Tin sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Apr 2019 to 06 Dec 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosomal Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

stannous chloride

IUPAC Name:

stannous chloride

Test animals

Species:

rat

Strain:

other: out-bred Han Wistar rats

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 202 to 261 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: wire topped, solid bottomed cages, with three animals of the same sex per cage
- Diet: ad libitum, 5LF2 EU Rodent Diet
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 23 April 2019 to 05 July 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

1-3 days

Frequency of treatment:

single dose

Post exposure period:

16 and 42 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes

Positive control(s):

Cyclophosphamide (CPA) 20 mg/kg.

Examinations

Tissues and cell types examined:

Bone marrow sampled: 16 hours (Day 2 - all dose groups) or 42 hours (Day 3 -
vehicle and high dose group only) after administration

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Femurs were removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL phosphate buffered saline (PBS) into appropriately labelled centrifuge tubes. The tubes were centrifuged at (200 g for 10 minutes) and the supernatant was carefully removed and cell pellets resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and placed in incubator set to 37°C for 30 minutes. The tubes were agitated approximately halfway through this period. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (200 g, 10 minutes) and resuspension. This procedure was repeated several times (centrifuging at 1250 g, 2-3 minutes) until the cell pellets were clean.

METHOD OF ANALYSIS:
Scoring was carried out using a light microscope at an appropriate magnification. Slides from vehicle and positive control animals were checked to for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with the dosing of the study. All animals per group were analysed. Mitotic Index was measured in 1000 cells per animal (including animals from positive
control groups) to assess any evidence of toxicity. Where possible, two hundred metaphases from each animal were analysed for chromosome aberrations. Only cells with 40-42 chromosomes were considered
acceptable for analysis of structural aberrations. Any polyploid or endoreduplicated cells observed during this search was noted and recorded separately. Classification of aberrations was based on the scheme described by ISCN (ISCN, 1995). Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid with no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment. Observations were recorded on raw data sheets with the microscope stage co-ordinates of any aberrant cell. Slide analysis was performed by a competent analyst trained in the applicable Lab standard operating procedures. The analyst was physically located remote from the facility, but was subject to lab management and GLP control systems (including QA inspection). All slides and raw data generated by the remote analyst were returned to the lab for archiving on completion of analysis.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic/aneugenic damage if:
1. A statistically significant increase in the proportion of cells with structural chromosome aberrations (excluding gaps) occured at one or more concentration and/or sample time
2. The proportion of cells with structural aberrations in individual animals at such a point exceeded the normal range
3. A dose-response trend in the proportion of cells with structural chromosome aberrations (where more than two dose levels were analysed) was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met and target tissue exposure has been confirmed.
Results which only partially satisfied the above criteria were dealt with on a case-bycase basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels.

Statistics:

The proportion of cells with structural chromosome aberrations excluding gaps will be compared with the proportion in vehicle controls by using Fisher’s exact test. In addition, a Cochran-Armitage Trend Test will be performed to aid determination of concentration response relationships. Probability values of p≤0.05 will be accepted as significant.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs

Vehicle controls validity:

valid

Remarks:

historical

Negative controls validity:

valid

Remarks:

historical

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In the Range-Finder Experiment, groups of three male and three female Han Wistar rats were dosed with the test item at 500, 1000 or 2000 mg/kg (dose volume 10 mL/kg). At 500 and 1000 mg/kg all animals showed limited piloerection on day 1 with no other signs of toxicity observed. At 2000 mg/kg all animals exhibited mild clinical signs including hunched posture, piloerection and decreased activity on day 1 with piloerection continuing in 4 animals on day 2 and 3 animals on day 3. From the data it was considered that the recommended test guideline regulatory maximum dose of 2000 mg/kg was suitably tolerated and was therefore selected as the high dose for the Main Experiment. Additional lower doses of 1000 and 500 mg/kg were also tested. As there was no substantial differences in toxicity noted between the sexes the Main Experiment was conducted in male animals only.

Applicant's summary and conclusion

Conclusions:

It is concluded that Tin dichloride did not induce chromosome aberrations in the bone marrow cells of male treated rats at dose levels of 500, 1000 and 2000 mg/kg (a recommended maximum dose in accordance with current test regulatory guidelines) following a single oral administration with sampling at 16 hours (500, 1000 and 2000 mg/kg) or 42 hours (2000 mg/kg) post dose.

Executive summary:

Tin dichloride was tested for its ability to induce chromosome aberrations in the bone marrow of treated rats according to OECD test guideline 475 and in compliance with GLP.

 

Analysis of Data – Structural Chromosome Aberration Data

Treatment of male rats with Tin dichloride for 16 hours resulted in frequencies of cells with structural chromosome aberrations (excluding gaps) that were similar to and not significantly (p≤0.05) higher than those observed in the concurrent vehicle controls for all dose groups. Individual aberration frequencies for the majority of all Tin dichloride dosed animals (all dose groups) were similar to those observed in the vehicle control group and which were consistent with historical vehicle control data ranges. No statistically significant increase in chromosome aberrations was observed at a dose of 2000 mg/kg at the 42 hour sample time with frequencies of cells with structural chromosome aberrations (excluding gaps) generally similar to values observed within the concurrent vehicle control group and consistent with historical vehicle control data ranges. These data indicated no evidence of any test article dose effect on chromosome aberration induction.

 

Analysis of Data – Numerical Aberration Data

Frequencies of cells with numerical aberrations for all doses (16 and 42 hour samples) were similar to those observed within the concurrent vehicle control groups. Individual numerical aberration frequencies fell within historical vehicle control range (HCR) data for all treated animals.

It is concluded that Tin dichloride did not induce chromosome aberrations in the bone marrow cells of male treated rats at dose levels of 500, 1000 and 2000 mg/kg (a recommended maximum dose in accordance with current test regulatory guidelines) following a single oral administration with sampling at 16 hours (500, 1000 and 2000 mg/kg) or 42 hours (2000 mg/kg) post dose.