Hajime Kojima

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.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

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.

Experimental study on skin sensitization potencies and cross‐reactivities of hair‐dye‐related chemicals in guinea pigs

In screening patch testing of hairdressers with occupational contact dermatitis, multiple positive reactions to hair dye‐related chemicals, such as p‐phenylenediamine (PPD), p‐toluenediamine ·  2HCI (PTD) and p‐aminophenol (PAP), a fabric dye p‐aminoazobenzene (PAB), and a tar dye Sudan III, were frequently encountered. To investigate individual skin sensitization potency and the cross‐reactivities among above chemicals, a guinea pig maximization test with the above 5 chemicals was performed. In each group, 6 animals were induced with one of the chemicals at 0.1% concentration by intradermal injection and at 1.0% by topical application. The animals were challenged with all 5 chemicals in concentrations of dilution by 10 from 0.1% to 0.001%. Under the conditions of 0.1% challenges, similar sensitization potencies were observed in PPD (6/6), PTD (6/6), PAP (5/6) and PAB (6/6) groups, but no positive reactions were elicited in the Sudan III group. The cross‐reactivities to PPD were confirmed in the animals challenged with PTD (6/6), PAP (6/6), PAB (6/6) and Sudan III (3/6). In the PTD‐induced group, positive responses to cross‐challenges were elicited by PPD (5/6), PAP (3/6), PAB (5/6) and Sudan III (1/6). The cross‐reactivities to PAP were observed only with PPD (2/5) and PAB (5/5). PAB‐induced animals responded only to PPD (1/6). The results indicate that all these chemicals except Sudan III are strong sensitizers. Their cross‐reactivities are different in sensitized conditions, respectively. The cross‐reactivities to PPD were higher than those to PTD, PAP and PAB.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (8) Evaluation of cytotoxicity tests on SIRC cells

Two common assays, the neutral red uptake assay (SIRC-NRU) and the crystal violet staining assay (SIRC-CVS), were evaluated as alternatives to the Draize eye irritation test (Draize test).The cytotoxicity of thirty-eight cosmetic ingredients as well as a physiological saline solution was determined on SIRC cells at five to seven laboratories. SIRC-NRU and SIRC-CVS were performed according to the common standard operating procedure (SOP). The 50% effective concentration (EC(50)) was determined for each ingredient. The EC(50) of SIRC-CVS was similar to that of SIRC-NRU, showing a strong correlation (r=0.995). The coefficient of variation (CV) of EC(50) which represents the interlaboratory reproducibility of SIRC-NRU was 32.1%, whereas that of SIRC-CVS was 32.8%. The logarithmically transformed EC(50) values showed a strong correlation with the maximal average Draize total score (MAS) (SIRC-NRU: r=-0.816 (n=30), SIRC-CVS: r=-0.805 (n=29)). Both methods could be applied to water-insoluble substances and dyes. However, strong acids, alkanolamines and alcohols had a tendency to deviate from the linear regression lines which were obtained from the in vivo and in vitro data for both methods in the present study. These results suggest that cytotoxicological testing on SIRC cells may provide an alternative method to the Draize test for cosmetic ingredients.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test

The haemolysis test using sheep red blood cells (RBC) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the test according to the standard operating procedure (SOP). Thirty-eight cosmetic ingredients and isotonic sodium chloride solution were used as test substances in this validation study. The concentrations of the test substances that induced 50% haemolysis (HC(50) value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC(50) values were not obtained for coloured or water-insoluble (turbid) substances. Three acids caused denaturation of haemoglobin leaked from RBC and consequently interfered with the determination of the HC(50) value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation coefficient and Spearmans rank correlation between the HC(50) value and maximum average Draize total score (MAS) were -0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 15 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS50) could be correctly classified by this method. These results suggest that the haemolysis test might be applied to cosmetic ingredients as a screening method to distinguish strong irritants that directly affect the cell membrane permeability and do not disturb spectrophotometrical determination of haemoglobin. In order to evaluate the potential for eye irritation of cosmetic ingredients, a combination of haemolysis with other methods based on different mechanism should be employed to improve the predictability.

Increase of β2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. β-actin, β-catenin, and β1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but β2-integrin expression markedly increased. Graphical abstract THP-1 cells were seeded in T-75, or CVM, or CVM added PMA 10 ng/mL; and β-actin(A), β-catenin(B), β1-integrin(C) and β2-integrin(D) expression were measured (N = 3).

A catch‐up validation study of an in vitro skin irritation test method using reconstructed human epidermis LabCyte EPI‐MODEL24

Three validation studies were conducted by the Japanese Society for Alternatives to Animal Experiments in order to assess the performance of a skin irritation assay using reconstructed human epidermis (RhE) LabCyte EPI‐MODEL24 (LabCyte EPI‐MODEL24 SIT) developed by the Japan Tissue Engineering Co., Ltd. (J‐TEC), and the results of these studies were submitted to the Organisation for Economic Co‐operation and Development (OECD) for the creation of a Test Guideline (TG). In the summary review report from the OECD, the peer review panel indicated the need to resolve an issue regarding the misclassification of 1‐bromohexane. To this end, a rinsing operation intended to remove exposed chemicals was reviewed and the standard operating procedure (SOP) revised by J‐TEC. Thereafter, in order to confirm general versatility of the revised SOP, a new validation management team was organized by the Japanese Center for the Validation of Alternative Methods (JaCVAM) to undertake a catch‐up validation study that would compare the revised assay with similar in vitro skin irritation assays, per OECD TG No. 439 (2010). The catch‐up validation and supplementary studies for LabCyte EPI‐MODEL24 SIT using the revised SOPs were conducted at three laboratories. These results showed that the revised SOP of LabCyte EPI‐MODEL24 SIT conformed more accurately to the classifications for skin irritation under the United Nations Globally Harmonised System of Classification and Labelling of Chemicals (UN GHS), thereby highlighting the importance of an optimized rinsing operation for the removal of exposed chemicals in obtaining consistent results from in vitro skin irritation assays. Copyright

Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators

A previous multi-center validation study demonstrated high transferability and reliability of reactive oxygen species (ROS) assay for photosafety evaluation. The present validation study was undertaken to verify further the applicability of different solar simulators and assay performance. In 7 participating laboratories, 2 standards and 42 coded chemicals, including 23 phototoxins and 19 non-phototoxic drugs/chemicals, were assessed by the ROS assay using two different solar simulators (Atlas Suntest CPS series, 3 labs; and Seric SXL-2500V2, 4 labs). Irradiation conditions could be optimized using quinine and sulisobenzone as positive and negative standards to offer consistent assay outcomes. In both solar simulators, the intra- and inter-day precisions (coefficient of variation; CV) for quinine were found to be below 10%. The inter-laboratory CV for quinine averaged 15.4% (Atlas Suntest CPS) and 13.2% (Seric SXL-2500V2) for singlet oxygen and 17.0% (Atlas Suntest CPS) and 7.1% (Seric SXL-2500V2) for superoxide, suggesting high inter-laboratory reproducibility even though different solar simulators were employed for the ROS assay. In the ROS assay on 42 coded chemicals, some chemicals (ca. 19-29%) were unevaluable because of limited solubility and spectral interference. Although several false positives appeared with positive predictivity of ca. 76-92% (Atlas Suntest CPS) and ca. 75-84% (Seric SXL-2500V2), there were no false negative predictions in both solar simulators. A multi-center validation study on the ROS assay demonstrated satisfactory transferability, accuracy, precision, and predictivity, as well as the availability of other solar simulators.