Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial mutagenicity study (OECD guideline 471): non-mutagenic

A mammalian chromosome aberration study (OECD guideline 473): non-clastogenic

A mammalian mutagenicity study (OECD guideline 476): non-mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Nov 2017-10 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
24, 76, 240, 760, 2400 ug/ml. Justification for top dose: No toxicity observed at 1600 ug/ml. At and above 3200 ug/ml plate the intensity of the bacterial background lawn was severely reduced associated with severe reduction in the mean number of revertant clones.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Evaluation criteria:
To determine a positive result, there should be a dose related increrase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the testi item either in the presence or absence of metabolic activation system. The test will be judged postive if the increase in mean revertants at the peak of the dose respomse is equal to or greater than two times the mean vehicle control values for strains TA98, TA100 or WP2uvrA(pKM101) or equal to or greater than three times the mean vehicle control values for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respecitive threshold cited above or a non-dose responsive increase that is equal to or greated than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Test item was found to be stable in DMSO for 48 hours at room temperature at the concentrations of 25 and 50000 ug/ml.
The test item did not precipitate on the basal agar plates at any of the tested doses
The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for all tester strains when conpared to the respective vehicle control plates, either in the presence or absence of the metabolic activation.
Positive control substances tested simultaneously produced more than a 3-fold increase in the mean nembers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as intensity of the bacterial background lawn of all the tester strains was compared to that of the respective vehicle control plates.
Conclusions:
The test item 4-allyveratrole (Methyl Eugenol) was not mutagenic in the bacterial reverse mutation test.
Executive summary:

The objective of this study was to determine the potential of 4 -allylveratrole (Methyl Eugenol) and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).The study procedures were based on the OECD 471 guideline.

Methyl eugenol was soluble in DMSO at 50mg/mL on sonication, and was found to be stable in DMSO for 48 hours at room temperature at the fortification levels of 25 an 50 000 ug/mL.

In a preliminart toxicity test to selct test doses for the mutaton assay, the test item did not show toxicity to the tester strain up to 1600ug/plate as the intensity of the bacterial background lawn was comparable to the vehicle control plates. Similarly, the mean number of revertant colonies was more or less comparable to the vehicle constrol plates at these test doses. However, at and above 3200ug/plate the intensity of the bacterial background lawn was severily reduced associated with a severe reduction in the mean number of revertant colonies. Based o these observations, a highest dose of 2400ug/plate was tested in the mutation assay.

At the concentrations used, 24, 76, 240, 760, 2400 ug/ml, the test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously proving the proper functionality of the test.

In conclusion, based on the results of this study it is concluded that 4 -allylveratrole (Methyl Eugenol) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 April 2019 - 15 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Name of Test Item : Methyl Eugenol
Chemical Name (IUPAC) : 4-allyl-1,2-dimethoxybenzene
CAS No. : 93-15-2
Physical Appearance (with color) : Pale yellow liquid
Batch No. : 40003010419
Purity as per (Certificate of Analysis) : 99.24%
Batch Produced by (Name and Address) : YASHO INDUSTRIES LIMITED
Plot No. 2514/2515, Phase IV,
G.I.D.C., Vapi-396195, Gujarat, India
Date of Manufacture : Feb 2019
Date of Expiry : Jan 2021
Storage Conditions : Ambient (21 to 29°C)
Test Item Code by Test Facility : D819-001
Species / strain / cell type:
primary culture, other: primary cell cultures derived from healthy human donor.
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human peripheral lymphocytes from the blood of healthy, young, non-smoking donors 24 (Female) and 29 (Male)with no known recent exposure to genotoxic chemicals or radiation were used.
- Suitability of cells: The primary cell cultures of human whole blood were selected on the basis of growth ability in culture, stability of the karyotype. This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events. Further as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test system.

For lymphocytes:
- Sex, age and number of blood donors: 24 (Female) and 29 (Male)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI Media supplemented with 10% FBS and antibiotics (1% Penicillin-Streptomycin) at 37±1ºC with 5±1% CO2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- method of preparation of S9 mix : Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar Rats induced with intraperitoneal injection of sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) : Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain.
Test concentrations with justification for top dose:
Dose range finding test:
Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosomal aberration test. The concentrations selected for initial cytotoxicity test were 0.0312, 0.0625, 0.125, 0.25 and 0.5 µL/mL.
Set No. Metabolic activation Treatment details Duration of treatment for Initial cytotoxicity test
1 +S9 Vehicle control/Test item 3 Hours & 30 minutes
2 - S9 Vehicle control/Test item 3 Hours & 30 minutes
3 - S9 Vehicle control/Test item 22 Hours

Chromosomal aberration test:
Based on the results of cytotoxicity test, the concentrations selected for the chromosomal aberration test were 0.125, 0.25 and 0.5 µL/mL of Methyl Eugenol as low, mid and high concentrations respectively.
Set No. Metabolic Activation Treatment Duration
1. +S9 Vehicle control/test item/positive control (10 µg/mL of Cyclophosphamide Monohydrate) 3 Hours and 20 minutes
2. -S9 Vehicle control/test item/positive control (0.05 µg/mL Mitomycin-C) 3 Hours and 20 minutes
3. -S9 Vehicle control/Test item/Positive control (0.05 µg/mL Mitomycin-C) 20 Hours and 49 minutes
Vehicle / solvent:
- Vehicle:dimethyl sulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:

- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:

- Exposure duration/duration of treatment: both with metabolic activation (+S9) (set 1) for 3 to 6 hours, without metabolic activation (-S9) - set 2 for 3 to 6 hours and without metabolic activation (-S9) for 20 to 24 hours set 3 at 37±1ºC and 5±1% CO2
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Before 1 to 3 hours of harvesting, colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation of 1 to 3 hours with colchicine, cell suspension was collected to pre labeled tubes and centrifuged for 10 minutes at 1500 rpm.
Pellets were mixed with 3 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation(-S9), treatment/group and slide number. The slides was air dried.
Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes.

SLIDE EVALUATION
All slides including vehicle control, treatment and positive controls of chromosomal aberration test were coded before evaluation.
Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data.
Gaps were recorded separately and reported but not included in the total aberration frequency.



- OTHER:
Evaluation criteria:
Acceptance of a test is based on the following criteria:
• The concurrent negative/vehicle controls should ideally be within the 95% control limits of the distribution of the laboratory’s historical negative/vehicle control database.
• Concurrent positive controls should produce a statistically significant increase compared with the concurrent negative/vehicle control and it positive controls should induce responses that are compatible with those generated in the historical positive control data base.
• Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analyzable concentrations) was analyzed.
• The criteria for the selection of concentrations for chromosomal aberration test was fulfilled.

Interpretation of Results:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:

• At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.

• The increase is dose-related when evaluated with an appropriate trend test.

The test item is then considered to be able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all experimental conditions examined:
• None of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.

• There is no concentration-related increase when evaluated with an appropriate trend test.

• All results are inside the distribution of the historical vehicle control data.
The test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p < 0.05) and the statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
heavy precipitation was observed at 1 and 2 µL/mL in the precipitation test
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: . no change in pH was observed in any of the concentration tested.
- Data on osmolality: not available
- Possibility of evaporation from medium: not evaluated
- Water solubility: Test item was soluble in dimethyl sulphoxide at 200 µL/mL.
- Precipitation and time of the determination: Precipitation test was conducted at 0.0156 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours 7 minutes of incubation, heavy precipitation was observed at 1 and 2 µL/mL.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Initial cytotoxicity test
Cell cultures treated with Methyl Eugenol at the concentrations of 0.0312, 0.0625, 0.125, 0.25 and 0.5 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) showed reduction in mitotic index and the observed values were 0.78%, 1.04%, 2.74%, 3.13% and 3.92% respectively. In the absence of metabolic activation (short term treatment 3 to 6 hours) the obtained reduction in mitotic index was 0.65%, 1.17%, 2.21%, 3.12% and 3.64% respectively. In the absence of metabolic activation (long term treatment 20 to 24 hours) the obtained reduction in mitotic index was 0.78%, 1.04%, 2.09%, 3.13% and 3.92% respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.3% of aberrated cells which was statistically significant compared to the vehicle control (0.67%). The reduction in mitotic index was 6.30% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.67% of aberrated cells which was statistically significant to the vehicle control (0.67%). The reduction in mitotic index observed was 7.54% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (0.67%). The reduction in mitotic index observed was 8.70% when compared with the vehicle control for long term treatment.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test item was soluble in dimethyl sulphoxide at 200 µL/mL. Precipitation test was conducted at 0.0156 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours 7 minutes of incubation, heavy precipitation was observed at 1 and 2 µL/mL. Mild and moderate precipitation was observed at 0.25 and 0.5 µL/mL respectively. No precipitation was observed in any other concentrations tested up to 0.0156 µL/mL. no change in pH was observed in any of the concentration tested. Hence 0.5 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. the other concentrations selected were 0.25, 0.125, 0.0625 and 0.0312 µL/mL of test item.
Initial cytotoxicity test

                                                                   INDIVIDUAL DATA OFCHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

Set No.

Treatment

Concentrations (µL/mL)

Replicate

Mitotic Index

Aberrations

Total No. of Aberrations

Total No. of Aberrations without Gaps

Total no. of Aberrant cells

Total % of Aberrated cells

Gaps

Breaks

Exchanges

Fragments

Ring

Deletion

Dicentric

Total No. of Cells

Total No. of MC

Mitotic Index

Percentage of MI

Chromatid

Chromosome

Chromatid

Chromosome

Chromatid

Chromosome

Set 1 (+S9)

(3 to 6 hours)

Vehicle control

-

R1

520

39

0.0750

7.50

 -

1

 -

 -

1

1

1

0.67

R2

512

38

0.0742

7.42

 -

 -

 -

1

 -

 -

 -

 -

1

1

1

0.67

Positive control (Cyclophosphamide Monohydrate)

10µg/mL

R1

515

34

0.0660

6.60

 -

 -

3

5

 -

2

5

2

1

 -

18

18

16

10.67

R2

515

38

0.0738

7.38

1

 -

1

8

 -

1

5

1

 -

17

16

15

10.00

Methyl Eugenol

0.125

R1

514

38

0.0739

7.39

 -

 -

1

 -

 -

 -

1

1

1

0.67

R2

518

37

0.0714

7.14

1

1

 -

 -

2

1

1

0.67

0.25

R1

508

38

0.0748

7.48

 -

 -

1

 -

1

 -

2

2

2

1.33

R2

519

36

0.0694

6.94

1

1

 -

 -

 -

2

2

2

1.33

0.5

R1

517

38

0.0735

7.35

 -

 -

 -

 -

0

0

0

0.00

R2

516

36

0.0698

6.98

 -

 -

 -

1

 -

1

 -

-

 -

2

2

2

1.33

MI: Mitotic Index, MP: Metaphase Plates,R1: Replicate 1, R2: Replicate 2, +S9: With metabolic activation.

150 metaphases evaluated per replicate.

 

 

APPENDIX 2 (Contd…). INDIVIDUAL DATA OF CHROMOSOMAL ABERRATIONS AND MITOTIC INDEX

Set No.

Treatment

Concentrations (µL/mL)

Replicate

Mitotic Index

Aberrations

Total No. of Aberrations

Total No. of Aberrations without Gaps

Total no. of Aberrant cells

Total % of Aberrated cells

Gaps

Breaks

Exchanges

Fragments

Ring

Deletion

Dicentric

Total No. of Cells

Total No. of MC

Mitotic Index

Percentage of MI

Chromatid

Chromosome

Chromatid

Chromosome

Chromatid

Chromosome

Set 2

(-S9)

(3 to 6 hours)

Vehicle control

-

R1

518

39

0.0753

7.53

 -

 -

 -

 -

1

 -

1

1

1

0.67

R2

519

38

0.0732

7.32

 -

 -

1

 -

 -

 -

1

 -

2

2

1

0.67

Positive control (Mitomycin C)

0.05µg/mL

R1

520

35

0.0673

6.73

1

 -

4

6

 -

2

5

1

19

18

16

10.67

R2

528

37

0.0701

7.01

2

1

3

4

 -

2

8

 -

2

22

19

16

10.67

Methyl Eugenol

0.125

R1

532

38

0.0714

7.14

 -

1

 -

 -

 -

1

1

1

0.67

R2

504

37

0.0734

7.34

 -

1

 -

 -

 -

1

1

1

0.67

0.25

R1

526

36

0.0684

6.84

-

 -

 -

2

 -

2

2

2

1.33

R2

509

38

0.0747

7.47

 -

 -

1

1

 -

 -

2

2

2

1.33

0.5

R1

521

38

0.0729

7.29

 -

 -

 -

1

1

 -

2

2

2

1.33

R2

529

37

0.0699

6.99

 -

1

 -

1

 -

 -

2

1

1

0.67

MI: Mitotic Index,MP: Metaphase Plates, R1: Replicate 1, R2: Replicate 2, -S9: Without metabolic activation.

150 metaphases evaluated per replicate.

 


APPENDIX 2 (Contd…). INDIVIDUAL DATA OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

Set No.

Treatment

Concentrations (µL/mL)

Replicate

Mitotic Index

Aberrations

Total No. of Aberrations

Total No. of Aberrations without Gaps

Total no. of Aberrant cells

Total % of Aberrated cells

Gaps

Breaks

Exchanges

Fragments

Ring

Deletion

Dicentric

Total No. of Cells

Total No. of MC

Mitotic Index

Percentage of MI

Chromatid

Chromosome

Chromatid

Chromosome

Chromatid

Chromosome

Set 3

(-S9)

(20 to 24 hours)

Vehicle control

-

R1

503

38

0.0755

7.55

1

1

1

 -

3

3

2

1.33

R2

511

39

0.0763

7.63

 -

 -

 -

 -

 -

0

0

0

0.00

Positive control       (Mitomycin C)

0.05µg/mL

R1

522

35

0.0670

6.70

2

1

3

5

1

2

5

 -

2

21

18

15

10.00

R2

503

36

0.0716

7.16

1

2

8

3

5

 -

1

20

16

16

10.67

Methyl Eugenol

0.125

R1

518

37

0.0714

7.14

 -

1

 -

 -

 -

1

 -

2

2

2

1.33

R2

509

39

0.0766

7.66

 -

 -

 -

1

 -

 -

 -

 -

1

1

1

0.67

0.25

R1

517

38

0.0735

7.35

 -

2

 -

 -

2

2

2

1.33

R2

502

37

0.0737

7.37

1

 -

1

 -

2

2

2

1.33

0.5

R1

511

38

0.0744

7.44

 -

 -

1

 -

1

1

 -

3

3

2

1.33

R2

504

36

0.0714

7.14

1

 -

 -

1

 -

 -

 -

2

2

2

1.33

MI: Mitotic Index, MP: Metaphase Plates, R1: Replicate 1, R2: Replicate 2, -S9: Without metabolic activation.

150 metaphases evaluated per replicate.

Conclusions:
Based on the results obtained, the test item, Methyl Eugenol is considered as non-clastogenic up to the concentration of 0.5 µL/mL both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The test item, Methyl Eugenol obtained fromYasho Industries Limited was evaluated for chromosomal aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473. Test item was soluble in dimethyl sulphoxide at 200 µL/mL.Precipitation test was conducted at 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 7 minutes of incubation, heavy precipitation was observed at 1 and 2 µL/mL, mild and moderate precipitation was observed at 0.25 and 0.5 µL/mL respectively. No precipitation was observed in any other concentrations tested up to 0.0156 µL/mL. No change in pH was observed in any of the concentration tested hence, 0.5 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.25, 0.125, 0.0625 and 0.0312 µL/mL of test item.

In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 0.65 to 3.92 at 0.0312, 0.0625, 0.125, 0.25 and 0.5 µL/mL. As the percentage reduction in MI was not more than 45±5% at 0.5 µL/mL, same has been selected as the highest concentration for the chromosomal aberration test. Other concentrations tested will be 0.25 and 0.125 µL/mL. 

In the chromosomal aberration test, the cells were treated withMethyl Eugenolat the concentrations of 0.125, 0.25 and 0.5 µL/mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.The observed mean percent aberrated cells at 0.125, 0.25 and 0.5 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.33 and 0.67 respectively. Similarly, the observed mean percent aberrated cells at 0.125, 0.25 and 0.5 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.33 and 1.0 respectively.

The observed mean percent aberrated cells at 0.125, 0.25 and 0.5 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.0, 1.33 and 1.33 respectively.Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.3% of aberrated cells which was statistically significant compared to the vehicle control (0.67%). The reduction in mitotic index was 6.30% when compared with the vehicle control for short term treatment.

Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.67% of aberrated cells which was statistically significant to the vehicle control (0.67%). The reduction in mitotic index observed was 7.54% when compared with the vehicle control for short term treatment.Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (0.67%). The reduction in mitotic index observed was 8.70% when compared with the vehicle control for long term treatment.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 May 2019 - 9 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
the study integrity was not adversely affected by the deviations.
GLP compliance:
yes
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:
Name of Test Item : Methyl Eugenol
Chemical Name (IUPAC) : 4-allyl-1,2-dimethoxybenzene
CAS No. : 93-15-2
Physical Appearance (with color) : Pale yellow liquid
Batch No. : 40003010419
Purity (Declared by sponsor and/or as per Certificate of Analysis) : 99.24%
Batch Produced by (Name and Address) : YASHO INDUSTRIES LIMITED
Plot No. 2514/2515, Phase IV,
G.I.D.C., Vapi – 396195,
Gujarat, India
Date of Manufacture : Feb 2019
Date of Expiry : Jan 2021
Storage Conditions : Ambient (21 to 29°C)
Test Item Code by Test Facility : D819-001
Target gene:
Hprt locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO AA8 , American Type Culture Collection (ATCC)
- Suitability of cells: CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting In vitro Mammalian gene mutation Test.

For cell lines:
- Absence of Mycoplasma contamination: Cells free of mycoplasma will be used for the experiment.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 10% FBS with antibiotics (1% streptomycin and penicillin) and incubated at 37±1°C and 5±1% CO2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Based on the results of solubility, pH and precipitation tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test. Five concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 µL/mL of the test item were tested in an initial cytotoxicity test.

For tests with exogenous metabolic activation, 1 mL of S9 mix was added to all the flasks. A volume of 100 µL of vehicle/different concentrations of test item was added to tetra plate cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 40 minutes at 37±1oC with 5±1% CO2.
For tests without exogenous metabolic activation, a volume of 100 µL of vehicle/different concentrations of test item was added to tetra plate culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 40 minutes at 37±1oC with 5±1% CO2.
Post incubation period (Set 1 and 2), medium from each flask was aspirated and monolayer was washed with DPBS. Cells were trypsinized. Trypsinization was stopped by adding culture media followed by collecting the media with cells.
Set No. Metabolic activation Treatment details Duration of treatment
1 +S9 Vehicle control/Test item 3 hours and 40 minutes
2 -S9 Vehicle control/Test item 3 hours and 40 minutes
Tetra plate treatments were pooled and collected in prelabelled tubes and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was resuspended in media.
Each treatment replicate was plated in triplicate with cell concentration of 200 cells / 5 mL media in 25 cm2 flasks and incubated at 37±1oC with 5±1% CO2 for 9 days.
Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually.
Vehicle / solvent:
- Vehicle: DMSO
- Justification for choice of solvent/vehicle: The test substance was dissolved in DMSO. Test substance concentrations were used within 2 hours after preparation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
3 µg/ml
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/ml
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
FOR GENE MUTATION:
• Since the test item did not precipitated at 0.25 µL/mL, slight precipitation was observed in 0.50 µL/mL. Hence 0.50 µL/mL was considered as the highest concentration in the initial cytotoxicity test.
• As the test item was not cytotoxic up to 0.25 µL/mL in the initial cytotoxicity test, the concentration of 0.25 µL/mL was selected as the highest Concentration for testing in the Gene mutation test.
• Four concentrations i.e. 0.03125, 0.0625, 0.125 and 0.25 µL/mL were selected for gene mutation test, based on initial cytotoxicity test.

Gene mutation test was carried out as described in section 6.8 and 6.9.2. Each treatment group was maintained with tetra plate cultures. Cells were exposed to the test item for 3 hours and 1 minute for both with exogenous metabolic activation and without exogenous metabolic activation respectively in the gene mutation test at 37±1oC with 5±1% CO2.
The treatment schedule for the experiment was maintained as indicated in the following table:
Set No. Metabolic Activation Treatment Duration
1 +S9 Vehicle Control/Test item/Positive Control (3 µg/mL of Benzo(a)pyrene) 3 hours and 1 minute
2 - S9 Vehicle Control/Test item/Positive Control (1 µg/mL of 4 Nitroquinoline N-oxide) 3 hours and 1 minute
Tetra plate treatments were pooled into a pre labeled tube and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained.
Each treatment replicate was plated in triplicate with cell concentration of 200 cells/5 mL media in 25 cm2 flasks and incubated at 37±1oC with 5±1% CO2 for 8 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytotoxicity level was determined using the following formulae:
Adjusted Cloning Efficiency (ACE) = CE × No. of cells at the end of treatment / No. of Cells at the beginning of the treatment

Relative Survival (RS) = ACE (Treated)/ACE (Vehicle Control) × 100

Cloning efficiency (CE) is the percentage of cells plated at a low density that are able to grow into a colony that can be counted.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant Frequency of each treatment was calculated using the following formula:
Mutant Frequency (MF) = Cloning efficiency of mutant colonies in selective medium / Cloning efficiency in non-selective medium
Cloning efficiency = Number of colonies / Number of cells plated.
MF is expressed as mutants per 2 × 10 6 clonable cells.


- OTHER:

Evaluation criteria:
Acceptance of test is based on the following criteria:
• Concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
• Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
• Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
• Adequate number of cells and concentrations are analyzable (according to OECD guidelines for testing of chemicals, No. 476).
• Criteria for selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.


Statistics:
Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance. The statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.25 µL/mL in the both presence of metabolic activation and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 5.5% to 91.74% and in the absence of metabolic activation, the RS values ranged from 4.67% to 95.33% at concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.50 µL/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Comparision with historical control data of spontaneous mutations frequencies of the solvent and positive controls was made.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.25 µL/mL in the both presence of metabolic activation and absence of metabolic activation. In the gene mutation test, there was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
Test item Methyl Eugenol was miscible in DMSO at 200 µL/mL. The precipitation and pH tested at concentrations up to 2 µL/mL. Post 3 hours of incubation, no change in pH and precipitation was observed at the concentrations tested at and up to 0.5 µL/mL. Precipitation observed at 2 µL/mL, moderate precipitation was observed at 1 µL/mL, slight precipitation was observed at 0.5 µL/mL. On the basis of the results 0.5 µL/mL was selected as the highest concentration for the initial cytotoxicity test.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

  SUMMARY OF GENE MUTATION TEST

Set No.

Treatment

Concentration (µL/mL)

Average Colony Count ± SD

Cloning Efficiency in selective media

Cloning Efficiency in non-selective media

Total number of Mutant Colonies/ 2×106cells

Mutant Frequency/ 2×106cells

Set 1 +S9

Vehicle Control (DMSO)

-

184.67

±

5.13

0.0000105

0.92

21

22.83

Methyl Eugenol

0.03125

176.00

±

6.56

0.0000105

0.88

21

23.86

0.0625

170.00

±

2.00

0.0000100

0.85

20

23.53

0.125

172.33

±

6.66

0.0000110

0.86

22

25.58

0.25

170.33

±

7.09

0.0000110

0.85

22

25.88

Benzo(a)pyrene                  (Positive Control)

3 µg/mL

174.67

±

4.51

0.0001120

0.87

224

257.47**

Set 2 -S9

Vehicle Control (DMSO)

-

186.67

±

10.41

0.0000105

0.93

21

22.58

Methyl Eugenol

0.03125

174.67

±

9.29

0.0000110

0.87

22

25.29

0.0625

181.33

±

4.04

0.0000115

0.91

23

25.27

0.125

175.67

±

7.09

0.0000115

0.88

23

26.14

0.25

176.67

±

14.05

0.0000110

0.88

22

25.00

4 Nitroquinoline -oxide      (Positive Control)

1 µg/mL

175.67

±

4.04

0.0001115

0.88

223

253.41**

+S9: with metabolic activation; -S9: without metabolic activation;                                                                                                                      

Note: Cloning efficiency = 200 cells plated for each replicate.  

 **: Statistically significant (p˂0.05). 

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non- selective medium.

                      HISTORICAL DATA

     Vehicle-DMSO

 

With Metabolic Activation

(3 to 6 hours)

Without Metabolic Activation

(3 to 6 hours)

Mean Data ofMutant Frequency/2x106Cells

24.51

25.43

Standard

Deviation

2.81

1.89

Margin of Error

1.95

1.31

Upper bound

26.46

26.74

Lower bound

22.56

24.12

 

     Positive Control-Benzo(a)pyreneand4- Nitroquinoline N-oxide

 

With Metabolic Activation

(3 to 6 hours)

[Benzo(a)pyrene]

Without Metabolic Activation

(3 to 6 hours)

[4 Nitroquinoline N-oxide]

Mean Data ofMutant Frequency/2x106Cells

261.94

264.60

Standard

Deviation

27.28

18.52

Margin of Error

17.82

12.10

Upper bound

279.76

276.70

Lower bound

244.12

252.50

 

Conclusions:
Based on the results obtained, the test item, Methyl Eugenol is considered as non-mutagenic at and up to the concentration of 0.25 µL/mL both in the presence and absence of metabolic activation under the tested laboratory conditions.
Executive summary:

The test item Methyl Eugenol was evaluated to identify gene mutations induced using genetic endpoints to measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT) using the gene mutation test in CHO AA8 cells.

The test item was found miscible in dimethyl sulphoxide (DMSO) at 200 µL/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 3 hours of incubation, no change in pH and precipitation was observed at the concentrations tested at and up to 0.5 µL/mL. Slight precipitation was observed at 0.5 µL/mL. On the basis of the results 0.5 µL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 µL/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the Relative Survival is greater than 10% at 0.25 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on the result 0.25 µL/mL was selected as highest concentration for gene mutation test.

The gene mutation test was conducted at concentrations of 0.03125, 0.0625, 0.125 and 0.25 µL/mL using DMSO as a vehicle in four plates / group in the presence and absence of metabolic activation (3 to 6 hours). Test item, resulted in mutant frequencies of 23.53 to 25.88 per 2×106 cells in the presence of metabolic activation with 22.83 per 2×106cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 25.00 to 26.14 per 2×106cells were observed with 22.58 per 2×106cells in the vehicle control. There was no statistically significant increase in the number of mutant colonies observed when compared with vehicle control at any of the tested concentrations. There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 22.61% to 93.91% and in the absence of metabolic activation the RS values ranged from 18.42% to 91.23% respectively.

Application of the positive control, 3 µg/mL of Benzo (a) pyrene, resulted in a RS value of 73.04% in the presence of metabolic activation, and a mutant frequency of 257.47per 2×106cells which was statistically significant when compared with the vehicle control. Treatment with the positive control, 1 µg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 71.05% in the absence of metabolic activation and a mutant frequencyof 253.41 per 2×106 cells and was statistically significant when compared with that of vehicle control.

                                                                                                                                   

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

AMES TEST

The objective of this study was to determine the potential of 4 -allylveratrole (Methyl Eugenol) and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium;TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli)strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).The study procedures were based on the OECD 471 guideline.

Methyl eugenol was soluble in DMSO at 50mg/mL on sonication, and was found to be stable in DMSO for 48 hours at room temperature at the fortification levels of 25 an 50 000 ug/mL.

In a preliminary toxicity test to select test doses for the mutation assay, the test item did not show toxicity to the tester strain up to 1600ug/plate as the intensity of the bacterial background lawn was comparable to the vehicle control plates. Similarly, the mean number of revertant colonies was more or less comparable to the vehicle control plates at these test doses. However, at and above 3200ug/plate the intensity of the bacterial background lawn was severily reduced associated with a severe reduction in the mean number of revertant colonies. Based o these observations, a highest dose of 2400ug/plate was tested in the mutation assay.

At the concentrations used, 24, 76, 240, 760, 2400 ug/ml, the test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously proving the proper functionality of the test.

In conclusion, based on the results of this study it is concluded that 4 -allylveratrole (Methyl Eugenol) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

CYTOGENICITY STUDY

Methyl Eugenol obtained fromYasho Industries Limited was evaluated for chromosomal aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473.Test item was soluble in dimethyl sulphoxide at 200 µL/mL.Precipitation test was conducted at 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 7 minutes of incubation, heavy precipitation was observed at 1 and 2 µL/mL, mild and moderate precipitation was observed at 0.25 and 0.5 µL/mL respectively. No precipitation was observed in any other concentrations tested up to0.0156 µL/mL. No change in pH was observed in any of the concentration tested hence, 0.5 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.25, 0.125, 0.0625 and 0.0312 µL/mL of test item.

In initial cytotoxicity test, the percentage reduction in Mitotic Index was in the range of 0.65 to 3.92 at 0.0312, 0.0625, 0.125, 0.25 and 0.5 µL/mL. As the percentage reduction in MI was not more than 45±5% at 0.5 µL/mL, same has been selected as the highest concentration for the chromosomal aberration test. Other concentrations tested will be 0.25 and 0.125 µL/mL. 

In the chromosomal aberration test, the cells were treated with Methyl Eugenol at the concentrations of 0.125, 0.25 and 0.5 µL/mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.The observed mean percent aberrated cells at 0.125, 0.25 and 0.5 µL/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.33 and 0.67 respectively. Similarly, the observed mean percent aberrated cells at 0.125, 0.25 and 0.5 µL/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.33 and 1.0 respectively.

The observed mean percent aberrated cells at 0.125, 0.25 and 0.5 µL/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.0, 1.33 and 1.33 respectively.Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.3% of aberrated cells which was statistically significant compared to the vehicle control (0.67%). The reduction in mitotic index was 6.30% when compared with the vehicle control for short term treatment.

Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.67% of aberrated cells which was statistically significant to the vehicle control (0.67%). The reduction in mitotic index observed was 7.54% when compared with the vehicle control for short term treatment.Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (0.67%). The reduction in mitotic index observed was 8.70% when compared with the vehicle control for long term treatment.

MUTAGENICITY STUDY

The test item Methyl Eugenol was evaluated to identify gene mutations induced using genetic endpoints to measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT) using the gene mutation test in CHO AA8 cells.

The test item was found miscible in dimethyl sulphoxide (DMSO) at 200 µL/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 3 hours of incubation, no change in pH and precipitation was observed at the concentrations tested at and up to 0.5 µL/mL. Slight precipitation was observed at 0.5 µL/mL. On the basis of the results 0.5 µL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 µL/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the Relative Survival is greater than 10% at 0.25 µL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on the result 0.25 µL/mL was selected as highest concentration for gene mutation test.

The gene mutation test was conducted at concentrations of 0.03125, 0.0625, 0.125 and 0.25 µL/mL using DMSO as a vehicle in four plates / group in the presence and absence of metabolic activation (3 to 6 hours).Test item, resulted in mutant frequencies of 23.53 to 25.88 per 2×106cells in the presence of metabolic activation with 22.83 per 2×106 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 25.00 to 26.14per 2×106cells were observed with 22.58 per 2×106 cells in the vehicle control. There was no statistically significant increase in the number of mutant colonies observed when compared with vehicle control at any of the tested concentrations. There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 22.61% to 93.91% and in the absence of metabolic activation the RS values ranged from 18.42% to 91.23% respectively.

Application of the positive control, 3 µg/mL of Benzo (a) pyrene, resulted in a RS value of 73.04% in the presence of metabolic activation, and a mutant frequencyof 257.47per 2×106cellswhich wasstatistically significant when compared with the vehicle control.Treatment with the positive control, 1 µg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 71.05% in the absence of metabolic activation and a mutant frequencyof253.41per 2×106cellsand wasstatistically significant when compared with that of vehicle control.

Justification for classification or non-classification

No classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.