2-(2-oxo-5-(1,1,3,3-tetramethylbutyl)-2,3-dihydro-1-benzofuran-3-yl)-4-(1,1,3,3-tetramethylbutyl)phenyl acetate

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:

02.06. - 18.07.1999

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:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Remarks:

NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-770-1

EC Name:

-

Cas Number:

216698-07-6

Molecular formula:

C32 H44 O4

IUPAC Name:

2-[2-oxo-5-(2,4,4-trimethylpentan-2-yl)-2,3-dihydro-1-benzofuran-3-yl]-4-(2,4,4-trimethylpentan-2-yl)phenyl acetate

Details on test material:

- Description: White solid
- Purity: 98.7%
- Storage condition of test material: At room temperature in the dark

Method

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver prepared five days after i.p. treatment of male adult Wistar rats with 500 mg/kg bw Aroclor 1254 (20% solution in corn oil; w/v).

Test concentrations with justification for top dose:

Doses:
Preliminary toxicity test in TA100 and WP2UvrA strain: 0, 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate tested in the absence and presence of S9-mix. Solvent was 0.1 mL DMSO. 3 plates per dose and control.
Main test (1st and 2nd experiment):
standard plate test 0, 10, 33, 100, 333, 1000 µg/plate tested in the absence and presence of S9-mix. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2UvrA. Solvent was 0.1 mL DMSO. 3 plates per dose and control.

Vehicle / solvent:

DMSO (0.1 mL)
- Justification for choice of solvent/vehicle: The test substance was insoluble in water but soluble in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: Sodium azide; 9-aminoacridine; daunomycine; methylmethanesulfonate, 4-nitroquinoline N-oxide. With S9-mix: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: colony growth

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Mean and standard deviation from 3 plates/concentration were calculated.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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 examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 and WP2 uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first experiment of the mutation test. The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards. Precipitation of test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards in tester strain TA100 and WP2 uvrA. To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. No reduction of the bacterial background lawn and no decrease in the number of revertants were observed.

MAIN TEST
Based on the results of the dose range finding study, the test substance was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation experiments. The first mutation experiment was performed with the strains TA1535, TA 1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2UvrA. Precipitation occurred in the top agar at concentrations of 333 and 1000 µg/plate. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate in all tester strains. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results of the range finder and Experiment I

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	8	11	4	4	17	17	70	98	8	9
3							90	72	9	10
10	11	11	4	3	15	20	74	94	11	14
33	9	8	4	4	1	21	79	91	8	10
100	9	10	3	3	15	21	77	94	7	10
333	14	7	2	3	12	20	79	76	5	11
1000^SP	9	10	3	4	15	17	82	78	10	14
3330^SP							85	81	9	8
5000^MP							86	73	6	12
positive control*	310	568	253	426	441	872	907	1372	901	118

Results of Experiment II

	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	9	9	5	4	16	23	100	98	12	11
10	12	12	5	4	15	28	91	90	10	12
33	10	11	9	5	16	24	98	106	8	9
100	12	7	8	6	15	21	108	95	10	10
333	8	13	5	5	16	21	89	86	11	11
1000^SP	10	10	6	3	15	25	105	99	11	7
positive control*	366	484	243	471	562	853	908	1130	536	373

SP: slight precipitate

MP: moderate precipitate

*Positive Controls

without metabolic activation:

sodium azide: 1 µg/plate (TA 1535)

9-aminoacridine: 60 µg/plate (TA 1537)

daunomycine: 4 µg/plate (TA 98)

methylmethanesulfonate: 650 µg/plate (TA 100)

4-nitroquinoline N-oxide: 10 µg/plate (WP2uvrA)

with metabolic activation:

2-aminoanthracene: 2.5µg/plate (TA 1537, TA 1535, TA 98), 1 µg/plate (TA100), 5 µg/plate (WP2uvrA)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2UvrA in two independent experiments. These tests were following GLP guidelines and were based on the OECD testing guideline 471. In the dose range finding test, the test article was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2UvrA. At this dose level no toxicity was observed. Precipitation occurred on the plates at dose levels of 1000 µg/plate and above. In the mutation assays, the test substance was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. The test article precipitated on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. No dose-related increase in the number of revertant both in the absence and presence of S9-metabolic activation was observed. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.