OECD TG 442C / 442D / 442E: Field Experience with 2o3 DA and In Vitro Alternatives in Skin Sensitization

Skin sensitization assessment is an indispensable component of product safety in the cosmetics, pharmaceuticals, pesticides, and medical device industries. Traditionally, this assessment was conducted using tests on animal models (LLNA, GPMT, Buehler); however, in line with the 3R principles and scientific advancements, validated in chemico and in vitro methods have come to the forefront. At the center of this transformation is OECD TG 442C (DPRA), OECD TG 442D (KeratinoSens™) and OECD TG 442E (h-CLAT), along with the 2o3 Defined Approach (DA) that combines these three tests.

A significant portion of the skin sensitization studies we have conducted in our laboratory over the past two years has involved these three in chemico/in vitro methods. In particular, the complete ban on animal testing under the EU Cosmetics Regulation has made these tests mandatory for manufacturers. However, our field experience shows that all three tests have their own unique strengths and weaknesses, and that the 2o3 DA yields reliable results only when interpreted correctly.

OECD TG 442C (DPRA): In Chemico Measurement of Protein Binding

OECD TG 442C is an in chemico test targeting the first key event (KE1) of the skin sensitization AOP. Sensitizing substances are typically electrophilic in nature and form covalent bonds with the nucleophilic portions of skin proteins (cysteine and lysine residues). DPRA uses synthetic peptides (containing cysteine and lysine) to model this reaction and measures the decrease in peptide concentration via HPLC following incubation with the test substance [citation:1][citation:2].

The most common issue we encounter when performing DPRA in our laboratory is the solubility of the test substance. The protocol specifies that the test substance should be soluble at a concentration of 100 mM; however, this is not always possible with lipophilic samples or those containing particles. In an assessment conducted under the UK REACH framework, it was reported that some substances could not be tested in DPRA due to the lack of a suitable solvent, and this situation led to a return to animal testing [citation:5]. Our solution is to report the result as “indeterminate” for substances with low solubility and to evaluate them in conjunction with the other components of 2o3 DA. Furthermore, the DPRA is not suitable for metal compounds and substances exhibiting oxidative properties; this limitation is clearly stated in our reports [citation:8].

The most significant advantage of the DPRA is that it is fast and cost-effective; however, it is not sufficient on its own to determine sensitization potential. Therefore, it is used as a source of information within the 2o3 DA.

OECD TG 442D (KeratinoSens™): Measurement of Keratinocyte Activation

OECD TG 442D is an in vitro test targeting the second key event (KE2) of the AOP. KeratinoSens™ is a reporter cell line created by transfecting the human keratinocyte cell line (HaCaT) with the luciferase gene. Following 48-hour exposure to the test substance, activation of the Nrf2-ARE pathway is measured by luciferase induction [citation:3][citation:15].

In our field experience, KeratinoSens™ demonstrated high accuracy, particularly with cosmetic raw materials. However, one case caught our attention: In a plant extract sample, the KeratinoSens™ result fell within the “indeterminate” range (Imax value between 1.35- and 1.67-fold). According to 2o3 DA guidelines, in this case, the other two tests (DPRA and h-CLAT) must be consistent and highly reliable [citation:12]. The DPRA test was negative, while the h-CLAT test was positive; due to this inconsistency, the 2o3 DA result was reported as “indeterminate.” We informed the client that the sensitization potential of this extract could not be definitively classified and that additional information (e.g., chemical characterization or in silico modeling) was required. This case demonstrates how critical it is to correctly interpret the “indeterminate” results for 2o3 DA.

OECD TG 442E (h-CLAT): Measurement of Dendritic Cell Activation

OECD TG 442E covers in vitro tests targeting the third key event (KE3) of the AOP. Three methods are defined within this guideline: h-CLAT, U-SENS™, and IL-8 Luc. h-CLAT measures the increase in the expression of the surface markers CD54 and CD86 in a human monocyte cell line (THP-1) using flow cytometry [citation:4][citation:10].

The most important point we consider when performing h-CLAT in our laboratory is that negative results for substances with a Log P > 3.5 have low reliability [citation:12]. This is due to lipophilic substances not dissolving sufficiently in the cell culture medium. An active ingredient in a pesticide (with a Log P value of 4.2) yielded a negative result in the h-CLAT; however, we did not interpret this result on its own as “not a sensitizer.” DPRA and KeratinoSens™ results were also considered within the scope of 2o3 DA, and since both were positive, the final result was reported as “sensitizer.” This is a concrete example of why h-CLAT should not be used in isolation.

The table below provides a comparative summary of the key features of the OECD TG 442C, 442D, and 442E guidelines.

2o3 Defined Approach: Integrated Interpretation of Three Tests

2o3 aims to determine skin sensitization potential without the use of animals by combining DA, DPRA, KeratinoSens™, and h-CLAT results. Under this approach, accepted under OECD TG 497, the basic rule is as follows: If at least two of the three tests are positive, the substance is classified as a “sensitizer”; if at least two are negative, it is classified as “not a sensitizer.” However, if one of the results falls within the “indeterminate” range or if the limitations of the tests come into play, the DA result may be “indeterminate” [citation:12].

When applying the 2o3 DA in our laboratory, we report the results of each test separately and present the final DA result along with our rationale. For example, for a cosmetic preservative, the DPRA was positive, the KeratinoSens™ was positive, and the h-CLAT fell within the indeterminate range. The two positive results were sufficient for the DA to conclude that the substance was a “sensitizer,” even though the h-CLAT result was indeterminate. We informed the client that this substance must be classified as GHS Category 1 and that the statement “May cause an allergic skin reaction” must be used on the label.

The most significant advantage of the 2o3 DA is that it completely eliminates the use of animals and is accepted by regulatory authorities. However, our field experience shows that the correct interpretation of DA’s “ambiguous” results is critical. According to UK REACH data, for some substances, in vitro tests have proven insufficient, leading to a return to animal testing; the primary reasons include solubility issues, UVCB substances, and incompatibility with test systems [citation:5]. For this reason, our laboratory thoroughly evaluates the physicochemical properties of the test substance before each study and verifies the applicability of the 2o3 DA in advance.

Application of the 2o3 DA and Field Observations in Our Laboratory

In our GLP-certified laboratory, we offer integrated testing under the 2o3 DA framework for tests covered by OECD TG 442C, 442D, and 442E. Our testing process begins with a comprehensive preliminary evaluation: the test substance’s solubility, lipophilicity (Log P), chemical class, and potential interferences are examined. Based on this information, a decision is made regarding the applicability of the 2o3 DA.

Substances we have evaluated using the 2o3 DA over the past two years include cosmetic preservatives, plant extracts, active ingredients in pesticides, and medical device extracts. The most common challenge we face is testing complex mixtures, such as plant extracts. Tests such as DPRA and KeratinoSens™ provide limited information for complex mixtures; in such cases, the most appropriate approach is to report the results as “indeterminate” and recommend additional information to the client (e.g., chemical characterization or in silico modeling).

Another observation from the field is the difficulty of applying 2o3 DA to medical device extracts. The ISO 10993-10:2021 standard for medical devices specifies GPMT, Buehler, or LLNA for sensitization assessment; however, 2o3 DA may not always be suitable due to the complexity of device extracts. In such cases, we work with the client to determine the most appropriate testing strategy and, when necessary, also evaluate the in vivo testing option.

Frequently Asked Questions

One of the most common questions we receive regarding OECD TG 442C, 442D, and 442E is whether 2o3 DA alone is sufficient. The answer depends on the characteristics of the test substance. For substances with good solubility, no metal content, and a Log P value below 3.5, 2o3 DA is generally sufficient. However, for substances with solubility issues, UVCB substances, or complex mixtures, DA may yield an “indeterminate” result; in such cases, additional information is required.

Regarding the test duration, the total duration of the three tests is generally 3–4 weeks (including reporting). The DPRA takes 1 week, KeratinoSens™ takes 2 weeks, and h-CLAT takes 2–3 weeks; however, since the tests can be conducted in parallel, the total duration is reduced. As for which test is suitable for which substance, the DPRA provides information specifically for substances with high protein reactivity, KeratinoSens™ for substances showing keratinocyte activation, and h-CLAT for substances with the potential to activate dendritic cells. However, they should not be used in isolation but rather as part of the 2o3 DA.

In skin sensitization assessment, the 2o3 DA is a modern approach that eliminates the use of animals and is recognized by regulatory authorities. With our tests fully compliant with OECD TG 442C, 442D, and 442E standards, we evaluate the sensitization potential of your products according to the highest scientific standards. Our GLP-certified laboratory, with its validated test systems, experienced staff, and field expertise, is your reliable partner for skin sensitization testing needs. To demonstrate your product’s sensitization profile, contact us.