Tetrabutyltin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 9 June 2004 to 1 August 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW, and MAFF

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutyltin

EC Number:

215-960-8

EC Name:

Tetrabutyltin

Cas Number:

1461-25-2

Molecular formula:

C16H36Sn

IUPAC Name:

tetrabutylstannane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Tetrabutyltin (CAS No, 1461-25-2)
- Physical state: colourless liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen
- Other: Date received 19 May 2004

Method

Target gene:

Mutant strains of Salmonella and E. coli that are unable to synthesize either histidine or tryptophan were exposed to test material to determine if the material is a mutagenic agent that can cause the bacteria to undergo a reverse mutation to histidine or tryptophan independence.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Salmonella was incapable of synthesizing histidine.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: E. coli was incapable of synthesizing tryptophan

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

PRELIMINARY TEST:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

EXPERIMENT 1
Salmonella strains: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E. coli strain WP2uvrA-: 50, 150, 500, 1500 and 5000 µg/plate
Additional dose levels were included where applicable to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.

EXPERIMENT 2
Salmonella strains: (except TA98) with S9: 15, 50, 150, 500, 1500 and 5000 µg/plate
E. coli strain WP2uvrA- and TA 98 with S9: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in distilled water and only partially soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. The test material was fully soluble in acetone at the same concentration.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: in triplicate

Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control, and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate for each bacterial strain and for each concentration of test material both with and without S9 mix. All plates were incubated at 37 degrees C for 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

Reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency to all of the Salmonella strains, initially at 500 and 1500 µg/plate in the absence and presence of S9, respectively. No toxicity was noted to E.coli strain WP2uvrA- at any test material dose level either with or without S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. These findings were not indicative of toxicity sufficiently severe to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate; this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The study was carried out under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate for the Salmonella strains and 50 to 5000 µg/plate for E. coli strain WP2uvrA. The experiment was repeated on a separate day using an amended dose range based on results from Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels were included (where applicable) to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency to all of the Salmonella strains, initially at 500 and 1500 µg/plate in the absence and presence of S9 respectively. No toxicity was noted to E. coli strain WP2uvrA at any test material dose level either with or without S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. These findings were not indicative of toxicity sufficiently severe to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed at and above 1500 µg/plate; this did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.