* Health and Environment Laboratories, Eastman Kodak Company, Rochester, New York 146526272;
Covance Inc., Vienna, Virginia, and
Eastman Chemical Company, Kingsport, Tennessee
Received November 9, 1999; accepted February 4, 2000
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ABSTRACT |
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Key Words: DEHP; di(2-ethylhexyl) phthalate; chronic toxicity; testes; pancreas; kidney.
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INTRODUCTION |
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The value of these studies for assessment of risk for human health from chronic exposure is limited based on the low numbers of animals used and the high mortality. The NTP study, although well conducted with sufficient numbers of animals focused on identifying target organs for carcinogenicity, did not include in-depth evaluation of chronic toxicity. Yet there is a need to determine the noncarcinogenic effects based on the widespread use of DEHP as a plasticizer in vinyl products (ATSDR, 1993). In addition, the ATSDR identifies food as a source of exposure to DEHP, although exposure from foods may be limited because DEHP is not used in food wrap. The ATSDR has estimated the total exposure from all sources to be 0.25 mg/person/day. This does not include exposure from medical devices such as vinyl tubing and blood bags, which can be higher (Huber et al., 1996
) but intermittent. In addition, the route of exposure influences the extent of hydrolysis and metabolism (Huber et al., 1996
), which may affect the toxicity.
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. 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. As part of those studies, the noncarcinogenic chronic effects of exposure were also evaluated. Those data are presented here and their results compared with dose levels at which peroxisome proliferation occurred. 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 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
). Therefore, it is of interest to compare the noncarcinogenic, chronic effects of DEHP to levels of peroxisome proliferation. 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 (nulliparous) Fischer-344 rats (CFD(F344)CrlBR) obtained from Charles River Laboratories, Inc. (Raleigh, NC) 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/hour were maintained in the room housing the animals.
Treatment levels for bioassay.
The animals were assigned to the 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 one-way analysis of variance (ANOVA). At randomization, the weight variation of the animals selected did not exceed two 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. Rats were divided into five groups consisting of 80, 50, 55, 65, and 80 animals per sex for Groups 15, respectively. Dosage levels, selected based on a 13-week study that indicated that exposure to 12,500 ppm would result in overt toxicity based on body weight changes, were 0, 100, 500, 2500, or 12,500 ppm DEHP in the diet (Groups 15, respectively). These groups were treated continuously for up to 104 weeks. A set of 10 animals per sex from Groups 1, 4, and 5 were sacrificed after week 78 for histopathology. All surviving 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 rats were observed for mortality and moribundity twice daily. A thorough physical examination was conducted at each weighing interval. A careful cageside observation for obvious indications of toxic effects was performed once daily.
Body weights, organ weights, 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, lungs, spleen, kidneys, testes, and uterus were measured for each animal. Organ weights were not measured for intercurrent deaths.
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. If possible, the same animals were bled at each time period. Whole blood samples were analyzed for red blood cell count (RBC), total and differential white blood cell counts, hemoglobin (Hgb), hematocrit (Hct), reticulocyte count, and platelet count. At necropsy, bone marrow smears were made for determination of myeloid-to-erythroid ratio. Serum samples were analyzed for albumin, total 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, ketones, microscopic examination of sediment, occult blood, pH, protein, specific gravity, and urobilinogen. In addition, the concentration of creatinine was determined to compare with serum creatinine for creatinine clearance. 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 10 animals per sex from Groups 1, 4, and 5 were sacrificed after week 78 for histopathology. All surviving animals were terminated during week 105. 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, and intercurrent deaths were evaluated microscopically. Target tissues and gross lesions 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 analysis of variance followed by a Dunnett's t-test. Tumor incidence was compared by the 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 12,500-ppm male and female rats were significantly lower at all or most intervals from week 1 to week 105 compared to the control group (Fig. 1). No consistent differences in body weight were observed for the other groups. Mean body weight changes for the groups were 226 ± 19.9, 229 ± 26.8, 223 ± 24.3, 216 ± 18.9, and 192 ± 28.1 g for males in Groups 15, respectively, and 149 ± 22.3, 155 ± 17.5, 166 ± 15.4, 153 ± 23.7, and 126 ± 15.3 g for females in Groups 15, respectively. Weight gains for Group 5 (12,500 ppm) were significantly lower than for the controls. Mean weekly food consumption values for 12,500 ppm male and female rats exhibited occasional differences from the values for the control groups, but there was no obvious dose- or treatment-related pattern. Overall mean food consumption values for males in Groups 15 were 4324 ± 217.8, 4334 ± 219.9, 4286 ± 288.0, 4361 ± 275.9, and 4368 ± 323.8, respectively, and 3380 ± 185.3, 3446 ± 140.7, 3411 ± 149.4, 3354 ± 189.6, and 3300 ± 180.3, respectively, for females. Based on the average daily feed consumption, the dose of DEHP was 5.8, 28.9, 146.6, and 789.0 mg/kg/day, respectively, for males; and 7.3, 36.1, 181.7, and 938.5 mg/kg/day, respectively, for females.
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The incidence of MNCL was compared among groups and to the historical control values of 128/420 males and 82/424 for females from the laboratory (same strain, age, supplier, and laboratory over a 5-year period). MNCL was significantly higher for the 2500- and 12,500-ppm male groups compared with the concurrent control, although no difference was seen for females. Compared with the historical control values, the incidence of MNCL for the 100-ppm female groups and 2500-ppm male group were significantly higher. Thus, although treatment appears to have increased the incidence of MNCL relative to the concurrent controls, no treatment-related effect is evident when compared with the historical control values. There were no other treatment-related lesions in any other organs. The thyroid gland, a suspected target organ for DEHP (Hinton et al., 1986; Price et al., 1987
), failed to demonstrate any treatment-related effect.
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DISCUSSION |
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The minor alterations in clinical chemistry and hematology coincide with increased peroxisome proliferation in the liver. In addition, the alterations are of such low magnitude that the clinical relevance is questionable. Increases in BUN and albumin in the absence of increases in other chemistries are not routinely associated with adverse effects on any target organ, but increases in serum protein levels following DEHP exposure have been reported. Hinton and coworkers (Hinton et al., 1985) showed that Wistar rats fed 1000 or 2000 mg/kg DEHP for up to 28 days had increased levels of protein P2, but lower levels of
1.2,
1.3,
1.5,
1.8, and ß3 protein. Because the protein levels were determined by electrophoresis, comparison of these changes to the results observed in the present study is difficult. Poon et al. (1997) found that male, but not female, Sprague-Dawley rats exposed to 5000 ppm for 13 weeks had higher albumin levels, but there was no effect on BUN. Thus, increases in BUN and albumin have not been consistently observed following DEHP exposure, possibly because previous studies did not expose animals to high dose levels for extended periods of time. The biologic significance of increased BUN and albumin is uncertain, but both may be linked to metabolic processes in the liver, a target organ for DEHP. It is conceivable that increases in metabolic enzymes that occur following exposure to DEHP have an impact on synthesis of albumin and urea (Youssef and Badr, 1998
).
Effects on hematologic parameters were also subclinical, but appeared to be related to treatment. RBC, hemoglobin, and hematocrit values were lower for the 2500 and 12,500-ppm groups, dose levels where peroxisome proliferation was increased. Similar decreases in red blood cell parameters have been observed by Gray et al. (1977) after exposure to 10,000 and 20,000 ppm for up to 17 weeks, by Chu et al. (1981) after 28 days of treatment with 1600 or 6400 ppm DEHP in the diet, and by Poon et al. (1997) after 13 weeks of exposure to 5000 ppm. Although the results of this study in combination with previous studies suggest RBC destruction, there was no evidence of hemosiderin or compensatory erythropoiesis in any study. Serum bilirubin levels were not increased, and there was no evidence of hemoglobin in the urine. In addition, Haberman et al. (1968) found that DEHP did not hemolyze human erythrocytes. There was, however, pigmentation of the Kupffer cells and kidneys observed in the present study. It is not clear if the pigmentation was related to red blood cell loss and increase in porphyrin or to other factors. The pigment may represent deposition of lipofuscin in the hepatocyte. 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. However, lipofuscin has not been described in kidneys of rats exposed to 2% DEHP for 4 weeks (Ohno et al., 1982).
The incidence of spongiosis hepatis was increased for male rats, but not female rats, and only at dose levels where peroxisome proliferation was increased. This lesion has been associated with treatment with potent hepatocarcinogens such as dimethylnitrosamine and nitrosomorpholine (Bannasch et al., 1981; Zerban and Bannasch, 1983
) but not with DEHP (Kluwe et al., 1982
). The etiology of this lesion was thought to reflect altered metabolism in the liver (Zerban and Bannasch, 1983
), but previous studies have focused on male rats only. The data presented here indicate that this lesion does not occur in female rats even though there are changes in metabolism induced by exposure to DEHP (David et al., 1999
). The difference between these two results may be in the potency of the carcinogen used. Zerban and Bannasch used N-nitrosomorpholine and dimethylnitrosamine, both potent initiators, to induce spongiosis hepatis. DEHP is not an initiator of carcinogenicity, and although it has promoting properties in mice, DEHP is not a promotor in female F344 rats (Ward et al., 1986
). In addition, the dose of N-nitrosomorpholine and dimethylnitrosamine used were relative low (~0.3 mg/kg/day), whereas the dose of DEHP that resulted in spongiosis hepatis was
146 mg/kg/day. Thus, there is an association between a higher incidence of spongiosis hepatis and exposure to high dose levels of DEHP for male rats, albeit through an unknown mechanism. The fact that female rats do not exhibit this lesion calls into question whether this lesion is related to exposure to DEHP alone or whether DEHP exacerbated a background lesion.
Another lesion associated with male rats was pancreatic acinar cell adenoma. Although the incidence of this lesion was increased only for the high-dose male group, it was only slightly above the historical incidence of this lesion in male F344 rats (Goodman et al., 1979). Nonetheless, pancreatic acinar cell adenoma has been reported following long-term exposure to DEHP, although not statistically increased and only in male rats (Kluwe et al., 1982
), and other peroxisome proliferators such as Wy-14,643 (Obourn et al., 1997
). Concomitant changes in serum bilirubin and other clinical chemistries were seen following Wy-14,643 exposure, which suggested that cholestasis was linked to the formation of pancreatic adenoma (Obourn et al., 1997
). The results from the present study do not support the hypothesis that these serum chemistry changes are a causal link to pancreatic acinar cell adenoma (the NTP study did not evaluate serum chemistry). However, it is possible that changes in liver metabolism play a role in the etiology of pancreatic acinar cell adenoma. Control rats treated with corn oil alone have a higher incidence of pancreatic acinar cell adenoma than do their counterpart controls from feeding studies (Haseman et al., 1998
). DEHP, like other peroxisome proliferators and lipids, interacts with the peroxisome proliferator-activated receptor (PPAR), which leads to alterations in metabolism in the liver (Eagon et al., 1994
). A recent study by Fan et al. (1998) demonstrated that high levels of fatty acids induce a variety of metabolic pathways in mice. Therefore, it seems possible that pancreatic acinar cell adenoma is the result of metabolic changes in the liver.
Several other lesions were associated with treated male rats only. An increased incidence of mononuclear cell leukemia (MNCL), mineralization of the renal papilla, castration cells in the pituitary, and bilateral aspermatogenesis were observed in male rats receiving high doses of DEHP. MNCL is a common finding in rats of this strain (Haseman et al., 1998) and was reported in the NTP study (Kluwe et al., 1982
), although the incidences among groups in that study were not significantly different. Ganning et al. (1990) did not report an increase in MNCL in Sprague-Dawley rats treated for 2 years with up to 20,000 ppm DEHP in the diet. Thus, this lesion is not consistently observed following long-term exposure to DEHP, and its relevance to humans has been questioned given the high background incidence for F-344 rats (Caldwell, 1999
).
Mineralization of the renal papilla was also seen exclusively in male rats, and although the background incidence of this lesion was substantial, exposure to DEHP increased the incidence and severity of the lesion. Mineralization in the kidneys is not uncommon for male rats (Greaves and Faccini, 1992) and is a lesion that has been associated with hyaline droplet formation and renal tumors (Hard et al., 1993
). High levels of urinary protein and
2u-globulin may lead to hyaline droplet formation (Lehman-McKeeman and Caudill, 1992a
,b
; Swenberg et al., 1989
), and exposure of F-344 male rats to high-dose levels of di-isononyl phthalate has been shown to precipitate
2u-globulin (Caldwell et al., 1999
). Lesions and tumors that occur through an
2u-globulin mechanism are not considered relevant to human risk assessment (Alison et al., 1994
; Swenberg et al., 1989
). Whether the observation of mineralization in the study presented here is associated with
2u-globulin is not clear. The presence of mineralization of the renal papilla is consistent with precipitation of
2u-globulin, especially when it is confined to male rats (Hard et al., 1993
). Hyaline droplets have not been observed following treatment of rats with DEHP, nor are there data indicating that DEHP precipitates
2u-globulin. In fact, Alvares et al. (1996) and Corton et al. (1997) showed that induction of PPAR
by Wy-14,643, ciprofibrate, gemfibrozil, and di-n-butyl phthalate downregulates the production of
2u-globulin. Thus, the mechanism for mineralization is not clear.
What is clear is that mineralization may not be the cause of the increases in kidney weight observed for high-dose males and females because female rats did not have a higher incidence of renal papillar mineralization compared with controls. Neither mineralization of the renal papilla, renal tubule pigmentation, nor any other kidney lesion examined was associated with the increase in kidney weight. In addition, previous studies of long-term oral exposure to DEHP have not demonstrated specific treatment-related kidney lesions (Carpenter et al., 1953; Chu et al., 1981
; Gray et al., 1977
; Kluwe et al., 1982
; Shaffer et al., 1945
), although there was evidence of peroxisome proliferation in the kidneys of rats following exposure to 2% DEHP for 4 weeks (Ohno et al., 1982
), to 1 g/kg for 21 days (Cimini et al., 1994
), and 10,000 ppm for 18 months (Price et al., 1987
). Crocker et al. (1988) reported increased type C cyst formation following treatment with DEHP for 12 months. No increase in the incidence of cysts was observed for the study presented here, however. Thus, in the absence of associated kidney lesions, the most likely hypothesis for increased kidney weights is that they reflect peroxisome proliferation, which was largely a reversible phenomenon (Cimini et al., 1994
; David et al., 1999
).
Finally, lesions of aspermatogenesis, interstitial cell tumors of the testes, and castration cells in the pituitary gland were hypothalamic-gonadal in origin. Male F-344 rats are known to develop interstitial cell tumors of the testes (Goodman et al., 1979; Haseman et al., 1998
; Prentice and Meikle, 1995
). The fact that this tumor had a high incidence for control male rats was therefore not surprising. In addition, the decreased incidence of this lesion observed for high-dose male rats was previously seen in the NTP study (Kluwe et al., 1982
). The decrease in interstitial cell tumors was concomitant with an increased incidence of castration cells in the pituitary and an increased incidence of bilateral aspermatogenesis, although these events may be unrelated. The mechanism for testicular tumors is thought to be linked to decreased levels of testosterone in aging rats (Alison et al., 1994
; Prentice and Meikle, 1995
). Without the negative feedback of testosterone on the hypothalamus, hypothalamic gonadotropins continue to stimulate Leydig cells, which in turn stimulate Sertoli cells to produce LHRH (Nolte et al., 1995
). The increased level of testicular LHRH activates the LHRH receptors on the Leydig cells. This phenomenon is apparently unique to the rat (Alison et al., 1994
; Prentice and Meikle, 1995
). The ability of DEHP to disrupt this process is likely at the level of the Sertoli cell. Gray and Gangolli (1986) and Sjöberg et al. (1986) demonstrated that doses of 10002800 mg/kg caused Sertoli cell damage. The secretion of inhibin from the Sertoli cell has a negative feedback on gonadotropin release from the pituitary (Culler and Negro-Villar, 1990
; Kitahara et al., 1991
). The secretion of a LHRH-like protein from the Sertoli cell stimulates the Leydig cells to secrete testosterone (Alison et al., 1994
; Prentice and Meikle, 1995
). Thus, damaged Sertoli cells that result in decreased inhibin and LHRH-induced testosterone secretions from the testes promote the formation of castration cells in the pituitary (Almeida et al., 1989
). DEHP may also act directly on the Leydig cell to reduce testosterone secretion (Jones et al., 1993
; Oishi, 1985
). Gray et al. (1977) reported castration cells in the pituitary following exposure to as little as 2000 ppm DEHP for 17 weeks. Thus, the presence of castration cells in the pituitary appears to be an effect of DEHP on the Sertoli cell and indicates a role in feedback on the pituitary. Likewise, the high incidences of aspermatogenesis found in the 2500- and 12,500-ppm male groups are a phenomenon that has been known for some time (Gray et al., 1977
) and is believed to be associated with damage to the Sertoli cell. The lack of aspermatogenesis for the 2500-ppm group at week 78 suggest that this lesion is age related at the 500- and 2500-ppm dose levels rather than treatment related. In addition, other species such as hamsters (Gray et al., 1982
) and primates are resistant to the testicular effects of DEHP (Kurata et al., 1998
). Therefore, the relevance of this lesion to human exposure to DEHP is questionable.
In conclusion, 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, and aspermatogenesis. There was also an increased severity or incidence of age-, species-, or strain-related lesions. The increased incidence of spongiosis hepatis correlated with increased palmitoyl CoA oxidase activity, but the incidence of other lesions was increased only at the highest dose level of 12,500 ppm. Aspermatogenesis did not reflect peroxisome proliferation as suggested by Ward et al. (1998). The no-observable-adverse-effect level is considered to be 500 ppm, which is equivalent to a daily dose of 28.936.1 mg/kg/day. By comparison, nonmedical lifetime human exposure levels are estimated to be 330 µg/kg/day (Doull et al., 1999) using a total nonoccupational exposure of 0.25 mg/person/day (ATSDR, 1993
).
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NOTES |
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