Reaction mass of dodecyl 4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannaicosanoate and tetradecyl 4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannadocosanoate

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:

17 May 2012 to 03 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: read-across target

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to DOT bis-(ethylmaleate) (CAS 68109-88-6), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: 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

Key result

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:

valid

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines.

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:

Ethyl 9,9-dioctyl-4,7,11-trioxo-3,8,10-trioxa-9-stannatetradeca-5,12-dien-14-oate

EC Number:

268-500-3

EC Name:

Ethyl 9,9-dioctyl-4,7,11-trioxo-3,8,10-trioxa-9-stannatetradeca-5,12-dien-14-oate

Molecular formula:

C28H48O8Sn

IUPAC Name:

ethyl (Z,Z)- 9,9-dioctyl-4,7,11-trioxo-3,8,10-trioxa-9-stannatetradeca-5,12-dien-14-oate

Test material form:

other: liquid (unspecified)

Details on test material:

- Name of test material (as cited in study report: ethyl 9,9-dioctyl-4,7,11-trioxo-3,8,10-trioxa-9-stannatetradeca-5,12-dien-14-oate
- Physical state: extremely pale yellow liquid
- Storage condition of test material: approximately 4 °C in the dark under nitrogen

Method

Target gene:

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

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiments 1 and 2
0, 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 dimethyl sulphoxide at 50 mg/mL; it was soluble in dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. Acetone was selected as the most appropriate vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (plate incorporation)
2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli), 0.1 mL of culture of the appropriate strain, 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation) were thoroughly mixed and the mix was immediately poured onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.05 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 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 plates were within acceptable ranges and if the results of the positive controls met the criteria for a positive response. Furthermore, all tester strains must have demonstrated the required characteristics as determined by their respective strain checks. There should be a minimum of four non-toxic test material dose levels and no evidence of excessive contamination.

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: 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

Key result

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:

valid

Additional information on results:

PRE-STUDY CHECKS
The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

PRELIMINARY TOXICITY TEST
The test material was non-toxic to the strains of bacteria used (TA 100 and WP2uvrA). The test material formulation and S9-mix used in the experiment were both shown to be sterile.

MUTATION TEST
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically 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 or exposure method. Small statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Results for the negative controls were considered to be acceptable. Furthermore, all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Table 1: Experiment 1 - Mean Number of Revertants

Dose (µg/plate)		TA100		TA1535		WP2uvrA		TA98		TA1537
		-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix
0	Mean	115	118	24	12	38	51	35	36	13	12
	SD	13.3	14.6	4.5	0.0	4.6	6.4	5.3	6.1	4.4	3.5
50	Mean	103	103	20	9	42	47	32	31	9	12
	SD	6.1	8.1	1.0	2.0	1.7	6.2	7.5	2.0	5.9	1.2
150	Mean	112	113	22	10	43	45	24	27	8	12
	SD	0.6	2.1	1.7	4.2	4.6	5.5	1.2	6.7	1.0	0.6
500	Mean	99	99	22	12	31	36	35	28	13	8
	SD	4.5	4.5	2.6	1.0	5.2	8.5	3.8	10.3	6.0	5.0
1500	Mean	108 P	107 P	23 P	11 P	42 P	41 P	30 P	30 P	12 P	8 P
	SD	1.7	4.0	3.5	1.7	5.6	10.5	10.5	4.0	8.0	6.4
5000	Mean	96 P	75 P	22 P	11 P	35 P	41 P	30 P	34 P	10 P	11 P
	SD	8.1	6.2	1.5	2.0	3.6	6.1	3.2	2.5	1.2	4.5
Positive Control	Substance (µg/plate)	ENNG 3	2AA 1	ENNG 5	2AA 2	ENNG 2	2AA 10	4NQO 0.2	BP 5	9AA 80	2AA 2
	Mean	409	1513	141	288	550	427	137	195	561	278
	SD	82.4	77.9	9.9	24.1	28.1	19.4	12.7	3.1	202.6	3.1

 

Table 2: Experiment 2 - Mean Number of Revertants

Dose (µg/plate)		TA100		TA1535		WP2uvrA		TA98		TA1537
		-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix
0	Mean	110	99	22	11	40	39	19	31	12	10
	SD	10.6	20.1	4.9	3.8	9.0	6.7	4.0	10.2	4.0	5.2
50	Mean	97	106	23	12	45	50*	19	32	8	12
	SD	2.9	10.1	0.6	3.1	0.0	4.6	4.0	7.0	4.6	7.2
150	Mean	100	112	20	10	35	46	30	31	13	8
	SD	12.9	11.6	4.6	1.2	4.0	4.2	7.6	6.8	5.5	1.0
500	Mean	108	103	21	15	43	48	16	28	10	11
	SD	20.3	5.0	2.0	4.6	11.6	7.0	4.6	4.5	4.0	3.2
1500	Mean	104 P	105 P	14 P	12 P	33 P	46 P	20 P	20 P	12 P	14 P
	SD	3.8	11.7	6.6	0.0	2.1	2.1	2.3	1.2	0.0	2.1
5000	Mean	103 P	91 P	20 P	9 P	32 P	52 P*	15 P	24 P	17 P	8 P
	SD	14.2	0.6	3.6	2.6	3.0	4.0	8.5	7.8	1.7	3.1
Positive Control	Substance (µg/plate)	ENNG 3	2AA 1	ENNG 5	2AA 2	ENNG 2	2AA 10	4NQO 0.2	BP 5	9AA 80	2AA 2
	Mean	466	1474	180	280	596	341	129	669	463	236
	SD	24.9	187.6	48.6	29.3	47.4	31.9	17.1	41.4	87.2	19.7

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4 -nitroquinoline-1 -oxide

9AA = 9 -aminoacridine

2AA = 2 -aminoanthracene

BP = benzo(a)pyrene

P = precipitate

* = p ≤ 0.05

SD = standard deviation

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was not mutagenic to S. typhimurium strains TA1535, TA1537, TA98 and TA100 and in the E. coli strain WP2uvrA in both the absence and presence of metabolic activation.

Executive summary:

The mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames Test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14. During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material using both the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test material precipitate (creamy and particulate in appearance) was noted at and above 1500 µg/plate, though this observation did not prevent the scoring of revertant colonies. No toxicologically 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 or exposure method. Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the presence of S9-mix at 50 and 5000 µg/plate in Experiment 2. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 and 5000 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.