Biological assays for irritant, tumor-initiating and tumor-promoting activities

Abstract

A versatile standardized protocol of initiation/promotion of skin is described using 7,12-dimethylbenz[a]anthracene as initiator and diterpene esters as promoters in 28 female NMRI mice per promoter (or initiator) dose group including appropriate positive and negative controls ("standardized initiation/promotion protocol 28" or "protocol 28"). To monitor the weekly tumor responses, the tumor rate Tr and the tumor yield Ty were recorded per dose group collectively as a measure of the tumor-promoting (or initiating) activity of individual compounds; similarly the general health status of the group was controlled twice weekly by collective weighing of the dose group and recorded daily by the survival rate Sr. Synoptic consideration of Tr, Ty and the promoter dose (or initiator dose) at a set time period of exposure was used for the semiquantitative estimation of the relative tumor-promoting (or initiating) potency, referring to a high-potency promoter (or initiator) as standard. The full scope of utilization of "protocol 28" to determine comparable individual promoting activities and -at 24 weeks of exposure--relative tumor-promoting potencies (rtpp24) is demonstrated in typical examples with 12-O-tetradecanoylphorbol 13-acetate (TPA) as well as various other phorbol esters as promoters. For estimation of promoting potencies TPA is rated a rtpp24 of + + + +. The other phorbol derivatives exhibit rtpp24 between 0 and + + + +. In this manner, "protocol 28" was verified in 1000 promoter dose groups (i.e. 28,000 mice) to determine tumor-promoting activities of individual diterpene esters and partly also their rtpp24. Grading of tumor-promoting potencies of diterpene esters in skin by rtpp24 proved useful for evaluating the relative environmental risk involved in exposure to various diterpene esters as well as in comparative mechanistic investigations including structure/activity relationships. As an example, the correlation of irritancy and tumor-promoting potency of certain diterpene esters in epithelial tissue, postulated previously from essentially qualitative data, is verified in semiquantitative terms by comparison--as a typical example--of the irritancy of phorbol esters on the ear and of the corresponding rtpp24 on the back skin of NMRI mice.