Dioctyltin dilaurate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

26 March 2002 to 10 April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions. Since the study was conducted with the read across substance, dioctyltin oxide (DOTO), it has been assigned a reliability score of 2.

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:

according 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:

Dioctyltin oxide

EC Number:

212-791-1

EC Name:

Dioctyltin oxide

Cas Number:

870-08-6

Molecular formula:

C16H34OSn

IUPAC Name:

Stannane, dioctyloxo-

Test material form:

solid: crystalline

Details on test material:

- Physical state: Powder, white crystalline.
- Storage condition of test material: -20 °C in the dark.

Method

Target gene:

Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.

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):

- Type and identity of media: Stored as nutrient broth cultures with 7.4 % DMSO at <60 °C.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: rfa-, uvrB-, +R-factor

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Stored as nutrient broth cultures with 7.4 % DMSO at <60 °C.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: rfa+, uvrA-, -R-factor

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.
Definitive test: 0, 62, 185, 556, 1667 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Methanol.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
2 mL molten top agar (0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium stains, and supplemented with 0.05 mM tryptophane for the E.coli WP2 uvrA strain), maintained at 46 °C. 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test material solution, or the solvent or positive control substance and 0.5 mL S9-mix for the plates with metabolic activation or 0.5 mL sodium phosphate 100 mM (pH 7.4) for 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
- Exposure duration: 48-72 hours at 37 °C.

NUMBER OF REPLICATIONS: The tests were performed in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was assessed as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

Evaluation criteria:

The mutagenicity study was considered valid if the mean colony counts of the control values of the strains were within acceptable ranges, if the results of the positive controls met the criteria for a positive response, and if no more than 5 % of the plates were lost through contamination or other unforseen events.
A test material was considered to be positive if the increase in the mean number of revertant colonies on the test plates was concentration-related or if a reproducible two-fold or more increase was observed compared to that of negative control plates.
A test material was considered to be negative in the bacterial gene mutation test if it produced neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test material was found to precipitate at all concentrations.

RANGE-FINDING/SCREENING STUDIES: A dose range finding test was performed with TA 98 in both the absence and 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 both in the absence and presence of S9-mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A slightly more dense background lawn of the bacterial growth was observed at the dose levels at which the slight increases were observed; however, these occurred alongside precipitation of the test material.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Results of Range Finding Test

Dose µg/plate		TA 98
		Without S9-mix	With S9-mix
0		38	52
		36	46
		29	48
	Mean	34	49
	SD	5	3
0.3		46	57
		48	61
		40	46
	Mean	45	55
	SD	4	8
0.8		46	69
		44	57
		26	60
	Mean	39	62
	SD	11	6
2.3		52	52
		42	55
		34	57
	Mean	43	55
	SD	9	3
7		27	59
		42	59
		46	60
	Mean	38	59
	SD	10	1
21		31	53
		27	42
		36	47
	Mean	31	47
	SD	5	6
62		42	56
		33	47
		40	60
	Mean	38	54
	SD	5	7
185		45	58
		36	63
		39	46
	Mean	40	56
	SD	5	9
556		34	38
		39	45
		35	40
	Mean	36	41
	SD	3	4
1667		45	59
		44	57
		39	68
	Mean	43	61
	SD	3	6
5000		37	57
		41	52
		38	50
	Mean	39	53
	SD	2	4
Positive control		1170	1090
		1225	1051
		1201	1074
	Mean	1199	1072
	SD	28	20

*The positive controls employed are detail in table 1.

Table 3: Results of the Definitive Test

Dose µg/plate		TA 1535		TA 1537		TA 98		TA 100		E. coli
		Without S9-mix	With S9-mix	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix
0		15	17	8	22	29	77	165	156	**	31
		21	28	13	6	32	51	186	189	23	26
		16	15	13	15	27	36	163	175	37	25
	Mean	17	20	11	14	29	55	171	173	30	27
	SD	3	7	3	8	3	21	13	17	10	3
62		15	20	5	15	16	60	151	170	40	46
		18	24	15	14	27	40	148	166	46	39
		20	22	12	8	24	51	173	198	40	42
	Mean	18P	22	11	12	22	50	153	178	42*	42
	SD	3	2	5	4	6	10	14	17	3	4
185		8	23	11	16	25	57	159	181	35	41
		23	24	6	4	28	55	149	161	36	34
		20	13	4	9	28	56	211	182	31	47
	Mean	17P	20	7	10	27	56	173	175	34*	41
	SD	8	6	4	6	2	1	33	12	3	7
556		17	20	9	9	31	38	183	144	49	37
		30	23	9	8	26	53	168	155	36	33
		14	25	8	11	29	35	170	159	30	49
	Mean	20P	23	9	9	29	42	174	153	38*	40
	SD	9	3	1	2	3	10	8	8	10	8
1667		20	11	5	6	48	31	146	162	33	50
		28	20	18	8	35	37	146	152	41	38
		22	31	18	12	36	85	150	170	67	27
	Mean	23P	21	14	9	40	51	147	161	47*	38
	SD	4	10	8	3	7	30	2	9	18	12
5000		31	20	8	11	31	40	153	166	46	49
		34	19	7	14	51	42	137	161	46	39
		30	28	11	15	26	48	131	161	42	41
	Mean	32*	22	9	13	36	43	140	163	45*	43
	SD	2	5	2	2	13	4	11	3	2	5
Positive control		442	365	1053	211	1105	634	744	1245	287	698
		444	345	863	212	1185	738	704	940	221	677
		427	374	913	216	1186	555	708	1012	242	715
	Mean	438	361	943	213	1159	642	719	1066	250	697
	SD	9	15	98	3	46	92	22	159	34	19
P: Precipitation of test material. * : Precipitation of test substance and slightly more dense background lawn of bacterial growth than in concomitant control plates. **: Not counted; no bacteria on the agar plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the test, the test material was found to be non-mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in the Escherichia coli strain WP2 uvrA, either in the absence or presence of metabolic activation (S9-mix).

Executive summary:

The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames test) in accordance with standardised guidelines OECD 471 and EPA OPPTS 870.5100. The cultures were exposed to 0, 62, 185, 556, 1667 and 5000 µg/plate. Precipitation of the test material was observed at a number of the concentrations tested. In some of the plates slight dose-dependent increases in the number of revertants were noticed, however this was accompanied by an increased background lawn and precipitation. This was therefore not attributed to the mutagenic potential of the test material. Under the conditions of the test, the test material was found to be non-mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in the Escherichia coli strain WP2 uvrA, either in the absence or presence of metabolic activation (S9-mix).