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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria. Weight of evidence. Experimental study following method equivalent to OECD guideline 471. Methyl eugenol was not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation.

In vitro gene mutation study in bacteria. Weight of evidence. Experimental study following method equivalent to OECD guideline 471. Methyl eugenol was not mutagenic with Salmonella typhimurium strains TA100, TA98, 1535, TA1537, TA1538 and Escherichia coli strain WP2 uvrA with and without metabolic activation.

In vitro DNA repair study in bacteria. Weight of evidence. Methyl eugenol was positive in the DNA-repair test (Rec assay) with B. subtilis strains M45 Rec- and H17 Rec+ without metabolic activation.

In vitro mammalian sister chromatid exchange. Weight of evidence: Experimental study following method equivalent to OECD guideline 479. Methyleugenol was positive in the in vitro SCE test with CHO cells in the presence of S9 metabolic activation.

In vitro mammalian chromosome aberration. Weight of evidence: Experimental study following method equivalent to OECD guideline 473. Methyleugenol was negative for chromosome aberrations with and without metabolic activation.

In vitro mammalian comet assay. Weight of evidence: Methyleugenol was positive in an in vitro comet assay performed in cultured Chinese hamster V79 cells.

In vitro mammalian micronuleus test. Weight of evidence: Methyleugenol was negative in an in vitro micronucleus test performed in cultured Chinese hamster V79 cells.

In vitro unscheduled DNA synthesis in hepatocytes. Weight of evidence: Methyleugenol induced a positive response in two in vitro unscheduled DNA synthesis assays performed with rat and mice hepatocytes.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
This information was not provided
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor 1254-induced male Sprague-Dawley rats (RLI) or male Syrian hamsters (HLI).
Test concentrations with justification for top dose:
Main test (2 experiments):
Trial 1: 0 (solvent control), 3, 10, 33, 100, 333 and 666 μg/plate with S9 mix, and 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate without S9 mix.
Trial 2: 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate with and without S9 mix.
The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: insoluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine: -S9: TA98; 2-aminoanthracene: +S9: all strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation assay

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

SELECTION AGENT (mutation assays): the lack of amino-acid (Histidine) in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2 independent tests with 3 plates/dose/strain.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was evidenced by one or more of the following phenomena: appearance of his- pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.




Evaluation criteria:
A positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
No statistical methods were used.

Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate (-S9) and 666 μg/plate (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate (-S9 and +S9 HLI, trial1) and toxic at 666 μg/plate (+S9)
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:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate and 666 μg/plate (+S9)
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:
cytotoxicity
Remarks:
Slight toxicity at 333 μg/plate (-S9) and 666 μg/plate (+S9 RLI) and toxic at 666 μg/plate (+S9 HLI)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary range-finding study was conducted with TA100 in the presence and absence of S9.

Table 1: Mutagenicity of Methyleugenol in Salmonella typhimurium (a)

Strain

Dose [µg/plate]

Revertants/plate b

-S9

+10% hamster S9

+10% rat S9

Trial 1

Trial 2

Trial 1

Trial 2

Trial 1

Trial 2

TA100

0

120 ± 3.2

90 ± 6.4

106 ± 4.4

103 ± 8.7

111 ± 7.8

98 ± 8.1

3

101 ± 0.0

86 ± 3.5

 

90 ± 8.0

 

95 ± 5.3

10

100 ± 6.4

93 ± 4.0

106 ± 4.6

89 ± 6.1

93 ± 3.3

94 ± 2.7

33

114 ± 8.3

93 ± 10.7

109 ± 4.6

90 ± 6.8

109 ± 4.4

92 ± 4.3

100

105 ± 11.6

96 ± 2.7

116 ± 7.0

80 ± 14.4

110 ± 0.7

91 ± 7.6

333

29 ± 8.2c

16 ± 13.1c

99 ± 4.2

78 ± 1.0

95 ± 9.4

97 ± 2.6

666

 

 

38 ± 38.0c

 

0 ± 0.0c

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control d

358 ± 7.0

388 ± 4.3

1469 ± 61.2

1111 ± 49.2

534 ± 46.0

351 ± 22.9

TA1535

0

24 ± 2.6

20 ± 3.5

10 ± 2.4

12 ± 2.1

10 ± 2.2

9 ± 0.6

3

37 ± 0.6

20 ± 2.3

 

8 ± 0.9

 

6 ± 0.0

10

33 ± 2.0

21 ± 2.3

13 ± 3.5

8 ± 2.3

8 ± 0.9

7 ± 0.3

33

37 ± 7.5

22 ± 3.3

14 ± 1.5

9 ± 2.8

9 ± 0.0

9 ± 2.6

100

32 ± 3.5

26 ± 2.7

11 ± 2.2

10 ± 3.7

6 ± 1.5

7 ± 1.0

333

5 ± 5.0c

2 ± 0.7c

10 ± 2.1

9 ± 2.3

4 ± 0.3

8 ± 0.9

666

 

 

4 ± 2.6c

 

0 ± 0.0c

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

375 ± 15.5

410 ± 15.2

381 ± 7.9

369 ± 20.8

146 ± 2.8

168 ± 21.2

TA1537

0

5 ± 0.6

5 ± 0.3

6 ± 1.0

5 ± 0.3

7 ± 0.7

6 ± 0.9

3

6 ± 1.5

3 ± 0.9

 

9 ± 1.5

 

8 ± 2.1

10

4 ± 1.2

3 ± 0.9

5 ± 1.2

6 ± 0.9

5 ± 0.9

4 ± 1.0

33

5 ± 0.3

4 ± 1.2

5 ± 1.5

5 ± 1.2

4 ± 0.3

9 ± 1.5

100

4 ± 1.5

4 ± 0.6

6 ± 1.5

5 ± 1.0

7 ± 0.0

7 ± 1.2

333

0 ± 0.0c

3 ± 0.03c

3 ± 1.2

4 ± 1.3

6 ± 0.3

5 ± 2.2

666

 

 

Toxic

 

0 ± 0.0c

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

184 ± 4.7

521 ± 48.1

424 ± 75.9

426 ± 15.5

157 ± 8.7

124 ± 11.9

TA98

0

15 ± 0.6

16 ± 1.7

25 ± 2.2

31 ± 3.7

17 ± 3.6

20 ± 4.1

3

15 ± 0.7

13 ± 2.2

 

31 ± 4.0

 

27 ± 0.9

10

15 ± 1.0

14 ± 0.9

27 ± 3.5

28 ± 2.3

24 ± 2.7

23 ± 2.6

33

15 ± 0.9

13 ± 1.8

31 ± 5.8

26 ± 1.2

29 ± 2.0

20 ± 2.3

100

14 ± 3.5

13 ± 0.9

28 ± 0.7

29 ± 6.0

20 ± 3.5

29 ± 5.5

333

0 ± 0.0c

3 ± 3.0c

15 ± 7.7c

21 ± 2.7

29 ± 5.0

19 ± 0.3

666

 

 

Toxic

 

Toxic

 

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

626 ± 20.6

412 ± 8.2

1274 ± 85.7

1362 ± 55.5

473 ± 34.3

444 ± 76.4

a Study performed at SRI International. The detailed protocol is presented by Mortelmans et al. (1986). 0 μg/plate dose was the solvent control.

b Revertants are presented as mean ± the standard error from three plates.

c Slight toxicity

d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 4-nitro-o-phenylenediamine (TA98), and 9-aminoacridine (TA1537). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Conclusions:
Methyl eugenol was not mutagenic with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation.

Executive summary:

In a bacterial reverse mutation assay following a method similar to OECD guideline 471, the test item methyl eugenol diluted in Dimethylsulfoxide (DMSO) was tested with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation (S9) using the preincubation method. Based on results of a preliminary assay, for the main test two independent experiments and 3 replicates of each were conducted with doses of 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate with and without S9 mix. Also a dose of 666 μg/plate with S9 mix was used in the first experiment. Concurrent solvent and positive controls were included in every experiment and their responses were considered valid. The test item did not show mutagenic activity in any of the bacterial strains tested.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium and tryptophan-requiring gene in Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from male Sprague-Dawley rat liver
Test concentrations with justification for top dose:
0, 30, 60, 120 and 300 μg/plate.
The highest dose used showed killing on bacteria based on previous examination with dissecting microscope.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-aminoanthracene (2-AA):
Remarks:
Note: quantities of positive controls in µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation method (-S9) and preincubation method described by Yahagi et al. (1975) (+S9).

DURATION
- Exposure duration: 48-72 h (Not specified for the test substance)

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants (revertant bacteria) can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3-5 (Not specified for the test substance)

DETERMINATION OF CYTOTOXICITY
- Method: degree of survival of bacteria was observed using a dissecting microscope.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 300 μg/plate)
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:
cytotoxicity
Remarks:
(at 300 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 300 μg/plate)
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:
cytotoxicity
Remarks:
(at 300 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 300 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 300 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Results of mutagenicity tests in S. typhimurium and in E. coli WITHOUT S9

Sample

Dose a
( µg/plate)

Revertants/plate (mean ± SD)

Salmonella typhimurium strains

Escherichia coli

TA100

TA1535

TA98

TA1537

TA1538

WP2 uvra

DMSO

50 µL

99 ± 6

8 ± 2

24 ± 4

4 ± 2

15± 3

55 ± 6

Positive control

b

769± 54

266 ± 34

660 ± 30

822± 88

301 ± 33

452 ± 68

Methyl eugenol

30

104± 3

11 ± 4

23 ± 7

4 ± 2

13 ± 3

54 ± 7

60

108± 12

11 ± 4

25 ± 4

5 ± 1

15 ± 4

54 ± 5

120

110± 12

11 ± 3

31 ± 2

5 ± 2

16 ± 3

53 ± 7

300

73± 9

8 ± 2

20 ± 3

2 ± 1

11 ± 3

35 ± 1

a) All test compounds showed killing on bacteria at the highest doses used (examined with a dissecting microscope).

b) TA100 (AF-2, 0.02); TA1535 (Na N3, 0.5); TA98 (AF-2, 0.1); TA1537 (9-AAc, 80); TA1538 (2 -NF, 2); WP2 uvrA (AF-2, 0.01).

Table 2: Results of mutagenicity tests in S. typhimurium and in E. coli WITH S9

Sample

Dose a
( µg/plate)

Revertants/plate (mean ± SD)

Salmonella typhimurium strains

Escherichia coli

TA100

TA1535

TA98

TA1537

TA1538

WP2 uvra

DMSO

50 µL

106 ± 9

10 ± 3

32 ± 4

7 ± 2

21± 4

59 ± 5

Positive control

b

431 ± 92

158 ± 45

180 ± 7

125 ± 37

154 ± 17

753 ± 95

Methyl eugenol

30

98 ± 6

8 ± 2

32 ± 3

4 ± 2

25 ± 5

65 ± 9

60

111 ± 10

9 ± 3

33 ± 5

5 ± 2

24 ± 7

63± 5

120

105 ± 8

8 ± 3

30 ± 3

5 ± 1

20 ± 6

69 ± 8

300

91 ± 12

7 ± 2

29 ± 2

5 ± 2

18 ± 7

63 ± 0

a) All test compounds showed killing on bacteria at the highest doses used (examined with a dissecting microscope).

b) TA100 (BP, 5); TA1535 (2-AA, 5); TA98 (BP, 5); TA1537 (2-AA, 5); TA1538 (2-AA, 2); WP2 uvrA (2-AA, 80).

Conclusions:
Methyl eugenol did not show any mutagenic effect in bacteria under test conditions with and without metabolic activation.

Executive summary:

Methyl eugenol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535, TA1537, TA1538 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to Ames method. Based on growth inhibition examined in a preliminary test, the concentrations used in the main test were: 0, 30, 60, 120 and 300 μg/plate for all strains with and without metabolic activation. The assay without S9 was performed by the plate incorporation method and the assay with S9 was conducted by the pre-incubation method described by Yahagi et al. (1975). DMSO was used as solvent. Negative (solvent) and positive controls were used in the test and showed expected results. Under test conditions, methyl eugenol was found not mutagenic in all strains tested with and without metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: DNA-repair test (Rec assay) with Bacillus subtilis performed as described by Kada et al. (1980). Comparison of the repair-competent (H17 Rec +) and repair-deficient (M45 Rec-) strains for zones of killing.
- Short description of test conditions: Spores of H17 Rec + or M45 Rec- were poured onto plates with 10 ml of molten nutrient agar to prepare spore-agar plates. Test samples were dissolved in ethanol immediately before use. Sample solutions (20 µL) were pipetted onto sterile filter-paper disks (8 mm in diameter), which were carefully placed on the spore-agar plate.
- Parameters analysed / observed: After incubation of the plate for 20-24 h at 37ºC, the zones of killing (diameter of growth inhibition zone - diameter of disk) with both strains (Rec + and Rec -) were measured and the difference between them was taken as the rec effect.
GLP compliance:
no
Type of assay:
Bacillus subtilis recombination assay
Target gene:
Not applicable
Species / strain / cell type:
bacteria, other: Bacillus subtilis strains H17 Rec+ and M45 Rec−
Remarks:
(bacteria donated by Dr. T. Kada, National Institute of Genetics, Mishima, Shizuoka)
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from PCB-treated male Sprague-Dawley rats according to Ames et al. (1975).
Test concentrations with justification for top dose:
1.0 mg/disk
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(ethanol)
True negative controls:
yes
Remarks:
(chloramphenicol)
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: Sample solutions (20 µL) were pipetted onto sterile filter-paper disks (8 mm in diameter), which were carefully placed on the spore-agar plate.

DURATION
- Exposure duration: 20-24 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 2x105 spores per plate (number lower than 2x106 stated by Kada et al. (1980) in order to increase sensitivity).
Evaluation criteria:
Difference between zones of killing of each strain (Rec + and Rec -) greater than 4 mm was taken as evidence of preferential killing of Rec- cells.
Key result
Species / strain:
bacteria, other: Bacillus subtilis strains H17 Rec+ and M45 Rec−
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
bacteria, other: Bacillus subtilis strains H17 Rec+ and M45 Rec−
Metabolic activation:
with
Remarks on result:
not determinable
Remarks:
(DNA-repair test with S9 were not successful)

Table 1. Results of DNA-repair test with B. subtilis strains

Compound

Dose (mg/disk)

Zone of killing (rmm) a

 

M45 Rec-

H17 Rec+

Difference b
(Rec- -
 Rec +)

Controls

 

 

 

 

Ethanol (solvent control)

20.0

0.0

0.0

0.0

Chloramphenicol (negative control)

2.5 x 10-3

14.0

14.5

-0.5

Mitomycin C (positive control)

2.5 x 10-5

16.2

3.6

12.6

Test compound

 

 

 

 

Methyl eugenol

1.0

16.3

9.8

6.5

a Mean values from 3 independent Expts.

b Difference greater than 4 mm was taken as evidence of preferential killing of Rec- cells.

Conclusions:
Methyl eugenol was positive in the DNA-repair test (Rec assay) with B. subtilis strains M45 Rec- and H17 Rec+ without metabolic activation.
Executive summary:

Methyl eugenol was tested in the DNA-repair test (Rec assay) with Bacillus subtilis strains M45 Rec- and H17 Rec+, performed as described by Kada et al. (1980), with and without metabolic activation (S9). Test item dissolved in ethanol was pipetted onto sterile filter-paper disk at 0.1 mg test item/disk and placed on the previously prepared spore-agar plate. After incubation of the plate for 20-24 h at 37°C, the zones of killing (diameter of growth inhibition zone - diameter of disk) with both strains (Rec + and Rec -) were measured and the difference between them was taken as the rec effect. Difference of the mean values of 3 replicates greater than 4 mm was taken as evidence of preferential killing of Rec- cells and thus of positive response. Methyl eugenol was found positive in this Rec assay without metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
not specified
Remarks:
(not reported)
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy’s 5A medium with 10% fetal calf serum, L-glutamine, and antibiotics.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
- Without S9: 5, 17, 50, 167 µg/mL
- With S9:17, 50, 167 μg/mL (Trial 1) and 50, 167, 250 μg/mL (Trial 2).

The doses tested were limited by toxicity to 233 μg/mL.
Vehicle / solvent:
- Solvents used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 26 hours (-S9) and 2 hours (+S9).
- Expression time (cells in growth medium): 28 h (with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 28 h (with and without S9)

SPINDLE INHIBITOR (cytogenetic assays): colcemid (2 hours with and without S9)

STAIN (for cytogenetic assays): Hoechst 33258 and Giemsa.

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After incubation, cells were harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50



Evaluation criteria:
An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses resulted in a determination that the trial was positive.
Statistics:
Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points (Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses resulted in a determination that the trial was positive. A statistically significant trend (P<0.005) in the absence of any responses reaching 20% above background led to a call of equivocal.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 233 μg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 233 μg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Result obtained in the 2 trials

Table 1: Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by Methyleugenol a

Compound

Concentration
(µg/mL)

Total Cells Scored

Total
Chromosomes

Total SCEs

SCEs/ Chromosome

SCEs/ Cell

Hrs in BrdU

Relative Change of SCEs/ Chromosomeb

-S9

 

 

 

 

 

 

 

 

Summary: Negative

 

 

 

 

 

 

 

 

Dimethylsulfoxidec

 

50

1,044

392

0.37

7.8

26.0

 

Mitomycin-Cd

0.001

50

1,046

549

0.52

11.0

26.0

39.78

 

0.004

10

210

182

0.86

18.2

26.0

130.82

Methyleugenol

5

50

1,046

373

0.35

7.5

26.0

-5.03

 

17

50

1,039

405

0.38

8.1

26.0

3.81

 

50

50

1,044

448

0.42

9.0

26.0

14.29

 

167

0

 

 

 

 

 

 

 

 

 

 

 

P=0.013e

 

 

 

+S9

 

 

 

 

 

 

 

 

Trial 1

 

 

 

 

 

 

 

 

Summary: Positive

 

 

 

 

 

 

 

 

Dimethylsulfoxide

 

50

1,047

359

0.34

7.2

26.0

 

Cyclophosphamided

0.125

50

1,048

675

0.64

13.5

26.0

87.84

 

0.500

10

208

204

0.98

20.4

26.0

186.04

Methyleugenol

17

50

1,048

469

0.44

9.4

26.0

30.52*

 

50

50

1,045

421

0.40

8.4

26.0

17.50*

 

167

50

1,044

607

0.58

12.1

26.0

69.57*

 

500

0

 

 

 

 

 

 

 

 

 

 

 

P<=0.000

 

 

 

Trial 2

 

 

 

 

 

 

 

 

Summary: Positive

 

 

 

 

 

 

 

 

Dimethylsulfoxide

 

50

1,050

398

0.37

8.0

26.0

 

Cyclophosphamide

0.125

50

1,046

588

0.56

11.8

26.0

48.31

 

0.500

10

208

207

0.99

20.7

26.0

162.56

Methyleugenol

50

50

1,047

430

0.41

8.6

26.0

8.35

 

167

50

1,047

477

0.45

9.5

26.0

20.19*

 

250

50

1,048

548

0.52

11.0

26.0

37.95*

 

 

 

 

 

P<=0.000

 

 

 

* Positive response (P≥20% increase over solvent control)

a Study was performed at Sitek Research Laboratories. The detailed protocol is presented by Galloway et al. (1987). SCE=sister chromatid exchange; BrdU=bromodeoxyuridine

b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells

c Solvent control

d Positive control

e Significance of SCEs/chromosome tested by the linear regression trend test versus log of the dose

Conclusions:
Methyleugenol was positive in the in vitro SCE test with CHO cells in the presence of S9 activation enzymes.

Executive summary:

Methyleugenol was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges (SCEs) by method reported by Galloway et al. (1987) and following a protocol similar to OECD TG 479. The test material dissolved in DMSO was tested in the presence and absence of metabolic activation in concentrations of 5, 17, 50 and 167 µg/mL (-S9); 17, 50 and 167 μg/mL (+S9, trial 1) and 50, 167 and 250 μg/mL (+S9, trial 2). The highest dose was limited by toxicity. Concurrent solvent control (DMSO) and positive controls (mytomicin-C without S9 and Cyclophosphamide with S9) were also tested. After total incubation of 28 h and exposure times to test material of 26 h without S9 and 2 h with S9, cells were harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. Colcemid (2 hours exposure) was used as spindle inhibitor with and without S9. 50 second-division metaphase cells were scored for frequency of SCEs/cell from each dose level. An SCE frequency 20% above the concurrent solvent control value at two or more doses was considered as a positive response. Methyleugenol induced SCEs at least at 2 doses in each of the two trials conducted with S9 while no significant increase in SCEs was observed without S9 at any dose tested. Thus, under these test conditions, methyleugenol was considered positive in the in vitro SCE test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
only 200 well-spread methaphase cells per concentration were scored.
GLP compliance:
not specified
Remarks:
(not reported)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a medium with 10 % foetal calf serum, L-glutamine and antibiotics.

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
50, 108 and 233 µg/mL.
The doses tested were limited by toxicity to 233 μg/mL.
Vehicle / solvent:
- Solvents used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 14.7 hours (-S9) and 2 hours (+S9).
- Fixation time (start of exposure up to fixation or harvest of cells): 16.7 hours (-S9) and 12.5 hours (+S9).

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After incubation, cells were harvested by mitotic shake-off, fixed, and stained with Giemsa.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200

Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). For a single trial, a statistically significant (P≤0.05) difference for one dose point and a significant trend (P≤0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call (Galloway et al., 1987).
Statistics:
To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P≤00.05) difference for one dose point and a significant trend (P≤00.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 233 μg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Methyleugenol (a)

Compound

Concentration (µg/mL)

Total cells scored

Number of Aberrations

Aberrations /cell

Cells with Aberrations (%)

-S9

 

 

 

 

 

Harvest time: 16.7 hours

 

 

 

 

 

Summary : negative

 

 

 

 

 

Dimethylsulfoxideb

 

200

1

0.01

0.5

Mitomycin-Cc

0.4

25

30

1.2

56.0

Methyleugenol

50

200

2

0.01

1.0

 

108

200

2

0.01

1.0

 

233

200

0

0.00

0.0

 

500

0

 

 

 

 

 

 

 

 

P=0.726d

+S9

 

 

 

 

 

Harvest time: 12.5 hours

 

 

 

 

 

Summary : negative

 

 

 

 

 

Dimethylsulfoxide

 

200

3

0.02

1.5

Mitomycin-C

20

25

15

0.60

40.0

Methyleugenol

50

200

3

0.02

1.5

 

108

200

8

0.04

4.0

 

233

200

10

0.05

4.5

 

500

0

 

 

P=0.015

a Study was performed at Sitek Research Laboratories. The detailed protocol is presented by Galloway et al. (1987).

b Solvent control

c Positive control

d Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

Conclusions:
Methyleugenol was negative for chromosome aberrations with and without metabolic activation.

Executive summary:

Methyleugenol was tested in cultured Chinese hamster ovary (CHO) cells for chromosomal aberrations by method reported by Galloway et al. (1987) and following a protocol similar to OECD TG 473. The test material disssolved in DMSO was tested in the presence and absence of metabolic activation in concentrations of 50, 108 and 233 µg/mL. The highest dose was limited by toxicity. Concurrent solvent control (DMSO) and positive controls (mytomicin-C without S9 and Cyclophosphamide with S9) were also tested. After total incubation of 16.7 h without S9 and 12.5 h with S9 and exposure times to test material of 14.5 h without S9 and 2 h with S9, cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. Colcemid (2 hours exposure) was used as spindle inhibitor with and without S9. 200 first-division metaphase cells were scored at each dose level. No significant induction of chromosomal aberrations occurred following incubation with methyleugenol in either the presence or the absence of S9. Thus, under these test conditions, methyleugenol was considered negative in the chromosomal aberration test.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Single cell gel electrophoresis (comet assay), performed according to the method of Gedik et al. (C. M. Gedik, S. G. Wood and A. R. Collins, Free Radical Res., 1998, 29, 609–615).

GLP compliance:
not specified
Remarks:
(not reported)
Type of assay:
comet assay
Target gene:
No applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Deutsche Sammlung für Mikroorganismen und Zellkultur (DSMZ), Braunschweig, Germany.
- Suitability of cells: Since parental V79 cells are devoid of Cytochrome P450 (CYP) activity, the genotoxicity results in the absence of the S9 mix could be due to either the direct acting activity of the test compound or possibly through the formation of reactive oxygen species. Therefore this test used in addition the DNA repair enzyme fpg, which primarily detects 8-oxo-7,8-dihydroguanine, to enhance mechanistic information to potential oxidative DNA damage.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco’s modified Eagle’s médium (DMEM) low glucose (1g L-1) (PAA, Coelbe, Austria). Cell culture medium was supplemented with 10% fetal calf serum (FCS; PAA, Coelbe, Austria), 1% penicillin/streptomycin (Pen/Strep, InvitrogenTM Life Technologies, Karlsruhe, Germany).
Cells were cultured at 37ºC in a water-saturated atmosphere containing 5% CO2.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
5, 10, 25, 50, 75 and 100 µM.
No growth inhibitory properties were observed up to 100 µM and 72 h of incubation using the sulforhodamine B (SRB) assay.
Vehicle / solvent:
- Solvents used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO, 0.5% (v/v))
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: menadione (10 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

-Cell density at seeding: 1 x 106 or 500,000 in 5 mL medium containing 10% FCS.

DURATION
-Preincubation period: V79 cells were spread into Petri dishes (6 cm diameter) and allowed to grow for 24 h prior to treatment.
- Exposure duration: 1 h and 24 h

STAIN (for cytogenetic assays): ethidium bromide (40 µL per coverslip, 20 µg mL-1)

NUMBER OF REPLICATIONS: 3 independent experiments each performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Aliquots (70 µl = 70 000 cells) were centrifuged (5 min, 200 x g). The resulting cell pellet was resuspended in 65 µL low melting agarose and distributed onto a frosted glass microscope slide, precoated with a layer of normal melting agarose. The slides were coverslipped and kept at 4ºC for 10 min to allow solidification of the agarose.
After removing the cover glass, slides were immersed for 1 h at 4ºC in lysis solution (89 mL lysis stock solution; 2.5 mM sodium chloride, 100 mM EDTA, 10 mM tris, 1% (w/v) N-laurylsarcosin sodium salt; 1 mL Triton-X-100, 10 mL DMSO).
For the additional detection of oxidative DNA damage, slides were washed three times in enzyme buffer (40 mM HEPES pH 8.0, 0.1 M potassium chloride, 0.5 mM EDTA, 0.2 mg mL-1 bovine serum albumin), covered with 50 µL of either enzyme buffer or fpg enzyme and incubated for 30 min at 37ºC.
Subsequently, DNA was allowed to unwind (300 mM NaOH, 1 mM EDTA, pH 13.5, 20 min, 4ºC) followed by horizontal gel electrophoresis at 4ºC for 20 min (25 V, 300 mA). Thereafter, the slides were washed three times with 0.4 M Tris-HCl, pH 7.5 and stained with ethidium bromide.

NUMBER OF CELLS EVALUATED: 100 individual cells per concentration (50 per slide) in each experiment.

DETERMINATION OF CYTOTOXICITY:
In parallel to the comet assay, viability of the cells was determined by trypan blue exclusion.


Evaluation criteria:
Fluorescence microscopy was performed with a Zeiss Axioskop 50/AC (λex = 546 ± 12 nm; λem ≥ 590 nm). Slides were subjected to computer-aided image analysis (Comet Assay IV System, Perceptive Instruments, Suffolk, Great Britain), scoring 50 images per slide randomly picked from each electrophoresis. For each concentration of drug two slides were independently processed and analyzed. The results were parameterized with respect to tail intensity (intensity of the DNA in the comet tail calculated as percentage of overall DNA intensity in the respective cell).
Statistics:
Differences between treatments were analyzed statistically by using Student’s t-test for unpaired data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

No additional DNA strand breaks were detected following fpg treatment. These findings suggest that methyleugenol does not induce oxidative DNA damage in the V79 cell line.

Conclusions:
Methyleugenol was positive in an in vitro comet assay performed in cultured Chinese hamster V79 cells.



Executive summary:

In an in vitro comet assay performed according to the method of Gedik et al (1998), cultured Chinese hamster V79 cells were exposed to methyleugenol dissolved in DMSO with and without enzyme fpg in order to evaluate also the potential oxidative DNA damage. V79 cells were treated for 1 h or 24 h with the solvent control (DMSO, 0.5% (v/v)), positive control (10 µM menadione) or the test compound in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. The concentrations used in the test were 5, 10, 25, 50, 75 and 100 µM. 3 independent experiments were performed in duplicate. 100 individual cells per concentration (50 per slide) were processed and analysed in each experiment. Methyleugenol significantly induced DNA damage at concentrations >10 µM after 1 h of incubation without an increase in efficacy after 24 h. No additional DNA strand breaks were detected following fpg treatment meaning that methyleugenol does not induce oxidative DNA damage. Thus, under these test conditions, methyleugenol was found positive in an in vitro comet assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
not specified
Remarks:
(not reported)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
No applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Deutsche Sammlung für Mikroorganismen und Zellkultur (DSMZ), Braunschweig, Germany.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco’s modified Eagle’s médium (DMEM) low glucose (1g L-1) (PAA, Coelbe, Austria). Cell culture medium was supplemented with 10% fetal calf serum (FCS; PAA, Coelbe, Austria), 1% penicillin/streptomycin (Pen/Strep, InvitrogenTM Life Technologies, Karlsruhe, Germany).
Cells were cultured at 37ºC in a water-saturated atmosphere containing 5% CO2.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
10, 25, 50 and 100 µM.
No growth inhibitory properties were observed up to 100 µM and 72 h of incubation using the sulforhodamine B (SRB) assay.
Vehicle / solvent:
- Solvents used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO, 0.5% (v/v))
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

-Cell density at seeding: 100000 or 50000 cells were seeded into Petri dishes (Φ60 mm).

DURATION
-Preincubation period: 24 h
- Exposure duration: 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 44 h (24 h exposure time and 20 h post-incubation)

STAIN (for cytogenetic assays): 0.1% 4’,6-diamidino-2-phenylindole-dihydrochloride (DAPI) solution

NUMBER OF REPLICATIONS: 3 independent experiments each performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After post-incubation, cells were fixed with 3 mL Carnoys fixation solution (methanol/acetic acid 3:1) per Petri dish at -20ºC for 1 h. For staining, fixation solution was removed and cells were incubated for 10 min with a 0.1%4’,6-diamidino-2-phenylindole-dihydrochloride (DAPI) solution at -20ºC, washed with ice-cold methanol and air-dried. Petri dishes were coverslipped and the dried cell layers were then covered with about 50 µL melted Kaiser’s glycerol gelatine and cover slips were fixed on top.

NUMBER OF CELLS EVALUATED: 2000 individual cells per concentration (1000 per Petri dish) in each experiment.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Fluorescence microscopy




Evaluation criteria:
Fluorescence microscopy was performed with a Zeiss Axioskop.
A positive response was determined if significant increase (Student’s t-test: p < 0.01 or p < 0.001) in micronuclei frequency was observed at any tested concentration compared to the solvent control.
Statistics:
Differences between treatments were analyzed statistically by using Student’s t-test for unpaired data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Additional information on results:
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see table below
- Indication whether binucleate or mononucleate where appropriate: N/A


Table 1. Number of micronucleated cells after treatment of V79 cells with various concentrations of methyleugenol or the positive control mitomycin C for 24 h and compound-free post-incubation for additional 20 h. (a)

 

Miconucleated cells per 1000 cells

Substance

 

10µM

25µM

50µM

100µM

DMSO (0.5%)

13.7 ± 4.4

 

 

 

 

Mitomycin C (0.6 mM)

90.2  ± 13.0***

 

 

 

 

Methyleugenol

 

13.5 ± 4.3

13.3 ± 1.8

12.5 ± 4.8

11.7 ± 4.3

(a) Data represent the mean ± SD of at least three independent experiments.

Conclusions:
Methyleugenol was negative in an in vitro micronucleus test performed in cultured Chinese hamster V79 cells.



Executive summary:

An in vitro Micronucleus test was performed with methyleugenol dissolved in DMSO following a method comparable to OECD TG 487. Chinese hamster V79 cells were previously incubated for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Cultured V79 cells were treated for 24 h and post-incubated for additional 20 h with the solvent control (DMSO, 0.5% (v/v)), positive control (0.6 µM Mitomycin C) or the test compound in concentrations of 10, 25, 50 and 100 µM. 3 independent experiments were performed in duplicate. 2000 individual cells per concentration (1000 per Petri dish) were processed and analysed using fluorescence microscopy in each experiment. No significant increase in micronuclei frequency was observed at any tested concentration compared to the solvent control. Thus, methyleugenol was negative in the in vitro micronucleus test.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
not specified
Remarks:
(not reported)
Type of assay:
other: unscheduled DNA synthesis (UDS)
Target gene:
No applicable
Species / strain / cell type:
hepatocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Fischer 344 rats (155--240 g), purchased from Harlan Olac Ltd (Bicester, Oxon, UK).
- Suitability of cells: The average viability of hepatocytes, as determined by trypan blue exclusion, was 85.7 ± 3% (range from 82 to 90%; n = 8).
- Sex, age and number of blood donors if applicable: Male rats

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 containing 10-4 M-hydrocortisone-21-sodium succinate, 10-6 M-insulin, 5% foetal bovine serum and 50 µg gentamicin/ml.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
1, 10, 100, 1000 and 10000 µM
Vehicle / solvent:
- Solvents used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-acetamidofluorene (2-AAF)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

- Cell density at seeding (if applicable): 6.7 x 106 viable cells/dish

DURATION
-Preincubation period: 4 h (RPMI medium) + 1 h (fresh medium + hydroxyurea).
- Exposure duration: 17 h

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Hepatocytes were seeded on to 90-mm tissue culture dishes in 9ml culture medium and incubated at 37°C (95% O2; 5% CO2). After 4hr the medium was replaced with 10 ml fresh medium containing 10mM-hydroxyurea. After incubation for a further hour, 10µL [3H]thymidine and 40 µL of the test compound in DMSO were added to each plate.

NUMBER OF CELLS EVALUATED:
After incubation for 17 h, the medium was removed from the dishes and the remaining cells were washed twice with ice-cold phosphate buffered saline containing 2 mM-thymidine. The cells were scraped off the plates using a rubber policeman, and cells from the duplicate plates were pooled. The hepatocytes were pelleted by centrifugation at 15g; the nuclei were isolated, and DNA was extracted according to the procedure of Althaus et al. (1982) and modified by Howes et al. (1986). Samples (2 x 0.5 ml) of the final solutions of DNA in 5% trichloroacetic acid were mixed with 3 ml scintillation fluid (Ecoscint; Manville, NJ, USA) and counted for radioactivity using a Packard Minaxi liquid scintillation spectrometer.

DNA assay: The DNA was assayed using a modification of the fluorometric method of Kissane and Robins (1958).

DETERMINATION OF CYTOTOXICITY
- Method: Cell viability was assessed by determining the leakage of cytoplasmic lactate dehydrogenase (LDH) from cells cultured overnight.





Evaluation criteria:
The UDS response following treatment of hepatocytes with the test item was determined by measuring the amount of [3H]thymidine incorporated into the nuclear DNA during the repair process. In a typical experiment, the amount of [3H]thymidine incorporated into the nuclear DNA of control cell cultures was 145 dpm/µg DNA. Results were expressed as a ratio of treated over control (DMSO) cultures. A ratio of 1.5 and above was considered to be a positive response.
Key result
Species / strain:
hepatocytes: Male Fischer 344 rats
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at concentrations ≥ 5x10-3 M)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Methyleugenol induced dose-related increases in UDS up to 2.7 times the control (see the graph below).
Conclusions:
Methyleugenol induced a positive response in an in vitro rat hepatocyte unscheduled DNA synthesis assay.


Executive summary:

Methyleugenol was tested in an in vitro rat hepatocyte unscheduled DNA synthesis assay following a method similar to the OECD Guideline 482. Primary hepatocytes were isolated from Male Fischer 344 rats. Cultures of hepatocytes were incubated in duplicate with the test material dissolved in DMSO for 17 hours at concentrations of 0 (solvent control), 1e-6, 1e-5, 1e-4, 1e-3 and 1e-2 mol/L. A response was considered positive when the result of the treated cultures was at least 1.5 times higher than the control (DMSO). The test material induced a marked and dose-dependent response in the test. A concentration of 5 x 10-3 M and above was toxic to the cells. Positive control induced an expected response which was considered valid. Based on these results, methyleugenol was demonstrated to be an UDS inducer in vitro.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
not specified
Remarks:
(not reported)
Type of assay:
other: unscheduled DNA synthesis (UDS)
Target gene:
No applicable
Species / strain / cell type:
hepatocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Male Fischer 344 rats (175–250 g) supplied by Hilltop Lab Animals, Scottdale, PA, and female B6C3F1 mice (21–27 g), by Harlan Sprague Dawley, Indianapolis, IN.
- Suitability of cells: Viability of the cells as determined by trypan blue exclusion exceeded 85%.
- Sex, age and number of blood donors if applicable: Male rats and female mice.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were suspended in William’s medium E supplemented with bovine calf serum (10% for rats, 1% for mice), and l-glutamine (200 mM) at 5 x 105 cells/ml.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.1, 0.5, 1, 5, 10, 50, 100 and 500 µM
Vehicle / solvent:
- Solvents used: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoflorene (2-AF)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

- Cell density at seeding (if applicable): 5 x 105 cells/ml

DURATION
- Exposure duration: 18 h

STAIN (for cytogenetic assays): hematoxylin and eosin stain

NUMBER OF REPLICATIONS: 3 for rat and 2 for mice (n=3-6 independent experiments)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Isolated hepatocytes were plated at 5 x105 cells per well on Thermanox coverslips and allowed to attach for 2 h. After treatment and determination of gross evidence of cytotoxicity, the cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting.

NUMBER OF CELLS EVALUATED: Twenty cells per slide, with three slides per dose level were counted when posible.

DETERMINATION OF CYTOTOXICITY
- Method: Cell viability was assessed by determining the leakage of cytoplasmic lactate dehydrogenase (LDH).

OTHER:
Modulation experiments were conducted by the addition to the wells containing methyleugenol, 2000 µM of cyclohexane oxide (CHO), an epoxide that competes for the detoxifying enzyme epoxide hydrolase, or 15 µM pentachlorophenol (PCP), an inhibitor of sulfation.




Evaluation criteria:
Results were quantified using an Artek colony counter. Slides were examined under oil immersion. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results were expressed as mean grain counts per nucleus. Positive grain counts indicated UDS. A compound was defined as positive if there was an increase in positive grain counts over two or more doses.


Key result
Species / strain:
hepatocytes: Male Fischer 344 rats and female B6C3F1 mice
Metabolic activation:
not applicable
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: methyleugenol showed a relatively flat cytotoxicity curve. For rat hepatocytes, LDH leakage remained at 50% from 600 to 3000 µM. Mouse hepatocytes showed a similar flat curve up to 1000 µM with an increase of 80–90%
LDH leakage at 3000 and 5000 µM.


In rat hepatocytes, methyleugenol caused UDS at concentrations between 10 and 500µM. Methyleugenol did not demonstrate a classic dose–response curve in rat hepatocytes, instead showing its highest incidence of UDS at 10µM with 43 mean net grain counts per nucleus. Mean net grain counts then gradually fell as the concentration of methyleugenol was increased. At higher concentrations, counts of about 20 mean grains per nucleus were observed (see graph below).

In mouse hepatocytes, methyleugenol caused UDS at concentrations between 5 and 500 µM, with peak UDS at 10 µM and with counts gradually falling as the concentration increased (see graph below).

UDS results with rat and mouse hepatocytes incubated with methyleugenol showed that the positive net grain counts seen with 10µM methyleugenol remained at similar levels in the presence of 2000µM CHO. This suggests that while the epoxide may have other biological consequences, implied by the increase in cytotoxicity, it does not contribute to the genotoxicity of methyleugenol However, in the presence of 15µM of PCP, mean net grain counts were reduced nearly to control levels. This finding strongly suggests that a pathway involving sulfation is important in the genotoxicity of methyleugenol.

Conclusions:
Methyleugenol induced a positive response in an in vitro unscheduled DNA synthesis assay tested with rat and mice hepatocytes.


Executive summary:

Methyleugenol was tested in an in vitro unscheduled DNA synthesis assay following a method similar to the OECD Guideline 482. Primary hepatocytes were isolated from male Fischer 344 rats and female B6C3F1 mice. Cultures of hepatocytes were incubated in triplicate (rats) or duplicate (mice) with the test material dissolved in DMSO for 18 hours at concentrations of 0 (solvent control), 0.1, 0.5, 1, 5, 10, 50, 100 and 500 µM. 60 hepatocytes per culture were evaluated for UDS per concentration. A positive difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count (from 3 neighbouring areas of cytoplasm in the same cell) indicated UDS. A positive response in the test was considered if there was an increase in positive grain counts over two or more doses. In rats, methyleugenol caused UDS at concentrations between 10 and 500 µM, without a classic dose–response curve. In mice, methyleugenol caused UDS at concentrations between 5 and 500 µM, with peak UDS at 10 µM and with counts gradually falling as the concentration increased. In the range of the tested concentrations methyleugenol was relatively non-cytotoxic. Positive control induced an expected response which was considered valid. Based on these results, methyleugenol was positive in the UDS assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In-vivo micronucleus test. Weight of evidence: Test method comparable to OECD 474. Methyleugenol was negative in the mammalian erythrocyte micronucleus test.

In vivo mammalian comet assay. Weight of evidence: Experimental study following method equivalent to OECD guideline 489. In a comet assay it was found that methyleugenol induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of methyleugenol in rodents.

In vivo mammalian DNA damage . Weight of evidence: Methyleugenol, administered in doses of 0.25 - 3 µmol, caused DNA adduct levels of 72.7 pmol/mg DNA. DNA adducts of at least 15 pmol/mg DNA are needed for statistically significant tumourigenesis potential.

In vivo mammalian β-catenin mutations. Weight of evidence: Hepatocellular adenomas and carcinomas were seen in mice treated with methyleugenol, most of which had beta-catenin mutations (67%) compared to 9% of mutations in spontaneous tumours seen in control animals.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 July 1988 - 13 October 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
(Food and Drug Administration (FDA) Good Laboratory Practice)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 6 weeks (female), 7 weeks (male)
- Weight at study initiation: 21.1-23.2 g (males) and 18.4-19.5 g (females)
- Housing: 1 per cage; Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly; Bedding: Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly; Racks: Stainless steel (Lab Products Inc., Maywood, NJ), changed and rotated every 2 weeks.
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly.
- Water (e.g. ad libitum): Tap water (Birmingham municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum.
- Acclimation period: 11 days (female) or 10 days (male)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day room fluorescent light
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% aqueous methylcellulose
- Justification for choice of solvent/vehicle: not reported.
- Concentration of test material in vehicle: 1, 3, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw /day
- Lot/batch no. (if required): 874544
- Purity: USP/FCC grade

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle was prepared by mixing methylcellulose and heated, deionized water with a magnetic stirrer to form a 0.5% solution, which was then cooled. Methyleugenol was slowly added to the 0.5% methylcellulose and mixed for 2 minutes using a homogenizer fitted with an anaerobic generator. The anaerobic generator was removed and the mixture was stirred with a magnetic stirrer for 1 hour.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days per week
Post exposure period:
None
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
(vehicle control)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
No

Tissues and cell types examined:
peripheral blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses were selected for the 14-week repeated dose toxicity study (NTP TR491, 2000) at the end of which peripheral blood samples were taken for this micronucleus study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Peripheral blood smears were obtained from B6C3F1 male and female mice after 14 weeks of treatment. Smears were immediately prepared and fixed in absolute methanol.

DETAILS OF SLIDE PREPARATION: The methanol-fixed slides were stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983) and coded.

METHOD OF ANALYSIS: Slides were scored at 630x or 1000x magnification by epifluorescence microscopy. Criteria for identification of MN were those of Schmid [1976] with the additional requirement that MN exhibit the fluorescence emission characteristic of the fluorescent stain used (orange with green [540nm] excitation with Hoechst/pyronin stain). Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated method described by Jauhar et al. [1988], in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Micronucleus frequency scores were based on 10,000 NCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 5,000 erythrocytes.
A set of fixed slides from mice maintained on 0.2% urethane in drinking water was available for random selection of three slides per micronucleus experiment. These slides from urethane-treated mice were stained, coded, and scored along with the experimental slides, thereby providing a positive control for staining and scoring.

Evaluation criteria:
The micronucleus results were tabulated as the mean frequency of micronucleated erythrocytes per 1000 cells per animal, plus or minus the standard error of the mean among animals within a treatment group. An individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single dose group was less than or equal to 0.025 divided by the number of dose groups.
Statistics:
The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group [Margolin and Risko, 1988; Margolin et al., 1990]. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an unadjusted one-tailed Pearson x2 test that incorporated the calculated variance inflation factor for the study.
In this test, an individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single dose group was less than or equal to 0.025 divided by the number of dose groups. A final call of positive for micronucleus induction was preferably based on reproducibly positive trials (as noted above). Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects. The percentage of PCEs among total erythrocytes was determined by an analysis of variance on ranks (classed by sex), and individual dosed groups were compared with the concurrent solvent control with a t-test on ranks.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated NCEs.
- Ratio of PCE/NCE (for Micronucleus assay): no alteration of the percentage of PCEs among total erythrocytes.

Table 1: Micronucleated Erythrocyte Frequencies in Peripheral Blood of B6C3F1 Mice from NTP Subchronic Toxicity Study (a,b)

Compound

Dose (mg/kg)

Number of Mice with Erythrocytes Scored

Micronucleated NCEs/ 1,000 NCEs

P valuec

PCEs (%)

Male

 

 

 

 

 

Methyleugenol

0

10

0.81+-0.10

 

1.40+-0.05

 

10

10

0.70+-0.10

0.8230

1.02+-0.06

 

30

10

0.78+-0.10

0.5935

1.51+-0.08

 

100

10

0.84+-0.05

0.4077

1.61+-0.11

 

300

10

0.63+-0.06

0.9364

1.18+-0.12

 

1000

10

0.65+-0.02

0.8992

1.16+-0.11

Trend testd

 

 

P=0.915

 

 

ANOVAe

 

 

 

 

P < 0.001

 

 

 

 

 

 

Female

 

 

 

 

 

Methyleugenol

0

9

0.46+-0.09

 

1.27+-0.15

 

10

10

0.45+-0.06

0.5555

1.15+-0.13

 

30

9

0.43+-0.06

0.6160

1.38+-0.13

 

100

10

0.54+-0.08

0.2151

1.41+-0.09

 

300

10

0.45+-0.08

0.5908

1.37+-0.10

 

1000

9

0.63+-0.08

0.0620

1.21+-0.13

Trend testd

 

 

P=0.027

 

 

ANOVAe

 

 

 

 

P = 0.599

a Values are mean ± SEM. Summary conclusions presented as (male/female).

b NCE, normochromatic erythrocytes; % PCE, percentage of polychromatic erythrocytes in 5,000 total erythrocytes.

c Comparison of treated group to control, x2-test, significant at P ≤ 0.025 per number of test chemical dose groups.

d One-tailed trend test, significant at P ≤ 0.025.

e Analysis of variance, significant at P ≤ 0.025.

Conclusions:
Methyleugenol was negative in the mammalian erythrocyte micronucleus test.

Executive summary:

An in-vivo micronucleus test was performed with methyleugenol following a method comparable to OECD TG 474. Groups of 10 male and 10 female B6C3F1 mice were administered test item dissolved in 0.5% methylcellulose in concentrations of 0, 10, 30, 100, 300, or 1,000 mg/kg bw by gavage 5 days per week during a 14-week repeated dose toxicity study. At the end of this study, peripheral blood samples were obtained from male and female mice and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Micronucleus frequency scores were based on 10,000 NCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 5,000 erythrocytes. Methyleugenol did not increase the frequency of micronucleated NCEs in peripheral blood and did not alter the percentage of PCEs among total erythrocytes (an indication of bone marrow toxicity). Thus, a negative result is obtained from the mammalian erythrocyte micronucleus test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
yes
Remarks:
In addition to the standard comet assay, enzyme-modified Comet assay was also conducted for detecting oxidative DNA damage.
GLP compliance:
not specified
Remarks:
(information not reported)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NCTR breeding colony (4 weeks of age)
- Housing: two per cage; cages were arranged in close proximity in order to minimize possible effects due to cage placement.
- Diet (e.g. ad libitum): NIH-31R Purina Rodent Chow (pellets), ad libitum.
- Water (e.g. ad libitum): Millipore-filtered drinking water, ad libitum.
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 55-65%.
- Photoperiod (hrs dark / hrs light): 12 light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: methylcellulose
- Purity: USP/FCC grade
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: MEG solutions were prepared in 0.5% aqueous methylcellulose.
Duration of treatment / exposure:
3 and 24 hours (initial experiment)
1, 3, 6 and 8 hours (second experiment)
Frequency of treatment:
Single exposure
Post exposure period:
No
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
solvent control
Dose / conc.:
400 mg/kg bw (total dose)
Remarks:
Initial experiment
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
Initial experiment
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
Second experiment
No. of animals per sex per dose:
Initial experiment: 3
Second experiment: 4
Control animals:
yes, concurrent vehicle
Positive control(s):
methylmethanesulfonate (MMS) dissolved in phosphate-buffered saline, pH 7.4 (PBS).
- Route of administration: gavage
- Doses / concentrations: 100 mg/kg bw
Tissues and cell types examined:
Initial experiment: liver, kidney, bladder, lung, and bone marrow
Second experiment: liver, bone marrow and bladder
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The choice of doses was based on previous studies that evaluated the genotoxicity of estragole, a derivative of MEG (Muller et al., 1994); in addition, Organization for Economic Cooperation and Development guidelines recommend 2000 mg/kg bw as the maximum acute dose for toxicological evaluations conducted in rodents (OECD, 2001).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were euthanised either 3 and 24 hours after treatment (selected based on general recommendation for conduting in vivo Comet assays), or 1, 3, 6 and 8 hr after treatment (selected to maximise the chances of detecting an early genotoxic response). The organs/tissues (liver, kidney, bladder, lung, and bone marrow) were aseptically removed and stored in cold tissue dissociation buffer for isolation of single cells. For all the samples, cell viability was measured by Trypan Blue dye exclusion and the % cell viability for tissues ranged from 75 to 95% irrespective of the treatment and tissue dissociation method used.

DETAILS OF SLIDE PREPARATION: 100 µL of the single-cell suspensions derived from different organs were mixed with 900 µL 1% low–melting-point agarose in PBS at 37ºC, and 200 µL of this suspension were applied to microscope slides previously coated with 1% agarose.
For detecting oxidative DNA damage, cells were embedded in agarose on microscope slides and stored in the lysis buffer as with the standard assay. After removing the slides from the lysis buffer, they were washed 3 x for 5 min each with an enzyme buffer. After the last wash, excess liquid was removed, and 200 µL of enzyme buffer alone, enzyme buffer containing formamidopyrimidine DNA glycosylase (Fpg) or enzyme buffer containing endonuclease III (Endo III) were applied to slides prepared from each treatment group. The slides were kept in a moist box and incubated at 37ºC for 45 min for Endo III and reference slides (enzyme buffer alone), 30 min for Fpg.

METHOD OF ANALYSIS: Two slides were scored from each tissue/treatment/ sampling time; 100 cells were selected randomly from each slide and scored using a system consisting of a Nikon 501 fluorescent microscope and Comet IV digital imaging software (Perceptive Instruments, Wiltshire, UK). Percent DNA (% DNA) in tail, defined as the fraction of DNA in the tail divided by the total amount of DNA associated with a cell multiplied by 100, was used as the parameter for DNA damage analysis.

In addition to comet assay, gene expression analysis was performed on liver samples from the MEG time-course experiment using the rat DNA damage and repair array (SABiosciences, Frederick, MD). Liver samples were used because liver is a major target organ for tumor induction by MEG in rats and because studies with [14C]-labeled MEG indicate that the liver is highly exposed immediately following oral dosing of rats with MEG (NTP, 2000) and was thus the most likely place to search for a detectable signal.
Evaluation criteria:
A P-value of less than 0.05 was considered statistically significant.
Statistics:
Oxidative DNA damage calculation was based on the method described in the protocol by Dusinska (2000). Briefly, the control gels (no enzyme treatment) provide an estimate of the background of DNA SB. The enzyme-treated gels reveal SB and oxidized bases (SB + OX). Assuming a linear dose-response while using % DNA in tail or other arbitrary units, subtraction of SB from SB + OX gives a measure of oxidized pyrimidines/altered purines.
DNA damage measured as % DNA in tail was analyzed as a function of dose or time by one-way ANOVA followed by the Holm-Sidak test to evaluate the differences in DNA damage among groups. Because the standard deviations of the DNA damage tended to increase with the magnitude of the response, a logarithmic transformation was performed before conducting the analyses. A P-value of less than 0.05 was considered statistically significant.
For gene expression, ANOVA with Tukey’s post hoc test was applied for gene-specific as well as time-point comparisons. To identify differentially expressed pathways and to determine functionally related sets of genes that are expressed differently in different experimental groups, MANOVA method (Tsai and Chen, 2009) was used in this study. The level of significance was set at a P-value of ≤ 0.05.
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Comet assay:
For rats exposed to 400 or 1000 mg/kg bw methyl eugenol, no statistically significant differences in DNA damage were observed compared with responses in the vehicle control group. The DNA damage caused by methyl eugenol exposure was either equal to or slightly but not significantly higher than control values, and the responses were not affected appreciably by the sampling times (3 or 24 hours).
At the higher dose of 2000 mg/kg measured at 1, 3, 6, and 8 h following the treatment, DNA damage was similar to the control at all time points and for all tissues. The only exception was in the bone marrow, where a slight but statistically significant (p < 0.05)
increase in DNA damage was observed at the 8-h sampling time. Due to the high turn over rate of bone marrow cells, this increase was interpreted as a coincidence instead of a real increase in DNA damage.
- Modified Comet assay - oxidative damage:
Endo III digestion, which cleaves DNA at oxidized pyrimidines, resulted in higher levels of % DNA in tail for liver samples from methyl eugenol-treated rats compared with liver from controls, at both the 6- and 8-h sampling times.
- Gene expression:
A decrease in expression of most genes was observed 3 h after MEG treatment. This decrease or suppression was unexpected and may suggest that inhibition of DNA damage repair may be one of the potential mechanisms for MEG-induced genotoxicity and carcinogenicity.
Conclusions:
In a comet assay it was found that methyleugenol induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of methyleugenol in rodents.

Executive summary:

In an in vivo study, methyleugenol was evaluated for DNA damage in rats using the standard Alkaline Comet Assay, the oxidative Comet Assay for detecting oxidative DNA damage and also a gene expression analysis associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg bw and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung after 3 h and 24 h. In addition, rats were given a single oral dose of 2000 mg/kg bw, and Comet assays were performed with liver, bone marrow, and bladder after 1, 3, 6, and 8 h. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. With one exception (bone marrow at 8 h), no DNA damage was detected in the standard Comet Assay. In the oxidative Comet Assay, whereas no DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6 and 8 h sampling times. Gene expression analysis on the livers from exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that methyleugenol exposure is associated with suppression of DNA repair gene expression and the induction of oxidative DNA damage which may be at least partially responsible for methyleugenol-induced genotoxicity in rodents. The study concludes that further studies specifically analyzing a free radical pathway are necessary to confirm the genotoxic mode of action of methyleugenol in rodent carcinogenesis.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Method not validated
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: 32P-Post-labelling analysis of DNA adducts formed in the livers of male mice (B6C3F1) after treatment with test item.
GLP compliance:
not specified
Remarks:
(information not reported)
Type of assay:
other: Estimation of DNA adducts formation
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Offspring male mice (B6C3F1) used in this test was obtained from pregnant C57B1 mice that had been mated with C3H/He males.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: National Institute for Medical Research, Mill Hill, London (dams)
- Housing: singly in plastic cages (dams)
- Diet (e.g. ad libitum): standard laboratory diet, ad libitum (dams)
- Water (e.g. ad libitum): ad libitum (dams)
- Acclimation period: at least 7 days before the litters were born (dams)
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Trioctanoin
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 0.01, 0.01, 0.04 and 0.04 mol/L
- Amount of vehicle (if gavage or dermal): 25, 50, 25 and 75 µL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test solutions were prepared by dissolving the test substance in vehicle trioctanoin.
Duration of treatment / exposure:
1, 7 and 21 days after the final injection. Mice were treated on days 1,8, 15 and 22 and killed on days 23, 29 and 43 after birth.
Frequency of treatment:
weekly
Post exposure period:
No
Dose / conc.:
0.25 other: µmol
Dose / conc.:
0.5 other: µmol
Dose / conc.:
1 other: µmol
Dose / conc.:
3 other: µmol
No. of animals per sex per dose:
3 per killing time.
Control animals:
yes, concurrent vehicle
Positive control(s):
none
Tissues and cell types examined:
liver
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Not reported.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Offspring male mice (B6C3F1) were injected i.p. with 0.25, 0.5, 1.0 and 3.0 µmol of test compound on days 1,8, 15 and 22, respectively, after birth; day 1 was
defined as the first 24 h after birth.Groups of 3 male mice were killed on days 23, 29 and 43 (i.e., 1, 7 and 21 days after the final injection) and the livers removed and weighed. DNA was isolated from the pooled livers by a rapid solvent-extraction procedure (Randerath, K. et al., 1984). Yields of 1.0-1.6 mg DNA/g liver were obtained.

DNA digestion and 32P-labelling: DNA (1 µg) was digested to deoxyribonucleoside 3' -monophosphates with micrococcal nuclease and spleen phosphodiesterase. The standard 32P-labelling conditions were: in a total volume of 10 µL, 0.17 µg DNA digest (0.5 nmol), 3 units T4 polynucleotide kinase, 150 µCi [γ-32P]ATP together with 0.6 nmol nonradioactive ATP, and 1.0 µM of a solution of 0.1 M Bicine-NaOH, 0.1 M MgCl2, 0.1 M dithiothreitol, 10 mM spermidine (pH 9.0). The labelling reaction was terminated after 25 min at 37ºC by the addition of potato apyrase (40 mU) and a mixture of deoxyribonucleoside 3',5'-biphosphates (dpNp's) (8 µg).

Mapping of 32P-labelled adducts: Removal of the labelled normal nucleotides from the test item adducts and resolution of the 32P-adducts was achieved by a modification of the combined reversed-phase and polyethyleneimine (PEI)-cellulose thin layer chromatography method.

METHOD OF ANALYSIS: The 32P-adducts were detected by autoradiography of the chromatogram and assayed by cutting the areas containing adduct spots from the (PEI)-cellulose sheet and counting them for radioactivity in a scintillation counter.
The incorporation of 32P into normal nucleotides in the samples was determined by chromatographing a portion of the labelled digest on PEI-cellulose with 80 mM ammonium sulphate and counting the radioactivity in the dpNp spot. Adduct levels were then estimated from the amounts of radioactivity incorporated into the resolved adducts relative to that incorporated into the normal nucleotides.
Evaluation criteria:
At least 15 pmol/mg DNA in male B6C3F1 mice are required for statistically-significant tumour formation (Miller E.C., et al., 1983).
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
RESULTS OF DEFINITIVE STUDY
At all three ages, highest levels of adduct formation were seen with methyleugenol with a maximum of 72.7 pmol/mg DNA (1 adduct in 41 300 nucleotides) at 23 days.
When the level of adducts formed by methyleugenol present at 43 days were corrected for liver growth, it was apparent that the majority of the adducts seen at the earlier time points persist for at least 21 days. It is considered to be unlikely that the formation
and partial removal of adducts had occurred sufficiently rapidly for substantially higher levels of adducts to be bound at times intermediate between those that have been analysed.


Table 1. Covalent binding of methyleugenol to DNA in B6C3F1 mouse liver

 

23 days

29 days

43 days

Tumour incidence
(% tumour bearing
animals)c

Av. no.
hepatomas/
mousec

 

pmol compound
bound/mg DNAa

Mean liver
weight (g)

pmol compound
bound/mg DNAb

Mean liver
weight (g)

pmol compound
bound/mg DNAb

Mean liver
weight (g)

Methyleugenol

72.7 ± 10.7

0.37

37.1 ± 9.7

0.87

25.6 ± 6.2

1.33

96

3.2

a Mean ± S.D. of at least three determinations.

b Mean ± S.D. of at least two determinations.

c Data from Miller E.C., et al., 1983. In this study uninjected control mice had a tumour incidence of 28% (average 0.5 hepatomas/mouse), and solvent-treated controls tumour incidence of 41% (0.5 hepatomas/mouse).

Conclusions:
Methyleugenol, administered in doses of 0.25 - 3 µmol, caused DNA adduct levels of 72.7 pmol/mg DNA. DNA adducts of at least 15 pmol/mg DNA are needed for statistically significant tumourigenesis potential.
Executive summary:

A 32P-Post-labelling analysis of DNA adducts formed in the livers of newborn male mice (B6C3F1) was performed after i.p. injection of methyleugenol with 0.25, 0.5, 1.0 and 3.0 µmol on days 1, 8, 15 and 22 after birth, respectively. 3 mice per killing time were sacrificed and their liver DNA isolated on days 23, 29 and 43, and analysed by a modified 32P-post-labelling procedure. At all three ages, highest levels of adduct formation were seen with methyleugenol with a maximum of 72.7 pmol/mg DNA (1 adduct in 41 300 nucleotides) at 23 days. After correction for liver growth it was estimated that most of these adducts were still present at 43 days. DNA adducts of at least 15 pmol/mg DNA are needed for statistically significant tumourigenesis potential (Miller, E.C., et al., 1983).

Endpoint:
genetic toxicity in vivo, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Method not validated
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Analysis of beta-catenin mutations in hepatocellular neoplasms.
- Parameters analysed / observed: hepatocellular adenomas and carcinomas from B6C3F1 mice treated with methyleugenol were examined for genetic alterations in H-ras, p53, and beta-catenin, genes that have been shown to be altered in human cancers. After beta-catenin mutations were found in these neoplasms, the neoplasms were examined for overexpression of beta-catenin protein by Western blot hybridization analysis and immunohistochemical methods.
GLP compliance:
not specified
Remarks:
(information not reported)
Type of assay:
other: Analysis of beta-catenin mutations in hepatocellular neoplasms
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 24.2-24.4 g (males) and 19.1-19.3 g (females) recorded on first week of the study.
- Fasting period before study: not specified
- Housing: 1 (males) or 5 (females) per cage. Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed twice weekly for females and once weekly for males; Bedding: Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed twice weekly for females and once weekly for males; Racks: Stainless steel (Lab Products Inc., Maywood, NJ), changed and rotated every 2 weeks.
- Diet (e.g. ad libitum): ad libitum. NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly.
- Water (e.g. ad libitum): ad libitum. Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI).
- Acclimation period: 11 days (males) or 12 days (females).

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 72º ± 3º F
- Humidity (%): 50% ± 15%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% methylcellulose
- Justification for choice of solvent/vehicle: Not reported
- Concentration of test material in vehicle: 3.7, 7.5 and 15 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
- Lot/batch no. (if required): 876672 and 946150
- Purity: not specified.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle was prepared by mixing methylcellulose and heated, deionized water with a magnetic stirrer to form a 0.5% solution, which was then cooled. Methyleugenol was slowly added to 0.5% methylcellulose and then diluted to the required volume with additional 0.5% methylcellulose while being stirred continuously.
Duration of treatment / exposure:
105 weeks
Frequency of treatment:
5 days per week
Post exposure period:
No
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
(vehicle control)
Dose / conc.:
37 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
none
Tissues and cell types examined:
hepatocellular neoplasms
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the high mortality rate of the 1000 mg/kg group, the lower mean body weight gains of the 300 mg/kg groups, and the hepatotoxic effects in the 300 and 1000 mg/kg groups in a 14-week study, the highest dose selected in this 2-year gavage study in mice was 150 mg/kg.

METHOD OF ANALYSIS:
Approximately equal numbers of hepatocellular neoplasms from the 3 dose groups (37, 75, and 150 mg/kg) of methyleugenol-treated and control B6C3F1 mice were used for this study. DNA isolation from these tumors and normal liver from B6C3F1 mice has been described previously (Devereux et al., 1993; Marmur, 1961).
Mutation Identification: Single-strand conformation polymorphism (SSCP) analysis was carried out on polymerase chain reaction (PCR) products corresponding to exon 2 of H-ras and exon 2 of beta-catenin.
Immunohistochemistry: Hepatocellular neoplasms were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin. Localization of p53 protein expression and beta-catenin expression was investigated using a rabbit polyclonal anti-p53 antibody (CM5 from Vector Laboratories) and a polyclonal goat anti-beta-catenin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Nonimmune rabbit IgG (Jackson Immuno Research Laboratories, West Grove, PA) was used as the negative control at equivalent conditions in place of the primary antibody.


Evaluation criteria:
Frequency of beta-catenin mutations in tumors after chemical treatment compared to frequency in spontaneous tumors.
Statistics:
Chi-Square analysis was used to compare the frequency of beta-catenin mutations in tumors after chemical treatment to frequency in spontaneous tumors.

Key result
Sex:
male/female
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
RESULTS OF DEFINITIVE STUDY
Twenty-nine methyleugenol-induced liver neoplasms from B6C3F1 mice were examined for molecular alterations in exon 2 of the beta-catenin gene, the region that contains potential phosphorylation sites for the glycogen-serine kinase-3β (GSK-3β) enzyme. Twenty (69%) of the neoplasms exhibited mutations consisting of 18 point mutations and 2 deletion mutations (Table below). In contrast, of 22 spontaneous liver neoplasms, only two (9%) had mutations. All of the point mutations affected codons 32, 33, 34, or 41, sites that are targeted for phosphorylation by the GSK-3β kinase or that are involved in ubiquitination of the protein and are important in regulation of β-catenin turnover. Eleven different base substitutions were represented among the four mutant codons, and there was no preponderance of G to T or G to A mutations. The relationship between frequency of β-catenin mutations and methyleugenol dose in the liver neoplasms also was examined. Nine of 12 neoplasms (75%) from the 37 mg/kg dose group, six of nine (67%) from the 75 mg/kg dose group, and five of eight (63%) from the 150 mg/kg dose group had β-catenin mutations, indicating that there was no association between mutation frequency and dose level. Mutations of β-catenin were detected almost equally in adenomas and carcinomas, suggesting that mutation of this gene is an early event in hepatocellular tumorigenesis.

Some of the neoplasms also were evaluated for expression of the β-catenin protein. Four methyleugenol induced hepatocellular neoplasms with mutations analyzed by Western blot hybridization showed accumulation of β-catenin protein, while two neoplasms without mutations had lower expression, similar to that observed for the normal liver control. In addition, 16 methyleugenol-induced liver neoplasms were examined by immunohistochemistry and eight of 10 neoplasms with mutations demonstrated some positive staining for the β-catenin protein. Strong positive staining of an altered hepatocellular focus from a methyleugenol-treated mouse provided further evidence that upregulation and accumulation of β-catenin occur early in tumorigenesis. Staining within the cells was localized to cell membranes, although some cytoplasmic staining was visible in some of the neoplasms.

These same methyleugenol-induced liver neoplasms were analyzed by immunohistochemistry for mutations in codon 61 of the H-ras protooncogene and altered p53 protein expression that would be indicative of a mutation in p53. However, no H-ras mutations were identified, and no p53 overexpression was detected in any of these neoplasms. These results indicate that H-ras protooncogene activation and p53 mutations are probably not involved in the liver neoplasm response to methyleugenol treatment in B6C3F1 mice.




Table 1. Summary of beta-Catenin Mutations in Hepatocellular Neoplasms from B6C3F1 Mice in the 2-Year Gavage Study of Methyleugenol

Tumor group

Frequency

Codon

Mutation

Bases

N

Amino acid

Control

2/22 (9%)

32

GAT to GCT

A to C

1

Asp to Ala

 

33

TCT to TTT

C to T

1

Ser to Phe

 

 

 

 

 

 

Methyleugenol

20/29 (69%)a

32

GAT to GTT

A to T

5

Asp to Val

 

 

32

GAT to TAT

G to T

1

Asp to Tyr

 

 

32

GAT to GGT

A to G

3

Asp to Gly

 

 

32

GAT to CAT

G to C

1

Asp to His

 

 

33

TCT to TAT

C to A

1

Ser to Tyr

 

 

33

TCT to TTT

C to T

1

Ser to Phe

 

 

34

GGA to AGA

G to A

1

Gly to Arg

 

 

34

GGA to CGA

G to C

1

Gly to Arg

 

 

34

GGA to GTA

G to T

2

Gly to Val

 

 

41

ACC to ATC

C to T

1

Thr to Ile

 

 

41

ACC to GCC

A to G

1

Thr to Ale

 

 

5-10

Deletions

 

2

 

a P<0.001 when comparing mutation frequency to controls.

N=number of neoplasm samples with that mutation

Asp=aspartate; Val=valine; Tyr=tyrosine; Gly=glycine; His=histidine; Ser=serine; Phe=phenylalanine; Arg=arginine; Thr=threonine; Ile=isoleucine; Ala=alanine

Executive summary:

In a 2-year carcinogenicity study conducted with B6C3F1 mice orally exposed to methyleugenol, increases in the incidences of hepatocellular adenomas, hepatocellular carcinomas, and hepatoblastomas were observed in all dosed groups of male and female mice. In this study, hepatocellular adenomas and carcinomas from those treated mice were examined for genetic alterations in H-ras, p53, and beta-catenin, genes that have been shown to be altered in human cancers. After beta-catenin mutations were found in these neoplasms, the neoplasms were examined for overexpression of beta-catenin protein by Western blot hybridization analysis and immunohistochemical methods. 29 methyleugenol-induced liver neoplasms were examined for molecular alterations in exon 2 of the beta-catenin gene and 20 (69%) of the neoplasms exhibited mutations consisting of 18 point mutations and 2 deletion mutations. In contrast, of 22 spontaneous liver neoplasms used as control group, only two (9%) had mutations. There was no association between mutation frequency and dose level. Mutations of β-catenin were detected almost equally in adenomas and carcinomas, suggesting that mutation of this gene is an early event in hepatocellular tumorigenesis. No H-ras mutations were identified, and no p53 overexpression was detected in any of these neoplasms. These results indicate that H-ras protooncogene activation and p53 mutations are probably not involved in the liver neoplasm response to methyleugenol treatment in B6C3F1 mice. Thus, there is an indication that methyleugenol caused alterations in the beta-catenin gene, which contributes significantly to the development of hepatocellular neoplasms.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

In vitro gene mutation in bacteria:

In a bacterial reverse mutation assay following a method similar to OECD guideline 471, the test item methyl eugenol diluted in Dimethylsulfoxide (DMSO) was tested with Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 with and without metabolic activation (S9) using the preincubation method. Based on results of a preliminary assay, for the main test two independent experiments and 3 replicates of each were conducted with doses of 0 (solvent control), 3, 10, 33, 100 and 333 μg/plate with and without S9 mix. Also a dose of 666 μg/plate with S9 mix was used in the first experiment. Concurrent solvent and positive controls were included in every experiment and their responses were considered valid. The test item did not show mutagenic activity in any of the bacterial strains tested.

Methyl eugenol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535, TA1537, TA1538 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to Ames method. Based on growth inhibition examined in a preliminary test, the concentrations used in the main test were: 0, 30, 60, 120 and 300 μg/plate for all strains with and without metabolic activation. The assay without S9 was performed by the plate incorporation method and the assay with S9 was conducted by the pre-incubation method described by Yahagi et al. (1975). DMSO was used as solvent. Negative (solvent) and positive controls were used in the test and showed expected results. Under test conditions, methyl eugenol was found not mutagenic in all strains tested with and without metabolic activation.

In vitro DNA repair test in bacteria:

Methyl eugenol was tested in the DNA-repair test (Rec assay) with Bacillus subtilis strains M45 Rec- and H17 Rec+, performed as described by Kada et al. (1980), with and without metabolic activation (S9). Test item dissolved in ethanol was pipetted onto sterile filter-paper disk at 0.1 mg test item/disk and placed on the previously prepared spore-agar plate. After incubation of the plate for 20-24 h at 37°C, the zones of killing (diameter of growth inhibition zone - diameter of disk) with both strains (Rec + and Rec -) were measured and the difference between them was taken as the rec effect. Difference of the mean values of 3 replicates greater than 4 mm was taken as evidence of preferential killing of Rec- cells and thus of positive response. Methyl eugenol was found positive in this Rec assay without metabolic activation.

Kada et al. (1980) reported that carcinogens that were not detected by their mutagenicities in the Ames test showed positive responses in the DNA-repair test with B. subtilis, as it is the case of methyl eugenol.

In vitro mammalian sister chromatic exchange:

Methyleugenol was tested in cultured Chinese hamster ovary (CHO) cells for induction of sister chromatid exchanges (SCEs) by method reported by Galloway et al. (1987) and following a protocol similar to OECD TG 479. The test material dissolved in DMSO was tested in the presence and absence of metabolic activation in concentrations of 5, 17, 50 and 167 µg/mL (-S9); 17, 50 and 167 μg/mL (+S9, trial 1) and 50, 167 and 250 μg/mL (+S9, trial 2). The highest dose was limited by toxicity. Concurrent solvent control (DMSO) and positive controls (mytomicin-C without S9 and Cyclophosphamide with S9) were also tested. After total incubation of 28 h and exposure times to test material of 26 h without S9 and 2 h with S9, cells were harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. Colcemid (2 hours exposure) was used as spindle inhibitor with and without S9. 50 second-division metaphase cells were scored for frequency of SCEs/cell from each dose level. An SCE frequency 20% above the concurrent solvent control value at two or more doses was considered as a positive response. Methyleugenol induced SCEs at least at 2 doses in each of the two trials conducted with S9 while no significant increase in SCEs was observed without S9 at any dose tested. Thus, under these test conditions, methyleugenol was considered positive in the in vitro SCE test.

In vitro mammalian chromosome aberration:

Methyleugenol was tested in cultured Chinese hamster ovary (CHO) cells for chromosomal aberrations by method reported by Galloway et al. (1987) and following a protocol similar to OECD TG 473. The test material disssolved in DMSO was tested in the presence and absence of metabolic activation in concentrations of 50, 108 and 233 µg/mL. The highest dose was limited by toxicity. Concurrent solvent control (DMSO) and positive controls (mytomicin-C without S9 and Cyclophosphamide with S9) were also tested. After total incubation of 16.7 h without S9 and 12.5 h with S9 and exposure times to test material of 14.5 h without S9 and 2 h with S9, cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. Colcemid (2 hours exposure) was used as spindle inhibitor with and without S9. 200 first-division metaphase cells were scored at each dose level. No significant induction of chromosomal aberrations occurred following incubation with methyleugenol in either the presence or the absence of S9. Thus, under these test conditions, methyleugenol was considered negative in the chromosomal aberration test.

In vitro mammalian comet assay:

In an in vitro comet assay performed according to the method of Gedik et al (1998), cultured Chinese hamster V79 cells were exposed to methyleugenol dissolved in DMSO with and without enzyme fpg in order to evaluate also the potential oxidative DNA damage. V79 cells were treated for 1 h or 24 h with the solvent control (DMSO, 0.5% (v/v)), positive control (10 µM menadione) or the test compound in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. The concentrations used in the test were 5, 10, 25, 50, 75 and 100 µM. 3 independent experiments were performed in duplicate. 100 individual cells per concentration (50 per slide) were processed and analysed in each experiment. Methyleugenol significantly induced DNA damage at concentrations >10 µM after 1 h of incubation without an increase in efficacy after 24 h. No additional DNA strand breaks were detected following fpg treatment meaning that methyleugenol does not induce oxidative DNA damage. Thus, under these test conditions, methyleugenol was found positive in an in vitro comet assay.

In vitro mammalian micronucleus test:

An in vitro Micronucleus test was performed with methyleugenol dissolved in DMSO following a method comparable to OECD TG 487. Chinese hamster V79 cells were previously incubated for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Cultured V79 cells were treated for 24 h and post-incubated for additional 20 h with the solvent control (DMSO, 0.5% (v/v)), positive control (0.6 µM Mitomycin C) or the test compound in concentrations of 10, 25, 50 and 100 µM. 3 independent experiments were performed in duplicate. 2000 individual cells per concentration (1000 per Petri dish) were processed and analysed using fluorescence microscopy in each experiment. No significant increase in micronuclei frequency was observed at any tested concentration compared to the solvent control. Thus, methyleugenol was negative in the in vitro micronucleus test.

In vitro unscheduled DNA synthesis in hepatocytes:

Methyleugenol was tested in an in vitro rat hepatocyte unscheduled DNA synthesis assay following a method similar to the OECD Guideline 482. Primary hepatocytes were isolated from Male Fischer 344 rats. Cultures of hepatocytes were incubated in duplicate with the test material dissolved in DMSO for 17 hours at concentrations of 0 (solvent control), 1e-6, 1e-5, 1e-4, 1e-3 and 1e-2 mol/L. A response was considered positive when the result of the treated cultures was at least 1.5 times higher than the control (DMSO). The test material induced a marked and dose-dependent response in the test. A concentration of 5 x 10-3 M and above was toxic to the cells. Positive control induced an expected response which was considered valid. Based on these results, methyleugenol was demonstrated to be an UDS inducer in vitro.

In another in vitro unscheduled DNA synthesis assay, methyleugenol was tested with primary hepatocytes isolated from male Fischer 344 rats and female B6C3F1 mice. Cultures of hepatocytes were incubated in triplicate (rats) or duplicate (mice) with the test material dissolved in DMSO for 18 hours at concentrations of 0 (solvent control), 0.1, 0.5, 1, 5, 10, 50, 100 and 500 µM. In rats, methyleugenol caused UDS at concentrations between 10 and 500 µM, without a classic dose–response curve. In mice, methyleugenol caused UDS at concentrations between 5 and 500 µM, with peak UDS at 10 µM and with counts gradually falling as the concentration increased. In the range of the tested concentrations methyleugenol was relatively non-cytotoxic. Positive control induced an expected response which was considered valid. Based on these results, methyleugenol was positive in the UDS assay.

In-vivo mammalian micronucleus test:

An in-vivo micronucleus test was performed with methyleugenol following a method comparable to OECD TG 474. Groups of 10 male and 10 female B6C3F1 mice were administered test item dissolved in 0.5% methylcellulose in concentrations of 0, 10, 30, 100, 300, or 1,000 mg/kg bw by gavage 5 days per week during a 14-week repeated dose toxicity study. At the end of this study, peripheral blood samples were obtained from male and female mice and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Micronucleus frequency scores were based on 10,000 NCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 5,000 erythrocytes. Methyleugenol did not increase the frequency of micronucleated NCEs in peripheral blood and did not alter the percentage of PCEs among total erythrocytes (an indication of bone marrow toxicity). Thus, a negative result is obtained from the mammalian erythrocyte micronucleus test.

In-vivo mammalian comet assay:

Methyleugenol was evaluated for DNA damage in rats using the standard Alkaline Comet Assay, the oxidative Comet Assay for detecting oxidative DNA damage and also a gene expression analysis associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg bw and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung after 3 h and 24 h. In addition, rats were given a single oral dose of 2000 mg/kg bw, and Comet assays were performed with liver, bone marrow, and bladder after 1, 3, 6, and 8 h. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. With one exception (bone marrow at 8 h), no DNA damage was detected in the standard Comet Assay. In the oxidative Comet Assay, whereas no DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6 and 8 h sampling times. Gene expression analysis on the livers from exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that methyleugenol exposure is associated with suppression of DNA repair gene expression and the induction of oxidative DNA damage which may be at least partially responsible for methyleugenol-induced genotoxicity in rodents. The study concludes that further studies specifically analyzing a free radical pathway are necessary to confirm the genotoxic mode of action of methyleugenol in rodent carcinogenesis.

In-vivo mammalian DNA damage:

A 32P-Post-labelling analysis of DNA adducts formed in the livers of newborn male mice (B6C3F1) was performed after i.p. injection of methyleugenol with 0.25, 0.5, 1.0 and 3.0 µmol on days 1, 8, 15 and 22 after birth, respectively. 3 mice per killing time were sacrificed and their liver DNA isolated on days 23, 29 and 43, and analysed by a modified 32P-post-labelling procedure. At all three ages, highest levels of adduct formation were seen with methyleugenol with a maximum of 72.7 pmol/mg DNA (1 adduct in 41 300 nucleotides) at 23 days. After correction for liver growth it was estimated that most of these adducts were still present at 43 days. DNA adducts of at least 15 pmol/mg DNA are needed for statistically significant tumourigenesis potential (Miller, E.C., et al., 1983).

In vivo mammalian β-catenin mutations:

In a 2-year carcinogenicity study conducted with B6C3F1 mice orally exposed to methyleugenol, increases in the incidences of hepatocellular adenomas, hepatocellular carcinomas, and hepatoblastomas were observed in all dosed groups of male and female mice. In this study, hepatocellular adenomas and carcinomas from those treated mice were examined for genetic alterations in H-ras, p53, and beta-catenin, genes that have been shown to be altered in human cancers. After beta-catenin mutations were found in these neoplasms, the neoplasms were examined for overexpression of beta-catenin protein by Western blot hybridization analysis and immunohistochemical methods. 29 methyleugenol-induced liver neoplasms were examined for molecular alterations in exon 2 of the beta-catenin gene and 20 (69%) of the neoplasms exhibited mutations consisting of 18 point mutations and 2 deletion mutations. In contrast, of 22 spontaneous liver neoplasms used as control group, only two (9%) had mutations. There was no association between mutation frequency and dose level. Mutations of β-catenin were detected almost equally in adenomas and carcinomas, suggesting that mutation of this gene is an early event in hepatocellular tumorigenesis. No H-ras mutations were identified, and no p53 overexpression was detected in any of these neoplasms. These results indicate that H-ras protooncogene activation and p53 mutations are probably not involved in the liver neoplasm response to methyleugenol treatment in B6C3F1 mice. Thus, there is an indication that methyleugenol caused alterations in the beta-catenin gene, which contributes significantly to the development of hepatocellular neoplasms.

Justification for classification or non-classification

Based on the available information, methyleugenol should be classified as Mutagen Category 2, H341 in accordance with CLP Regulation (EC) no 1272/2008.