Tin bis(2-ethylhexanoate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro assays (Ames, cytogenicity/ chromosomal aberrations and HGPRT) were negative with tin bis(2 -ethylhexanoate).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Report lacks information on metabolic activation system, characterisation of test material, individual plate counts, mean number of revertant colonies per plate, and positive and negative controls used.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

An additional Spot Plate Test was conducted against each indicator organism using the undiluted test material.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat liver induced by AROCLOR 1254

Test concentrations with justification for top dose:

0.5, 5, 100 and 500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard solvent

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar spot plate test and overlay plate test

Spot Plate Test
Approximately 10e8 cells from a 16-hour culture of each indicator strain are added to separate test tubes containing 2.0 ml of molten overlay agar supplemented with biotin and a trace of histidine. For nonactivation tests, the contents of the tube is then poured over the surface of a sterile petri dish containing approximately 30 ml of hardened base agar and allowed to solidify. In activation tests, just prior to pouring, an aliquot of the reaction mixture (0.5 ml containing the 9000 x g liver homogenate) is added to each of the overlay agar tubes which are then mixed and poured over the base agar. When all plates are hardened, the test materials, solvent and positive controls are spotted on individual platesof each bacterial strain, with and without activation, in 10 uL quantities. The plates are then covered to prevent photoreactivity of chemicals and within one hour the plates are transferred to a darkened incubator where they are held at 37 deg C for 48-72 hours.

Overlay Plate Test
Approximately 10e8 cells from a 16-hour culture of each indicator strain are added to separate test tubes containing 2.0 ml of molten agar supplemented with biotin and a trace of histidine. For non-activation tests, at least four dose levels of the test compound are added to the contents of the appropriate tubes and poured over the surfaces of selective agar plates. In activation tests, a minimum of four different concentrations of the test chemical are added to the appropriate tubes with cells. Just prior to pouring, an aliquot of reaction mixture (0.5 ml containing the 9000 x g liver homogenate) is added to each of the activation overlay tubes, which are then mixed and the contents poured over the surface of a minimal agar plate and allowed to solidify. The plates are incubated for 48-72 hours at 37 deg C and scored for the number of colonies growing on each plate.

The number of colonies on each plate are counted using a Model C111 Automated Colony Counter.

Evaluation criteria:

Because the procedures used to evaluate the mutagenicity of the test chemical are semi-quantitative, the criteria used to determine positive effects are inherently subjective and are based primarily on a historical database. Most data sets are evaluated using the following criteria:
1. Strains TA-1535, TA-1537, and TA-1538
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered t o be mutagenic.
2. Strains TA-98 and TA-100
If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and two to three times the solvent control value for strain TA-98 is considered to be mutagenic. For these strains , the dose response increase should start at approximately the solvent control value.
3. Pattern
Because TA-1535 and TA-100 were both derived from the same parental strain (G-46) and because TA-1538 and TA-98 were both derived from the same parental strain (03052), there is a built - in redundancy in the microbial assay. In general, the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain , e.g. TA-1537, responds to a mutagen in non-activation tests, it will generally do so inactivation tests. (The converse of this relationship is not expected). While similar response patterns are not required for all mutagens, they can be used to enhance the reliabilityof an evaluation decision.
4. Reproducibility
If a chemical produces, a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data loses significance.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test material failed to exhibit mutagenic activity in both the activation and non-activation systems and is considered not to be mutagenic under the conditions employed.

Executive summary:

The test substance was prepared in DMSO at concentrations ranging from 0.5 - 500 µg/plate. The Ames bacterial assay was conducted both with and without metabolic activation. An additional Spot Plate test was conducted against each indicator organism using the undiluted test material. No evidence of genetic activaity was observed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 24, 2016 to November 1, 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test Material Name: Stannous Octoate
Chemical Name: 2-Ethylhexanoic acid, Tin(2+) Salt (2:1)
Synonyms: Stannous Octoate S-26, Tin bis(2-ethylhexanoate)
Lot/Reference/Batch Number: A072E2N001
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 96.9% by gas chromatography with identification by nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy (Zhang, 2016).
Test Material Stability Under Storage Conditions: Stannous octoate was determined to be stable for 2 weeks at 55°C which is equivalent to 24 months under ambient storage conditions (Schoeb and Linehan, 2011).

Target gene:

Chromosomal aberration assay utilizing rat lymphocytes.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Species and Sex: Rats (Male)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: 10-12 weeks

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley rats

Test concentrations with justification for top dose:

Cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 253.2 μg stannous octoate per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol (CAS No.64-17-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: cyclophosphamide monohydrate (CP, CAS No. 6055-19-2)

Details on test system and experimental conditions:

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), from several rats was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM L-glutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

In Vitro Metabolic Activation System:
S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley rats was purchased from a commercial source, and stored at -100°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a "mix" (O'Neill et al., 1982). The mix consisted of 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium was 1/5 of the concentrations stated above.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in ethanol up to 405.1 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was dissolved in ethanol and further diluted (1: 100) in medium. This technique has been shown to be an effective method for detecting various in vitro clastogens in this test system. All dosing units were expressed in μg/ml. MMC was dissolved in treatment medium, and CP was dissolved in distilled water.

Dose Level Selection:
The cultures were treated with various concentrations of the test material and the selected concentration of the positive control chemicals. Soluble materials were tested up to 10 mM, 2000 μg/ml or 2 μl/ml, whichever was the lowest. Test materials with limited solubility were tested up to or beyond their limit of solubility. In some cases, more than one insoluble concentration was tested to ascertain whether toxicity would occur at higher insoluble concentrations. The other concentrations tested were separated by a factor of 2 to 3.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay A1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by gas chromatography with tandem mass spectrometry (GC/MS-MS). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was conducted on all stock concentrations.

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Evaluation criteria:

Evaluation Criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the vehicle controls. The aberration frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. A test chemical was considered positive in this assay if it induced a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was outside the control limits of the laboratory historical vehicle control range. A test chemical was considered negative in this assay if it did not induce a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal.

Statistics:

Statistical Analysis:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed (Armitage, 1971).
Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells was pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 and based on the exposure time.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

4.0, 15.8 and 31.7 μg/ml dose levels

Cytotoxicity / choice of top concentrations:

other: Increasing toxicity observed with increasing dose levels.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

7.5, 25.0 and 60.0 μg/ml dose levels

Cytotoxicity / choice of top concentrations:

other: Increasing toxicity observed with increasing dose levels.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

0.25, 0.5 and 1.0 μg/ml dose levels

Cytotoxicity / choice of top concentrations:

other: Slight toxicity observed at lowest dose, very slight toxicity at the middle dose and an increased level of toxicity observed at the highest dose level evaluated.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 4051 μg/ml of the test material and medium containing 1% ethanol were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.41, osmolality = 446 mOsm/kg H2O; treatment medium with 1% ethanol, pH = 7.42, osmolality = 437 mOsm/kg H2O).

Remarks on result:

other: 4-Hour Treatment

Assay A1:

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 4.0, 7.9, 15.8, 31.7, 63.3, 126.6, and 253.2 μg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 2.0 μg/ml. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 145.1 to 83.1% of the target.

Short Treatment:

In the absence of S9, the cultures displayed excessive toxicity at the top two target concentrations (i.e., 126.6 and 253.3 μg/ml) as exhibited by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 3.4 to 106.8% compared to the vehicle control values. Based upon these results, this portion of the assay was repeated in a separate assay, due to not achieving the recommended level of cytotoxicity (i.e., 60 + 10% reduction in mitotic index).

In the presence of S9, again excessive toxicity was observed in the cultures treated with the top two target concentrations (i.e., 126.6 and 253.3 μg/ml) as exhibited by the lack of observable mitotic figures. The relative mitotic indices of the remaining cultures ranged from 6.5 to 97.8% compared to the vehicle control values.

Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 4.0, 15.8, and 31.7 μg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the presence of S9 activation.

Among the cultures treated with the positive control chemicals for 4 hours, 4 μg/ml of CP was selected for evaluation of aberrations in the presence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures (4-hour treatment, with S9) as compared to the vehicle control values. In the presence of S9, cultures treated with the test material at target concentrations of 4.0, 15.8, and 31.7 μg/ml had aberrant cell frequencies of 1.3, 0.0, and 1.0%, respectively as compared to the vehicle control value of 0.3%. Statistical analysis of these data did not identify significant differences between the vehicle control and any of the test material-treated cultures with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in CP (+S9, 4-hour treatment) cultures was 39.3% (Table 5) which was within the control limits of the laboratory historical positive control range.

Continuous Treatment:

Cultures treated continuously for 24 hours in the absence of S9 activation had excessive toxicity at the three highest target concentrations (i.e., 63.3, 126.6, and 253.2 μg/ml) as exhibited by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 4.5 to 381.8% relative to the vehicle control value. The relative mitotic indices of test material-treated cultures were inflated due to the lack of mitotic indices observed in the vehicle controls. Based upon these results this portion of the assay was repeated, due to the inability of evaluating the test material-related cytotoxicity relative to the vehicle control treatment (i.e., 60 + 10% reduction in mitotic index) and the limited number of observable mitotic figures present in the vehicle controls.

Assay B1:

Based on toxicity data concluded in the initial assay (Assay A1) cultures were treated in the absence of S9 for 4 hours at target concentrations of 0 (vehicle control), 5.0, 7.5, 15.0, 20.0, 25.0, 30.0, and 60.0 μg/ml. Cultures were treated for 24 hours at target concentrations of 0 (vehicle control) 1.25, 2.5, 5.0, 7.50, 10.0, 15.0, and 30.0 μg/ml (data in study file). Due to poor lymphocyte growth in all cultures, slides were unable to be evaluated for mitotic indices and chromosome aberrations in the absence of S9, in the short (4-hour) and continuous treatment (24-hour) conditions. Therefore, these conditions were repeated in a separate assay.

Assay C1:

In the absence of S9 (4-hour treatment condition) target concentrations were unchanged from the previous repeat. However, in the 24 hour treatment conditions the concentrations were slightly adjusted to target concentrations of 0 (vehicle control), 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 μg/ml (data in study file). Due to poor lymphocyte growth in all cultures, slides were unable to be evaluated for mitotic indices and chromosome aberrations in the absence of S9, in the short (4-hour) and continuous treatment (24-hour) conditions. Therefore, these conditions were repeated in a separate assay.

Assay D1:

Cultures were treated with the test material for 4 hours in the absence of S9 activation at target concentrations of 0 (vehicle control), 5.0, 7.5, 15.0, 20.0, 25.0, 30.0, and 60.0 μg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 at target concentrations of 0 (vehicle control), 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 μg/ml.

Short Treatment:

In the absence of S9, moderate to no toxicity was observed with relative mitotic indices ranging from 48.6 to 101.4% (Table 6). Based upon these results, cultures treated with 0 (vehicle control), 7.5, 25.0, and 60.0 μg/ml in the absence of S9 were selected for determination of chromosomal aberration frequencies and the incidence of polyploidy. The cultures treated with the positive control chemical, 0.5 μg/ml MMC were evaluated for chromosomal aberrations and the incidence of polyploidy.

There were no significant increases in the incidence of polyploid cells in test material treated cultures compared to the vehicle control values. In the absence of S9, cultures treated with the test material at target concentrations of 7.5, 25.0, and 60.0 μg/ml had aberrant cell frequencies of 0.0, 1.0, and 0.3%, respectively, compared to the vehicle control value of 0.3%. Statistical analysis of these data did not identify significant differences between the vehicle control and any of the test material-treated cultures without S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in MMC (-S9, 4-hour treatment) cultures was 27.3%, which was within the control limits of the laboratory historical positive control range.

Continuous Treatment:

Cultures treated continuously for 24 hours exhibited excessive toxicity at the top target concentration (i.e., 10.0 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures displayed relative mitotic indices of 21.3 to 97.9%. Based upon these results targeted concentrations of 0 (vehicle control) 0.25, 0.5, and 1.0 μg/ml were selected for evaluations of chromosomal aberrations and the incidence of polyploidy. The cultures treated with the positive control chemical, 0.05 μg/ml MMC were evaluated for chromosomal aberrations and the incidence of polyploidy.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

The frequency of aberrant cells in the vehicle control was 0.0% and the corresponding values at concentration levels of 0.25, 0.5, and 1.0 μg/ml were 0.3, 0.7, and 0.3%, respectively. There were no statistically significant differences between the test material treated cultures and the vehicle control values and all values were within the control limits of the laboratory historical vehicle control range.

A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC (0.05 μg/ml) treated cultures was 13.0%, which was within the control limits of the laboratory historical positive control range.

Conclusions:

It was concluded that under the experimental conditions used, stannous octoate was negative in this in vitro chromosomal aberration test.

Executive summary:

Stannous octoate (2-Ethylhexanoic acid, Tin(2+) Salt (2:1)) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 253.2 μg stannous octoate per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The duration of treatment was 4 hours without and with S9 and 24 hours without S9. The analytically determined concentrations of stannous octoate in the dose preparations ranged from 145.1 to 83.1% of the targeted values. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 4.0, 15.8, and 31.7 μg/ml in the presence of S9 and 0 (vehicle control), 7.5, 25.0, and 60.0 μg/ml in the absence of S9 and cultures treated for 24 hours with 0 (vehicle control), 0.25, 0.5, and 1.0 μg/ml were analyzed.

There were no significant increases in the frequency of cells with aberrations administered stannous octoate in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells. Based upon these results, stannous octoate was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 24, 2016 to October 4, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Test Material Name: Stannous Octoate
Chemical Name: 2-Ethylhexanoic acid, Tin(2+) Salt (2:1)
Synonyms: Stannous Octoate S-26, Tin bis(2-ethylhexanoate)
Lot/Reference/Batch Number: A072E2N001
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 96.9% by gas chromatography with identification by nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy (Zhang, 2016).
Test Material Stability Under Storage Conditions Stannous octoate was determined to be stable for 2 weeks at 55°C which is equivalent to 24 months under ambient storage conditions (Schoeb and Linehan, 2011).

Target gene:

The gene for Hgprt is located on the mammalian X-chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED:
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge
National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4
cell line was selected as the test system for this study because it is sensitive to mutagens,
has a low background mutant frequency, and is readily available. Stock cultures were
stored at approximately -80°C or below. The cultures were periodically checked for
mycoplasma contamination (American Type Culture Collection, Manassas,
Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture
lab-ware under standard conditions of approximately 5% CO2 in air at 37°C in a
humidified incubator.

MEDIA USED:
The cells were routinely maintained in Ham's F-12 nutrient mix supplemented with 5%
(v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum, 25 mM HEPES,
antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml;
fungizone, 0.25 μg/ml), and an additional 2 mM L-glutamine. Treatment medium was
the above-mentioned medium without serum. The selection medium used for the
detection of Hgprt- mutants was Ham's F-12 nutrient mix without hypoxanthine,
supplemented with 10 μM 6-thioguanine, 5% serum, 25 mM HEPES, 2 mM L-glutamine,
and the above-mentioned antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 3.75 to 140.0 μg/ml in the absence and presence of S9. The highest concentration was based on the cytotoxicity of the test material.

Vehicle / solvent:

Ethanol (CAS No. 64-17-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 20-methylcholanthrene (20-MCA, CAS No. 56-49-5)

Details on test system and experimental conditions:

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in ethanol up to 405.1 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was first dissolved in ethanol and further diluted (1:100) in medium to obtain the desired concentrations as recommended in the test guidelines. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981). EMS was dissolved in treatment medium. 20-MCA was dissolved first in DMSO and further diluted in the treatment medium. All dosing units were expressed in μg/ml.

Treatment Procedure:
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 10^6 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with treatment medium, S9 mix (when applicable), the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++ and Mg++ free).

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay B1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by gas chromatography with tandem mass spectrometry (GC/MS-MS). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was conducted on all stock concentrations.

In Vitro Metabolic Activation:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium is 1/5 of the concentrations stated above.

Evaluation criteria:

Evaluation Criteria:
For an assay to be acceptable, the mutant frequency in positive controls should be significantly higher than the vehicle controls. The mutation frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. The test chemical was considered positive if it induced a statistically significant, dose-related increase in mutant frequency, and the mutant frequency was outside the control limit of the laboratory historical vehicle control range. The test chemical was considered negative if it did not induce a statistically significant, dose-related increase in mutant frequency, and the mutant
frequency was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the vehicle and positive controls.

Statistics:

Statistical Analysis:
The frequency of mutants per 10^6 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts were assumed to follow a Poisson distribution, therefore, the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence of S9 excessive toxicity was seen at 27.0 μg/ml and not plated to determine mutation frequency. Remaining cultures had RCS values from 16.2 to 101.2% compared to the vehicle control. With S9, RCS values ranged from 25.6 to 99.5%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

There was moderate toxicity observed in the absence of S9 with RCS values ranging from 12.0 to 110.4% compared to the vehicle control. In the presence of S9, RCS values showed moderate toxicity with values ranging from 23.2 to 100.3%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 4051.0 μg/ml of the test material and medium containing 1% ethanol were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.35, osmolality = 479 mOsm/kgH2O; treatment medium with 1% ethanol, pH = 7.34, osmolality = 487 mOsm/kgH2O).

Assay A1 – Preliminary Toxicity Assay:

In the preliminary toxicity assay, the test material was tested at concentrations of 0.0 (vehicle control), 1.0, 2.0, 4.0, 7.9, 15.8, 31.7, 63.3, 126.6 and 253.2 μg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration tested was based upon the limitations imposed by the solubility of the test material in treatment medium. The highest two concentrations (126.6 and 253.2 μg/ml) precipitated in the treatment medium, as observed at the end of treatment. The treated cultures without S9 activation showed excessive toxicity at the four highest concentrations (i.e., 31.7, 63.3, 126.6 and 253.2 μg/ml). The remaining cultures had relative cell survival (RCS) values ranging from 66.3 to 117.5%. In the presence of S9 activation, excessive toxicity was observed at the two highest concentrations (i.e., 126.6 and 253.2 μg/ml). The remaining cultures had RCS values ranging from 25.1 to 116.9%. Based upon the results of this assay, concentration levels of 0.0 (vehicle control), 7.5, 15.0, 20.0, 25.0, 30.0, and 60.0 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and 0.0 (vehicle control), 7.5, 15.0, 25.0, 50.0, 70.0, 90.0, and 120.0 μg/ml in the presence of S9.

Assay B1 – Initial Mutagenicity Assay:

In the initial mutagenicity assay (Assay B1), in the absence of S9 excessive toxicity was observed at the three highest concentrations (i.e., 25.0, 30.0, and 60.0 μg/ml) and therefore cells were not plated to determine mutation frequency. The remaining cultures had RCS values ranging from 41.1 to 94.0%. In the presence of S9, RCS values ranged from 15.1 to 92.7%. As a result of a technical error cultures treated with target concentrations of 50.0, 70.0, and 90.0 μg/ml were lost and thus were unable to be evaluated for mutation frequency. There was assumed to be an error upon dosing in which the 120.0 μg/ml concentration and 70.0 μg/ml concentration were inadvertently interchanged based upon the observed RCS values. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 at the remaining concentration levels were not significantly different from the concurrent vehicle control values.

As a result of the excessive toxicity observed in the absence of S9, resulting in less than four analyzable doses, and the technical errors in the presence of S9, the initial mutagenicity assay B1 was repeated at target concentrations of 0.0 (vehicle control), 3.75, 7.5, 15.0, 20.0, 23.0, and 27.0 μg/ml in the absence of S9 and 0.0 (vehicle control), 7.5, 15.0, 25.0, 50.0, 70.0, 90.0, and 120.0 μg/ml in the presence of S9.

Assay B2 – Repeat Initial Mutagenicity Assay:

In the repeat initial mutagenicity assay (Assay B2), in the absence of S9 excessive toxicity was observed at the highest concentration (i.e., 27.0 μg/ml) and thus was not plated to determine mutation frequency. The remaining cultures had RCS values ranging from 16.2 to 101.2% compared to the vehicle control. In the presence of S9, RCS values ranged from 25.6 to 99.5%. Due to a technical error one replicate of the positive control (8 μg/ml MCA) was lost and thus not evaluated for mutation frequency. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 at all concentration levels were not statistically significantly different from the concurrent vehicle control values. Mutant frequencies of the vehicle controls were comparable to the laboratory historical vehicle control values, despite one replicate slightly exceeding the control limits of the laboratory historical vehicle control range. Based on the results of the repeat initial toxicity assay a confirmatory mutagenicity assay was conducted utilizing identical target concentrations analyzed in the repeat mutagenicity assay with the exclusion of the lowest concentration (i.e., 7.5 μg/ml) and an additional higher concentration (i.e., 140.0 μg/ml) in the presence of S9.

Assay C1 – Confirmatory Mutagenicity Assay:

In the confirmatory assay (Assay C1), the concentrations ranged from 3.75 to 27.0 μg/ml in the absence of S9 and 15.0 to 140.0 μg/ml in the presence of S9. There was moderate toxicity observed in the absence of S9 with RCS values ranging from 12.0 to 110.4% compared to the vehicle control. In the presence of S9, RCS values showed moderate toxicity with values ranging from 23.2 to 100.3%. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values. Mutant frequencies of the vehicle controls were comparable to the laboratory historical vehicle control values, despite one replicate slightly exceeding the control limits of the laboratory historical vehicle control range. In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutation frequencies. The mutant frequencies exhibited by the cultures treated with the positive control chemicals were comparable to the laboratory historical positive control values while slightly exceeding the control limits of the laboratory historical positive control range. These data confirmed the adequacy of the experimental conditions for detecting induced mutations. The analytically observed concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 87.6 to 130.5% of target.

Conclusions:

It was concluded that under the experimental conditions used, stannous octoate was negative in this in vitro CHO/HGPRT forward gene mutation assay.

Executive summary:

Stannous octoate (2-Ethylhexanoic acid, Tin(2+) Salt (2:1)) was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 3.75 to 140.0 μg/ml in the absence and presence of S9. The highest concentration was based on the cytotoxicity of the test material. The analytically determined concentrations of stannous octoate in the dose preparations ranged from 87.6 to 130.5%. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e. ethanol). There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. Cultures treated with the positive control chemicals had significantly higher mutant frequencies. Based upon these results, stannous octoate was considered to be negative in this in vitro CHO/HGPRT forward gene mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Three in vitro assays (Ames, cytogenicity/ chromosomal aberrations and HGPRT) were negative with tin bis(2 -ethylhexanoate), so that neiter an in vivo-follow up nor potential classification according to Regulation (EC) No 1272/2008 are warranted.