Tsuneaki Nakamura

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.

Validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals.

Short time exposure (STE) test is a cytotoxicity test in SIRC cells (rabbit corneal cell line) that assesses eye irritation potential following a 5-min chemical exposure. This validation study assessed transferability, intra- and inter-laboratory reproducibility, and predictive capacity of STE test in five laboratories (supported by Japanese Society for Alternatives to Animal Experiments). Sodium lauryl sulfate, calcium thioglycolate, and Tween 80 were evaluated, in triplicate, using 5%, 0.5%, and 0.05% concentrations in physiological saline, to confirm transferability. Good transferability was noted when similar mean relative viabilities and rank classifications were obtained in all five laboratories and were comparable to data from test method developing laboratory. Good intra- and inter-laboratory reproducibility was obtained with four assay controls (three solvents and one positive control), and four assay controls and 25 chemicals, respectively. STE irritation category based on relative viability of a 5% solution of 25 blinded test chemicals showed good correlation with Globally Harmonized System (GHS) categories (NI; I: Cat. 1 and 2). The STE prediction model, using relative viability of the 5% and 0.05% solutions, provided an irritation rank (1, 2, or 3) that had a good correlation (above 80%), or predictive capacity, with GHS irritation ranks in all laboratories. Based on these findings, the STE test is a promising alternative eye irritation test that could be easily standardized.

Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients. (2) Chorioallantoic membrane (CAM) test.

A chorioallantoic membrane (CAM) assay evaluates the blood vessel reaction and damage to the CAM of a fertilized hens egg. Two types of CAM assays, the hens egg test-chorioallantoic membrane (HET-CAM) method and the chorioallantoic membrane-trypan blue staining (CAM-TB) method, were evaluated as alternative methods to the Draize eye irritation test (Draize test). The validation project was composed of three test phases in which 10, 15 and 14 test chemicals, respectively, were evaluated. The test procedure of the five independent laboratories was controlled under the same standard operating procedure (SOP). The interlaboratory variation was relatively high for both methods. However, the rank correlation was relatively high among the values obtained by the five laboratories. The variation associated with the CAM-TB method was smaller than that of the HET-CAM method, which requires macroscopic observation, suggesting that the objectivity and quantitativeness differs between the assay systems. The average values using these two methods were compared with the maximum average Draize total score (MAS). The correlation coefficient (r) between the HET-CAM scores and the MAS was 0.688. This suggests that a simple linear regression may not be appropriate for HET-CAM. However, the Spearmans rank correlation coefficient (rs) was relatively high (rs=0.802). In contrast, the CAM-TB test results showed a good correlation with the MAS when the test chemicals were classified according to their physical properties (r=0.801, liquid and r=0.926, powder). These results suggest that both the HET-CAM and CAM-TB methods may present alternative method of evaluation of eye irritation despite problems of interlaboratory reproducibility.

Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

SummaryThe purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.

Immunocytochemical and enzymecytochemical studies on the intracellular transport mechanism of secretory immunoglobulin A and lactoferrin in human salivary glands

The intracellular transport mechanism of secretory immunoglobulin A (sIgA) has been immunocytochemically defined in human submandibular glands. To examine the properties of the intracytoplasmic vesicles which contain IgA, the enzyme labeled antibody method for SC and IgA and Novikoffs method for acid phosphatase (ACPase) activity were employed on the same sections. The intracytoplasmic vesicles containing IgA and SC in the serous acinar cells were free of ACPase activity and were thus distinguishable from lysosomes. Neither IgA nor SC was localized in the secretory granules. Lactoferrin was localized in the secretory granules and glandular lumen of the serous acinar cells, but not in the cytoplasmic vesicles, which were also free of ACPase activity. These findings suggests that the transport of sIgA was performed by intracytoplasmic vesicles and that lactoferrin is discharged from secretory granules into the lumen and finally makes a “rendezvous” with sIgA in the lumen of acinar cells.

Interlaboratory Validation of the In Vitro Eye Irritation Tests for Cosmetic Ingredients. (6) Evaluation of MATREXTM

MATREX(TM) is a test system for evaluating eye irritation potential, using the living dermal model (LDM). The LDM consists of normal human dermal fibroblasts in a contracted collagen lattice, which eventually forms a three-dimensional structure. This system has several advantages. It can be applied to insoluble substances and does not require sterile conditions for operation. In the present study, MATREX was introduced as an alternative to the Draize eye irritation test (Draize test) for cosmetics ingredients. MATREX was evaluated through a three-phase series interlaboratory validation as part of a joint project of the National Institute of Health Sciences (NIHS) and Japan Cosmetic Industry Association (JCIA). Toxicity for LDM was mainly evaluated by cytotoxicity, the indicator was EC(50) (concentration that inhibits the viability of the cell to 50% of control) value. Additionally, MATREX score indicating the grade of cytotoxicity was also introduced in the third phase of the validation study. Both test procedures were controlled under the same standard operating procedure (SOP), at all the participating laboratories. A total of 39 test substances both water-soluble and -insoluble were examined. LDM was applicable to almost all substances that could be evaluated by the Draize test. Furthermore interlaboratory variance was relatively low. The correlation coefficient between the EC(50) value and the maximal average Draize total score (MAS) was -0.672. The MATREX score was closely related to the EC(50) value. Moreover, the MATREX scoring method showed a similar prediction ability for eye irritation potential to the EC(50) method. Thus, the MATREX scoring method, a simplified EC(50) method, appears to be a viable alternative to the current EC(50) measurement method. The present results demonstrate the possibility that the MATREX system would form part of a prediction system of Draize test results.

Second-phase validation study of short time exposure test for assessment of eye irritation potency of chemicals.

A Short Time Exposure (STE) test is a cytotoxicity test that uses SIRC cells (rabbit corneal cell line) to assess eye irritation potency following a 5-min chemical exposure. This second-phase validation study assessed the predictive capacity of the STE test using 40 coded test substances at three laboratories. A Validation Management Team (VMT) then evaluated the predictivity of the STE test for United Nation (UN) Globally Harmonized System (GHS) categories using 63 test substances including the results of the first-phase validation study. The STE test can assess not only the severe or corrosive ocular irritants (corresponding to the UN GHS Category 1) but also non-irritant (corresponding to UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. The predictivity by STE test, however, was insufficient for identification of UN GHS categories (Category 1, Category 2, or Non Category). These results suggest that the STE test can be recommended as an initial step in a top-down approach to identification of severe irritants and test substances that require classification for eye irritation (UN GHS Category 1) as well as an initial step in a bottom-up approach to identification of test substances that do not require classification for eye irritation (UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. On the other hand, the STE test is not considered adequate for the identification of mild or moderate irritants (i.e., UN GHS Categories 2A and 2B) and severe irritants (UN GHS Category 1).

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (11) EYTEXTM

EYTEX(TM) is an in vitro test system for predicting the ocular irritation potential of chemicals and formulations. This method was evaluated as an alternative method to the Draize eye irritation test (Draize test) for the eye irritation potential of several cosmetic ingredients in a three-phase validation study conducted at five to seven laboratories. Thirty-nine test substances were used as coded samples. The test procedures were controlled under the same standard operating procedure (SOP) at all participating laboratories. The interlaboratory coefficient of variation (CV) was 20.8%. The correlation coefficient between EYTEX scores and the maximal average Draize total score (MAS) was 0.313. Irritancy classifications were established based on the results of 54 EYTEX tests and the EYTEX/Draize equivalent was calculated. Thirty-eight EYTEX test results concurred with the results of the Draize test, substantial equivalence was 70.4%. These results indicate that EYTEX provides a rough method of classification rather than providing absolute values. The present results also indicate that EYTEX has the following characteristics: (1) intensely coloured substance may not be compatible; (2) some cationic surfactants may be underestimated; (3) EYTEX can be applied to most test substances under the same conditions as the in vivo tests.

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.