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Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2011-09-19 to 2011-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- blood from three healthy, non-smoking female volunteers (age: 25 to 39 years old) was used for each experiment.
- no donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals.
- all donors are non-smokers, not heavy drinkers of alcohol and not taking any form of medication (contraceptive pill excluded).
The measured cell cycle time of the donors used falls within the range 13 ± 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in centrifuge tubes by placing 0.4 mL of pooled heparinised blood into HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen phytohaemagglutinin (reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1°C for approximately 48 hours and rocked continuously.
Metabolic activation:
with and without
Metabolic activation system:
S-9: prepared from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Range-finder:
- 5.442, 9.070, 15.12, 25.19, 41.99, 69.98, 116.6 194.4, 324.0, 540.0, 900.0, and 1500 µg/mL (with metabolic activation; 3 hour treatment and 17 hour recovery)
- 0.07254, 0.1209, 0.2016, 0.3359, 0.5599, 0.9331, 1.555, 2.592, 4.320, 7.200, 12.00, and 20.00 µg/mL (without metabolic activation; 3 hour treatment and 17 hour recovery or 20 treatment without recovery)

Experiment 1:
- 0.4, 0.8, 1.1, 1.4, 1.7, 2.0, 2.3, 2.6, 2.9, 3.2, 3.6, 4.0, and 5.0 µg/mL (without metabolic activation; 3 hour treatment and 17 hour recovery)
- 15.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 115.0, 130.0, 150.0, and 200.0 µg/mL (with metabolic activation; 3 hour treatment and 17 hour recovery)

Experiment 2:
- 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 8.0, and 10.0 µg/mL (without metabolic activation; 20 treatment without recovery)
- 25.0, 50.0, 75.0, 100.0, 120.0, 140.0, 160.0, 180.0, 200.0, 225.0, 250.0, and 300.0 µg/mL (with metabolic activation; 3 hour treatment and 17 hour recovery)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Test article stock solutions were prepared by formulating stannous sulfate, crystalline under subdued lighting in purified water, with the aid of vortex mixing, to give the maximum concentrations. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 mm 0.2 μm pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within approximately 5 hours of initial formulation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Positive control without metabolic activation: concentration: 2.5 and 5.0 µg/mL; vehicle: anhydrous analytical grade DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive control with metabolic activation: concentration: 6.25 and 12.5 µg/mL; vehicle: anhydrous analytical grade DMSO
Details on test system and experimental conditions:
CYTOTOXICITY RANGE-FINDER
Immediately prior to treatment, all 3+17 hour +S-9 cultures were pelleted (approximately 300 g, 10 minutes) and 0.9 mL of culture medium removed to give a final pre-treatment volume of 8.5 mL.
S-9 mix or KCl (0.5 mL/culture) was added appropriately. Cultures were treated with the test article or vehicle control (1.0 mL/culture). Positive control treatments were not included. The final culture volume was 10 mL. Cultures were incubated at 37 ± 1°C for the designated exposure time (3 hours or 20 hours).

MAIN EXPERIMENTS
Immediately prior to Experiment 1 treatment, all vehicle and test article treated cultures were pelleted (approximately 300 g, 10 minutes) and 0.9 mL of culture medium removed to give a final pre-treatment volume of 8.5 mL. Immediately prior to Experiment 2 treatment, all positive control cultures had 0.9 mL culture medium added to give a final pre-treatment volume of 9.4 mL.
S-9 mix or KCl (0.5 mL/culture) was added appropriately. Cultures were treated with the test article or vehicle control (1.0 mL/culture) or positive controls (0.1 mL/culture).
The final culture volume was 10 mL. Cultures were incubated at 37 ± 1°C for the designated exposure time (3 hours or 20 hours).

For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (37 ± 1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin.

Please refer for the treatment schedule of the cytotoxicity range finder and main experiments (table 1) to the field "Any other information on materials and methods incl. tables" below.

HARVESTING
Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 μg/mL to arrest dividing cells in metaphase. At the defined sampling time cultures were centrifuged at approximately 300 g for 10 minutes; the supernatant was carefully removed and cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37 ± 1ºC for 15 minutes. Cells were fixed by dropping the KCl suspension into cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, two to three minutes) until the cell pellets were clean.

SLIDE PREPARATION
Lymphocytes were kept in fixative at 2-8ºC prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading and several drops of suspension were transferred on to clean microscope slides. Slides were flamed, as necessary. Slides were dried on a hot plate (approximately 80 - 100°C) and stained in filtered 4% (v/v) Giemsa in pH 6.8 Gurr’s buffer for 5 minutes. The slides were rinsed, dried and mounted with coverslips using DPX.

SELECTION OF CONCENTRATIONS FOR MAIN EXPERIMENTS
Slides from the cytotoxicity Range-Finder Experiment were examined and the mitotic index (MI) determined.
MI is a measure of the proliferative state of the culture at a particular moment in time and was calculated as follows:
MI = (number of cells in mitosis / total number of cells observed) x100

Mitotic inhibition (MIH) is calculated as:
MIH (%) = [1 - (mean MIT/mean MIC)] x 100%
(where T = treatment and C = vehicle control)
Slides from sufficient concentrations from each treatment group were scored to determine whether chemically induced MIH had occurred. This is defined as a clear decrease in MI compared with vehicle controls, (based on at least 1000 cells counted, where possible), and is preferably concentration-related.
A suitable range of concentrations was selected for the Main Experiments based on these toxicity data.

SELECTION OF CONCENTRATIONS FOR CHROMOSOME ANALYSIS (MAIN EXPERIMENTS ONLY)
Slides were examined for MI to determine whether chemically induced mitotic inhibition had occurred.
The highest concentrations for chromosome analysis from cultures sampled at 20 hours were ones at which at least (or approximately) 50% mitotic inhibition had occurred. Slides from the highest selected concentration and at least two lower concentrations were taken for microscope analysis, such that a range of mitotic inhibition from maximum to little was covered.
For each treatment regime, two vehicle control cultures were analysed for chromosome aberrations. Positive control concentrations, which gave satisfactory responses in terms of quality and quantity of mitoses and extent of chromosomal damage, were analysed.

SLIDE ANALYSIS
Slides from positive control treatments in Experiment 1 were checked to ensure that the system was operating satisfactorily.
Where appropriate, one hundred metaphases from each slide were analysed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) were noted on a slide, analysis was terminated. Only cells with 44 to 48 chromosomes were considered acceptable for analysis. Any cell with more than 48 chromosomes (that is, polyploid, hyperdiploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately. Structural aberrations were classified according to the ISCN scheme (ISCN, 1995)*.
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.
Chromosome aberration analysis was not conducted on slides generated from the Range-Finder treatments.

ANALYSIS OF RESULTS
The numbers of aberrant cells in each culture were categorised as follows:
1. cells with structural aberrations including gaps
2. cells with structural aberrations excluding gaps
3. polyploid, endoreduplicated or hyperdiploid cells.

The totals for category 2 in vehicle control cultures were compared with the current historical vehicle control (normal) ranges to determine whether the assay was acceptable. The totals for category 2 in test article treated cultures were also compared with normal ranges. The statistical significance of increases in the percentage of cells with structural aberrations for any data set was only taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentrations exceeds the normal range. The proportions of cells in categories 1 and 3 were also examined in relation to normal ranges.

Acceptance criteria:
The assay was considered valid if all the following criteria were met:
1. the binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures
2. the proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range
3. at least 160 cells out of an intended 200 were suitable for analysis at each concentration, unless 10 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis
4. the positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated structural aberration cell frequencies (excluding gaps) that clearly exceeded the current historical vehicle control ranges.

*Reference:
- ISCN (1995) An International System for Human Cytogenetic Nomenclature. Editor Felix Mitelman; S Karger, Switzerland
Evaluation criteria:
The test article was considered to induce clastogenic events if:
1. a proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05)
3. there was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered positive if all of the above criteria were met.
The test article was considered negative if none of the above criteria were met.
Results which only partially satisfied the above criteria were to be dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al, 1990)*.

Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, effects occurring only at high or very toxic concentrations and the types and distribution of aberrations.

*Reference:
- Scott D, Dean B J, Danford N D and Kirkland D J (1990) Metaphase chromosome aberration assays in vitro. Basic Mutagenicity Tests; UKEMS recommended procedures. Kirkland D J (Ed), pp 62-86
Statistics:
The statistical method used was Fisher's exact test (Richardson et al, 1989)*. Probability values of p ≤ 0.05 were accepted as significant.

*Reference:
- Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D O and Phillips B (1989) Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Kirkland D J (Ed) Cambridge University Press, pp 141-154.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: no marked changes in pH and osmolality were observed at the highest concentration tested under all treatment conditions in range-finder (1500 μg/mL), compared to the concurrent vehicle controls.

- Water solubility: preliminary solubility data indicated that stannous sulfate, crystalline was soluble in water for irrigation (purified water) at concentrations of at least 22.60 mg/mL. The solubility limit in culture medium was less than 565.0 μg/mL, as indicated by visible precipitation at this concentration 20 hours after test article addition. A maximum concentration of 1500 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to a precipitating concentration.

- Precipitation: during the range finder precipitation was observed for the following concentrations (at end of treatment incubation period and at harvest): 540.0, 900.0, and 1500 µg/mL (with metabolic activation; treatment: 3 hours; recovery: 17 hours)

MAIN EXPERIMENTS
1) Structural aberrations
Treatment of cells with stannous sulfate, crystalline for 3+17 hours in the absence of S-9 in Experiment 1 resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle control cultures. Numbers of aberrant cells (excluding gaps) fell within the 95th percentile of the normal range (0-3% aberrant cells) with the exception of one culture at the highest concentration analysed (5.000 μg/mL) in which 6% aberrant cells were observed, which fell outside the observed range of 0-4%.
Treatment of cells for 20+0 hours in the absence of S-9 in Experiment 2 resulted in frequencies of cells with structural aberrations that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle control cultures at the highest two concentrations analysed (4.500 and 5.000 μg/mL, giving 42% and 57% mitotic inhibition, respectively). Numbers of aberrant cells (excluding gaps) exceeded the normal range in both cultures at 5 μg/mL and in single cultures at 4.000 and 4.500 μg/mL.
Treatment of cells in the presence of S-9 in Experiment 1 resulted in frequencies of cells with structural aberrations that were slightly elevated, compared to those observed in concurrent vehicle control cultures, at all concentrations analysed. Numbers of aberrant cells (excluding gaps) exceeded the normal range of 0-3% aberrant cells in single cultures at all three concentrations analysed (7%, 6% and 5% at 80.00, 130.0 and 200.0 μg/mL, respectively). The aberrant cell frequencies (excluding gaps) in the replicate cultures fell within the normal range, but the mean frequencies exceeded the 95th percentile of the normal range at 80.00 and 200.0 μg/mL.
Treatment of cells in the presence of S-9 in Experiment 2 resulted in frequencies of cells with structural aberrations that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle control cultures at the highest three concentrations analysed (100.0, 140.0 and 160.0 μg/mL, giving 39%, 53% and 57% mitotic inhibition, respectively). Numbers of aberrant cells (excluding gaps) exceeded the normal range in both cultures analysed at these concentrations.

2) Numerical aberrations
No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent vehicle controls and the normal range, were observed in cultures treated with stannous sulfate, crystalline in the absence and presence of S-9.
Conclusions:
Interpretation of results: positive

It is concluded that stannous sulfate, crystalline induced structural chromosome aberrations in cultured human lymphocyte cells when tested for 3+17 hours in the presence of S-9 and for 20+0 hours in the absence of S-9. In the same test system, stannous sulfate, crystalline did not induce structural chromosome aberrations when tested up to toxic concentrations for 3+17 hours in the absence of S-9.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosomal Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
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
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
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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.
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.
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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Introductory remarks on genetic toxicity

According to ECHA “Guidance “on information requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance” (Version 2.4, February 2014), existing data should be qualified by its (i) adequacy, (ii) reliability and (iii) relevance. Especially the reliability of data must be considered when assessing its usefulness for hazard/risk assessment purposes. The guidance suggests applying the rating according to Klimisch. According to this rating scheme, existing references are rated according to (i) the adherence to different test guidelines (compared with today's standards) (ii) the proper characterisation of the test substance (in terms of purity, physical characteristics, etc.) (iii) the level of sophistication of techniques/procedures.

A number of OECD Guidelines on genetic toxicology testing have recently been withdrawn from the portfolio of OECD test guidelines, such as:

477: Sex-Linked Recessive Lethal test in Drosophila melanogaster

478: Genetic Toxicology: Rodent dominant Lethal Test

479: in vitro Sister Chromatid Exchange in Mammalian Cells

480: Saccharomyces cerevisiae, Gene mutation assay

482: DNA Damage and Repair, Unscheduled DNA synthesis in Mammalian Cells.

According to the qualification criteria for existing data as stated above, tests conducted according to these above mentioned guidelines have a minor contribution to the overall assessment, since newer and more up-to-date test guidelines exist. Existing tests conducted according to the more up-to-date guidelines have therefore been rated with a higher reliability and were subsequently considered with a higher contribution to the overall assessment of genetic toxicity of the category substance. Further, tests which do not directly address the endpoint genetic toxicity were considered of minor relevance, such as DNA damage in bacteria tested according to the rec assay. This assay only measures differential killing and is not a mutation assay.

For some references, only short abstracts are available. Due to the limited information content, these references were not considered for hazard assessment purposes and rated with reliability 4 “not assignable” (Maciel VB (2011), Anonymous (1987)).

Some references investigate the direct interaction of tin salts with isolated DNA in aqueous systems (Caldeira-de-Araujo A (1996), de Mattos JCP (2005), de Mattos JCP (2000)). Such study design does not allow a conclusive statement on the genetic toxicity relevant for chemicals hazard assessment, since it completely excludes natural barriers such as cell membranes, cellular defence mechanisms, such as exocytosis and further biological responses relevant for cellular response towards a chemical insult. Consequently, these references are considered not to contribute to any relevant extent to the assessment of genetic toxicity of divalent tin salts.

 

In vitro genetic toxicity

Gene mutation in bacteria

Stannous methane sulfonate (purity not reported) was tested unequivocally negative independently in two unpublished study reports in bacterial reverse mutation assays (Burr, AD 1988; Grötsch, W 1986). S. typhimurium tester strains TA98, TA100, TA1535, TA1537 were exposed to doses up to 5000 µg/plate (5 doses) in the presence and absence of a metabolic activation system. Tests were conducted according to OECD 471 (1983) and under GLP in triplicate and two independent experiments, using the plate incorporation method. Cytotoxicity characterised by reduced background lawn or a decrease in number of revertants was seen in all strains at the highest dose in one study. Positive control substances significantly increased the number of revertants, demonstrating the sensitivity of the test system. A significant increase of revertant colonies was not observed in any of the assays performed with any of the tester strains with or without metabolic activation. In one study, an isolated significant increase was observed in the second experiment at the highest dose in strain TA1535, TA1537, TA98 (-S9) and TA1537 (+S9). This isolated finding was not reproduced in the first experiment, did not follow a dose-response relationship and is likely being caused by the automatic scoring system. Thus, this artefact was not considered to represent a test-item induced response of the test system and was therefore considered not biologically relevant. Both studies were conducted in accordance with test guidelines, were conducted under GLP and fulfil the requirements for chemical risk assessments. Experiments and results are presented in detail, whereas the purity of the test item was not reported. Both references are considered reliable with restrictions.

Stannous dichloride (anhydrous, purity not reported) was tested in a bacterial reverse mutation assay using a modified pre-incubation method (Pungartnik, C 2005). Tester strains S. typhimurium TA97, TA98, TA100, TA102 and E. coli IC203, IC188 were exposed to eight doses of stannous dichloride up to 5mM without metabolic activation, only 5 doses were evaluated. It is unclear whether the dose referred to the test item concentration in the pre-incubation tube or on the plate. The methods section describes a pre-incubation method, using strains TA97, TA98, TA100, TA102, whereas only results for IC188, IC 203 and TA102 were reported. Cytotoxicity was not measured or reported. A slight increase in revertants (2fold above negative control values) was observed in IC203 at the highest concentration. Since negative and positive historical control data were not reported, a conclusive interpretation of the results cannot be given. Due to the unclear experimental procedure, the incomplete reporting and the questionable dose selection, this reference is not considered reliable for the chemical safety assessment.

Simmon (1978) investigated the mutagenic effects of stannous dichloride dihydrate (purity not reported) in a bacterial reverse mutation assay using strains TA98, TA100, TA1535, TA1537, TA1538 and E. coli WP2(uvrA). The test was performed equivalent to OECD TG 471, but without GLP. The bacteria were incubated in duplicate using the plate incorporation protocol at doses of 0.3 to 10000µg/plate with and without metabolic activation in two independent experiments. Six doses from 33.3 to 10000µg/plate were evaluated for the presence of revertants. The highest dose produced cytotoxicity by reduction of the number of revertants per plate in all strains tested. The positive control substance caused a significant increase in revertant rates, demonstrating the sensitivity of the test system. Stannous dichloride dihydrate did not show an increase in revertant rates in any of the tester strains with or without metabolic activation. The results were summarised and published in a peer-reviewed journal (Prival, MJ 1991). Experiments and results presented in detail, the purity of the test item was not reported. Thus, the combination of both references is considered reliable with restrictions.

Stannous fluoride (identity and purity not reported) was tested in a programme for bacterial reverse mutation and induction of micronuclei in the bone marrow of rats and mice (reported below) (Gocke, E. et al. 1981). Five tester strains of S. typhimurium TA98, TA100, TA1535, TA1538 were incubated with the test item at five doses up to 3600µg/plate with and without metabolic activation. Details on parallel cultures and the number of independent experiments are missing. Results were only presented graphically for TA100 with an unclear axis annotation (doses given as µM, whereas methods section gives doses as µg/plate). No results are reported for strains TA98, TA1535, TA1538. The experiments with metabolic activation show a dose-dependent increase in revertants but only a 1.6-fold increase at the maximum dose. There was no such increase in the experiments without metabolic activation. Contrary to the authors’ interpretation, these findings unequivocally show a non-mutagenic activity of stannous fluoride, since the revertant rate clearly stays below a 2-fold increase. The reference is not considered reliable for hazard and risk assessment purposes due to several experimental and reporting deficiencies as detailed above.

Mortelmans, K (1986) investigated the mutagenic potential of stannous chloride (purity: >99%) in a bacterial reverse mutation assay using S. typhimurium strains TA98, TA100, TA1535, TA1537 at 5 doses between 3.3 and 333 µg/plate. Tests were conducted in triplicates in two independent experiments using the preincubation assay. The maximum concentration was limited by signs of toxicity, i.e. reduced background lawn and reduced numbers of revertant colonies. Concurrent positive and negative controls were added, S9-mix from rat and hamster were used as metabolic activation systems. The authors only reported the results of the second experiment, since these were allegedly in agreement with the results of the first experiment. Stannous chloride did not produce an increase in revertant frequencies when tested up to cytotoxic concentrations with or without metabolic activation in any of the tester strains. The publications reports sufficient details on the experimental procedure for a comprehensive assessment. However, only aggregated results or historical control data were reported which renders the study reliable with restrictions.

Overall, stannous (II) salts show a consistently negative response in the bacterial reverse mutation assay – in assays judged reliable, there was no increase in the mutation frequency in any of the tester strains with or without metabolic activation when tested up to cytotoxic or limit concentrations. It is therefore concluded that stannous (II) salts are not mutagenic in the bacterial reverse mutation assay.

 

Typically, bacterial reverse mutation assays are of limited relevance for metals risk assessment, since the uptake of many metal ions by bacteria is considered to be poor, and thus the sensitivity of bacterial test systems on the detection of the mutagenic potential of dissolved metal ions appears to be low (cf. REACH Guidance on information requirements and chemical safety assessment, Chapter R.7a (Version May 2008), Page 390; HERAG fact sheet No 5 Mutagenicity, Chapter 2.1). However, it has been shown that tin cations in fact interact with bacterial cells and become systemically available (Attramadal A, 1980; Camosci DA, 1984,Ferretti GA 1982). Consequently, studies on bacterial reverse mutations can be considered relevant for the assessment of genetic toxicity of tin salts.

 

In vitro mammalian cell gene mutation

Stannous chloride dihydrate (purity not reported) was assayed for the ability to induce mutations at the thymidine kinase (tk) locus (5-trifluorothymidine [TFT] resistance) in L5178Y mouse lymphoma cells (Myhr BC et al. 1991). This work was a part of the US NTP work programme on the investigation of carcinogenic and genotoxic effects of stannous dichloride. The cells were exposed to 6 concentrations of 10-100µg/mL in triplicate cultures for 4 hours with and without metabolic activation in two independent experiments. During the exposure, precipitation was observed at concentrations of 80µg/mL and above; an acidic pH shift was observed at 50µg/mL and above. The relative total growth ranged from an average of 35% without S9 and 47% with S9 at the highest concentration. No relevant increase in mutation frequency could be observed in any of the cultures. Some erratic increase in MF was observed, but without any dose response correlation or reproducibility in parallel cultures, thus were considered of no biological relevance. Since no significant increase in mutation colonies was observed, a colony sizing was not performed. Experiments and results are presented in detail, the study design was similar to OECD 476, but without GLP and without further characterisation of the test item. The reference is therefore considered reliable with restrictions.

In conclusion, stannous dichloride does not show signs of increased mutation frequencies in mammalian cells when tested up to precipitating concentrations in the mouse lymphoma assay with or without metabolic activation. It is therefore concluded that tin(II) chloride is non-mutagenic in the in vitro mammalian gene mutation assay.

 

In vitro mammalian chromosome aberration/micronucleus tests

Damati A et al. (2014) investigated the clastogenic effect of five different tin substances (tin(II)acetate, purity not given; tin(II) chloride, purity not given; tin(II) ethylhexanoate, purity ≥95%; tin(II) oxalate, purity 98%; tin(II) oxide, purity >99%) for the induction of micronuclei in human peripheral blood lymphocytes using a study design similar to OECD 487. Blood samples were obtained from healthy non-smoking young donors. Two replicate cultures were exposed to concentrations of 0, 1, 5, 10, 20, 50 and 75 µM for 31 hours; Cyto-B was added 3 hours after exposure. In total, 4,000 binucleated cells per dose were scored for the presence of micronuclei, and 2,000 cells per dose were scored for the calculation of the CBPI. Effects on pH and osmolality were not checked. The results are summarised in graphical format only, which does not facilitate a quantitative analysis. However, the authors state a cytotoxicity at the highest concentration for the test items as follows: tin(II)acetate: not analysable due to cytotoxicity; tin(II) chloride: 64.75%; tin(II) ethylhexanoate: not analysable due to cytotoxicity; tin(II) oxalate: 45.78%; tin(II) oxide: 21%. The highest concentration of tin(II) oxide showed signs of precipitation. None of the test items showed an increase of the micronucleus frequency in any of the tested concentrations. Positive control MN frequencies were reported to be in accordance with published MN frequencies (data not shown). Negative control MN frequencies were in accordance with historical control MN frequencies from other labs. The maximum effective dose to achieve a cytotoxicity of 55% ±5% was not achieved with tin(II) oxalate, which might be explained by a masking of the tin cation by oxalate ligands, leading to a less active form. The maximum concentration for tin(II) oxide was limited by signs of test item precipitations. The experimental design is reported in detail and the summarised results were presented in graphical format as well as textually in sufficient detail for an assessment. The lack of compliance in not achieving the targeted cytotoxicity at the highest concentration or addition of a metabolic activation system does not change the overall conclusion. Due to the minor shortcomings, the reference is considered reliable with restrictions.

In an unpublished study report, stannous sulfate (purity 99%) was tested in an in vitro chromosomal aberration assay in human peripheral blood lymphocytes (Lloyd M 2012). Duplicate cultures of pooled blood lymphocytes from three female healthy donors were exposed to stannous sulfate at concentrations of 2-5 µg/mL (-S9) and 25-200 µg/mL (+S9). Changes for osmolality or pH were checked in the cytotoxicity range finding experiments. In two independent main experiments with 3 and 20 hours exposure duration, chromosome aberration frequencies were scored in three out of four concentrations. No marked changes in osmolality or pH were observed in the range finding experiments up to concentrations of 1500µg/mL. In the first experiment with 3 hours exposure duration and S9, an increase in chromosomal aberrations frequency exceeding the historical control range was observed. However, the increase is not dose-dependent and, if viewed in isolation, is considered equivocal. The highest concentrations showed 45% and 44% cytotoxicity without and with metabolic activation, respectively. In the second experiments, a statistically significant and dose-dependant increase in the aberration frequency also exceeding the historical control range, was observed in the 3 hour treatment with S9 and the 20 hour treatment without S9 mix. Positive and negative control aberration frequencies were within the range of historical control data. Stannous sulfate was therefore considered clastogenic under the conditions of this assay by induction of structural chromosome aberrations when tested for 3 hours in the presence of S9 and for 20 hours in the absence of S9. The study is considered valid and relevant without restriction.

Gulati DK (1989) investigated the ability of stannous chloride (purity not reported) for the induction of chromosome aberrations and sister chromatid exchanges. This work was a part of the US NTP work programme on the investigation of carcinogenic and genotoxic effects of stannous dichloride:

- in the chromosomal aberration assay, CHO cells were exposed to concentrations of 0, 100, 500, 750µg/mL (Experiment I, -S9), 0, 25, 50, 100µg/mL (Experiment II, -S9), 0, 750, 1000, 1500µg/mL (+S9) in singular cultures. MMC was used as positive control substance without metabolic activation and cyclophosphamide was used as positive control substance with metabolic activation. Deviating statements on the incubation duration were found throughout the report, whereas the methods section states an incubation duration of 10 hours without metabolic activation and 11 hours with metabolic activation, the results table state an incubation duration of 18 and 19 hours and the discussion section states an incubation duration of 12 and 2 hours, respectively. Colcemid was added 2 hours prior to harvest. Cells were Giemsa stained and 200 metaphase cells were scored. There is no record on how cytotoxicity was determined. Precipitation was observed at concentrations at 500 µg/mL and above. Stannous chloride showed a significant increase of chromosomal aberrations in CHO cells with and without metabolic activation. The study shows a number of experimental and reporting deficiencies. It is unclear for how long the cells were incubated, since the reported durations deviate significantly. Cytotoxicity was not measured, which does not allow any correlation between cytotoxicity and clastogenicity. The authors report test item precipitation at 500µg/mL and above, however testing was performed well above precipitation concentrations which is not in-line with guideline recommendations. The precipitation of stannous chloride also brings into question the test item identity, since stannous chloride has a water solubility of 178 g/L. Taken together, this experimental part of the publication is not considered reliable for chemical safety assessment.

- in the sister chromatid exchange assay, CHO cells were exposed to concentrations of 0, 1.6, 5, 16, 50µg/mL (Experiment I, -S9), 0, 75, 100, 125, 150µg/mL (Experiment II, -S9), 0, 25, 50, 75, 100µg/mL (Experiment III, -S9), 0, 16, 50, 160, 500µg/mL (Experiment I, +S9), 0, 160, 500, 750, 1000µg/mL (Experiment II, +S9) in singular cultures. MMC was used as positive control substance without metabolic activation and cyclophosphamide was used as positive control substance with metabolic activation. Cells were treated for a duration of 2 hours prior to the addition of BrdU (final concentration 10µM) and further incubated for 24 hours.After the 2 hour exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS and 10 µM BrdUrd was added. Cells were incubated for an additional 26 hours, with colcemid present for the final 2-3 hours of incubation. Two to three hours after addition of colcemid, cells were harvested by mitotic shake-off. Cells were treated in a hypotonic solution, fixed and stained. Fifty second-division metaphase cells were scored per dose for the incidence of sister chromatid exchange.Cytotoxicity was not measured, which does not allow any correlation between cytotoxicity and clastogenicity. The authors report test item precipitation at 500µg/mL and above, however testing was performed well above precipitation concentrations which is not in-line with test guideline recommendations. The precipitation of stannous chloride also brings into question the test item identity, since stannous chloride has a water solubility of 178 g/L. Taken together, this experimental part of the publication is not considered reliable for chemical safety assessment.

In a testing series, the US Food and Drug Administration commissioned the conduct of in vitro and in vivo genetic toxicity tests in rats with stannous chloride (purity not reported) at Litton Bionetics (Anonymous, 1974; in vivo results discussed further below). In an in vitro mammalian cell chromosome aberration test stannous chloride (purity not reported) was tested for its ability to induce structural aberrations in human embryonic lung cells (WI-38). Cells were exposed to concentrations of 0.1, 1, 10µg/mL without metabolic activation in triplicate cultures for 24 hours. Cells were harvested by shaking when sufficient mitoses were observed, fixed and stained. Mitotic inhibition was scored as a measure of cytotoxicity. A total of 100 metaphases per dose were scored for chromosomal aberrations. No test item precipitation was observed. The positive control substance produced a significant increase of the CA frequency. A slight increase in the aberration frequency was observed at the high dose, bus still in the range of normal levels. The testing series in this report was conducted prior the implementation of harmonised test guidelines, such as the OECD technical guidelines. However, the study design follows generally accepted scientific principles and was conducted following an equivalent study design as foreseen in the first OECD 473 (1983), OECD 475 (1984) and OECD 478 (1984). The study was conducted prior to the implementation of the good laboratory practice principles in 1978. Due to the missing test item characterisation, the low number of metaphases scored per dose and the unclear cytotoxicity determination this study is considered reliable with restriction and should only be used in a weight-of-evidence approach.

Overall, tin(II) salts show an equivocal outcome in in vitro clastogenicity tests. Tin(II) acetate, chloride, ethylhexanoate oxalate did not elicit an increase in the micronucleus frequency, whereas tin(II) sulfate exhibited a significant increase in the chromosome aberration frequency in human peripheral blood lymphocytes. The positive findings with tin(II) sulfate were primarily observed with metabolic activation, which was not applied in the micronucleus test with 5 difference tin(II) substances. Tin(II) chloride did not show any clastogenic effects (small colony formation) in the mouse lymphoma assay in L5178Y cells.

 

Summary entry – assays for DNA damage in vitro

Several studies on in vitro DNA damage studies in mammalian cells (involving comet assays) were identified which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA guidance on information requirements, chapter R.7.7. Most of the references are on mechanistic investigations to examine specific mechanisms of tin salts and are not suitable to address the endpoint of in vitro genetic toxicity. These studies are discussed below only briefly for information purposes only, and were included in the IUCLID merely as a summary entry:

McLean JRN (1983) investigated the uptake and DNA damage of tin(II) chloride (purity not reported) in human white blood cells. Cells were treated with different tin(II) chloride concentrations for 30 min at 0 and 37°C and the fluorescence quenching effect of DNA strand breaks on a double stranded DNA-specific dye, ethidium bromide was measured. The authors did not correlate the fluorescence quenching with cytotoxicity, and fluorimetric analysis is no a direct measure of DNA damage. Authors did not sufficiently demonstrate that direct and test item specific DNA damage was measured.

McLean JRN (1983) investigated the DNA damaging effect of tin(II) chloride (purity not reported) in CHO cells. Cells were treated with different tin(II) chloride concentrations (50-550µM equals 9.5-104mg/L) for 60 min at 37°C. DNA damage was measured by an alkaline sucrose gradient. Authors did not correlate the sucrose gradient with cytotoxicity. Any effects on osmolality or pH were not measured. The authors did not sufficiently demonstrate that direct and test item specific DNA damage was measured.

Olivier Ph (1987) tested the cytotoxic effect of stannous chloride dihydrate (purity not reported) in a SOS chromotest in E. coli PQ37 and PQ35. The SOS chromotest is not a direct measure of genetic toxicity but rather a test on sequential cytotoxicity.

Dantas FJS (2002) investigated the cytotoxic and DNA damaging effect of stannous chloride dihydrate (purity note reported) on K562 cells. Cells were treated with stannous dichloride at concentrations of 0-886µM up to 24 hours. Authors observed dose-dependent cytotoxicity with a max of <20% viability at the highest concentration. A similar DNA damaging effect dependency was observed in the comet assay. Authors did not correlate cytotoxicity with increased DNA damage observed in the comet. Since levels of cytotoxicity above 50% are known to cause positive findings in the comet assay, the observed DNA damaging effect by stannous dichloride was a direct succession of cytotoxicity.

Viau CM (2009) investigated the cytotoxic and DNA damaging effect of stannous chloride dihydrate (purity note reported) on V79 cells. Cells were treated with stannous dichloride at concentrations of 50-1000µM for 2 hours and further incubated for 24 or 48 hours. Authors observed dose-dependent cytotoxicity with a max of <25% viability at the highest concentration. A similar DNA damaging effect dependency was observed in the comet assay. The authors did not correlate cytotoxicity with increased DNA damage observed in the comet. Since levels of cytotoxicity above 50% are known to cause positive findings in the comet assay, the observed DNA damaging effect by stannous dichloride was a direct succession of cytotoxicity.

Cabral REC (1998) investigated the cytotoxicity / cell survival of stannous dichloride (purity not reported) on bacterial strains with lacking DNA repair genes. Cultures were treated with concentrations of 6.25, 12.5, 25, 50 µg/mL and the subsequent survival was measured. Cytotoxicity is not a direct measure of mutagenic activity or DNA damage. The authors did not sufficiently demonstrate that direct and test item specific DNA damage was measured.

Bernardo-Filho M (1994) investigated the cytotoxicity / cell survival of stannous dichloride (purity not reported) on E. coli K12 by measuring colony forming ability and SOS responses such as filamentation. Cultures were treated with concentrations of 5, 10, 25, 50, 75 µg/mL and the subsequent survival or filamentation was measured. Neither cell survival nor cell filamentation are a direct measure of DNA damage or mutagenic activity. The authors did not sufficiently demonstrate that direct and test item specific DNA damage was measured.

Swierenga SHH, (1983) described in a very brief publication the effects of tin(II) chloride (purity not reported) DNA damage and initiation of DNA synthesis in human white blood cells and primary rat hepatocytes. The experimental procedure and results are presented very briefly, which does not allo a comprehensive assessment of the study design and the results. Tin(II) chloride was given in a single dose, which does not allow the investigation of a dose-response relationship.

 

Summary entry - unsuitable test systems in vitro

Several studies were identified which do not fulfil the relevance, reliability and adequacy criteria as foreseen by the ECHA guidance on information requirements, chapter R.7.7. DNA damage in bacteria, induction of SCE in mammalian cells, or tests in yeasts or drosophila are no longer recommended as part of regulatory testing by many agencies worldwide and there are no up-to-date OECD guidelines for their conduct. Interpretation of the relevance of both positive and negative results from such tests is therefore unclear and was not used for the current assessment. The studies are discussed below in brief for information purposes only and were included in the IUCLID as summary entry.

Viau CM (2006): genotoxic effects of stannous chloride in Saccharomyces cerevisiae (as discussed above).

 

Overall conclusion on in vitro genetic toxicity

There was no evidence for mutagenic activity of divalent tin salts in bacterial reverse mutation assays or in a mouse lymphoma assay up to the maximum concentration limited by cytotoxicity. Consequently, divalent tin salts can be considered non-mutagenic in suitable in vitro test systems.

There is an equivocal evidence in vitro for clastogenic activity of divalent tin salts in the mammalian cell micronucleus and mammalian chromosome aberration assay. Positive findings were primarily in the presence of metabolic activation and at elevated concentrations. The underlying mechanism for positive findings in the presence of metabolic activation remain unclear – one may safely assume that divalent tin cations are not metabolised. No aneugenic activity was observed in an in vitro micronucleus assay. Consequently, no conclusive statement can be made on the clastogenic effects of divalent tin salts.

 

In vivo genetic toxicity tests

In vivo chromosome aberration/micronucleus test

Gocke E. et al (1981) treated 2 male and 2 female NMRI mice per dose group at 0 and 24 hrs with doses of 0, 9.8, 19.6, 39.5 mg stannous fluoride/kg bw via i.p. injection. Bone marrow smears were prepared at 30 hrs, and 1,000 polychromatic erythrocytes were scored per animal. No increase in the MN PCE frequency could be observed in the bone marrow of mice, the MN PCE frequency (2.5-2.9‰) was within the range of the negative control animals (30 parallel experiments showed a MN-PCE of 1.0 to 3.1‰). It is therefore concluded that stannous chloride is non-clastogenic in the bone marrow of mice. The reference shows deficiencies in the study design as well as in reporting. Animals were treated twice after 0 and 24 hrs and sacrificed after 30 hrs, i.e. 6 hrs after final treatment. This period is too short for any clastogenic events to develop – usually sacrifice between 18 and 24 hrs after final administration is foreseen for bone marrow analysis to allow cell to undergo at least one complete cell cycle. The test item was given via an unphysiological route of exposure. Whereas this route might be relevant for pharmaceutical applications, it is considered of limited relevance for the hazard assessment of industrial chemicals. The experimental procedures for cell collection, preparation and staining is not given at all, no further details on the test animals, such as age, weight, housing conditions were presented. The number of animals per dose group is too low, which compromises the statistical robustness of the results. Based on the above given experimental and reporting shortcomings, this reference is not considered reliable for hazard and risk assessment purposes.

In an unpublished in vivo micronucleus test according to OECD 474 (1983) and under GLP, the clastogenic potential of stannous methane sulfonate (purity not reported) was tested (Jenkinson PC, 1989). In a dose range finding experiment the maximum tolerated dose in albino CFLP mice was determined to be 1000 mg/kg bw, based on mortalities observed at doses of 2000 and 5000 mg/kg bw. In the main experiment groups of 5 male and 5 female animals were given the maximum tolerated dose of 1000 mg/kg bw via single gavage. Animals were sacrificed 24, 48 and 72 hours after exposure, the bone marrow of femur isolated fixed and stained. A total of 1000 polychromatic erythrocytes (PCE) were scored per animals for the presence of micronuclei. In addition, the PCE/NCE ratio was determined by scoring 1000 normochromatic erythrocytes (NCE), for the assessment of target organ toxicity. There were no significant increases in the MN-PCE frequency in any of the treatment groups. An isolated increase in the MN frequency exceeding the negative control frequencies was observed in one male animal sacrificed after 24 hours (13/1000); a re-scoring gave a frequency of 6/1000, 9/1000 and 8/1000. The negative control animals showed a MN-PCE frequency of 0/1000 to 5/1000; the positive control animals showed a MN-PCE frequency of 10/1000 to 41/1000. Both the negative and positive control data appear to be high in comparison with up to date control data from other testing laboratories. No target organ toxicity by a change of the PCE/NCE ratio could be observed in any of the treatment groups. The fluctuation of the re-scoring results for one male animal showing significant increase in MN-PCE frequency might be indicative for less experienced staff scoring the cells. Based on the above given criteria the study is considered reliable with restriction and should only be used in a weight of evidence approach.

In an unpublished study, tin(II) methane sulfonate (purity not reported) was tested for the induction of micronuclei in mice (Korn WD, 1986). The non-GLP study was conducted in accordance with OECD 474 (1983). In a dose-range finding experiment 2 animals per dose were given intraperitoneal injections of 50, 100, 200, 300 mg/kg bw, the lowest dose did not cause lethality but signs of toxicity and was therefore considered as MTD. In the main experiment young NMRI mice, 5 males and 5 females per dose group, were given an aqueous solution of tin(II) methane sulfonate at a dose of 50 mg/kg bw via i.p. injection and sacrificed 24, 48 and 72 hours post exposure. Negative control animals received an equal amount of water, positive control animals received 40 mg/kg bw Endoxan (i.e. Cyclophosphamide), both groups were sacrificed 24 hours after exposure. The bone marrow was isolated from femora, washed, stained and analysed. A total of 1000 polychromatic erythrocytes (PCE) were scored for presence of micronucleated polychromatic erythrocytes (MN-PCE), the ratio of polychromatic to normochromatic erythrocytes (NCE) was calculated on the base of 1000 cells. A negative control MN-PCE frequency of 0.8% was given as laboratory historical control; the MN-PCE frequency of the negative control animals ranged from 0.1 to 0.9% in this study. The PCE/NCE ratio significantly increased 72 hours after exposure, showing bone marrow toxicity. No biologically relevant increase in the MN-PCE frequency was observed in any animals at any time point (range from 2/1000 to 13/1000). An isolated increase in the MN frequency exceeding the negative control frequencies was observed in two male animals sacrificed after 24 hours (13/1000), this was not observed in other male or female animal analysed at that time point. It is therefore concluded that this isolated increase is of no biological relevance. Cyclophosphamide induced a significant increase in the MN-PCE frequency (range from 55/1000 to 118/1000), confirming the sensitivity of the used animal strain. Both the negative and positive control data appear to be high in comparison with recent control data from other testing laboratories (negative mean: 0.8-1.2‰). Based on the results, tin(II) methane sulfonate is considered non-clastogenic in the bone marrow of i.p. exposed mice. The unphysiological i.p. route of administration is considered as worst-case exposure route, since it bypasses physiological barriers and elimination mechanisms, which would be observed with physiological routes of administration. Due to the high MN-PCE control frequencies, the missing GLP status and test item characterisation, this study is considered reliable with restriction and should only be used in a weight of evidence approach.

In a very briefly documented publication, the clastogenic effect of stannous chloride (purity >99%, confirmed by gas chromatography) in pregnant mice was investigated (El-Makawy AI, 2008). Groups of 5 males and 10 females were given oral doses stannous chloride dihydrate of 2, 10 and 20 mg/kg bw/day. The maximum dose was based on a WHO human MTD of 2 mg/kg bw. After 3 weeks of treatment, male and female animals were cohabitated in a 2:1 ratio. The presence of a vaginal plug was considered as day of gestation and female animals were treated until GD18. The male animals were apparently not examined further. Animals received an i.p. injection of colchicine (dose not reported) prior to sacrifice, the femora were dissected and subsequently the bone marrow was isolated, washed and stained. It is unclear whether the cells were treated in a hypotonic solution prior to staining. The chromosomes of 100 metaphases per animal were scored for the presence of structural or numerical aberrations. The authors did not provide any details on clinical signs of toxicity, gross necropsy observations or changes in body weight. The authors report a statistically significant and dose-dependent increase in the frequency of chromosomal aberrations (with and without gaps) in the bone marrow of pregnant female mice (CA without gaps: control 1.9, low dose 2.2, mid dose 9.0, high dose 13.0; only summarised data available). The aberration frequency in the control animals is 6-20 fold above the historical control CA frequencies observed in other laboratories (0.09-0.31). Overall, the reference is considered highly unreliable due to severe experimental and reporting deficiencies. It is quite implausible that the authors determined the purity of the test item using gas chromatography – this method is technically completely unsuitable for the analysis of inorganic salts. The authors also do not provide sufficient details on the experimental procedure for the bone marrow preparation. Details on clinical signs of toxicity, gross necropsy observations or changes in body weight of the treated animals are completely lacking, which prohibits a correlation of toxicity with clastogenicity. It remains unclear why male animals were exposed for a duration of at least 3 weeks but not further investigated. The CA frequencies of the control animals is 6-20 fold above historical control CA frequencies observed in other laboratories. In summary, the results presented in this publication appear implausible when compared with other in vivo studies, the experimental performance and reporting raises doubts on the reliability of the outcome. Consequently, the publication is not considered reliable for chemical hazard assessment purposes.

In a publication series, Shelby MD et al. (1993, 1995) investigated the clastogenic effect of stannous chloride (purity not reported) in mice. This work was a part of the US NTP work programme on the investigation of carcinogenic and genotoxic effects of stannous dichloride:

- in the micronucleus experiments 5 male B6C3F1 mice per dose group received stannous chloride suspended in corn oil via intraperitoneal injection at doses of 0, 26.3, 52.5, 105 mg/kg bw/day in the first experiment and 0, 52.5, 105, 157.5, 210 mg/kg bw/day in the second experiment. The maximum tolerated dose was determined in a dose range finding experiment. In the first experiment, the test item was administered on three consecutive days, animals were euthanized 24 hours after the third administration, bone marrow smears were prepared, fixed and stained. The frequency of MN-PCE among 2000 PCE was determined. The second experiment was conducted after negative findings in the first experiment and was following the same experimental design but a different dosing. DMBA was administered via i.p. injection in corn oil as positive control, solvent was administered as negative control. The vehicle corn oil is somewhat inappropriate for an inorganic salt which could better be dissolved in water or aqueous buffer systems. One mortality occurred in the first experiment in the low dose group and two mortalities occurred in the second experiment in the high dose group. The cause of death in both experiments was not investigated further, but the fatalities in the second experiment might be indicative for an exceedance of the MTD (see also Korn WD, 1986 who reported fatalities at 100mg/kg bw and above in a single i.p. injection in NMRI mice). Animals showed a dose dependent decrease in the %PCE ratio, indicating bone marrow toxicity, thus target organ exposure can be anticipated. The median negative control MN-PCE frequencies out of 50 parallel studies also reported in the publication was 2.15‰ (min: 1.1‰, max: 4.6‰, 90thpercentile: 3.5‰) and thus 2-3 fold above recent MN-PCE negative historical control data of other laboratories (mean: 0.8-1.2‰). The positive control substance caused elevated MN-PCE frequencies of 6.93‰ ±2.59 (range 2.00-12.17‰) over a range of 14 studies; no further details were reported. Stannous chloride did not show an increase in the MN-PCE frequency at any dose, up to the maximum administered dose in both experiments. Based on the results, stannous chloride is considered non-clastogenic in the bone marrow of i.p. exposed mice. The unphysiological i.p. route of administration is considered as worst-case exposure route, since it bypasses physiological barriers and elimination mechanisms, which would be observed with physiological routes of administration. Due to the unusually elevated MN-PCE control frequencies, the inappropriate vehicle, the missing GLP status and test item characterisation, this study is considered reliable with restriction and should only be used in a weight of evidence approach.

- in the chromosomal aberration experiments 8 male B6C3F1 mice per dose group received stannous chloride suspended in corn oil via intraperitoneal injection at doses of 0, 37.5, 75, 150 mg/kg bw/day in the first experiment and 0, 112.5, 150 mg/kg bw/day in the second experiment. The vehicle corn oil is considered inappropriate for an inorganic salt, which could be dissolved in water or aqueous buffer systems. The maximum tolerated dose was determined in a dose range finding experiment. Two hours prior sacrifice, animals were given an i.p. injection of colchicine. Animals were euthanised 17 and 36 hours after administration in the first experiment and 36 hours after administration in the second experiment. Bone marrow was extracted from both femora, cells were treated in a hypotonic solution, fixed and stained. In total 50 metaphases per animal were analysed for the presence of structural or numerical aberrations. Positive control was performed but not reported. Two mortalities occurred in the first experiment in the low and mid dose group respectively. The cause of death was not investigated further. The median negative control CA frequencies out of 24 parallel studies also reported in the publication was 1.56% (min: 0.2%, max: 4.75%, 90thpercentile: 2.4%) were 5-8 fold above the recent CA negative historical control data of other laboratories (mean: 0.09-0.31%, range 0-2%). The positive control values could not be evaluated due to missing data. Stannous chloride showed a weak statistical significant increase in the aberration frequency in the high dose of the first (4.5% ±0.73) and second (5.5% ±0.98) experiment at a sampling time of 36 hours. However, the positive finding in the first experiment lacks a dose-response relationship. The positive finding in the second experiment could not be repeated in a second trial; due to the performance of only two doses a conclusive trend test could not be performed. The outcome of this study is difficult to interpret. The isolated positive findings in the high dose group animals appear to be test item related. However, due to (i) the lacking dose response relationship in the first experiment (ii) failure of repeatability in the second experiment and (iii) clear negative findings in parallel micronucleus experiments in the same animals at even higher doses, the overall conclusion is that stannous chloride is considered non-clastogenic in the bone marrow of i.p. exposed mice. The unphysiological i.p. route of administration is considered as worst-case exposure route, since it bypasses physiological barriers and elimination mechanisms, which would be observed with physiological routes of administration. Due to the unusual elevated %CA control frequencies, the missing GLP status and test item characterisation, this study is considered reliable with restriction and should only be used in a weight of evidence approach.

In a publication by De Mattos JCP et al. (2012) the clastogenic effect of stannous chloride dihydrate (purity not reported) in rats was studied. Adult male Wistar rats (250-300g bw, 5 animals per dose group) were given a 100µL single intravenous injection of 500µg/mL (equivalent to 50µg per animal and 12.5-15µg/kg bw). Negative control animals received 0.9% NaCl solution, positive control animals received 50mg/kg cyclophosphamide via i.v. injection. Animals were sacrificed 36 hours post exposure, femora dissected, the bone marrow extracted and the cells were stained. A total of 2000 polychromatic erythrocytes were analysed per animal. An analysis of the PCE/NCE ratio was not performed. Animals dosed with stannous dichloride did not show an increased frequency of MN-PCE 36 hours post exposure. The reference shows several shortcomings and is not considered compliant with the study design as foreseen in the OECD 474: (i) Adult animals were used instead of young animals as foreseen in the OECD guideline; (ii) Single dose treatment is appropriate in case a limit does of 2000mg/kg bw is administered, however the animals were exposed to a single dose of max. 15µg/kg bw which does not allow an analysis of a dose-response relationship; (iii) Target organ exposure by determination of the PCE/NCE ratio or presence of general signs of toxicity was not determined; hence target organ exposure could not be demonstrated; (iv) The number of animals per dose group is too low in order to demonstrate statistical robustness, whereas the guideline foresees at least 5 animals per dose and sex. Based on the above given shortcomings and the missing GLP status, the reference is not considered reliable and therefore unsuitable for chemical hazard assessment.

In a testing series, the US Food and Drug Administration commissioned the conduct of in vitro and in vivo genetic toxicity tests in rats with stannous chloride (purity not reported) at Litton Bionetics (Anonymous, 1974; in vitro results discussed further above):

- in an in vivo chromosome aberration test, 5 adult Sprague-Dawley rats were assigned in each treatment group. Animals received two types of treatment schedules: acute treatment (single treatment; sacrifice of animals at 6, 24, and 48 hours after treatment) at doses of 4, 40, 400mg/kg bw (Tier I), at a dose of 1300mg/kg bw (Tier II) or subacute treatment (5 treatments 24 hours apart; sacrifice 6 hours after last treatment) at doses of 4, 40, 400mg/kg bw. The dose of 400mg/kg bw was reported to be the LD5. The test item was dissolved in 0.85% saline and given via gavage. Positive control animals received a single i.p. injection of triethylene amine. Bone marrow cells were extracted from the femora, washed, fixed and stained. A total of 50 metaphases per animal were scored for the presence of structural aberrations, the mitotic index was determined by counting at least 500 cells. The acute study did not show aberration frequencies exceeding the normal range value (1-3%). The aberration frequencies in the positive control animals were significantly higher than in the negative control animals, demonstrating the sensitivity of the animal strain for chromosomal damage. The subacute study showed a non dose-dependent increased frequency of chromosomal aberrations but still within the normal range values.

- in a dominant lethal assay in male and female Sprague Dawley rats, the clastogenic effects of stannous chloride (purity not stated) was investigated. Animals received two types of treatment schedules: acute treatment (single treatment; sacrifice of animals at 6, 24, and 48 hours after treatment) at doses of 4, 40, 400mg/kg bw (Tier I), at a dose of 1300mg/kg bw (Tier II) or subacute treatment (5 treatments 24 hours apart; sacrifice 6 hours after last treatment) at doses of 4, 40, 400mg/kg bw. The dose of 400mg/kg bw was reported to be the LD5. The test item was dissolved in 0.85% saline and given via gavage. Positive control animals received a single i.p. injection of triethylene amine. Following treatment, the males were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Two virgin female rats were housed with a male for 5 days. These two females were removed and housed in a cage until killed. The male was rested for two days and then two new females were introduced to the cage. Females were killed using CO2 at 14 days after separating from the male, and at necropsy the uterus was examined for deciduomata (early deaths), late foetal deaths and total implantations. Corpora lutea, early foetal deaths, late foetal deaths and total implantations per uterine horn were recorded. Stannous chloride did not induce a detectable increase in dominant-lethal mutations in either male or female germ cells of rats. It is concluded that stannous chloride does not induce chromosome aberration in the rat germ-cell stages tested.

The testing series in this report was conducted prior the implementation of harmonised test guidelines, such as the OECD technical guidelines. However, the study design follows generally accepted scientific principles and was conducted following an equivalent study design as foreseen in the first OECD 473 (1983) and OECD 475 (1984) and OECD 478 (1984). The study was conducted prior to the implementation of the good laboratory practice principles in 1978. Due to the missing test item characterisation, the low number of metaphases scored per animal in the in vitro CA assay and the low number of animals used per dose group this study is considered reliable with restriction and should only be used in a weight of evidence approach.

In vivo DNA damage studies

In a publication by De Mattos JCP et al. (2012), the DNA damaging effect via Comet assay of stannous chloride dihydrate (purity not reported) in rats was observed. Adult male Wistar rats (250-300g bw, 5 animals per dose group) were given a 100µL single intravenous injection of 500µg/mL (equivalent to 50µg per animal and 12.5-15µg/kg bw). Negative control animals received 0.9% NaCl solution, positive control animals received 50mg/kg cyclophosphamide via i.v. injection. Blood was collected by cutting the tail tip directly before the treatment and 3 and 24 hours thereafter. Cells were treated directly transferred to LMP agarose gels, incubated in a lysis solution and subsequently in alkaline electrophoresis buffer. Comets were evaluated via a four class rating (no migration to tail two-time longer than nucleus), 50 nuclei were evaluated. It is unclear whether this is related to per animal or per dose group. An analysis of the cytotoxicity was not performed. An approx. 2-fold increase in DNA damage was observed in the peripheral blood of animals after 3 hours. The reference shows several shortcomings and is not considered compliant with the study design as foreseen in the OECD 474: (i) Adult animals were used instead of young animals as foreseen in the OECD guideline; (ii) Single dose treatment is appropriate in case a limit does of 2000mg/kg bw is administered, however the animals were exposed to a single dose of max. 15µg/kg bw which does not allow an analysis of a dose-response relationship; (iii) cytotoxicity not determined; hence correlation of DNA damage and cytotoxicity cannot be performed; (iv) the number of animals per dose group is too low in order to demonstrate statistical robustness; (v) the experimental procedure is not described in sufficient detail in order to e.g. determined the number of cells examined per animal (vi) the evaluation of the comets by comparing tail length with nucleus diameter is unusual, commonly tail length, %DNE in tail or tail moment is determined. Based on the above given shortcomings and the missing GLP status, the reference is not considered reliable and unsuitable for chemical hazard assessment.

 

Summary entry - unsuitable test systems in vivo

The references contained in the summary entry unsuitable test systems represent studies in drosophila melanogaster (Fourman P (1994), Tripathy NK (1990), Mitchell (1973)). These references are of limited value for risk assessment purposes,since this test system is no longer recommended as part of regulatory testing by many agencies worldwide and there is no up-to-date OECD guideline for its conduct. The existing OECD test guideline 477 was withdrawn on 2ndApril 2014. Interpretation of the relevance of both positive and negative results from such tests is therefore unclear and was not used for the current assessment.The information contained therein was included for information purposes only.

 

Conclusion on in vivo genetic toxicity

The in vivo data for divalent tin salts present a consistent pattern: in none of the reliable studies did the administration of divalent tin salts either via intraperitoneal injection or via the oral route in rats and mice showed any increase of chromosomal aberrations or micronuclei formation in the bone marrow, or an increase of dominant lethal mutations. Doses were limited by toxicity. Some isolated increases of CA or MN frequencies lacked dose-response and could not be repeated in parallel experiment. Consequently, these findings were considered to be of no biological relevance. It is therefore concluded that divalent tin salts do not cause clastogenic or aneugenic events in animals.

 

 

Conclusion

The available data on genetic toxicity allow a conclusive statement on the genetic toxicity for divalent tin salts. Irrespective of the reporting quality of the publications, both positive and negative findings are reported in in vitro as well as in vivo test systems.

Following a rigorous relevance and reliability screening, it can be concluded that divalent tin salts do not show any clastogenic potential. The references discussed underin vitroclastogenicity do not show a consistent pattern on the induction of chromosome and the overall conclusion was equivocal. However, high-qualityin vivocytogenicity studies with divalent tin salts via intraperitoneal injection and oral administration in mice and rats did not show an increase of micronuclei formation or chromosome aberration up to the maximum tolerated dose. This finding is supported by a negative dominant lethal test in rats after single and repeated oral administration in rats. Also, there was no evidence for any mutagenic activity in bacterial reverse mutation or mammalian gene mutation assays. Consequently, divalent tin salts are considered not to induce gene mutations.

Overall, there is no consistent evidence of induction of genetic toxicity with relevance to humans for divalent tin salts.

Short description of key information:  The available data on genetic toxicity allow a conclusive statement on the genetic toxicity for divalent tin salts. Irrespective of the reporting quality of the publications, both positive and negative findings are reported in in vitro as well as in vivo test systems.  Following a rigorous relevance and reliability screening, it can be concluded that divalent tin salts do not show any clastogenic potential. The references discussed under in vitro clastogenicity do not show a consistent pattern on the induction of chromosome and the overall conclusion was equivocal. However, high-quality in vivo cytogenicity studies with divalent tin salts via intraperitoneal injection and oral administration in mice and rats did not show an increase of micronuclei formation or chromosome aberration up to the maximum tolerated dose. This finding is supported by a negative dominant lethal test in rats after single and repeated oral administration in rats. Also, there was no evidence for any mutagenic activity in bacterial reverse mutation or mammalian gene mutation assays. Consequently, divalent tin salts are considered not to induce gene mutations.  

Overall, there is no consistent evidence of induction of genetic toxicity with relevance to humans for divalent tin salts.  

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Justification for non-classification

The in vivo data for divalent tin(II) salts present a consistent pattern. In reliable and guideline compliant studies as discussed above, combining chromosome aberration and micronucleus endpoints up to toxic doses in mice and rats, divalent tin(II) salts did not show any test item induced genetic toxicity.

None of the in vitro genotoxicity studies rated as reliable showed any effect in bacterial reverse mutation assays, in mammalian cell gene mutation test (TK assay). Mammalian cell chromosome aberration or micronucleus tests returned an equivocal outcome. Since higher-tier in vivo data should take precedence over in vitro data, these equivocal findings were sufficiently addressed by in vivo data and shown not to occur.

The classification criteria acc. to regulation (EC) 1272/2008 as germ cell mutagen are not met, thus no is classification applicable.

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