Skin Sensitisation


  1. ECVAM validated test methods
  2. Test methods under validation by ECVAM
  3. Development and optimisation of alternative methods




Skin sensitisers are substances able to elicit an allergic response following contact with the skin, termed allergic contact dermatitis (ACD) in humans.

Sensitisation evolves in two phases: the first phase is the induction of specialised immunological memory in an individual following exposure to an allergen. The second phase is elicitation, i.e. the production of a cell-mediated allergic response by exposure of a sensitised individual to the same allergen.

The assessment of the skin sentisation potential represents an important component of the safety assessment of substances, relevant in the context of several EU regulations aiming at the protection of human health and the environment.

Officially accepted animal test methods for skin sensitization potential assessment include the Mouse Local lymph Node Assay (LLNA) and its non-radioactive modifications (LLNA-DA and the LLNA-BrdU Elisa), the Guinea Pig Maximisation Test by Magnusson & Kligman (GPMT) and the Buehler occluded patch test in the guinea pig (see Table). The mouse and guinea pig methods differ with respect to the endpoints used; whereas the mouse LLNA measures the responses provoked during the induction of sensitisation, the two guinea pig tests measure challenge induced elicitation reactions in previously sensitised animals.

The Local Lymph Node assay is considered a reduction and refinement method compared to the traditional guinea pigs tests since it provides advantages in terms of animal welfare.


Test Method


Test Method

OECD Test Guideline

Skin sensitisation phases

Animal species

Guinea Pig Maximisation Test (GPMT)

B.6 Skin Sensitisation

TG 406

Induction + elicitation

Guinea pig

Buehler Test

B.6 Skin Sensitisation

TG 406

Induction + elicitation

Guinea pig

Local Lymph Node Assay

B.42 Skin Sensitisation: Local Lymph Node Assay

TG 429



Local Lymph Node Assay: DA


TG 442A



Local Lymph Node Assay: BrdU Elisa


TG 442B



At present there are no formally validated and regulatory adopted alternative methods for skin sensitization. Given the complexity of the biological mechanisms underlying the acquisition of skin sensitisation it is proposed that, in the near future, no single non-animal test method will be able to replace the currently used regulatory animal tests, instead a combination of methods addressing key mechanisms of the sensitization adverse outcome pathway (AOP) (*) will be needed to achieve full replacement.

See: Adler S. et al., Alternative (non-animal) methods for cosmetics testing: current status and future prospects – 2010. Report coordinated by the European Centre for the Validation of Alternative Methods. Archives of Toxicology 2011, 85, 367.

(*) For AOP: see OECD Testing of chemicals Series, No. 168: The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins: Part 1Part 2.

Alternative test methods and approaches

1. ECVAM validated test methods

EURL ECVAM is considering mechanistically relevant non-animal test methods that can contribute to reduce and replace the use of laboratory animals for skin sensitisation testing.

At the end of 2012 the EURL ECVAM Scientific Advisory Committee (ESAC) released its opinions on the scientific validity of two methods: the Direct Peptide Reactivity Assay (DPRA) and the KeratinoSens .The DPRA test method underwent a EURL ECVAM coordinated validation study whereas the KeratinoSens  was submitted to EURL ECVAM for peer review upon finalization of an industry-led ring trial. The DPRA addresses the process of haptenation, i.e. the covalent binding of low-molecular weight substances (haptens) to skin proteins which is considered to be the molecular initiating event of the skin sensitisation AOP. The KeratinoSens  addresses the activation of the antioxidant/electrophile response element (ARE)-dependent pathway in keratinocytes, a biological mechanism covered by the second key event of the skin sensitisation AOP. Both test methods provide mechanistic information considered relevant for the assessment of the skin sensitisation potential of chemicals. They are proposed to be used within non-animal integrated approaches for skin sensitisation testing together with complementary information. EURL ECVAM views on the scientific validity of the DPRA and the KeratinoSens  test methods and on their possible regulatory use are provided in the respective EURL ECVAM Recommendations (see DPRA; KeratinoSens  ).

As described in the pdf icon EURL ECVAM Strategy for Replacement of Animal Testing for Skin Sensitisation Hazard Identification and Classification ongoing activities are aimed at exploring how information from the above mentioned methods can be used within integrated approaches to support the identification of skin sensitisers and non-sensitisers and to classify skin sensitisers according to subcategories 1A and 1B of the Globally Harmonised System of Classification and Labelling of Chemicals (GHS).

In relation to EURL ECVAM activities towards refinement and reduction, the scientific validity of the Local Lymph Node Assay (LLNA) was endorsed by the ESAC in 2000. The LLNA provides an alternative method for identifying potential skin sensitising test substances. The LLNA is an in vivo method with the potential to reduce the number of animals required to assess the allergenic contact sensitising activity and offering a substantial refinement (less pain and distress) of the way in which those animals are used compared to other guinea pig methods (i.e. TG 406). The LLNA should be applicable for testing any test substances unless there are properties associated with these materials that may interfere with the accuracy of the LLNA as specified in the testing guideline. 

In April 2007 the ESAC endorsed the scientific validity of the reduced LLNA (r-LLNA), a revised protocol of the full Local Lymph Node Assay, to further reduce the number of animals used to distinguish between skin sensitisers and non-sensitiser chemicals. According to TG 429, the r-LLNA should not be used for hazard classification of skin sensitising test substances when dose-response information is needed since it only provides a positive-negative answer. The performance of the r-LLNA as assessed by the independent peer review has been subsequently confirmed by an evaluation performed by ECVAM on a larger set of LLNA data used to register new chemicals between 1981 and 2008 in the New Chemicals Database, which was used as a notification scheme for new chemical substances, manufactured or imported in the European Union (See: Angers-Loustau A, Tosti L, Casati S. The regulatory use of the Local Lymph Node Assay for the notification of new chemicals in Europe. Regul Toxicol Pharmacol. 2011 Aug;60(3):300-7). 

ECVAM, in collaboration with ICCVAM and JaCVAM, developed internationally harmonised LLNA Performance Standards which were endorsed by ESAC on its meeting in November 2008. The LLNA Performance Standards enable to fasten the evaluation of the validation status of new or modified test methods that are similar to the LLNA (for example non-radioactive variants of the LLNA that might further promote the use of the LLNA worldwide). The LLNA performance standards include: 1) a description of the essential test method components used to judge similarity, 2) a list of reference chemicals to be used for the evaluation and 3) a description of the accuracy and reliability performance values that should be met by the similar method to be considered scientifically valid.  

ECVAM participated and contributed to the ICCVAM-led activities on the evaluation of non-radioactive versions of the LLNA, the LLNA BrdU-ELISA and the LLNA-DA, and to the evaluation of the usefulness and limitations of the LLNA for potency categorization of chemicals (see NICEATM-ICCVAM web site).


Contribution to international adoption of test guidelines for skin sensitisation testing

EURL ECVAM is playing a leading role at the OECD in the development of Test Guidelines (TG) on test methods which underwent formal evaluated by EURL ECVAM. Work is currently being undertaken to consolidate draft TGs for the DPRA and the KeratinoSens  (for this latter in collaboration with the  Swiss Federal Office of Public Health).

In addition, EURL ECVAM is leading a project at the OECD on the development of a Guidance Document on the “Evaluation and Application of Integrated Approaches to Testing and Assessment for Skin Sensitisation” with the ultimate aim to facilitate globally harmonised approaches for skin sensitisation assessment.

In the past, ECVAM supported and contributed to the OECD activities related to the revision of OECD Test Guidelines No. 429 which now includes the r-LLNA, the LLNA performance standards and a revised protocol which reduces animal use by 20% compared to the original protocol.  In addition ECVAM contributed to the adoption by the OECD of two non-radioactive versions of the LLNA, the LLNA:DA (OECD TG 442A) and the LLNA:BrdU Elisa (OECD TG 442B).


Related documentation


Local Lymph Node Assay (LLNA)
  • ESAC statement on the Local Lymph Node Assay pdf icon
  • ESAC Statement on reduced Local Lymph Node Assay (rLLNA) pdf icon
  • ESAC Statement on the Scientific Validity of the LLNA Performance Standards pdf icon 
  • ECVAM Performance Standards for the Local Lymph Node Assay (LLNA) pdf icon 


2. Test methods under validation by ECVAM

EURL ECVAM is currently evaluating mechanistically relevant test methods for their reliability (transferability, within and between laboratory reproducibility) and preliminary predictive capacity in view of their future use as part of integrated approaches for skin sensitization hazard assessment.


The human Cell Line Activation Test (h-CLAT) which was formally evaluated in a EURL ECVAM-coordinated validation study in collaboration with the Japanese Center for the Validation of Alternative Methods (JaCVAM) is currently undergoing peer review. The h-CLAT quantifies the induction of protein markers, associated with DC maturation in vivo, on the surface of Dendritic Cell-like cell lines following exposure to the chemical, it is therefore addressing one of the biological mechanisms covered by key event 3 of the skin sensitisation AOP.



3. Development/ optimisation / improvement of alternative of test methods

From 2005 to 2011 EURL ECVAM has partnered the EU 6th Framework Program sponsored integrated project Sens-it-iv which aim was to develop "in vitro" alternatives to animal tests used for the risk assessment of potential skin or lung sensitizers.  EURL ECVAM was leading WP1 on compound selection and contributing to WP8 on in vitro assay development and to WP9 on dissemination of information and technology transfer. Sens-it-iv aimed to develop in vitro methods, to be used as part of a testing strategy, up to a level to be ready to enter pre-validation. This implies that test methods are developed using an appropriate set of compounds/materials allowing performing a preliminary evaluation of their performance. To achieve this goal, one of the main activities of WP1 was to identify a training set of reference compounds to be used for test method development (link to Sens-it-iv website relevant newsletter). 

In addition to this, in a joint effort with Colipa (now Cosmetics Europe) EURL ECVAM identified a set of chemicals to be used for the development/validation of alternative methods for skin sensitisation (see: Casati S, Aeby P, Kimber I, Maxwell G, Ovigne JM, Roggen E, Rovida C, Tosti L, Basketter D. Selection of chemicals for the development and evaluation of in vitro methods for skin sensitisation testing).

Relevant EURL ECVAM workshop reports

  • The Development of Novel Approaches to the Identification of Chemical and Protein Respiratory Allergens. Progress made from the Conclusions and Recommendations of ECVAM Workshop 60. E.Roggen et al.(2008). ATLA 36, 591-598. pdf icon
  • An Evaluation of Performance Standards and Nonradioactive Endpoints for the Local Lymph Node Assay. ECVAM Workshop Report 65. Basketter et al. (2008). ATLA 36, 243-257 pdf icon
  • Chemical Reactivity Measurement and the Predictive Identification of Skin Sensitisers. ECVAM Workshop Report 64. Gerberick et al. (2008). ATLA 36, 215-242. pdf icon
  • Chemical Respiratory Allergy: Opportunities for Hazard Identification and Characterisation. ECVAM Workshop Report 60. Kimber et al. (2007). ATLA 35, 243-265. pdf icon
  • Skin Sensitisation and Epidermal Disposition: The Relevance of Epidermal Disposition for Sensitisation Hazard Identification and Risk Assessment. ECVAM Workshop Report 59. Basketter et al. (2007). ATLA 35, 137-154. pdf icon
  • Dendritic Cells as a Tool for the Predictive Identification of Skin Sensitisation Hazard. ECVAM Workshop Report 51. Casati et al. (2005). ATLA 33, 47-62 pdf icon