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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2006 to 19 May 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
other: Japanese Ministry of Labor, Notifications No. 77 and 67
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Reference substance name:
α,3,3-trimethylcyclohexylmethyl formate
EC Number:
246-735-2
EC Name:
α,3,3-trimethylcyclohexylmethyl formate
Cas Number:
25225-08-5
IUPAC Name:
1-(3,3-dimethylcyclohexyl)ethyl formate

Method

Target gene:
S. typhimurium TA1535: hisG46 rfa uvrB; S. typhimurium TA1537: hisC3076 rfa uvrB; S. typhimurium TA 1538: hisD3052 rfa uvrB; S. typhimurium TA98: hisD3052 rfa uvrB pKMl01; S. typhimurium TA100: hisG46 rfa uvrB pKM101; E. coli WP2 uvrA: trp
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:
rat S9
Test concentrations with justification for top dose:
Dose-range finding test 1: a total of 6 doses consisting of 5000 μg/plate as the highest dose and 5 lower doses diluted with a geometric progression of 4 were employed.
Dose-range finding test 2 and main test: the highest dose at 78.1 μg/plate without S9 and 313 μg/plate with S9 and each lower 5 doses diluted with a geometric progression of 2.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in distilled water at 50 mg/mL but it was soluble in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable as there was no change in colour no heat generation at room temperature within 2 hours after preparation.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide
Remarks:
TA98, TA 100, and WP2uvrA without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine x 2 HCl
Remarks:
TA1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 48 hours

NUMBER OF REPLICATIONS: triplicate for the negative control group and duplicate per dose for the test substance and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition
Evaluation criteria:
The test substance was judged positive when the number of revertant colonies increased to twice or more that in the negative control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all other cases it was judged negative.
Statistics:
No statistical methods were used.

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:
cytotoxicity
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test 1 the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9 and >313 μg/plate with S9. In the dose-range finding test 2 the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9, >156 μg/plate in TA100, TA1535, TA98 and TA1537 and >313 μg/plate in WP2uvrA with S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main test the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9, >156 μg/plate in TA100, TA1535, TA98 and TA1537 and >313 μg/plate in WP2uvrA with S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The substance was concluded to give negative results in the Ames test.
Executive summary:

Genotoxicity of CP Formate in prokaryotes was studied in a GLP-compliant study performed according to the protocol similar to OECD guidelines 471 and 472 in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2uvrA, with and without metabolic activation. The experiments were performed by preincubation method. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9, > 156 μg/plate in TA100, TA1535, TA98 and TA1537 and > 313 μg/plate in WP2uvrA with S9. The numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all test strains, both with and without metabolic activation. Therefore the substance was concluded to give negative results in the Ames test.