Carvacrol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2017-23 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Carvacrol

EC Number:

207-889-6

EC Name:

Carvacrol

Cas Number:

499-75-2

Molecular formula:

C10H14O

IUPAC Name:

2-methyl-5-(propan-2-yl)phenol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wanxiang International Flavors & Fragrances Pte., Ltd.; 2016111001
- Expiration date of the lot/batch:10 November 2018
- Purity test date:10-11-2016
- Purity: 99.91%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8C), protected from light and humidity

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β- naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 5000; 2500; 1000; 316; 100, 31.6, 10 μg/plate
Main test (1): 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate
Main test (2): 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). The test item was insoluble in Distilled water at 100 mg/ml concentration. The test item was soluble at this concentration using DMSO, DMF (clear solution was detected in both cases). Based on the results, DMSO was selected as vehicle (solvent) for the study.

Controls

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

Remarks:

TA98: 4-nitro-1,2-phenylenediamine (NPD) without activation; All strains: 2-aminoanthracene (2AA) with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

DURATION
- Preincubation period: 20min at 37ºC
- Exposure duration: 48±1 hours

NUMBER OF REPLICATIONS: Triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in background lawn

Evaluation criteria:

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and
Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and
TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a
reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only
determining factor for a positive response.

Results and discussion

Test results

Test results 1

Species / strain:

other: All strains

Remarks:

Main test (1)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: All strains

Remarks:

Main test (2)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

158.1 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was detected on the plates in the main tests in all examined strains with and without metabolic activation at higher concentrations.

RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
No precipitate of the test item was detected in the Preliminary Range Finding Test. Inhibitory, cytotoxic effect of the test item (absent, reduced and sligtly reduced background lawn development, pinpoint colonies) was detected in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98, TA100 strains at 5000, 2500 and 1000 μg/plate concentrations with and without metabolic
activation. The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.


HISTORICAL CONTROL DATA
- Positive historical control data: Yes with ranges, means and standard deviation (Period of 2011-2015) Appendix 6
- Negative (solvent/vehicle) historical control data: Yes with ranges, means and standard deviation (Period of 2011-2015) Appendix 6

Applicant's summary and conclusion

Conclusions:

The test item CARVACROL had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

In a reverse gene mutation assay in bacteria (16_399-007M), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to Carvacrol (99.91%) in DMSO at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0 μg/plate (direct plate incorporation; experiment 1) and 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β- naphthoflavone-induced rat liver S9).

Carvacrol 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.