KeratinoSens™ Skin Sensitization Assay

Understand potential skin sensitization using our in vitro KeratinoSens™ skin sensitization assay performed in accordance with OECD guidance.

Skin sensitization assessment using KeratinoSens™ is in our portfolio of in vitro topical services for the cosmetics/personal care, chemical and pharmaceutical industries.

Assessment of skin sensitizing potential using the reporter cell line, KeratinoSens™

The Keap1-Nrf2-ARE pathways have been shown to be major regulator of cytoprotective responses to oxidative stress or electrophilic compounds. These pathways are also known to be involved in the cellular processes in skin sensitization.^1,2,3
The KeratinoSens™ assay uses an immortalized adherent human keratinocyte cell line (HaCaT cell line), transfected with a selectable plasmid to quantify luciferase gene induction as a measure of activation of Keap1-Nrf2-antioxidant/electrophile response element (ARE)¹ and has been validated as a useful in vitro system for assessing the skin sensitizing potential of compounds.
In February 2014, KeratinoSens™ was recommended by EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) for use within an integrated strategy for skin sensitization testing. An OECD test guideline was released in February 2015.⁴
Cyprotex offer the KeratinoSens™ assay in accordance with the OECD guidelines.

Small electrophilic substances such as skin sensitizers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g., covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes

⁴OECD Guideline for the Testing of Chemicals. Draft Proposal for a New Test Guideline: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, May 2014

Protocol

KeratinoSens™ skin sensitization assay protocol

Cell Line	KeratinoSens™ cell line
Analysis	Induction of luciferase reporter gene expression and cell viability
Test Article Concentrations	12 concentrations in triplicate in 3 independently performed experiments Highest concentration: 2000 µM (according to OECD guideline, dependent on customer requirements)
Time Points	48 hrs
Quality Controls	Vehicle control: 1% DMSO (vehicle) Positive control: cinnamic aldehyde Negative control: sodium dodecyl sulfate
Data Delivery	Dose response curves for cell viability and luciferase reporter gene expression MEC (minimum effective concentration), AC₅₀, IC₃₀ and maximum response (%) for cell viability EC_1.5, AC₅₀ and maximum response (I_max) for luciferase gene expression as well as sensitising potential classification

Data

Data from Cyprotex's KeratinoSens™ skin sensitization assay

Figure 1
KeratinoSens™ cells stained with a Hoechst 33342 (nuclear stain; blue) and Phalloidin (filamentous actin (F-actin); red).

Compound	In vivo	Classification	EC_1.5* (µM)	IC₅₀ (µM)	Classification	*EC_1.5 (µM)**	IC₅₀ (µM)
		Literature Data^4,5,6			Cyprotex Data
Non-sensitizing compounds
Isopropanol	Non-sensitizer	Negative	>1000	>1000	Negative	>1000	>1000
Salicylic acid	Non-sensitizer	Negative	>1000	>1000	Negative	>1000	>1000
Lactic acid	Non-sensitizer	Negative	>1000	>1000	Negative	>1000	>1000
Glycerol	Non-sensitizer	Negative	>1000	>1000	Negative	>1000	>1000
Sodium dodecyl sulphate	Non-sensitizer	Negative	NR	NR	Negative	>31	54
Sensitizing compounds
Cinnamyl alcohol	Weak	Positive	25-175	>1000	Positive	119	>1000
Ethylene glycol dimethacrylate	Weak	Positive	5-125	>500	Positive	39	810
2-Mercaptobenzothiazole	Moderate	Positive	50-250	>500	Positive	1187	1025
Methyldibromo glutaronitrile	Strong	Positive	<20	20-100	Positive	9.1	25
4-Methylaminophenol	Strong	Positive	<12.5	20-200	Positive	3.3	15
2,4 Dinitro-1-chlorobenzene	Extreme	Positive	<12.5	5-20	Positive	1.5	8
Cinnamic aldehyde	Weak	Positive	NR	NR	Positive	13	100
2,3 Butanedione	Weak	Positive	<100	NR	Positive	54	370

* EC_1.5 represents the concentration for which gene induction is above the 1.5-fold threshold (i.e., 50% enhanced gene activity is obtained).

NR = not reported

Table 1
Data from the KeratinoSens™ assay for 13 compounds with comparison to literature data including the OECD draft guidelines.^4,5,6

The KeratinoSens™ data illustrated in Table 1 comprises of eight sensitizing compounds and five non-sensitizing compounds. All compounds were predicted correctly when compared with the in vivo classification, with previously published data and the proficiency compounds of the OECD draft guidelines.

Figure 2
Data from the KeratinoSens™ assay for the skin sensitizers cinnamic aldehyde and ethylene glycol dimethacrylate. The upper graphs illustrate the activation of the luciferase reporter with increasing concentrations of test article. TThe lower graphs illustrate MTT data which are used to assess cytotoxicity of the test article. The points on the upper graphs are excluded if they exceed the cytotoxicity limit and these points are illustrated as open blue circle.

References

¹ Emter R et al., (2010) Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro. Toxicol Appl Pharmacol 245(3); 281-290
² Natsch A et al., (2010) The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers – Functional relevance and hypothesis on innate reactions to skin sensitizers. Toxicol Sci 113(2); 284-292
³Dinkova-Kostova AT et al., (2005) The role of Keap1 in cellular protective responses. Chem Res Toxicol 18(12); 1779-1791
⁴OECD Guideline for the Testing of Chemicals. In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, Adopted February 2015
⁵Bauch C et al., (2012) Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol 63(3); 489-504
⁶ Natsch A et al., (2011) The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories. Toxicol In Vitro 25(3); 733-744