Post on 27-Mar-2015
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).
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