Iso(C10-C14)alkyl (3,5-di-tert-butyl-4-hydroxyphenyl)methylthioacetate

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:

1988-02-02 to 1988-08-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Iso(C10-C14)alkyl (3,5-di-tert-butyl-4-hydroxyphenyl)methylthioacetate

EC Number:

404-800-4

EC Name:

Iso(C10-C14)alkyl (3,5-di-tert-butyl-4-hydroxyphenyl)methylthioacetate

Cas Number:

118832-72-7

Molecular formula:

C30H52O3S

IUPAC Name:

11-methyldodecyl 2-{[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]sulfanyl}acetate

Details on test material:

- Physical state: liquid
- Analytical purity: commercial grade
- Storage condition of test material: room temperature

Method

Target gene:

This test system permits the detection of point mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium and in tryptophan-requiring strain of Escherichia coli.

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Range in the cytotoxicity test: 0.08 - 5000 µg/ 0.1 mL
Range in the mutagenicity test without and with microsomal activation: 5 - 5000 µg/ 0.1 mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylformamide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98 and TA 1538: 2-nitrofluorene; TA 100, E.coli WP2 uvrA: N-ethyl-N'-nitro-N-nitrosoguanidine; TA 1535: sodium azide; TA 1537: 9(5)-aminoacridine hydrochloride monohydrate

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 98, TA 100, TA 1537 and TA 1538: benzo(a)pyrene; TA 1535, E.coli WP2uvrA: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3 per experiment, two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: colony count

TEST PERFORMANCE
A preliminary toxicity test on strain TA100 was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 mL. The protocol used was the same as in the mutagenicity test. Accordingly, the concentration of 5000 µg/0.1 mL was used as the highest in the mutagenicity test and the tests were performed with the following concentrations of the test substance without and with microsomal activation: 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL. The substance was dissolved in the negative control item dimethylformamide. Each Petri dish contained: 1) approx. 20 mL of minimum agar (agar, (bacteriological grade) plus salts and glucose), 2) 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture in 2.0 mL of soft agar. For the Salmonella strains the soft agar was composed of: 100 mL of 0.6% agar solution with 0.6% NaCL and 10 mL of an aqueous solution of 1-histidine, 0.5 mM and +biotin 0.5 mM. For the E. coli strain the 1-histidine and the +biotin were replaced by 1- tryptophan, 0.5 mM in bidistilled water. In the experiments without microsomal activation 0.5 mL of M/10 sodium phosphate buffer was added. In the experiments in which the substance was metabolically activated, the buffer was replaced by 0.5 mL of an activation mixture. 1 mL activation mixture contained: 0.1 mL S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 and 0.9 mL of a solution of co-factors.

Evaluation criteria:

Criteria for a positive response:
The test substance is considered to be mutagenic in this test system if one or both of the following conditions are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 1535, TA 1537, TA 1538 and E.coli WPuvrA.
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain S. typhimurium TA 100.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis was not performed.

Results and discussion

Test results

Species / strain:

other: S.typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 312.5 µg/0.1 ml and above the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES:
Nine concentrations of the test substance ranging from 0.08 to 5000 µg/0.1 ml were tested to determine the highest concentration to be used in the mutagenicity assay. No cytotoxicity was observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment I

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2 uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	16	26	94	96	13	8	6	13	10	15	23	24
5	18	24	103	113	12	11	6	13	8	21	24	20
10	12	29	99	97	16	10	5	16	8	18	18	20
50	9	27	92	104	13	11	11	13	8	21	22	27
100	19	30	102	94	14	11	5	12	6	15	16	20
500	16	23	100	107	20	9	6	10	9	19	21	22
1000	18	28	108	109	14	9	7	17	6	15	16	24
5000	20	26	105	96	7	13	9	10	7	13	18	21
solvent control	16	25	106	112	14	10	9	18	9	17	25	21
positive control A	156	236	384	978	311	97	27	159	224	155	546	465
positive control B	245	271	783	912	495	85	377	133	399	107	1263	581

Experiment II

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2 uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	15	29	109	106	19	9	5	8	8	16	27	24
5	19	34	110	92	14	6	6	6	6	12	27	20
10	16	31	108	86	17	11	3	7	13	15	26	21
50	21	28	114	84	13	4	8	12	6	17	28	16
100	17	30	115	97	14	7	6	9	6	15	31	17
500	22	29	109	89	17	11	5	5	11	16	32	25
1000	18	36	104	88	12	8	6	6	7	17	32	16
5000	20	32	108	80	15	9	5	7	9	18	25	20
solvent control	17	22	121	93	17	8	8	14	10	19	24	21
positive control A	142	340	561	1083	290	72	16	208	242	173	524	372
positive control B	250	276	843	1395	415	58	183	114	361	198	1189	456

Positive controls:

Without S9 mix:

TA 98, TA 1538: 2-nitrofluorene, 1.0 (A) and 2.0 (B) µg/0.1 mL dimethylsulfoxide

TA 100: 4N-ethyl-N'-nitro-N-nitrosoguanidine, 3.0 (A) and 5.0 (B) µg/0.1 ml dimethylsulfoxide

TA 1535: sodium azide, 0.5 (A) and 1.0 (B) µg/0.1 ml bidistilled water

TA 1537: 9(5)- aminoacridine hydrochloride monohydrate, 40 (A) and 80 (B) µg/0.1 mL dimethylsulfoxide

WP2 uvrA: N-ethyl-N'-nitro-N-nitrosoguanidine, 2.0 (A) and 5.0 (B) µg/0.1 ml dimethylsulfoxide

With S9-Mix:

TA 98, TA 100, TA 1537, TA 1538: benzo(a)pyrene, 5.0 (A) and 10 (B) µg/0.1 ml dimethylsulfoxide

TA 1535: 2-aminoanthracene, 2.0 (A) and 4.0 (B) µg/0.1 mL dimethylsolfoxide

WP2 uvrA: 2-aminoanthracene, 40 (A) and 80 (B) µg/0.1 ml dimethylsulfoxide

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

A GLP-compliant Salmonella typhimurium and E. coli reverse mutation assay was carried out according to OECD Guideline 471. Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 as well as E. coli strain WP2uvrA were tested for the induction of base-pair substitutions or frameshift mutations. The experiment was conducted in the presence and absence of a rat liver S-9-microsomal activation system and at concentrations of 5-5000 μg per plate. The experiment was repeated independently for confirmation. At concentrations of 312.5 µg/0.1 ml and above the substance precipitated in soft agar. No evidence of a mutagenic potential was observed. In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed no marked deviations. Cytotoxicity did not occur up to and including the highest dose tested. Based on this result, no evidence of the induction of point mutations was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.