2,6-di-tert-butyl-4-(4,6-bis(octylthio)-1,3,5-triazin-2-ylamino)phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(E.coli was not included, only few details on experimental procedure, in experiments with metabolic activation a positive control was only investigated in TA1535)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-di-tert-butyl-4-(4,6-bis(octylthio)-1,3,5-triazin-2-ylamino)phenol

EC Number:

213-590-1

EC Name:

2,6-di-tert-butyl-4-(4,6-bis(octylthio)-1,3,5-triazin-2-ylamino)phenol

Cas Number:

991-84-4

Molecular formula:

C33H56N4OS2

IUPAC Name:

2,6-di-tert-butyl-4-(4,6-bis(octylthio)-1,3,5-triazin-2-ylamino)phenol

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine-auxotrophic

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254 and a solution of co-factors

Test concentrations with justification for top dose:

25, 75, 225, 675, and 2025 µg/0.1 ml

Vehicle / solvent:

- Solvent used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below for details

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C in darkness

NUMBER OF REPLICATIONS: 2 independent experiments, each with 3 plates each concentration or control group

POSITIVE CONTROLS:
no metabolic activation:
- Strain TA 98: Daunorubicin-HCl (5 and 10 µg/0.1 ml phosphate buffer)
- Strain TA 100: 4-nitroquinoline-N-oxide (0.125 and 0.25 µg/0.1 ml phosphate buffer)
- Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (3 and 5 µg/0.1 ml phospahte buffer)
- Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (50 and 100 µg/0.1 ml DMSO)
with metabolic activation:
- Starin TA 1537: Cyclophophamide (250 µg/0.1 ml phosphate buffer)

Evaluation criteria:

A test substance was generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 225 µg/0.1 ml and above the substance precipitated in soft agar.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tab. 1: Salmonella/Mammalian-Microsome Mutagenicity Test: First experiment without microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -06 -25)

Compound	Concentration	TA 98	TA 100	TA 1535	TA 1537
Test substance	Control	20	152	8	2
	25 µg/0.1 ml	16	126	11	3
	75 µg/0.1 ml	18	131	5	2
	225 µg/0.1 ml	13	134	10	4
	675 µg/0.1 ml	14	123	7	2
	2025 µg/0.1 ml	10	142	5	4
Daunorubicin-HCl	Control	21
	5 µg/0.1 ml	675
	10 µg/0.1 ml	1110
4-Nitroquinoline-N-oxide	Control		147
	0.125 µg/0.1 ml		661
	0.25 µg/0.1 ml		1083
N-Methyl-N'-nitro- N-nitrosoguanidine	Control			10
	3 µg/0.1 ml			350
	5 µg/0.1 ml			1352
9(5)Aminoacridine hydrochloride	Control				3
	50 µg/0.1 ml				254
	100 µg/0.1 ml				866

Tab. 2: Salmonella/Mammalian-Microsome Mutagenicity Test: First experiment with microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -06 -25)

Compound	Concentration	TA 98	TA 100	TA 1535	TA 1537
Test substance	Control	28	154	6	7
	25 µg/0.1 ml	31	145	9	5
	75 µg/0.1 ml	33	153	17	6
	225 µg/0.1 ml	37	137	9	3
	675 µg/0.1 ml	23	152	9	10
	2025 µg/0.1 ml	26	155	14	6
Cyclophosphamide	Control			6
	250 µg/0.1 ml			384

Tab. 3: Salmonella/Mammalian-Microsome Mutagenicity Test: Second experiment without microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -07-09)

Compound	Concentration	TA 98	TA 100	TA 1535	TA 1537
Test substance	Control	21	166	11	5
	25 µg/0.1 ml	20	147	10	3
	75 µg/0.1 ml	26	151	12	6
	225 µg/0.1 ml	22	152	12	7
	675 µg/0.1 ml	20	151	14	4
	2025 µg/0.1 ml	21	135	17	4
Daunorubicin-HCl	Control	31
	5 µg/0.1 ml	629
	10 µg/0.1 ml	1025
4-Nitroquinoline-N-oxide	Control		160
	0.125 µg/0.1 ml		693
	0.25 µg/0.1 ml		1176
N-Methyl-N'-nitro- N-nitrosoguanidine	Control			14
	3 µg/0.1 ml			489
	5 µg/0.1 ml			1820
9(5)Aminoacridine hydrochloride	Control				5
	50 µg/0.1 ml				164
	100 µg/0.1 ml				985

Tab. 4: Salmonella/Mammalian-Microsome Mutagenicity Test: Second experiment with microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -07-09)

Compound	Concentration	TA 98	TA 100	TA 1535	TA 1537
Test substance	Control	45	159	13	11
	25 µg/0.1 ml	39	160	13	7
	75 µg/0.1 ml	42	163	13	13
	225 µg/0.1 ml	43	156	14	10
	675 µg/0.1 ml	41	144	16	14
	2025 µg/0.1 ml	35	165	9	9
Cyclophosphamide	Control			9
	250 µg/0.1 ml			502

Applicant's summary and conclusion

Conclusions:

No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal activation was detectable.

Executive summary:

In a bacterial reverse mutation assay conducted similar to OECD Guideline 471 the test substance was analysed for mutagenicity by the detection of point mutations in bacteria (histidine-auxotrophic mutants of Salmonella typhimurium). To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were also performed with the addition of an activation mixture (rat liver microsomes and co-factors). The strains TA 98, TA 100, TA 1535, and TA 1537 were treated with the test substance dissolved in DMSO in doses of 25, 75, 225, 675, and 2025 µg/0.1 ml (plate incorporation test). A negative control (vehicle) and appropriate positive control substances were also evaluated. At concentrations of 225 µg/0.1 ml and higher the test compound precipitated in soft agar. No dose-related biologically relevant increase or doubling in the number of his+ revertants was observed in plate incorporation test in any of the tested strains with or without metabolic activation. Effects on cytotoxicity were not given. According to the results of the present study, the test substance is not mutagenic in the bacterial reverse mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.