Mitsuteru Masuda

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (1) Overview of the validation study and Draize scores for the evaluation of the tests

A three-step interlaboratory validation of alternative methods to the Draize eye irritation test (Draize test) was conducted by the co-operation of 27 organizations including national research institutes, universities, cosmetic industries, kit suppliers and others. Twelve alternative methods were evaluated using 38 cosmetic ingredients and isotonic sodium chloride solution. Draize tests were conducted according to the OECD guidelines using the same lot of test substances as was evaluated in the alternative tests. Results were as follows. (1) Variation in Draize scores was large near the critical range (maximal average Draize total scores (MAS)=15-50) for the evaluation of cosmetic ingredients. (2) Interlaboratory variation was relatively small for the alternative tests. The mean coefficients of variation (CV%) were less than 50 for all assays except for the hens egg-chorioallantoic membrane test (HET-CAM), chorioallantoic membrane-trypan blue staining test (CAM-TB) and haemoglobin denaturation test (HD). The CV% of these three methods came into the same range as the other tests when non-irritants were excluded from the data analysis. (3) Results for acids (pH of 10% solution <2.5), alkalis (pH of 10% solution >11.5) and alcohols (lower mono-ol) in cytotoxicity tests clearly deviated from the other samples in the comparison of cytotoxicity with Draize results. (4) Pearsons correlation coefficients (r) between results from cytotoxicity tests using serum and MAS were -0.86 to -0.92 for samples excluding acids, alkalis and alcohols. (5) When the samples were divided into liquids and powders, r of CAM-TB increased from 0.71 for all samples to 0.80 and 0.92, respectively. (6) Spearmans rank correlation coefficients between the results of alternative methods and MAS were relatively high (r>0.8) in the case of HET-CAM and CAM-TB. Those for cytotoxicity tests were high if the data for acids, alkalis and alcohols were excluded (SIRC-CVS: r=0.945, SIRC-NRU: r=0.931, HeLa-MTT: r=0.926, CHL-CVS: r=0.880). Exclusion of data for powdered samples also increased the coefficient of HET-CAM and CAM-TB to 0.831 and 0.863, respectively. These results suggest that no single method can constitute an evaluation system applicable to all types of test substances by itself. However, several methods will be useful for the prediction of eye irritation potential of cosmetic ingredients if they are used with clear understanding of the characteristics of those methods.

Study of a rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate.

We have developed an in vivo micronucleus (MN) test that uses rat skin as the target organ. Sample preparation involves cold-treating the epidermis with trypsin, peeling it off with a fine forceps, treating it in hypotonic solution, and staining it with acridine orange (A.O.). We evaluated the assay using mitomycin C (MMC) and methyl methanesulfonate (MMS) as model clastogens, applying them as single and repeat treatments. Both chemicals induced a significant, dose-dependent increase in MN frequency in basal cells. One treatment per day for 3 days was optimal for MN induction.

Biodegradation of 2-sulfonatofatty acid methyl ester (α-SFMe)

The biodegradation pathways of α-SFMe were determined based on changes in the chemical structure of C 14 -α-SFMe by IR, NMR and HPLC analyses in the MITI test. Microbial attack on the surfactant structure was initiated by ω-oxidation to form a carboxyl group and continued with β-oxidation, causing the removal of two carbons at a time, to form a temporary intermediate, monomethyl α-sulfosuccinate. Degradation subsequently occurred by desulfonation

Experimental Study for the Development of an in vitro Test for Contact Allergens

We have previously reported an in vitro hapten-specific sensitization method using Pam-212 cells (in vitro sensitization test) to identify the potential effectiveness of contact allergens. In the present study, we conducted comparison studies of 11 allergens and 2 irritants in order to evaluate the method as an alternative predictive test. The guinea pig maximization test (GPMT) was developed based on the test described by Magnusson and Kligman. Our assay was carried out as follows: we treated Pam-212 cells with 13 test chemical solutions, while T cells and macrophages of BALB/c mice were cultured with hapten-conjugated Pam-212 cells for 5 days. After incubation, 105 T cells were stimulated with mitomycin-C-treated spleen cells conjugated with chemicals. Three days later, the [3H]methyl thymidine incorporation was counted. The results of the GPMT were in agreement with those reported in previous studies except for benzocaine. In our GPMT experiments, benzocaine was negative, but it had been classified as a moderate sensitizer in previous studies. Our assay detected extreme, strong and moderate sensitizers as previously classified by the GPMT. They could be summarized as follows: three of five chemicals classified as moderate sensitizers, and 100% of strong or extreme sensitizers were detected by both the GPMT and the in vitro sensitization test. No irritants showed a positive reaction in our assay. These results support the view that the sensitivity of our in vitro test may be equivalent to that of the GPMT and may be useful as a rapid and objective allergen screening test.

Biodegradation of 2-sulfonato fatty acid methyl ester (α-SFMe) : identification of microorganisms isolated from activated sludge and their capability for degrading α-SFMe

Three bacterial strains, A, B and C, were isolated from activated sludge as 2-sulfonato-fatty-acid-methyl-ester (α-SFMe)-degrading microorganisms. From the results of morphological, physiological and biochemical studies, and analyses of 16S rRNA gene sequences, isolate A was identified as Agrobacterium tumefaciens while B and C were Pseudomonas putida, respectively. To demonstrate their capability for the ultimate biodegradation of α-SFMe, the degradation kinetics have been investigated using C14-α-SFMe and 2-14C-labeled C16-α-SFMe. The biodegradation was determined by measuring dissolved organic carbon (DOC) and released SO42−, in the shake-culture test, and evolved 14CO2 in the modified Organisation for Economic Co-operation and Development (OECD) test. In the shake culture test with C14-α-SFMe, DOC removal was progressive throughout the test. Liberation of inorganic sulfate started after DOC removal and then rapidly increased. During the 14CO2 evolution tests, the mineralization of radiolabeled carbon started quickly and reached about 80% of the initially added radioactivity at the end of the tests. The results obtained indicated that all of the isolates had the capability for ultimately degrading α-SFMe through the oxidation of the alkyl carbons and desulfonation (cleavage of the C-S linkage).