Cellular and molecular hypoxic response in common carp (Cyprinus carpio) exposed to linear alkylbenzene sulfonate at sublethal concentrations.

Linear alkylbenzene sulfonate (LAS) is an anionic surfactant commonly used in cleaning agents such as laundry detergents. Trace amounts of LAS are released into environmental waters after processing in wastewater treatment plants after the use of this chemical. Acute toxicity of LAS has been well-studied using various organisms, and its effects are particularly well known in fish. LAS damages fish gill morphology and induces mucous excretion from these organs. LAS also causes hematological changes. These observations suggest that LAS might induce hypoxic conditions in fish. However, the connections between hypoxia and hematological changes at the cellular and molecular levels remain unknown. Common carp were exposed to LAS at concentrations of 625, 1250, and 2500 µg/L for 96 h. A total of 9-10 fish were sampled at the end of the exposure period for each concentration. For hematological analysis, carp blood was sampled from the caudal vein. Gill tissue was used for real-time PCR analysis to evaluate transcriptional changes of hypoxia-induced genes. The number of normal red blood cells and the number of immature red blood cells were significantly decreased and increased, respectively, in fish exposed to 2500 µg/L LAS. The hypoxic marker genes hypoxia inducible factor 1α, myoglobin 1, and erythropoietin 2 were upregulated in these fish. Our results suggest that LAS decreases erythrocyte numbers and induces hypoxic conditions. In addition, LAS-exposed fish increase production of immature erythrocytes and upregulate myoglobin expression in gills to improve oxygen transport and absorption. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 122-130, 2017.

Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose.

Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo. Five groups of rats were orally administered a single dose of glycidol ranging from 0 to 75mg/kg bw, and diHOPrVal levels were measured 24h after administration. A dose-dependent increase in diHOPrVal levels was observed with high linearity (R(2)=0.943). Blood sampling at different time points (1, 10, 20, or 40days) from four groups administered glycidol at 12mg/kg bw suggested a linear decrease in diHOPrVal levels compatible with the normal turnover of rat erythrocytes (life span, 61days), with the calculated first-order elimination rate constant (kel) indicating that the diHOPrVal adduct was chemically stable. Then, we measured the second-order rate constant (kval) for the reaction of glycidol with N-terminal valine in rat and human hemoglobin in in vitro experiments with whole blood. The kval was 6.7±1.1 and 5.6±1.3 (pmol/g globin per µMh) in rat and human blood, respectively, indicating no species differences. In vivo doses estimated from kval and diHOPrVal levels were in agreement with the area under the (concentration-time) curve values determined in our earlier toxicokinetic study in rats. Our results indicate that diHOPrVal is a useful biomarker for quantification of glycidol exposure and for risk assessment.

The reconstructed skin micronucleus assay in EpiDerm™: reduction of false-positive results - a mechanistic study with epigallocatechin gallate.

The high rate of false-positive or misleading results in in vitro mammalian genotoxicity testing is a hurdle in the development of valuable chemicals, especially those used in cosmetics, for which in vivo testing is banned in the European Union. The reconstructed skin micronucleus (RSMN) assay in EpiDerm™ (MatTek Corporation, USA) has shown promise as a follow-up for positive in vitro mammalian genotoxicity tests. However, few studies have explored its better predictive performance compared with existing in vitro assays. In the present study, we followed the protocol of the RSMN assay and used eight chemicals to compare micronucleus (MN) induction with EpiDerm™ with that in normal human epidermal keratinocytes (NHEKs), both derived from human skin. The assessments of EpiDerm™ conformed to those of in vivo MN assay, whereas those of NHEKs did not. The effect of cell differentiation status on MN induction was further addressed using a model compound, epigallocatechin gallate (EGCG), which is a major component of green tea extract that shows positive results in in vitro mammalian genotoxicity assays via oxidative stress and negative results in in vivo MN studies. RSMN assay in an underdeveloped epidermal model, EpiDerm-201™ (MatTek Corporation), showed a negative result identical to that in EpiDerm™, indicating that the barrier function of keratinocytes has limited impact. Analysis of the gene expression profile of both EpiDerm™ and NHEKs after EGCG treatment for 12h revealed that the expression of genes related to genotoxic response was significantly induced only in NHEKs. Conversely, antioxidative enzyme activities (catalase and glutathione peroxidase) in EpiDerm™ were higher than those in NHEKs. These results indicate that EpiDerm™ has antioxidant properties similar to those of a living body and is capable of eliminating oxidative stress that may be caused by EGCG under in vitro experimental conditions.

Predictive performance for human skin sensitizing potential of the human cell line activation test (h-CLAT).

BACKGROUND: Recent changes in regulatory restrictions and social opposition to animal toxicology experiments have driven the need for reliable in vitro tests for predicting the skin sensitizing potentials of a wide variety of industrial chemicals. Previously, we developed the human cell line activation test (h-CLAT) as a cell-based assay to predict the skin sensitizing potential of chemicals, and showed the correspondence between the h-CLAT and the murine local lymph node assay results. OBJECTIVES: This study was conducted to investigate the predictive performance of the h-CLAT for human skin sensitizing potential. MATERIALS/METHODS: We selected a total of 66 test chemicals with known human sensitizing potential, and tested all chemicals with the h-CLAT. We then evaluated the performance of the h-CLAT in predicting human sensitizing potential. RESULTS AND CONCLUSION: Forty-five of 51 tested sensitizers were positive in the h-CLAT, indicating relatively high sensitivity. Also, 10 of 15 non-sensitizers were correctly detected as negative. The overall agreement between human data and h-CLAT outcome was 83%. Furthermore, the h-CLAT could accurately predict the human sensitizing potential of 23 tested chemicals that were amines, heterocyclic compounds, or sulfur compounds. Our data indicate the utility of the h-CLAT for predicting the human skin sensitizing potential of a variety of chemicals.

A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA).

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential.

An interlaboratory study of the short time exposure (STE) test using SIRC cells for predicting eye irritation potential.

We have developed the short time exposure (STE) test using a rabbit corneal cell line (SIRC cells) as an alternative eye irritation test. The STE test uses relative viability as the endpoint after cells are exposed to the test material at 5%, 0.5%, and 0.05% concentrations for 5 minutes. In this interlaboratory study, 2 laboratories conducted the test using 70 raw materials in order to evaluate transferability, between-laboratory reproducibility, and predictive capacity of the STE test as an alternative eye irritation test. Transferability was assessed using saline as a negative control and 0.01% sodium lauryl sulfate as a positive control. The relative viabilities obtained for the 2 laboratories were almost the same. Therefore, transferability was considered to be excellent. The 2 laboratories showed similar relative viabilities for all 70 raw materials at each test concentration. The correspondence rates of the eye irritation categories (irritants and nonirritants) were over 97% for each concentration tested, exhibiting high between-laboratory reproducibility. The correspondence rates for predicting eye irritation potential of undiluted raw materials and a diluted solution (10%) were over 85% at each laboratory for the 5% and 0.05% test concentrations in the STE. Lastly, the correspondence rate for the rank classification by the STE test prediction model at each laboratory was over 72%, and the correspondence rate became almost 90% when acids, amines, and alcohols were excluded from the analysis. From the above data, excellent transferability, high between-laboratory reproducibility, and high predictive capacity of the STE test were observed in the interlaboratory study by 2 laboratories.

Involvement of hydrogen peroxide in chromosomal aberrations induced by green tea catechins in vitro and implications for risk assessment.

Catechins, which are polyphenol compounds found in abundance in green tea, have elicited high interest due to their beneficial effects on health. Catechins have also been demonstrated to induce chromosomal aberrations in vitro, although no clastogenicity was confirmed in studies in vivo. We investigated the mechanism of catechin-induced chromosomal aberrations in CHL/IU cells. Addition of catalase suppressed chromosomal aberrations, indicating involvement of hydrogen peroxide (H2O2). We confirmed that substantial amounts of H2O2 are generated when catechins are incubated under in vitro culture conditions, whereas, interestingly, extremely low amounts of H2O2 were detected when catechins were incubated at the same concentration in water. Generation of H2O2 increased steeply above pH 6, indicating that pH is a key factor in determining how much H2O2 is generated via catechins in vitro. Our assessment indicates that humans have practically non-existent exposure to H2O2 when catechins are ingested in a beverage. Polyphenols, including catechins, are known to act as antioxidants due to their reducing potential. However, under in vitro culture conditions, catechins are thought to act primarily as pro-oxidants by reducing ambient or dissolved oxygen to form H2O2. Based on the above observations, we conclude that in vitro culture conditions as currently employed are inappropriate to address genotoxicity concerns regarding polyphenols, including catechins.

Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells.

Using SIRC (rabbit corneal cell line) cells, we developed an alternative eye irritation test: the short time exposure (STE) test. This STE test is a cytotoxicity test using physiological saline or mineral oil as the test solvent. Evaluation exposure time is short (5 min), which is similar to actual exposure situations, and uses the cell viability (CV) at a constant concentration as the endpoint for irritation potential. First, in order to confirm the usefulness of this STE test in assessing eye irritation potential of chemicals, 51 raw materials were tested and the correlation between CV in the STE test and the eye irritation score in the Draize test was examined. For the undiluted raw materials tested in the Draize test, the 5% test concentration in the STE test gave irritation classes that correlated well with the irritation classes from the Draize test (accuracy: 89.6%). For those materials tested as a 10% solution in the Draize test, STE irritation classes with 0.05% test concentration corresponded well with the Draize irritation classes (accuracy: 80.0%). Next, using the cell viabilities at these two concentrations, the STE prediction model (PM) was developed. A score of 1 or 2 was given for the results from each tested concentration in the STE test and Draize test. The scores from each test were then summed to yield a 3-level (Rank 1: minimally irritant, Rank 2: moderate irritant, Rank 3: severe irritant) eye irritation potential classification. Rank classification in the STE test showed a good correlation mostly to that in the Draize test (irritation class correspondence rate: 70.2%, but after exclusion of data of alcoholic materials, the rate was 91.7%). In most cytotoxicity test, the cytotoxicity of acids and amines is generally underestimated due the use of medium as the solvent. This is the result of the buffering capacity of the media. On the other hand, the STE test could predict the eye irritation potential by evaluating the chemical with a 5% test concentration. Eleven water insoluble materials such as toluene, octanol, and hexanol could be evaluated by using mineral oil as test solvent in the STE test. The STE test demonstrated itself to be simple, promising, have great potential, be of value, and to be an easily standardized alternative eye irritation test.

A retrospective evaluation method for in vitro mammalian genotoxicity tests using cytotoxicity index transformation formulae.

Although in vitro chromosomal aberration tests and micronucleus tests have been widely used for genotoxicity evaluation, false-positive results have been reported under strong cytotoxic conditions. To reduce false-positive results, the new Organization for Economic Co-operation and Development (OECD) test guideline (TG) recommends the use of a new cytotoxicity index, relative increase in cell count or relative population doubling (RICC/RPD), instead of the traditionally used index, relative cell count (RCC). Although the use of the RICC/RPD may result in different outcomes and require re-evaluation of tested substances, it is impractical to re-evaluate all existing data. Therefore, we established a method to estimate test results from existing RCC data. First, we developed formulae to estimate RICC/RPD from RCC without cell counts by considering cell doubling time and experiment time. Next, the accuracy of the cytotoxicity index transformation formulae was verified by comparing estimated RICC/RPD and measured RICC/RPD for 3 major chemicals associated with false-positive genotoxicity test results: ethyl acrylate, eugenol and p-nitrophenol. Moreover, 25 compounds with false-positive in vitro chromosomal aberration (CA) test results were re-evaluated to establish a retrospective evaluation method based on derived estimated RICC/RPD values. The estimated RICC/RPD values were in good agreement with the measured RICC/RPD values for every concentration and chemical, and the estimated RICC suggested the possibility that 12 chemicals (48%) with previously judged false-positive results in fact had negative results. Our method enables transformation of RCC data into RICC/RPD values with a high degree of accuracy and will facilitate comprehensive retrospective evaluation of test results.

Modified Ames test using a strain expressing human sulfotransferase 1C2 to assess the mutagenicity of methyleugenol.

INTRODUCTION: Several alkenylbenzenes, including methyleugenol (ME), are present in a wide range of botanicals and exhibit carcinogenic and mutagenic properties. Negative results are generally obtained for alkenylbenzenes in standard in vitro genotoxicity tests, including the Ames test. A lack of mutagenicity observed in such tests is thought to result from impaired metabolic activation of alkenylbenzenes via hydroxylation, with subsequent sulfoconjugation to its ultimate mutagenic or carcinogenic form. Although recent studies have reported the mutagenicity of hydroxylated ME metabolites in the Ames test using modified TA100 strains expressing human sulfotransferases (SULTs), to our knowledge, the detection of ME mutagenicity has not yet been reported. FINDINGS: Using strain TA100-hSULT1C2, which expresses human SULT1C2, we optimized the protein content of S9 Mix and the pre-incubation time required to promote metabolic activation in the Ames test. This procedure enabled us to obtain a positive response with ME. CONCLUSIONS: We established Ames-test conditions enabling the detection of ME-induced mutagenicity, using a strain expressing human SULT1C2 in the presence of induced-rat S9 Mix. This simple approach will help assess the mutagenicity of other alkenylbenzenes and related chemicals.

Comprehensive retrospective evaluation of existing in vitro chromosomal aberration test data by cytotoxicity index transformation.

New OECD test guidelines have been issued, in which the cytotoxicity index relative cell count (RCC) is replaced with a new index, RICC or RPD (relative increase in cell count/relative population doubling), with the goal of reducing the high proportion of false positive results in in vitro chromosomal aberration tests. Using a mathematical approach to estimate new indices from the RCC, we constructed an evaluation flow that quantitatively estimates how often the previous test conclusions change when applying the updated cytotoxicity criteria. The new evaluation flow was applied to a retrospective evaluation of 285 chemicals in two databases. The effects of the employment of new cytotoxicity indices are investigated at a large scale. Using the new evaluation flow, 90 chemicals were estimated as positive, 39 were designated as estimated negative (13 probably negative and 26 possibly negative), and 140 were designated as negative. Moreover, we also applied a prioritization index to indicate the likelihood of a chemical being re-evaluated as negative and assigned priorities for testing. Most of the chemicals that were designated as estimated negative and had negative results in the in vivo micronucleus tests were considered as false-positives that would be correctly judged under the new test guideline. Furthermore, statistical analysis of the frequency of estimated negatives revealed that the results for Ames-positive chemicals, especially those with a strong response, are unlikely to change. Therefore, we concluded that the new indices would likely reduce the proportion of false positive results and not increase the proportion of false negative results. This study is the first report of a comprehensive re-evaluation of test results in terms of new cytotoxicity indices. The evaluation flow we have developed facilitates efficient retrospective evaluation of genotoxicity.

Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats.

The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action.

Synthesis and reactions of a novel 3,4-didehydropyroglutamate derivative.

Some reactions such as catalytic hydrogenation, Diels-Alder reaction, cyclopropanation, dihydroxylation, and Michael addition of a novel 3,4-didehydropyroglutamate derivative, in which the carboxylic group is protected as an ABO ester, are examined and found to take place in a stereospecific manner giving 3- and/or 4-substituted pyroglutamate derivatives without loss of enantiomeric purity at the alpha-position.

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells.

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals.

Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals.

Recent changes in regulatory restrictions and social views against animal testing have accelerated development of reliable alternative tests for predicting skin sensitizing potential and potency of many chemicals. Lately, a test battery integrated with different in vitro tests has been suggested as a better approach than just one in vitro test for replacing animal tests. In this study, we created a dataset of 101 test chemicals with LLNA, human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and in silico prediction system. The results of these tests were converted into scores of 0-2 and the sum of individual scores provided the accuracy of 85% and 71% for the potential and potency prediction, compared with LLNA. Likewise, the straightforward tiered system of h-CLAT and DPRA provided the accuracy of 86% and 73%. Additionally, the tiered system showed a higher sensitivity (96%) compared with h-CLAT alone, indicating that sensitizers would be detected with higher reliability in the tiered system. Our data not only demonstrates that h-CLAT can be part of a test battery with other methods but also supports the practical utility of a tiered system when h-CLAT and DPRA are the first screening methods for skin sensitization.

Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis.

Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.

Species differences in toxicokinetic parameters of glycidol after a single dose of glycidol or glycidol linoleate in rats and monkeys.

Glycidol fatty acid esters (GEs) have been identified as contaminants in refined edible oils. Although the possible release of glycidol (G) from GEs is a concern, little is known about the conversion of GEs to G in the human body. This study addressed the toxicokinetics of glycidol linoleate (GL) and G in male Crl:CD(SD) rats and cynomolgus monkeys. Equimolar amounts of GL (341 mg/kg) or G (75 mg/kg) were administered by gavage to each animal. G was found in both species after the G and GL administration, while plasma GL concentrations were below the lower limit of quantification (5 ng/ml) in both species. In rats, the administration of GL or G produced similar concentration-time profiles for G. In monkeys, the C(max) and AUC values after GL administration were significantly lower than those after G administration. The oral bioavailability of G in monkeys (34.3%) was remarkably lower than that in rats (68.8%) at 75 mg/kg G administration. In addition, plasma G concentrations after oral administration at three lower doses of GL or G were measured in both species. In monkeys, G was detected only at the highest dose of G. In contrast, the rats exhibited similar plasma G concentration-time profiles after GL or G administration with significantly higher G levels than those in monkeys. In conclusion, these results indicate that there are remarkable species differences in the toxicokinetics of GEs and G between rodents and primates, findings that should be considered when assessing the human risk of GEs.

Glycidol exposure evaluation of humans who have ingested diacylglycerol oil containing glycidol fatty acid esters using hemoglobin adducts.

Glycidol fatty acid esters (GEs) have been found as impurities in refined edible oils including diacylglycerol (DAG) oil, and concerns of possible exposure to glycidol (G), a known animal carcinogen, during digestion have been raised. We previously measured N-(2,3-dihydroxy-propyl)valine (diHOPrVal), a G hemoglobin adduct, for DAG oil exposed and non-exposed groups and showed there was no significant difference between them. In the present study, we conducted an additional analysis to verify the outcome of the previous report. The first experiment was designed as a matched case-control study to adjust variables with an increased sample size. The average levels of diHOPrVal were 6.9 pmol/g-globin (95%CI: 4.9-9.0) for 14 DAG oil exposed subjects and 7.3 pmol/g-globin (95%CI: 6.1-8.5) for 42 non-exposed volunteers, and no significant difference in levels was found between the two groups. In a second experiment, we compared the adduct levels of 12 DAG oil exposed subjects before and after discontinuing use of DAG oil, and found there was no significant change in diHOPrVal levels (from 7.1±1.1 to 7.5±1.4 pmol/g-globin). These results suggest that there was no increased exposure to G for humans who ingested DAG oil daily, although the evaluated population was limited.

A tiered approach combining the short time exposure (STE) test and the bovine corneal opacity and permeability (BCOP) assay for predicting eye irritation potential of chemicals.

For the evaluation of eye irritation, one in vitro alternative test may not completely replace the Draize test. Therefore, a tiered approach combining several in vitro assays, including cytotoxicity assays, is proposed in order to estimate the eye irritation potential of a wide range of chemical classes. The Short Time Exposure (STE) test, a relatively newer alternative eye irritation test, involves exposing Statens seruminstitut rabbit cornea (SIRC) cells for 5 min to two concentrations (5% and 0.05%) of test material. In the present study, we examined the predictive capacity of a tiered approach analyzing the results from the STE test and then the results of the bovine corneal opacity and permeability (BCOP) assay for assessing globally harmonized system (GHS) eye irritation rankings of various chemicals. The accuracy of predicting the GHS rankings was slightly improved when the tiered approach combination of STE test and BCOP assay was used compared to when the STE test irritation rank classification was used alone. Moreover, the under prediction rate was substantially improved when this tiered approach was used. From these results, the tiered approach of combining the data analysis of the STE test and BCOP assay might be a promising alternative eye irritation test strategy.

Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency.

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI=3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI=150 for CD86 (EC150), and for CD54 (EC200) with LLNA's EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R=0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 µg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.