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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Andersen and Jensen
Year:
1984
Bibliographic source:
Mutation Research

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: As mention below
Principles of method if other than guideline:
To evaluate the mutagenic potential of test chemical in Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97and TA98.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (IUPAC name): (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one- Common name: Menthone - Molecular formula: C10H18O- Molecular weight: 154.2512 g/mol- Smiles notation: C1([C@@H](CC[C@@H](C1)C)C(C)C)=O- InChl: 1S/C10H18O/c1-7(2)9-5-4-8(3)6-10(9)11/h7-9H,4-6H2,1-3H3- Substance type: Organic- Physical state: Liquid

Method

Target gene:
Histidine
Species / strain
Species / strain:
other: TA1537, TA1535, TA100, TA 97and TA98.
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate.
Test concentrations with justification for top dose:
0, 6.4, 32,160 or 800 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Negative controls:
not specified
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: For all strains, 2-anthramine with S9 served as a positive control.
Details on test system and conditions:
METHOD OF APPLICATION: in agar- Cell density at seeding (if applicable): no dataDURATION- Preincubation period: No data- Exposure duration: No data- Expression time (cells in growth medium): No data- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No data SELECTION AGENT (mutation assays): No data SPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: Each dosage was tested on 5 parallel plates and all the tests were performed on two separate occasions.METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No dataNUMBER OF CELLS EVALUATED: No dataNUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No dataCRITERIA FOR MICRONUCLEUS IDENTIFICATION: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data- Any supplementary information relevant to cytotoxicity: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data- OTHER: No data
Rationale for test conditions:
Not specified.
Evaluation criteria:
The mean number of revertants for n plates at each dose level was calculated. The test was considered to be positive if mean number of revertants for n plates at each dose level was greater than control.
Statistics:
Comparisons of the number of revertants per plate were done as t-tests after a square-root transformation of each number had been performed to give homogeneity of variance. The mean number of revertants for n plates at each dose level was calculated to be the squared value of the mean (y) of the square roots. The standard error of the mean was calculated as 2 y~[' s2/n where s 2 is the pooled variance of all individual square root values

Results and discussion

Test results
Species / strain:
other: strains TA1537, TA1535, TA100, TA 97 and TA98.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic effect were observed.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Definition of acceptable cells for analysis: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: On the basis of the toxicity study, the doses were selected for the main studyCYTOKINESIS BLOCK (if used)- Distribution of mono-, bi- and multi-nucleated cells: No dataNUMBER OF CELLS WITH MICRONUCLEI- Number of cells for each treated and control culture: No data- Indication whether binucleate or mononucleate where appropriate: No dataHISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:- Negative (solvent/vehicle) historical control data:ADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Any other information on results incl. tables

Table: 1

NUMBER OF REVERTANTS AFTER EXPOSURE TO THE TEST CHEMICAL IN THE SALMONELLA/MAMMALIAN-M1CROSOME TEST PRESENTED AS MEAN OF 10 PLATES ± S.E.

 

Dose

(µg/plate)

TA1535

TA100

TA1537

TA98

WITHOUT S9

WITH S9

WITHOUT S9

WITH S9

WITHOUT S9

WITH S9

WITHOUT S9

WITH S9

CONTROL

7 ± 1

8 ± 1

85 ± 4

114 ± 8

7 ± 1

11 ± 1

32 ± 3

33 ± 2

6.4

7 ± 1

10 ± 1

96 ± 4

114 ± 8

13 ±2

11 ± 1

25 ± 2

28 ± 2

32

7 ± 1

9 ± 1

92 ± 4

121 ± 8

12 ± 2

12 ± 1

31 ± 3

27 ± 2

160

5 ± 1

8 ± 1

97 ± 4

123 ± 8

8 ± 1

11 ± 1

28 ± 2

26 ± 2

800

7 ± 1

9 ± 1

59 ± 6

104 ± 7

4 ± 1

11 ± 1

13 ± 3

22 ± 2

Contrary to these results, menthone induced an increased number of revertants in the strain TA1537 without S9 at dose levels of 6.4 and 32 µg per plate (as per table). This increase was statistically significant (p≤0.01). But no effect at highest concentration.

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98 in the presence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/mammalian-microsome test was performed on Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98. The test material was exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate. DMSO was used as solvent control. Positive controls were also used for all strain respectively. The test was performed by standard plate method. The toxicity of the substances was tested in the tester strains at 10-7 dilutions. On the basis of the results obtained, the following concentrations of the test substance were selected for testing: 800, 160, 32 or 6.4 µg per plate. Each dosage was tested on 5 parallel plates. The mean number of revertants for n plates at each dose level was calculated. No mutagenic effects were observed in all strain except strain TA1537. Contrary to these results, test chemical induced an increased number of revertants in the strain TA1537 (dose levels of 6.4 and 32 µg per plate). But no effects were observed at the dose level of 160 or 800 µg/plate in strain TA1537 without S9. On the basis of observations made, the test chemical was considered to be non mutagenic in all strain at highest dose concentration and in the presence of S9 too in all strain. Hence the test material cannot be classified as gene mutant in vitro.