Codification of h-CLAT as an Alternative Skin Sensitization Test
In a nod to the growing global awareness of nature-orientation, environmental protection and animal welfare, Shiseido has established a safety assurance system based on animal-free alternatives to animal experiments and has discontinued the use of animal experiments in its cosmetic and quasi-drug development projects launched after March 2013. Shiseido also has been aggressively and vigorously lobbying relevant public authorities in foreign countries and regions to codify the alternatives to animal experiments as a validated method of experiment.
h-CLAT is the authorized alternative under such circumstances. Shiseido previously had developed its own proprietary alternative sensitization test method and applied for patents to cover the technical findings, which Shiseido has published. In the meantime, Shiseido collaborated with Kao Corporation, which was also undertaking similar studies, in a joint research and successfully developed an in vitro skin sensitization test called human cell line activation test (h-CLAT). After undergoing assessments in studies jointly conducted with other cosmetic companies in Japan and overseas, the test procedure has been fine-tuned and has become a globally accepted test method capable of protecting the safety of consumers worldwide. After a very long and stringent validation (validity verification) process in Europe, the h-CLAT was listed on Economic CO-operation and Development (OECD) test guideline in July 2016. In order to disseminate this test method, Shiseido will offer free of charge its proprietary core technology associated with h-CLAT to all outside institutions for use as a skin sensitization test that is being considered by the OECD guideline. Moreover, to encourage its use by outside contract research organizations, Shiseido will make public further details on the use of this technology and will be happy to answer any questions. Please see the following link for the protocol and principles.
History of Development of h-CLAT
- First presentation by Shiseido (The European Society of Contact Dermatitis)
- Joint research with Kao Corporation began. Technical information was actively made public
- Joint research with Cosmetics Europa began
- Joint research began among seven cosmetic companies under the jurisdiction of the MHLW
- Validation by the European Commission began
- Review by the OECD began. All institutions are permitted to use the method for free in an effort to promote alternatives to animal experiments
- Listed on OECD test guideline
Background and Principles in the Research on h-CLAT
Studies in the early 1990s showed that sensitizers increase the amount of cell surface proteins such as CD86 and MHC class II expressed on Langerhans cells, which play a role in antigen presentation in the skin. Taking note of this finding, Shiseido began developing test methods that apply this phenomenon. Langerhans cells, however, are present in only a very low number in the epidermis and become activated by the isolation procedure alone, thus creating challenges in developing a test that uses these cells. Scientists considered using dendritic cells instead, which have Langerhans cell-like characteristics and can be obtained from the peripheral blood. The need to use blood to prepare dendritic cells and the huge fluctuations in response depending on the donor, however, made it difficult to establish a highly practical test method that uses dendritic cells.
Scientists at Shiseido took note of THP-1, a human monocyte cell line derived from leukemia. Because it is easy to culture, THP-1 has been used in a large array of studies, and its characteristics are well known relative to those of other cell lines. Thus, it was selected as a replacement for dendritic cells.
For the protocol and principles of and references on h-CLAT assay, go to the ECVAM DataBase site below and type in “human cell line activation test” in the search box.
(You must register with ECVAM and create an account to view and download articles)
You may download the protocol evaluation sheet, among other papers.
Information from the ICCVAM Workshop
FAQ on h-CLAT
- Do test results vary depending on the source of THP-1 cells?
- Yes. The THP-1 cells from a particular cell bank are known to have mutations and have become unresponsive to some sensitizers. Please make sure the cells you will be using meet the criteria provided in the protocol.
- Does the positive control (DNCB) or the negative control (lactic acid) always provide stable test results?
- The positive and negative controls have been verified to test positive and negative, respectively, in most cases, unless there are technical or material issues.If your control does not perform as expected, there may be a problem. Please contact us at the following phone number or e-mail address for specifics.
- Do you provide hands-on guidance?
- First, please contact us at the following e-mail address for any problems in conducting the assay.
Inquiries regarding h-CLAT
For inquiries on technical matters:
Quality Assurance Center, Shiseido Co., Ltd.
For inquiries on patents:
Global Patent Group
Research Administration Department, Shiseido Co., Ltd.