An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures.
We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen sections using a modified methyl-green pyronine (MGP) staining procedure, was used as a marker of irritancy. A decrease in epidermal RNA after a 4-, 24- or 48-h exposure to a certain concentration of a test chemical equated to a MGP score of 3, 2 or 1, respectively. The MGP score was 0 if there was no keratinocyte cytotoxicity after a 48-h exposure. A minimum of three donors were used per chemical and the average MGP score was used to classify the chemical as irritant or not. Chemicals with an average MGP score > or =1.5 were classified as irritants (R38), at that concentration. Chemicals with a MGP score <1.5 were not classified (NC), at that concentration. The results obtained using human skin in vitro were compared with published data obtained using cultured porcine skin, the cutaneous Draize test (from this point referred to as the "rabbit skin irritation test") and volunteer studies. There was an excellent correlation between the classification of a chemical, as R38 or NC, based on hOSEC and results of volunteer studies. The hOSEC model predicted perfectly the irritation hazard of the 22 chemicals for which volunteer data were available. The porcine OSEC correctly predicted the classification of 21 of 22 (95%) chemicals and the rabbit skin irritation test correctly predicted the classification of 14 of 15 chemicals (93%) for which data were available. In conclusion, MGP staining of human skin explant cultures can be used to predicted human skin irritancy in vivo. In addition, the data validate the use of porcine skin as an alternative to human skin for screening for dermal irritants in vitro.
Department of Pharmacology, TNO-PML, Rijswijk, The Netherlands. j.j.l.jacobs@vet.uu.nl
J J L
JJ
Jacobs
C
C
Lehé
K D A
KD
Cammans
P K
PK
Das
G R
GR
Elliott
D000826:Animal Testing Alternatives; D000818:Animals; D002478:Cells, Cultured; D004396:Coloring Agents / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007509:Irritants / Q000145:classification / Q000633:toxicity*; D008739:Methyl Green / Q000737:chemistry*; D011754:Pyronine / Q000737:chemistry*; D012313:RNA / Q000032:analysis; D011817:Rabbits; D015203:Reproducibility of Results; D012867:Skin / Q000737:chemistry* / Q000187:drug effects* / Q000473:pathology; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D018675:Toxicity Tests
D004396:Coloring Agents; D007509:Irritants; D012313:RNA; D008739:Methyl Green; D011754:Pyronine