XCellR8’s animal-product-free adaptation of the KeratinoSens™ skin sensitisation assay was incorporated into OECD Test Guideline 442D following its development and validation.
The original KeratinoSens™ protocol, like many cell-based methods performed in other testing facilities, relied on foetal bovine serum in the culture medium. XCellR8 adapted the assay – as with all of our other cell-based assays – to fully animal-product-free conditions. We validated the new protocol against OECD proficiency chemicals and Performance Standards, and published the work in ALTEX in 2017.[1]
Regulatory acceptance followed, with the adapted method accepted for use in REACH registration dossiers before the animal-product-free protocol was incorporated into OECD TG 442D.[1,4]
Why was animal serum there in the first place?
KeratinoSens™ addresses Key Event 2 of the skin sensitisation Adverse Outcome Pathway: keratinocyte activation.
The assay uses a modified human keratinocyte cell line containing a luciferase reporter linked to the antioxidant response element pathway. When a sensitiser activates that pathway, the cells produce luciferase and generate a measurable luminescent signal.
It is an elegant way of detecting a biologically relevant response. However, the original culture conditions contained foetal bovine serum, which provided a broad mixture of growth factors and nutrients to support cell growth, as well as porcine trypsin to lift the cells from the base of the cell-culture plasticware.
The drawbacks of using these animal-derived products include well-documented animal-welfare concerns around their production,[2,3] as well as the presence of non-human components in a test designed to model a human physiological response.
Developing an animal-product-free version
The KeratinoSens™ method represents a great advancement in the replacement of animal testing. However, XCellR8’s mission is the full replacement of animal testing for cosmetics and their ingredients globally, motivated by an industry requirement to maximise the human relevance of in vitro models. Therefore, we adapted the KeratinoSens™ method to remove the animal-derived components from the culture system while retaining the performance required for regulatory use.
These adaptations replaced the foetal bovine serum with a commercially available, ethically sourced, pooled human serum (60–70 donors), and the porcine trypsin with a recombinant animal-free equivalent (TrypZean®).[1]
The KeratinoSens™ cell line adapted well to routine culture in human serum (in place of bovine serum), showing healthy morphology and comparable growth rates.[1]
The adapted, animal-product-free method was validated using the 10 proficiency chemicals detailed in Test Guideline 442D, and 11 additional chemicals listed in the OECD Performance Standards.[1] The resulting work was published in ALTEX in 2017.[1]
Results showed that the prediction for the 21 reference chemicals was identical to the VRM.[1]
Following regulatory review, the animal-product-free protocol was incorporated into OECD Test Guideline 442D.[4]
The method we use today is not an alternative version sitting outside the recognised guideline. The animal-product-free protocol forms part of the guideline itself, and because we sought inclusion of this adaptation into the Test Guideline framework, the animal-product-free adaptation is now globally available for use by other test facilities.[4] Subsequent adoption by other laboratories will collectively help to reduce the unnecessary use of a large volume of animal-derived serum, while maximising the relevance of the test to human safety.
Where KeratinoSens™ fits
Skin sensitisation is a complex biological process, so KeratinoSens™ is not used in isolation to represent the entire endpoint.
The OECD Adverse Outcome Pathway divides sensitisation into a sequence of Key Events. TG 442C addresses protein binding, TG 442D addresses keratinocyte activation, and TG 442E covers dendritic cell activation. Results from these methods can then be combined within integrated or Defined Approaches to support hazard identification and, increasingly, potency and risk assessment.[4,5]
This is important because it reflects how non-animal toxicology is developing more broadly. Rather than searching for a single assay that reproduces an entire animal study, different methods can interrogate specific biological mechanisms and be combined into a regulatory conclusion.
KeratinoSens™ is one piece of that wider strategy, but its history also demonstrates something else: replacing animal testing does not stop at removing the animal study itself. The reagents, culture conditions and materials used within the replacement method matter too. The success of the animal-product free KeratinoSensTM test offers a possible blueprint for the removal and replacement of foetal bovine serum and animal trypsin in other cell-based assays.
We’re proud that the animal-product-free conditions we developed are now part of the recognised method. At XCellR8, we run KeratinoSens™ to GLP under our MHRA accreditation, with data recognised across territories participating in the OECD Mutual Acceptance of Data system.
If you need skin sensitisation data and want to understand where KeratinoSens™ fits within your wider testing strategy, get in touch.
Email info@x-cellr8.com or call +44 (0)1925 607 134.
References
[1] Belot, N., Sim, B., Longmore, C., Roscoe, L. & Treasure, C. (2017). Adaptation of the KeratinoSens™ skin sensitization test to animal-product-free cell culture. ALTEX, 34(4), 560–564. DOI: 10.14573/altex.1701311.
[2] Jochems, C.E.A., van der Valk, J.B.F., Stafleu, F.R. & Baumans, V. (2002). The use of fetal bovine serum: Ethical or scientific problem? Alternatives to Laboratory Animals, 30(2), 219–227. DOI: 10.1177/026119290203000208.
[3] van der Valk, J. & Gstraunthaler, G. (2017). Fetal Bovine Serum (FBS) — A pain in the dish? Alternatives to Laboratory Animals, 45(6), 329–332. DOI: 10.1177/026119291704500611.
[4] OECD. Test No. 442D: In Vitro Skin Sensitisation — ARE-Nrf2 Luciferase Test Method. OECD Guidelines for the Testing of Chemicals, Section 4. Appendix IA, Annex 2: Adaptation of the KeratinoSens™ test method using human reagents to achieve xeno-free cell culture.
[5] OECD. Test No. 442C: In Chemico Skin Sensitisation — Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins; Test No. 442E: In Vitro Skin Sensitisation — Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation; and Guideline No. 497: Defined Approaches on Skin Sensitisation.
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