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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:
04 May, 1999 - 21 May, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
Not always dosed high enough. As experiment 1 or 2 shows toxicity in all strains, this deviation does not effect the outcome of the study.
Qualifier:
according to guideline
Guideline:
other: Methods for the determination of physio-chemical properties, toxicity and ecotoxicity: Annex V to Directive 79/831/EEC. B14. Salmonella typhimurium - Reverse mutation assay (1992).
Deviations:
yes
Remarks:
Not always dosed high enough. As experiment 1 or 2 shows toxicity in all strains, this deviation does not effect the outcome of the study.
Principles of method if other than guideline:
The proposals of the ICH working group for genotoxicity testing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde
EC Number:
257-942-2
EC Name:
1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde
Cas Number:
52475-86-2
Molecular formula:
C14H22O
IUPAC Name:
1-methyl-4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde
Constituent 2
Chemical structure
Reference substance name:
1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde
EC Number:
257-941-7
EC Name:
1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde
Cas Number:
52474-60-9
Molecular formula:
C14H22O
IUPAC Name:
1-methyl-3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde
Specific details on test material used for the study:
- Name of test material (as cited in study report): Precyclemone B
- Appearance: colorless liquid
- Analytical purity: 99.3 % (GLC, sum of two peaks)
- Lot/batch No.: 9000320077
- Expiration date of the lot/batch: 25-03-2000
- Storage condition of test material: refrigerator

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary test (without and with S9) TA100: 50, 158, 500, 1580 and 5000 µg/plate

Experiment 1: TA1535, TA97, TA98, TA100 and TA102:
Without and with S9-mix: 2, 6.32, 20, 63.2 and 200 µg/plate

Experiment 2: TA1535, TA97, TA98, TA100 and TA102:
Without and with S9-mix: 1, 3.16, 10, 31.6 and 100 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was miscible in DMSO and it is to be expected that no gross degradation is occuring when dissolved in DMSO for the test duration (≤ 6 hours).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 (TA100 and TA1535 1.0 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 (TA98 0.5 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: ICR-191 1 µg/plate in DMSO for TA97
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 (TA102 0.4 µg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, exp. 1) and preincubation (exp. 2)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain, duplicates for postive controls. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for TA1535 and TA98. For TA97, TA100 and TA102 a 1.5-fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation (Claxton et al. 1987) and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: small droplets were visible at dose levels of 1580 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 50 μg/plate and above in the absence of S9-mix. In the presence of S9-mix, toxicity was observed at dose levels of 158 μg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mutant frequencies of the controls were in the range of the historical control values and the data published in the literature (Maron and Ames, 1983; Levin et al., 1982).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
TA1535: without S9: 63.2 µg/plate and above and with S9: no toxicity
TA97: no toxicity
TA98: without and with S9: 200 µg/plate
TA100: without and with S9: 63.2 µg/plate and above
TA102: without and with S9: 63.2 µg/plate and above
Experiment 2:
TA1535: without S9: 10 µg/plate and above and with S9: no toxicity
TA97: without S9: 100 µg/plate and above and with S9: no toxicity
TA98: without S9: 10 µg/plate and above and with S9: no toxicity
TA100: without S9: 10 µg/plate and above and with S9: 100 µg/plate
TA102: without S9: 10 µg/plate and above and with S9: 100 µg/plate

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD guidelines and GLP principles.
Executive summary:

The genetic toxicity of the substance was assessed using Salmonella typhimurium TA97, TA98, TA100, TA102 and TA1535 strains, in accordance with OECD guidelines (OECD 471, 1997) and GLP principles. The first experiment was a standard assay, concentrations up to 200 µg/plate, and the second experiment was a preincubation assay with concentrations up to 100 µg/plate. This was based on a dose range test with TA100 showing toxicity at dose levels of 50 μg/plate and above in the absence of S9-mix. In the presence of S9-mix, toxicity was observed at dose levels of 158 μg/plate and above.

In the main test, toxicity was observed in several strains depending on strain and metabolic activation conditions.

All bacterial strains showed negative responses over the entire dose range, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments with and without metabolic activation up to the highest concentration. Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

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