PLEASE NOTE THAT TO OBTAIN DETAILS OF THE PROTOCOL YOU SHOULD REGISTER

TO OBTAIN DETAILED PROTOCOL YOU SHOULD FIRST REGISTER AT ECVAM SIS

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

O
N
-
L
I
N
E

I
N
V
I
T
T
O
X

Protocol no. 77 
THE COMPLEMENT PHOTOACTIVATION ASSAY

Complement activation that takes place when human plasma and the test compound are incubated in the presence of light is taken as an indication of potential phototoxicity of the test compound.

CONTACT

E-mail:dslado@ib.amwaw.edu.pl
Tel: +44-22- 6281041 ext. 71 
Fax: +48-22-6217543 

RATIONALE

One of the mechanisms underlying phototoxic reactions in human skin is the activation of the complement system. Complement activation products may cause phototoxic lesions directly (C3a, C4a, C5a, C5-9) and/or indirectly by activating dermal mast cells and by inducing the release of prostaglandins and interleukins from surrounding cells. The method described here permits the evaluation of the degree of complement activation by quantifying the specific complement fragments generated from plasma by phototoxic compounds in the presence of light. Increased complement activation compared to control (non-exposed) samples indicates that the test substance may produce phototoxic lesions/inflammation in human skin. 

BASIC PROCEDURE

The assay is performed using freshly isolated human plasma (centrifuged, heparinised blood). Plasma samples containing dissolved test compound are irradiated and the concentrations of iC3b complement fragments that are generated are measured using a commercialELISA kit (Quidel). 

TEST STASTUS

The protocol presents the standard operation procedure used in the EU/COLIPA Validation Study on Phototoxicity. 

CHEMICALS TESTED

Photosensitisers 
5-MOP 
6-methylcoumarin 
Acridine-hydrochloride 
Acridine-free base 
Amiodarone 
Anthracene 
Bergamot oil 
Bithionol 
Chlorpromazine 
Demeclocycline 
Eosine Yellowish 
Fenofibrate 
Furosemide 
Musk Ambrette 
Nalidixic acid-sodium salt 
Nalidixic acid-free acid 
Neutral red 
Norfloxacin 
Ofloxacin 
Promethazine 
Protoporphyrin IX-free acid 
Protoporphyrin IX-disodium 
Rose bengal 
Tetracycline Hydrochloride 
Tiaprofenic acid 
Non-photosensitisers
2-hydroxy-4methoxybenzophenon
Chlorhexidine dihydrochloride 
Hexachlorophene 
Ketoprofen 
PABA 
Penicilin G 
Sodium dodecyl sulphate (SDS) 
Sodium lauryl sulphate 

REFERENCES

  1. Holers V.M., Kinoshita T., and Molina H. (1992) The evolution of mouse and human complement C3-binding protein: divergence of form but conservation of function. Immunology Today 13: 231
  2. Lim H.W., and Gigli I. (1983) Complement-derived peptides in phototoxic reaction in Experimental and Clinical Photoimmunology (eds. R.A. Dayes and D. Spikes), Vol. 1 p. 82-93. CRC Press Inc.; Florida
  3. Lim H.W., Poh-Fitzpatrick M.B., and Gigli L. (1984) Activation of complement system in patients with porphyries after irradiation in vivo. J. Clin. Invest. 74: 1961 
  4. Morgan B.P. (1990) Complement and dermatological diseases. in Complement (ed. B.P. Morgan) Academic Press; San Diego, New York, London 
  5. Sladowski D., Steer S., Moore L., Glassborow R., Clothier R., and Balls M. (1993) Preliminary findings of an in vitro assay based on human complement activation for detection of chemicals with phototoxicity potential. ATLA 21: 509-512. 
  6. Torinuki W., and Tagami, H. (1986) Role of complement in chlorpromazine-induced phototoxicity. J. Invest. Derm. 86: 142 
IP-77 March 1997