Carvacrol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):. The substance Carvacrol did not induce mutagenicity in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and and E. coli WP2 uvr A in the presence or absence of rat liver S9 metabolic activation (OECD 471, GLP);

Chromosome aberration (mammalian cell cytogenetics assay; micronucleus test): Carvacrol did not induce any chromosome aberrations in L5178Y TK+/- mouse lymphoma cells in the presence or absence of rat liver S9 metabolic activation (OECD 487/GLP).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in CHO K1 Chinese hamster ovary cells exposed to Carvacrol (CAS No. 499-75-2) in the presence or absence of mammalian metabolic activation (Phenobarbital and ß-naphthoflavone-induced rat liver S9) (OECD 476/GLP).  

Link to relevant study records

Reference

Reference 1

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

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

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

Species / strain / cell type:

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

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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 2016 - 19 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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: At room temperature, protected from light and humidity

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France)
- Suitability of cells: L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test.
- Cell cycle length, doubling time or proliferation index: The average cell cycle time is approximately 10-12 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v).
- Periodically checked for Mycoplasma contamination: Yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 0.02, 0.2, 0.5, 2, 5, and 10 mM.

Main experiments without S9 mix:
0.006, 0.013, 0.025, 0.05, 0.10, 0.15, 0.20 and 0.25 mM in the first experiment,
0.003, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.10 mM in the second experiment.

Main experiment with S9 mix:
0.006, 0.013, 0.025, 0.05, 0.10, 0.15, 0.20 and 0.25 mM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Avaiable solubility data from Ames test (16_399-007M_Final Report)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 3 hr and 24 hr without S9; 3 hr with S9.
- Expression time (cells in growth medium): 3 hrs - 24 hrs recovery; 24 hrs - no recovery

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed.
Following the fixation, the cells were kept at +4°C for at least an overnight period.

Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), three dose-levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5% Giemsa.

NUMBER OF CELLS EVALUATED:1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
-micronuclei should be clearly surrounded by a nuclear membrane,
-the micronucleus area should be less than one-third of the area of the main nucleus,
-non-refractility of the micronuclei,
-micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
-micronuclei should be located within the cytoplasma of the cell,
-only mononucleated cells with a number of micronuclei  5 should be scored to exclude apoptosis and nuclear fragmentation.
Number of cells with micronuclei and number of micronuclei per cell were given separately for each treated and control culture.

Miller B.M., Pujadas E. and Gocke E. (1995) Evaluation of the micronucleus test in vitro using chinese hamster cells: results of four chemicals weakly positive in the in vivo micronucleus test. Environ. Mol. Mutagen. 26, 240-247.


DETERMINATION OF CYTOTOXICITY
- Method: Population Doubling

Evaluation criteria:

Each main experiment was considered valid if the following criteria were met:
-the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone mitotis),
-the mean frequency of micronucleated cells in the vehicle control should be consistent with (but not necessary within) control historical data of the Laboratory (Appendix 2). In any case, this frequency should be ≤ 5‰,
-a statistically significant increase in the frequency of micronucleated cells had to be obtained in the positive controls over the background frequency of the vehicle control cultures,
-adequate number of cells and concentrations were analysable .

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the Chi squared test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose-levels (Appendix 4). This statistical analysis was performed using SAS Enterprise Guide software.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3 hr (Firsr test)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3 hr (Second test)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

24 hr

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>0.05 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3 hr

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>0.10 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH & osmolaloty: See RANGE-FINDING/SCREENING STUDIES below
- Precipitation: No emulsion observed during any of the main experiments

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 300.44 mg/mL in the vehicle (DMSO) and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1502.2 µg/mL) was achievable. Thus, the dose-levels selected for the treatment of the preliminary test were 0.02, 0.2, 0.5, 2, 5, and 10 mM.
At the highest dose-level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 367 mOsm/kg H2O (383 mOsm/kg for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions and the dose-level of 10 mM could be selected as the highest dose-level for the main experiments.

An emulsion was observed in the culture medium at dose-levels ≥ 5 mM at the end of the 3-hour treatments and at the dose-level of 10 mM at the end of the 24-hour treatment.

Following the 3- and 24-hour treatments without S9 mix, a severe cytotoxicity was observed at dose-levels ≥ 0.2 mM, as shown by a 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a slight to severe cytotoxicity was observed at all tested dose levels, as shown by a 39 to 100% decrease in the PD.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes; May 2013 - December 2015
- Negative (solvent/vehicle) historical control data: Yes; May 2013 - December 2015

Conclusions:

Under the experimental conditions of the study, the test item, Carvacrol, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

In a mammalian cell cytogenetics assay [micronucleus test; 44629 MNV], L5178Y TK+/- mouse lymphoma cells were exposed to Carvacrol, (99.91%), in DMSO at concentrations of  0.006, 0.013, 0.025, 0.05, 0.10, 0.15, 0.20, 0.25 mM (first experiment; 3 and 24 hrs), 0.003, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.10 mM (second experiment; 3 hrs) without metabolic activation and 0.006, 0.013, 0.025, 0.05, 0.10, 0.15, 0.20, 0.25 mM for 3 hrs with metabolic activation (Aroclor 1254-induced rat liver S9).

Carvacrol was tested up to cytotoxic concentrations. Positive controls induced the appropriate response.  There was no evidence of any chromosome damage or damage to the cell division apparatus induced over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 487 (in vitro mammalian cell micronucleus test) for in vitro cytogenetic mutagenicity data.  

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2017 - 4 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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

Target gene:

hprt

Species / strain / cell type:

other: CHO-K1

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (Manassas, Virginia, United States)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium and incubated at 37oC ( 0.5 C) in a humidified atmosphere (5 0.3% CO2 in air).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes. Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
- Periodically 'cleansed' against high spontaneous background: Yes. ). Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 22 April 2016

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary tests:
5 hr +S9: 3.906 μg/mL- 2000μg/mL
5 hr -S9: 3.906 μg/mL- 2000μg/mL
24hr -S9: 3.906 μg/mL- 2000μg/mL

Main tests:
Assay 1
5 hr +S9: 250, 200, 175, 150, 125, 100, 50, 25 and 12.5 µg/mL
5 hr -S9: 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 µg/mL
Assay 2
5 hr +S9: 250, 200, 175, 150, 125, 100, 50, 25 and 12.5 µg/mL
24hr -S9: 150, 125, 100, 75, 50, 25, 12.5 and 6.25 µg/mL

Vehicle / solvent:

DMSO; based on the available information from another study performed at the Test Facility (CiToxLAB study code: 16/399-007M)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Assay 1 - 5 hrs in the presence and absence of S9
Assay 2 - 5 hrs in the presence of S9 and 24 hrs in the absence of S9
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: Duplicate cultures in both assays

STAINING TECHNIQUE USED: After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.


DETERMINATION OF CYTOTOXICITY
- Method: Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle) control. Other parameters measuring cytotoxicity like relative survival after treatment (comparing the cell number determined after treatment to the vehicle control) or relative total growth (comparing the growth rate during the entire expression period to the vehicle control) were also calculated.

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data was also taken into consideration if necessary).

According to the relevant OECD No. 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

Statistics:

The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If the obtained result were significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.

The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel software (R-squared values were calculated for the log concentration versus the mutation frequency).

In the statistical analysis, negative trends were not considered significant.

Species / strain:

other: CHO-K1

Remarks:

Assay 1 (5 hrs)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: CHO-K1

Remarks:

Assay 2 (5 hrs)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

125 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: CHO-K1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There were no large changes in pH and osmolality after treatment in any cases.
- Precipitation: In Assays 1 and 2, insolubility / minimal amount of insolubility was detected in the final treatment medium at the end of the treatment in the 200-250 µg/mL or 125-250 µg/mL concentration ranges with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of
S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. Based on the available information about the cytotoxic potential of the test item, the highest test concentration in the preliminary test was 2000 µg/mL. Tabulated results of the preliminary experiment are given in Appendix 3.

Insolubility and cytotoxicity were detected in the preliminary experiment. The concentrations selected for the main experiments were based on the cytotoxicity data to cover the range from cytotoxicity to no or little cytotoxicity according to the relevant OECD guideline. Lower test concentrations were separated by factor of two, but more closely spaced concentration were selected in the expected cytotoxic concentration range. At least eight concentrations were selected for the main experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in harmony with the historical data in all assays (Appendix 15, updated 17-10-17).

Conclusions:

In an in vitro mammalian cell gene mutation assay performed in CHO K1 Chinese hamster ovary cells at the hprt locus, Carvacrol was not mutagenic in the presence or absence of metabolic activation.

Executive summary:

In an in vitro gene mutation study in mammalian cells (16/399-015C), CHO K1 Chinese hamster ovary cells were exposed to Carvacrol (99.91%) in DMSO at concentrations of 250, 200, 175, 150, 125, 100, 50, 25 and 12.5 µg/mL and 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 µg/mL (experiment 1) for 5 hrs with and without Phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation respectively and 250, 200, 175, 150, 125, 100, 50, 25 and 12.5 µg/mL and 150, 125, 100, 75, 50, 25, 12.5 and 6.25 µg/mL (experiment 2) for 5 and 24 hours in the presence/absence of metabolic activation respectively.

Carvacrol was tested up to the limits of cytotoxicity.The positive controls induced the appropriate response. 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 476 for in vitro cytogenetic gene mutation data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Rat bone marrow chromosome aberration assay: Carvacrol significantly induced structural and total chromosome abnormalities at all concentrations (10, 30, 50 and 70 mg/kg bw) and treatment times (6, 12, 24 hrs) when compared with the control in Sprague Dawley rats (OECD 475).

The results from this study were disregarded due to methodological deficiencies (only 2 animals per sex were used per dose and the route of administration (intraperitoneal) is not recommended by the OECD guideline).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Only 2 animals per sex were used and the route of administration (intraperitoneal) is not recommended by the OECD guideline.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

no

Type of assay:

other: Cytogenicity / bone marrow chromosome aberration

Specific details on test material used for the study:

Purity: 98%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Medical Sciences, Experimental Research and Application Center of Cukurova University., Turkey
- Age at study initiation: 12–16 weeks
- Housing: he plastic cages (32 3 46 3 18 cm3) were used for handling the rats in Genetic Laboratory

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

6, 12, and 24 hr

Frequency of treatment:

Once

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

70 mg/kg bw/day (nominal)

No. of animals per sex per dose:

2 males
2 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Urethane (Sigma, U2500)
- Route of administration: IP
- Doses / concentrations: 400 mg/kg b.w.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

To arrest mitosis, colchicine (3 mg/kg bw) was injected intraperitoneally 2 h before the animals were sacrificed by cervical dislocation. The femurs were stripped proximally, and the bone marrow was aspirated in 4 mL of 0.9% NaCl (37C). The suspension was centrifuged for 10 min at 1200 rpm, and the bone marrow pellet was resuspended in 0.4% KCl at 37C for 30 min, and then fixed in cold methanol: glacial acetic acid (3:1) for 20 min at room temperature. The treatment with fixative was repeated two times. Then the cells were spread on glass slides and air-dried. The slides were stained with Giemsa (5% in Sorensen buffer) for 15 min.

Evaluation criteria:

One hundred well spread metaphases per animal (totally 400 metaphases per group) were examined at 1000X magnification for the occurrence the structural and numerical chromosome aberrations. The MI was also determined by scoring 3000 cells from each animal.

Statistics:

The t test were used for the statistical significance for the percentage of structural, numerical and total CA and MI. Dose response relationships were determined from the correlation and regression coefficients for the percentage of structural, numerical and total CA and MI.

Sex:

male/female

Genotoxicity:

positive

Remarks:

6 hrs

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male/female

Genotoxicity:

positive

Remarks:

12 hrs

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male/female

Genotoxicity:

positive

Remarks:

24 hrs

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Table I shows the in vivo results of carvacrol in bone marrow cells of rats. Carvacrol significantly induced the structural and total chromosome abnormalities (CA) in all the concentrations and treatment times when compared with control. Carvacrol showed the similar effects with the positive control urethane on induction of the percentage of structural and total CA at the highest concentrations (70 mg/kg bw) for 12 h treatment period. In addition, carvacrol induced the numerical CA at the highest two concentrations (50 and 70 mg/kg bw) for 24 h treatment period. There were dose-dependent effects on induction of structural, numerical and total CA, for carvacrol was capable of inducing the structural chromosome aberrations (especially the chromatid type abnormalities) and numerical chromosome aberrations (especially the polyploidy).

Carvacrol decreased the mitotic index (MI) in all the concentrations and treatment times when compared with control. Carvacrol showed similar effects with the positive control urethane on decreasing the MI at 70 mg/kg bw for 6 h, at 30 and 50 mg/kg bw for 12 h and at all concentrations for 24 h treatment periods. The decreasing of the MI was in a dose-dependent manner and these were statistically significant.

Conclusions:

In a disregarded rat bone marrow chromosome aberration assay, Carvacrol significantly induced the structural and total chromosome abnormalities in all the concentrations and treatment times when compared with control.

Executive summary:

In a rat bone marrow chromosome aberration assay (Azirak, 2008), groups of male and female Sprague-Dawley rats (4/dose) were treated intraperitoneally with Carvacrol (98%) at doses of 10, 30, 50 and 70 mg/kg bw. Bone marrow cells were harvested at 6h, 12h, and 24h post-treatment. The vehicle was DMSO.

There were signs of cytotoxicity during the study via the % mitotic index. Carvacrol was tested at an adequate dose. The positive control induced the appropriate response. Carvacrol significantly induced the structural and total chromosome abnormalities (CA) in all the concentrations and treatment times when compared with control. Carvacrol showed similar effects with the positive control urethane on induction of the percentage of structural and total CA at the highest concentrations (70 mg/kg bw) for 12 h treatment period. In addition, carvacrol induced the numerical CA at the highest two concentrations (50 and 70 mg/kg bw) for 24 h treatment period. There were dose-dependent effects on induction of structural, numerical and total CA, for carvacrol was capable of inducing the structural chromosome aberrations (especially the chromatid type abnormalities) and numerical chromosome aberrations (especially the polyploidy).

This study is classified as not acceptable. This study does not satisfy the requirement for Test Guideline OECD 475 for Mammalian Bone Marrow Chromosome Aberration data, due to methodological deficiencies (only 2 animals per sex were used per dose and the route of administration (intraperitoneal) is not recommended by the OECD guideline).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

In vitro

In a reverse gene mutation assay in bacteria (OECD 471/GLP), 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.

In a mammalian cell cytogenetics assay [micronucleus test; OECD 487/GLP], L5178Y TK+/- mouse lymphoma cells were exposed to Carvacrol, (99.91%), in DMSO at concentrations of  0.006, 0.013, 0.025, 0.05, 0.10, 0.15, 0.20, 0.25 mM (first experiment; 3 and 24 hrs), 0.003, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.10 mM (second experiment; 3 hrs) without metabolic activation and 0.006, 0.013, 0.025, 0.05, 0.10, 0.15, 0.20, 0.25 mM for 3 hrs with metabolic activation (Aroclor 1254-induced rat liver S9). Carvacrol was tested up to cytotoxic concentrations. Positive controls induced the appropriate response.  There was no evidence of any chromosome damage or damage to the cell division apparatus induced over background.

In an in vitro gene mutation study in mammalian cells (OECD 476/GLP), CHO K1 Chinese hamster ovary cells were exposed to Carvacrol (99.91%) in DMSO at concentrations of 250, 200, 175, 150, 125, 100, 50, 25 and 12.5 µg/mL and 200, 175, 150, 125, 100, 75, 50, 25 and 12.5 µg/mL (experiment 1) for 5 hrs with and without Phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation respectively and 250, 200, 175, 150, 125, 100, 50, 25 and 12.5 µg/mL and 150, 125, 100, 75, 50, 25, 12.5 and 6.25 µg/mL (experiment 2) for 5 and 24 hours in the presence/absence of metabolic activation respectively. Carvacrol was tested up to the limits of cytotoxicity.The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

In vivo

In a rat bone marrow chromosome aberration assay (OECD 475), groups of male and female Sprague-Dawley rats (4/dose) were treated intraperitoneally with Carvacrol (98%) at doses of 10, 30, 50 and 70 mg/kg bw. Bone marrow cells were harvested at 6h, 12h, and 24h post-treatment. The vehicle was DMSO. There were signs of cytotoxicity during the study via the % mitotic index. Carvacrol was tested at an adequate dose. The positive control induced the appropriate response. Carvacrol significantly induced structural and total chromosome abnormalities (CA) at all the concentrations and treatment times when compared with control. Carvacrol showed similar effects with the positive control urethane on induction of the percentage of structural and total CA at the highest concentrations (70 mg/kg bw) for 12 h treatment period. In addition, carvacrol induced the numerical CA at the highest two concentrations (50 and 70 mg/kg bw) for 24 h treatment period. There were dose-dependent effects on induction of structural, numerical and total CA, for carvacrol was capable of inducing the structural chromosome aberrations (especially the chromatid type abnormalities) and numerical chromosome aberrations (especially the polyploidy).The results from this study were disregarded due to methodological deficiencies (only 2 animals per sex were used per dose and the route of administration (intraperitoneal) is not recommended by the OECD guideline).

All three in vitro studies were clearly negative. The results from the in vivo rat bone marrow chromosome aberration assay were disregarded due to methodological deficiencies. The results from these studies are suitable for use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Carvacrol (CAS No. 499 -75 -2) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.