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).

Optimal patch application time in the evaluation of skin irritation.

We investigated the optimum application for evaluating skin irritation response by using samples of irritants commonly used as additives in cosmetics and other common household products. We studied 47 volunteers (16 men and 31 women). We selected three types of surfactant, one moisturizer, one anti-infective agent and one oil solution. Using Finn chambers on Scanpor tape, we performed the patch test. A total of 0.015 mL of each sample was applied to the Finn chamber. For liquids, circular filter paper was soaked in 0.015 mL of the sample. Samples were placed on the upper back of participants, and closed for 4, 24 or 48 h. A patch application time of 24 h is sufficient to detect primary skin irritation from irritants in cosmetics and other common household products. In addition, we found that skin irritation reactions were strongest at 24 h after patch removal and that the reaction tended to be weaker at 48 h after patch removal. Patch testing to evaluate irritants should be performed by means of a 24-h patch test with a follow-up reading at 24 h after patch removal. An application time of 24 h places less of a burden on patients than a 48-h patch test.

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.

Lack of skin carcinogenicity of topically applied titanium dioxide nanoparticles in the mouse.

This study was conducted to examine the post-initiation carcinogenic potential of coated and uncoated titanium dioxide nanoparticles (CTDN and UCTDN) using a mouse medium-term skin carcinogenesis bioassay. For this purpose, 5, 10 and 20mg/animal doses of CTDN or UCTDN were applied to mouse skin in the post-initiation phase (up to 20 weeks) in a two-stage skin carcinogenesis model using 7 week old CD1 (ICR) female mice. 7,12-dimethylbenz[a]anthracene (DMBA) and 12-o-tetradecanoylphorbol 13-acetate (TPA) were used as the initiator and a positive control promoter, respectively. Pentalan 408 served as a vehicle control. No changes in survival rate, general condition and body weight related to the test materials were observed. On macroscopic observation, 1-2 nodules/group on the skin were observed in each group applied CTDN and UCTDN as well as the control group after DMBA initiation. The nodules were histopathologically diagnosed as squamous cell hyperplasia, sebaceous gland hyperplasia, squamous cell papilloma and keratoacanthoma. CTDN and UCTDN experiments, while enlargement of the mandibular, pancreatic, lumbar region and inguinofemoral lymph nodes, spleen and thymus was observed in mice given 5 and 10mg but not 20mg, the lack of dose-dependence suggests no biological significance. In the present study, CTDN and UCTDN applied in post-initiation stages at doses of up to 20mg/mouse did not increase the development of nodules, and thus it was concluded that titanium dioxide nanoparticles do not possess post-initiation potential for mouse skin carcinogenesis.

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.

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.