Buehler test

See also: Guinea pig maximisation test

The Buehler test is an in vivo test to screen for substances that cause human skin sensitisation (i.e. allergens). It was first proposed by Edwin Vernon Buehler in 1965^[1] and further explained in 1980.^[2] The method has been codified under the Organisation for Economic Co-operation and Development (OECD) Test Guideline 406^[3] and the U.S. Environmental Protection Agency (EPA) Office of Chemical Safety and Pollution Prevention (OCSPP) guideline 870.2600^[4].

The Buehler Test is designed to detect substances capable of inducing delayed contact hypersensitivity (Type IV allergic reactions). It employs a closed-patch technique, in which test substances are applied to the skin under occlusive conditions. The test consists of three key phases: a preliminary irritation screen, an induction phase, and a challenge phase, followed by scoring of dermal reactions.^[5]

Preliminary Irritation Screening

A range-finding or pilot study is performed prior to the main test to determine appropriate non-irritating concentrations. The concentration selected for induction should not cause more than slight irritation (erythema score ≤ 1), while the challenge concentration must not exceed very slight irritation (erythema score ≤ 0.5).^[5][3]

Main Test Procedure

• Induction Phase: A group of 20 guinea pigs receives topical applications (0.4 mL or 0.4 g) of the test substance under occlusion once weekly for three consecutive weeks. A control group of 10 animals remains untreated during this phase.
• Rest Period: A recovery period of 10 to 14 days follows to allow for a potential immune response to develop.
• Challenge Phase: All animals are then challenged on a previously untreated skin site with the same concentration of the test substance. Observations are made 24 and 48 hours after application.

Interpretation

A positive sensitization response is defined by the appearance of a score of ≥1.0 in the test group that exceeds any response seen in the control group. The key metrics used are:

• Incidence: The percentage of test animals showing a positive reaction at either 24 or 48 hours post-challenge. A material is typically considered a sensitizer if ≥15% of animals respond.^[5]
• Severity: Calculated as the mean erythema score per group. If the test group shows more pronounced responses than the control, a rechallenge may be warranted to confirm sensitization.^[5]

New Approach Methodologies (NAMs)

OECD-approved non-animal test guidelines support the evaluation of skin sensitization by targeting Key Events in the Adverse Outcome Pathway (AOP). These New Approach Methodologies (NAMs) are used in an Integrated Testing Strategy (ITS) that follows a “2 out of 3” approach; at least two out of the three validated assays must provide concordant results to classify a substance as a skin sensitizer. ^[6][7] Non-animal test guideline methods include the Direct Peptide Reactivity Assay (DPRA; OECD 442C), KeratinoSens (OECD 442D), and the Human Cell Line Activation Test (h-CLAT; OECD 442E).