Eugenol

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of eugenol was evaluated in 2 LLNA studies (one key and one supporting) and one supporting maximization study in guinea pigs. The LLNA studies were compared to OECD Guideline No. 429 while the maximization study was compared to OECD Guideline No. 406. None of the studies were conducted according to Good Laboratory Practices (GLP).

 

In the key LLNA assay, 4 female CBA/Ca mice were administered 25 µL of eugenol to the dorsal surface of both ears at concentrations of 0 (vehicle of DEP:ethanol at a ratio of 1:3), 2.5, 5, 10, 25, 50% once/day for 3 consecutive days (Lalko and Api, 2006). The use of a positive control compound was not reported. The study authors concluded that eugenol is a potential skin sensitizer at concentrations at and above 10.0%. Further details were not reported by the study authors.

 

In a supporting LLNA study, female CBA/JN mice (4/group) were administered 25μL of eugenol to the dorsal surface of both ears at concentrations of 1, 6, 15, or 30% in an acetone:olive oil (4:1) vehicle for 3 consecutive days (Takeyoshi et al.,2004). The use of negative/vehicle controls and a positive control compound was not reported. A positive response was noted at an eugenol concentration of 30%, indicating that the compound is sensitizing at this level. Further details were not reported by the study authors.

 

In a supporting maximization study, 10 female Hartley guinea pigs were given eugenol at a concentration of 5% (in an olive oil vehicle). For the induction phase, eugenol was administered via the intradermal and epicutaneous routes and for the challenge phase via the epicutaneous route (under occlusive conditions) (Takeyoshiet al.,2004). The use of negative/vehicle controls and a positive control compound was not reported. According to the methodology of Magnusson and Kligman, the sensitization response rate was 20%, indicating that eugenol is a mild sensitizer. Further details were not reported by the study authors.

 

Migrated from Short description of key information:
Eugenol demonstrated evidence of skin sensitization potential in mice in a key and supporting local lymph node assay (LLNA) at concentrations of ≥ 10% (Takeyoshi et al., 2004; Lalko and Api, 2006). In addition, a guinea pig maximization test indicated that eugenol was mildly sensitizing at a concentration of 5% (Takeyoshi et al., 2004).

Justification for selection of skin sensitisation endpoint:
Key study in compliance with OECD 429 guideline and GLP

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Under the REACH regulation there is no standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitization. However, guidance document Chapter R.7.a endpoint specific guidance (paragraphs 7.3.5-7.3.9) describes how to use human and non-human data. As regards non-human data there is:

·   No definitive guidance on use of QSARs

      

·   No specific in vitro method

·   The role on LLNA, cytokine fingerprints, total IgE/specific IgE methods are described. However, it is widely appreciated that, in both humans and animals, the accurate evaluation of antibody responses against chemical allergens in the form of hapten–protein conjugates can be technically demanding and highly variable between laboratories.

·      Assessment should be case-by-case

Nonetheless, the registrant recognises that further information can be requested beyond the information mentioned in Annexes VII to X of REACH, if there is a concern that a given substance may constitute a risk to human health or the environment, and further information is needed to clarify such concern. The substance has been identified as a weak skin sensitizer and hence there is potential for a risk of respiratory sensitization effects following inhalation exposure; either as a cause of respiratory sensitization or as a route of exposure to elicit dermal and/or respiratory effects in an individual that is already sensitized via the dermal exposure route.

The case-by-case risk assessment for the potential for respiratory sensitization for this substance is addressed as follows.

Prevalence.Compared with contact allergens, of which a few thousand have been identified, far fewer chemicals have been implicated as having the potential to cause sensitization of the respiratory tract, the number being no more than 80, all are associated with occupational exposures (Kimber and Dearman, 1997; Bakerly et al., 2008; Health and Safety Executive, 2001; Baur, 2013; Baur and Bakehe, 2014).

Mechanistic Factors.Most chemicals are too small to induce an adaptive immune response. To acquire immunogenic potential they must form stable associations with protein (hapten–protein conjugates). Mechanistic chemistry studies have revealed that chemical respiratory allergens can be assigned to one of six electrophilic mechanistic domains, with harder (stronger) electrophilic mechanisms such as acylation being more prevalent than softer (weaker) mechanisms, with the hypothesis being that the harder nucleophile lysine is the favoured biological nucleophile for sensitization of the respiratory tract (Enoch et al., 2012). Eugenol is not-predicted to react with skin proteins directly, but may act as a pro-hapten (OECD toolbox v3.1). The substance or its’ metabolites have been demonstrated to react via covalent binding with a cysteine peptide under the metabolic conditions of the peroxidase peptide reactivity assay (PPRA) (Gerberick, 2009), which is the first step of the adverse outcome pathway (AOP) of sensitization. The possible importance of lysine reactivity (in comparison to cysteine binding) in sensitization of the respiratory tract by chemical allergens is supported by some in chemico andin vitrostudies (Hopkins et al., 2005; Lalko et al., 2011, 2012, 2013b). Hence, as eugenol metabolites preferentially react with cysteine it is unlikely to be a respiratory sensitizer, which seem to be those chemicals that are more likely to react with lysine.

Antigenic Response.A positive response in the LLNA does not imply that a chemical will cause respiratory sensitization because the immune responses induced by contact allergens and chemical respiratory allergens begin to diverge in a qualitative sense after the initial activation of T lymphocytes (Cochrane et al., 2015). Chemical respiratory allergens result in the development of a selective Th2-type immune response characterised by the increased expression of type 2 cytokines such as IL-4, IL-5 and IL-13. In contrast, under the same conditions, skin sensitising chemicals elicit Th1-selective immune responses.

Threshold Effects.There is evidence that thresholds of elicitation can be defined for IgE mediated allergies and in the case of human respiratory sensitization to proteins there is evidence for thresholds even if it is not currently possible to be specific in numerical terms (Basketter et al., 2010, 2012; Peters et al., 2001; Sarlo, 2003).

As an example for chemical respiratory sensitizers, over the past 30 years evidence has accumulated of occupational asthma associated with the use of glutaraldehyde, including the involvement of IgE antibody, and in particular in endoscopy, radiography and pathology suites. The evidence suggests that brief exposures to high levels of glutaraldehyde are required to induce allergic sensitization, this is consistent with the probability that peak exposures to chemicals may drive sensitization (Arts et al., 2006; Vyas et al., 2000).

Experimental Evidence.A study was conducted with patients with confirmed contact allergy to isoeugenol (an analogue of eugenol) or hydroxyisohexyl-3-carboxaldehyde (HICC). These patients were exposed to the chemical to which they were sensitized by inhalation using an exposure chamber, with skin contact being shielded by protective clothing. No significant changes in lung function were observed suggesting the absence of respiratory sensitization (Schnuch et al., 2010). This study provides evidence that individuals already sensitised to a substance via the dermal route do not experience symptoms of sensitization when exposed via the inhalation route. Also of relevance is a study of lung function among employees in the fragrance industry (Dix, 2013). A group of workers exposed to fragrance materials during production or similar operations was compared with a non-exposed control group of office workers. There were no significant differences in lung function as determined by measurement of forced expiratory volume, forced vital capacity of peak expiratory flow (Dix, 2013). The relevance of these studies and other work has been recently reviewed (Basketter and Kimber, 2015).

Consumer Exposure Levels.Irrespective of whether sensitization is acquired via dermal contact or inhalation exposure, current mechanistic understanding of allergic sensitization is such that it can be assumed there will be threshold effects for immunological priming, both in the skin and respiratory tract. This means that for both the skin and respiratory tract there will be levels of exposure below which sensitization will fail to develop (Cochrane et al, 2015). Furthermore, inhalation exposure may, in some circumstances at least, induce immunological tolerance rather than priming or sensitization (Kimber and Dearman, 2002). The consumer exposure levels to the substance are considered to be low.

Worker Exposure.The substance is identified as a skin sensitizer and the workplace environmental controls, standard personal protection equipment and safety procedures (risk management measures) are considered adequate to limit the risk of respiratory sensitization in workers.

Conclusion.The current identified mechanisms of dermal and respiratory sensitization may be sufficiently different, and exposure levels are significantly lower than the probable minimum threshold level required for induction and/or elicitation to prevent any risk of respiratory sensitization. This conclusion is based on various elements of scientific evidence that together constitute a robust argument and obviate the need to conduct further specific studies to investigate the potential for respiratory sensitization of this substance.

References

Arts, J.H.E., Mommers, C., de Heer, C., 2006. Dose-response relationships and threshold levels in skin and respiratory allergy. Crit. Rev. Toxicol. 36, 219–251.

Bakerly, N.D., Morre, V.C., Vellore, A.D., Jaakkola, M.S., Robertson, A.S., Burge, P.S., 2008. Fifteen-year trends in occupational asthma: data from the shield surveillance scheme. Occup. Med. 58, 69-174.

Baur, X., 2013. A compendium of causative agents of occupational asthma. J. Occup. Med. Toxicol. 8, 1-8.

Baur, X., Bakehe, P., 2014. Allergens causing occupational asthma: an evidence based evaluation of the literature. Int. Arch. Occup. Environ. Health 87, 339-363.

Basketter, D.A., Broekhuizen, C., Fieldsend, M., Kirkwood, S., Mascarenhas, R., Maurer, K., Pedersen, C., Rodriguez, C., Schiff, H.-E., 2010. Defining occupational consumer exposure limits for enzyme protein respiratory allergens under REACH. Toxicology 268, 165–170.

Basketter, D.A., Berg, N., Kruszewski, F.H., Sarlo, K., Concoby, B., 2012. Relevance of sensitization to occupational allergy and asthma in the detergent industry. J. Immunotoxicol. 9, 314–319.

Basketter, D.A. and Kimber, I., 2015. Fragrance sensitisers: Is inhalation an allergy risk? Regul. Toxicol. Pharmacol. 73(3): 897-902.

Cochrane, SA., Arts, J.H.E., Ehnes, C., Hindle, S., Hollnagel, H.M., Poole, A., Suto, H., and Kimber, I., 2015. Thresholds in chemical respiratory sensitization. Toxicology, 333, 179-194.

Dix, G.R., 2013. Lung function in fragrance industry employees. Occup. Med. (Lond.) 63, 377-379.

Enoch, S.J., Seed, M.J., Roberts, D.W., Cronin, M.T., Stocks, S.J., Agius, R.M., 2012. Development of mechanism-based structural alerts for respiratory sensitization hazard identification. Chem. Res. Toxicol. 25, 2490–2498.

Gerberick GF1, Troutman JA, Foertsch LM, Vassallo JD, Quijano M, Dobson RL, Goebel C, Lepoittevin JP. Investigation of peptide reactivity of pro-hapten skin sensitizers using a peroxidase-peroxide oxidation system. Toxicol Sci. 2009, 112(1):164-74.

Hopkins, J.E., Naisbitt, D.J., Kitteringham, N.R., Dearman, R.J., Kimber, I., Park, B.K., 2005. Selective haptenation of cellular and extracellular protein by chemical allergens: association with cytokine polarization. Chem. Res. Toxicol. 18, 375–381.

HSE (Health and Safety Executive), 2001. Asthmagen? Critical Assessments of the Evidence for Agents Implicated in Occupational Asthma. UK Health and Safety Executive.

Kimber, I., Dearman, R.J., 1997. Chemical respiratory allergy: an introduction. In: Kimber, I., Dearman, R.J. (Eds.), Toxicology of Chemical Respiratory Hypersensitivity. Taylor & Francis, London, UK, pp. 1-6.

Kimber, I., Dearman, R.J., 2002. Chemical respiratory allergy: role of IgE antibody and relevance of route of exposure. Toxicology 181–182, 311–315.

Lalko, J.F., Kimber, I., Dearman, R.J., Gerberick, G.F., Sarlo, K., Api, A.M., 2011. Chemical reactivity measurements: potential for characterization of respiratory chemical allergens. Toxicol. In Vitro 25, 433–445.

Lalko, J.F., Kimber, I., Gerberick, G.F., Foertsch, L.M., Api, A.M., Dearman, R.J., 2012. The direct peptide reactivity assay: selectivity of chemical respiratory allergens. Toxicol. Sci. 129, 421–431.

Lalko, J.F., Dearman, R.J., Gerberick, G.F., Troutman, J.A., Api, A.M., Kimber, I., 2013a. Reactivity of chemical respiratory allergens in the peroxidase peptide reactivity assay. Toxicol. In Vitro 27, 651–661.

Lalko, J.F., Kimber, I., Dearman, R.J., Api, A.M., Gerberick, G.F., 2013b. The selective peptide reactivity of chemical respiratory allergens under competitive and noncompetitive conditions. J. Immunotoxicol. 10, 292–301.

Peters, G., Johnson, G.Q., Golembiewski, A., 2001. Safe use of detergent enzymes in the workplace. Appl. Occup. Environ. Hyg. 16, 389–396.

Sarlo, K., 2003. Control of occupational asthma and allergy in the detergent industry. Ann. Allergy Asthma Immunol. 90, 32–34.

Schnuch, A., Oppel, E., Oppel, T., Rommelt, H., Kramer, M., Riu, E., Darsow, U., Przybilla, B., Nowak, D., Jorres, R.A., 2010. Experimental inhalation of fragrance allergen in predisposed subjects: effects on skin and airways. Br. J. Dermatol. 162, 598-606.

Vyas, A., Pickering, S.A.C., Oldham, L.A., Francis, H.C., Fletcher, A.M., Merrett, T., Niven, R.M., 2000. Survey of symptoms respiratory function, and immunology and their relation to glutaraldehyde and other occupational exposures among endoscopy nursing staff. Occup. Environ. Med. 57, 752–759.

Migrated from Short description of key information:
A robust evaluation of the available evidence for skin and respiratory sensitization, focussing on the mechanisms of dermal and respiratory sensitization. A conclusion is reached, based on various elements of scientific evidence, that together constitute a robust argument and obviate the need to conduct further specific studies to investigate the potential for respiratory sensitization of this substance.

Justification for selection of respiratory sensitisation endpoint:
The current identified mechanisms of dermal and respiratory sensitization may be sufficiently different, and exposure levels are significantly lower than the probable minimum threshold level required for induction and/or elicitation to prevent any risk of respiratory sensitization. This conclusion is based on various elements of scientific evidence that together constitute a robust argument and obviate the need to conduct further specific studies to investigate the potential for respiratory sensitization of this substance.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

The submission substance has an EC3 value of >2% (EC3 = 5.4%) and a response rate of 20% in a GPMT study. As a result, the substance does meet the criteria for Category 1B classification according to Regulation (EC) No 1272/2008, Annex I section 3.4.

No direct scientific data are available on the substance to address respiratory sensitisation. However, a scientific argument using mechanistic information has been constructed and is detailed in the discussion of the endpoint summary. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.4.