In Vitro Assays
The purpose of this study is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2.
Freshly harvested bovine corneas from the eyes of cattle are utilized in the BCOP test method in order to evaluate the ocular irritancy and corrosive potential of a test chemical by assessing quantitative measurements of:
(1) Changes in corneal opacity, measured with an opacimeter
(2) Corneal permeability, measured by a visible light spectrophotometer
Both measurements are used to calculate an In Vitro Irritation Score (IVIS), which is used to assign a classification category.
The objective of this study is to determine the potential for ocular irritation using OECD Guidelines for the Testing of Chemicals, Nos. 492 and 437, which serve as alternatives to the traditional Draize methodology. The use of two internationally-validated, OECD-accepted non-animal tests can provide a GHS categorization for the vast majority of test articles. The combination of the Bovine Corneal Opacity and Permeability Test and EpiOcular™ Eye Irritation Test followed by a Weight-of-Evidence analysis of the results allows for the classification of GHS Category 2.
In Vitro Assays
The Corrositex® assay is performed using a kit produced and distributed by In Vitro International. The kit contains tubes of proprietary buffers, a Chemical Detection System (CDS), and components used to make synthetic proteinaceous macromolecular bio‐barriers. After preliminary testing to determine if the test article is compatible with the Corrositex® assay (qualification) and to determine cut‐off times (categorization), the test article is applied topically on prepared bio‐barriers set atop vials of CDS. The amount of time it takes for the test article to penetrate the bio‐barriers and cause a visually detectable change in the CDS (breakthrough time) can be used to determine the UN Packing Group or the UN GHS subcategory (classification).
Upon exposure to skin sensitizers, the KeratinoSens™ Test measures activation of Keap1-Nrf2-antioxidant/electrophile response element (ARE). These tests use an immortalized, adherent, human keratinocyte cell line (HaCaT) that was transfected with a plasmid to monitor luciferase gene induction.
The DPRA is an in chemico method that quantifies cysteine‐ or lysine‐containing peptide depletion following 24 hours incubation with the test article (TA). Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the TA to one of four reactivity classes used to support the discrimination between sensitizers and sonosensitizers.
This study is designed to evaluate local responses to extracts and complies with the standards set forth in the current International Organization for Standardization (ISO) International Standard 10993-23:2021 (E) Tests for Irritation.
Contact dermal sensitization is an immunological process where the host, through repeated skin exposure, acquires a specific allergic sensitivity to a substance. In the Buehler model, contact dermal sensitivity is manifested as increased erythema.
Non-Guideline Eye and Skin Safety Tests
In Vitro Eye Irritation
Alternative Eye Irritation
This test is used to determine the potential for ocular irritation using an alternative to the Draize methodology. The methodology is based on that described in An Improved CAM Assay for Predicting Ocular Irritation Potential, Bagley, D.M., Rizvi, P.Y., Kong, B.M., and De Salva, S.J. (1988), Alternative Methods in Toxicology, Vol. 6, Progress in In vitro Toxicology, pp. 131-138, and "Bovine Corneal Opacity and Permeability Test: An In Vitro Assay of Ocular Irritancy,” (1994); Gautheron, Pierre; Dukic, Martine; Alix, Danielle and Sina, Joseph F.; Fundamental and Applied Toxicology 18, 442-449, and will include an analysis based on the current OECD Guideline for the Testing of Chemicals No. 437.
The PorCORA is an ex vivo alternative assay developed by MB Research that utilizes isolated, freshly harvested corneas from the eyes of swine in order to evaluate the corrosive potential of a test chemical by assessing:
(1) Sodium fluorescein stain retention, visualized using a transilluminator
(2) Time of sodium fluorescein clearance (up to 21 days post‐exposure)
The porcine corneas are maintained in cell culture medium with antibiotics for at least 21 days to model the Draize Rabbit Eye Test.