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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:
16-10-2012 to 30-10-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
EC Number:
251-020-3
EC Name:
[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one
Cas Number:
32388-55-9
Molecular formula:
C17H26O
IUPAC Name:
1-((3R,3aR,7R,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulen-5-yl)ethanone
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Methyl Cedryl Ketone
- Physical state: Liquid
- Purity test date: 31-07-2012
- Lot/batch No.: 0100172226
- Expiration date of the lot/batch:31-07-2014

- Stability under test conditions: The stability of Methyl Cedryl Ketone and the stability and homogeneity of Methyl Cedryl Ketone in the vehicle were not determined as part of this study.
- Storage condition of test material: Room temperature, in the dark

Method

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 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Commercially available S9 fraction (Trinova Biochem GmbH, Germany) supplemented with co-factors.Lot No.: 2907 (Date of preparation: 15 March 2012) – used in test 1; Lot No.: 2943 (Date of preparation:18 May 2012) – used in test 2.
Test concentrations with justification for top dose:
Mutation Test - Experiment 1 (direct plate incorporation): 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiment 2 (pre-incubation): 0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of Methyl Cedryl Ketone was assessed at 50 mg/mL in dimethyl sulphoxide
(DMSO) and was found to be soluble. DMSO (HPLC grade) was, therefore, used as the vehicle for this study.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
NaN3: 2 µg/plate TA100 and TA1535; AAC: 50 µg/plate TA1537; NQO: 2 µg/plate WP2 uvrA (pKM101); 2-NF:2 µg/plate for strain TA98.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
With metabolic activation
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
AAN: 5 µg/plate TA100 and TA1535, 10 µg/plate WP2 uvrA (pKM101); B[a]P:5 µg/plate for TA98 and TA1537
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Mutation Test - Experiment 1: direct plate incorporation method;
Mutation Test - Experiment 2 : preincubation method

DURATION
- Preincubation period: 30 minutes (Mutation Test - Experiment 2)
- Exposure duration: approximately 72 hrs (both methods)

NUMBER OF REPLICATIONS:3 (Experiments 1 & 2)

DETERMINATION OF CYTOTOXICITY
- Method: Substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

Statistical analysis can be performed if results fail to satisfy the criteria for a clear “positive” or “negative” response; see Statistics below.

It is acceptable to conclude an equivocal response if no clear results can be obtained after statistical analysis.
Statistics:
If the results obtained satisfy the criteria for a clear “positive” or “negative” response, no statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Biological importance will be considered along with statistical significance. In general,treatment-associated increases in revertant colony
numbers below two or three times those of the vehicle controls are not considered biologically important.

MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I. de G., ROBINSON,
W.D. and TWEATS, D.J. (1989) Analysis of data from microbial colony assays in:
KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing.
Report. Part III. Statistical Evaluation of Mutagenicity Test Data, pp.26-65. Cambridge
University Press, Cambridge.

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:The mean revertant colony counts for the vehicle controls were within or close to the current
historical control range for the laboratory (Period: 1 May 2007 to 30 April 2012; Appendix I).



Any other information on results incl. tables

Study report attachments:

Table 1 Results obtained in the absence of metabolic activation - test 1 (MBB0003)

Table 2 Results obtained in the presence of metabolic activation - test 1 (MBB0003)

Table 3 Viability counts obtained in test 1 (MBB0003)

Table 4 Results obtained in the absence of metabolic activation - test 2 (MBB0003)

Table 5 Results obtained in the presence of metabolic activation - test 2 (MBB0003)

Table 6 Viability counts obtained in test 2 (MBB0003)

Appendix 1 Historical Control Data (MBB0003)

Applicant's summary and conclusion

Conclusions:
It was concluded that Methyl Cedryl Ketone showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

In a reverse gene mutation assay in bacteria (MBB0003), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A (pKM101) were exposed to Methyl Cedryl Ketone in DMSO at concentrations of 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (direct plate incorporation; experiment 1) and 0, 50, 150, 500, 1500 and 5000 µg/plate (pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (rat S9). Methyl Cedryl Ketone was tested up to the limit concentration (5000 µg/plate).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.