* Health and Environment Laboratories, Eastman Kodak Company, 1100 Ridgeway Ave., Rochester, New York 14652-6272;
Covance, Inc., Vienna, Virginia; and
Eastman Chemical Company, Kingsport, Tennessee
Received May 23, 2000; accepted August 18, 2000
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ABSTRACT |
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Key Words: DEHP; di(2-ethylhexyl)phthalate; chronic toxicity; liver; kidneys; testes.
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INTRODUCTION |
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Since dietary administration represents the most widespread exposure, an assessment of the potential toxicity from dietary sources is appropriate. Data for rats have been published (Carpenter et al., 1953; David et al. 2000
; Harris et al. 1956
), but relatively few long-term studies of DEHP have been performed using mice. The NTP conducted a chronic oncogenicity study in the 1980s, demonstrating liver tumors after 2 years of exposure to 3000 and 6000 ppm (Kluwe et al., 1982
), but few other effects were observed. There were no "toxic" lesions in the liver. Ward et al. (1986) reported that male B6C3F1 mice exposed to 3000, 6000, or 12,000 ppm DEHP for 18 months had substantially lower body weight at 6000 ppm (20%) and 3000 ppm (10%). Cytoplasmic eosinophilia of the liver and hepatomegaly were reported at all dose levels. Renal tubular degeneration, necrosis and cystic hyperplasia were seen in the kidneys, and some animals at 6000 ppm had degeneration of the seminiferous tubules. Other organs were not evaluated, and there was no assessment of clinical chemistry or hematology. The value of having data for mice is that it provides information on distinguishing species-specific effects from test substance-specific effects.
Long-term studies of the chronic effects of DEHP in mice and rats were recently undertaken to help evaluate the correlation of peroxisome proliferation, cell proliferation, and hepatocarcinogenicity in rodents (David et al., 1999). Dose levels of 2500 and 12,500 ppm resulted in an increased incidence of hepatocellular neoplasm, hepatomegaly, and palmitoyl CoA oxidation in rats. Palmitoyl CoA oxidation was increased 1.71.9 times control for the 2500-ppm groups, and 3.65.1 times control for the 12,500-ppm dose groups. Dose levels of 500, 1500, and 6000 ppm resulted in an increased incidence of hepatocellular neoplasm, hepatomegaly, and palmitoyl CoA oxidation in mice. Palmitoyl CoA oxidation was increased 2.24 times control for the 500-ppm groups, 2.76.8 times control for the 1500-ppm groups, and 7.615.0 times control for the 6000-ppm dose groups. As part of those studies, the noncarcinogenic chronic effects of exposure were also evaluated. We previously reported that the noncarcinogenic effects of long-term oral exposure to DEHP were limited to minor alterations in clinical chemistry and hematology, increased incidence of liver lesions (pigment in Kupffer cells, spongiosis hepatis), pancreatic acinar cell adenoma, aspermatogenesis, and castration cells in the pituitary gland for rats (David et al., 2000
). DEHP exposure exacerbated age-, species- or strain-related lesions such as mineralization of the renal papilla and chronic progressive nephropathy in male rats. Those effects and the effect levels were compared with dose levels at which peroxisome proliferation occurred. Lesions such as pancreatic acinar-cell hyperplasia and spongiosis hepatis have been associated with exposure to peroxisome proliferators such as WY-14,643 (Obourn et al., 1997
) or weak hepatocarcinogens (Bannasch et al., 1981
) in rat. Ward et al. (1998) suggested that testicular and renal lesions were unrelated to peroxisome proliferation, based on a study in which PPAR-knockout mice given 20,000 ppm DEHP in the diet developed testicular and renal lesions similar to those found in the wild type. Other effects may also be unrelated.
Bilateral aspermatogenesis in the testes, castration cells in the pituitary gland, spongiosis hepatis, and pancreatic acinar cell adenoma were observed for 12,500 ppm male rats. Aspermatogenesis and spongiosis hepatis were observed for 2500 ppm-dosed male rats, and aspermatogenesis was seen in 500-ppm male rats. Kupffer cell pigmentation and renal tubule pigmentation were seen only in male and female high-dose rats (12,500 ppm), although peroxisome proliferation was also apparent at the next lower dose level (2500 ppm). The increased incidence of spongiosis hepatis correlated with increased palmitoyl CoA oxidase activity, but the incidence of pancreatic acinar-cell adenoma was increased only at the highest dose level. Thus, spongiosis hepatis, Kupffer cell pigmentation, renal tubule pigmentation, and pancreatic acinar-cell adenomas may be associated with high levels of peroxisome proliferation. The data for mice are presented here for comparison.
Animals were evaluated for evidence of progressive alterations in clinical chemistry, hematology, and urine parameters. Tissues were examined after 78 and 104 weeks for histopathology. These results are useful for assessing the risk from oral exposure and may provide some insight into the association of noncarcinogenic effects with peroxisome proliferation.
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MATERIALS AND METHODS |
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Animals.
Four-week old male and female B6C3F1 mice (B6C3F1/CrlBR), obtained from Charles River Laboratories, Inc. (Portage, MI), were used for the study following a 2-week acclimation period. Animals were maintained on tap water and powdered PMI #5002 chow (Purina Mills, Inc., Richmond, VA). Animal husbandry conformed to standards outlined in the Guide for the Care and Use of Animals (National Research Council, 1996) using temperatures at 72 ± 6°F with a relative humidity of 50 ± 20%. A 12-h light/12-h dark cycle and 10 or greater air changes/h were maintained in the room housing the animals.
Treatment levels for bioassay.
The animals were assigned to study using a computerized weight-randomization program, which first eliminated the animals with extreme body weights, then selected the random assignment that produced homogeneity of variance and means by Bartlett's test and 1-way analysis of variance (ANOVA). At randomization, the weight variation of the animals selected did not exceed 2 standard deviations of the mean body weight for each sex, and the mean body weight for each group of each sex was not statistically different. Mice were divided into 5 groups of 70, 60, 65, 65, and 70 for Groups 15, respectively. Dosage levels were 0, 100, 500, 1500, or 6000 ppm DEHP, given in the diet (Groups 15, respectively), based on the results of a 13-week study. These groups were treated continuously for up to 104 weeks. A set of 1015 animals per group was terminated during Week 79. The remaining animals were terminated during Week 105. Diets containing DEHP were mixed weekly during the study, and the concentration of DEHP in the diet was verified periodically by high-performance liquid chromatography (HPLC) analysis.
Mortality and clinical observations.
The mice were observed for mortality and moribundity twice daily. A thorough physical examination was conducted at each weighing interval. A careful cage-side observation for obvious indications of toxic effects was performed once daily.
Body weights, organ weight, and food consumption.
Body weights and food consumption were measured weekly for Weeks 117, and once every 4 weeks thereafter. At necropsy, the terminal (fasted) body weight and the weights of the brain (with stem), lungs, liver, spleen, kidneys (paired), testes (paired with epididymides), and uterus were measured.
Clinical pathology.
Blood was collected under anesthesia from the retro-orbital sinus of 10 fasted animals per sex per group during Weeks 26, 52, 78, and 104 for clinical chemistry and hematology analyses. Whole blood samples were analyzed for counting red blood cells (RBC), total and differential white blood cells (WBC), reticulocytes, and platelets; as well as hemoglobin (Hgb) and hematocrit (Hct), counts. If possible, blood was collected from the same animals at each respective interval. Serum samples were analyzed for albumin, protein, calcium, phosphorous, urea nitrogen (BUN), creatinine, glucose, sodium, potassium, chloride, bilirubin, gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Urine samples from the same animals were collected overnight for evaluation of appearance (color and turbidity); bilirubin, glucose, and ketone levels; and microscopic examination of sediment, occult blood, pH, protein, specific gravity, and urobilinogen. Hematology analyses (RBC, total WBC, Hgb, and Hct) were performed using a Coulter Counter Model S-Plus IV (Coulter Co., Hialeah, FL). Differential leukocyte, reticulocyte, and platelet counts were determined from blood smears. Serum and urine chemistry analyses were performed using a BMD/Hitachi 704/737 Chemistry Analyzer (Boehringer Mannheim Diagnostics, Indianapolis, IN). Semiquantitative urinalysis determinations were performed using Ames Multistix (Miles, Inc., Diagnostic Division, Elkhart, IN) or equivalent. Specific gravity was determined with a refractometer.
Histopathology.
A set of 1015 animals per sex from all groups was sacrificed after Week 78 for histopathology studies. All surviving animals were terminated after 104 weeks of treatment. Animals were food-fasted overnight, weighed, given an intraperitoneal injection of sodium pentobarbital, and exsanguinated. All tissues listed in the U.S. EPA Health Effects Testing Guideline for Combined Chronic Toxicity/Oncogenicity Study (40 CFR 798.3320) from the high-dose and control groups were evaluated microscopically. Target tissues from other groups were also examined. The tissues were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for microscopic examination.
Statistics.
Body weights, feed consumption, clinical chemistry, hematology, and organ weights were analyzed by ANOVA, followed by a Dunnett's t-test. Tumor incidence was compared by Fisher's exact test. A probability of 0.05 was used to determine significance.
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RESULTS |
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Mean body weights for 6000-ppm male mice were significantly decreased compared with the control group for most of the study (Fig. 1). For female mice, significantly lower body weight means were noted occasionally for Group 5 (Fig. 2
). Mean body weight changes for the groups were 10.5 ± 2.7, 11.4 ± 2.3, 11.7 ± 1.9, 10.8 ± 2.7, and 5.8 ± 2.5 g for males in Groups 15, respectively, and 11.8 ± 1.9, 12.8 ± 2.0, 13.0 ± 1.9, 12.5 ± 2.4, and 10.5 ± 2.4 g for females in Groups 15, respectively. Weight gain for Group-5 males (6000 ppm) was significantly lower than for the controls.
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Clinical Chemistry
There were no significant differences in clinical chemistry or hematology at the interim sampling periods (data not shown). At termination, no toxicologically significant changes in clinical chemistry parameters were observed for mice exposed to DEHP. The serum potassium level for the male 6000-ppm group was significantly lower when compared with controls (Table 1). In addition, the concentration of potassium in the urine demonstrated a trend to lower concentrations at higher-dose levels (data not shown). However, the volumes of urine for the higher-dose-level groups were also higher, such that there was no apparent difference in urinary excretion of potassium. Therefore, the decrease in serum potassium was not considered to be toxicologically meaningful. Mean ALT value for the 1500-ppm group was substantially higher than values for controls, but these were not statistically significant because of the large standard deviation for the groups. Similar results were seen for female mice. Because of limitations in the volume of blood collected, total protein, albumin, and globulin values are insufficient for good comparison among groups, although the mean values do not suggest any effect in these parameters.
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DISCUSSION |
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Although few long-term studies of DEHP have been performed using mice, some of the effects observed in the study presented here were described by Ward et al. (1986). In that study, male B6C3F1 mice exposed to 3000, 6000, or 12,000 ppm DEHP for 18 months had substantially lower body weights at 6000 ppm (20%) and 3000 ppm (10%). Cytoplasmic eosinophilia of the liver and hepatomegaly were reported at the 12,000-ppm dose level. Renal tubular degeneration, necrosis, and cystic hyperplasia (lesions resembling chronic progressive nephropathy) were seen in the kidneys of all treated groups, and some animals at 6000 ppm had degeneration of the seminiferous tubules. Those results are consistent with the lesions observed in the study presented here. How closely linked these lesions are to cellular events associated with peroxisome proliferation is not clear. Pigmentation and eosinophilia may represent deposition of lipofuscin in the hepatocyte known to occur with peroxisome proliferators. Lake et al. (1987) observed a "golden-brown" pigment in hepatocytes stained with hematoxylin and eosin, which was identified, using a different stain, as lipofuscin. Ward et al. (1998) observed an absence of hepatocyte pigmentation and cytoplasmic eosinophilia in PPAR-knockout mice treated with 12,000 ppm DEHP for 24 weeks whereas these liver lesions were observed in Sv/129 wild-type mice following up to 16 weeks of exposure. There was also an absence of peroxisome proliferation in the PPAR-knockout mice whereas peroxisome proliferation was observed in the wild-type mice. Those data suggest that pigmentation and eosinophilia are associated with peroxisome proliferation. However, pigmentation in hepatocytes and cytoplasmic eosinophilia of hepatocytes were not observed in the study presented here at all dose levels where peroxisome proliferation was significantly increased. This information implies that there is a threshold in mice for peroxisome proliferator-induced noncarcinogenic liver effects.
The relationship of increased incidence of hypospermia to peroxisome proliferation is also not clear. Ward et al. (1998) evaluated whether peroxisome proliferation was a causative factor by studying the testicular effects in PPAR-knockout mice treated for up to 24 weeks with 12,000 ppm DEHP. Their data suggest that peroxisome proliferation plays some role in the onset of testicular toxicity, but not in the severity or extent of cellular damage. Focal tubular degenerative lesions of the testes were seen in wild-type mice and spermatogenesis was diminished by 8 weeks; whereas PPAR-knockout mice had normal testes at 8 weeks but lacked "normal indicators of spermatogenesis" only in the outer portion of the testis. Severe tubular degeneration was observed in the PPAR-knockout mice at 24 weeks (interim time periods were not investigated). The data from the study presented here support the hypothesis that testicular lesions and hypospermia are time-dose dependent relative to the increase in peroxisome proliferation. Male mice receiving 1500 ppm of DEHP for 78 weeks had no signs of hypospermia, whereas 30% of the animals treated for 104 weeks had hypospermia. All male mice receiving 6000 ppm of DEHP for 78 weeks had hypospermia. Peroxisome proliferation in these animals was 2.7 times control for the 1500-ppm groups and 7.6 times control for the 6000-ppm group. At 500 ppm, where the testes appeared normal, peroxisome proliferation was only 2.2 times control, although there was a slight decrease in relative testis weight. The significance of the decrease in testes weights is unclear since there was no histopathology at this dose level. By contrast, Ward et al. (1998) observed at least minimal hypospermia in PPAR-knockout mice given 12,000 ppm DEHP that exhibited a decrease in testes weights.
For the kidney, Ward et al. (1986) imply that lesions were dose- and time-dependent. However, the data from the study presented here indicate that the relationship must occur prior to Week 78. Based on the incidence of chronic progressive nephropathy among control animals at Week 78, this lesion is considered to be naturally occurring. It is also clear that exposure to high levels of DEHP (300 mg/kg) exacerbated the severity, but the mechanism is not clear. Ward et al. (1990) described increased severity of focal nephropathy in B6C3F1 mice exposed to 6000 ppm DEHP for 78 weeks. Ward et al. (1998) also evaluated the association of nephropathy with peroxisome proliferation using PPAR-knockout and wild-type mice. In that study, Ward and coworkers described nephropathy and severe cystic renal tubules in Sv/129 wild-type mice exposed to 12,000 ppm for 16 weeks. However, PPAR
-knockout mice had minimal focal renal tubular lesions at 4 and 8 weeks, and diffuse and mildly cystic kidneys at 24 weeks. Thus, the nephropathy observed in mice is influenced by PPAR
or sequelae of peroxisome proliferation, but are not entirely responsible for that effect. Peroxisome proliferation has been observed in the kidneys of rats following exposure to 20,000 ppm DEHP for 4 weeks (Ohno et al., 1982
), to 1 g/kg for 21 days (Cimini et al., 1994
), and 10000 ppm for 18 months (Price et al., 1987
). Thus, in the absence of associated kidney lesions, the most likely hypothesis for increased kidney weights is that they reflect peroxisome proliferation, which was observed in the liver at this dose level (David et al., 1999
).
Comparing the study presented here for mice to data generated for rats, it appears that there are clear differences in the responses of mice and rats to DEHP exposure. Absent in mice were several rat-specific lesions with questionable relevance to humans. For example, hematologic changes (decreased erythrocyte count, hemoglobin, and hematocrit), mononuclear cell leukemia, spongiosis hepatis, mineralization of the renal tubules, and pancreatic acinar-cell adenoma were not observed in mice but were observed in rats. Many of these lesions (leukemia, spongiosis hepatis, mineralization of the renal tubules, and pancreatic acinar-cell adenoma) appear to be associated with rat-specific mechanisms such as 2u-globulin nephropathy (David et al., 2000
). The lack of effect in mice given even higher dose levels of DEHP reinforces the hypothesis of species-specificity for these lesions.
In conclusion, exposure of mice to high levels of DEHP resulted in minimal signs of toxicity: histopathologic changes in the liver and testes similar to those observed for rats. Also observed was exacerbation of naturally occurring lesions in the liver and kidneys such as those observed for rats. All lesions occurred at dose levels where peroxisome proliferation was 2.7 times control. This is in contrast to the effects in rats, where testicular lesions occurred at a dose level of minimal peroxisomal enzyme activity. A dose level of 500 ppm (98.5116.8 mg/kg/day) was identified as a no-observed-adverse-effect level for noncarcinogenic effects. By comparison, nonmedical lifetime human exposure levels are estimated to be 330 µg/kg/day (Doull et al., 1999
) using a total non-occupational exposure of 0.25 mg/person/day (ATSDR, 1993).
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NOTES |
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REFERENCES |
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