All proficiency substances were purchased from Sigma-Aldrich and selected based upon the criteria detailed in Annex II of OECD TG 442E and supporting information provided in the European Union Reference Laboratory for Alternative Methods to Animal Testing (ECVAM) Human Cell Line Activation Test (h-CLAT) Validation Study Report.
These were conjugated to FITC for use in the h-CLAT in order to detect changes in the cell surface markers.
Based upon these results, THP-1 cells used in the animal-product-free h-CLAT were seeded at 0.2x[10.sup.6] cells/ml for both 48 and 72 hour pre-incubations as there was a low likelihood of cell overgrowth (> 1x[10.sup.6] cells/ml) during this time period.
The adapted animal-product-free version of the h-CLAT was used to test the 10 proficiency panel substances from OECD TG 442E that are detailed in Section 2.1.
Figure 2 and 3 show representative examples of the CD54 and CD86 data produced for each of the 6 substances that are known sensitizers using the h-CLAT method as specified in the OECD guideline.
Figure 4 and 5 show representative examples of the CD54 and CD86 data produced for each of the 4 substances that are known non-sensitizers using the h-CLAT method as specified in the OECD guideline.
In addition, we suggest a decision rule for applying the BR to a combination of the DPRA, LuSens and the h-CLAT in the 2-out-of-3 ITS.
The three non-animal test methods DPRA, LuSens and h-CLAT were developed to address the three key events of the AOP in order to assess a substance's skin sensitization potential.
The experimental sets to which the BR concept was applied in order to identify borderline substances consisted of 199 substances for the DPRA, 79 for LuSens, 40 for h-CLAT and 22 substances for LLNA, see Bauch et al.
The human Cell Line Activation Test (h-CLAT) (Ashikaga et al., 2006, 2010; Sakaguchi et al., 2006, 2010) determines the dendritic cell activating potential by measuring the induction of the expression of the cell surface markers CD54 and CD86 after treatment with a test substance relative to concurrent vehicle controls in immortalized human monocytic leukemia THP-1 cells as surrogate dendritic cells.
According to this approach, 2 out of 3 concordant test results using the DPRA, ARE-NrF2 luciferase method, and the h-CLAT determine the prediction.
The BR around the classification threshold of the h-CLAT was calculated using test results from 13 substances tested during routine (in house) test applications, yielding 528 individual measurements covering different substances and concentrations (see Appendix 1, Tab.