Subchronic 90-day feeding studies were conducted in male and female (F-344) rats on highly refined white mineral oils and waxes representative of those used for food applications. The goal was to help clarify the mixed results found in other toxicity studies with laboratory animals. Seven white oils and 5 waxes were fed at dietary doses of 20,000, 2,000, 200, and 20 ppm and compared with control groups on untreated diet; toxicity was assessed at 90 days and also after a reversal period of 28 days and/or 85 days. Higher molecular-sized hydrocarbons (microcrystalline waxes and the higher viscosity oils) were without biological effects. Paraffin waxes and low-to midviscosity oils produced biological effects that were inversely related to molecular weight, viscosity, and melting point; oil type and processing did not appear to be determinants. Biological effects were more pronounced in females than in males. Effects occurred mainly in the liver and mesenteric lymph nodes and included increased organ weights, microscopic inflammatory changes, and evidence for the presence of saturated mineral hydrocarbons in affected tissues. Inflammation of the cardiac mitral valve was also observed at high doses in rats treated with paraffin waxes. Further studies are required to elucidate the mechanism for the responses observed and the relevance of these inflammatory responses in the F-344 rat to other species, including humans.

A 90-day study was conducted to compare the effects of dietary administration of a food-grade white oil in female Fischer-344 (F-344) and Sprague-Dawley-derived (CRL:CD) rats. Animals were fed a low viscosity (15 mm2/sec at 40 degrees C) paraffinic white oil (designated as P 15[H]) at 0, 0.2, or 2.0% of the diet for 30, 61, or 92 days. There were no significant adverse clinical observations or unscheduled deaths. In the F-344 rats, occasional treatment-related changes were seen in hematology and clinical chemistry parameters. At necropsy, mesenteric lymph nodes were enlarged, and there was an increase in absolute and relative liver, mesenteric lymph node, and spleen weights as compared to controls. Histopathologic effects included hepatic and mesenteric lymph node microgranulomas and mesenteric lymph node histiocytosis. In CRL:CD rats, the only effects noted were accumulations of chronic inflammatory cells in the liver at the high dose only, without the formation of discrete microgranulomas. A dose-related increase in mineral hydrocarbon (MCH) material in the liver and mesenteric lymph nodes was observed in both F-344 and CRL:CD rats. Although increased, liver MhC content was significantly less (approximately 50%) in CRL:CD rats than the levels detected in the F-344 rats. Mesenteric lymph node MHC levels did not differ significantly between the strains. This study demonstrated strain differences among rats in histopathologic effects of white oil, with the CRL:CD rat essentially showing no response compared to the F-344 rat.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study evaluated the effect of di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX) activity, and replicative DNA synthesis in several rodent species with differing susceptibilities to peroxisome proliferator-induced hepatic tumorigenesis. A low (non-tumorigenic) and high (tumorigenic) dietary concentration of DEHP was administered to male F344 rats for 1, 2, 4, and 6 weeks. Additionally, a previously non-tumorigenic dose (1000 ppm) and tumorigenic dose of DEHP (12,000 ppm), as determined by chronic bioassay data, were examined following 2 weeks dietary administration. Male B6C3F1 mice were fed the non-tumorigenic concentration, 500 ppm, and the tumorigenic concentration, 6000 ppm, of DEHP for two and four weeks. The hepatic effects of low and high concentrations of DEHP, 1000 and 6000 ppm, were also examined in male Syrian Golden hamsters (refractory to peroxisome proliferator-induced tumorigenicity). In rat and mouse liver, a concentration-dependent increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed at the earliest time point examined. Concurrent to these observations was an inhibition of GJIC. In hamster liver, a slight increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed. However, these effects were not of the same magnitude or consistency as those observed in rats or mice. Furthermore, DEHP had no effect on GJIC in hamster liver at any of the time points examined (2 and 4 weeks). HPLC analysis of DEHP and its primary metabolites, mono-2-ethylhexyl phthalate (MEHP), and phthalate acid (PA), indicated a time- and concentration-dependent increase in the hepatic concentration of MEHP. At equivalent dietary concentrations and time points, the presence of MEHP, the primary metabolite responsible for the hepatic effects of DEHP, demonstrated a species-specific response. The largest increase in the hepatic concentration of MEHP was observed in mice, which was greater than the concentration observed in rats. The hepatic concentration of MEHP was lowest in hamsters. Hepatic concentrations of DEHP and phthalic acid were minimal and did not correlate with concentration and time. Collectively, these data demonstrate the inhibition of hepatic GJIC and increased replicative DNA synthesis correlated with the observed dose- and species-specific tumorigenicity of DEHP and may be predictive indicators of the nongenotoxic carcinogenic potential of phthalate esters.

Several phthalate esters, compounds used as plasticizers in a variety of commercial products, have been shown to induce hepatic tumors in rodents. In this study, the comparative effects of phthalate monoesters on inhibition of gap junctional intercellular communication and induction of peroxisomal beta-oxidation were assessed in primary cultured hepatocytes from rats, mice, hamsters, cynomolgus monkeys, and humans. A human liver cell line was also utilized. Eight monoesters examined included mono-2-ethylhexyl phthalate (MEHP), mono-n-octyl phthalate (MNOP), mono-isononyl phthalate (MINP, 3 types, -1, -2, and -3), mono-isoheptyl phthalate (MIHP), mono-isodecyl phthalate (MIDP), and mono-(heptyl, nonyl, undecyl) phthalate (M711P). Gap junctional intercellular communication was measured 4 and 24 h after treatment by lucifer yellow dye coupling. Gap junctional intercellular communication was inhibited in rat and mouse hepatocytes by all eight monoesters in a concentration-dependent manner. In most cases, gap junctional intercellular communication was significantly reduced at the lowest concentrations tested (50 pM). Inhibition of gap junctional intercellular communication in rodent cells was substantially reversed within 24 h of monoester removal. In contrast, cell-to-cell communication was not inhibited in hamster, cynomolgus, or human hepatocytes or in a human liver cell line at any concentration examined. In rat hepatocytes, peroxisomal beta-oxidation was elevated after treatment with MEHP, MINP, MIHP, and MIDP but not MNOP or M711P, and with all but MIHP in mouse hepatocytes. The eight phthalates produced no marked change on peroxisomal beta-oxidation in hepatocytes from other species. These data provide additional evidence that the toxicological effects of phthalate esters are species specific.

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The tumor promotion stage of chemical carcinogenesis has been shown to exhibit a persistence of cellular effects during treatment and the reversibility of these changes upon cessation of treatment. Inhibition of gap-junctional intercellular communication and increased replicative DNA synthesis appear to be important in this process. The present study assessed the persistence and reversibility of gap-junctional intercellular communication inhibition, peroxisomal proliferation, and replicative DNA synthesis in livers from male F344 rats and B6C3F1 mice. Dietary administration of 20,000 mg/kg DEHP to male rats for 2 weeks decreased intercellular communication (67% of control) and enhanced replicative DNA synthesis (4.8-fold over control). Elevation of the relative liver weight and the induction of peroxisomal beta oxidation were also observed following treatment with 20,000 mg/Kg DEHP for 2 weeks. Following DEHP administration at a dose of 6000 mg/kg for 18 months, inhibition of gap-junctional intercellular communication persisted, and the relative liver weight and induction of peroxisomal beta oxidation remained elevated in both rats and male B6C3F1 mice. Treatment of rats and mice with phenobarbital for 18 months (500-mg/kg diet) also produced an increase in relative liver weight and a decrease in cell-to-cell communication. In recovery studies in which DEHP was administered to male F344 rats for 2 weeks and then withdrawn, the relative liver weight, rate of peroxisomal beta oxidation, increase in replicative DNA synthesis, and inhibition of gap-junctional intercellular communication returned to control values within 2 to 4 weeks after DEHP treatment ceased. Recovery studies with phenobarbital produced similar results. The primary active metabolite of DEHP, mono-2-ethylhexyl phthalate (MEHP), was detected in the livers of animals treated with DEHP for greater than 2 weeks. However, it could not be detected after removal of DEHP from the diet for 2 weeks. This study demonstrated that inhibition of gap-junctional intercellular communication, along with indicators of peroxisomal proliferation, including increased relative liver weight and enhanced peroxisomal beta oxidation, persist while DEHP treatment continues but reverses when treatment is stopped. Studies with phenobarbital produced a similar pattern of response.

Renal xenobiotic metabolism can result in production of electrophiles or free radicals that may covalently bind macromolecules or initiate lipid peroxidation. The mechanisms of renal xenobiotic metabolism may vary in different anatomical regions. Kidney cortex contains a cytochrome P-450 system while medulla contains a prostaglandin endoperoxidase. Recently cysteine conjugated-lyase has been implicated in production of reactive intermediates. Metabolic activation may be amplified by accumulation of xenobiotics within renal cells due to tubular concentrating and/or secretory mechanisms. Additionally, renal xenobiotic detoxicification can occur by conjugation with glucuronide, sulfate or glutathione.

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