Thyme, Thymus zygis, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The thymus zygis oil is considered as a substance similar to thymus vulgaris oil because they contain both the same major constituant (51% of thymol for Thymus zygis oil and 39% of thymol for Thymus vulgaris oil).
They also contain both the same (second) major constituant (16% of para cymene for Thymus zygis oil and 18% of para cymene for Thymus vulgaris oil).
They also contain both the same (third) major constituant (8.7% of gamma terpinene for Thymus zygis oil and 11% of gamma terpinene for Thymus vulgaris oil).
Moreover, they belong to the same botanical family of the Lamiaceae and the same botanical genus (Thymus), so to the same category.
Hence, a read across approach can be used.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 / 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.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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.

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	0	81.3	6.65	1.3
300	100	5.56	7	82	2.64	2
400	102	1.00	9	83	4.35	3
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

Conclusions:

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Thyme, Thymus vulgaris, ext.

EC Number:

284-535-7

EC Name:

Thyme, Thymus vulgaris, ext.

Cas Number:

84929-51-1

Molecular formula:

not relevant for a UVCB substance

IUPAC Name:

Thyme, Thymus vulgaris, ext.

Test material form:

liquid: volatile

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Applicant's summary and conclusion

Conclusions:

Thymus vulgaris essential oil had no mutagenic or DNA-damaging activity in either the Ames test (strains TA1535, TA1537, TA98, TA100, with and without metabolic activation, 0.25 and 0.5 µl essential oil/plate) or Bacillus subtilis rec-Assay (10 µl and 30 µl essential oil)(Zani et al., 1991).
As a similar substance, it is predicted that Thymus zygis essential oil has no mutagenic or DNA-damaging activity also.