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4 Di(2-ethylhexyl) Phthalate John T. James, Ph.D. Johnson Space Center Meclical Sciences Division Houston, Texas PHYSICAL AND CHEMICAL PROPERTIES Di(2-ethylhexyl) phthalate (DEHP) is a colorless, viscous liquid with a slight odor. (See Table 4-1 for more details.) OCCURRENCE AND USE DEHP became a commercial product in 1933 and is widely used as a plasticizer in many materials, especially polyvinyl chloride (Schmid and Slatter 1985). It is also used in insect repellents, lacquers, and rocket pro- pellants. During the NASA/Mir program, DEHP was found in spacecraft recycled wafer at an average concentration of 2 micrograms per liter (~g/L), with a high of 28 ~g/L (Pierre et al.1999). The humidity condensate in the shuttle has been found to contain DEHP, which is sometimes called dioctyl phthalate, at concentrations up to 460 ~g/L (Straub et al. 1995). By com- parison, concentrations up to 3-4 ~g/L have been reported in river water in Japan and Europe (WHO 1992). Concentrations in catfish from rivers in the southern United States have been reported to be as high as 3,200 fig per kilogram (kg) (Mayer et al.1972). Oral exposure ofthe general population, 121

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122 Spacecraft Water Exposure Guidelines TABLE 4-1 Physical and Chemical Properties of DEHP Formula Synonyms CH 2-CH ~ /~ 1 CO O -CH 2-CH -CH 2-CH 2-CH 2-CH LOO O -CH 2-CH -CH 2-CH 2-CH 2-CH \/ 1 f:H2-CH 3 CAS registry no. Molecular weight Melting point Boiling point Solubility Density C24H38O4 Di(2-ethylhexyl) phthalate (DEHP), dioctyl phthalate, 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester 1 17-81-7 390.56 -50C 387C 45 ~g/L in water at 20C (Leyder and Boulanger 1983); values from 285 ~g/L to 360 ~g/L have been reported at room temper- ature (WHO 1992) 0.99 g/mL primarily from residues in food, has been estimated at 30 ~g/kg per day Aid, which is approximately 2 milligrams (mg)/d for a 70-kg person (Doull et al. 1999~. The presence of DEHP in food and water contained in plastic could affect the amount of DEHP ingested by astronauts. Food repackaged by NASA is generally placed in laminated containers that have polyethylene as their innermost component. Polyethylene should not leach any DEHP; however, that does not completely preclude the possibility of leaching from the packaging in which the food was shipped or in foods flown in their original packaging. The amount of food-borne DEHP ingested by the crew should be small and certainly no more than that ingested by earthbound people eating food mostly from commercial packaging. Water is not stored in containers made with DEHP. TOXICOKINETICS Primary targets for DEHP toxicity are the liver and testes. Effects in the pituitary, thyroid, ovaries, andblood have also been explored. When inves- tigating DEHP's toxicity, it is important to consider significant differences

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Di(2-ethylhexyl) Phthalate . . 123 In species and organ response. The toxicokinetic discussion will focus on the relevance of DEHP's carcinogenic and reproductive effects in rodents to human health. The toxicokinetics of DEHP are remarkably complex and depend on many different factors. Absorption of an oral dose of DEHP is related to the chemical's metabolism to readily absorbed compounds. Its metabolism in the gut is saturable, so the amount of each metabolite ab- sorbed depends on the dose of DEHP. Further metabolism of the absorbed compounds depends on the species and age of the subject. Absorption An oral dose of DEHP can be absorbed in the small intestine as un- metabolized DEHP or as mono~ethy~hexyI) phthalate (MEHP) and 2-ethyI- hexanol; the relative proportions of parent compound and metabolites ab- sorbed depend on the dose (Astill 1989~. In general, plasma levels peak approximately 1-3 hours (h) after oral exposure. Minimal retention of the compound is observed (NTP 2000~. At low doses, most ofthe DEHP given to rats is hydrolyzed by pancreatic lipase to MEHP; however, above a threshold dose, unmetabolized DEHP is absorbed and reaches the liver (Albro 1986~. A larger portion of a low dose is absorbed compared with the portion of a higher dose that is absorbed. For example, the "area under the curve" for DEHP and its metabolites in marmoset blood for the first 24 h after oral administration of DEHP was only doubled when the dose was increased from 100 mg/kg to 2,000 mg/kg (Rhodes et al. 1986~. This sug- gests that the absorption of DEHP and MEHP from the gut are saturable processes. A key observation regarding absorption of DEHP is that in primates, including humans, much less of an oral dose of DEHP (in milligrams per kilogram of body weight) is absorbed and hydrolyzed to MEHP than in rodents (ICI 1982; Shell 1982; Schmid and Schlatter 1985; Rhoades et al. 1986~. For example, at an oral dose of 2,000 mg/kg, marmoset tissues are exposed to approximately the same levels of DEHP and its metabolites as are rat tissues after a dose of only 200 mg/kg (Rhodes et al. 1986~. This result may be due to the marmoset's reduced lipase activity in the gut, where absorption of DEHP is facilitated. In a comparative study of mon- keys, rats, and mice, Astill (1989) reported that the monkeys hydrolyze substantially less of a gavage dose of DEHP at 100 mg/kg than did either of the rodent species.

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124 Spacecraft Water Exposure Guidelines Distribution The tissue distribution of DEHP and its metabolites has received limited study. Rats end marmosets were given 14 massive oral doses of ~4C-labeled DEHP at 2,000 mg/kg in corn oil, and selected tissues were removed for study. The distribution of radiolabel in the blood, liver, kidneys, and testes of the rat and marmoset showed a similar tissue pattern; however, the abso- lute levels in the marmoset/issues were one-fifth to one-tenth of those found in the comparable rat tissues (Rhodes et al. 1986~. In another study, rats, dogs, and miniature swine were given unlabeled DEHP at 50 mg/kg for 21-28 ~ and then given a final dose of ~4C-labeled DEHP at 50 mg/kg (Ikeda et al. 1980~. Animals were sacrificed 4 h, 1 4, and 4 ~ later, and the distribution of radiolabel in tissue and body fluids was measured. Of the tissues studied, the labeling was highest in rat liver, dog muscle, and swine fat at the 4-h time point; the pattern of highest labeling changed slightly at 4 ~ to rat lung, dog muscle, and swine fat (Ikeda et al. 1980~. Other tissues studied included the brain and kidneys; however, distribution to the testes was not measured. Metabolism The metabolism of DEHP has been studied in great detail, revealing approximately 30 metabolites in various species (ATSDR 1993~. Qualita- tively, the metabolites are similar in most species studied, but there are important quantitative differences in DEHP metabolism. These quantitative metabolic differences may help explain the interspecies differences in peroxisome proliferation (PP) and in the induction of cancer; however, the data are not completely consistent in implicating specific metabolites. In this section, the metabolism of DEHP will be depicted in summary form, and species differences in the maj or pathways will be considered. It is these species differences that will have the greatest bearing on the rationale used to develop human exposure limits. The metabolism of DEHP is summarized in Figure 4-1 . In the gut, and in many other tissues, DEHP is hydrolyzed by intestinal lipases to MEHP and 2-ethy~hexanol (2EH) so that very little free DEHP, when given in moderate doses, is excreted by any species. Following oral exposure in rodents, the majority of DEHP is absorbed in the form of MEHP because of rapid hydrolysis by gut lipases. The fraction of free MEHP excreted is large in guinea pig urine (72%), intermediate in monkeys and mice urine

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Di(2-ethylhexyl) Phthalate 125 (17-18%), low in hamster urine (4.5/O), and almost absent in rat urine (Albro 1986; Rhodes et al. 1986; Astill 1989~. In a relatively minor path- way, 2EH is oxidized to 2-ethy~hexanoic acid (2EHA) and several ketoacids, which are found in human urine (Albro and Corbett 1978~. In most species studied, the hydrolysis of MEHP to phthalic acid is not a ma- jor pathway. MEHP is hydrolyzed to phthalic acid by enzymes in liver microsomes and is found free in the urine of rodents; hamsters and mice excrete more than rats or guinea pigs (Albro et al. 1982; Albro 1986~. In humans and other primates, the portion of conjugated metabolites in urine from side-chain oxidation is 60-65%; guinea pigs, hamsters, and mice have a smaller portion present as conjugates; and rats seem to excrete very little conjugated metabolites in their urine (Albro et al. 1982; Schmid and Schlatter 1985). Two human volunteers were given 30 mg of DEHP once and 10 mg/d for 4 d. Their urinary metabolites were quantified by gas chromatography and mass spectrometry (Schmid and SchIatter 1 98 5~. The maj or metabolites found in hydrolyzed urine, in decreasing concentration, were IX, V, VI, VII, IV, and I.~ MEHP was also found in the urine. In the single-dose study, 1 1-15% of the dose was recovered in the urine in about 50 h, and the vast majority of that was excreted in the f~rst 20 h. When the four repeated doses were given, 15-25% was recovered over 5 d. The toxicologic significance of the various DEHP metabolites com- pared with the parent compound differs depending on species and toxic end point. For example, attempts to relate specif~c metabolites to PP, and pre- sumably to potential induction of liver cancer, have involved several ofthe many MEHP oxidation products, including 2-ethyI-3-carboxypropyl phtha- late (I), 2-ethyl-5-oxyhexyl phthalate (VI), and 2-ethyl-5-hydroxyhexyl phthalate (IX) (Lhuguenot et el. 1985; Mitchell et al. 1985a; Astill 1989~. One would hope to f~nd a metabolite that is present at relatively high con- centrations in species susceptible to PP and at low concentrations in less susceptible species. Furthermore, in vitro confirmation of the compound's ability to cause PP would be helpful in selecting the proximate metabo- ~The designation in Roman numerals is per the convention of Albro et al. (1973,1981,1982), and translates as follows: I, 2-ethyl-3-carboxypropyl phthalate; IV, 2-carboxymethylhexyl phthalate; V, 2-ethyl-5-carboxypentyl phthalate; VI, 2- ethyl-5-oxyhexyl phthalate; VII, 2~2-hydroxyethyl~hexyl phthalate; VIII, 2-ethyl-4- hydroxyhexyl phthalate; IX, 2-ethyl-5-hydroxyhexyl phthalate.

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126 Phthalic Acid Spacecraft Water Exposure Guidelines DEHP side chain oxidations glucuronic acid conjugation MEPH Oxidized Products Conjugated Products 2-Ethy~hexanof Acids and Keloacids FIGURE 4-1 Overall metabolism of DEHP. See page 123 for details ofthe oxida- tion products proposed by Albro et al. (1982~. liters). Unfortunately, the search for such a metabolite has resulted in obser- vations that cannot be explained by such a simple approach. In comparing monkeys, rats, and mice, the metabolites found at low concentrations in monkeys and at high concentrations in rats and mice were I and VI (Astill 1989~. Compound IX, a precursor of VI, was found in roughly comparable fractions in the three species. Using cultured rat hepatocytes and an enzy- matic marker of PP, Mitchell et al. (1985a) found that compound I had little effect on the marker, whereas compounds VI and IX induced the marker approximately 1 O-fold. The little data available on humans suggest that VI and IX are major metabolites found in urine and I is a minor metabolite found in urine (Albro et al. 1982; Schmid and Schiatter 1985~. Humans and other primates are thought to be resistant to PP induced by agents that are capable of inducing peroxisomes in rats (Doull et al. 1999~. This might be because of the low level of the peroxisome proliferator-activated recep- tor-alpha (PPARa) in primate liver (Parkinson 1996~. To further complicate the picture, the relationship between liver cancer and PP may not be direct for DEHP. Rather, the ability to induce a persistent increase in replicative DNA synthesis seems to correlate better with cancer induction (Marsman etal.l988~.

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Di(2-ethylhexyl) Phthalate 127 The interspecies differences in PP caused by DEHP can be partially explained by proposed differences in high- and low-dose kinetics related to the abundance of PPARa (Holder and Tugwood 1999~. The relationship of PP to lipid homeostasis can also be explained by differences in the acti- vation thresholds for the two processes. Below is a diagram (Figure 4-2), adapted from two figures in Holden and Tugwood (1999), modeling how the interspecies differences in PP susceptibility can be understood. A PP compound, such as MEHP, enters the hepatocyte nucleus and causes the heterodimerization of PPARa with another nuclear receptor called retinoid X receptor. This dimmer binds to DNA at the peroxisome proliferator response element, which is a repeat of a TGACCT-like sequence. In rats, this sequence is known to promote acy! CoA oxidase and bifunctional dehydrogenase/dehydratase, which effect the PP process. Similarly, PPARa can activate genes for apolipoprotein and lipoprotein lipase, which control lipid homeostasis. The relative thresholds for activation ofthese processes (illustrated by the vertical arrow in Figure 4-2) suggest that PP in rats and mice has a much higher threshold in terms of PPARa concentration than does lipid metabolism. The PPARa concentration in human hepatocytes is believed to be regulated upstream of and expressed at only 5-10% of that in rodent hepatocytes; therefore, most humans do not have sufficient PPARa to enable the PP process. These observations are supported by investigations showing lack of PP in humans taking pharmaceutical agents known to induce PP in rodents. Nonhuman primate studies are largely supportive PP was not observed in marmosets exposed at up to 2,500 mg/kg/d or in cynomolgus monkeys dosed at up to 500 mg/kg/d (Short et al. 1987; Kurata et al. 1998~. The DEHP-exposed marmosets, however, had increases inperoxisomal volume. There is some concern that certain people may have suff~cient PPARa to exceed the minimum threshold for PP (Holder and Tugwood 1999~. This is illustrated by the darkened region on the PPARa arrow in Figure 4-2. The PPARa knockout mouse model has also provided relevant mecha- nistic information for other DEHP-induced toxicants. For example, though it is resistant to DEHP-induced hepatic effects, the PPARa knockout mouse has been shown to be susceptible to kidney, developmental, and testicular toxicities (Peters et al. 1997~. The role of other PPARs, such as beta or gamma, in mediating organ system toxicity is unclear; however, research has identif~ed specif~c metabolites of DEHP, such as MEHP, but not not 2EHA, as able to activate PPAR-gamma (Maloney et al. 1999~.

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128 MEHP ~ ~ ~ _ > pp Homeostasis Spacecraft Water Exposure Guidelines [PPARa] Rat Mouse Human FIGURE 4-2 Nucleus of ahepatocyte showing how PPARa concentration canplay a key role in determining species susceptibility to PP compounds such as MEHP and how lipid metabolism is related to PP through DNA activation. Source: Adapted from Holden and Tugwood 1999. Specific DEHP metabolites have been evaluated for their ability to cause developmental toxicity and/or reproductive toxicity. MEHP is a de- velopmental and reproductive toxicant and is the suspected critical metabo- lite responsible for testicular toxicity. The evidence for this conclusion is discussed in the reproductive toxicity section. Metabolites 2EH and 2EHA have been shown to produce developmental toxicity in multiple rodent species, but the reproductive effects ofthose metabolites is less well charac- terized (NIP 2000~. Phthalic acid has also been shown to be a developmen- tal toxicant in rodents but is less potent than other tested metabolites (NIP 2000~.

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Di(2-ethylhexyl) Phthalate 129 Elimination The primary routes of elimination of DEHP and its metabolites are the feces and the urine. Two human subjects given a single oral dose of DEHP (30 mg/kg) excreted an average of 13% as urinary metabolites (Schmid and Slatter 1985~. Those same subjects excreted an average of 20% after doses of 10 mg/kg/d for 4 d. A physiologically based pharmacokinetic (PBPK) model was used to predict the elimination of MEHP from the blood of rats given a dose of DEHP or MEHP (Keys et al. 1999~. Flow-limited, diffusion-limited, and pH-trapping models were tested for their ability to fit blood elimination profiles. The best predictions from a single model came from the pH-trapping model; however, that does not mean that the other mechanisms are not involved to some extent in the elimination of MEHP from blood. The pH-trapping model postulates that MEHP can diffuse across mem- branes only in the un-ionized state and that once MEHP enters a cell it converts to MEHP-, which is slowly converted back to MEHP. Thus, the MEHP is trapped in a cell in the ionic form under conditions of suitable pH. When radiolabeled DEHP was administered intravenously to rats, the elimination of radioactivity in blood was very rapid (Schultz and Rubin 1973~. The elimination was at feast biphasic, with elimination half-lives of 4.5-9 minutes (min) and 22 min. respectively. TOXICITY SUMMARY The toxicity of DEHP has been studied in several species, for many different end points, by various routes of administration, and for a variety of exposure times. The intent of this section is to summarize the studies that might be useful for setting SWEGs for DEHP. For example, only oral studies with a feeding or water protocol were considered, unless only other types of studies were available for a given exposure time or could demonstrate relevant species differences in susceptibility. Oral gavage studies tend to exaggerate the toxic effect of a compound because of the bolus nature ofthe dose. The primary target organs of DEHP are the testes and liver, at least in rodents. Different species can have important differ- ences in their responses to oral ingestion of DEHP, and absorption of DEHP may depend on the vehicle used to deliver the dose.

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130 Spacecraft Water Exposure Guidelines Acute Toxicity (1-5 d) There is a report that attempts to relate the acute toxicity of DEHP from a single dose to toxicity from"chronic" (12-week twk]) doses (Lawrence et al.1975~. Using intraperitoneal injections 5 d/wk and LD50 (dose lethal to 50/O of subjects) values as the basis for comparison, these investigators found that DEHP was 28 times more toxic chronically than it was acutely (the LD50 for acute exposure was 28 times smaller). This ratio was the highest of any ofthe eight phthalate esters tested and primarily is due to the low acute toxicity of DEHP compared with the other phthalate esters. The oral LD50s for a single administration of DEHP were 31 g/kg (21 -45 g/kg) in male Wistar rats and 34 g/kg (25-46 g/kg) in male rabbits (Shaffer et al. 1945~. The authors attribute the deaths, which tended to be delayed for several days after the dosing, to liver and kidney injury. Other oral LD50s have been derived from unpublished data from Union Carbide and include the following: guinea pigs, 26,000 mg/kg; rats, 34,000 mg/kg; and mice, 34,000 mg/kg (Krauskopf 1973~. Those LD50 values place the acute toxic- ity of DEHP below that of smaller dialkyl phthalates and above that of larger dialkyl phthalates (Krauskopf 1973~. Repeated administration of DEHP was lethal to rabbits and guinea pigs at 2 g/kg/d when administered for up to 7 d, but the same dose was not lethal to rats and mice (Parmar et al.1988~. Young rats are more suscepti- ble than older rats to DEHP administered over 5 d (Dostal et al. 1987~. Wistar rats given a diet containing DEHP at 20,000 ppm for 3 d showed a 34/O decrease in serum T4, but there seemed to be a recovery, because the decrease after 10 d was only 15% (Hinton et al.1986~. The reduced T4 was also evident after 21 d of exposure at 10,000 parts per million (ppm). In addition, certain liver enzymes were increased after only 3 d of ingestion of DEHP at 20,000 ppm (Hinton et al. 1986~. In a multi-timed study, Wistar rats were given nominal doses of 50, 200, and 1,000 mg/kg/d in their diets and were sacrificed from 3 d to 9 months (mo) after the start of the dosing (Mitchell et al. 1985b). At sacri- fice, the livers were removed and subjected to a number of biochemical, DNA, and histopathologic evaluations. The liver weights of males were increased 26% after 3 d at the highest dose, but the two lower doses did not cause significant hypertrophy until 14 dofingestioninmales. Females were somewhat less susceptible to DEHP-induced liver hypertrophy. Other observations in males ingesting DEHP for 3 d included increased PP (top two doses), transient increased bile flow and damage of the bile canaliculi

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Di(2-ethylhexyl) Phthalate 131 (top two doses), increased DNA synthesis (top two doses), and changes in various tissue enzymes (one to three dose levels). In general, females were less susceptible to injury or changes than males. The lowest dose, 50 mg/kg/d, was without adverse effect after 3 ~ of ingestion. In another study, after 3 ~ of consuming food spiked with DEHP at 20,000 ppm, the liver weights of male Wistar rats were increased 46% compared with the liver weights of controls (Mann et al.l985~. Light mi- croscopy revealed an increased number of mitotic figures in the liver, and electronmicroscopy of the liver revealed PP. A number of hepatocyte en- zymes were also changed after only 3 ~ of DEHP ingestion at this high level. Apparently, a single human ingestion of 10 g of DEHP caused gastric distress and catharsis, whereas 5 g did not elicit symptoms (Shaffer et al. 1945~. Short-Term Toxicity (6-30 d) Observations of toxic effects in this range of exposure times are often made as interim observations during a much longer study. At 2 wk into a 79 wk study, male Wistar rats given DEHP at 2% in their food showed a 60% increase in liver weights and induction of enzymes associated with PP and hydrogen peroxide metabolism (assessed first at 4 wk) (Tamura et al. 1990~. In another study, Wistar rats given DEHP at 2% in their food for 1 wk showed severe atrophy ofthe testes (43 TO weight decrease) accompanied by high testicular testosterone and low testicular zinc (Oishi and Hiraga 1980~. In addition, liver weights increased by 27% and kidney weights decreased by 18% on an absolute organ weight basis (Oishi and Hiraga 1980~. Because most ofthe studies have involved rat exposures, it is essential to know the relevance of those studies to human exposures to DEHP. In the absence of human data, exposures of other primates can be useful. One such study, by Rhodes et al. (1986), compared the short-term toxicity of DEHP in marmosets and rats. Both species were given 2 g/kg/d as single oral doses in corn oil for 14 consecutive days. Testicular atrophy, hepato- megaly, and reduced body-weight gain were found in the rats; however, only reduced body-weight gain was found in the marmosets. The induction of peroxisomes and associated enzymes was found in rat liver, but not in the livers of the marmosets. Absorption studies suggested that DEHP is not as readily absorbed from the marmoset gut as from the rat gut. Even when the

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158 Spacecraft Water Exposure Guidelines Ingestion for 10 d Mann et al. (1985) found evidence of testicular injury in rats ingesting DEHP in their food for 10 d. The paper focused almost entirely on liver effects; however, the testes of rats exposed at approximately 1,600 mg/kg/d weighed only 2.07 g, whereas the control testes weighed an average of 2.28 g. The authors did not consider this difference statistically significant, but there was clearly a difference in weight at 21 ~ of exposure. Because a decrease in testicular weight is a very insensitive end point for testicular injury, 1,600 mg/kg/d was established as a LOAEL. The 10-d AC for change in testicular weights was calculated as follows: 10-d AC = (1,600 mg/kg/d x 70 kg) (2.8 L/d x 10 x 3) = 1,300 mg/L. The factors of 10 and 3 are for extrapolation from a LOAEL to a NOAEL and for species extrapolation, respectively. For male reproductive effects, the weight of evidence suggests that adult humans are much less susceptible than rodents to DEHP; therefore, the usual species extrapolation factor of 10 was reduced to 3. Ingestion For 100 d The ACs for this time of exposure were set to avoid potential testicular injury and hematotoxicity on the basis of the results of Poon et al. (1997~. Rats were fed DEHP in their diets for 13 wk at concentrations of 0, 5, 50, 500, and 5,000 ppm (Poon et al.1997~. Thyroid changes were found in the highest-dosed animals, but rodent thyroids are thought to provide an overly sensitive model of the human thyroid (McCIain 1992~. Sertoli cell vacuol- ization was found in seven of 10 ofthe rats ingesting 500 ppm (38 mg/kg/d, Table 4-2~; hence, the NOAEL was set at the next lower dose of 3.7 mg/kg/ d. The 100-d AC for potential testicular injury was calculated as follows: 100-d AC = (3.7 mg/kg x 70 kg) (2.S x 3 x 1.1) = 28 mg/L. The factor of 1.1 was used to compensate for the study being only 90 4, whereas the AC is for 100 d. The factor of 3 is for species extrapolation from rodents to humans for male reproductive effects. The data were not suitable for BMD analysis because ofthe poor fit ofthe dose-response data by standard approaches and the difficulty in applying the lesion-severity

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Di(2-ethylhexyl) Phthalate 159 data in the analysis (see Table 4-2~. The subcommittee recommended against attempting a time extrapolation of this value to a 1,000-d ingestion period because good chronic data are available. A 9/O reduction in red cell counts was noted in the group of male rats given DEHP at 5,000 ppm for 13 wk (Poon et al. 1997~. The next lower group (500 ppm) did not show a statistically significant change. This amount of DEHP in food is approximately equivalent to 40 mg/kg/d, which was taken as the NOAEL for hematologic effects. The AC for hemotoxicity was calculated as follows: 100-d AC = (40 mg/kg/d x 70 kg) (2.8 L/d x 10 x 3 x 1.1) = 30 mg/L. The factors of 10,3, and 1.1 are for interspecies differences, spaceflight effects, and time differences between rat exposures and potential human exposures, respectively. Ingestion for 1,000 d Kluwe et al. ~ 1982) reported seminiferous tubular degeneration in F-344 rats and B6C3F~ mice fed DEHP-spiked food for 103 wk. Rats and mice in their respective high dose groups showed the lesion; however, rats in the group fed 320 mg/kg/d did not show the lesion. Using the BRIDLE of 211 mg/kg/d (Table 4-3) as a NOAEL (rats were more sensitive than mice), the AC for testicular effects was estimated as follows: 1,000-d AC = (211 mg/kg/d x 70 kg) (2.8 L x 3) = 1,760 mg/L. This AC clearly is not consistent with the 28 mg/L estimated for avoidance oftesticular effects for a 100-d ingestion period using the 13-wk SD rat data from Poon et al. (1997~. Poon et al. (1997) reported mild Sertoli cell vacu- olization at 500 ppm (40 mg/kg/d), but minimal to mild seminiferous tubu- lar atrophy was reported only in the highest dose (5,000 ppm, or about 400 mg/kg/d). The subcommittee and the original investigators recommended that 3.7 mg/kg/d be considered the NOAEL in the Poon et al. (1997) study. The difference in the findings is probably related to differences in the depth of assessment of the testicular injury and perhaps also to differences in the inherent susceptibilities of the two strains of rat and in the age at exposure. Increases in the incidence of bilateral aspermiogenesis were found in F- 344 rats exposed to DEHP in their feed for 104 wk (Table 4-5) (David et al.

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160 Spacecraft Water Exposure Guidelines 2000~. The study gave a NOAEL of 5.8 mg/kg/d, which results in an AC as follows: 1,000-d AC = (5.8 mg/kg/d x 70 kg) (2.8 L/d x 3) = 48 mg/L. The NOAEL approach can be compared with the BMD analysis. The dose-response curve reversed at intermediate doses, making the BMD anal- ysis subject to large uncertainty. The resulting BMDLo~ values spanned a range of several orders of magnitude; however, the three models with the highest p values gave consistent, intermediate results. The intermediate BMDLo~ results were as follows: the logistic model gave 3.0 mg/kg/d, the probit model gave 3.6 mg/kg/d, and the quantal-linear model gave 2.2 mg/kg/~. In this situation, a BMDLo~ of 2.2 mg/kg/d was used for a conser- vative risk assessment. That value is somewhat below the range of 3.7 mg/kg/d to 14 mg/kg/d suggested as the NOAEL for reproductive effects in rats (NIP 2000~. The AC calculation, using a species extrapolation factor of only 3, was as follows: 1,000-d AC = (2.2 mg/kg/d x 70 kg) (2.8 L/d x 3) = 18 mg/L. This AC is consistent with the 100-d AC of 28 mg/kg/d derived from the data of Poon et al. (1997) for prevention of Sertoli cell vacuolization. Chronic renal inflammation was noted in a group of male mice fed DEHP at 6,000 mg/kg in feed for 103 wk. but was not observed in a group fed 3,000 mg/kg in feed (Kluwe et al.1982~. The authors estimate that the mean ingestion of DEHP in the unaffected group was 670 mg/kg/~. A BMDLo~ of 71 mg/kg/d was established as a NOAEL for kidney effects. The AC for kidney effects was estimated as follows: 1,000-d AC = (71 mg/kg/d x 70 kg) (2.8 L/d x 10) = 177 mg/L. The factor of 10 is for interspecies differences. Clearly, the kidney is not very sensitive to oral ingestion of DEHP. Functional Reproductive Toxicity Aside from the testicular atrophy caused in rodents by DEHP ingestion, a functional impairment has been demonstrated in mice (Meinick et al. 1987~. Functional impairment of reproduction was demonstrated in CD-1

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Di(2-ethylhexyl) Phthalate 163 mice at exposures of 0.1% and 0.3/0 in feed, but not at 0.01%. This inges- tion exposure at 0.01% is equivalent to a dose of approximately 18 mglkg/d and was chosen as a NOAEL. The AC for functional reproductive toxicity was calculated as follows: AC = (18 mg/kg/d x 70 kg) (2.8 L/d x 10) = 45 mg/L. This is applicable to both males and females, because the crossover mating of the mice showed that the effect was mediated through either sex. This AC is higher than those determined from other toxic end points for 100 ~ or 1,000 ~ of ingestion, so the calculations for functional reproductive toxicity were not shown in Table 4-1 1 (above). RECOMMENDATIONS The toxicity of DEHP has been broadly studied, especially in rodents; however, the relevancy of rodent studies to human toxicity requires further investigation. Although DEHP has been shown to be a liver carcinogen in rodents, mechanistic studies suggest that this finding is of minimal rele- vance to human risk assessment. DEHP has been shown to be a develop- mental and reproductive toxicant in multiple rodent species. In particular, exposure during early development presents a special concern for male reproductive toxicity. However, the purpose ofthis document is to evaluate relevant risks for occupational exposures for adult, nonpregnant astronauts. Pharmacokinetic studies have suggested significant species differences in absorption kinetics following oral ingestion between humans and rodents. Combined, these findings suggest that the risk assessment for SWEGs have erred on the conservative side. Additional quantitative toxicity data defin- ing the age and kinetic differences in cross-species effects of oral DEHP would help to further refine these assessments. REFERENCES Albro, P.W. 1986. Absorption, metabolism, and excretion of di(2-ethylhexyl) phthalate by rats and mice. Environ. Health Perspect. 65:293-8. Albro, P.W., R.E. Chapin, J.T. Corbett, J. Schroederm and J.L. Phelps. 1989. Mono-2-ethylhexyl phthalate, a metabolite of di-~2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats. Toxicol. Appl. Pharmacol. 100:1 93-200.

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164 Spacecraft Water Exposure Guidelines Albro, P.W., J.T. Corbett, J.L. Schroeder, S. Jordan, and H.B. Matthews. 1982. Pharmacokinetics, interactions with macromolecules and species differences in metabolism of DEHP. Environ. Health Perspect. 45: 19-25. Albro, P.W., J.R. Hass, C.C. Peck, D.G. Odam, J.T. Corbett, J.F. Bailey, H.E. Blatt, and B.B. Barrett. 1981. Identification of the metabolites of di-~2-ethylhexyl) phthalate in urine from the African green monkey. Drug Metab. Dispos. 9:223-5. Albro, P.W., R. Thomas, and L. Fishbein. 1973. Metabolism of diethylhexyl phthalate by rats isolation and characterization of the urinary metabolites. J. Chromatogr. 76:321-330. Ashby, J., F.J. De Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter, and M.D. Shelby, eds. 1985. Evaluation of short-term tests for carcinogens. Report of the programme on chemical safety's collaborative study on in vitro assays. New York, NY: Elsevier Science. Astill, B.D. 1989. Metabolism of DEHP: Effects of prefeeding and dose variation, and comparative studies in rodents and the cynomolgus monkey. Drug Metab. Rev. 21:35-53. ATSDR (Agency for Toxic Substances and Disease Registry).1993. Toxicological profile for di(2-ethylhexyl) phthalate. TP92/05. U.S. Department of Health and Human Services, Washington, DC. Berman, E., and J.W. Lasky. 1993. Altered steroidogenesis in whole-ovary and adrenal culture in cycling rats. Reprod. Toxicol. 7:349-358. Butterworth, B.E., E. Bermudez, T. Smith-Oliver, L. Earle, R. Cattaley, J. Martin, J.A. Popp, S. Strom, R. Jirtle, and G. Michalopoulos.1984. Lack of genotoxic activity of di(2-ethylhexyl) phthalate in rat and human hepatocytes. Carcinogenesis 5:1329-1335. Carpenter, C.P., C.S. Weil, and H.F. S myth. 1953. Chronic oral toxicity of di~ethylhexyl) phthalate for rats, guinea pigs, and dogs. Arch. Ind. Hyg. 8:219-226. Crocker, J.F.S., S.H. Safe, and P. Acott. 1988. Effects of chronic phthalate expo- sure on the kidney. J. Toxicol. Environ. Health 23:433-44. David, R.M., M.R. Moore, M.A. Cifone, D.C. Finney, and D. Guest. 1999. Chronic peroxisome proliferation and hepatomegaly associated with the hepatocellular tumorigenesis of di(2-ethylhexyl) phthalate and the effects of recovery. Toxicol. Sci. 50: 195-205. David, R.M., M.R. Moore, D.C. Finney, and D. Guest. 2000. Chronic toxicity of di(2-ethylhexyl~phthalate in rats. Toxicol. Sci. 55:433-443. Dostal, L.A., W.L. Jenkins, and B.A. Schwetz.1987. Hepaticperoxisomeprolifera- tion and hypolipidemic effects of di(2-ethylhexyl) phthalate in neonatal and adult rats. Toxicol. Appl. Pharmacol. 87:81 -90. Doull, J., R. Cattley, C. Elcombe, B.G. Lake, J. Swenberg, C. Wilkinson, G. Wil- liams, M. van Gemert. 1999. A cancer risk assessment of di(2-ethylhexyl) phthalate: Application ofthe new U.S. EPA risk assessment guidelines. Regul. Toxicol. Pharmacol. 29:327-57.

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Di(2-ethylhexyl) Phthalate 165 EPA (U.S. Environmental Protection Agency). 1992a. Fed. Reg. 57~138~:31791- 31792. EPA (U.S. Environmental Protection Agency). 1992b. Drinking Water Criteria Document for Phthalic Acid Esters (Final). Page VIII-48, revised. U.S. Envi- ronmental Protection Agency, Washington, DC. August 1992. EPA (U.S. Environmental Protection Agency). 1998. National Primary Drinking Water Regulations, Technical Fact Sheet on Di(2-ethylhexyl) Phthalate. Of lice of Ground Water and Drinking Water, U. S. Environmental Protection Agency, Washington, DC. Ganning, A.E., U. Brunk, and G. Dallner.1984. Phthalate esters and their effect on the liver. Hepatology 4:541-7. Ganning, A.E., M.J. Olsson, U. Brunk, and G. Dallner. 1991. Effects of prolonged treatment with phthalate ester on rat liver. Pharmacol. Toxicol. 68:392- 401. Gray, T.J.B., and S.D. Gangolli.1986. Aspects ofthe testicular toxicity of phthalate esters. Environ. Health Perspect. 65:229-235. Hinton, R.H., F.E. Mitchell, A. Mann, D. Chescoe, S.C. Price, A. Nunn, P. Grasso, and J.W. Bridges.1986. Effects of phthalic acid esters on the liver and thyroid. Environ. Health Perspect. 70: 195-200. Holden, P.R., and J.D. Tugwood. 1999. Peroxisome proliferator-activated receptor alpha: role in rodent liver cancer and species differences. J. Mol. Endocrinol. 22:1-8. ICI. 1982. Di(2-ethylhexyl) phthalate: A comparastive subacute toxicity study in the rat and marmoset. TSCATS 215194, Doc. I.D. 87-8220040.Bridgewater, NJ: ICI Americas, Inc. IRIS (Integrated Risk Information System). 1993. Di(2-ethylhexyl) Phthalate. National Library of Medicine, U.S. Department of Health and Human Services, Washington, DC [Online]. Available: http://www.epa.gov/iris/subst/0014.htm. Ikeda, G.J., P.P. Sapienza, J.L. Couvillion, T.M. Farber, and E.J. van Loon. 1980. Comparative distribution, excretion and metabolism of di(2-ethylhexyl) phthalate in rats, dogs, and miniature pigs. Food Cosmet. Toxicol.18:637-642. Keys, D.A., D.G. Wallace, T.B. Kepler, and R.B. Conolly. 1999. Quantitative evaluation of alternative mechanisms of blood and testes disposition of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in rats. Toxicol. Sci. 49:172-185. Kluwe, W.M., J.K. Haseman, J.F. Douglas, and J.E. Huff.1982. The carcinogenic- ity of dietary di(2-ethylhexyl) phthalate (DEHP) in Fischer 344 rats and B6C3F1 mice. J. Toxicol. Environ. Health 10:797-815. Kornburst, D.J., T.R. Barfknecht, P. Ingram, and J.D. Shelburne. 1984. Effect of di(2-ethylhexyl) phthalate on DNA repair and lipid peroxidation in rat hepatocytes and on metabolic cooperation in Chinese hamster V-79 cells. J. Toxicol Environ. Health 13 :99- 116. Krauskopf, L.G.1973. Studies on the toxicity of phthalates via ingestion. Environ. Health Perspect. 3 :61-72. Kurata, Y., F. Kidachi, M. Yokoyama, N. Toyota, M. Tsuchitani, and M. Katoh. 1998. Subchronic toxicity of di(2-ethylhexyl) phthalate in common marmosets:

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166 Spacecraft Water Exposure Guidelines Lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia. Toxicol. Sci. 42:49-56. Lake, B.G., S.L. Kozlen, J.G. Evans, T.J.B. Gray, P.J. Young, and S.D. Gangolli. 1987. Effect of prolonged administration of clofibric acid and di(2-ethylhexyl) phthalate on hepatic enzyme activities and lipid peroxidation in the rat. Toxi- cology 44:213-228. Lawrence, W.H., M. Malik, J.E. Turner, A.R. Singh, and J. Autian.1975. Atoxico- logical investigation ofsome acute, short-term, and chronic effects of adminis- tering di(2-ethylhexyl) phthalate (DEHP) and other phthalate esters. Environ. Res. 9:1-11. Leyder, F., and P. Boulanger.1983. Ultraviolet absorption, aqueous solubility, and octanol-water partition for severalphthalates. Bull. Environ. Contam. Toxicol. 30:152-157. Lhuguenot, J.-C., A.M. Mitchell, G. Milner, E.A. Lock, and C.R. Elcombe.1985a. The metabolism of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in rats: In vivo and in vitro dose and time dependency of metabolism. Toxicol. Appl. Pharmacol. 80:11-22. Maloney, E.K., and D.J. Waxman. 1999. Trans activation of PPAR-alpha and PPAR-gamma by structurally diverse environmental chemicals. Toxicol. Appl. Pharmacol. 161:209-218. Mann, A.H., S.C. Price, F.E. Mitchell, P. Grasso, R.H. Hinton, and J.W. Bridges. 1985. Comparison of the short-term effects of di(2-ethylhexyl) phthalate, di~n-hexyl) phthalate, and di~n-octyl) phthalate in rats. Toxicol. Appl. Pharmacol. 77:116-132. Marsman, D.S., R.C. Cattley, J.G. Conway, and J.A. Popp. 1988. Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepato- carcinogenicity ofthe peroxisome proliferators di(2-ethylhexyl) phthalate and [4-chloro-6-~2,3-xylidino)-2-pyrimidinylthio~acetic acid in rats. Cancer Res. 48:6739-44. Marx, J. 1990. Animal carcinogen testing challenged. Science 250:743-5. Mayer, F.L., D.L. Stalling, and J.L. Johnson.1972. Phthalate esters as environmen- tal contaminants. Nature (London) 238:411-3. McClain, R.M. 1992. Thyroid gland neoplasia: Non-genotoxic mechanisms. Toxicol. Lett. 64/65:397-408. Melnick, R.L., R.E. Morrissey, and K.E. Tomaszewski. 1987. Studies by the Na- tional Toxicology Program on di(2-ethylhexyl) phthalate. Toxicol. Ind. Health 3:99-118. Mitchell, A.M., J.-C. Lhuguenot, J.W. Bridges, and C.R. Elcombe.1985a. Identifi- cation of the proximate peroxisome proliferator~s) derived from di(2-ethyl- hexyl) phthalate. Toxicol. Appl. Pharmacol. 80:23-32. Mitchell, F.A., S.C. Price, R.H. Hinton, P. Grasso, and J.W. Bridges.1985b. Time and dose-response study ofthe effects on rats ofthe plasticizer di(2-ethylhexyl) phthalate. Toxicol. Appl. Pharmacol. 81:371-392. Morton, S.J. 1979. The Hepatic Effects of Dietary DEHP. Ph.D. Thesis at Johns Hopkins University, Baltimore, MD

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168 Spacecraft Water Exposure Guidelines Schultz, C.O., and R.J. Rubin. 1973. Distribution, metabolism, and excretion of di-2-ethylhexyl phthalate in the rat. Environ. Health Perspect. 3:123-9. Shaffer, C.B., C.P. Carpenter, and H.F. S myth, Jr.1945. Acute and subacute toxic- ity of di(2-ethylhexyl) phthalate with note upon its metabolism. J. Ind. Hyg. Toxicol. 27: 130-5. Shell Oil Co.1982. Bis(2-ethylhexyl) phthalate: Toxicokinetics of 14-day subacute oral administration to rats and marmosets. TCATS:OTS 0539135, Doc. I.D. 88-920002040. Shell Oil Co., Houston, TX. Short, R.D., E.C. Robinson, A.W. Lington, and A.E. Chin. 1987. Metabolic and peroxisome proliferation studies with di(2-ethylhexyl) phthalate in rats and monkeys. Toxicol. Ind. Health 3:185-195. Sjoberg, P., N.G. Lindqvist, and L. Ploen.1986. Age-dependent response ofthe rat testes to di(2-ethylhexyl) phthalate. Environ. Health Perspect. 65:237-42. Smith-Oliver, T., and B.E. Butterworth. 1987. Correlation of the carcinogenic potential of di(2-ethylhexyl) phthalate with induced hyperplasia rather than with genotoxic activity. Mutat. Res. 188 :21 -8. Straub, J.E., J.R. Schultz, W.F. Michalek, and R.L. Sauer.1995. Further character- ization and multifiltration treatment of shuttle humidity condensate. SAE- ICES Paper 951685. Warrendale, PA: Society of Automotive Engineers. Takagi, A., K. Sai, T. Umemura, R. Hasegawa, andY. Kurokawa.1990. Significant increase in 8-hydroxydeoxyguanosine in liver DNA of rats following short-term exposure to the peroxisome proliferators di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate. Jpn. J. Cancer Res. 81 :213-215. Tamura, H., T. Iida, T. Watanabe, and T. Suga. 1990. Long-term effects of hypolipidemic peroxisome proliferator administration on hepatic hydrogen peroxide metabolism in rats. Carcinogenesis 11 :445-450. Tomita, I., Y. Nakamura, N. Aoki, and N. Inui. 1982. Mutagenic/carcinogenic potential of DEHP and MEHP. Environ. Health Perspect. 45:119-125. Treinen, K.A., W.C. Dodson, and J.J. Heindel.1990. Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulose cell cultures. Toxicol. Appl. Pharmacol. 106:334- 340. Tyl, R.W., C.J. Price, M.C. Marr, and C.A. Kimmel.1988. Developmental toxicity evaluation of dietary di(2-ethylhexyl) phthalate in Fischer 344 rats and CD-1 mice. Fundam. Appl. Toxicol. 10:395-412. WHO (World Health Organization). 1992. Environmental Health Criteria 131, Diethylhexyl Phthalate. Geneva: WHO. Zacharewski, T.R., M.D. Meek, J.H. Clemons, Z.F. Wu, M.R. Fielden, and J.B. Matthews. 1998. Examination of the in vitro and in vivo estrogenic activities O f eight commercial phthalate e sters. Toxicol. S ci . 46: 282 - 93.