Tin sulphate

Endpoint summary

Description of key information

Repeated dose toxicity: via oral route

OECD 408 conducted with the analogous substance Tin(II) chloride - Adverse effects on body weight and/or body weight gain in males, NOAEL considered 2500 ppm (equivalent to 175.7 mg/kg bw/day) for males and 6000 ppm for females (equivalent to 669.5 mg/kg bw/day) (Holalagoudar, 2021)

Repeated dose toxicity: via inhalation route

OECD 412 conducted with the analogous substance Tin(II) oxide - NOAEC was considered to be 9.19 μg/L (Walker, 2015)

Repeated dose toxicity: via dermal route

Due to the classification Skin Sens 1 intensive risk management measures are in place to avoid significant skin contact. Hence, a study via dermal route is not performed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

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

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)

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

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:

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.

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.

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.

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, 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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

175.7 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Remarks:

corresponding Dose-reange finder

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Adopted: 7 September 2009

Deviations:

yes

Remarks:

significant variance in dose and humidity

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

EC Methods for the determination of toxicity. Annex to Commission Directive
92/69/EC (Official Journal No. L 38 A/140), Part B, Method B.8: Repeated dose
(28 days) toxicity (inhalation).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHanTM:WIST strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation: 69 to 76 days
- Weight at study initiation:
Males: 245 to 281 g
Females: 180 to 203 g
- Housing: 5 animals of the same sex/cage; Polycarbonate cages with a stainless steel mesh lid, changed at appropriate intervals; Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): ad libitum (removed overnight before blood sampling for haematology or blood chemistry and during the period of exposure).
- Water (e.g. ad libitum): ad libitum (except during exposure)
- Acclimation period: At least 12 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Diet: Rat and Mouse No. 1 Maintenance Diet.
Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Mean value of humidity in
- Group 3: 38.6 %
- Group 4: 33.8 /(min: 27.7)

- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 2.7 - <= 3 µm

Geometric standard deviation (GSD):

2.54

Remarks on MMAD:

GSD: 2.41 - 2.54

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: snout only exposure

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 h exposure

Frequency of treatment:

5 days/week

Dose / conc.:

2.3 mg/m³ air (nominal)

Remarks:

achieved 2.44 µg/L

Dose / conc.:

9.1 mg/m³ air (nominal)

Remarks:

achieved 9.19 µg/L

Dose / conc.:

80 mg/m³ air (nominal)

Remarks:

achieved 87.9 µg/L

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: In a previous preliminary inhalation toxicity study (Huntingdon Life Sciences Study Number URA0001) tin monoxide was administered to rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 2.28, 10.6 and 41.8 μg/L. These exposure levels were well tolerated and there were no adverse findings. Consequently, in order to establish signs of toxicity, the high target exposure level for this study has been selected as 80 μg/L. The low and intermediate target exposure levels remained the same as those used on the previous study (2.3 and 9.1 μg/L). The intermediate exposure level will investigate toxicity at the German occupational exposure limit (German TRGS 900 Sn(II) compounds). The low exposure level was not expected to cause any toxicity.
- Rationale for animal assignment (if not random): Randomly allocated on arrival.
- Fasting period before blood sampling for clinical biochemistry: removed overnight before blood sampling for
haematology or blood chemistry

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started and for each week throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Morphology:
Anisocytosis
Macrocytosis
Microcytosis
Hypochromasia
Hyperchromasia

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following sequence of statistical tests was used for body weight, blood oxygen saturation, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

For clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable. For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs observed during the detailed weekly physical examination. Immediately after dosing, wet fur was observed for a majority of animals and red staining of the head was observed for a single animal on Day 1. These are both common findings associated with the method of restraint and are considered not to be treatment related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight gain was reduced for males exposed to 9.19 and 87.9 μg/L of tin monoxide (0.6X and 0.4X control values respectively). A single male exposed to 87.9 μg/L (Animal 16), experienced a body weight loss of 37 g over the 4 week exposure period. Reduced group mean body weight reduction was evident for all females exposed to tin monoxide (0.6, 0.4 and 0.8X control values, with increasing exposure concentration).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly reduced for males exposed to 87.9 μg/L during the first two weeks of treatment compared to concurrent control and pretreatment values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L (1.2X control values for both groups) and all females exposed to tin monoxide (1.3X control values for both groups). Calculation and statistic not traceable. Other changes from control were small, inconsistent between sexes or did not show a clear relationship to treatment and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment related effects. Calculation and statistic not traceable. Other changes from control were small, inconsistent between sexes or did not show a clear
relationship to treatment and were therefore attributed to normal biological variation.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean lung and bronchi weights (adjusted for terminal body weight) were statistically significantly greater than control for all animals exposed to tin monoxide (up to 2.1X control for males and up to 2.4X control for females). Other changes from control were small, inconsistent between sexes or did not show a clear relationship to treatment and were therefore attributed to normal biological variation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 4 weeks of treatment revealed changes in the lungs, tracheobronchial and mediastinal lymph nodes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with tin monoxide were present within the lungs, tracheobronchial and mediastinal lymph nodes, larynx and kidneys. In addition, one male
animal had findings within the liver, stomach, duodenum and jejunum.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Blood oxygen saturation
Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L (1.5X control values) and both sexes exposed to 87.9 μg/L (1.4 and 1.6X control values for males and females respectively). Statistical analysis was not performed for females due to the limited number of results available for females exposed to 2.44 μg/L.

Bioavailability
Two hours after the completion of exposure in Week 4, there was a dose related increase in tin concentration in the plasma of animals exposed to 9.19 and 87.9 μg/L. For animals
exposed to 87.9 μg/L, group mean tin plasma concentration values were 30.0 ng/mL for
males and 15.0 ng/mL for females. For animals exposed to 9.19 μg/L, quantifiable levels of tin were present in the plasma of a single male and female (approximately 10 ng/mL). For the remaining animals exposed to 9.19 μg/L and all animals exposed to 2.44 μg/L, tin plasma concentrations were below the lower limit of quantification. Tin was not detectable in any of the Control group samples.

Details on results:

Histopathological changes related to treatment were observed within the lungs, the tracheobronchial and mediastinal lymph nodes, larynx and kidneys. In the lungs, accumulation of pigmented and flocculent material within alveoli correlated with macroscopic discolouration. Together with pigmented and flocculent material within the epithelium and lamina propria of the larynx, this was considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. This was variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT in the lungs which was indicative of irritation by the test article. The combined findings in the lungs also correlated with increased group mean lung and bronchi weights for all animals exposed to tin monoxide. Similar changes, at a lesser severity (showing dose-dependence), were seen within the lungs of animals dosed with 2.44 or 9.19 μg/L, with the exception of increased BALT, which was absent in these animals. Histopathological changes were also observed in the tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), and kidneys (increased tubular pigment). Findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of the test article. This correlated with enlarged tracheobronchial lymph nodes, and to some extent, enlarged mediastinal lymph nodes. Histopathological findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose response.

Dose descriptor:

NOAEL

Effect level:

9.19 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.

Executive summary:

A GLP-compliant subacute inhalation toxicity study in rats according to OECD Guideline 412 was performed with the test item tin monoxide. The objective of this study was to assess the systemic toxic potential of tin monoxide in a 4 week (6 hours per day, 5 days per week) inhalation study in RccHanTM:WIST rats. The study included three dose groups and one control group (air control) with 5 rats/sex in each group. The analytical exposure concentrations were: 0, 2.44, 9.19 and 87.9 µg/L. The achieved levels were 106, 101 and 110% of the target concentrations for Groups 2, 3 and 4 respectively. The Mass Median Aerodynamic Diameters for Groups 2, 3 and 4 are within the ideal range (1-3 μm) for a repeat dose inhalation study. There were no treatment related clinical signs observed during the detailed weekly physical examination. Lower body weight gain was evident for males exposed to 9.19 μg/L and 87.9 μg/L and for all females exposed to tin monoxide. Food consumption was also slightly reduced for males exposed to 87.9 μg/L. Two hours after the completion of exposure in Week 4, tin was detected in the plasma of a single male and female exposed to 9.19 μg/L and in all animals exposed to 87.9 μg/L. Tin was not quantifiable in control animals or animals exposed to 2.44 μg/L. Group mean lung and bronchi weights (adjusted for terminal body weight) were greater than control for all animals exposed to tin monoxide. Histopathological changes related to treatment were observed within the lungs (the accumulation of pigmented and flocculent material within alveoli, variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT), tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), larynx (pigment accumulation within the epithelium and lamina propria) and kidneys (increased tubular pigment). Observations within the larynx and lungs were considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. Accompanying findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of test article. Findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose-related response. Amongst animals dosed with 2.44 μg/L, findings were restricted to the lungs and tracheobronchial lymph nodes. A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L and all females exposed to tin monoxide. Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L and both sexes exposed to 87.9 μg/L.

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 August 2014 - 13 November 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Remarks:

main study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Due to the preliminary nature of this study no specific regulations or guidelines are applicable.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
- Purity, including information on contaminants, isomers, etc.:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
- Reactivity of the test material with the incubation material used (e.g. plastic ware):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel:
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment:

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:

Species:

rat

Strain:

Wistar

Remarks:

RccHan(TM) Wistar rats

Details on species / strain selection:

Strain/Species RccHan™:WIST rat.
Supplier: Harlan (UK) Ltd

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species RccHan™:WIST rat.
Supplier: Harlan (UK) Ltd
Number of animals: 15 males and 15 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: 11 days before commencement of treatment.
Age of the animals at start of treatment: 61 to 67 days old.
Weight range of animals at the start of treatment: Males: 225 to 260 g, Females: 159 to 181 g

Allocation: Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages: Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal housing, diet and water supply
Environmental. control
Rodent facility Restricted entry - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between r.ooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 19-23 ºC and 40-70 %.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal accommodation: Cages Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: The cages constituting each group were blocked together by sex on separate batteries.
Number of animals per cage: Three of the same sex.

Bedding: Wood based bedding which was changed at appropirate intervals each week.

Environmental enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

Diet supply
Diet: Rat and Mouse No. 1 Maintenance Diet.
Availability: Non-restricted (removed overnight before blood sampling for haematology or blood chemistry, urine collection and during dosing).

Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted (except during urine collection and dosing).

Route of administration:

inhalation: dust

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 3.2 - <= 4.8 µm

Geometric standard deviation (GSD):

2.58

Remarks on MMAD:

Group 2 - Target concentration = 2.3 µg/L - Achieved Exposure level = 2.28 µg/L = MMAD = 4.8 µm - geometric standard diameter = 2.74
Group 3 - Target concentration = 9.1 µg/L - Achieved Exposure level = 10.6 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.51
Group 4 - Target concentration = 36.3 µg/L - Achieved Exposure level = 41.8 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.49

The achieved levels were 99, 116 and 115 % of the target concentrations for Groups 2, 3 and 4 respectively. The MMAD values for Groups 3 and 4 are slightly above the ideal range of 1 to 3 μm with 79 to 80 % of the particles within the respirable range (< 7 μm). The Group 2 MMAD value was higher with a lower percentage of particles within the respirable range (64 %).

Details on inhalation exposure:

The animals on study were acclimated to the method of restraint, over a 5 day period immediately preceding the first test substance exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mass Median Aerodynamic Diameter:
The MMAD values for Groups 3 and 4 are slightly above the ideal range of 1 to 3 μm with 79 +/- 80 % of the particles within the respirable range (<7 μm). A jet mill was used for Groups 3 and 4 to reduce the average particle size, however it was not practical to fit a jet mill on the Group 2
system, this is considered to be the reason for the higher MMAD value of 4.8 μm and lower percentage of particles within the respirable range (64 %).

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5

Dose / conc.:

2.28 mg/m³ air (analytical)

Remarks:

(nominal: 2.3 µg/L = 2.3 mg/m³)

Dose / conc.:

10.6 mg/m³ air (analytical)

Remarks:

(nominal: 9.1 µg/L = 9.1 mg//m²)

Dose / conc.:

41.8 mg/m³ air (analytical)

Remarks:

(nominal: 36.3 µg/L = 36.3 mg/m³)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: yes (12 hours)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):

This study was designed to assess the systemic toxic potential of tin monoxide in a 1 week (6 hours per day, 5 days a week) inhalation study in RccHan™;WIST rats.
The study design was as follows: Group 1 - Control - 0 µg/L (3 males & 3 females), Group 2 - Tin Monoxide - 2.3 µg/L - (3 males & 3 females), Group 3 - Tin Monoxide - 9.1 µg/L (3 males & 3 females), Group 4 - Tin Monoxide - 36.3 µg/L (3 males & 3 females)
Animals received air only or the test substance, tin monoxide by inhalation for 1 week (6 hours per day, 5 days a week).
During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, bioavailability, blood oxygen saturation, organ weight, macropathology and histopathology investigations were undertaken.

Positive control:

none

Observations and examinations performed and frequency:

Clinical observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

Signs associated with dosing:
Detailed observations were recorded daily at the following times in relation to dose administration:
- Pre-exposure observation
- During exposure however, observation is severely restricted due to tube restraint
- As each animal is returned to its home cage
- As late as possible in the working day

Clinical signs: A detailed weekly physical examination was performed on each animal to monitor general health.

Body weight: The weight of each animal was recorded daily from Day -5 (Day P3), throughout the study and before necropsy.

Food consumption: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily from Day -5 (Day P3) throughout the study.

Haematology, peripheral blood:
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasion: Week 1: All animals.
Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.3 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC),
Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)
Platelet count (Plt)
Morphology:,Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of:
- Prothrombin time (PT) - using IL PT-Fibrinogen reagent.
- Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Haematology, bone marrow: Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period.
Fixation: Smears were air dried and subsequently fixed in methanol.
Retention Slides were retained by the Department of Biomarkers, Bioanalysis and Clinical Sciences.
Analysis: At the discretion of the pathologist, examination of smears to further evaluate other findings was not considered necessary; the smears are retained in the archives.

Blood chemistry: Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:
Occasion Animals: Week 1 - All animals

Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.5 mL, Groups 2 and 3 and 0.6 mL Groups 1 and 4) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Copper (CU) – Groups 1 and 4 only (non-GLP), Iron (Fe).

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

Urinalysis
Animals were placed in an individual metabolism cage overnight, without food or water. Urine samples were collected over approximately 16 hours at the following occasion: Week 1 - All animals
The individual samples were examined for the following characteristics:
Using manual methods:
- Clarity and Colour (App) - by visual assessment
- Volume (Vol) - using a measuring cylinder
- pH - using a pH meter
- Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips, interpreted using a Clinitek®500 instrument: Ketones (Keto); Bilirubin/bile pigments (Bili); Blood pigments (UBld);
Using a Roche P Modular analyser: Protein (T-Prot); Creatinine (T-Creat); Glucose (T-Gluc)

Sacrifice and pathology:

Terminal Procedures
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule: Study animals were killed following treatment on Day 5.
Method of kill: Overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.
Sequence: To allow satisfactory inter-group comparison.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed as detailes in the field Any other information on materials and methods

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for study animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid.
Eyes: In Davidson’s fluid.
Bone marrow smears: See below.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. A single section was prepared from each of the tissues required.
Full List All animals killed or dying prematurely.
Study animals of Groups 1 and 4 killed at the scheduled necropsy.
Routine staining Sections were stained with haematoxylin and eosin.

Light microscopy
Tissues preserved for examination were examined as follows:
Category: Scheduled kill - Animals: All animals of Groups 1 and 4, Tissues: All specified
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Other examinations:

Bioavailability (Non-GLP)
The sampling schedule was as follows: At termination Group 1 and Group 4: Time of sampling (hours after completion of dosing): 2 hours; Parameter: Tin
Samples were collected under terminal anaesthesia.
Blood sample site: Cardiac puncture.
Anaesthetic: Isoflurane.
Anticoagulant: Lithium heparin.
Blood volume: 0.6 mL.
Treatment of samples: Gently inverted several times and then placed on an automatic mixer for at least 2 minutes continuously at ambient temperature.
Centrifugation conditions At 1300 g for 10 minutes at 21 °C.
Number of aliquots per sample: One
Temporary storage conditions: Plasma was processed at ambient temperature and stored frozen (approximately -20 ˚C) as soon as practicable following completion of blood sampling completion of blood sampling
Final storage conditions: Deep frozen (approximately -20 ºC).
Fate of plasma samples: Despatched to a Responsible Scientist on solid carbon dioxide.

Blood oxygen saturation
The sampling schedule was as follows: at Day 5 Group 1 and Group 4 animals
Blood sample site: Tail vein.
Anticoagulant: Lithium heparin.
Blood volume: 0.3 mL.
Samples were analysed without delay.
Each sample was used to measure the following parameter using a blood gas analyser system (ABL80 CO-OX FLEX).
-- Oxygen saturation (sO2)

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analysed at each timepoint separately:
- Body weight, using gains over appropriate study periods
- Haematology
- Blood chemistry
- Urinalysis
- Organ weights, absolute and adjusted for terminal body weight

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs observed during the detailed weekly physical examination.
Signs associated with the administration procedure included wet fur and red staining of the head on return to the home cage. These signs are associated with the method of restraint used.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in body weights.
There was a body weight loss for all animals on Day 4. This is considered due to all animals having food and water withdrawn overnight on Day 3 for blood and urine sampling. Body weight gains were evident for all animals on Day 5.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related changes in food consumption.
Reduced food consumption was evident for all animals from Days 3 to 4. This is considered due to all animals having food withdrawn overnight on Day 3 for blood and urine sampling. Food consumption had recovered for all animals on Days 4 to 5.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Group mean neutrophil counts were statistically significantly high for males exposed to 41.8 μg/L (1.8X control values). Neutrophil counts were also high for males and females exposed to 2.28, 10.6 μg/L. However, a few individual values for each sex, in each group, were close to control values. Due to the lack of a dose-related response in females, it is not considered a treatment related effect in females.
Other changes from control were small, inconsistent between sexes and/or did not show clear dose relationships.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Group mean inorganic phosphorus levels where high for males exposed to 41.8 μg/L (1.3X control values) and all females exposed to tin monoxide (1.3X, 1.3X and 1.4X control values for females exposed to 2.28, 10.6 and 41.8 μg/L respectively), all attaining statistical significance.
Other changes from control including those that attained statistical significance, were small, inconsistent between sexes and/or did not show clear dose relationships.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment related effects on urinalysis parameters.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight adjusted lung and bronchi weights were statistically significantly high for females exposed to 10.6 and 41.8 μg/L (both 1.2X control values).
Group mean body weight adjusted spleen weights were higher for all males exposed to tin monoxide (1.2, 1.4 and 1.3X control values for males exposed to 2.28, 10.6 and 41.8 μg/L respectively), with males exposed to 41.8 μg/L attaining statistical significance.
All other differences from control including those that attained statistical significance, were generally small and are not considered to be of toxicological significance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 1 week of treatment revealed the followingbchanges in the tracheobronchial lymph nodes.
Tracheobronchial lymph nodes
Enlargement was seen in two male animals treated with 41.8 μg/L, one female animal treated with 2.28 μg/L and one female animal treated with 10.6 μg/L.
The nature and incidence of all other findings were consistent with the commonly seen background of macroscopic changes and were considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment related findings
There were no microscopic findings that were considered to be related to treatment with tin monoxide.

Incidental findings
All histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Blood oxygen saturation levels appeared to be lower than control values for all males exposed to 41.8μg/L. However, the blood gas analyser reported that there were errors which may have affected the results for two of the males exposed to 41.8 μg/L (animal numbers 10 and 11). For the remaining male exposed to 41.8μg/L, the blood oxygen saturation level was lower than control (approximately 0.7X). Blood oxygen saturation levels for females exposed to 41.8 μg/L were all similar to control values.

Bioavailability (non-GLP): Tin concentrations in plasma for controls were all below the limit of quantification (< 5 ng/mL) for the test analysis. Tin concentrations in plasma for males and females exposed to 41.8 μg/L were all approximately 2-3X greater than the lower limit of quantification.

Dose descriptor:

NOAEL

Effect level:

> 41.8 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The exposure was well tolerated and resulted in no adverse findings.

Discussion

The test article, tin monoxide, was administered by snout-only inhalation administration, for 6 hours a day for 5 consecutive days, at achieved exposure levels of 2.28, 10.6 and 41.8 μg/L. There were no unscheduled deaths or treatment related effects on clinical signs, body weight, food consumption, urinalysis parameters and histopathology findings. Slightly higher body weight adjusted lung and bronchi weights were evident for females exposed to 10.6 and 41.8 μg/L. In the absence of a similar effect in males or any correlating histopathological changes, these were considered to be of no toxicological importance. Slightly higher body weight adjusted spleen weights were evident for all males exposed to tin monoxide.

Whilst histopathological correlates for these changes could not be made as spleens were not examined microscopically, the absence of any corresponding macropathology or clinical pathology changes, this is not considered to be related to treatment.

Analysis of tin monoxide concentrations in plasma for males and females exposed to 41.8 μg/L indicated that the test article was present in plasma 2 hours after the completion of exposures.

However, this work was not performed to the principles of GLP and as such, no formal conclusions can be drawn from this data.

A reduction in blood oxygen saturation levels was observed in one male exposed to 41.8 μg/L. However, due to the lack of conclusive results from the remaining two males exposed to 41.8 μg/L, a relationship to treatment cannot be confirmed for males. Blood oxygen saturation results for females were similar to control values.

An increase in neutrophil counts was evident for males exposed to 41.8 μg/L. The slight increases in inorganic phosphorous levels for males exposed to 41.8 μg/L, and all females exposed to tin monoxide, were of uncertain relationship to treatment.

Conclusion

It is concluded that snout only exposure to tin monoxide to RccHan™;WIST rats for six hours each day for five days, at achieved exposure levels of 2.28, 10.6 or 41.8 μg/L, was well tolerated and resulted in no adverse findings. The results therefore demonstrate that exposing animals to at least the same exposure levels that were used in this study would be suitable for use in a four week toxicity study.

Conclusions:

Snout only exposure to tin monoxide to RccHan™;WIST rats for six hours each day for five days, at achieved exposure levels of 2.28, 10.6 or 41.8 μg/L, was well tolerated and resulted in no adverse findings.

Executive summary:

This study was designed to assess the systemic toxic potential of tin monoxide in a 1 week (6 hours per day, 5 days a week) inhalation study in RccHan™;WIST rats. It was conducted as a Dose-range finder for the envisaged OECD 412, which was then conducted.

The study design was as follows: Group 1 - Control - 0 µg/L (3 males & 3 females), Group 2 - Tin Monoxide - nominal 2.3 µg/L = analytically verifeid 2.28 µg/L (3 males & 3 females), Group 3 - Tin Monoxide - nominal 9.1 µg/L = analytically verified 10 .6 µg/L (3 males & 3 fenales), Group 4 - Tin Monoxide - nominal 36.3 µg/L = analytically verified 41.8 µg/L (3 males & 3 females). Animals received air only or the test substance, tin monoxide by inhalation for 1 week (6 hours per day, 5 days a week).

During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, bioavailability, blood oxygen saturation, organ weight, macropathology and histopathology investigations were undertaken.

Concernign the Mass Median aerodynamic diameter, the following is reported: Group 2 - Target concentration = 2.3 µg/L - Achieved Exposure level = 2.28 µg/L = MMAD = 4.8 µm - geometric standard diameter = 2.74, Group 3 - Target concentration = 9.1 µg/L - Achieved Exposure level = 10.6 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.51, Group 4 - Target concentration = 36.3 µg/L - Achieved Exposure level = 41.8 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.49, The achieved levels were 99, 116 and 115 % of the target concentrations for Groups 2, 3 and 4 respectively. The MMAD values for Groups 3 and 4 are slightly above the ideal range of 1 to 3 μm with 79 to 80% of the particles within the respirable range (<7 μm). The Group 2 MMAD value was higher with a lower percentage of particles within the respirable range (64 %).

Group mean body weight adjusted lung and bronchi weights high for females exposed to 10.6 and 41.8 μg/L. Group mean body weight adjusted spleen weights were higher for all males exposed to tin monoxide.

Compared with control, higher group mean neutrophil counts was evident for males exposed to 41.8 μg/L. Compared with control, higher group mean inorganic phosphorous levels were evident for males exposed to 41.8 μg/L and all females exposed to tin monoxide. Group mean body weight adjusted lung and bronchi weights were statistically significantly high

for females exposed to 10.6 and 41.8 μg/L. Group mean body weight adjusted spleen weights were higher for all males exposed to tin monoxide. Enlargement of the tracheobronchial lymph nodes was evident for two males exposed to

41.8 μg/L, one female exposed to 2.28 μg/L and one female exposed to 10.6 μg/L. Tin was present in plasma 2 hours after the completion of dosing for all male and female animals exposed to 41.8 μg/L. However, this work was not performed to GLP.

Conclusion

It is concluded that snout only exposure to tin monoxide to RccHan™;WIST rats for six hours each day for five days, at achieved exposure levels of 2.28, 10.6 or 41.8 μg/L, was well tolerated and resulted in no adverse findings. The results therefore demonstrate that exposing animals to at least the same exposure levels that were used in this study would be suitable for use in a four week toxicity study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

9.19 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Remarks:

corresponding Dose-reange finder

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Adopted: 7 September 2009

Deviations:

yes

Remarks:

significant variance in dose and humidity

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

EC Methods for the determination of toxicity. Annex to Commission Directive
92/69/EC (Official Journal No. L 38 A/140), Part B, Method B.8: Repeated dose
(28 days) toxicity (inhalation).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHanTM:WIST strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation: 69 to 76 days
- Weight at study initiation:
Males: 245 to 281 g
Females: 180 to 203 g
- Housing: 5 animals of the same sex/cage; Polycarbonate cages with a stainless steel mesh lid, changed at appropriate intervals; Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): ad libitum (removed overnight before blood sampling for haematology or blood chemistry and during the period of exposure).
- Water (e.g. ad libitum): ad libitum (except during exposure)
- Acclimation period: At least 12 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Diet: Rat and Mouse No. 1 Maintenance Diet.
Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Mean value of humidity in
- Group 3: 38.6 %
- Group 4: 33.8 /(min: 27.7)

- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 2.7 - <= 3 µm

Geometric standard deviation (GSD):

2.54

Remarks on MMAD:

GSD: 2.41 - 2.54

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: snout only exposure

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 h exposure

Frequency of treatment:

5 days/week

Dose / conc.:

2.3 mg/m³ air (nominal)

Remarks:

achieved 2.44 µg/L

Dose / conc.:

9.1 mg/m³ air (nominal)

Remarks:

achieved 9.19 µg/L

Dose / conc.:

80 mg/m³ air (nominal)

Remarks:

achieved 87.9 µg/L

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: In a previous preliminary inhalation toxicity study (Huntingdon Life Sciences Study Number URA0001) tin monoxide was administered to rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 2.28, 10.6 and 41.8 μg/L. These exposure levels were well tolerated and there were no adverse findings. Consequently, in order to establish signs of toxicity, the high target exposure level for this study has been selected as 80 μg/L. The low and intermediate target exposure levels remained the same as those used on the previous study (2.3 and 9.1 μg/L). The intermediate exposure level will investigate toxicity at the German occupational exposure limit (German TRGS 900 Sn(II) compounds). The low exposure level was not expected to cause any toxicity.
- Rationale for animal assignment (if not random): Randomly allocated on arrival.
- Fasting period before blood sampling for clinical biochemistry: removed overnight before blood sampling for
haematology or blood chemistry

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started and for each week throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Morphology:
Anisocytosis
Macrocytosis
Microcytosis
Hypochromasia
Hyperchromasia

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following sequence of statistical tests was used for body weight, blood oxygen saturation, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

For clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable. For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs observed during the detailed weekly physical examination. Immediately after dosing, wet fur was observed for a majority of animals and red staining of the head was observed for a single animal on Day 1. These are both common findings associated with the method of restraint and are considered not to be treatment related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight gain was reduced for males exposed to 9.19 and 87.9 μg/L of tin monoxide (0.6X and 0.4X control values respectively). A single male exposed to 87.9 μg/L (Animal 16), experienced a body weight loss of 37 g over the 4 week exposure period. Reduced group mean body weight reduction was evident for all females exposed to tin monoxide (0.6, 0.4 and 0.8X control values, with increasing exposure concentration).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly reduced for males exposed to 87.9 μg/L during the first two weeks of treatment compared to concurrent control and pretreatment values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L (1.2X control values for both groups) and all females exposed to tin monoxide (1.3X control values for both groups). Calculation and statistic not traceable. Other changes from control were small, inconsistent between sexes or did not show a clear relationship to treatment and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment related effects. Calculation and statistic not traceable. Other changes from control were small, inconsistent between sexes or did not show a clear
relationship to treatment and were therefore attributed to normal biological variation.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean lung and bronchi weights (adjusted for terminal body weight) were statistically significantly greater than control for all animals exposed to tin monoxide (up to 2.1X control for males and up to 2.4X control for females). Other changes from control were small, inconsistent between sexes or did not show a clear relationship to treatment and were therefore attributed to normal biological variation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 4 weeks of treatment revealed changes in the lungs, tracheobronchial and mediastinal lymph nodes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with tin monoxide were present within the lungs, tracheobronchial and mediastinal lymph nodes, larynx and kidneys. In addition, one male
animal had findings within the liver, stomach, duodenum and jejunum.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Blood oxygen saturation
Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L (1.5X control values) and both sexes exposed to 87.9 μg/L (1.4 and 1.6X control values for males and females respectively). Statistical analysis was not performed for females due to the limited number of results available for females exposed to 2.44 μg/L.

Bioavailability
Two hours after the completion of exposure in Week 4, there was a dose related increase in tin concentration in the plasma of animals exposed to 9.19 and 87.9 μg/L. For animals
exposed to 87.9 μg/L, group mean tin plasma concentration values were 30.0 ng/mL for
males and 15.0 ng/mL for females. For animals exposed to 9.19 μg/L, quantifiable levels of tin were present in the plasma of a single male and female (approximately 10 ng/mL). For the remaining animals exposed to 9.19 μg/L and all animals exposed to 2.44 μg/L, tin plasma concentrations were below the lower limit of quantification. Tin was not detectable in any of the Control group samples.

Details on results:

Histopathological changes related to treatment were observed within the lungs, the tracheobronchial and mediastinal lymph nodes, larynx and kidneys. In the lungs, accumulation of pigmented and flocculent material within alveoli correlated with macroscopic discolouration. Together with pigmented and flocculent material within the epithelium and lamina propria of the larynx, this was considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. This was variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT in the lungs which was indicative of irritation by the test article. The combined findings in the lungs also correlated with increased group mean lung and bronchi weights for all animals exposed to tin monoxide. Similar changes, at a lesser severity (showing dose-dependence), were seen within the lungs of animals dosed with 2.44 or 9.19 μg/L, with the exception of increased BALT, which was absent in these animals. Histopathological changes were also observed in the tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), and kidneys (increased tubular pigment). Findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of the test article. This correlated with enlarged tracheobronchial lymph nodes, and to some extent, enlarged mediastinal lymph nodes. Histopathological findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose response.

Dose descriptor:

NOAEL

Effect level:

9.19 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.

Executive summary:

A GLP-compliant subacute inhalation toxicity study in rats according to OECD Guideline 412 was performed with the test item tin monoxide. The objective of this study was to assess the systemic toxic potential of tin monoxide in a 4 week (6 hours per day, 5 days per week) inhalation study in RccHanTM:WIST rats. The study included three dose groups and one control group (air control) with 5 rats/sex in each group. The analytical exposure concentrations were: 0, 2.44, 9.19 and 87.9 µg/L. The achieved levels were 106, 101 and 110% of the target concentrations for Groups 2, 3 and 4 respectively. The Mass Median Aerodynamic Diameters for Groups 2, 3 and 4 are within the ideal range (1-3 μm) for a repeat dose inhalation study. There were no treatment related clinical signs observed during the detailed weekly physical examination. Lower body weight gain was evident for males exposed to 9.19 μg/L and 87.9 μg/L and for all females exposed to tin monoxide. Food consumption was also slightly reduced for males exposed to 87.9 μg/L. Two hours after the completion of exposure in Week 4, tin was detected in the plasma of a single male and female exposed to 9.19 μg/L and in all animals exposed to 87.9 μg/L. Tin was not quantifiable in control animals or animals exposed to 2.44 μg/L. Group mean lung and bronchi weights (adjusted for terminal body weight) were greater than control for all animals exposed to tin monoxide. Histopathological changes related to treatment were observed within the lungs (the accumulation of pigmented and flocculent material within alveoli, variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT), tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), larynx (pigment accumulation within the epithelium and lamina propria) and kidneys (increased tubular pigment). Observations within the larynx and lungs were considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. Accompanying findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of test article. Findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose-related response. Amongst animals dosed with 2.44 μg/L, findings were restricted to the lungs and tracheobronchial lymph nodes. A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L and all females exposed to tin monoxide. Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L and both sexes exposed to 87.9 μg/L.

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

9.19 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral - Holalagoudar, 2021

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 was supported 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).

 

 Repeated dose toxicity: inhalation – Walker 2015

A GLP-compliant subacute inhalation toxicity study in rats according to OECD Guideline 412 was performed with the test item tin monoxide. The objective of this study was to assess the systemic toxic potential of tin monoxide in a 4 week (6 hours per day, 5 days per week) inhalation study in RccHanTM:WIST rats. The study included three dose groups and one control group (air control) with 5 rats/sex in each group. The analytical exposure concentrations were: 0, 2.44, 9.19 and 87.9 µg/L. The achieved levels were 106, 101 and 110% of the target concentrations for Groups 2, 3 and 4 respectively. The Mass Median Aerodynamic Diameters for Groups 2, 3 and 4 are within the ideal range (1-3 μm) for a repeat dose inhalation study. There were no treatment related clinical signs observed during the detailed weekly physical examination. Lower body weight gain was evident for males exposed to 9.19 μg/L and 87.9 μg/L and for all females exposed to tin monoxide. Food consumption was also slightly reduced for males exposed to 87.9 μg/L. Two hours after the completion of exposure in Week 4, tin was detected in the plasma of a single male and female exposed to 9.19 μg/L and in all animals exposed to 87.9 μg/L. Tin was not quantifiable in control animals or animals exposed to 2.44 μg/L. Group mean lung and bronchi weights (adjusted for terminal body weight) were greater than control for all animals exposed to tin monoxide. Histopathological changes related to treatment were observed within the lungs (the accumulation of pigmented and flocculent material within alveoli, variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT), tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), larynx (pigment accumulation within the epithelium and lamina propria) and kidneys (increased tubular pigment). Observations within the larynx and lungs were considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. Accompanying findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of test article. Findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose-related response. Amongst animals dosed with 2.44 μg/L, findings were restricted to the lungs and tracheobronchial lymph nodes. A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L and all females exposed to tin monoxide. Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L and both sexes exposed to 87.9 μg/L.

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.

Justification for classification or non-classification

To comply with the ECHA Final Decision (SEV-C-2114560739-35-01/F, dated 13 June 2018), the need for classification of the registered substance as STOT RE 2 for the hematological and cardiovascular systems was re-evaluated by a newly conducted repeated-dose toxicity study according to current standards. The STOT RE 2 classification for the hematologic and cardiovascular systems was based on the results from a previous repeated-dose toxicity study (De Groot, 1973), which can be considered to be of limited reliability; the study was conducted prior adoption of related standardised testing guidelines, and not in accordance with GLP.

 

In the now available reliable oral sub-chronic toxicity study (90 days) in rats according to OECD 408 and in compliance with GLP (Holalagoudar, 2021), no changes in blood parameters, thus no adverse effects on the cardiovascular system, or other target organs were observed. Thus, the results reported in a previous non-guideline repeated-dose toxicity study (De Groot, 1973), were not reproduced. Based on the outcome of the OECD 408 study (Holalagoudar, 2021), it can therefore be concluded that no target organ toxicity is to be expected after repeated administration of the registered substance via the oral route, thus no classification is necessary.

 

Based on effects in the lungs (indicative of irritation) reported in a subacute repeated-dose toxicity study by inhalation administration to rats according to OECD TG 412 on tin monoxide (Walker, 2015), the registered substance is classified as STOT RE 1 (respiratory system), H372, according to Regulation (EC) No 1272/2008.