Within Great Britain, just over 3.6 million scientific procedures were conducted on animals in 2009. Although one percent less than 2008, this was the second highest total since current statistics began in 1988. Primate use decreased overall by seven percent, although new world primate use increased substantially. For the first time genetically altered animals outnumbered normal animals. 67% of all procedures did not utilise any form of anaesthesia. The profound animal welfare implications of these factors are discussed.

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Two reconstructed human skin models, Episkin^SM and EpiDerm^TM, have been approved as alternative membranes for skin corrosive/irritation experiments due to their close correlation with animal skin. Such reconstructed human skin models were evaluated as alternative membranes for skin permeation experiments. Seven drugs with different lipophilicities and almost the same molecular weight were used as test penetrants. Relationships were investigated between permeability coefficients (P values) of the seven drugs through six kinds of reconstructed cultured human skin models and human skin. A fairly good relationship in P values was observed between TESTSKIN^TM LSE-high (LSE-high) or EpiDerm^TM and human skin, suggesting that these reconstructed human skin models could be used as alternative membranes for skin permeation experiments. However, the partition parameter, KL, and diffusion parameter, DL^-2 in these reconstructed human skin models were different to those of human skin. Especially, KL values in reconstructed human skin models were very different to those in human skin, even for LSE-high and EpiDerm^TM. Therefore, suitable reconstructed human skin models should be carefully selected on a case by case basis.

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We have developed the human Cell Line Activation Test (h-CLAT) as an in vitro skin sensitization test. In this study, in order to examine whether h-CLAT can predict the skin sensitizing potential of a variety of preservatives, the ring study was performed in two independent laboratories. We selected a total of 16 preservatives, which have been used in cosmetic products at one time, and two different solvents, physiological saline (saline) and dimethyl sulfoxide (DMSO). According to h-CLAT protocol, expression of CD86 and CD54 on THP-1 cells was measured by flow cytometry after 24 h treatment with each chemical at 8 doses. The skin sensitizing potential for 13 of 16 preservatives was correctly predicted in one laboratory and 12 of 16 preservatives in the other. These data indicate relatively high concordance with the animal tests. Furthermore, five preservatives with sensitizing potential were tested using both solvents to compare the effect of saline and DMSO on the predicting capacity of h-CLAT. The sensitizing potential of these preservatives was correctly identified in both saline and DMSO. Our results from this small-scale ring study demonstrate the utility of h-CLAT for detecting the preservatives with skin sensitizing potential.

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We are conducting a Japanese ring study to develop the human Cell Line Activation Test (h-CLAT). The aim of this study is to confirm whether the h-CLAT can predict skin sensitization for oxidative hair dyes. In addition, we studied the effect of test chemical fluorescence on prediction performance. h-CLATs were independently performed in two laboratories for eight chemicals, yielding good reproducibility between the laboratories. Good concordance was obtained between LLNA results and study chemicals. p-Phenylenediamine, which exhibits fluorescence at around 530 nm, which is in the h-CLAT measurement range, was correctly evaluated as positive by h-CLAT. It was possible to compensate for the influence of fluorescence in CD86/CD54 expression measurements, suggesting that substances exhiviting autofluorescence, such as oxidative hair dyes, can also be evaluated correctly. While a notably high, concentration-dependent CD54 expression was observed with Bandrowski's base, this expression was not observed with p-phenylenediamine, a result which suggests that the sensitization responses of these two dyes may differ. In conclusion, it is suggested that h-CLAT is useful for evaluating the skin sensitization potential of oxidative hair dyes.

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A ring study was conducted to examine whether the human Cell Line Activation Test (h-CLAT) can predict the skin sensitization potential of fragrance materials with low solubility and high volatility. Seven fragrance materials which had previously been evaluated for skin sensitization potential in vivo (local lymph node assay; LLNA) were selected. In addition, to investigate the influence of volatility or solubility of test materials, we performed the assay with or without sealing of the plate or supersonic ultrasonic wave treatment of the sample solution. All experiments were performed independently in two laboratories.
The reproducibility between the two laboratories was 100%. The accuracy with respect to LLNA was 86%: diethyl phthalate gave a false positive in the tests of both laboratories. Sealing the plate had little effect on the CV75 (75% cell viability) or CD86/CD54 RFI (relative fluorescence intensity of CD86/54 expression) values. Ultrasonic wave treatment of the sample solution altered the turbidity and CV75 values in some cases, but did not affect the CD86/CD54 RFI values. Our results indicate that h-CLAT is useful for evaluation of the skin sensitization potential of fragrance materials.

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A skin-mimicking model membrane system consisting of a silicone membrane and laminated dialysis membranes as a skin model based on artificial membranes was prepared and used to study a drug interaction involving protein binding. Bovine serum albumin (BSA) solution was placed between the dialysis membranes to mimic the protein leaching in inflamed skin and flurbiprofen (FP), selected as a model drug, was applied to the surface of the laminated membranes. A linear relationship between the FP permeation rate through and the FP concentration in the laminated membranes suggested that the unbound FP concentration in each region can be calculated from the permeation rate in the membrane system. The FP concentration in the laminated membranes decreased depending on the concentration of BSA placed in the membranes. When ketoprofen (KP) was introduced into the microdialysis probe to inhibit the BSA binding of FP during the permeation process, the FP permeation through the laminated membranes and the free concentration in BSA solution in the laminated membranes were increased. This result was due to a competitive interaction of FP and KP involving protein binding. Such simultaneous permeation-binding of two drugs is difficult to study in vivo and in vitro. Our experimental system will help researchers to understand such complicated process and, therefore, reduce the use of experimental animals in the development of new skin products.

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The contents of education at dental colleges and university faculties of dentistry vary among countries. In Japan, dental curriculum are completed in 6 years. The contents of curricula that dental students must undergo are extensive, as they must take many examinations including the national examination for the qualification of dentists. Therefore, animal experiments are performed in a very small scale with limited contents at Japanese dental colleges and faculties of dentistry. However, measures to reduce or substitute contents of animal experiments in undergraduate dental education have begun to be evaluated, as in medical education.

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