U-SENS™ Skin Sensitization Assay

Background Information

The U-SENS™ Skin Sensitization Assay is an in vitro-based method that assesses the third key event in the skin sensitization adverse outcome pathway (AOP) - activation and mobilisation of cutaneous dendritic cells (DCs) following exposure to test articles.
The assay measures upregulation of the co-stimulatory molecule CD86 on the surface of human histiocytic lymphoma cells (U937), a characteristic marker of immune activation that is implicated in the induction of allergic contact dermatitis.
Despite being monocytic in phenotype, U937 cells recapitulate DC-like responses in vitro when exposed to sensitizing agents while affording the advantages of a standardised testing approach i.e. enhanced reproducibility (90%)¹ and robust predictive capacity (accuracy – 86%).^{1, 2}
These metrics are comparable to those obtained for the h-CLAT assay (80 and 85% respectively),^{3, 4} another methodology commonly used to measure DC activation as part of OECD guideline 442E.
Screening can be performed upon a wide range of test articles including single ingredients, preservatives, dyes and surfactants. Approved adaptations to the method can also be readily applied to facilitate the screening of test articles that exhibit intrinsic fluorescence.
At Cyprotex, all U-SENS™ testing is performed in accordance with guidelines outlined in OECD 442E⁵ and has been validated in-house using the recommended proficiency panel.
The EURL ECVAM recommends using the U-SENS™ assay as part of an integrated approach to testing and assessment (IATA).³As a result, it is advised that complementary assays such as Direct Peptide Reactivity assay (DPRA) and KeratinoSens™ assay are performed alongside U-SENS™ assay to provide a more complete assessment of the potential for a test article to cause skin sensitization. To support this IATA strategy, Cyprotex also offers these OECD guideline methods as part of a comprehensive in vitro skin sensitization package.

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Protocol

U-SENS™ Skin Sensitization Assay Protocol

Data

Data from Cyprotex's U-SENS™ Skin Sensitization Assay

Figure 1
Dose response curves showing the CD86 expression profiles of resorcinol (A), a sensitizer, and lactic acid (B), a non-sensitizer. Red dotted lines represent the EC₁₅₀threshold used within OECD test guideline 442E to determine whether a test article exhibits skin sensitizing potential. Both proficiency substances were negative with regards to cytotoxicity and generated a CV₇₀value that exceeded the maximum concentration tested (200µg/mL).

Table 1

Data generated by in-house validation studies using the proficiency substances recommended in OECD 442E test guideline. Comparison of CV₇₀ (cell viability) and EC₁₅₀(CD86 expression) values with both the reference ranges stipulated in the guideline and in vivo data^{1, 2} demonstrate that the U-SENS™ assay can accurately differentiate between sensitizing and non-sensitizing compounds when performed at Cyprotex. The data shown represents a single run out of the three independent runs that were conducted, which were all concordant in nature. NR is attributed to parameters where no response was observed below the maximum concentration tested i.e. 200µg/mL.

References

_{1) Alépée, N., Piroird, C., Aujoulat, M., Dreyfuss, S., Hoffmann, S., Hohenstein, A., Meloni, M., Nardelli, L., Gerbeix, C., Cotovio, J. (2015). Prospective multicentre study of the U-SENS test method for skin sensitization testing. Toxicol. In Vitro 30, 373-382.

2) Piroird, C., Ovigne, J.M., Rousset, F., Martinozzi-Teissier, S., Gomes, C., Cotovio, J., Alépée, N. (2015). The Myeloid U937 Skin Sensitization Test (U-SENS) addresses the activation of dendritic cell event in the adverse outcome pathway for skin sensitization. Toxicol. In Vitro 29, 901-916.

3) EC EURL ECVAM (2012). human Cell Line Activation Test (h-CLAT) Validation Study Report. Accessible here.}_{4) Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. (2013). Predictive performance of the human Cell Line Activation Test (h-CLAT) for lipophilic with high octanol-water partition coefficients. J. Toxicol. Sci. 38, 599-609.

5) OECD (2024). Annex 2: In vitro skin sensitization: U937 cell line activation test (U-SENS™). Test No. 442E: In Vitro Skin Sensitisation. Accessible at OECD website}