Eugenol

Carcinogenicity

Description of key information

The carcinogenic potential of eugenol was evaluated in key studies in male and female B6C3F1 mice and male and female F344/N rats (NTP, 1983) and in a supporting study in female CD-1 mice (Miller et al., 1983).  In rats, there was no evidence of carcinogenicity in either male or females compared to Controls. In the key mouse study, dietary eugenol at concentrations of 0 (control), 3,000, or 6,000 ppm for 103 weeks resulted in a dose-dependent increase in the incidence of hepatocellular adenomas and carcinomas (combined; statistically significant at the high-dose in females); however, eugenol is concluded not to be carcinogenic in mice, based on a number of considerations including a lack of a dose-response relationship in the increase in hepatocellular tumours in male mice and high and variable incidences of adenomas or carcinomas (combined) within the historical range for B6C3F1 mice.  In the key rat study, dietary administration of eugenol at concentrations of 0 (control), 3,000, or 6,000 ppm in males and 0 (control), 6,000, or 12,500 ppm in females did not result in an increase in tumour incidence in either males or females.  In the supporting mouse study, dietary administration of 0.5% eugenol did not result in an increase in the incidence of hepatic tumors through the 20-month dosing period.  
IARC Monographs, Volume 36, reports that no cell transformation data are available on eugenol.
No inhalation or dermal carcinogenicity studies have been conducted.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

Based on the data, the evidence indicates that eugenol is not a carcinogen in rodents. This conclusion is consistent with that of the recent review of the toxicology of eugenol conducted by the European Food Safety Authority, which concluded that eugenol is devoid of carcinogenic activity (EFSA, 2009). Given the quality of the studies, and considering the results of the studies while taking into account historical control incidences, rodent and human metabolism of eugenol, the results of the key and several reliable in vitro and in vivo genotoxicity assays, and structure-activity relationships, eugenol is concluded to be “unclassifiable” with respect to carcinogenicity. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.6.

Additional information

The carcinogenic potential of eugenol was investigated in 2 key studies in mice and rats (NTP, 1983) and a supporting study in mice (Miller et al.,1983). All 3 studies were evaluated against OECD Guideline No. 451 and none of the studies were conducted according to Good Laboratory Practices (GLP). In the key rat (F344/N strain) study, dietary administration of eugenol at concentrations of 0 (control), 3,000, or 6,000 ppm to males and 0 (control), 6,000, or 12,500 ppm to females for 104 weeks did not result in any test article-related changes in mean body weight, mean food consumption, or survival. In addition, there was no evidence of carcinogenicity in either male or female rats compared to controls (NTP, 1983). In the key mouse (B6C3F1 strain) study, dietary administration of eugenol at concentrations of 0 (control), 3,000, or 6,000 ppm did not result in any test article-related changes in mean body weights, mean food consumption, and survival in either males or females (NTP, 1983). The data show that there were increases in the incidences of hepatocellular adenomas, hepatocellular carcinomas, and the combination of hepatocellular adenomas and carcinomas in males (see Table 1). This finding was statistically significant at the low-dose level but not at the high-dose level. Moreover, the incidence of either tumour type alone at the high-dose level was not statistically significantly different from the control group. In female mice, the incidence of hepatocellular adenomas and carcinomas (combined) was statistically significant only at the high-dose; although a dose-response trend was seen, the incidences of adenomas or carcinomas alone were not statistically significant at either dose. Under these experimental conditions, the authors concluded that there was equivocal evidence of carcinogenicity since eugenol caused increased incidences of both carcinomas and adenomas of the liver in male mice at the 3,000 ppm dietary level and because eugenol was associated with an increase in the combined incidences of hepatocellular carcinomas or adenomas in female mice. The authors did not utilise a consideration of historical control data in their conclusion.

Therefore, on the basis of scientific considerations eugenol is concluded not to be carcinogenic in mice or rats, based on the following:

1.   A lack of carcinogenic activity of eugenol in rats in the 2-year oral carcinogenicity study;

2.   A statistically significant increase in hepatocellular tumours in male mice at the low-dose level, but not at the mid- and high-dose level, indicating a lack of a dose-response relationship; 

3.   The incidences of adenomas or carcinomas (combined) in the study were within the historical range for this strain of mouse (i.e., B6C3F1) in both males and females (see Table 1), a strain that is known to have a high and variable incidence of spontaneous hepatocellular tumours (Haseman et al.,1985; Battershill and Fielder, 1998; Haseman et al.,1998). While a statistically significant increase in the combination of hepatocellular adenoma or carcinoma was noted in female mice at the highest dose, the incidence (18%) was within the historical control range of 0 to 20% (Haseman et al.,1985);

4.  Evidence from in vivo studies in mice that eugenol does not bind to DNA (no formation of hepatocellular DNA adducts)(Phillips et al.,1984; Phillips, 1990), likely due to the presence of detoxification processes and/or mechanisms (i.e., glutathione conjugation), as exemplified in the report of Thompson et al. (1991) who noted that incubation of eugenol with isolated rat hepatocytes resulted in the formation of sulphate, glucuronide, and glutathione conjugates. As glutathione stores were depleted, binding to proteins and cytotoxicity ensued.  Conversely, restoration of glutathione by addition of N-acetylcysteine prevented the onset of cytotoxicity;

5.   Metabolism studies indicating that >95% of an administered dose of eugenol is eliminated in the urine as the glucuronide and sulphate conjugates in both rodents and humans, with only minor formation of other metabolites which are, ultimately, also readily conjugated and excreted in the urine, suggesting that rapid metabolism would prevent the formation or prolonged residency of reactive intermediates and/or toxic metabolites at doses that do not saturate detoxification pathways (JECFA, 2006a; JECFA, 2006b); and

6.   A lack of structural alerts for genotoxic and non-genotoxic carcinogenicity.

7. The key and several reliable in vitro and in vivo genotoxicity studies all provide negative result for mutagenicity and clastogenicity.

8. Eugenol is considered to be non-carcinogenic whereas methyl eugenol has consistently been shown to be carcinogenic in animal studies. Methyl eugenol has been shown to be metabolised by allylic 1'-hydroxylation followed by sulphation leading to an unstable genotoxic metabolite. The 1'-hydroxy metabolite is considered to be the proximate carcinogen. Studies using rat, mouse and human liver microsomes have shown that eugenol is less likely to form its' 1'-hydroxy metabolite than is methyl eugenol. Furthermore, in the presence of glucoronide the 1'-hydroxy metabolite of eugenol is so rapidly detoxified that it's presence could not be detected in human or mouse liver S9 and only a very small amount in rat liver S9.

Therefore, it may be concluded that methyl eugenol metabolism leads to the formation of the 1'hydroxy proximate carcinogen whereas eugenol metabolism leads predominantly to a non-genotoxic glucuronide conjugate. Consequently, methyl eugenol is not a valid analogue of eugenol.

Table 1          Historical Liver Tumour Incidences in B6C3F1 Mice Compared to Liver Tumour Incidences From a 103-Week Study in B6C3F1 Mice^a

	Males		Females
Liver Tumour Type	Historical Control Range (%)	(%) Tumour Incidences from NTP (1983) Study for Eugenol; 3,000 ppm^b	Historical Control Range (%)	(%) Tumour Incidences from NTP (1983) Study for Eugenol; 6,000 ppm^c
Adenoma	0 to 44	26	0 to 18	6
Carcinoma	8 to 32	40	0 to 15	12
Adenoma or carcinoma	14 to 58	56	0 to 20	18
Source: Haseman et al.,1985 ^aHistorical control ranges are based on NTP carcinogenesis studies in untreated B6C3F1 mice. ^bValues for males of the low-concentration (i.e., 3,000 ppm) group. Statistically significant increases were not noted in the high-concentration (i.e., 6,000 ppm) group. ^cValues for females of the high-concentration (i.e., 6,000 ppm) group. Statistically significant increases were not noted in the low-concentration (i.e., 3,000 ppm) group.

In the supporting mouse (CD-1 strain) study designed specifically to investigate the development of hepatomas, administration of 0.5% eugenol in the diet (with or without 0.05% phenobarbital in the drinking water) for 20 months did not result in the development of hepatic tumours (Miller et al.,1983). 

Based on the data from the 2 key studies and the supporting study, the evidence indicates that eugenol is not a carcinogen in rodents. This conclusion is consistent with that of the recent review of the toxicology of eugenol conducted by the European Food Safety Authority, which concluded that eugenol is devoid of carcinogenic activity (EFSA, 2009). In its review of eugenol, JECFA (2006b) discussed the hepatic findings in the mouse study and noted not only the sensitivity of the mouse strain, but also the potential effect of other factors such as housing of the animals. JECFA concluded that the hepatic neoplasms in the NTP study are "not relevant to the safety of eugenol in humans".

References

Battershill JM, Fielder RJ. Mouse-specific carcinogens: An assessment of hazard and significance for validation of short- term carcinogenicity bioassays in transgenic mice. Hum Exp Toxicol 1998;17:193-205.

EFSA. Scientific Opinion of 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.

Haseman JK, Hailey JR, Morris RW. Spontaneous neoplasm incidences in Fischer 344 rats and B6C3F(1) mice in two-year carcinogenicity studies: a National Toxicology Program update. Toxicol Pathol 1998;26:428-441.

Haseman JK, Huff JE, Rao GN, Arnold JE, Boorman GA, McConnell EE. Neoplasms observed in untreated and corn oil gavage control groups of F344/N rats and (C56BL/6N X C3H/HeN)F(1) (B6C3F1) mice. J Natl Cancer Inst 1985;75:975-984.

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/

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.

Thompson DC, Constantin-Teodosiu D, Moldeus P. Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes. Chem Biol Interact 1991;77:137-147.

Justification for selection of carcinogenicity via oral route endpoint:
Key studies in rats and mice were concluded to show no evidence of carcinogenicity. No cell transformation data are available for eugenol. Substances similar to eugenol, that have been reported as carcinogenic to rodents (e.g. methyl eugenol) are considered to be non-relevant analogues because of critical differences in their metabolism pathway.