Chlorotrioctylstannane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2002 to 28 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Chlorotrioctylstannane

EC Number:

219-969-8

EC Name:

Chlorotrioctylstannane

Cas Number:

2587-76-0

Molecular formula:

C24H51ClSn

IUPAC Name:

chlorotrioctylstannane

Test material form:

liquid

Specific details on test material used for the study:

- Appearance: Colourless liquid
- Density: 1.04
- Storage conditions of test material: at < -18 °C, in the absence of light
- Date of receipt: 7 October 2002
- Date of expiry: 31 July 2004

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Frozen stocks of each strain were checked for histidine requirement and for sensitivity to ampicillin, crystal violet and UV radiation.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Frozen stocks were checked for tryptophan requirement and for sensitivity to ampicillin, crystal violet and UV radiation.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- Range Finding Test: 0, 0.3, 0.8, 2.3, 7, 21, 62, 185, 556, 1667 and 5000 µg/plate (TA 98 only)
- Main Test: 62, 185, 556, 1667 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO. Test material was dissolved in DMSO at 50 mg/mL. A colourless, slightly viscous solution was obtained.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, N-ethyl-N-nitrosourea

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
To 2 mL molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophan for the E. coli strain), maintained at 46 °C, were added subsequently: 0.1 mL of a fully grown culture of the appropriate strain, 0.1 mL of the appropriate test material solution or of the negative or positive control substance solution, and 0.5 mL S9-mix for with metabolic activation or 0.5 mL sodium phosphate 100 mM (pH 7.4) without metabolic activation.
- The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose).

DURATION
The plates were incubated at ca. 37 °C for 48 - 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of background lawn

EXAMINATIONS
- The his+ and trp+ revertants were counted

Evaluation criteria:

A test material is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.
A test material is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Positive results from the bacterial reverse mutation test indicate that a test material induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test material is not mutagenic in the tested strains.
Both numerical significance and biological relevance are considered together in the evaluation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic at the highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TEST
The test material was not toxic and no precipitation was observed at any concentration.

MUTAGENICITY TEST
The test material was toxic to strain TA 1537 at the highest concentration, as was evidenced by a decrease in the mean number of revertant colonies. Precipitation of the test material was observed at 1667 and 5000 µg/plate.
In both the absence and the presence of S9-mix and in all strains, the test material did not cause more than a two-fold increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.
The mean number of his + and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Any other information on results incl. tables

Table 1: Summary of Mutagenicity Experiment

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 62 185 556 1667 5000	191 189 192 165 183 174	20 18 25 20 23 18	28 32 32 36 41 32	40 34 32 32 33 36	17 15 12 12 14 9
+	Solvent 62 185 556 1667 5000	183 185 194 215 191 208	17 18 26 15 19 14	39 38 37 39 25 33	53 63 65 61 59 50	27 23 22 20 14 10
Positive Controls
-	Name	SA	SA	NENN	2NF	9AA
	Concentration (µg/plate)	1	1	100	2	80
	Mean no. colonies/plate	811	652	190	1740	2040
+	Name	2AA	2AA	2AA	2AA	BP
	Concentration (µg/plate)	2	2	80	2	4
	Mean no. colonies/plate	3351	618	1885	1576	272

SA = Sodium azide

NENN = N-ethyl-N-nitrosourea

2NF = 2-Nitrofluorene

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and US EPA OPPTS 870.5100 under GLP conditions.

The test material was examined for mutagenic activity using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, the tryptophan-requiring Escherichia coli strain WP2 uvrA, and a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix).

The test material was dissolved in DMSO. A dose range finding test was performed with TA98 both in the absence and the presence of S9-mix with ten different concentrations of the test material, ranging from 0.3 - 5000 µg/plate. The test material was not toxic at any concentration.

In the main bacterial reverse mutation test, the test material was evaluated at concentrations of 62, 185, 556, 1667 and 5000 µg/plate. Negative controls (solvent) and positive controls were run simultaneously with the test material.

The test material was toxic to strain TA 1537 at the highest concentration, as was evidenced by a decrease in the mean number of revertant colonies.

In both the absence and the presence of S9-mix and in all strains, the test material did not cause more than a two-fold increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.

The mean number of his + and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.