Comparison of the Mutagenicity and Teratogenicity of Cyclophosphamide and Its Active Metabolites, 4-Hydroxycyclophosphamide, Phosphoramide Mustard, and Acrolein1

Cyclophosphamide must be metabolically activated to have maximal mutagenic or teratogenic activity. The first step in this activation is hydroxylation to 4-hydroxycyclophosphamide; this metabolite breaks down to form two cytotoxic metabolites, phosphoramide mustard and acrolein. In this report, the mutagenicity and teratogenicity of Cyclophosphamide, 4-hydroperoxycyclophosphamide (which forms 4-hydroxycyclophos phamide spontaneously in solution), phosphoramide mustard, and acrolein were compared. Mutagenicity was assessed using a Salmonella typhimurium TA 1535 test system; teratogenicity was studied in rats on Day 20 of gestation after intraamniotic injection of drug on Day 13. The activation of Cyclophospha mide to mutagenic metabolites was dependent on the presence of liver microsomes and reduced nicotinamide adenine dinucleotide phosphate while both phosphoramide mustard and 4hydroperoxycyclophosphamide were mutagenic without acti vation, with the latter being the most potent. The third metab olite, acrolein, was bacteriotoxic at low concentrations; it was not mutagenic in the absence and only very weakly mutagenic in the presence of liver microsomes. All four compounds tested were teratogenic. The malformations produced by Cyclophos phamide, 4-hydroperoxycyclophosphamide, and acrolein in cluded edema, hydrocephaly, open eyes, cleft palate, micrognathia, omphalocele, bent tail, and forelimb and hindlimb defects, whereas phosphoramide mustard produced only hy drocephaly and tail, forelimb, and hindlimb defects. 4-Hydroperoxycyclophosphamide and its breakdown product, acrolein, were more potent as teratogens than either Cyclophosphamide or phosphoramide mustard. Cyclophosphamide produced mal formations in both the injected and contralateral uninjected fetuses while the other three compounds all produced malfor mations only in the injected fetuses. Thus, the site of activation of Cyclophosphamide to a teratogen is probably maternal. Because acrolein plays a major role in the teratogenicity of Cyclophosphamide but is only weakly, if at all, mutagenic, the teratogenicity and mutagenicity of metabolites of this drug are dissociable.