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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:
15 August 2000 - 29 September 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl acetate
EC Number:
201-036-1
EC Name:
[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl acetate
Cas Number:
77-54-3
Molecular formula:
C17H28O2
IUPAC Name:
[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl acetate

Method

Target gene:
Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED- Source of cells: Dr. Bruce N. Ames, University of California, Berkeley, California, USAMEDIA USED- Type and identity of media including CO2 concentration if applicable:- Properly maintained: [yes/no]- Periodically checked for Mycoplasma contamination: [yes/no]- Periodically checked for karyotype stability: [yes/no)- Periodically 'cleansed' against high spontaneous background: [yes/no]
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix
Test concentrations with justification for top dose:
Experiment 1:
- TA98, TA100, TA102 & TA1537 (with and without S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
- TA1535 (without S9): 50, 150, 500, 1500 and 5000 µg/plate
- TA1535 (with S9): 15, 50, 150, 500 and 1500 µg/plate
Experiment 2:
- TA98 & TA100 (with and without S9): 50, 150, 500, 1500 and 5000 µg/plate
- TA102 (with and without S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
- TA1535 & TA1537 (without S9): 50, 150, 500, 1500 and 5000 µg/plate
- TA1535 & TA1537 (with S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 3:
- TA102 (with S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
The dose levels were chosen based on the outcome of an initial (cyto)toxicity test.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: standard solvent
Controls
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: Alleexperiments were performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: assessment of the reduction in the number of revertant colonies and diminution of the background lawn
Rationale for test conditions:
Standard plate incorporation test conditions.
Evaluation criteria:
A significant increase in number of mutant colonies is considered a mutagenic response. A dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels supports this. An additional general rule is that two-fold (or more) increases in mean revertant numbers must be observed between the solvent control and a specific dose level.
Statistics:
An estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was made using a X2-test (Mohn and Ellenberger, 1977).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound on the plates was observed at 1500 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES: Initial toxicity test was performed to determine dose levels.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of spontaneous revertants observed using each of the live strains was close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cytotoxicity was observed in the presence of S9-mix towards the strains TA1537 and TA102 at 5000 µg/plate and in the absence of S9-mix towards the strain TA102 at 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance was determined to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed according to the standard plate-incorporation assay, in the absence and presence of S9-mix. The dose levels were selected based on an initial toxicity test. Adequate negative and positive controls were included. A significant increase in the mutation frequency of the tester strain TA102 in the presence of a metabolic activation system was seen in one of three experiments. This was not observed in the absence of S9-mix. The substance did not induce a significant increase in the number of revertant colonies in any of the other S. typhimurium strains (TA1535, TA1537, TA98 and TA100), both in the absence and presence of S9-metabolic activation. The significant increase in mutation frequency for TA102 was only evident in 1 out of 3 tests which have been performed with a metabolizing system, not found to be dose-dependent and also not two-fold as compared to the solvent control. Also the mean mutant frequency at each of the dose levels was still within the historical control range for this strain. Based on this, the overall result for strain TA102 is considered negative, as well as for the other strains. Based on these results, the substance was found to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of the CLP Regulation (1272/2008/EC).