1. In vitro 3T3 NRU Phototoxicity test:
Ann De Smedt

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

3. 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

4. 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

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

6. 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

7. 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??

8. 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 ?

9. 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: 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?

10. 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?

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

12. Thank you!

13. Back-up slides

14. 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