A low cost and quick assay system has been developed using the free-living nematode Caenorhabditis elegans to determine the effects of Kampo medicines (traditional Japanese medicines) on life span. A key characteristic of this system is the use of a fer-15 mutant, which is sterile when grown at 25°C fowing to the production of spermatids that fail to activate into spermatozoa. This prevented the production of progeny, which would otherwise complicate life span analyses. In addition, liquid culture medium permitted easy handling of the test subjects. We employed this system to examine the longevity effects of 26 Kampo medicines and crude drugs. Of these, Rhei Rhizoma was found to extend the life span and acts as an anti-oxidant that suppresses superoxide anion generation from mitochondria.
The European Centre for the Validation of Alternative Methods (ECVAM) is currently supporting formal validation studies of in vitro tests for predicting skin irritancy and corrosivity, including tests employing two human skin models, EPISKINTM and EpiDermTM. When skin models are used to evaluate skin irritancy, it is important that suitable chemical application procedures are used. We evaluated the skin irritancy of 44 chemicals using the postincubation method (10-minutes treatment and 18-hours post-treatment incubation) that we originally developed to predict skin irritancy which is similar to a refined protocol for EPISKINTM proposed in an ECVAM validation study. The sensitivity, specificity, and accuracy obtained with the MTT reduction assay-based prediction model were 77.8%, 76.9%, and 77.3%, respectively while the corresponding values obtained with the interleukin-1α secretion assay-based prediction model were 61.1%, 92.3%, and 79.5%, respectively. Combining these endpoints indicated a clear increase in sensitivity and accuracy to 94.4% and 81.8%, respectively. Vitrolife-SkinTM showed a basic utility for irritancy testing by this method and it is possible to confidently predict skin irritancy, provided that an appropriate chemical application procedure is used.
The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization test based on the enhancement by sensitizers of CD86 and/or CD54 expression on THP-1 cells. The aim of this study is to confirm the transferability and reproducibility of the h-CLAT protocol. Seven Japanese laboratories participated in this h-CLAT ring study. First, two well-known sensitizers (dinitrochlorobenzene (DNCB) and nickel sulfate (Ni)) and one non-sensitizer (sodium lauryl sulfate (SLS)) were evaluated at each laboratory with the same protocol at the same application dose. All laboratories correctly evaluated the skin sensitization potential of these three chemicals. Next, four sensitizers and one non-sensitizer were tested as a second trial. There were two false-negatives (ethylene diamine and eugenol) in some laboratories. Finally, chemicals tested in the second trial were re-evaluated with doses individually determined by each laboratory as a third trial. The results were almost the same as the results obtained when all the laboratories tested the same application doses. These results suggest that for more precise evaluation of difficult samples (e. g., unstable or water-insoluble chemicals), modifications of the protocol and prediction model are needed. However, the protocol was easily transferred to all laboratories and there were only a few false-negatives among 56 tests (8 chemicals at 7 laboratories).
As shown in OECD test guidelines 430 and 431, the human skin epidermal assay and Transcutaneous Electrical Resistance Test (TER) were validated and peer reviewed as an alternative method to corrosivity testing; however, these methods have not been used widely in Japan. The problems related to techniques and evaluation are not clear. Therefore, we performed a validation study of EPI-200 (EpiDermTM), a 3-dimensional cultured epidermal model and Vitrolife-SkinTM, a 3-dimensional cultured skin model made in Japan as a catch-up validation trial of alternatives for skin corrosivity testing using 13 chemicals including a positive control: 10% potassium hydroxide solution in Japan. From the obtained data, we identified the potential of utilizing these models to evaluate the corrosivity of a chemical.