Vinclozolin (Vin CAS 50471-44-8), a dicarboximide fungicide, has been reported be an antagonist of androgen receptor binding in rats, effects that could result in decreased fertility, defects in reproductive organs or cancer. It is thought that Vin activation to the metabolites 2-[[(3,5-dichloro-phenyl)carbamoyl]oxy]-2-methyl-3-butanoic acid (M1, CAS 119209-27-7), 3',5-dichloro-phenyl-2-hydroxy-2-methylbut-3-enanilide (M2, CAS 83792-61-4), and 3,5-dichtorobenzenamine (M3, CAS 626-43-7) could induce similar toxicity in workers. A potential biomonitoring method was developed to measure tissue levels and evaluated using Dutch Belted rabbits after exposure to Vin, either as a single dermal dose or multiple dermal doses. Vin (100 mg/kg) was applied to the skin in 100 microL DMSO. Plasma samples were taken at 0, 6, 8, 12; 24, and 48 hr after a single dose or weekly during multiple dose exposure. Skin from dermal application sites and testes were excised at termination, homogenized and filtered. A BenchMate robotic workstation was used for automation of sample preparation. Bond Elute 500 mg C8 SPE columns were conditioned with acetone, MeOH and H2O pH 7. Samples were loaded onto columns, rinsed twice with H2O. A fraction containing >96% of Vin and Vin metabolites was collected in 1.5-mL acetone, and reduced to 1 mL for GC-MS STM analysis (Hewlett-Packard quadruple using a 12 m X 0.2 mm Ultra I column). Chromatographic standards M1 and M 2 were synthesized by alkaline hydrolysis of Vin and isolated by HPLC. Vin, M1, M2., and M3 were detected in both plasma and tissues, but animal variation was problematic. After a single application Vin plasma levels were highest, at 6 hr where as levels of M1, M2, and M3 were highest at 12 hr. After 4 weeks of dosing Vin levels in plasma were 162.2 n.g/mL - consistent with testes levels. M2 was detected in the testes and skin levels of Vin were about 10 fold that of plasma. Thus, Vin and Vin metabolite internal exposure levels can be rapidly quantified in a single assay using an automated system.
The Toxicologist. Society of Toxicology 38th Annual Meeting, March 14-18, 1999, New Orleans, Louisiana