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

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

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

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

Inter-laboratory study of short time exposure (STE) test for predicting eye irritation potential of chemicals and correspondence to globally harmonized system (GHS) classification.

Short time exposure (STE) test using rabbit corneal cell line (SIRC) cells was developed as an alternative eye irritation test. STE test uses relative viability as the endpoint after cells are exposed to the test material at constant concentrations for 5 min. In this inter-laboratory study with 3 laboratories, 44 chemicals with a wide range of classes were evaluated for the transferability, between-lab reproducibility and predictive capacity of the STE test as an alternative eye irritation test. Globally harmonized system (GHS) classification based on Draize eye irritation test data was used as the comparative in vivo data. Transferability was assessed using standard chemicals (sodium lauryl sulfate, calcium thioglycolate, and Tween 80) and the coefficient variations (CVs) of relative viabilities between 3 labs were less than 0.13. The irritation category (Irritant or Non irritant) at each test concentration (5% and 0.05%) in STE test was the same in 3 laboratories for all 44 tested chemicals. The predictive capacity irritation category classification between STE test and GHS were compared, and a good correlation was confirmed (accuracy was 90.9% at all laboratories). In addition, the STE rankings of 1, 2, and 3 classified by the prediction model (PM) based on the relative viability at two concentrations (5% and 0.05%) were highly correlated with the GHS ranks of non-irritant, category 1, and category 2, respectively (accuracy was 75.0% at all laboratories). These results suggest that the STE test possessed easy transferability, reproducibility, good predictive performance.

Further evaluation of the skin micronucleus test: results obtained using 10 polycyclic aromatic hydrocarbons.

The standard in vivo micronucleus (MN) test detects clastogenicity in hematopoietic cells and is not suitable for detecting chemicals that target the skin. Previously, we have developed an in vivo rodent skin MN test that is simple to perform and can be applied to several laboratory animals, including the hairless mouse-a species whose use simplifies the procedure of skin testing. In this paper, we report new data that confirms the predictive ability of the test. Following the application of 10 polycyclic aromatic hydrocarbons (7,12-dimethylbenz[a]anthracene; 3-methylcholanthrene; benzo[a]pyrene; dibenz[a,h]anthracene; benz[a]anthracene; dibenz[a,c]anthracene; chrysene; benzo[e]pyrene; pyrene; anthracene) with various degrees of genotoxicity to the dorsal skin of hairless mice, we found that these compounds caused MN production that in general correlated with their reported carcinogenicity. We believe that this test will be useful in detecting skin clastogens that test negative when analyzed using the standard micronucleus test.

Toxicity studies of tetradecanoic acid, 2-sulfo-, 1-methylester, sodium salt (C14-MES).

We evaluated the toxicity of tetradecanoic acid methyl ester sodium salt (C14-MES), a major component of fabric detergents, following the test guidelines of the Organization for Economic Cooperation and Development. The rat acute oral LD(50) was 1,000 mg/kg in males and 500 mg/kg in females. Applying the combined repeated dose and reproductive/developmental toxicity screening test (ReproTox), we exposed groups of Crj:CD (SD) IGS rats to C14-MES in the diet at concentrations of 0, 0.3, 0.6, or 1.2%. We observed decreases in fibrinogen levels and longer prothrombin time at the 1.2% treated level in females and decreases in serum triglyceride levels in both sexes at the 0.6% and 1.2% treatment levels, but the effects were not clinically significant. The no-observed-effect-level (NOEL) for repeated dose toxicity was 0.3% (175 mg/kg body weight/day for males, 249 for females). The NOEL for reproduction/developmental toxicity was 1.2% (740 mg/kg for males, 1039 for females). C14-MES was negative in the reverse gene mutation assay and the chromosomal aberration test and did not induce skin sensitization in the guinea pig maximization test. These data confirm that C14-MES is of low hazard potential.

Further evaluation of an in vivo micronucleus test on rat and mouse skin: results with five skin carcinogens.

In a previous paper, we presented a practical in vivo micronucleus (MN) test that used rat skin as the target organ. To evaluate the test, as well as to determine the reproducibility and applicability of the method to mice, we used it to test the effect of five skin carcinogens (N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P)) on rat and mouse skin. All five compounds significantly and dose-dependently increased the MN frequencies in the basal cells of the chemical-treated skin. These results indicated the reproducibility of the test results and also the applicability of the test to mice as well as rats.