Joseph Kubilus

New epidermal model for dermal irritancy testing.

An interlaboratory comparison of a new model of the human epidermis (EpiDerm) was conducted using a range of anionic and non-ionic surfactants and surfactant-containing final formulations. The toxicity of the materials was estimated by MTT conversion, using both concentration (EC(50)) and time (ET(50)) protocols. A range of 16 compounds was tested on different production lots of EpiDerm following storage periods of 1 and 2 days (after shipping) at MatTek and at two independent testing laboratories, Microbiological Associates (MA), USA and Scotland, UK. The EC(50) and ET(50) values were compared and the least squares fit lines with resulting correlation coefficients (r) calculated. Correlation of in vitro results to human clinical chamber irritation and repeat handwash testing gave r values ranging from 0.977 to 0.993 and comparison of the results obtained in the independent laboratories with the site of manufacture was good (MA, USA, r = 0.84; MA, UK, r = 0.74). The model appears to have utility in predicting clinically observed dermal irritation in vitro which is reproducible in different laboratories and after transatlantic shipping, such that it is worthy of further investigation.

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants. The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semisolid, solid or waxy. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37 degrees C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42 h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations.

Development of an EpiDerm™ in vitro Skin Irritation Test (SIT) for the Globally Harmonized System (GHS) of classification and labeling of chemicals

Carcinogen classification involves two interrelated determinations: evaluation of strength of evidence and consideration of all other relevant information (weight of evidence analysis). Under GHS, carcinogens are categorized as either known/presumed carcinogens (Category 1) or suspected carcinogens (Category 2). Category 1 is subdivided further based on whether the evidence for classification is mostly from human or animal data. See Table 1 below for hazard categories and hazard communication elements for carcinogens.