Search

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 μm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.

Three-dimensional (3D) cultured hepatocyte capable of maintaining liver-specific function in an in vivo state over a relatively long period of time have drawn attention as a new method for evaluating the metabolic process, hepatotoxicity and enzyme induction potential of drugs. When human hepatocytes were seeded on a plate for spheroid formation, and cell morphology and albumin secretion were examined, hepatocyte spheroid was stably maintained for at least 21 days after seeding. As a result of drug exposure to this spheroid, sequential metabolic reactions by Phase I and Phase II enzymes and metabolic reactions peculiar to only humans were observed. Moreover, when several drugs were exposed to spheroids and hepatotoxicity was evaluated, stable values were obtained for the 50% inhibitory concentration (IC50) of albumin secretion at 14 and 21 days. The IC50 values of most of the tested drugs were lower than in conventional assays, suggesting that the reported evaluation methods might underestimate hepatotoxicity. Furthermore, examination of mRNA expression level and activity of various cytochrome P450 (CYP) after exposure of typical inducers of CYPs to hepatocyte spheroid resulted in a significant increase in the expression level and activity of each. From these results, it was shown that this 3D hepatocyte spheroid system is suitable for follow-up of metabolic processes, long-term tests of hepatotoxicity and enzyme activity induction potential of drugs.

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein-derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar-concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight-concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar-concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar-concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight-concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu2+ promoted NAC oxidation significantly. When 0.25 μm of EDTA was added in the presence of Cu2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

The NanoCulture Plate (NCP) is a novel microstructural plate designed as a base for the three-dimensional culture of cells/tissues. This study examined whether or not the metabolic capability of human primary hepatocytes is well maintained during culture on NCPs. The hepatocytes formed aggregates after seeding and their ATP content was well maintained during culture for 21 days. Expression of CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 mRNAs was detected throughout the 21-day culture period. Addition of CYP substrate drugs (midazolam, diclofenac, lamotrigine and acetaminophen) resulted in the formation of multiple metabolites with a corresponding decrease in the amounts of the unchanged compounds. The inducers omeprazole, phenobarbital and rifampicin increased the levels of CYP1A2, 2B6 and 3A4 mRNAs by 110-fold, 12.5-fold and 5.4-fold, respectively, at day 2, compared with control human hepatocytes. CYP activities were also increased at 2 days after inducer treatment (CYP1A2, 2.2-fold; CYP2B6, 20.6-fold; CYP3A4, 3.3-fold). The results indicate that the hepatocyte spheroids on NCP have detectable and inducible metabolic abilities during the 7-day culture period.

In vitro safety tests may be used as replacements for animal tests owing to their accuracy and high-throughput performance. However, several in vitro skin sensitization tests produce false-negative results such as acid anhydride. Here, we investigated the relationship between false-negative results of acid anhydride and its hydrolysis by aqueous vehicle. Differences in the pattern of hydrolysis for phthalic anhydride (PAH) due to addition of 1 drop of stock solution of PAH in liquid paraffin (LP) dispersion medium and PAH in DMSO were analyzed in a cell-free system. The results showed that use of LP dispersion medium stabilized the concentration of PAH in water over 5min by sustained-release, although almost all PAH converted to phthalic acid in water within 5min using DMSO. Additionally, treatment of THP-1 cells with PAH and phthalic acid using LP dispersion medium for 5min resulted in a 32-fold increase in IL-8 release for PAH as compared with that in the vehicle control. In contrast, for PAH using aqueous vehicle and phthalic acid using LP dispersion medium, there were no significant increases in IL-8 release. Similarly, using LP dispersion medium, trimellitic anhydride significantly increased IL-8 release was observed.

Some drugs induce cytochrome P450s (CYPs), and thus may cause increased metabolic toxicity from concomitantly administered agents. Hence, we need a means of evaluating the potential of compounds to cause drug-induced liver injury (DILI) under conditions where inducers of CYP1A2 are present. Here, we present a system for evaluating CYP1A2-mediated metabolic toxicity using three-dimensional (3D) cultures of primary human hepatocyte spheroids treated with the CYP1A2 inducer omeprazole (OPZ). As a test substrate, we employed dacarbazine (DTIC), which causes toxicity during the metabolic process. We measured cell viability, CYP1A2 mRNA expression level and metabolism of DTIC, as well as several markers of hepatic function, i.e. albumin secretion, urea secretion, and aspartate aminotransferase (AST) leakage. Markers of hepatic function were significantly decreased by addition of OPZ and DTIC even under conditions where the cell viability was largely unchanged. This experimental system sensitively detected CYP1A2-mediated metabolic toxicity. Therefore, the developed system should be helpful for evaluating the potential of compounds to cause DILI under conditions where inducers of CYP1A2 are present.

The human cell line activation test (h-CLAT) is a skin sensitization test that measures the expression of cell surface proteins CD86 and CD54 to evaluate the skin sensitization potential of test chemicals. However, some skin irritants have been reported to induce dramatically high CD54 expression leading to false-positive h-CLAT results. Furthermore, CD54 expression is strongly induced by cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, or danger signals that activate its signaling pathways. In this study, we focused on the relationship between CD54 expression and the Nucleotide binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome, a protein complex that plays a pivotal role in intra-cellular inflammation. We observed the activation of caspase-1 and production of IL-1β after exposure of THP-1 cells to 2,4-dinitrochlorobenzene (DNCB, sensitizer), octanoic acid (OA, non-sensitizer), and salicylic acid (SA, non-sensitizer), implying NLRP3 activation. These observations confirmed the activation of the inflammasome by CD54-only positive chemicals. CD54 expression, induced by OA and SA, was suppressed by potassium chloride, a typical inhibitor of NLRP3 inflammasome activation. These results suggested that the NLRP3 inflammasome may be activated in THP-1 cells resulting in the expression of CD54, and subsequently leading to false-positive results.

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.

Recently, animal testing has been affected by increasing ethical, social, and political concerns regarding animal welfare. Several in vitro safety tests for evaluating skin sensitization, such as the human cell line activation test (h-CLAT), have been proposed. However, similar to other tests, the h-CLAT has produced false-negative results, including in tests for acid anhydride and water-insoluble chemicals. In a previous study, we demonstrated that the cause of false-negative results from phthalic anhydride was hydrolysis by an aqueous vehicle, with IL-8 release from THP-1 cells, and that short-time exposure to liquid paraffin (LP) dispersion medium could reduce false-negative results from acid anhydrides. In the present study, we modified the h-CLAT by applying this exposure method. We found that the modified h-CLAT is a promising method for reducing false-negative results obtained from acid anhydrides and chemicals with octanol-water partition coefficients (LogKow) greater than 3.5. Based on the outcomes from the present study, a combination of the original and the modified h-CLAT is suggested for reducing false-negative results. Notably, the combination method provided a sensitivity of 95% (overall chemicals) or 93% (chemicals with LogKow > 2.0), and an accuracy of 88% (overall chemicals) or 81% (chemicals with LogKow > 2.0). We found that the combined method is a promising evaluation scheme for reducing false-negative results seen in existing in vitro skin-sensitization tests. In the future, we expect a combination of original and modified h-CLAT to be applied in a newly developed in vitro test for evaluating skin sensitization.

It was believed that high molecular weight molecules including proteins cannot penetrate the skin. However, protein penetration through disrupted/ruptured skin has been reported recently, thus carrying the potential for inducing an allergic response. We used the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, to assess the allergic potential of proteins by measuring levels of CD86 and CD54 in the human monocytic leukemia cell line THP-1. Six allergens including ovalbumin (OVA) and human serum albumin (HSA; negative control) upregulated CD86 and/or CD54; a false-positive result was obtained using HSA. This was caused by lipopolysaccharide (LPS) contamination. Naturally derived materials often include LPS at various concentrations and may influence protein induction of CD86 and CD54. Additionally, polymyxin B, an LPS inhibitor, could not completely overcome the effect of LPS. Therefore, if test proteins contain ≥0.1 EU/mL LPS, their allergenic potency will not be assessed accurately using h-CLAT. These data show that naturally occurring materials or those derived from living organisms should be evaluated for their LPS content. It is important to confirm the applicability of in vitro methods such as h-CLAT for assessing the allergenic potency of naturally occurring proteins; our findings can be a foundation for future studies.

We recently developed a novel Vitrigel‐eye irritancy test (EIT) method. The Vitrigel‐EIT method is composed of two parts, i.e., the construction of a human corneal epithelium (HCE) model in a collagen vitrigel membrane chamber and the prediction of eye irritancy by analyzing the time‐dependent profile of transepithelial electrical resistance values for 3 min after exposing a chemical to the HCE model. In this study, we estimated the predictive performance of Vitrigel‐EIT method by testing a total of 118 chemicals. The category determined by the Vitrigel‐EIT method in comparison to the globally harmonized system classification revealed that the sensitivity, specificity and accuracy were 90.1%, 65.9% and 80.5%, respectively. Here, five of seven false‐negative chemicals were acidic chemicals inducing the irregular rising of transepithelial electrical resistance values. In case of eliminating the test chemical solutions showing pH 5 or lower, the sensitivity, specificity and accuracy were improved to 96.8%, 67.4% and 84.4%, respectively. Meanwhile, nine of 16 false‐positive chemicals were classified irritant by the US Environmental Protection Agency. In addition, the disappearance of ZO‐1, a tight junction‐associated protein and MUC1, a cell membrane‐spanning mucin was immunohistologically confirmed in the HCE models after exposing not only eye irritant chemicals but also false‐positive chemicals, suggesting that such false‐positive chemicals have an eye irritant potential. These data demonstrated that the Vitrigel‐EIT method could provide excellent predictive performance to judge the widespread eye irritancy, including very mild irritant chemicals. We hope that the Vitrigel‐EIT method contributes to the development of safe commodity chemicals. Copyright © 2015 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd., The sensitivity, specificity and accuracy of Vitrigel‐EIT method in comparison to GHS were 90.1%, 65.9% and 80.5%, respectively. In case of eliminating nine chemicals showing pH 5 or lower, those were improved to 96.8%, 67.4% and 84.4%, respectively. Meanwhile, nine of 16 false‐positive chemicals were classified irritant by EPA and immunohistologically confirmed to have an eye irritant potential. These data demonstrated that the Vitrigel‐EIT method could provide excellent predictive performance to judge the widespread eye irritancy, including mild irritant chemicals.

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events.

In vitro generated human skin equivalents are generating interest as promising tools in basic study, as alternatives to animal testing, and for clinical applications in regenerative medicine. For prediction of skin irritation and corrosion, three-dimensional human skin equivalents consisting of differentiated human keratinocytes (KCs) have been developed and some models have been internationally accepted. However, more delicate assessments using full-thickness skin models, such as skin sensitization tests, cannot be performed due to the lack of a dermis containing fibroblasts or appendages. In a previous study, we developed dermo-epidermal human skin equivalents (DESEs) using a cell coating technique, which employs cell surface coating by layer-by-layer assembled extracellular matrix (ECM) films. The DESEs with dermis consisting of normal human dermal fibroblasts (NHDFs) and epidermis consisting of human KCs were easily fabricated by using this technology. In this study, the constructed DESEs were evaluated as an alternative skin for skin permeation and irritation tests. A good relationship of permeability coefficient of chemicals was observed between the DESEs and human skin data. We investigated whether the DESEs, a new in vitro skin model, are capable of identifying skin irritant and nonirritant substances among 20 reference chemicals. It was confirmed that the DESEs are applicable to skin irritation testing as defined in the European Centre for the Validation of Alternative Methods (ECVAM) Performance Standard (OECD Test Guideline 439). We further studied the construction of DESEs with density-controlled blood capillary networks using human umbilical vein endothelial cells (HUVECs). The results suggest that DESEs allowing incorporation of skin appendages are more promising alternatives to animal testing and can be applied to the design of physiologically relevant in vitro skin models.

The reactive oxygen species (ROS) assay was developed by Onoue and Tsuda (Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances. Pharm Res. 2006;23:156–64) and is a high-throughput, high-performance system for predicting the phototoxic potential of pharmaceutical substances. This assay has been proposed for use as a component in an integrated photosafety testing strategy to evaluate the phototoxicity potential of pharmaceuticals and other test substances. The reproducibility and predictivity of the ROS assay is sufficient to support its use in an integrated photosafety testing and decision strategy for drug research and development. In this strategy, negative results in the ROS assay would not require further testing in animals or other tests; positive, weakly positive, and inconclusive results would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity Assay (OECD. Test Guideline 432), as accepted by ICH S10 in 2013. The use of the ROS assay could potentially provide significant savings in time, cost, and reduced animal use for photosafety assessments.

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.

We previously reported that the time-dependent relative changes of transepithelial electrical resistance (TEER) after exposing four different chemicals to a human corneal epithelium (HCE) model were well correlated to the potential of ocular irritancy. Meanwhile, we recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils equivalent to connective tissues in vivo as a three-dimensional culture tool. The CVM chamber is useful for biomedical assays and immunohistology using cryosections that are inappropriate to be performed using the conventional Millicell chamber with a polyethylene terephthalate membrane. In this study, we aimed to develop a new eye irritancy test (EIT) method called "Vitrigel-EIT method" that can facilitate to briefly and accurately estimate the widespread irritancy of test chemicals by applying the TEER assay system to a HCE model fabricated in the CVM chamber. HCE-T cells (a HCE-derived cell strain) were cultured in the CVM chamber for 6 days, and consequently, the Vitrigel-HCE model possessing the following characteristics of HCE in vivo was formed: six cell layers with specific protein expressions and their barrier function. Time-dependent profiles of TEER values after exposing 30 test chemicals to the HCE model were converted into the scores of three indexes (time lag, intensity, and plateau level), and each chemical was successfully classified into irritant or nonirritant category by utilizing the criteria for the indexes, resulting in the excellent correlation with Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification (sensitivity: 100%, specificity: 75%, accuracy: 90%). These data suggest that the widespread eye irritancy of chemicals can be predicted without false negatives by the Vitrigel-EIT method. Interestingly, the disruption of tight junctions was immunohistologically observed after exposing not only irritants but also three compounds classified as nonirritant by GHS but found positive in our Vitrigel-EIT method confirming a possible mild irritant property.

Introduction The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. Methods The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. Results The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. Discussion In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

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 x 2HCl (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.

The neutral red uptake (NRU) assay was included as part of the COLIPA international validation trial of in vitro alternatives to the Draize eye irritation test. In a blind trial, 55 substances were tested at four laboratories. Following testing, a prediction of the in vivo Draize modified maximum average score (MMAS) for each substance was made by each laboratory using a prediction model relating mean NR(50) value (concentration causing 50% reduction in NRU from that of untreated control cells) to MMAS. Following statistical analysis of the results and breaking of the code, assessment of the results and further analysis was carried out by the participating laboratories. This paper presents the conclusions with regard to the NRU assay. The initial trial analysis indicated that the interlaboratory reproducibility of results of the NRU assay was good. However, there was a poor correlation between observed and predicted MMAS (using the proposed prediction model) when all the test substances were analysed together (r=0.246). Data analysis of subsets of substances indicated that the best predictions were for pure surfactants only (r=0.843) although this data did not fit within the limits of the prediction model. The NRU assay therefore appears to have limited use as a complete Draize replacement. A further examination of the COLIPA trial data may identify combinations of assays which may be more useful than the individual assays which, like NRU, have been shown to be poor predictors of eye irritation.

The present interlaboratory validation study was performed in order to evaluate the use of Chinese hamster lung cell lines that employs crystal violet staining (CHL–CVS) as an alternative cytotoxicity test to the Draize eye irritation test (Draize test) for cosmetic ingredients. Ten substances, nine of which were surfactants, were evaluated at seven laboratories in the first phase of the validation study; 15 substances including dyes and lipids were evaluated at seven laboratories in the second phase of the validation study; 14 substances including acids and alkalis were evaluated at four laboratories in the third phase of the validation study. The logEC50 values obtained for CHL–CVS were compared with the maximal average Draize total score (MAS) for a 10% (w/v) solution of 38 cosmetic ingredients as well as isotonic sodium chloride solution. The interlaboratory coefficient of variation (CV) for EC50s was 35.6%, which was considered to be within a tolerable range. The correlation coefficient and the Spearman's rank correlation coefficient between the in vitro and in vivo tests were −0.729 and 0.709, respectively. The prediction ability of the proposed method was assessed from the linear regression line for a MAS cut-off point of 15. According to this analysis, four substances (two alcohols and two acids) were determined to be false negative. The present study revealed the following characteristic factors of this method: (1) CHL–CVS could be applied to all the test substances including dyes and lipids in this study; (2) The results for medium-insoluble substances varied according to the laboratory; (3) The correlation between the in vivo and in vitro data for acids and alcohols (lower mono-ol) differed from that of the other substances. These results suggested that the CHL–CVS might have a potential to predict the Draize MAS if definite criteria can be established for the compounds to be applicable.

A reactive oxygen species (ROS) assay was previously developed for photosafety evaluation of pharmaceuticals, and the present multi-center study aimed to establish and validate a standard protocol for ROS assay. In three participating laboratories, two standards and 42 coded chemicals, including 23 phototoxins and 19 nonphototoxic drugs/chemicals, were assessed by the ROS assay according to the standardized protocol. Most phototoxins tended to generate singlet oxygen and/or superoxide under UV–vis exposure, but nonphototoxic chemicals were less photoreactive. In the ROS assay on quinine (200 µm), a typical phototoxic drug, the intra- and inter-day precisions (coefficient of variation; CV) were found to be 1.5–7.4% and 1.7–9.3%, respectively. The inter-laboratory CV for quinine averaged 15.4% for singlet oxygen and 17.0% for superoxide. The ROS assay on 42 coded chemicals (200 µm) provided no false negative predictions upon previously defined criteria as compared with the in vitro/in vivo phototoxicity, although several false positives appeared. Outcomes from the validation study were indicative of satisfactory transferability, intra- and inter-laboratory variability, and predictive capacity of the ROS assay. Copyright © 2012 John Wiley & Sons, Ltd.

The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5-bromo-2′-deoxyuridine (BrdU) incorporation (LLNA:BrdU-ELISA). The LLNA:BrdU-ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU-ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut-off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU-ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd.

In November 2005, the Japanese Center for the Validation of Alternative Methods (JaCVAM) was established as a part of the Division of Pharmacology at the National Center for Biological Safety and Research affiliated with Japan's National Institute of Health Sciences (NIHS). JaCVAM facilitates the validation, peer-review, and international harmonization of alternative to animals testing. Key objectives of JaCVAM are: 1) facilitate 3R's(*), prioritizing Reduction and Replacement, and 2) to ensure new test methods are validated, peer reviewed, officially accepted by the regulatory agencies, and made internationally compatible. In this paper, JaCVAM's current activities and future directions are shown in the validation and peer review of alternatives to testing for skin irritation, eye irritation, phototoxicity, skin sensitization, acute toxicity, genotoxicity and endocrine disruptor screening. (*) 3R's for animal testing (Reduction, Refinement, Replacement).

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.

The cytotoxicity test of neutral red (NR) uptake in normal rabbit corneal epithelial cells (CornePack®) was validated as an alternative method to the Draize rabbit eye irritation test (Draize test). We tested 38 cosmetic ingredients as well as isotonic sodium chloride solution in three phases of the validation study. The test procedures were controlled among participating laboratories under a common standard operating procedure (SOP). The concentration of test substances that showed a 50% reduction in NR uptake relative compared with controls (median NR uptake concentration: NR50, μg/ml) was determined and compared with in vivo Draize scores. Six laboratories participated in the first phase of the validation study, seven in the second, and five in the third. The average interlaboratory coefficient of variation (CV) was 32.9%. The correlation and rank correlation coefficients between the maximal average Draize total scores (MAS) and NR50 were −0.583 and 0.587, respectively. When the anionic detergents were excluded from analysis, the correlation coefficient increased to −0.738. When the cut-off point for positive and negative irritation was set at MAS of 15 and the predictability of this method was assessed by liner regression line, six substances (two acids, two alkanolamines and two alcohols) were false negative. Through this project, it appeared that CornePack, supplied in kit form with frozen secondary cultured cell in serum-free medium, could provide an effective, highly sensitive and simple preliminary screen for determining the cytotoxicity of substances. These results suggested that CornePack might have the potential to predict the MAS if definite criteria can be established for the compounds to be applicable. However, it is important to understand the nature of CornePack responses since its NR50 profile was quite different from other cytotoxicity assays.

Interlaboratory validation of the haemoglobin denaturation (HD) test on 38 cosmetic ingredients was conducted by five to eight participating laboratories. The HD test was evaluated as an alternative method to the Draize eye irritation test (Draize test) based on three indices of protein denaturation: the test substance concentration that induces 50% HD of the positive control (RDC(50)), a relative HD rate at 1% of the test substance (1%RDR) and a relative change in maximum absorption wavelength (1% lambdamax). The coefficients of variation associated with a positive HD test among the participating laboratories were within an acceptable range for practical application. The in vitro test results were in relatively good agreement with the Draize test. The correlation coefficient (r) between the in vivo maximal average Draize total score (MAS) and log (RDC(50)), 1%RDR and 1% lambdamax were -0.91, 0.67 and 0.79, respectively. The results revealed several limitations associated with the HD test: (1) the HD test cannot be applied to coloured test substances with a strong absorption, around 418nm; (2) water-insoluble test substances cannot be evaluated by RDC(50) or 1%RDR; (3) the HD test cannot be applied to strong acids that exceed the buffering capacity of a phosphate buffer solution; (4) the HD test cannot be used to determine the potential for eye irritation caused by factors other than protein denaturation, for example, polyoxyethylene octylphenylether (10 E.O.). Thus, the HD test alone is not appropriate for predicting eye irritation potential. Nevertheless, the good agreement between the HD test results and in vivo irritation scores as well as the ease of application suggest that this test may play an important role in a test system to determine eye irritation potential.

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 hen's 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% solution2.5), alkalis (pH of 10% solution11.5) and alcohols (lower mono-ol) in cytotoxicity tests clearly deviated from the other samples in the comparison of cytotoxicity with Draize results. (4) Pearson's 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) Spearman's rank correlation coefficients between the results of alternative methods and MAS were relatively high (r0.8) 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.

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 (HC50 value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC50 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 HC50 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 Spearman's rank correlation between the HC50 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 (MAS⩾50) 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.

This study evaluates seven alternative assays carried out on the main ingredients in cosmetics to determine which battery is the best set and what is the best predictor of the maximal Draize rabbit eye irritation scores (MDESs). The assays consisted of the maximal primary Draize rabbit skin irritation scores (MDSSs), a cytotoxicity test on neutral red uptake using Chinese hamster lung cells (NR-EC 50 ), a cytotoxicity test on MTT using normal skin fibroblasts (MTT-EC 50 ), the hen's egg test-chorioallantoic membrane test using fertile chicken eggs (HET-CAM), a haemolysis test using red blood cells from Wistar rats (HC 50 ), a protein denaturation test using haemoglobin from bovine (HDR), and pH. We tested 10% solutions of 24 tested chemicals, that is, 20 surfactants, three solvents and formaldehyde, to select from the assays a best set for prediction (x) and to obtain the best predictor [f(x)] based on the prediction sum of squares criterion. The selected set consisted of NR-EC 50 and HDR, and the resultant best predictor was f(x) = 74.0 − 29.52 log(NR-EC 50 ) + 0.87 HDR. This predictor achieved a high score of 89.6% of the contribution ratio. For two of the 24 test chemicals, an inconsistency occurred in the criticality of corneal damage between the observed and predicted Draize eye scores, although this is not considered to affect significantly the overall results.

To clarify the relationship between eye irritancy and cytotoxic potential induced by irritant materials, we made lenses coated with standard reference materials (SRMs) prepared from various amounts of zinc diethyldithiocarbamate (ZDEC) and polyurethane (PU). Zinc diethyldithiocarbamate was classified as a mild irritant by the Draize eye irritation test. When ZDEC-SRM coated contact lenses were applied to rabbit eyes, Draize scores increased in proportion to both the ZDEC and PU concentrations used for coating. Furthermore, correlation with the cytotoxic potential (gamma = -0.93) was better than with lactate dehydrogenase (LDH) activities of tears from rabbit eyes wearing these coated lenses (gamma = 0.78). In conclusion, in vivo eye irritancy induced by wearing lenses could be estimated quantitatively with the cytotoxic potentials using a colony assay. Furthermore, we could compare different sensitivities caused by the same set of SRMs among three different sites of tissue. As a result, the order of sensitivity was eye > muscle >> skin.

We tested the desmutagenic effect of hot water extracts of various oolong teas and some green teas. The desmutagenic effect was tested by a modification of the Ames method using Salmonella typhimurium TA 98 and TA 100 strains and a chromosome aberration test using Chinese hamster lung (CHL) cells.Three kinds of oolong teas and 2 kinds of green teas showed a desmutagenic effect against mutagenicity induced by benzo [a] pyrene. Taiwanese oolong tea also showed a desmutagenic effect on mutagenicity induced by extracts of gasoline vehicle exhaust gas and cooked salmon, 1, 6-dinitropyrene, Trp-P-2 (3-amino-1-methyl-5H-pyrido[4, 3-b] indole) and IQ (2-amino-3-methylimidazo [4, 5-f] quinoline).Oolong teas that were treated at 30°C for a month or were heated at 121°C for 20min showed the same desmutagenic effect. Moreover, this desmutagenic effect was also detected on activated Trp-P-2. These results indicated that the desmutagenic effect of oolong tea was not due to a metabolic inactivation of mutagens.