The potential toxicologic and oncogenic effects of spinosad, a natural fermentation product with insecticidal properties, were investigated. The 13-week toxicity study consisted of groups of 10 CD-1 mice/sex provided diets containing 0, 0.005, 0.015, 0.045, or 0.12% spinosad (Study 1). The 0.12% group was terminated on Test Day 44 due to mortality and overt clinical signs of toxicity. An 18-month chronic oncogenicity study consisted of groups of 50 CD-1 mice/sex provided diets containing 0, 0.0025, 0.008, or 0.036% spinosad (Study 2). Two interim groups of 10 mice/sex/group were terminated after 3 and 12 months. Females given 0.036% were terminated on Day 455 due to markedly lower body weights and feed consumption, as well as excessive mortality. Because of the early termination of the female high-dose group, additional groups of 10 male and female mice (12-month interim necrospy) and 50 male and female mice (18-month necropsy) were provided diets containing 0, 0.0008, or 0.024% spinosad (Study 3) to fully assess potential chronic toxicity and oncogenicity. Standard toxicologic parameters were evaluated consistent with existing regulatory guidelines. The primary effect in the 13-week and 18-month studies was intracellular vacuolation of histiocytic and epithelial cells in numerous tissues and organs at doses of > or = 0.015%. The histological vacuolation corresponded to ultrastructural lysosomal lamellar inclusion bodies. This alteration was consistent with phospholipidosis, a condition that results from accumulation of polar lipids in lysosomes. Lesions with no apparent direct relation to vacuolation were hyperplasia of the glandular mucosa of the stomach, skeletal muscle myopathy, bone marrow necrosis, and anemia with associated splenic hematopoiesis. The incidence of tumors in mice given spinosad was not increased relative to controls at any dose level. The no observed effect level for the 13-week study was 0.005% (6 mg/kg/day) spinosad, and for the chronic toxicity/oncogenicity study was 0.008% (11 mg/kg/day) spinosad for male and female CD-1 mice.

The (6,(9)(10)(11). Reaction at the guanine site is stereoselective both in vivo (6) and in vitro (9, 12) and results from asymmetries in the secondary structure of DNA (12). Minor adducts between racemic anti-BPDE [(+)anti-BPDE] and adenine (9, 10, 13), cytosine (9, 10), and the N-7 position of guanine (14) have been reported.(±)anti-BPDE is unstable in aqueous media and readily undergoes hydrolysis to form isomeric 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (tetrols). Hydrolysis of (+)anti-BPDE and the syn diastereomer (in which the 7-hydroxyl group is cis to the epoxide) occurs by general acid catalysis (15)(16)(17). Furthermore, product analyses and ionic strength effects have led to similar proposals for the mechanism of (±)anti-BPDE hydrolysis (16,17). This mechanism involves a rapid equilibrium between general acid catalyst and (±)anti-BPDE, followed by proton transfer and carbonium ion formation in the rate-determining step (rds).DNA catalyzes hydrolysis of (+)anti-BPDE (18,19), and in a recent study the acid dependence of this reaction was investigated at pH 6.5-7.5 (20). The results indicated that hydrolysis in the presence of DNA was also an acid-catalyzed process (20). A model has been proposed for DNAcatalyzed hydrolysis and covalent binding in which a carbonium ion formed in the rds serves as a common intermediate for both reactions (21). The rate of DNA-catalyzed hydrolysis may be important to the total level of covalent adduct obtained and, thus, to the genesis of tumors induced by chemical carcinogens. We have, therefore, investigated the mechanisms of these reactions in an in vitro model system utilizing calf thymus DNA. Our results suggest that a carbonium ion is formed in the rds for each process, but the activated intermediates for hydrolysis and covalent binding are formed in different domains. Kinetic results support our model that carbonium ion precursors to covalent adducts are derived from physically intercalated hydrocarbons, while tetrols are formed from carbonium ions generated on the outside of the DNA helix.MATERIALS AND METHODS Synthesis. (±)anti-BPDE was synthesized as previously described (10, 12). The preparation of 3H-labeled (±)anti-BPDE has also been reported (10).Chemicals. Calf thymus DNA was obtained from Sigma. All other chemicals were obtained from commercial sources and were reagent quality or higher grade purity.Hydrolysis Kinetics. Hydrolysis of (+)anti-BPDE was followed spectrophotometrically (15-17), both at an absorption band of the hydrocarbon (345.5 nm) and at the long-wavelength red-shifted transition representing the BPDE-DNA intercalation complex (centered at 353 nm) (22, 23). Firstorder plots resulted in straight lines, indicating that hydrolysis was measured under pseudo-first-order conditions. Lines were fitted by least-squares analysis and the resulting slopes were used to determine pseudo-first-order rate constants. Replicate values for rate constants were within 5%.Covalent Binding Assays. Covalent binding of (+)anti-BPDE was meas...

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