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Toxicological information

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:
2005-08-03 to 2005-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
4-Vinylcyclohexen,
Test substance No.: 05/0544-1
98.1 % (see analytical report, dated September 2, 2005)
Colorless liquid

Method

Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
obtained from Merck on September 9, 1991
Additional strain / cell type characteristics:
other: tryptophan auxotroph
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
obtaines from KNOLL AG on October 30, 1989
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9; male Sprague-Dawley rats
Test concentrations with justification for top dose:
Experiment I : 0, 20, 100, 500, 2500, 5000 µg/plate, Standard plate test with and without S-9 mix, 3 test plates per dose or per control
Experiment II: 0, 50, 100, 150, 200, 250 µg/plate, Standard plate test with and without S-9 mix, 3 test plates per dose or per control
Experiment III: 0, 1.25, 2.5, 5, 10, 20 µg/plate, Standard plate test with and without S-9 mix, 3 test plates per dose or per control
Vehicle / solvent:
DMSO (Dimethyl sulfoxid, CAS No. 67-68-5; purity > 99 %)
Controls
Untreated negative controls:
no
Remarks:
vehicle will be used as negative reference item
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
for details see "Details on test system and conditions"
Positive control substance:
other: for details see "Details on test system and conditions"
Remarks:
with metabolic activation: 2-aminoanthracene, strains: TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate, dissolved in DMSO strain: Escherichia coli WP2 uvrA: 60 µg/plate, dissolved in DMSO
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Standard plate test with and without S-9 mix
- Metabolic activation assay: Arochlor 1254 induced rat liver S9, 5 male Sprague-Dawley rats receive single intraperitoneal injection of 500 mg
Arochlor 1254, 5 days before sacrifice
ADMINISTRATION
- Dosing: at least 5 concentrations up to 5000 µg/plate
- Data : 3 independent experiments with and without metabolic activation
Experiment I : 0, 20, 100, 500, 2500, 5000 µg/plate, Standard plate test with and without S-9 mix, 3 test plates per dose or per control
Dose selection and evaluation are based on the findings of the 1st experiment
Experiment II: 0, 50, 100, 150, 200, 250 µg/plate, see above
Experiment III: 0, 1.25, 2.5, 5, 10, 20 µg/plate, see above
- Number of plates: 3 per dose or per control
- Positive and negative control groups and treatment:
- positive without metabolic activation:
strains TA 1535, TA 100: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 µg/plate
strain TA 98: 4-nitro-o-phenylendiamine (NOPD), 10 µg/plate
strain TA 1537: 9-aminoacridine (AAC), 100µg/plate
strain E. coli WP2 uvrA: 4-nitroquinoline-N-oxide (4-NQO), 5 µg/plate
- positive with metabolic acivation
strains TA 1535, TA 100, TA 1537, TA 98: 2-aminoanthracene, 2.5 µg/plate, dissolved in DMSO
strain Escherichia coli WP2 uvrA: 2-aminoanthracene, 60 µg/plate, dissolved in DMSO
- negative control: solvent control: DMSO for all strains
- Incubation time: 48 - 72 h at 37 °C in the dark

Evaluation criteria:
Toxicity:
-toxicity detected by decrease in number of revertants, clearing or diminution of the background lawn, reduction of the titer (recorded for all test groups)
Solubility:
-precipitation recorded
Acceptance criteria (experiment is considered valid if following criteria are met:)
- number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain
-sterility controls revealed no indication of bacterial contamination
- positive control both with and without S9 mix induced a significant increase in number of revertants within the range of the historical control data or above
- titer of viable bacteria was at least 10 exp8 / ml
Assessment criteria:
-Test is considered positive if a dose related and reproducible increase in number of revertant colonies is observed i.e. about doubling of spontaneous mutation rate in at least one tester strain either without or with metabolic activation.
- The test item is generally considered nonmutagenic in this test if:
The number of revertants for all test strains were within the historical negative control range under all experimental conditions in two independ experiments
Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory; mean number of revertant colonies per plate and standard deviation are determined

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Bacteriotoxic effects was observed depending on the strain and test conditions from about 20 µg - 50 µg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ
No precipitation of the test substance was found.
Bacteriotoxic effects was observed depending on the strain and test conditions from about 20 µg - 50 µg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

no further information

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the test results of the present study, the test substance 4-Vinylcyclohexen is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Executive summary:

The test substance 4-Vinylcyclohexen was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of serveral bacterial strains, Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2, in a reverse mutation assay. The study was carried out according to OECD method 471 in compliance with the current OECD Principles of Good Laboratory Practice. Dose levels covering a total range of 1.25 µg - 5000 µg/plate, in a standard plate test both with and without the addition of a metabolising system (Arochlor 1254 induced rat liver S9 mix) were employed.

A bacteriotoxic effects was observed depending on the strain and test conditions from about 20 µg - 50 µg/plate onward.

A relevant increase in the number of his + or trp + revertants was not observed either without S-9 mix or after addition of a metabolizing system.

According to the test results of the present study, the test substance 4-Vinylcyclohexen is not mutagenic in the Salmonella typhimurium/Escherichiacoli reverse mutation assay under the experimental conditions chosen here.