Eugenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: not mutagenic in S. typhimurium TA98, TA 100, TA 1535, TA 1537, TA1538 and E. coli WP2 (OECD 471, GLP, K, rel. 1)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Equivalent to accepted guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

reliability scoring based on 1997 guideline

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine or tryptophan

Species / strain / cell type:

other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

polychlorinated biphenyl (PCB) induced S9 from male Sprague-Dawley rats

Test concentrations with justification for top dose:

60, 120, 300 and 600 μg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Without S9: in agar (plate incorporation)
With S9: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: 3 to 5 plates per test

Evaluation criteria:

no data

Statistics:

no data

Species / strain:

other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Eugenol was not mutagenic under the conditions of the assay.

Executive summary:

The mutagenic potential of eugenol was evaluated in a bacterial reverse mutation assay. Eugenol (60, 120, 300 and 600 μg/plate) did not induce a significant increase in revertant numbers either with Salmonella tester strains (TA98, TA100, TA1535, TA1537, TA1538) or with E. coli WP2uvrA in the absence or presence of S9.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Micronucleus test (bone marrow, rats: negative (eq. to OECD 474, non-GLP, K, rel. 2)

- Mouse transgenic assay: negative (eq. to OECD 488, non-GLP, K, rel.2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP and only 4 animals used per dose level.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Non-GLP and only 4 animals used per dose level.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 100-120 g

(Further details were not reported.)

Route of administration:

oral: gavage

Vehicle:

Water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Watery suspension

Duration of treatment / exposure:

Half the dose was administered at 30 hours and the remainder at 6 hours prior to sacrifice.

Frequency of treatment:

Half the dose was administered at 30 hours and the remainder at 6 hours prior to sacrifice.

Post exposure period:

Sacrifice was 6 hours following administration of second half of the dose.

Remarks:

Doses / Concentrations:
335 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
670 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1340 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

4/dose

Control animals:

yes

Positive control(s):

Triethylenemelamine
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 1 mg/kg bw

Tissues and cell types examined:

Polychromatic erythrocytes; 1000 PCE scored/animal

Details of tissue and slide preparation:

Details were not reported but based on standard method of Schmid (1975).

Evaluation criteria:

Not reported.

Statistics:

Chi-square test.

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

other: - unclear if vehicle control or negative control was used.

Negative controls validity:

other: - unclear if vehicle control or negative control was used.

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
The genotoxic potential of eugenol was assessed in a bone marrow micronucleus assay in rats. Eugenol was administered via the oral (gavage) route at doses of 335, 670, or 1,340 mg/kg as divided doses at 30 and 6 hours prior to harvesting of the bone marrow. Eugenol was negative in this micronucleus assay.

Executive summary:

The genotoxic potential of eugenol was assessed in a bone marrow micronucleus assay in rats. Eugenol was administered via the oral (gavage) route at doses of 335, 670, or 1,340 mg/kg as divided doses at 30 and 6 hours prior to harvesting of the bone marrow. Eugenol was negative in this micronucleus assay. In the REACH guidance r7a integrated testing strategy for mutagenicity, the in vivo micronucleus assay may be used to follow-up in vitro clastogenicity postive results. Therefore, this study may be used to fulfil the Annex VIII section 8.4.2 requirement for a gene mutation study in mammalian cells, which also states in column 2 that the in vitro study is not required if adequate data from an in vivo cytogenicity test are available.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Deviations:

yes

Remarks:

Age of animals: 15 weeks at beginning of treatment instead of 8-12. Exposure duration: 58 days instead of 28. Laboratory proficiency not verified.

GLP compliance:

not specified

Remarks:

The laboratory that performed the study is an Internationally recognized research center. It is assumed that the study was performed to a GLP-equivalent standard.

Type of assay:

transgenic rodent mutagenicity assay

Specific details on test material used for the study:

The purity of the Eugenol sample was reported as 99% and being sourced from Janssen Chimica (Tilburg).

Species:

mouse

Strain:

other: λ-lacZ-transgenic mouse strain 40.6

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TNO Centre for Animal Research, Rijswijk, The Netherlands
- Age at study initiation: 15 weeks old
- Weight at study initiation: appox. 25 g
- Assigned to test groups randomly: Not reported
- Fasting period before study: Not reported
- Housing: sterilized Makrolon cages with a grid cover of stainless steel and with a bedding of sterilized softwood chips
- Diet (e.g. ad libitum): pelleted purified diet. Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 40-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: Not reported

Route of administration:

oral: feed

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
5% of Eugenol premix was prepared using 12% of eugenol and 88% of corn oil
<=> 0.6% nominal concentration, eq. to 0.4% actual concentration.
Correspondance in mg/kg bw/day: 1000 mg/kg bw/day (nominal) / 580 mg/kg bw/day (actual)

DIET PREPARATION
- Rate of preparation of diet (frequency): twice a week
- Mixing appropriate amounts with (Type of food): pelleted purified diet
- Storage temperature of food: -20°C until use

Duration of treatment / exposure:

58 days

Frequency of treatment:

Continuous exposure

Post exposure period:

None

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Corresponding to 580 mg/kg bw/day (actual), calculated as concentration in ppm * (food consumption/mean body weight) = 4000 * (4.21/29).

No. of animals per sex per dose:

8 males per dose (among which 4 were given an i.p. injection of Benzoapyrene)

Control animals:

yes, concurrent no treatment

Positive control(s):

Benzo[a]pyrene 100 mg/kg bw given as an intraperitoneal dose on Day 10

Tissues and cell types examined:

Liver DNA.
The lacZ mutants were recovered by packaging of DNA isolated from liver into lambda phage, and expressed in E. coli C lacZrecA-galE- bacteria.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
According to the OECD TG 488, for an administration period of 28 days (i.e. 28 daily treatments), the limit dose is 1000 mg/kg body weight/day. This dose was the maximum nominal dose selected for this limit assay.
The achieved dose (actual) was ca. 580 mg/kg bw/day. This actual dose is considered to be the maximum tolerated dose level as the eugenol diet resulted in an apparent growth retardation in comparison with the control group.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Phase 1 - In vivo assay
Male mice were fed a diet containing either 0.4% (w/w) eugenol or a control diet for 58 days. On day 10, half of the mice in each group received an i.p. dose of 100 mg/kg b.w. B[a]P suspended in olive oil. The other animals were given olive oil only.
- Phase 2: DNA isolation and preparation of microsomes and cytosol
Livers were cut into pieces and homogenized in buffer (250 mM sucrose, 100 mM EDTA, pH 7.4); per gram liver 9 ml was used (all volumes are expressed per g liver as starting material). The homogenate was centrifuged (1000 × g, I 0 min, 4°C). The supernatant was used for preparation of microsomes and cytosol and the pellet for DNA isolation. The supernatant was first centrifuged at 9000 X g for 30 min at 4°C and then at 100 000 × g for 90 min at 4°C. The supernatant containing the cytosolic fraction was separated and the pellet containing the microsomal fraction was resuspended in 1 ml of 144 mM KC1 (pH 7.4). Cytosolic and microsomal fractions were quickly frozen on solid CO 2 and stored at -80°C until analysis.
Liver DNA was isolated as described by Roggeband et al. (1993). Extractions of DNA were carried out with equal volumes of phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) and chloroform/isoamyl alcohol 24: 1, v/v). The DNA was precipitated by addition of 0.1 volume of 3 M sodium acetate, pH 6.0 and 3 volumes of ethanol at -20°C, washed with 70% ethanol at -20°C and dried in vacuo. The DNA was dissolved in 700 I~1 TE buffer (10 mM Tris-HC1, pH 7.6, 1 mM EDTA) and used for the mutation assay. XDNA was rescued from the preparations by use of in vitro packaging extracts (Giga-Pack Gold II, Stratagene, La Jolla, CA, USA). For each sample, aliquots of DNA (5 µl) were mixed with half of the packaging extracts and incubated at 25°C for 6 h.
For determination of adducts, the DNA was dissolved in 3.0 ml TE buffer and treated with a mixture of 22.5 I~l RNase A (50 *µg/ml) and 22.5 µl RNase T1 (50 U/ml) for 90 min. Extractions of DNA were carried out with equal volumes of phenol, phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) and chloroform/isoamyl alcohol (24: 1, v/v). The DNA was precipitated, washed and dried as described above. The DNA was dissolved in 5 mM sodium acetate, pH 6.0.
- Phase 3: Mutation Assay
A culture of E. coli C lacZ-recA-galE- (Gossen et al., 1993) harbouring a gaIK- and galT- expressing plasmid (pAA119) was grown at 37°C to A709 = 0.6 in LB medium (Gibco BRL, Paisley, Scotland) supplemented with 10 mM MgSO 4, 0.2% maltose and 60 µg/ml ampicillin. Packaged DNA was added to 700 µl of the bacterial suspension and incubated for 16 min at 30°C. After incubation 10 µl of the phage/bacteria mix was used for titration. The remaining phage/bacteria mix was suspended in 5 ml top-agar containing 2% LB, 0.8% agar, 10 mM MgSO 4, 60 µg/ml ampicillin, 0.3% fructose, and 0.3% phenyl-13-D-galactopyranoside (P-gal). The mixture was poured onto 10-ml agar plates (containing 2% LB, 1.6% agar, 10 mM MgSO 4 and 0.3% fructose) and incubated overnight at 37°C. The resulting (mutant) plaques were picked out and tested on X-gal containing plates to confirm their mutant status.

METHOD OF ANALYSIS:
- 32P-postlabelling analysis for the detection of B[a]P-DNA adducts
The 32P-postlabelling assay was performed according to the nuclease P1 enhancement procedure (Reddy and Randerath, 1986).
All chromatographic steps were carried out in subdued light to avoid photochemical decomposition of DNA adducts. The 32P -labelled adducts were visualized by autoradiography at - 70°C for 15 h with Kodak XAR-5 film and intensifying screens.
- 32P-postlabelling analysis for the detection of eugenol-induced DNA adducts
Same as above except that the solvents for chromatography were adapted to the conditions suitable for alkenylbenzene-DNA adducts.
- Quantitative analysis of the adduct levels
The relative amount of radioactivity in the adduct spots was determined by use of a Phosphor Imager system from Molecular Dynamics (Sunnyvale, CA, USA). The amount of background radioactivity was determined in a blank area in the upper left comer of the chromatogram. The adduct levels were determined by comparison with standard samples analyzed concurrently with the chromatographic conditions for B[a]P-DNA adducts. The standards used for quantification were B[a]P diol-epoxide (BPDE) modified DNA samples in which the amount of B[a]P-DNA adducts had been determined by synchronous fluorescence spectrophotometry
- Biotransformation enzyme activities
Cytosolic and microsomal protein content was quantified by the method of Lowry et al., with bovine serum albumin as a standard (Lowry et ai., 1951). The activity of cytosolic glutathione S-transferase (GST) was determined with l-chloro-2,4-dinitrobenzene as a substrate as described by Habig et al. (1974). Microsomal glucuronyl transferase (GT) activity was determined with 4-chlorophenol and 4-hydroxybiphenyl as substrates as described by Mulder and van Doom (1975)

Evaluation criteria:

Statistical evaluation

Statistics:

Data are presented as mean _+ SD. Statistical differences were determined by two-way analysis of variance (ANOVA). Before analysis, data were transformed by log transformation to stabilize the variance. When the result was significant (p < 0.05), asymptotic pairwise t-tests with Bonferroni α-correction were performed (2-tailed) (Dixon, 1988).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Growth retardation, seen as reduced body weight gain

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Apparent growth retardation, seen as a reduction in body weight gain, in comparison with the control group although the differences were not statistically significant.
- Evidence of cytotoxicity in tissue analysed: none reported

Table 7.6.2/1 : lacZ mutant frequencies in liver of λ-lacZ-transgenic mice

Eugenol (% diet)	No. of phages analyzed	No. of mutants	MF (x10^6)	Group mean MF ± SD
0.0	521550	24	46.0	71.3 ± 25.3
	226062	24	16.2
	330030	23	69.7
	458280	29	63.3
0.4	266760	19	71.2	63.9 ± 22.1
	347130	12	34.6
	320625	20	62.4
	389025	34	87.4

0.0 BP 100 mg/kg bw	301815	36	119.3	185.2 ± 66.7
	195453	29	148.4
	184509	50	271.0
	415530	84	202.2

Conclusions:

Eugenol was not mutagenic in this study.

Executive summary:

In a Transgenic Rodent Somatic Gene Mutation Assays, male Muta^TM mouse (6 males) were fed a diet containing 580 mg/kg bw/day eugenol or a control for 58 days. The lacZ mutants were recovered by packaging of DNA isolated from liver into lambda phage and expressed in E. coli C lacZ recA-galE- bacteria. The vehicle was corn oil. A positive control group received benzo[a]pyrene at 100 mg/kg bw via the intraperitoneal route on day 10.

There were signs of toxicity (apparent growth retardation as shown by a reduced body weight gain) during the study. However, the difference compared to the control group was not statistically significant. Eugenol was tested at the maximum tolerated dose level in this study.

The positive control, Benzo[a]pyrene, induced the appropriate response. 

Eugenol was not mutagenic in this study.

This study is classified as acceptable and, even if not performed according to the OECD 488 TG (note that the study was performed several years before the TG was published), it is considerd to satisfy the requirements of the TG.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The potential of eugenol to cause genotoxicity was investigated in 3 key studies(1 in vitro and 2 in vivo) including 1 bacterial reverse mutation assays, 1 mammalian chromosome aberration test and 1 mammalian gene mutation assay. These three studies meet the minimum genotoxicity data requirements for this substance. In addition a number of in vitro and in vivo supporting assays are included in the evaluation. All of the key and supporting studies provided negative results. Several in vitro and in vivo studies were evaluated as unreliable and were disregarded; these studies presented a mix of negative and positive results, as claimed by the authors. However, all of these studies suffered from serious technical and reporting deficiencies and in some cases the results and conclusions were clearly contradicted by the more reliable studies.

For Annex VII section 8.4.1, in a key study and two supporting studies eugenol did not increase the reverse mutation rate when tested in various bacterial strains (including, but not limited to, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 and Escherichia coli strain WP2uvrA) either in the presence and/or absence of an externally-added metabolic activating system (S9).

For Annex VIII section 8.4.2 there is no key study for the in vitro cytogenicity endpoint. The available in vitro studies were considered unreliable, primarily because the dose levels used were too high, which is a major deviation from the relevant test guidelines in terms of dose setting, and caused excessive toxicity, and consequently they were disregarded. However, using a column 2 adaptation the in vitro data are not required if adequate data from an in vivo cytogenicity test are available. In this case there is a key oral gavage micronucleus study that is supported by six other studies of reliability grade 2, all of which provided negative results. These studies include micronucleus studies using the oral gavage, intraperitonel, and oral capsule dose routes, and a chromosome aberration study using oral capsules. A study waiver was included for the genetic toxicity in vitro cytogenicity endpoint.

For Annex VIII section 8.4.3, there is no key study for the in vitro mutagenicity endpoint. However, using a column 2 adaptation the in vitro data are not required if adequate data from an in vivo mutagenicity test are available. In this case there is a key mouse transgenic assay that is supported by a TEGA assay. These studies all provided negative results. A study waiver was included for the genetic toxicity in vitro gene mutation endpoint.

The conclusion regarding the genotoxic potential of eugenol has taken into account that:

-Studies of the metabolism of eugenol indicate rapid metabolism; any formation of putative reactive metabolites would be followed by Phase II conjugation reactions leading to rapid detoxification (JECFA, 2006a, b).

-In vivo studies showed no evidence of DNA binding by eugenol. While study results were mixed, some data suggest that eugenol or the purported eugenol metabolites eugenol quinone methide and hydroxychavicol may bind to DNA in vitro ( Bodell et al.,1998; Sakano et al.,2004); however, in vivo mouse studies all produced negative results (Phillips et al.,1984; Phillips, 1990). Moreover, while O-demethylation has been reported in rats (Sutton et al.,1985), the metabolism of eugenol does not result in the formation of the hydroxychavicol metabolite and in vivo studies in humans have indicated that the methoxy group of eugenol is resistant to cleavage in man (Fischer et al.,1990). The lack of adduct detection in the in vivo test systems is likely due to the presence of detoxification processes (i.e.,glutathione conjugation), which are likely not present to a significant extent in the in vitro test systems. These processes either conjugate parent eugenol or neutralize the oxidative metabolites that may be generated by microsomal metabolism.

-Eugenol shows no structural alerts for genotoxicity.

-Structural analogues of eugenol have also provided some data erroneously reported as positive. For example, Isoeugenol is reported as negative in the Ames bacterial mutation assay, negative in the CHO chromosome aberration test in vitro, negative in male mice in a micronucleus test but positive in female mice of the same study. However, it is clear from the data that this is a statistical correlation only, caused by a particularly low value in the female vehicle control group. When historical control data are included in the evaluation of the result then the study is clearly negative in both sexes. (NTP technical Report 551, 2010). It is considered therefore, that the data for eugenol are most relevant in the evaluation of the genotoxic potential of this substance and reference to any of the analogues raises questions about the reliability of the studies on those analogues. In particular it is considered that methyl eugenol is not a relevant analogue because of the difference in its metabolic pathway.

In summary, all of the reliable and relevant in vitro and in vivo genotoxicity studies show no evidence of genotoxic potential for eugenol.

The lack of genotoxicity is consistent with the finding of no substantive carcinogenic activity in mouse and rat oral bioassays, as reported by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2006b) and the European Food Safety Authority (EFSA, 2009).

References

Bodell WJ, Ye Q, Pathak DN, Pongracz K. Oxidation of eugenol to form DNA adducts and 8-hydroxy-2'- deoxyguanosine: Role of quinone methide derivative in DNA adduct formation. Carcinogenesis 1998;19:437-443.

EFSA. Scientific Opinion on the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from Commission on Consideration of eugenol and related hydroxyallybenzene derivatives evaluated by JECFA (65th meeting) structurally related to ring-substituted phenolic substances evaluated by EFSA in FGE.22 (2006) (question no EFSA-2008 -32L, published on 12 February 2009 by European Food Safety Authority). EFSA J. 2009:ON-965:1 -54. Available at http://www.efsa.europa.eu/en/efsajournal/pub/965.htm.

Fischer IU, Von Unruh GE, Dengler HJ. The metabolism of eugenol in man. Xenobiotica 1990;20:209-222.

JECFA. Evaluation of Certain Food Additives and Contaminants. Sixty-Fifth Report of the Joint FAO/WHO Expert Committee on Food Additives, June 7-16, 2005. (WHO Technical Report Series, no 934). Geneva, Switz.: World Health Organization (WHO); 2006a. Available at:http://whqlibdoc.who.int/trs/WHO_TRS_934_eng.pdf.

JECFA. Eugenol and related hydroxyallylbenzne derivatives\. In: Safety Evaluation of Certain Food Additives: Sixty-fifth Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). (WHO food additives series no. 56). Geneva, Switz.: World Health Organization (WHO), International Programme on Chemical Safety (IPCS); 2006b:155-200. Available at:http://www.inchem.org/documents/jecfa/jecmono/v56je09.pdf/

OECD.InVitroMammalian Cell Gene Mutation Test. In: OECD Guidelines for the Testing of Chemicals. (OECD Guideline no 476) [Adopted 21 July, 1997]. Paris, France: Organization for Economic Co-operation and Development (OECD); 1997a.

OECD. Mammalian Erythrocyte Micronucleus Test. In: OECD Guidelines for the Testing of Chemicals. (OECD Guideline no 474) [Adopted 21 July 1997]. Paris, France: Organization for Economic Co-operation and Development (OECD); 1997b.

Phillips DH. Further evidence that eugenol does not bind to DNA in vivo. Mutat Res. 1990;245:23-26.

Phillips DH, Reddy MV, Randerath K. (32)P-Post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally- occurring alkenylbenzenes. II. newborn male B6C3F1 mice. Carcinogenesis 1984;5:1623-1628.

Sakano K, Inagaki Y, Oikawa S, Hiraku Y, Kawanishi S. Copper- mediated oxidative DNA damage induced by eugenol: possible involvement of O-demethylation. Mutat Res. 2004;565:35-44.

Sutton D, Sangster S, Caldwell J. Dose-dependent variation in the disposition of eugenol in the rat. Biochem Pharmacol 1985;34: 465-466.

Justification for classification or non-classification

Harmonised classification:
The substance has no harmonised classification for genetic toxicity according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:
Based on the available data, no additional classification is proposed according to the CLP or the GHS.