine movements, total ambulations, total rears, and total distance travelled were observed varyingly during the initial interval until 40 minutes for females given 830, 2500, or 6000 ppm. These observations were considered not adverse in consideration that during the later observation interval, motor activity observations were comparable with concurrent control and moreover, no adverse clinical observation were observed in the study.

Immunological findings:

no effects observed

Description (incidence and severity):

No noteworthy difference was identified in the anti-KLH immunoglobulin G (IgG) or immunoglobulin M (IgM) titer kinetics of controls and test article-treated animals; any evidence of marked variation was attributed to high inter-animal variability and was not a dose-dependent effect. Therefore, the provision of test item in the rat diet did not result in any notable KLH TDAR changes considered immunotoxic in nature, which was supported by the lack of adverse clinical pathology findings.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No organ weight changes that suggested an effect of test item were recorded. Organ weight and organ weight ratio changes, including statistically significant changes, were attributed to normal biological variation and were considered not related to Stannous chloride as they were small in magnitude, not dose-dependent, inconsistent between sexes, due to normal inter-animal variability and/or lacked a microscopic correlate.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings that suggested treatment-related effects were recorded. Most tissues were macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings that suggested treatment-related effects were recorded. Most tissues were microscopically unremarkable and the findings observed were generally consistent with the usual pattern of findings in animals of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No treatment-related effect on the estrous stage was observed. The estrous stages observed in animals of test article groups were comparable with concurrent control. Also, no treatment-related effect on sperm parameters was observed.
The mean values of CASA parameters and sperm morphology in the test article treated groups were comparable to concurrent control group.
No treatment-related effect on thyroid hormone levels (TSH, T3, T4) was observed.

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, daily oral dietary administration of Stannous Chloride Anhydrous to Crl:WI(Han) rats for at least 90 days resulted in no test article-related deaths.
Adverse effects on body weight and/or body weight gain were observed for males given 6000 ppm, whereas the effect on body weight gain was non-adverse for females given 6000 ppm. A non-adverse increase in alkaline phosphatase (HALP) was observed for males given 6000 ppm and females given 2500 or 6000 ppm, and non-adverse effects on motor activities were observed for animals given ≥830 ppm. Therefore, based on the generated data, the no observed adverse effect level (NOAEL) was considered to be 2500 ppm for males (equivalent to 175.7 mg/kg/day) and 6000 ppm for females (equivalent to 669.5 mg/kg/day).

Executive summary:

The objective of this study was to determine the subchronic toxicity of the test article, Stannous Chloride Anhydrous, following daily oral (dietary) administration to the rat for at least 90 days for the determination of a no observed adverse effect level (NOAEL), and also to evaluate the immunotoxicity.

Male and female Crl:WI (Han) rats were assigned to four groups, and doses were given as indicated in the following table. For assessing subchronic repeated dose toxicity, animals were dosed via oral (dietary) administration, daily for at least 90 days. The control article (vehicle) was 5KB3 EU Rodent Diet (expanded, ground fine). Immunotoxicity animals were dosed with keyhole limpet (KLH) via intravenous (bolus) manual injection on Days 70 and 78 of the dosing phase at a volume of 2 mL/kg.

Four groups of ten, Crl:WI(Han) rats per sex were given Stannous Chloride Anhydrous mixed in diet at 0, 830, 2500, or 6000 ppm for the period of 90 days. Additionally, 6 rats/sex/group given Stannous Chloride Anhydrous mixed in diet at 0, 830, 2500, or 6000 ppm for the period of 90 days were used in the study for the determination of immunotoxicity.

Assessment of toxicity was based on mortality, clinical observations, functional battery observations, motor activity, body weights, food consumption, ophthalmic observations, and clinical and anatomic pathology. Blood samples were collected for toxicokinetic evaluation and anti-keyhole limpet hemocyanin (KLH) antibody analysis.

No test article-related mortality occurred. However, one male given 830 ppm was sacrificed in a moribund condition and the cause of demise was considered incidental. Stannous Chloride Anhydrous–related mild or moderate vocalization was observed from dosing day 10 in eight females given 6000 ppm. Vocalization was also observed in four control females, and four females and one female given 830 or 2500 ppm, respectively; as such this observation was considered not adverse. No Stannous Chloride Anhydrous-related ophthalmic observations were recorded. No Stannous Chloride Anhydrous-related effects on detailed clinical observations or quantitative assessments were observed.

A Stannous Chloride Anhydrous–related, nonadverse effect on motor activity was observed. Statistically significant increase and/or decrease in motor activities in males given over 2500 ppm or females given over 830 ppm were either of varying consistency or were comparable with concurrent control and moreover, no adverse clinical observation were observed in the study.

A Stannous Chloride Anhydrous–related decrease in body weight gain and/or body weight was observed for males given 2500 or 6000 ppm. A statistically significantly decreased mean cumulative body weight gain (Days 1 through 90) by 31% for males given 6000 ppm was considered adverse due to the magnitude resulting in decreased absolute body weight (up to 18%). However, the effect on body weight gain for males given 2500 ppm was considered not adverse as the reduction in cumulative body weight gain was due to decreased body weight gain in the first two weeks of treatment and subsequent body weight gains were comparable to the values noted for the control animals and no effects on absolute body weight were observed. The dose related decrease in mean cumulative body weight gain for females was considered not adverse due to the lack of statistical significance and missing effects on absolute body weight.

No Stannous Chloride Anhydrous-related effect on food consumption was observed.

The decrease in food consumption noted for males was transient and increased food consumption noted for females did not adversely effect the body weight, and no relevant clinical observations were noted. Mean test article consumption was 57.3, 175.7 and 449.0 mg/kg/day and 73.3, 234.1, and 669.5 mg/kg/day for males and females given 830, 2500, or 6000 ppm, respectively. The achieved Stannous Chloride Anhydrous consumption by animals was as expected. The average achieved consumption was approximately 8.5- or 2.5-fold lower for animals given 830 or 2500 ppm, respectively, compared to animals given 6000 ppm, and was approximately 3-fold lower for animals given 830 ppm, compared to animals given 2500 ppm. No treatment-related effect on the estrous stage of females or on sperm parameters were observed.

A test item-related slight but statistically significant increase in alkaline phosphatase (HALP) was observed for males given 6000 ppm (83 IU/L compared to 60 IU/L for control) and females given 2500 or 6000 ppm (50 and 58 IU/L, respectively compared to 31 IU/L for control). In the absence of relevant microscopic changes noted in the liver, muscle, kidney, or heart, this observation was considered not adverse effect of test article. No test item-related effect on hematology or urine parameters was observed. No test item-related effect on thyroid hormone was observed. Stannous Chloride Anhydrous in the rat diet did not result in any notable KLH T-cell dependent antibody response (TDAR) changes that were considered immunotoxic in nature, which wassupported by the lack of adverse clinical pathology findings.

No organ weight changes or macroscopic or microscopic findings were recorded that suggested Stannous Chloride Anhydrous-related effects.

In conclusion, daily oral dietary administration of Stannous Chloride Anhydrous to Crl:WI(Han) rats for at least 90 days resulted in no test article-related deaths.

Adverse effects on body weight and/or body weight gain were observed for males given 6000 ppm, whereas the effect on body weight gain was non-adverse for females given 6000 ppm. A non-adverse increase in alkaline phosphatase (HALP) was observed for males given 6000 ppm and females given 2500 or 6000 ppm, and nonadverse effects on motor activities were observed for animals given≥830ppm.

Therefore, based on the generated data, the no observed adverse effect level (NOAEL) was considered to be 2500 ppm for males (equivalent to 175.7 mg/kg/day) and 6000 ppm for females (equivalent to 669.5 mg/kg/day).