Bis(2-ethylhexyl) 2,2'-thiobisacetate

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:

04 October 2007 - 21 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) 2,2'-thiobisacetate

EC Number:

246-131-9

EC Name:

Bis(2-ethylhexyl) 2,2'-thiobisacetate

Cas Number:

24293-43-4

Molecular formula:

C20H38O4S

IUPAC Name:

2-ethylhexyl 2-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)acetate

Details on test material:

- Name of test material (as cited in study report): Di (2-ethylhexyl) thiodiglycolate
- Physical state: Clear yellow liquid
- Analytical purity: 97.0 %
- Lot/batch No.: 20705
- Expiration date of the lot/batch: 17 March 2008
- Storage condition of test material: Room temperature in the dark, over silica gel

Method

Target gene:

genes involved in histidine synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal preparation (S9)

Test concentrations with justification for top dose:

Sighting study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Main study: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: Test substance was insoluble in water, but fully soluble in DMSO at 50 mg/mL.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitro-N-nitrosoguanidine, Mitomycin C, 4-nitroquinoline-N-oxide, 9-aminoacridine, 2-Aminoanthracene, Benzo(a)pyrene, 1,8-Dihydroxanthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation);

DURATION
- Exposure duration: 48 hours at 37ºC

NUMBER OF REPLICATIONS:
Experiment 1: Five concentrations of the test material were assayed in triplicate against each tester strain.
Experiment 2: Methodology as for experiment 1, using fresh bacterial cultures, test material and control solutions.

NUMBER OF CELLS EVALUATED: 1 to 9.9 x 10(9) bacteria per mL.

DETERMINATION OF CYTOTOXICITY
growth of bacterial background lawn.

Evaluation criteria:

Several criteria for determining a positive result, such as dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Statistical significance and biological relevance were considered.

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

of 5000 µg/plate, at which precipitation occured.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

of 5000 µg/plate, at which precipitation occured.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test substance was not soluble in water
- Precipitation: A globular precipitate was observed at 5000 µg/plate. This did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
The dose range from 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
S. typhimurium TA 100 was used with and without metabolic activation.
No cytotoxicity was observed up to the limit concentration of 5000 µg/plate in S. typhimurium TA100.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the historical vehicle and positive controls were in the range of the concurrent controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The material was non-toxic to the strains of bacteria used up to the limit concentration of 5000 µg/plate as determined by the growth of the background lawn.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
-	0	78	12	219	16	10
-	50	78	12	228	13	11
-	150	76	17	237	14	4
-	500	78	15	226	19	7
-	1500	80	14	231	15	7
-	5000	84 P	14 P	231 P	11 P	7 P
Positive controls - S9	Name	ENNG	ENNG	MMC	4NQO	9AA
	Concentrations (μg/plate)	3	5	0.5	0.2	80
	Mean No. of colonies/plate (average of 3)	994	619	944	126	4601
		TA 100	TA1535	TA102	TA98	TA1537
+	0	76	11	269	29	15
+	50	80	12	255	27	8
+	150	82	13	263	27	10
+	500	81	10	256	21	13
+	1500	69	13	246	23	14
+	5000	70 P	13 P	234 P	19 P	7 P
Positive controls + S9	Name	2AA	2AA	DAN	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3)	981	158	931	301	445
Concurrent Negative control	0	71	14	264	17	11

 

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

MMC = mitomycin C

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

DAN = 1,8-Dihydroxanthraquinone

P = Precipitate

 

 

 

 

 

 

Table 2: Test Results of Experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
-	0	87	23	258	19	11
-	50	63	26	253	15	11
-	150	74	20	256	19	12
-	500	72	21	228	11	9
-	1500	69	18	254	11	4
-	5000	73 P	20 P	241 P	14 P	6 P
Positive controls - S9	Name	ENNG	ENNG	MMC	4NQO	9AA
	Concentrations (μg/plate)	3	5	0.5	0.2	80
	Mean No. of colonies/plate (average of 3)	672	760	1327	125	3573
		TA 100	TA1535	TA102	TA98	TA1537
+	0	70	15	254	22	9
+	50	89	19	256	20	13
+	150	95	22	236	25	12
+	500	73	23	256	23	7
+	1500	73	25	251	27	7
+	5000	85 P	14 P	243 P	28 P	12 P
Positive controls + S9	Name	2AA	2AA	DAN	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3)	757	194	1170	330	405
Concurrent Negative control	0	77	22	235	21	5

 

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

MMC = mitomycin C

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

DAN = 1,8-Dihydroxanthraquinone

P = Precipitate

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance was found to be non-mutagenic in bacteria.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material Di-(2-ethylhexyl) thiodiglycolate diluted in DMSO using the Ames plate incorporation method according to OECD Guideline No. 471 at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate for both of two consecutive experiments.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. A globular precipitate was observed at the highest dose, 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.