Assays

For over 50 years, MB Research Laboratories has provided eye and dermal hazard identification services to the specialty chemical, agrichemical, pharmaceutical, and consumer product industries. Since 1989, MB Research has been leading the development and promotion of alternative and in vitro assays, inventing, adopting, and adapting non-animal replacements. Modernize Today!

Eye Safety

In Vitro Assays

EpiOcular™ Eye Irritation Test (EIT)
OECD 492/GHS

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.

Alternative Assays

Bovine Corneal Opacity & Permeability (BCOP) Assay
OECD 437/GHS

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.

Bovine Corneal Opacity & Permeability Assay and EpiOcular™ Eye Irritation Test Combination Study (BCOP-EIT)
OECD 437/GHS, OECD 492/GHS

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.

Skin Safety

In Vitro Assays

Irritation

EpiDerm™ Skin Irritation Test (SIT)
OECD 439/GHS, ISO 10993-23
The purpose of this study is to provide classification of the dermal irritation potential of chemicals by using a three-dimensional human epidermis model, according to the OECD Guideline for the Testing of Chemicals No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. The EpiDerm™ SIT allows discrimination between irritants (Category 2) and non-irritants, in accordance with U.N. GHS classification.

Corrosion

EpiDerm™ Skin Corrosion Test (SCT)
OECD 431/GHS
The purpose of this study is to provide classification of the dermal corrosion potential of chemicals by using a three-dimensional human epidermis model, according to the OECD Guideline for the Testing of Chemicals No. 431, “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”. The EpiDerm™ SCT allows discrimination between non-corrosive and corrosive substances in accordance with U.N. GHS classification. Corrosivity classification can be further divided into either optional Sub-category 1A or a combination of Sub-categories 1B and 1C.
Corrositex®
OECD 435/GHS

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).

Sensitization

KeratinoSens™
OECD 442D, ISO 10993-10

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.

Human Cell Line Activation test (h-CLAT)
OECD 442E/GHS, ISO 10993-10
To determine the skin sensitization potential of a test article due to cell activation by the measurement of changes in the expression of dendritic cell surface markers (CD86, CD54) via flow cytometry. The THP-1 human monocytic cell line serves as the test system. The h-CLAT is recommended by EURL ECVAM as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between sensitizers (i.e., UN GHS Category 1) and non-sensitizers for the purpose of hazard classification and labeling, per OECD Guideline 442E “In Vitro Skin Sensitization: Human Cell Line Activation Test (h-CLAT).”
Direct Peptide Reactivity Assay (DPRA)
OECD TG 442C

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.

Additional Assays

Irritation

Irritation Test by Skin Exposure
ISO 10993-23
The purpose of this study is to determine the irritant or corrosive effects, if any, of a test article when applied to the skin of the test subject. This study is designed to comply with the standards set forth in the current International Organization for Standardization (ISO) International Standard 10993-23:2021(E) Tests for Irritation.
Irritation Test by Intracutaneous (Intradermal) Administration
ISO 10993-23

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.

Irritation/Corrosion

Acute Dermal Irritation and Corrosion
OECD 404, OCSPP 870.2500, DOT 49 CFR 173.137, FHSA 16 CFR 1500.41
The study is designed to determine the skin irritancy or corrosive potential of a test article. This determination is made by treating the skin of up to three test subjects for a three-minute, one-hour and/or four-hour exposure period and evaluating the skin at scheduled time points for up to 14 days.

Sensitization

Local Lymph Node Assay (LLNA) Flow Cytometry
OECD 429
To determine the sensitizing potential of topically applied test material utilizing the LLNA, and measurement of lymphocyte proliferation by BrdU incorporation (by flow cytometry). Our protocol is not a guideline assay but is designed to be compliant with OECD TG 429
Local Lymph Node Assay (LLNA-BrdU ELISA)
OECD 442, ISO 10993-10, OCSPP 870.2600
The local Lymph Node Assay (LLNA) is an alternative study to the Guinea Pig Sensitization Test, used for determining sensitization potential of materials. Following exposure to a sensitizing test substance, lymphocyte proliferation occurs in the local lymph node. The LLNA measures increased proliferation of lymphocytes in the auricular lymph nodes (which drain the site of exposure; ears). Proliferation is assessed by determining the incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into the DNA of lymph node cells using flow cytometric method.
Delayed Contact Dermal Sensitization Test – Buehler Assay
OECD 406, ISO 10993-10, OCSPP 870.2600

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.

Guinea Pig Maximization Test (GMPT)
OECD 406, ISO 10993-10
Contact dermal sensitization is an immunological process where the host, through repeated skin exposure, acquires a specific allergic sensitivity to a substance. In the Guinea Pig Maximization (GPMT) model, contact dermal sensitivity is manifested as increased erythema.

Non-Guideline Eye and Skin Safety Tests

In Vitro Eye Irritation

EpiOcular™ MTT Viability Assay (Time Course or Dose Response)
The EpiOcular™ MTT Viability Assay utilizes three-dimensional reconstructed human cornea-like epithelium (RhCE) tissues. The RhCE tissues are composed of primary human cells, which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that of the human cornea.

Alternative Eye Irritation

Hen’s Egg Test Chorioallantoic Membrane (HET-CAM)
The purpose of this assay is to determine the potential for ocular irritation using an alternative to the Draize methodology. The methodology is based on that described in INVITTOX 1992 No. 47: HET-CAM Test.
Chorioallantoic Membrane Vascular Assay (CAMVA) 10/14 Day
The chorioallantoic membrane (CAM) of a fertilized chicken egg serves as an analog to the human conjunctiva with remarkable structural similarities in vasculature. Varying concentrations of a test article are administered to the CAMs of sets of eggs and qualitative measurements of vascular changes are performed in order to evaluate eye irritation potential.
Bovine Corneal Opacity and Permeability (BCOP) Test and Chorioallantoic Membrane Vascular Assay (CAMVA)

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.

Porcine Corneal Opacity & Permeability Assay (PorCORA)

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.

In Vitro Skin Irritation

EpiDerm™ MTT Viability Assay (Time Course or Dose Response)
The EpiDerm™ MTT Viability Assay utilizes three-dimensional reconstructed human epidermis (RhE) tissues. Following exposure to a test article (TA) and incubation, tissue viability is measured via MTT reduction. The RhE tissues are composed of primary human cells, which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that of human skin.

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