In vitro 3T3 NRU Phototoxicity test: Ann De Smedt

Topics Background The in vitro 3T3 NRU Phototoxicity Test Test strategy & Results Post acceptance evaluation

Background Phototoxicity – is defined as a toxic acute response from a substance applied to the body which is either elicited or increased after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance. (OECD432). In vitro 3T3 NRU phototoxicity test determines the potential of chemicals to cause phototoxicity following exposure to UVA radiation (315 – 400 nm) EU guidance differs from FDA guidance

In vitro 3T3 NRU phototoxicity test I. Treatment of 3T3 cells Balb/c 3T3 cells X 24 h incubation 1 h treatment Seeding cells II. Irradiation 5J UVA 18-22 h incubation Neutral Red Uptake determination - UVA

In vitro 3T3 NRU phototoxicity test -UV + UV IC50 PIF=IC50/IC50 PROBABLE Phototoxic NO Phototoxic PIF<2 2<PIF<5 5<PIF

Testing strategy at J&J In vivo phototoxicity test in pigmented rats POSITIVE NO further testing NO Phototox label POSITIVE UV/VIS-Absorption and Skin and Eye accumulation or Topical application No further testing NEGATIVE In vitro 3T3 NRU phototoxicity test YES No further testing NEGATIVE

6 compounds tested in vivo 43 JNJ-compounds tested in vitro 6 compounds tested in vivo 30 compounds NEGATIVE (70 %) 13 compounds POSITIVE NEGATIVE False positive results??

ICH guidelines? High rate of false positive results Lead to a lot of follow-up work More in vivo tests needed Doubts about use of validated in vitro 3T3 NRU phototoxicity test Post-acceptance evaluation ?

Post-acceptance evaluation Importance of light source?  during validation study same source used Importance of UVA/UVB?  OECD guideline: not specified  EU guidance: 20/1  at J&J: 174/1 Light source Concentration of compound Maximum tested concentration:  OECD: 1000 g/ml  EU guidance: 100 g/ml or highest achievable Correlation in vitro results with in vivo results Is there any skin/eye absorption levels that triggers testing? Thresholds? What to do with insoluble compounds?

Post-acceptance evaluation validation study: mainly chemicals Compounds post-validation: a lot of pharmaceuticals tested  correlation with in vivo?  which in vivo test? what about 3D human skin models as follow-up studies? Tiered approach?

Post-acceptance evaluation Recent initiatives: EFPIA survey !! DIA Workshop (21-22 Nov, 2007, Amsterdam)

Thank you!

Back-up slides

Differences in guidelines (phototox) EMEA FDA Note for Guidance on Photosafety Testing (2002) Guidance for Industry: Photosafety Testing (2003) Encourage use of validated in vitro method on all photoreactive compounds bioavailable to skin or eye regardless of level of exposure. In vivo non-clinical studies  not warranted Possible clinical follow up  MED in volunteers Encourages use of traditional animal tests, perhaps followed by clinical studies on photoreactive compounds bioavailable in skin or eyes (at levels sufficient to cause photoirritation, clinical evidence or class effect). driven by Directive 86/609/EEC