Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans.

A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed male Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200mg/m(3). While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.

Dosimetry considerations in the enhanced sensitivity of male Wistar rats to chronic ethylene glycol-induced nephrotoxicity.

Male Wistar rats have been shown to be the most sensitive sex, strain and species to ethylene glycol-induced nephrotoxicity in subchronic studies. A chronic toxicity and dosimetry study was therefore conducted in male Wistar rats administered ethylene glycol via the diet at 0, 50, 150, 300, or 400 mg/kg/day for up to twelve months. Subgroups of animals were included for metabolite analysis and renal clearance studies to provide a quantitative basis for extrapolating dose-response relationships from this sensitive animal model in human health risk assessments. Mortality occurred in 5 of 20 rats at 300 mg/kg/day (days 111-221) and 4 of 20 rats at 400 mg/kg/day (days 43-193), with remaining rats at this dose euthanized early (day 203) due to excessive weight loss. Increased water consumption and urine volume with decreased specific gravity occurred at 300 mg/kg/day presumably due to osmotic diuresis. Calculi (calcium oxalate crystals) occurred in the bladder or renal pelvis at > or =300 mg/kg/day. Rats dying early at > or =300 mg/kg/day had transitional cell hyperplasia with inflammation and hemorrhage of the bladder wall. Crystal nephropathy (basophilic foci, tubule or pelvic dilatation, birefringent crystals in the pelvic fornix, or transitional cell hyperplasia) affected most rats at 300 mg/kg/day, all at 400 mg/kg/day, but none at < or =150 mg/kg/day. No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg/day groups. At 300 and 400 mg/kg/day, oxalate levels increased proportionally with the nephrotoxicity score supporting the oxalate crystal-induced nephrotoxicity mode of action. No treatment-related effects on the renal clearance of intravenously infused (3)H-inulin, a marker for glomerular filtration, and (14)C-oxalic acid were observed in rats surviving 12 months of exposure to ethylene glycol up to 300 mg/kg/day. In studies with naïve male Wistar and F344 rats (a less sensitive strain), a significant difference was observed in oxalate clearances between young rats (i.e. Wistar clearance < F344) but not in age-matched old rats. Regardless, the ratios of oxalate:inulin clearances in these two strains of rats, including those exposed to ethylene glycol, were all < 1, suggesting that a fraction of the filtered oxalate is reabsorbed. Other species, including humans, typically have clearance ratios >1 and are more effective at clearing oxalic acid by both glomerular filtration and active secretion. Thus, the lower renal clearance and kidney accumulation of oxalates in male Wistar rats enhances their sensitivity, which will be a factor in human risk assessments. The benchmark dose values (BMD05, BMDL05) were 170 mg/kg/day and 150 mg/kg/day for nephropathy, and 170 mg/kg/day and 160 mg/kg/day for birefringent crystals, using incidence times severity data in each case. The NOAEL of 150 mg/kg/day is the same as that reported after 16-week exposure and appears to be a threshold dose below which no renal toxicity occurs, regardless of exposure duration.

Subchronic toxicity study of dicyclopentadiene vapor in rats.

Fischer 344 rats were exposed by inhalation to 0, 1, 5 or 50 ppm dicyclopentadiene (DCPD) vapor 6 hr/day, 5 days/week for 13 weeks, followed by a 13-week recovery period. Animals were euthanized following completion of exposure at 2, 6, or 13 weeks and at postexposure weeks 4 or 13. No mortality, overt signs, body weight changes, hematologic or clinical chemistry values were related to DCPD exposure. In the high-exposure male rats, relative liver weights were significantly increased but with no accompanying histopathologic changes. Exposure to DCPD produced adverse kidney effects in male, but not female, rats as evidenced by the excretion of epithelial cells in the urine. Histologic changes were localized to the proximal tubules of the kidney and included increased accumulation of protein droplets, regenerative epithelium, and the presence of intraluminal proteinaceous material. In addition, several alterations in renal function were observed. Urinary Na+ excretion rates were decreased and urinary K+ excretion rates were increased throughout the exposure period; however, glucose was not present in the urine, and creatinine clearance was normal. The ability of the kidney to concentrate urine was also impaired. After the recovery period, many of the treatment-related kidney effects were not observed, including the presence of hyaline droplets in the proximal tubules and epithelial cells in the urine. These findings indicate an overall low degree of systemic toxicity following subchronic inhalation exposure of dicyclopentadiene at exposure levels up to 50 ppm. The only effect that was observed was a male rat-specific nephropathy that is characteristic of the hyaline droplet nephropathy produced by a diverse group of compounds.

Methyl isocyanate eight-day vapor inhalation study with Fischer 344 rats.

Groups of ten male and ten female Fischer 344 rats were exposed by inhalation 3.1, 0.6, 0.15, or 0.0 (control) ppm of methyl isocyanate (MIC) vapor 6 hr per day for 8 days (two 4-day sessions separated by a 2-day rest). Evaluation of toxic effects included body weight, food consumption, organ weights, and selected hematologic, ophthalmic, neurologic, gross anatomic, and histologic examinations. There were no deaths during the study. Rats of the 3.1 ppm exposure group had decreased body weights, food consumption, and blood oxygen saturation (males only). An increase in hemoglobin concentration (males only) and in lung weights (absolute and as a percentage of body weight) were also observed in the 3.1 ppm rats. Ophthalmic or neurofunctional behavior evaluations were negative for all MIC exposure groups. Only 3.1 ppm of MIC vapor resulted in lesions in the respiratory tract, 0.6 or 0.15 ppm did not. The types of lesions observed were inflammation and squamous metaplasia in the nasal cavity, trachea, and bronchi; inflammation of the bronchioles and alveoli; and submucosal fibroplasia of the bronchioles. No significant lesions were observed in tissues other than those of the respiratory tract in all MIC exposure groups. The results of this study indicate the current 0.02 ppm threshold limit value for MIC is not too high regarding toxicity.

Reproduction and fertility evaluations in CD rats following nitrobenzene inhalation.

A two-generation reproduction study was performed by exposure of Sprague-Dawley CD rats to concentrations of 40, 10, 1, or 0 (control) ppm of nitrobenzene (NB) vapor. No NB-related effects on reproduction were observed at 10 or 1 ppm. At 40 ppm, a decrease in the fertility index of the F0 and F1 generations occurred, which was associated with alterations in the male reproductive organs. Specifically, weights of testes and epididymides were reduced and seminiferous tubule atrophy, spermatocyte degeneration, and the presence of giant syncytial spermatocytes were observed. The only significant finding in the litters derived from rats exposed to 40 ppm was an approximate 12% decrease in the mean body weight of F1 pups on Postnatal Day 21. Survival indices were unaltered. To examine the reversibility of this selective effect on male gonads, the F1 males from the 40-ppm group were allowed a 9-week nonexposure period and mated to naive females. An almost fivefold increase in the fertility index was observed, indicating at least partial functional reversibility upon removal from NB exposure. Also, the numbers of giant syncytial spermatocytes and degenerated spermatocytes were greatly reduced. The results of this study support the selection of 10 ppm of NB as the no-observable-effect level for reproduction and fertility effects in rats.

2,4-Pentanedione: 9-day and 14-week vapor inhalation studies in Fischer-344 rats.

Fischer-344 rats, in groups of 10 males and 10 females, were exposed for 9 days (6 hr/day) to 2,4-pentanedione (2,4-PD) vapor at mean concentrations of 805, 418, 197, and 0 (control) ppm. No deaths occurred, and the only adverse signs were of sensory irritation (partial closure of eyelids, periocular and perioral wetness) at 805 ppm. Also at 805 ppm were decreased body and organ weights, lymphocytosis, and moderate inflammation of the nasal mucosa. At 418 ppm there was a decrease in body weight gain and mild inflammation of the nasal mucosa. Apart from minimal nasal mucosal inflammation, there were no effects at 197 ppm. In the subchronic (14-week) study, rats were exposed (6 hr/day; 5 days/week) to 650, 307, 101, and 0 (control) ppm of 2,4-PD vapor, using groups containing 20 males and 20 females, with half being sacrificed at the end of the exposure period and the remainder kept for a 4-week postexposure recovery period. An additional 10 males were added to the 650 and 0 ppm groups for glutaraldehyde perfusion and subsequent electron microscopic examination of sciatic nerves. At 650 ppm, all females and 10 of 30 males died between the second and sixth weeks of exposure. These animals had acute degenerative changes in the deep cerebellar nuclei, vestibular nuclei and corpora striata, and acute lymphoid degeneration in the thymus. Seven of 15 male survivors of the 650 ppm group (combined 14-week and recovery sacrifices) had gliosis and malacia in the same brain regions, minimal squamous metaplasia in the nasal mucosa, decreased body and organ weights, lymphocytosis, and minor alterations in serum and urine chemistries. No ultrastructural evidence of peripheral neuropathy was observed. Except for central neuropathy, many of the adverse effects at 650 ppm were less marked in the 4-week recovery animals. No deaths occurred at 307 ppm, but females had slightly decreased body weight gains, and in both sexes there were minor alterations in hematology, serum chemistry, and urinalysis parameters, which were not present in the 4-week recovery animals. Rats exposed to 101 ppm showed no differences from the control rats. Subchronic exposure to 650 ppm of 2,4-PD vapor causes serious adverse biological effects. Under these study conditions, the minimum-effects concentration was 307 ppm, and the no-adverse effects concentration was 101 ppm.

Acute inhalation studies with methyl isocyanate vapor. I. Methodology and LC50 determinations in guinea pigs, rats, and mice.

Groups of male and female Fischer 344 rats, B6C3F1 mice, and Hartley guinea pigs were exposed once for 6 hr to mean concentrations of 10.5, 5.4, 2.4, 1.0, or 0 (control) ppm of methyl isocyanate (MIC) vapor. Rats and mice were also exposed to 20.4 ppm of MIC. No deaths occurred in animals exposed to 2.4 or 1.0 ppm. The majority of deaths for the 20.4- and 10.5-ppm groups occurred during postexposure Days 1 through 3, while at 5.4 ppm deaths were observed throughout the 14-day postexposure period. The 6-hr LC50 values (with 95% confidence limits) were 6.1 (4.6 to 8.2) ppm for rats, 12.2 (8.4 to 17.5) ppm for mice, and 5.4 (4.4 to 6.7) ppm for guinea pigs. Notable clinical observations during and immediately following MIC exposure were lacrimation, perinasal/perioral wetness, respiratory difficulty (e.g., mouth breathing), decreased activity, ataxia, and hypothermia. The frequency of clinical signs decreased during the second postexposure week. Body weight losses were common in all species following MIC exposures of 2.4 ppm or greater. At 1.0 ppm, only female mice had body weight depression. Recovery of body weight loss was observed in the 5.4- (guinea pigs only), 2.4- and 1.0-ppm concentration groups. The lungs of all animals that died were discolored. Following microscopic examination of the respiratory tract, deaths were attributed to pulmonary edema and congestion. In a separate study, Fischer 344 rats and Hartley guinea pigs were exposed once for 4 hr to mean concentrations of 36.1, 25.6, 15.2, or 5.2 ppm of MIC vapor. In general, the results were similar to those of the single 6-hr exposure study.

Frequency, size and location of brain tumours in F-344 rats chronically exposed to ethylene oxide.

Increased frequencies of primary brain tumours were seen in male and female F-344 rats exposed to 100 or 33 ppm ethylene oxide (EO) vapour (for 6 hr daily on 5 days/wk for up to 2 yr) but no such increase was seen in rats similarly exposed to 10 ppm EO. The tumours that were seen (glial-cell tumours, granular-cell tumours and malignant reticuloses) were similar in appearance to those that develop spontaneously in F-344 rats, but the tumours associated with EO exposure at levels of 100 or 33 ppm were larger, and in at least six cases were thought to be the primary cause of death. Only two of the 23 tumours seen microscopically were detected by gross examination at necropsy, and brain weights were of minimal value in predicting the presence of tumours. Only three animals demonstrated abnormal neurological signs. These findings point to the need for thorough microscopic examination of the brains of rodents in chronic studies.

Monochlorodiisobutylene vapor: acute and 9-day inhalation studies in Fischer-344 rats and B6C3F1 mice.

Acute and 9-day repeated exposures to monochlorodiisobutylene (CDIB) were conducted in male and female Fischer-344 rats and B6C3F1 mice. The 4-hr LC50 values for these animals ranged between 1400 and 2100 ppm. Animals in the 9-day study were exposed at a mean concentration of 478, 97, or 25 ppm of CDIB for 6 hr per day. Treatment-related effects differed between species in this study. Body weight change was decreased in rats. Morphologic changes in the kidneys with accompanying polyuria and hematuria/hemoglobinuria were observed in male rats. The only effect observed at 25 ppm was a low incidence of hematuria/hemoglobinuria in male rats. Mice appeared unaffected by exposure to CDIB at levels as high as 478 ppm.

Brain tumors in F344 rats associated with chronic inhalation exposure to ethylene oxide.

Groups of F344 rats of each sex were exposed to either ethylene oxide (ETO) vapor (concentrations of 100, 33 or 10 ppm) or to room air for 6 hours daily, 5 days per week, for up to 2 years. Three representative sections of the brain from each rat were evaluated. Twenty-three primary brain tumors were found, two of which were in control animals. Increased numbers of brain tumors were seen in 100 ppm and 33 ppm ETO exposed male and female rats. Significant trend analyses were found for both males and females, indicating that, under the conditions of this study, ETO exposure above 10 ppm was related to the development of these brain tumors.

A subchronic inhalation study on the toxicologic potential of ethylene oxide in B6C3F1 mice.

Four groups of B6C3F1 mice, each containing 30 per sex, were exposed to ethylene oxide (EtO) vapor at target concentrations of 250, 100, 50, 10, or 0 ppm which are equivalent to 450, 180, 90, 18, and 0 mg/m3, respectively. The actual mean chamber concentrations were 236, 104, 48, 10, and 0 ppm, respectively. After an exposure regimen of 6 hr per day, 5 days per week, for 10 weeks (males), or 11 weeks (females), urine and blood samples were taken for clinical pathologic evaluation and selected tissues were weighed and examined by light microscopy. Statistically significant findings that could be indicative of a toxic response were observed in the 250-ppm exposure group. These included minimal changes in certain erythroid parameters, increased liver weight, decreased testicular weight, and decreased spleen weight which was noted also in the 100-ppm group. However, there were no microscopic findings to support or explain any of these apparently treatment-related effects. Results of a neuromuscular screening test indicated that certain reflex responses and locomotor activities were altered in the EtO-exposed animals. A dose-related trend of response in the 250-, 100-, and 50-ppm exposure groups was noted in the evaluation of locomotor function; however, because of the small sample size, it was not possible to determine a threshold concentration for this effect. There were no accompanying histopathologic alterations in muscle and central or peripheral nervous tissue.

A two-year inhalation study of the carcinogenic potential of ethylene oxide in Fischer 344 rats.

Fischer 344 rats were exposed to 100, 33, 10, or 0 ppm of ethylene oxide vapor (EtO) by inhalation for 6 hr per day, 5 days per week, for approximately 2 years. Inhalation of EtO resulted in a significant depression of body weight gain in the 100- and 33-ppm exposure groups and a significant increase in mortality in the 100-ppm group. Through 18 months of exposure to EtO, no statistically significant increases in tumor incidence were observed. After 18 months, the incidence of primary brain tumors was increased for both sexes. Statistical evaluation indicated a treatment-related response, particularly for the male rats, in the 100- and 33-ppm exposure groups. After 24 months of exposure, histologic findings confirmed hematologic evidence that exposure to EtO resulted in an increased prevalence of mononuclear cell leukemia, which is a neoplasm that is common in aged Fischer 344 rats. This increase was dose related and increased for each of the three exposure concentrations. The percentage of female rats with multiple neoplasms was also greater in all three exposure groups than in controls. Furthermore, in both the 100- and 33-ppm exposure groups, the percentage of female rats with at least one malignant neoplasm was increased. An increased frequency of peritoneal mesothelioma was treatment related in the male rats exposed to 100 or 33 ppm of EtO. This study has shown biologically significant adverse effects at all concentrations tested. The incidences of mononuclear cell leukemia, peritoneal mesothelioma, and primary brain tumors in the air-control rats were similar to those reported in the literature. The possible contribution of a sialodacryoadenitis viral outbreak (which occurred during the 15th exposure month) to the EtO exposure-related tumors is unknown, though unlikely.

Effects of daily interrupted nursing on body weight and survival of Fischer 344 rat pups.

The effects on Fischer 344 rat pup body weight and survival were investigated when the dam was separated from her litter and deprived of food and water, approximately 7 h per day, from day 5 to day 21 of the postnatal period. This separation procedure simulates an inhalation reproduction study in which only the dams are exposed to the test material during lactation. The dams and pups were observed daily for clinical signs, and the number of pups in each litter and their body weights were determined on days 4, 9, 14 and 21 after parturition. When pups and dams were so separated, there was no effect on pup survival, but the pup body weights were moderately lower for this group by the end of the postnatal observation period.

Ethylene glycol monobutyl ether: acute, 9-day, and 90-day vapor inhalation studies in Fischer 344 rats.

The acute 4-hr LC50 (with 95% confidence limits) for Fischer 344 rats was determined to be 486 (339 to 696) ppm of ethylene glycol monobutyl ether (EGBE) for males and 450 (315 to 645) ppm for females. Notable observations included loss of coordination, red stained urine, and enlarged discolored kidneys at 867 and 523 ppm. In a subsequent study, rats were exposed for 9 days (6 hr/day) to EGBE concentrations of 245, 86, 20, or 0 (control) ppm. There were significant depressions of red blood cell (RBC) count (approximately 20% below control values), hemoglobin (Hgb), and mean corpuscular hemoglobin (MCH) concentration and increases in nucleated erythrocytes, reticulocytes, and lymphocytes in males and females of the 245 ppm group. Decreased body weight gains and increased liver weights were also found. A 14-day postexposure recovery showed substantial reversal of the affected blood parameters. Similar, but less marked, hematologic effects were observed in rats exposed to 86 ppm of EGBE, while rats of the 20 ppm group were indistinguishable from controls. In a 90-day study, rats were exposed to EGBE concentrations of 77, 25, 5, or 0 ppm for 13 weeks (6 hr/day, 5 days/week). Slight, but statistically significant, decreases in RBC (13% below control) and Hgb, accompanied by an increase in MCH (11% above control) were observed in the 77 ppm-exposed females after 6 weeks. At the conclusion of the 90-day exposure regimen, the hematologic effects seen in the females had lessened (RBC was 7% below control) or returned to control value ranges. Furthermore, no treatment-related differences were found in body weight, organ weights, urine or serum chemistries, gross lesions, or microscopic lesions in males or females. There were no significant biological effects in rats exposed subchronically to 25 or 5 ppm. The subtle hematologic findings of these studies confirm the known RBC perturbations of EGBE.