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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:
24-72 hrs
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well documented study according to OECD 471 and OECD & French Government GLP. Study report includes, QA & GLP statement and C of A for test article. Study report does not include analytical verification of test substance concentration or stability in study media.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
The test substance LUPEROX 231 (1, I-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE) used in the study was supplied by Elf Atochem Deutschland.
Documentation supplied by the Sponsor identified the test substance as follows:
-name: LUPEROX 231 (1,I-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
- labelling: LUPEROX 231
-batch number: 530-9502-102
-labelling: 530-9502-102
-description: yellowish to colourless liquid
-quantity and container: 2 plastic flasks each containing 0.1 kg
-date of receipt: 18.5.95
-storage conditions: at room temperature and protected from light
-purity: 97.4%.
Data relating to the characterization of the test substance are documented in a test article description and results of analysis.

Method

Target gene:
The five strains (Ames et aI., 1975; Maron and Ames, 1983): TA 1535, TA 1537, TA 98, TA 100 and TA 102, are supplied by B.N. Ames' Laboratory (University of California, Berkeley, U.S.A.). They are stored in a cryoprotective medium containing 1 ml nutrient broth and 0.09 ml dimethylsulfoxide in a liquid nitrogen container.

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. In addition, to increase their sensitivity to mutagenic substances, additional mutations have been added:
-the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to
large molecules that do not penetrate the normal bacteria cell wall,
-the uvr B mutation is a deletion of a gene code for the DNA excision repair system, which renders the bacteria unable to use this repair
mechanism to remove the damaged DNA,
-the addition of the pKM 10 1 ampicillin resistant plasmidic R -factor in the strains T A 98, TAI00 and TAl 02 enhances their detection
sensitivity to some mutagens.
-in addition, the pAQl tetracycline resistant plasmidic factor has been added to the TAI02 strain.

Genotype of the bacterial strains
Strains Histidine mutation Additional mutations
TA 1535 His G46 rfa uvrB
TA 100 His G46 rfa uvrB Factor R
TA 102 His G 428 (pAQl) rfa Factor R
TA 1537 His C 3076 rfa uvrB
TA 98 His D 3052 rfa uvrB Factor R

The T A 1535, TAl 00 and TAl 02 strains are reverted by base-pair substitution mutagens and the T A 1537 and T A 98 strains by frameshift
mutagens. In addition, the TAl 02 strain detects oxidative mutagens.
Species / strain
Species / strain / cell type:
other: TA1535, TA100, TA102, TA1537, TA98
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500, 5000 ug/plate
Vehicle / solvent:
The vehicle was acetone, batch No. 074BR (Prolabo, 75011 Paris, France).
Controls
Untreated negative controls:
yes
Remarks:
acetone
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
other: Five known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C in distilled water), were used to check the sensitivity of the test system: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C, 2-anthramine
Details on test system and experimental conditions:
Rationale for dose selection
The top dose was selected according to the following criteria specified in international regulations:
• for non-toxic, freely soluble test substances, the top dose is 5000 ug/plate,
• for non-toxic, poorly soluble test substances, the top dose is the lowest precipitating dose.
• for toxic test substances, irrespective of solubility, the top dose is based on the level of toxicity: clearing of the bacterial lawn and/or reduction in the number of revertants when compared to the controls. However, precipitation should not interfere with the scoring of the test.

Treatment
The day before treatment, cultures were inoculated from frozen permanents: a crystal was sampled under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37°C for about 14 hours.

The tests were performed according to:
-direct plate incorporation method (both tests without S9 mix, first test with S9 mix): 0.05 ml of the test substance solution, 0.5 ml of S9 mix when required and 0.1 ml of the strain were mixed with 2 ml of overlay agar containing traces of the relevant amino-acid and biotin and maintained at 45°C.After rapid homogenization, the mixture was spread out on a Petri plate containing minimum medium.
-preincubation method (second test with S9 mix): 0.05 ml of the test substance solution, 0.5 ml of S9 mix and 0.1 ml of the strain were incubated for 60 minutes at 37°C prior adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.L, 75015 Paris, France).

Preliminary toxicity test
To assess the toxicity of the test substance to the bacteria, six doses (one plate/dose) were tested in the T A 98, TAl 00 and TAl 02 strains, with or without S9 mix.The sterility of the test substance was checked during this test and was found to be satisfactory.

Mutagenicity tests
In two independent tests, five doses of the test substance (three plates/dose) were tested on each strain, with or without S9 mix. A third test was performed with S9 mix.
During each test, the following controls were made using triplicate plates:
-vehicle control: strain treated with the vehicle,
-positive control: strain treated with the known mutagens previously mentioned.
The sterility of the S9 mix was checked during each test (before the beginning and at the end of the experiment) and was found to be satisfactory.
Evaluation criteria:
Treatment of results
During each test, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtainedin the presence of the vehicle), are presented in a table in the attached study report.
Acceptance criteria:
This study was considered valid because the following criteria were fully met:
• the number of revertants of the controls was within the range of our historical data,
• the number of revertants of the positive controls was higher than that of the controls and
within the range of our historical data.
Evaluation criteria:
Biological relevance of the results was considered first. In addition, the following criteria may be used as an aid for determining a positive response:
• a dose-related increase in the number of revertants,
and/or
• a reproducible increase in the number of revertants (i.e. a doubling in at least one strain when compared to that of the controls) for at least one of the doses.

Results and discussion

Test results
Species / strain:
other: TA1535, TA100, TA102, TA1537, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the reported experimental conditions, the test substance LUPEROX 231 (1, I-DI-(tertBUTYLPEROXY)- 3,3,5-TRIMETHYLCYCLOHEXANE) did not show mutagenic activity in the reverse mutation assay on Salmonella typhimurium.
Executive summary:

RESULTS

MUTAGENICITY TESTS: The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and within the range of our historical data. The top dose was selected according to the criteria specified in the international regulations. Since the test substance was non-toxic, freely soluble, the top dose was 5000 ug/plate. The selected range dose was: 312.5,625, 1250,2500 and 5000 ug/plate. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the five strains. Since an increase in the number of revertants was obtained at 312.5 and 625 ug/plate for the TA 1537 strain in the first test with S9 mix using the direct incorporation method, but not in the second test using the preincubation method, a third test with S9 mix using the direct incorporation method, was performed using the following doses: 125, 250, 500, 1000 and 2000 ug/plate. No increase was recorded.

CONCLUSION: Under the reported experimental conditions, the test substance LUPEROX 231 (1, I-DI-(tertBUTYLPEROXY)- 3,3,5-TRIMETHYLCYCLOHEXANE) did not show mutagenic activity in the reverse mutation assay on Salmonella typhimurium.