NTP technical report on the toxicity studies of Dibutyl Phthalate (CAS No. 84-74-2) Administered in Feed to F344/N Rats and B6C3F1 Mice.

Abstract

Dibutyl phthalate is a phthalate ester with extensive use in industry in such products as plastic (PVC) piping, various varnishes and lacquers, safety glass, nail polishes, paper coatings, dental materials, pharmaceuticals, and plastic food wrap. Concomitant with this extensive worldwide use is the high potential for human exposure to dibutyl phthalate in the workplace and the home environment through direct sources as well as indirectly, through contamination of water, air, and foodstuffs. Because existing toxicity information was considered inadequate, the effects of exposure to dibutyl phthalate were examined in male and female F344/N rats and B6C3F1 mice in 13-week feed studies. Furthermore, due to concern over the potential for pervasive exposure of humans to dibutyl phthalate, additional perinatal studies examined rats and mice exposed as pups in utero, for the 4 weeks of lactation, and for an additional 4 weeks postweaning. Additional studies examined the effects on rats of combining perinatal and adult subchronic exposure. Due to the recognized biologic activity of this and other phthalates, hepatic peroxisome proliferation during the in utero and lactational phases and testicular toxicity during the perinatal period were also examined. Finally, reproductive assessment by continuous breeding (including crossover mating trials and offspring assessment) and genetic toxicity studies were also conducted. In the maximum perinatal exposure (MPE) determination study in rats, dibutyl phthalate was administered in the diet to dams during gestation and lactation, and to the pups postweaning for four additional weeks, at concentrations of 0, 1,250, 2,500, 5,000, 7,500, 10,000, and 20,000 ppm. Decreased weight gains were noted in dams exposed to 20,000 ppm during gestation and to dams exposed to 10,000 ppm during lactation. The gestation index (number of live pups per breeding female) was significantly lower in the 20,000 ppm group than in the controls, and pup mortality in this group was marked (100% by Day 1 of lactation); however, survival was 89% or greater in all other treatment groups. The mean body weight of pups in the 10,000 ppm group at Day 28 of lactation was approximately 90% of the mean weight of control pups. Pups were weaned onto diets containing dibutyl phthalate at the same concentrations fed to dams. After an additional 4 weeks of dietary administration, final mean body weights of pups in the 10,000 ppm groups were 92% of the control value for males and 95% of the control value for females. Hepatomegaly (increased relative liver weight) was observed in males in all exposed groups and in females receiving 2,500 ppm or greater. No gross lesions were observed at necropsy. Moderate hypospermia of the epididymis was diagnosed in all male rats in the 7,500 and 10,000 ppm groups; mild hypospermia of the epididymis was diagnosed in 2 of 10 males in the 5,000 ppm group. No degeneration of the germinal epithelium was detected in the testis of these rats. Thus, although toxicologically important, the epididymal hypospermia was not considered to be life threatening, and 10,000 ppm was recommended as the MPE concentration for male and female rats. In the subsequent subchronic toxicity study of dibutyl phthalate with perinatal exposure, dams were administered diets containing 0 or the MPE concentration (10,000 ppm) during gestation and lactation, and weaned pups were administered the same diets as their dams received for an additional 4 weeks, until the beginning of the 13-week exposure phase. Male and female rats then received diets containing dibutyl phthalate at concentrations of 0, 2,500, 5,000, 10,000, 20,000, and 40,000 ppm for 13 weeks. No mortality or toxicity was observed in dams during the perinatal phase of the study; however, before pups were culled at 4 days postpartum, the percentage of live pups per litter was 86% to 93% that of the controls. Through weaning, litter weights of exposed pups ranged from 89% to 92% of the control values. Ten control and ten exposed pups per sex were examined at the time of trol and ten exposed pups per sex were examined at the time of weaning; hepatomegaly and markedly increased peroxisomal enzyme activities (approximately 9-fold greater than the control values) were observed in exposed pups. Body weights of the perinatally exposed pups remained lower than those of the controls throughout the 4-week period before the 13-week adult exposures began. During the 13-week adult exposure phase, the final mean body weight of males in the MPE: 0 ppm control group (MPE rats, returned to the base diet for 13 weeks), was 95% that of the controls. The body weight gain of females in the MPE:0 ppm group was greater than that of the unexposed controls, and the final body weights of these two groups were similar. Body weight gains of rats treated with dibutyl phthalate as adults decreased with increasing exposure concentration; for rats that received the MPE concentration followed by 40,000 ppm for 13 weeks, final body weights were 51% of the control value for males and 74% of the control value for females. Hepatomegaly apparently regressed in rats in the MPE:0 ppm groups but was observed in male rats receiving 5,000 ppm or greater and in females receiving 2,500 ppm or greater. In males that received 20,000 ppm as adults, testis and epididymal weights were less than in the controls; males in the 40,000 ppm group also had a lower testis weight than the controls. Results of hematologic analyses conducted at the end of the 13-week exposure period suggested a mild anemia in male rats administered 10,000 ppm or greater as adults and female rats administered 40,000 ppm as adults. Hypocholesterolemia and hypotriglyceridemia were observed in male and female rats at the higher exposure concentrations. Hypotriglyceridemia was detected in females receiving 20,000 or 40,000 ppm and in males receiving 10,000 ppm or greater. Elevations in alkaline phosphatase activities and bile acid concentrations in male and female rats receiving 20,000 or 40,000 ppm as adults were indicative of cholestasis. Microscopic examination revealed hepatocellular cytoplasmic alteration, consistent with glycogen depletion, in male and female rats receiving a concentration of 10,000 ppm or greater. In the liver of rats receiving 40,000 ppm, small, fine, eosinophilic granules were also observed in the cytoplasm of hepatocytes. Ultrastructural examination suggested the presence of increased numbers of peroxisomes. Lipofuscin accumulation was detected in rats that received 10,000 ppm or greater. Consistent with the regression of the hepatomegaly in rats in the MPE:0 and MPE:2,500 ppm groups, peroxisomal enzyme activity was not elevated in these groups. Marked elevations of peroxisomal enzyme activity were detected, however, in males receiving 5,000 ppm or greater and in females receiving 10,000 ppm or greater; at the 40,000 ppm concentration, the highest concentration tested, enzyme activities were approximately 20 fold greater than the control values. Histopathologic examination of the testes revealed degeneration of the germinal epithelium, a mild to moderate focal lesion in rats in the 10,000 and 20,000 ppm groups and a marked, diffuse lesion in all males receiving 40,000 ppm; at 40,000 ppm, an almost complete loss of the germinal epithelium resulted. Testicular zinc concentrations were lower in the 40,000 ppm group than in the controls, a finding consistent with the marked loss of germinal epithelium at this exposure concentration. Spermatogenesis was evaluated in rats in the 0, 2,500, 10,000, and 20,000 ppm groups; rats administered 20,000 ppm had fewer spermatid heads per testis than the unexposed controls, and epididymal spermatozoal concentration was less than that in the MPE:0 ppm group. For comparison with the perinatal subchronic study, a standard 13-week evaluation of the toxicity of dibutyl phthalate in male and female rats was also conducted. In this study, rats received dibutyl phthalate at the same dietary concentrations used in the 13-week exposure phase of the study with perinatal exposure: 0, 2,500, 5,000, 10,000, 20,000, and 40,000 ppm. No deaths occurred in the standard study. Markedly reduced final mean body weights were observed in males and females in the 40,000 ppm groups (45% and 73% of control body weights, respectively); final mean body weights of males receiving 10,000 ppm or greater and females receiving 20,000 ppm or greater were lower than those of the controls. Hepatomegaly was observed in males that received 5,000 ppm or greater and in females that received 10,000 ppm or greater. Testis and epididymal weights of males in the 20,000 and 40,000 ppm groups were lower than those of the controls. A minimal anemia was detected in male rats receiving 5,000 ppm or greater. Hypocholesterolemia was observed in male and female rats receiving 20,000 or 40,000 ppm, and hypotriglyceridemia was detected in males in all exposed groups and in females receiving 10,000 ppm or greater. Elevations in alkaline phosphatase activity and bile acid concentration in male and female rats were considered indicative of cholestasis. Morphologic evaluation again confirmed the toxicity of dibutyl phthalate to the liver and testes of rats. Microscopic examination of the liver revealed hepatocellular cytoplasmic alterations, consistent with glycogen depletion, in male and female rats receiving 10,000 ppm or greater. In the liver of rats in the 40,000 ppm groups, small, fine, eosinophilic granules were also observed in the cytoplasm of hepatocytes. Ultrastructural examination suggested the presence of increased numbers of peroxisomes, and peroxisomal enzyme activity was elevated in the livers of male and female rats administered 5,000 ppm or greater; the enzyme activities in the 40,000 ppm groups were approximately 13-fold greater than the control value for males and 32-fold greater than t