2-(2H-Benzotriazol-2-yl)-6-(1-methyl-1-phenylethyl)-4-(1,1,3,3-tetramethylbutyl)phenol

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:

03-Jan-1997 to 27-Jan-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 471)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 26-May-1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

CCR Cytotest Cell Research GmbH & Co. KG, 64380 Roftdorf, In den Leppsteinswiesen 19

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-600-5

EC Name:

-

Cas Number:

73936-91-1

Molecular formula:

C29H35N3O

IUPAC Name:

2-(2H-1,2,3-benzotriazol-2-yl)-6-(2-phenylpropan-2-yl)-4-(2,4,4-trimethylpentan-2-yl)phenol

Details on test material:

- Physical state: whitish, solid
- Analytical purity: 99.35-99.45 %
- Storage: at room temperature, away from light

Method

Target gene:

trp, his

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from phenobarbital (i.p.) and beta-naphthoflavone (p.o.) induced male Wistar rats

Test concentrations with justification for top dose:

33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in acetone. The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

solvent

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Remarks:

all controls were concurrent

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment I), preincubation (experiment II and IIb)

DURATION
- Preincubation period: 1 hour
- Exposure duration: 48 hours
- Expression time: at least 48 hours

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was assessed as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

POSITIVE CONTROLS
without S9 mix:
sodium azide for strains TA 1535, TA 100 (10 µg/plate)
4-nitro-o-phenylene-diamine for strains TA 98 (10 µg/plate) and TA 1537 (50 µg/plate)
methyl methane sulfonate for strain WP2uvrA (5 µl/plate)
with S9 mix:
2-aminoanthracene for strains TA 1535, TA 1537, TA 98, TA 100 (2.5 µg/plate) and WP2 uvrA (10 µg/plate)

Evaluation criteria:

The generally accepted conditions for the evaluation of the results were:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates
A test item was considered as positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration was induced. A test item producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any one of the test points was considered non-mutagenic in this system.
A test item was considered as mutagenic if in the strains TA 98, TA 100 and WP2 uvrA the number of reversions were at least twice as high and in the strains TA 1535 and TA 1537 were at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

The data were not evaluated statistically.

Results and discussion

Test results

Species / strain:

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

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar from 333.3 up to 5000.0 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with three plates each. According to the results of this pre-experiment the concentrations applied in the main experiments were chosen.

Any other information on results incl. tables

Experimental Result

Without S9 Mix

	Revertants/plate (mean from three plates)
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Concentration µg/plate	I	II	I	II	I	II	I	II	I	II
Negative control	17	18	11	11	20	17	141	104	44	28
Solvent control	16	15	9	9	18	18	139	102	39	32
Positive control	1167	1109	134	128	430	568	1213	1102	1086	993
33.3	16	13	11	7	21	14	147	103	34	37
100	16	16	8	9	20	16	142	108	42	39
333.3	17	14	12	7	24	13	146	89	39	33
1000	13	14	11	7	26	16	145	106	46	35
2500	17	10	8	8	27	13	134	94	39	35
5000	16	9	9	8	24	25	129	110	45	34

With S9 -Mix

	Revertants/plate (mean from three plates)
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Concentration µg/plate	I	II	I	II	I	II	I	II	I	II	IIa
Negative control	18	19	17	15	33	33	158	147	44	32	38
Solvent control	16	13	15	12	30	25	154	119	52	28	33
Positive control	275	118	92	70	1119	482	1244	604	236	146	174
33.3	17	16	13	9	24	26	151	131	47	30	32
100	17	18	14	14	30	24	138	119	53	30	31
333.3	17	14	16	12	27	28	149	137	58	41	39
1000	19	14	16	12	31	26	152	132	50	52	35
2500	17	16	16	14	27	32	139	156	57	67	38
5000	17	14	14	16	24	40	156	164	52	50	31

I = Experiment I (standard plate test)

II and IIa = Experiment II and IIa (preincubation method)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test article is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A GLP-compliant Ames study following OECD guideline 471 was performed to investigate the test article's potential to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and an additional experiment (experiment IIa) following the preincubation procedure with the Escherichia coli strain WP2 uvrA in the presence of metabolic activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in both experiments. No toxic effects occurred in the test groups with and without metabolic activation. No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed after treatment with test material at any dose level, neither in the presence nor in the absence of metabolic activation (S9 mix). A slight increase in the number of revertants occurred in the second experiment in strain WP2 uvrA in the presence of metabolic activation exceeding the threshold of twice the number of the corresponding solvent control at 2500 µg/plate. This effect could not be verified under identical conditions in an additional experiment (experiment IIa) in strain WP2 uvrA in the presence of metabolic activation. Therefore, the slight increase observed in the second experiment was judged to be caused by statistical fluctuations of the rather low numbers of revertant colonies and does not represent a mutagenic effect. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.