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Toxicological information

Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data

Data source

Reference Type:

Materials and methods

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

Test material

Test material form:
liquid: volatile
Specific details on test material used for the study:
Thymus vulgaris essential oil (Thyme oil)


Target gene:
The test strain used for the Ames test was Salmonella typhymurium strains TA100 (hisG46/rfa/∆ uvrB/pKM101), developed by Dr B.N.Ames of the university of California, Berkeley.
Species / strain
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Test concentrations with justification for top dose:
50, 100, 200, 300, 500, 1000 and 2000 µg/ml
Vehicle / solvent:
A mixture containing each test component in 0.1 ml of dimethyl sulfoxide (DMSO)
Details on test system and experimental conditions:
0.1 ml of the test strain cell culture in the early stationary phase and 0.5 ml of S9 mix was incubated at 37 °C for 20 min in each test tube with a shaking frequency of 120 strokes per minute. After incubation, 2 ml of 0.05 mM L-histidine/0.05 mM biotin molten top agar were added to each test tube, mixed and poured onto the surface of minimal glucose agar medium. The plate was incubated for 48 h at 37°C and the number of reverent colonies was counted.
The Ames test using the S9 fraction and without S9 mix (phosphate buffer in place of the S9 mix) for all the test compounds was performed on the same occasion. The S9 mix (0.5 ml) contained 0.05 ml of the S9 fraction and 0.45 ml of a cofactor solution (ISO 16240,. 2005.04.01).The S9 mix composed of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADPH, 4mM NADH, and 100 mM sodium phosphate (pH 7.4). The protein amount of each S9 fraction that was used was 1 mg/plate.
Evaluation criteria:
By using TA100, the mean values of negative controls were within the range of 80-180 mutant colonies per plate, the mean values of positive controls showed at least the induction rate of +100 colonies.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
All 4 essential oils in all 7 tested (Eugenia caryophyllata (Clove), Cinnamum zeylanicum (Cinnamon), Thymus vulgaris (Thyme) and Zataria multiflora) dilutions were negative in the Ames Salmonella reversion assay without S9 (microsomal mutagenesis assay), induction rates were from 0 to 13.
However all 3 essential oils in each 7 tested dilution except for Clove oil (Eugenia caryophyllata) were negative in the Ames Salmonella reversion assay with S9 (microsomal mutagenesis assay), induction rates were from 0 to 15.

Any other information on results incl. tables

                   Mutagenicity test results of Thyme


       with S9

       without S9

 Conc (µg/ml)  Mean*  SD  Induction rate  Mean  SD  Induction rate
 50 92  2.64  -1  81  4  1
 100  93  2.00  0  81  1.73  1
 200  93  1.00  81.3  6.65  1.3
 300  100  5.56  82


 400  102  1.00  9  83


 500  105  3.60  12  82 3.00   2
 1000 105   5.56  12  83 3.60   3
 2000  105  5.19  12  85 2.64   5

* Mean: Average of colonies in triplicate plates

Applicant's summary and conclusion

Thyme essential oil in concentrations of 50–2000 µg/ml did not show signs of mutagenicity in an Ames test using Salmonella typhymurium strain TA100 with and without rat liver S9.