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Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 resultsopen allclose all
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
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
1000SP 9 10 3 4 15 17 82 78 10 14
3330SP 85 81 9 8
5000MP 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
1000SP 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.