epaa meeting november 2007 1 in vitro 3t3 nru phototoxicity test: ann de smedt
TRANSCRIPT
EPAA meeting November 2007 1
In vitro 3T3 NRU
Phototoxicity test:
Ann De Smedt
EPAA meeting November 2007 2
Topics
Background
The in vitro 3T3 NRU Phototoxicity Test
Test strategy & Results
Post acceptance evaluation
EPAA meeting November 2007 3
Background
In vitro 3T3 NRU phototoxicity test determines the potential of chemicals to cause phototoxicity following exposure to UVA radiation (315 – 400 nm)
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).
EU guidance differs from FDA guidance
EPAA meeting November 2007 4
In vitro 3T3 NRU phototoxicity test
I. Treatment of 3T3 cells
II. Irradiation
5J UVA
- UVA18-22 h incubation
Neutral Red Uptake determination
Balb/c 3T3 cells
X
24 h incubation 1 h treatmentSeeding cells
EPAA meeting November 2007 5
Neutral Red Assay
0
25
50
75
100
125
150
0.1 1.0 10.0Concentrration (µg/ml)
% o
f c
on
tro
l
Neutral Red Assay
0
25
50
75
100
125
0.1 1.0 10.0 100.0Concentrration (µg/ml)
% o
f c
on
tro
l
-UV
+ UV
IC50
IC50
PIF=IC50/IC50
PROBABLEPhototoxic
NO Phototoxic
Phototoxic
PIF<2
2<PIF<5
5<PIF
In vitro 3T3 NRU phototoxicity test
EPAA meeting November 2007 6
Testing strategy at J&J
NO further testing
NO
No further testing
NEGATIVE
In vitro 3T3 NRU phototoxicity test
YES
UV/VIS-Absorption and
Skin and Eye accumulation
or Topical application
No further testing
NEGATIVE
Phototox label
POSITIVE
In vivo phototoxicity test
in pigmented rats
POSITIVE
EPAA meeting November 2007 7
Compounds Tested
43 JNJ-compounds tested in vitro
13 compounds
POSITIVE
NEGATIVE
False positive results??
6 compounds
tested in vivo
30 compounds
NEGATIVE(70 %)
EPAA meeting November 2007 8
High rate of false positive results
Doubts about use of validated in vitro 3T3 NRU
phototoxicity test
Lead to a lot of follow-up work
More in vivo tests needed
Post-acceptance evaluation ?
ICH guidelines?
EPAA meeting November 2007 9
Post-acceptance evaluation
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?
• 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
EPAA meeting November 2007 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?
EPAA meeting November 2007 11
EFPIA survey !!
DIA Workshop (21-22 Nov, 2007, Amsterdam)
Post-acceptance evaluation
Recent initiatives:
EPAA meeting November 2007 12
Thank you!
EPAA meeting November 2007 13
Back-up slides
EPAA meeting November 2007 14
Differences in guidelines (phototox)
Directive 86/609/EEC
Note for Guidance on Photosafety Testing (2002)
Guidance for Industry: Photosafety Testing (2003)
FDAEMEA
• 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