Identification of Allergens in Complex Mixtures and Products

  • Elena Giménez-ArnauEmail author
Living reference work entry


Identifying contact allergens among the myriad of chemicals used in commercial products is an extremely arduous task. This is even more difficult in the case of components of complex mixtures such as natural extracts and essential oils. Today, non-animal alternative methods validated to assess the sensitizing potential of chemicals are, overall, unable to completely predict the skin sensitization potential of these complex mixtures of chemicals. Moreover, toxicological profiles are very difficult to extrapolate from the mere sum of the profiles of the individual constituents as required today. In this context, hazard identification remains still one of the best solutions for risk assessment. This chapter presents a brief overview of methods allowing the identification of allergens in complicated chemical mixtures, going from bioassay-guided fractionation and combination with structure-activity relationships studies to more recent new techniques developed to trap and identify reactive intermediates in natural extracts during air oxidation. Practical applications are exposed such as the case of oak moss absolute. Also, the assessment of the sensitization potential of tea tree oil is a typical example of the challenges that toxicologists encounter in the safety evaluation of essential oils. Methods based on chemical reactivity and fluorescence trapping, basically studying the oil after aging and air oxidation, are discussed. Hence, a great deal of progress has been accomplished during the last years for the identification of chemicals culprit of the sensitizing potential of very complex chemical mixtures. That being said, the identification of allergens in complex mixtures and products is still a challenging puzzle.


Bioassay-guided fractionation Structure-activity relationships In chemico methods Fluorescence trapping Chemical reactivity Oak moss absolute Tea tree oil 


  1. 1.
    International Fragrance Association.
  2. 2.
    de Groot AC, Schmidt E (2016) Contact allergy to essential oils: general aspects. In: Essential oils. Contact allergy and chemical composition. CRC Press, Taylor & Francis Group, Boca Raton, pp 21–38Google Scholar
  3. 3.
    European Commission (2003) Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products – 7th Amendment to the European Cosmetics Directive. Off J Eur Union L66: 26–35Google Scholar
  4. 4.
    European Commission (2006) Regulation (EC) No 1907/2006 of the European Parliament and of the Council of the 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency. Off J Eur Union L396: 1–849Google Scholar
  5. 5.
    OECD (2015) OECD guideline for the testing of chemicals: In: Chemico skin sensitization: direct peptide reactivity Assay (DPRA). OECD testing guidelines 442c.
  6. 6.
    OECD (2015) OECD guideline for the testing of chemicals: in vitro skin sensitization: ARE-Nrf2 luciferase test method. OECD testing guidelines 442d.
  7. 7.
    OECD (2016) OECD guideline for the testing of chemicals: human cell line activation test (h-CLAT). OECD testing guidelines 442e.
  8. 8.
    Api AM, Lalko J (2006) Investigation of the dermal sensitization potential of various essential oils in the local lymph node assay. Food Chem Toxicol 44:739–746. Scholar
  9. 9.
    Andres E, Sá-Rocha M, Barrichello C, Haupt T, Ellis G, Natsch A (2013) The sensitivity of the KeratinoSens™ assay to evaluate plant extracts: a pilot study. Toxicol In Vitro 27:1220–1225. Scholar
  10. 10.
    Lang M, Giménez-Arnau E, Lepoittevin JP (2017) Is it possible to assess the allergenicity of mixtures based on in chemico methods? Preliminary results on common fragrance aldehydes. Flavour Fragr J 32:63–71. Scholar
  11. 11.
    Pieters L, Vlietinck AJ (2005) Bioguided isolation of pharmacologically active plant components, still a valuable strategy for the finding of new lead compounds? J Ethnopharmacol 100:57–60. Scholar
  12. 12.
    Houghton PJ, Raman A (1998) Laboratory handbook for the fractionation of natural extracts, 2nd edn. Springer, BerlinCrossRefGoogle Scholar
  13. 13.
    Mutterer V, Giménez-Arnau E, Lepoittevin JP, Johansen JD, Frosch PJ, Menné T, Andersen KE, Bruze M, Rastogi SC, White IR (1999) Identification of coumarin as the sensitizer in a patient sensitive to her own perfume but negative to the fragrance mix. Contact Dermatitis 40:196–199. Scholar
  14. 14.
    Frosch PJ, Pirker C, Rastogi SC, Andersen KE, Bruze M, Svedman C, Goossens A, White IR, Uter W, Giménez-Arnau E, Lepoittevin JP, Menné T, Johansen JD (2005) Patch testing with a new fragrance mix detects additional patients sensitive to perfumes and missed by the current fragrance mix. Contact Dermatitis 52:207–215. Scholar
  15. 15.
    Frosch PJ, Rastogi SC, Pirker C, Brinkmeier T, Andersen KE, Bruze M, Svedman C, Goossens A, White IR, Uter W, Giménez-Arnau E, Lepoittevin JP, Johansen JD, Menné T (2005) Patch testing with a new fragrance mix – reactivity to the individual constituents and chemical detection in relevant cosmetic products. Contact Dermatitis 52:216–225. Scholar
  16. 16.
    Dharmagunawardena B, Takwale A, Sanders KJ, Cannan S, Rodger A, Ilchyshyn A (2002) Gas chromatography: an investigative tool in multiple allergies to essential oils. Contact Dermatitis 47:288–292. Scholar
  17. 17.
    de Groot AC, Weijland JW (1992) Systemic contact dermatitis from tea tree oil. Contact Dermatitis 27:279–280. Scholar
  18. 18.
    Lepoittevin JP (2011) Molecular aspects in allergic and irritant contact dermatitis. In: Johansen JD, Frosch PJ, Lepoittevin JP (eds) Contact dermatitis, 5th edn. Springer, Berlin, pp 91–110CrossRefGoogle Scholar
  19. 19.
    Kao D, Chaintreau A, Lepoittevin JP, Giménez-Arnau E (2014) Mechanistic studies on the reactivity of sensitizing allylic hydroperoxides: investigation of the covalent modification of amino acids by carbon-radical intermediates. Toxicol Res 3:278–289. Scholar
  20. 20.
    Uter W, Geier J, Frosch P, Schnuch A (2010) Contact allergy to fragrances: current patch test results (2005–2008) from the Information Network of Departments of Dermatology. Contact Dermatitis 63:254–261. Scholar
  21. 21.
    Schulz H, Albroscheit G (1989) Characterization of oak moss products used in perfumery by high-performance liquid chromatography. J Chromatogr 466:301–306. Scholar
  22. 22.
    Thune P, Solberg Y, McFadden N, Staerfelt F, Sandberg M (1982) Perfume allergy due to oak moss and other lichens. Contact Dermatitis 8:396–400. Scholar
  23. 23.
    Bernard G, Giménez-Arnau E, Rastogi SC, Heydorn S, Johansen JD, Menné T, Goossens A, Andersen KE, Lepoittevin JP (2003) Contact allergy to oak moss: search for sensitizing molecules using combined bioassay-guided chemical fractionation, GC-MS, and structure-activity relationship analysis. Arch Dermatol Res 295:229–235. Scholar
  24. 24.
    Johansen JD, Bernard G, Giménez-Arnau E, Lepoittevin JP, Bruze M, Andersen KE (2006) Comparison of elicitation potential of chloroatranol and atranol–2 allergens in oak moss absolute. Contact Dermatitis 54:192–195. Scholar
  25. 25.
    Johansen JD, Andersen KE, Svedman C, Bruze M, Bernard G, Giménez-Arnau E, Rastogi SC, Lepoittevin JP, Menné T (2003) Chloroatranol, an extremely potent allergen hidden in perfumes: a dose-response elicitation study. Contact Dermatitis 49:180–184. Scholar
  26. 26.
    Rastogi SC, Bossi R, Johansen JD, Menné T, Bernard G, Giménez-Arnau E, Lepoittevin JP (2004) Content of oak moss allergens atranol and chloroatranol in perfumes and similar products. Contact Dermatitis 50:367–370. Scholar
  27. 27.
    International Fragrance Association (2008) Code of practice 43rd amendment. Standard on oak moss extracts.
  28. 28.
    European Commission, Scientific Committee on Consumer Products (2004) Atranol and chloroatranol present in natural extracts (e.g. oak moss and tree moss extract).
  29. 29.
    European Commission, Scientific Committee on Consumer Products (2008) Opinion on oak moss/tree moss (sensitisation only). risk/committees/04_sccp/docs/sccp_o_131.pdf
  30. 30.
    Nardelli A, Giménez-Arnau E, Bernard G, Lepoittevin JP, Goossens A (2009) Is a low content in atranol/chloroatranol safe in oak moss-sensitized individuals? Contact Dermatitis 60:91–95. Scholar
  31. 31.
    Mowitz M, Zimerson E, Svedman C, Bruze M (2013) Patch testing with serial dilutions and thin-layer chromatograms of oak moss absolutes containing high and low levels of atranol and chloroatranol. Contact Dermatitis 69:342–349. Scholar
  32. 32.
    Commission Regulation (EU) (2017) 2017/1410 of 2 August 2017 amending Annexes II and III to Regulation (EC) No 1223/2009 of the European Parliament and on the Council on cosmetic products. Off J Eur Union L202: 1–2Google Scholar
  33. 33.
    Chittiboyina AG, Avonto C, Rua D, Khan IA (2015) Alternative testing methods for skin sensitization: NMR spectroscopy for probing the reactivity and classification of potential skin sensitizers. Chem Res Toxicol 28:1704–1714. Scholar
  34. 34.
    Avonto C, Chittiboyina AG, Rua D, Khan IA (2015) A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers. Toxicol Appl Pharmacol 289:177–184. Scholar
  35. 35.
    de Groot AC, Schmidt E (2016) Tea tree oil. In: Essential oils. Contact allergy and chemical composition. CRC Press, Taylor & Francis Group, Boca Raton, pp 809–824Google Scholar
  36. 36.
    Scientific Committee on Consumer Products (2008) Opinion en tea tree oil, SCCP/1155/08Google Scholar
  37. 37.
    Aptula A, Patlewicz G, Roberts D (2005) Skin sensitization: reaction mechanistic applicability domains for structure-activity relationships. Chem Res Toxicol 18:1420–1426. Scholar
  38. 38.
    Karlberg A-T, Bergstrom MA, Borje A, Luthman K, Nilsson JL (2008) Allergic contact dermatitis-formation, structural requirements, and reactivity of skin sensitizers. Chem Res Toxicol 21:53–69. Scholar
  39. 39.
    Avonto C, Chittiboyina AG, Wang M, Vasquez Y, Rua D, Khan IA (2016) In chemico evaluation of tea tree essential oils as skin sensitizers: impact of the chemical composition on aging and generation of reactive species. Chem Res Toxicol 29:1108–1117. Scholar
  40. 40.
    Mori HM, Iwahashi H (2013) Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavine mononucleotide and ferrous ion. Free Radic Res 47:657–663. Scholar
  41. 41.
    Boche J, Runquist O (1968) Kinetics of the thermal rearrangement of ascaridole. J Org Chem 33:4285–4286. Scholar
  42. 42.
    Christoffers WA, Blömeke B, Coenraads P-J, Schuttelar M-LA (2013) Co-sensitization to ascaridole and tea tree oil. Contact Dermatitis 69:181–191. Scholar
  43. 43.
    Rudzki E, Rapiejko P, Rebandel P (2003) Occupational contact dermatitis, with asthma and rhinitis, from camomile in a cosmetician also with contact urticaria from both camomile and lime flowers. Contact Dermatitis 49:162. Scholar
  44. 44.
    Avonto C, Rua D, Lasonkar PB, Chittiboyina AG, Khan IA (2017) Identification of a compound isolated from German chamomile (Matricaria chamomilla) with dermal sensitization potential. Toxicol Appl Pharmacol 318:16–22. Scholar
  45. 45.
    Hansen L, Hammershøy O, Boll PM (1986) Allergic contact dermatitis from falcarinol isolated from Schefflera arboricola. Contact Dermatitis 14:91–93. Scholar
  46. 46.
    Avonto C, Chittiboyina AG, Sadrieh N, Vukmanovic S, Khan IA (2018) In chemico skin sensitization risk assessment of botanical ingredients. J Appl Toxicol 38:1047–1053. Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Dermatochemistry LaboratoryUniversity of Strasbourg-Institut de Chimie, Institut le Bel, CNRS UMR 7177StrasbourgFrance

Personalised recommendations