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Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1 (2004)

Chapter: Appendix 4: Di(2-ethylhexyl) Phthalate

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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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Suggested Citation:"Appendix 4: Di(2-ethylhexyl) Phthalate." National Research Council. 2004. Spacecraft Water Exposure Guidelines for Selected Contaminants: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/10942.
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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

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 -50°C 387°C 45 ~g/L in water at 20°C (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

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.

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

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.

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~.

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~.

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~.

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.

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

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

132 Spacecraft Water Exposure Guidelines bioavailability of the DEHP metabolites in target tissue is matched in the two species, the rat seems to respond more readily. In another study comparing the responses of rats and monkeys, mon- keys were found to be much less susceptible to PP induction by DEHP. Male F-344 rats and cynomolgus monkeys were treated with DEHP for 21 d. The average daily dose to specific groups of rats, administered in their feed, was approximately 11,105,670,1,200, or 2,100 mg/kg, whereas the daily dose by gavage to the monkeys was 500 mg/kg (Short et al.1987~. In rats fed 105 mg/kg/d, there were no increases in liver weights, but there were increases in two of three enzymatic markers for PP; at 670 mg/kg/d, liver weights increased 80°/O (Short et al. l 987~. In contrast, monkeys given gavage doses of 500 mg/kg/d showed no evidence of PP. The study by Mitchell et al. (1985b) included sacrifices at 7,14, and 28 ~ after the rats began ingesting DEHP-spiked food at nominal doses of 50, 200, or 1,000 mg/kg/~. By day 14, liver weights among male rats in the low-dose group were 22% above control-group liver weights; male rats in the middle group had liver weights 38°/O above controls; and male rats in the high-dose group had liver weights 69% above controls. However, at 28 4, only the high-dose group had statistically significant increases in liver weights. Females were less susceptible to the induction of increased liver weights. DNA synthesis was increased in the livers from all groups at 7 4, but was normal in all groups by 14 d. Various enzymes from liver tissue showed different activities from controls at various times after the doses, but the bile canalicular damage seen in male rats at 3 ~ was gone at 7 d. Based on the increases in liver weights, which were transient at 7-28 ~ in males exposed at the lowest dose, one can conclude that 50 mg/kg/d pro- duces a marginal adverse effect. The testes from the rats sacrificed at or before 28 d were not examined as part ofthis study. The "urogenital appara- tus" was examined in rats killed after 9 mo of exposure, but the findings were not included in the report (Mitchell et al. 1985b). A high dose of DEHP (20,000 ppm in the feed) has been used to evalu- ate the progression of effects induced in Wistar rats after 3-21 d of ingestion (Mann et al.1985~. In terms of weight changes reported in this experiment, the liver was more sensitive than the testes. Large increases (e.g., 140% after 21 d) in liver weight were found at all sacrifice times, whereas the only change in testicular weights was a decrease of 22%, and that was after 21 d. As discussed above, these investigators also report a number of morpho- logic and biochemical changes in the liver in response to DEHP ingestion, but morphologic and biochemical changes were not evaluated in the testes. The testes of young rats have been shown to be a target organ for short-term toxicity of DEHP at high doses (Gray and Gangolli 1986~. Ad-

Di(2-ethylhexyl) Phthalate 133 ministration of DEHP in corn oil (presumably by gavage) at 2,800 mg/kg/d for 10 ~ caused seminiferous tubular atrophy and reductions in the weights of seminal vesicles and prostate. These adverse effects occurred in 4- to 5- wk-old rats, but not in 15-wk-old rats. Although this gavage study is not directly useful for risk evaluation, it indicates that age can have a profound effect on susceptibility to DEHP-induced reproductive toxicity. When compared with the results of Mitchell et al. (1985b), this study suggests that in mature rats liver effects occur at much lower doses than do male repro- ductive effects. In a study of effects induced in monkeys given 14 consecutive doses of DEHP at 500 mglkgld by feeding tube, the findings were negative (Pugh et al.2000~. The test animals were four 2-y-old male cynomolgus monkeys. Animals were evaluated by observation of clinical signs, hematology, clini- cal chemistry, urinalysis, gross necropsy, and histopathology. There were no detectable effects on the liver, red blood cells, or testes. Subchronic Toxicity (30-1X0 d) The earliest subchronic study of DEHP administered orally was re- ported almost 60 y ago by Shaffer et al. (1945~. Male Wistar rats were fed DEHP at doses equivalent to 0,0.2,0.4,0.9, and 1.9 glkgld for 90 d. There was some degree of growth retardation in the three highest-dose groups, but none of the animals died during the study. Hematologic parameters were normal in all groups, and the only histopathologic effect was atrophy and degeneration in the testes of rats from the two highest-dose groups. In addition to the testes, the liver, kidneys, spleen, and heart were examined microscopically. The authors concluded that 0.2 glkgld caused no effect, and the only effect at 0.4 glkgld was growth retardation of unspecified magnitude. It is surprising, in light of more recent studies, that liver effects were not described. Two subchronic oral studies have been reported one in rats, and the other in marmosets. In the first study, Spague-Dawley rats were given DEHP in their diets at 0, 5, 50, 500, and 5,000 ppm for 13 wk (Poon et al. 1997~. Liver weights were increased 40°/O in males and 20% in females in the highest-dose group, but not in any of the other groups. Other changes confined to the highest-dose rats included increased serum albumin in males and females, reduced thyroid follicle size and colloid densities in males and females, decreased serum cholesterol in females, and a 9°/O average decrease in red cell count in males. Testicular changes in highest-dose rats included minimal to mild seminiferous tubule atrophy (9/10) and mild to moderate

134 Spacecraft Water Exposure Guidelines Sertoli cell vacuolization (9/10~. In addition, minimal Sertoli cell vacuol- ization was found in the 500-ppm group (7/10~. According to the study authors, the 13-wkNOAEL (no-observed-adverse-effect level) was 50 ppm, equivalent to 3.7 mg/kg/d (males). It is essential to decide whether the Sertoli cell vacuolization seen in rats that consumed DEHP in food for 13 wk was an adverse effect (Poon et al. 1997~. The histopathologic data are summarized in Table 4-2. The authors concluded that the Sertoli cell effects preceded the germ cell effects; therefore, the observation at 500 ppm should be considered an early, ad- verse effect. That conclusion is difficult to reconcile with the fact that no seminiferous tubule atrophy was observed in the 500-ppm males. Sertoli cells normally contain some vacuoles. Vacuolization of Sertoli cells repre- sents an enlargement of the smooth endoplasmic reticulum (SER) due to alterations in secretion or transport of proteins, disruption of ionic pumps, or changes in the cytoskeleton supporting the SER (Richburg and Boekelhiede 1997~. Each Sertoli cell serves a species-specif~c number of germ cells; in rats, approximately 22 germ cells adhere to each Sertoli cell. Germ cells are lost when the cell-to-cell contacts are lost, and that can lead to release of immature sperm cells. The mechanism ofthis loss is unknown; however, vacuolization ofthe Sertoli cells alone seems insufficient to cause loss of germ cells. In agreement with the authors ofthe study, the subcom- mittee advises that 3.7 mg/kg/d (50 ppm) be considered the NOAEL. To understand the significance of this pathology, and to understand DEHP's potential reproductive toxicity, it is important to consider the overall weight of scientific evidence that DEHP produces adverse reproductive effects. Over 70 reproductive studies and many good consensus documents discuss this research (NTP 2000~. The database includes evidence that DEHP is a reproductive toxicant in male rats, mice, guinea pigs, and ferrets and pro- duces structural changes in the testes, reduced fertility, and altered sperm dysfunction (NTP 2000~. Morphologic, functional, end biochemical assess- ment has shown that the Sertoli cells are cellular targets for adult and pre-adult exposures (NTP 2000~. In vitro studies with MEHP exposures in Sertoli-germ cell cultures support the hypothesis that MEHP inhibits prolif- eration and is a critical player in determining DEHP's testicular toxicity (NTP 2000~. Although there are sufficient data to demonstrate that DEHP is a reproductive toxicant in rodents, significant data gaps exist in determin- ing precise dose-response relationships (NTP 2000~. The NTP (2000) report used this weight- of-evidence in their assessment and determined that the data supported a NOAEL within the range of 3.7-14 mg/kg/d for male reproductive toxicity caused by oral exposure to DEHP in rats.

Di(2-ethylhexyl) Phthalate TABLE 4-2 Histopathology of Male Rats Fed DEHPa 135 DEHP in Diet (ppm) o Dose (mg/kg/d) (average) Siminiferous Tubule Atrophyb 1 (0.1) 3 (0.5) 1 (0.4) o Sertoli Cell Vacuolizationb o 5 50 500 5,000 0.4 3.7 37.6 375 o 9 (1.5) 4 (0.2) 4(0.5) 7(1.0) 9(2.4) aStudy duration was 13 wk. bAverage severity of lesions in all tissues examined is shown in parentheses. The first digit indicates the degree of injury (O = minimal, 1 = mild, 2 = moderate, 3 = severe). Dispersions of lesions were added fractionally to the integer (0.25 = focal, 0.5 = locally extensive, 0.75 = multifocal). For example, 1.25 = mild, focal; and 2.5 = moderate, locally extensive. Source: Data from Poon et al. 1997. Although the rodent response to ingested DEHP seems to have been studied thoroughly, the question remains is the rodent an appropriate model of the human response to DEHP? There are large differences in the absorption and metabolism of DEHP in primates and rodents (Rhodes et al. 1986~. According to a recent risk assessment on reproductive toxicity, "Absorption studies, as well as PBPK modeling, suggest that DEHP metab- olism to MEHP and its absorption from the human gut and marmoset gut saturates at a low dose relative to that ofthe rat" (NIP 2000~. A subchronic study in marmosets also suggests that rats are probably a poor model ofthe human response to DEHP. In that study, groups of four male and four female marmosets were administered DEHP via oral catheter at doses of 0, 100, 500, and 2,500 mg/kg/d for 91 consecutive days (Kurata et al. 1998~. High-dose males had significantly lower body-weight gains midway through the study, but not at the end of the study. The liver peroxisome number, volume density, morphology, and enzyme activity were unchanged by treatment; however, the mean peroxisome volume increased about 35°/O in the mid- and high-dose males. According to the authors, measurements associated with liver cytochrome P-450 tended to increase; however, there was no clear dose-response relationship. Atrophic testicular changes re- ported in rodent studies were not seen in the marmosets, even by electron microscopy. Testicular weights, testicular zinc, and blood testosterone were unchanged in all groups. There were no changes in blood chemistry or organ histopathology associated with DEHP treatment.

136 Spacecraft Water Exposure Guidelines Chronic Toxicity (0.5 y to lifetime) There have been several chronic ingestion studies of DEHP in rodents. Some studies focused on changes in specific organs (e.g., liver), while others attempted to discover the full range of adverse effects caused by DEHP. The discussion below is arranged chronologically, and DEHP-in- duced neoplastic and non-neoplastic effects are considered separately. Non-Neoplastic Lesions The earliest chronic oral study on DEHP came from the same laboratory as the early subchronic study cited above (Shaffer et al. 1945~. The chronic study is especially valuable because three species (rats, guinea pigs, and dogs) were exposed, although not for the same length of time. Male and female Sherman rats, beginning at 60 ~ of age, were exposed to DEHP in their food for 2 y at concentrations that resulted in doses of 0, 0.02, 0.06, and 0.2 g/kg/d (Carpenter et al. 1953~. Male and female guinea pigs were given food spiked with DEHP for 1 y; the doses approximated 0, 0.02, and 0.06 mg/kg/d (for the last 10 mo of the study). A few dogs (four controls, four exposed) were given DEHP in capsules at doses of 0.03 milliliters (mL)/kg/d for 19 ~ and then 0.06 mL/kg, 5 d/wk, for a total of 240 doses. A fairly thorough histopathologic evaluation of the animals was done after necropsy; however, hematology findings, which were negative, were re- ported only for the rats. Increased liver and kidney weights were reported in the high-dose rats, but no adverse effects were reported from the two lower-dose groups or in any of the guinea pigs or dogs. Despite a few irregularities in the study (e.g., lung infections causing excess mortality in control rats, problems with an unusually high number of litters in For control rats, and liver-weight increases in female guinea pigs that were not dose- related), the authors concluded that the three species are roughly compara- ble in sensitivity to production of neoplastic lesions following DEHP inges- tion, with a NOAEL of 0.06 g/kg/d or higher (Carpenter et al. 1953~. Nikonorow et al. (1973) reported the effects of the administration of DEHP in food to male and female Wistar rats at 0.35°/O for 12 mot The rats had decreases in body weights, increases in kidney weights, and liver en- largement. Blood cell counts were unchanged, and the histopathology of liver, kidney, and spleen were normal. This report is difficult to understand because of the different plasticizers and stabilizers studied, the different exposure times, and the incomplete information provided.

Di(2-ethylhexyl) Phthalate 137 A later chronic study, conducted in rats and mice by NIP (Kluwe et al. 1982), was done using concentrations at least 5-fold higher than the study by Carpenter et al. (1953) and revealed significantly different results, in- cluding the ability of DEHP to induce liver tumors (see discussion below). F-344 rats ingested DEHP at 0.32 g/kg/d or 0.67 g/kg/d (males) or 0.39 g/kg/d or 0.77 g/kg/d (females) from their food for 103 wk. The mean daily ingestions of DEHP by B6C3F~ mice were 0.67 g/kg/d or 1.32 g/kg/d (males) and 0.80 g/kg/d or 1.82 g/kg/d (females). Only the low-dose female mice showed a statistically significant decrease in survival, and the investi- gators did not attribute that to DEHP ingestion. The incidence of non-neo- plastic lesions in treated male rats and mice was found to exceed the inci- dence in their respective controls. Pituitary hypertrophy was found in 22 of 49 high-dose male rats (one of 46 controls) and seminiferous tubular degeneration ofthe testis was found in 43 of 48 high-dose male rats (one of 49 controls). Chronic kidney inflammation was found in 10 of 50 high- dose male mice (one of 50 controls) and seminiferous tubular degeneration was found in seven of 49 high-dose male mice (one of 49 controls). The benchmark dose (BMD) analyses ofthese data are shown in Tables 4-3 and 4-4 and in Figure 4-3, along with the statistically significant changes. There was one chronic time point in a study attempting to define the time- and dose-response relationships for male and female Wistar rats ad- ministered DEHP in food at 50, 200, and 1,000 mg/kg/d (Mitchell et al. 1985b). The study was focused primarily on changes in the liver. After 9 mo, body weights were lower in the two highest-dose male groups and in the highest-dose female group. Liver weights were from 18% to 40°/O higher in males of all dose groups and 17% to 31 % higher in the two high- est-dose female groups. Few changes occurred between the 28-d time point (see subchronic section above) and the 9-mo time point. After 9 mo, thy- roid alterations were reported, including basophylic deposits in the colloid and enlargement of the lysosomes. The response of females was qualita- tively similar to males, but females were clearly less susceptible to the liver effects caused by DEHP. In another study focused exclusively on changes in the rat liver induced by DEHP (and other compounds), the relationship between PP and lipid peroxidation was investigated (Lake et al. 1987~. Male Sprague-Dawley rats were administered DEHP at 2% in their diets for 2 y. They were then killed, and their livers were studied for various biochemical changes. Two ofthe three markers of lipid peroxidation showed large increases compared with control values. The study confirms the association between PP and lipid peroxidation; however, the role of enhanced lipid peroxidation in

138 Spacecraft Water Exposure Guidelines TABLE 4-3 BMD Analysis of Rat Data Showing Seminiferous Tubule Degenerationa Dose (mg/kg/d) Incidence o BMDo1 BMDLo1 (mg/kg/d) (mg/kg/d) 285 211 322 674 1/49 2/44 43/48b aA log-logistic model was used. Statistically significant change level. Abbreviations: BMDol, benchmark dose corresponding to a 1% risk; BMDLol, lower confidence limit on the BMD corresponding to a 1% risk. Source: Data from Kluwe et al. 1982. the hepatocarcinogenicity of DEHP remains to be demonstrated (Lake et al. 1987). In a study focused on the kidneys, Crocker et al. (1988) evaluated the potential for DEHP or residues from artificial kidneys to cause an effect in rats similar to the polycystic kidney disease noted at autopsy in patients who had undergone long-term hemodialysis. Rats (gender and strain un- specified) were administered DEHP at approximately 2 mg/kg (based on a human equivalent dose of 150 mg per 70 kg body weight) three times a week by feeding tube and were killed after 3, 6, 9, and 12 mot The study suggests that the dose may have been higher because of compression of the usual dialysis patient's exposure time of 5-7 y into a rat's life span, but that is not clear. DEHP apparently caused focal cystic changes in the kidneys of rats killed et the 12-mo time period, end the creatinine clearance was also reduced approximately 50°/O after 12 mot However, the reported kidney effects seem inconsistent with reports from other studies in which rats given much higher doses did not exhibit kidney injury. The study also involved an unusually high number of deaths (e.g., four of 20 control animals died). An in vitro study using cultured kidney cells showed that when cells are exposed to MEHP or 2-ethy~hexanoic acid, only the former causes a marked toxic effect (Rothenbacher et al. 1998~. In a study focused on the liver effects of DEHP in rodents, Canning et al. (1991) gave DEHP in food at concentrations of 0.02%, 0.2%, and2°/O for 102 wk. The two highest doses caused a reduction in body-weight gain, which was apparent before 24 wk. A number oftissue enzymes were stud- ied for the time course oftheir changes in response to accumulated exposure

Di(2-ethylhexyl) Phthalate TABLE 4-4 BMD Analysis of Mouse Data Showing Chronic Renal Inflammations 139 Dose (mg/kg/d) Incidence o BMDo1 BMDLo1 (mg/kg/d) (mg/kg/d) 71 672 1,325 1/50 2/48 1 0/5 ob 525 aA Weibull model was used, although a log-logistic model would give the same result. Both models provide an exact fit to the data because there are only three points. Statistically significant change level. Abbreviations: BMDol, benchmark dose corresponding to a 1% risk; BMDLol, lower confidence limit on the BMD corresponding to a 1% risk. Source: Data from Kluwe et al. 1982. to DEHP. PalmitoyI-Co-A dehydrogenase, a marker of PP, increased im- mediately in the liver homogenates from the highest-dose group; showed rapid increases in the middle-dose group; and showed a slow, near-linear increase to about 100% above the control value by 102 wk. Carnitine- acety~transferase, a mitochondrial enzyme, showed a pattern of increased activity similar to palmitoyI-Co-A dehydrogenase activity. Other enzymes showed transient spikes in activity during the time course of the study. In animals whose treatment was discontinued after 1 y, all enzyme levels return to normal within 2-3 wk after the end oftreatment.These results show the cumulative effects of DEHP ingestion on enzyme activities but also demonstrate the reversibility of the induced changes once the toxicant is withdrawn. Data from a chronic study in F-344 rats show that aspermiogenesis and possibly hematologic effects resulted from ingestion of DEHP in food (Da- vid et al. 2000~. The pertinent data and the BMD analysis for reduced red blood cell (RBC) counts is show in Table 4-5 and in Figure 4-4. The data on aspermiogenesis were difficult to use in BMD analysis because of a dose-response reversal in the intermediate doses. However, a BMDLo~ of 2.2 mg/kg/d was found using a quantal-linear model; this result was used for risk analysis. RBC counts at the highest dose had to be deleted to make the BMD approach work; however, the subcommittee advised against using the RBC databecause the results were not considered statistically orbiolog- ically significant.

140 0.35 0.3 0~00.25 0.2 ·°~0. 1 5 0.1 0.05 o Spacecraft Water Exposure Guidelines Weibull Model with 0.95 Confidence Level Weibull ~ .-~ ........ BMD,L . ............. 0 200 400 2 . ~-~ ......... ABED "- . 600 800 ..................... . Dose 1000 1200 1400 FIGURE 4-3 BMD analysis ofthe chronic renal inflammation data in Table 4-4. Source: Data from Kluwe et al. 1982. Neoplastic Lesions The chronic study by Kluwe et al. (1982) demonstrated an increased incidence of liver tumors in all exposed groups (Table 4-6~. The increases were significant when compared either pair-wise with controls or by a trend test. It is interesting that, in male rats, ingestion of DEHP caused a signif~- cant reduction in the incidence of neoplasms of the pituitary, thyroid, and testes. For example, the incidences of testicular neoplasms in control, low-dose, and high-dose groups were 47/49,42/44, and 11/4S, respectively (Kluwe et al. 1982~. In a more recent study, the relationship between PP, cell proliferation, and hepatocarcinogenesis was explored. For 104 wk. male and female F- 344 rats were fed a diet containing DEHP at 0, 100, 500,2,500, or 12,500 ppm, and male and female B6C3F~ mice were fed a diet containing DEHP at 0, 100, 500, 1,500, or 6,000 ppm (David et al. 1999~. In a satellite re- covery group, the highest-dose animals had treatment discontinued at 79 wk into the study and were evaluated at 104 wk. The total incidences of

Di(2-ethylhexyl) Phthalate TABLE 4-5 Toxic Effects of DEHP in F-344 Rats 141 Dose (mg/kg/d) o Incidence of A. . spermlogenesls 37/64 BMD Analysis on RBC (106/~1) Aspermiogenesis BMDo~ = 3.4 mg/kg/d BMDLo~ = 2.2 mg/kg/d 5.8 34/50 8.38 28.9 43/55a 8.22 Quantal-linear model 146.6 48/64 7.49 789 62/64 (8.01) aIncidence considered statistically significant alp < 0.05. Abbreviations: BMD, benchmark dose; BMDo~, benchmark dose corresponding to a 1% risk; BMDLo~, lower confidence limit on the BMD corresponding to a 1% risk; RBC, red blood cells. Source: Data from David et al. 2000. hepatocellular neoplasms are given in Table 4-7. Assessment of an enzyme marker of PP at an earlier time point in the study indicates that there was a minimum increase necessary for a significant increase in tumors. The au- thors concluded that a >70°/O increase is necessary for rats, and a >300°/O increase is necessary for mice. That cessation of treatment at 79 wk re- sulted in a reduction in the incidence of tumors at 104 wk suggests that DEHP has a promoting effect on cells that have been transformed. A dose of 500 ppm (29-36 mg/kg/~) for 104 wk did not increase liver weights, induce PP in the liver, or cause the incidence of liver tumors to increase in rats. For mice, dietary consumption of 100 ppm (19-24 mg/kg/~) did not cause increased liver weights, induce PP, or induce an increased incidence of liver tumors. The authors concluded that a threshold approach to calcu- lating the no-significant-risk dose for human exposure is appropriate (David et al. 1999~. Liver tumors induced by peroxisome proliferators such as DEHP have limited application to human toxicity for two fundamental reasons. First, the absorption of DEHP from the gut in humans is much lower than the absorbtion from the gut in rodents at the high concentrations necessary to induce tumors. This results in a much lower blood level of the proximate metabolite MEHP in humans, and much less of that metabolite is delivered to the target tissue. Second, the response of hepatocytes to a given level of MEHP is less likely to result in PP (and subsequent tumors) in humans because the level of PPARa is much lower than it is in rat hepatocytes (see

142 0.9 0.8 ,a) ~0.7 `, 0.6 IL Spacecraft Water Exposure Guidelines Quantal-Linear Model with 0.95 Confidence Level 0.5 0.4 , ..... . . . . . . . . . | Quantal Lin - ~ ~ - . - . ~ - . .......... . - ~ - -. .. At. .................. ~ . ................. . . .. ~ ...... . . - - . . . . - - ~ ..... ...... . . - - . . . . . . ...... 8MDL-.BMD ..................................................................................... . 0 100 200 300 400 Dose 500 600 700 800 FIGURE 4-4 BMD analysis of aspermiogenesis data given in Table 4-5. Source: Data from David et al. 2000. Figure 4-2~. The importance of PPARa and the role of PP in mediating the DEHP tumor response were demonstrated in PPARa knockout mice ex- posed to DEHP. None of the mice showed hepatotoxicity or tumor (Ward et al. 1998~. On the basis of such mechanistic information, IARC recently changed its classification of DEHP from "possibly carcinogenic to humans" to "not classifiable as to carcinogenicity in humans" (NIP 2000~. Genotoxicity DEHP and many of its metabolites have been tested for genotoxicity in mammalian-cell test systems, in bacterial mutagenicity test systems, and in non-mammalian eucaryotic systems. The results of that testing have been summarized, and the discussion below will not attempt to repeat that sum- mary (WHO 1992~. The goal of this section is to select representative reports in each of the areas of testing and place the overall results into the context of our understanding of the way DEHP induces liver tumors in rodents. The vast majority of bacterial mutagenicity studies have been negative for DEHP and its metabolites, MEHP, 2-ethy~hexanol, and phthalic acid.

Di(2-ethylhexyl) Phthalate 143 TABLE 4-6 Incidence of Liver Neoplasms in Rodents Ingesting DEHPa Species Gender Control Low Doseb High Doseb Rat Male 3/50 6/49 12/49 Female 0/50 6/49 13/50 Mouse Male 14/50 25/48 29/50 Female 1/50 12/50 18/50 aExposure duration was 103 wk. bThe doses vary eith species and gender. For actual doses, see Table 4-8. Source: Data from Kluwe et al. 1982. The exception is a report by Tomita et al. (1982) in which S. typhimurium TA100 strain showed revertants after DEHP orMEHP exposure and MEHP was positive in B. subti1/tis and E. co1/ti mutagenicity assays. DEHP has been evaluated in several species of fungi, and there is only one report of a posi- tive finding (Ashby et al.1985~. Similarly, mammalian cells evaluated for mutagenicity showed positive results in one out of 10 investigations using mouse lymphoma cells (Ashby et al. 1985~. Marginally positive results were found in collaborative Drosophi1/la mutagenicity testing, but only at specific intermediate concentrations (Ashby et al. 1985~. Generally, methods used to detect DNA interactions, including un- scheduled DNA synthesis (UDS) and single-strand breaks, have given negative results for DEHP in mammalian systems (WHO 1992~. In vitro results using hepatocytes, CHO cells, and HeLa cells have proven negative for DNA interactions except for an isolated example of UDS, which did not fit a dose-response relationship. The consensus in that case was that DEHP does not cause UDS (Ashby et al. 1985~. In viva studies of rat liver from animals exposed to oral doses of DEHP have likewise been negative. Even under conditions of prolonged administration and induction of peroxisomes,indices of DNA interactions have been negative (Butterworth et al.1984; Kornburst et al.1984~. One interesting exception is the finding that S-hydroxydeoxyguanosine was increased in hepatocytes from rats given DEHP at 600 mg/kg/d for 2 wk (Takagi et al. 1990~. Chromosomal changes, such as sister chromatic exchanges, caused by DEHP have been found to be absent in CHO cells. MEHP induced sister chromatic exchanges in three cell lines, but generally at cytotoxic doses (WHO 1992~. This cytotoxicity may be due to action of MEHP on cell membranes. DEHP can cause aneuploidy in mammalian cells and in fungi in vitro (WHO 1992~.

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Di(2-ethylhexyl) Phthalate 145 A number of in viva genotoxicity investigations of DEHP have shown negative results. Those include a chromosomal study of bone marrow cells from rats dosed with DEHP for 5 4, a micronucleus assay on blood cells from mice treated for 5 4, and dominant lethal studies in mice (WHO 1992~. In most of these tests, metabolites MEHP and 2-ethy~hexano! were also found to be negative. DEHP can stimulate DNA synthesis in rat hepatocytes immediately after dosing, and it has been postulated that this increased cell division rate might increase the probability that a DNA error caused by an endogenous mutagen will become an irreversible mutation (Smith-Oliver and Butterworth 1987~. Cells are known to be more vulnerable to irreversible alterations in their DNA when undergoing rapid division (Marx 1990~. Reproductive Toxicity Certainly the hepatotoxicity of DEHP is its most-studied toxicity as- pect; however, the effects of DEHP on the testes have also been thoroughly studied. Some aspects of the testicular toxicity have been discussed above as part ofthe results from short-term and subchronic rodent bioassays (Poon et al. 1997~. Depending on which study is used and how the end points are assessed, the susceptibility of rat livers to PP and rat testes to atrophy by DEHP are roughly comparable in adult animals. However, young male rats are much more susceptible to DEHP testicular injury than are mature rats. For example, in 4-wk-old rats given a 1 0-d oral treatment of DEHP at 2,800 mglkg/d, testes, seminal vesicles, end prostates weighed approximately 50°/O less than the same organs in control rats (Gray and Gangolli 1986~. When 1 5-wk-old rats were given the same treatment, the reductions in the weights ofthe three organs were all less than 10%, end the weights were not statisti- cally different from the weights of organs from control rats. MEHP, which is formed when DEHP is absorbed from the gut, is thought to be the metabolite responsible for testicular injury. If MEHP is given by intravenous infusion, the age-related differences in testicular toxic- ity seen in oral administration studies are not apparent (Sjoberg et al. 1986~. The increased toxicity of orally administered DEHP to young male rats may simply be because of their increased ability to absorb DEHP and convert it to MEHP in the process. Furthermore, cocultured Sertoli and germ cells are much more susceptible to germ-cell detachment by MEHP than by metabo- lites V, IV, and IX of DEHP (Gray and Gangolli 1986~. This in vitro, co- culture assay correlates well with the in viva testicular toxicity of a series

146 Spacecraft Water Exposure Guidelines of phthalate monoesters (Gray and Gangolli 1986~. The extremely limited ability of cultured Sertoli cells to metabolize MEHP to other metabolites also supports the hypothesis that MEHP is the cause of testicular injury (Albro et al. 1989~. Testicular atrophy caused by DEHP varies from one species to another. After reviewing the literature on DEHP-induced testicular atrophy, it was concluded that rats and guinea pigs are the most susceptible to testicular damage, mice are intermediate in susceptibility, and marmosets and ham- sters are least susceptible (WHO 1992~. There has been much less study devoted to female reproductive toxicity then to male reproductive toxicity. The reproductive toxicity of DEHP was assessed in the NTP Fertility Assessment by Continuous Breeding protocol. Male and female CD-1 mice were fed chow containing DEHP at PRO, 0.01%, 0.1%, and 0.3°/O for a 7-d premating period and a 98-d cohabitation period (Meinick et al. 1987~. There was complete suppression of fertility at 0.3°/O and significant reduction at 0.1%. For example, live pups per litter was 10.6 in controls and 5.2 in the 0.01% group. In crossover matings between 0.3°/O mice and 0°/O mice, there was a reduced number of live pups per litter whether the DEHP effects were mediated through males or fe- males. DEHP was a reproductive toxicant in both male and female CD-1 mice (Meinick et al. 1987~. Weights of both male and female reproductive organs were decreased in animals fed 0.3°/O, but organs from animals ex- posed to lower concentrations of DEHP were apparently not evaluated. There were no apparent effects on fertility in the group fed DEHP at 0.01% for 105 4, which, using Table 5 from Ty! et al. (1988), NASA estimates to be approximately 18 mg/kg/~. DEHP was one of eight commercial phthalate esters tested in a study comparing the results of in vitro and in vivo assays for estrogenic activity. The in vitro assay involved competitive ligand binding to the estrogen receptor, whereas the in viva assay involved uterotrophic and vaginal cornification assays on ovariectomized SD rats. DEHP was nonresponsive in all assays (Zacharewski et al. 1998~. In contrast, Berman and Laskey (1993) have shown that DEHP alters the release of progesterone, testoster- one, and estradio! in the culture medium from minced, cultured whole rat ovaries. Ovaries were removed from rats that had been given DEHP at gavage doses of 0 mg/kg/d or 1,500 mg/kg/d for 10 d and were in specific stages of their estrus cycle. The ovaries were cultured for 1 h, and the hor- mones were assayed from decanted culture medium. The results showed that DEHP altered steroid profiles so that proestrus appeared to be delayed. The mechanism responsible for DEHP effects on ovarian granulosa cells might be related to the mechanism causing effects on testicular Sertoli

Di(2-ethylhexyl) Phthalate 147 cells. MEHP, the presumptive active metabolite of DEHP, inhibits FSH- stimulated cAMP accumulation in both Sertoli cells and in granulosa cells. The endocrine and paracine support granulosa cells provide to the develop- ing ovum is analogous to the role Sertoli cells play for developing sperma- tozoa. When MEHP was added at 100 EM to cultured rat granulosa cells, the cAMP produced by FSH stimulation was 40°/0 less than the amount produced by cultured cells without MEHP treatment (Treinen et al. 1990~. In addition, the production of progesterone by granulosa cells cultured for 48 h was reduced 30°/0 by treatment with MEHP at onlylO micromolar (~M). Developmental Toxicity The developmental toxicity of DEHP has been studied in several spe- cies and by various routes of administration; however, there are few studies involving the oral exposures. Because absorption in the gut has a major effect on the metabolism of DEHP, the discussion will be confined to oral studies of developmental toxicity. The developmental toxicity of DEHP has been thoroughly evaluated in F-344 rats and CD-1 mice by administration in food over gestational days 0-20 (rats) and 0-17 (mice). The food concentrations were as follows: 0°/0, 0.5%, 1.0%, 1.5%, and 2.0% in rats; 0°/0, 0.025%, 0.05%, 0.10%, and 0.15% in mice (Ty! et al. 1988~. In rats, the three highest doses showed maternal toxicity and reduced fetal body weight per litter. The number and percentages of malformed fetuses per litter were not affected by DEHP treatment in any group. In contrast, DEHP caused increased incidences of fetal malformations in mice at the two highest doses, which were also toxic to the dams. However, at 0.05% there were increased incidences of malfor- mations in the absence of maternal toxicity. The NOAELs for embryo-fetal toxicity from this study were 0.025% for mice and 0.5% for rats (Ty! et al. 1988~. The NOAEL for developmental effects in CD-1 mice is equivalent to 44 mg/kg/d, which is well above the NOAEL for reproductive effects in the same strain (Meinick et al. 1987~. An NTP panel that reviewed the reproductive and developmental toxic- ity of DEHP reached a conclusion on the possible health effects in humans at current ambient exposure levels. For the general adult population, the panel expressed "minimal concern that ambient human exposures adversely affect adult human reproduction. This level of concern is not appreciably altered for adults medically exposed to DEHP or MEHP" (NTP 2000~. The same panel expressed "concern that tDEHP] exposure may adversely affect

148 Spacecraft Water Exposure Guidelines male reproductive tract development in healthy infants and toddlers, and that ambient oral exposures to pregnant or lactating women may adversely affect the development oftheir offspring." There is a large difference in the doses that are toxic to laboratory animals and those that are encountered by the general human population; however, the unique exposures to DEHP associated with medical devices need further evaluation (NTP 2000~. Spaceflight Effects DEHP induces a number oftoxic effects; however, except for effects on hematologic parameters, spaceflight-induced physiologic changes and the toxic effects of DEHP seem independent. One investigator reported a 9°/0 average decrease in RBC count in male rats ingesting DEHP at 5,000 ppm for 13 wk (Poon et al. 1997~. Synergistic Effects There are no known chemicals present in spacecraft water that would be expected to increase the toxicity of DEHP. One would expect many of the phthalate esters to be additive in their toxic effects. Loss of red cell mass encountered during spaceflight must be considered when risks from hematologic end points are examined. LIMITS SET BY OTHER ORGANIZATIONS Table 4-9 provides a list of the current standards for DEHP. There is considerable range in these values, and that reflects the rapidly expanding database on DEHP toxicity and the evolving understanding of the mecha- nisms of DEHP toxicity. Rationales Drinking Water Standard from the National Research Council When setting the acceptable daily intake (ADI) value shown in Table 4-9, the National Research Council (NRC) committee noted that DEHP can

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154 Spacecraft Water Exposure Guidelines cause the following toxic effects: liver function changes, reproductive toxic- ity, developmental effects, and cancer (NRC 1986~. The committee started with data on dialysis patients showing that 150 mg/wk (estimated amount) caused no detectable changes in the liver except that after 1 y there were significantly higher numbers of peroxisomes (Ganning et al. 1984~. This finding has been rejected as a starting point for risk assessment by others because only one patient showed the increase in liver peroxisomes, and the exposure may have included other peroxisome prolif-erators (ATSDR 1993~. For a 70-kg person, this estimated dose equates to an exposure of 0.3 mg/kg/~. The committee applied a safety factor of 10 (presumably converting aLOAEL tiowest-observed-adverse-effect level] into aNOAEL) and an interindividual variability factor of 10 to reach an ADI of 0.003 mg/kg/~. The committee determined that the NOEL for liver function changes was 50 ppm (3.3 mglkg/~) on the basis of a 7-d feeding study of rats (Morton 1979~. To this NOEL the committee applied a safety factor of 1,000, including a factor of 10 to compensate for the shortness ofthe study, to reach a value of 0.003 mg/kg/~. The committee also looked at a chronic rat study by Ganning et al. ~ 1984) and a rat teratogenicity study by Ruddick et al. (1981), but those led to higher ADIs than did the liver effects end point. The subcommittee noted that the NTP carcinogenesis bioassay pro- duced a 1 o-6 risk (upper 95°/O confidences at a lifetime dose of 0.03 mg/kg/d when the generalized multistage model for carcinogenesis was used. This model probably overestimates the cancer risk at low doses, because DEHP seems to be inactive on the liver at lower oral doses (i.e., there is a threshold for cancer). EPA's Maximum Contaminant Level It has proven difficult to trace the origin of the EPA's maximum con- taminant level (MCL) on DEHP. The appropriate pages of the Feclera1t Register (EPA 1992a) contain only a rationale for an MCL goal (MCLG) of zero because DEHP is an animal carcinogen. In a document entitled "Final Drinking Water Criteria Document for Phthalic Acid Esters" (EPA 1992b), an interim value was indicated on the basis of the most sensitive slope of the tumor incidence from the data of Kluwe et al. (1982~. Using the upper bound of the cancer risk from ingestion of 2 L of water per day for 70 y, with assumption of linearity, EPA estimated that the risk was 10-4 at 0.300 ~g/L, 10-5 at 0.030 ~g/L, and 10-6 at 0.003 vigil.

Di(2-ethylhexyl) Phthalate TABLE 4-9 Standards Set by Government Organizations 155 Value Water Equivalent Organization Standard (mg/kg/d) (mg/L)a Reference NRC ADI 0.003 0.1 NRC 1986 EPA MCL 0.006 EPA 1998 EPA MCLG 0 0 EPA 1992a EPA RfD (oral) 0.02 0.7 IRIS 1993 California 0.004 ATSDR 1993 Kansas 4.2 Maine 1.2 Massachusetts 0.01 Minnesota 0.04 Rhode Island 1.2 Assumes a 70-kg person consuming 2 L of water per day and no other source of DEHP. Abbreviations: ADI, acceptable daily intake; EPA, U.S. Environmental Protection Agency; MCL, maximum contaminant level; MCLG, maximum contaminant level goal; NRC, National Research Council; RfD, reference dose. EPA's Reference Dose The reference dose (R]D) for DEHP was based on the 1-y study of guinea pigs given DEHP in food at 0.13% or 0.04°/O (equivalent to 64 ma/ kg/d and 19 mg/kg/d, respectively) (Carpenter et al. 1953~. Guinea pigs appeared to be a more sensitive species than the Sherman rats exposed in a second study (Carpenter et al. 1953~. From the guinea pig study, 19 mg/kg/d was determined to be a LOAEL for increases in liver weights in females. Factors of 10 were applied for interspecies variation, inter- individual variation, and to compensate for the fact that the exposure was not lifetime. Thus, an RfD of 0.02 mg/kg/d was established. This is equiv- alent to a water concentration of 0.7 mg/L if all DEHP came from drinking 2 L of water per day. The RfD is not consistent with an MCL of 0.006 mg/L. Comparison to SWEGs for DEHP The proposed SWEG for 1,000 ~ of ingestion is 20 mg/L in water used

156 Spacecraft Water Exposure Guidelines for drinking and for reconstituting food (Table 4- 10~. This was based on BMD) analysis of the aspermiogenesis data from David et al. (2000~. The 1,000-d SWEG is well above the water-equivalent chronic RID of 0.7 mg/L. The 1,000-d SWEG is not consistent with an MCL of 0.006 mg/L. NTP's Human Reproductive Risk Assessment An expert review of DEHP health risks provides an important gauge for comparison with the SWEGs presented in this document (NTP 2000~. The expert panel expressed minimal concern for the health of general adult population, which they believe is exposed to ambient DEHP at up to 0.03 mg/kg/d (2.1 mg/d for a 70-kg person). Furthermore, the "minimal con- cern" was not appreciably altered for adults medically exposed to DEHP. Human medical procedures can result in exposures at up to 1,000 mg/y (2.7 mg/~) (NTP 2000, Table 6~. Combining the general population exposure and the exposure from medical procedures gives a dose of 4.8 mg/d, which the panel also described as a "minimal concern" (NTP 2000~. For persons ingesting 2.8 L/d of DEHP-contaminated water, the concentration in the water could be as high as 1.7 mg/L without exceeding the minimal concern criterion expressed by the panel. This assumes no other significant sources of DEHP exposure. Considering that the medical procedures mentioned in the NTP report could last several years and the normal ingestion period is for an adult lifetime, the 100-d SWEG of 30 mg/L and the 1,000-d SWEG of 10 mg/L are well above the levels discussed by the expert panel, which also considered the equivalent of roughly 2 mg/L ingestion to be of minimal concern (NTP 2000~. RATIONALE Setting specific human exposure limits for DEHP is difficult because although there is a plethora of data in rodents, sufficient mechanistic data suggest that significant interspecies differences affect the extrapolation of risk to human health. Recent data on the incidence of liver tumors in rats and mice suggest that a linear model ofthe log-dose vs risk, which was used by EPA (1992), is not applicable. The relevance of river tumors induced by DEHP to human risk assessment has been critically questioned (Doull et al. 1999~. In addition, the limited data available on primates suggest that the rodent is a poor model for hepatic changes caused by DEHP and is perhaps

Di(2-ethylhexyl) Phthalate TABLE 4-10 Spacecraft Water Exposure Guidelines for DEHP 157 Duration Concentration (mg/L) Target Toxicity 1 d 1,800 Gastric upset 10 d 1,300 Testicular injury 100 d 30 Hematotoxicity, testicular injury 1,000 d 20 Testicular injury also a poor model for testicular effects. Confounding all this is the variation in the target organ sensitivity of different animal models to DEHP-induced toxicity. It could be that the variation in sensitivity is due more to the indi- vidual investigator's determination to look for changes in specific organs (liver or testes), rather than a true difference in each model's susceptibility to DEHP. The approach below assumes that the rodent is an acceptable model for hematologic and testicular changes, but not for liver and thyroid changes. The rodent thyroid is thought to be a poor model for the human thyroid (Me CIain 1992~. A risk assessment will also be done for chronic renal inflammation seen in male mice (Kluwe et al. 1982~; however, renal toxicity seen in male rats will not be used for risk assessment. Guidelines promulgated by the National Research Council (2000) have been followed to select pertinent data and to analyze it for human health risks. Ingestion for 1 d There is one report that ingestion by a human of 10 g of DEHP caused gastric distress, but ingestion of 5 g was without apparent effect (Shaffer et al. 1945~. The 5-g dose can be considered a NOAEL. The fact that only a single person was involved is compensated for by the bolus nature of the dose. The same dose administered in water over a day is much less likely to cause gastric distress than a single large dose. The 1-d AC (gastric dis- tress) was calculated as follows: 1-d AC = 5,000 mg 2.8 L/d = 1,800 mg/L. This value is far above any that could be derived from adverse effects on animals.

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

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.

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.

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 167 Nikonorow, M., H. Mazur, and H. Piekacz. 1973. Effect of orally administered plasticizers and polyvinyl chloride stabilizers in the rat. Toxicol. Appl. Pharmacol. 26:253-9. NRC (National Research Council). 1986. Pp. 338-359 in Drinking Water and Health, Vol. 6. Washington, DC: National Academy Press. NRC (National Research Council). 2000. Methods for Developing Spacecraft Water Exposure Guidelines. Washington, DC: National Academy Press. NTP (National Toxicology Program).2000. NTP-CERHR Expert Panel Report on Di(2-ethylhexyl) Phthalate. Center for the Evaluation of Risks to Human Re- production, Alexandria, VA. Oishi, S., and K. Hiraga.1980. Testicular atrophy induced by phthalic acid esters: Effect on testosterone and Zn concentrations. Toxicol. Appl. Pharmacol. 53:35-41. Parkinson, A. 1996. Biotransformation of xenobiotics. Ch. 6 in Casarett and Doull's Toxicology, 5th Ed., C.D. Klaassen, M.O. Amdur, and J. Doull, eds. New York: McGraw-Hill. Parmar, D., S.P. Srivastava, and P.K. Seth. 1988. Effect of di(2-ethylhexyl) phthalate on hepatic mixed function oxidases in different animal species. Toxicol. Lett. 40:209-217. Peters, J.M., M.W. Taubeneck, C.L. Keen, F.L. Gonzalez.1997. Di(2-ethylhexyl) phthalate induces a functional zinc deficiency during pregnancy and teratogenesis that is independent of peroxisome proliferator-activated recep- tor-alpha. Teratology 56:311-316. Pierre, L.M., J.R. Schultz, R.L. Sauer et al. 1999. Chemical analysis of potable water and humidity condensate: Phase one final results and lessons learned. SAE-ICES Paper 1999-01 -2028. Warrendale, PA: Society of Automotive Engineers. Poon, R., P. Lecavalier, R. Mueller, V.E. Valli, B.G. Proctor, and I. Chu. 1997. Subchronic oral toxicity of di-n-octyl phthalate and di(2-ethylhexyl) phthalate in the rat. Food Chem. Toxicol.35:225-39. Pugh Jr., G., J.S. Isenberg, L.M. Kamendulis et al. 2000. Effects of di-isononyl phthalate, di-2-ethylhexyl phthalate, and clofibrate in cynomolgus monkeys. Toxicol. Sci. 56: 181 -8. Rhodes, C., T.C. Orton, I.S. Pratt, P.L. Batten, H. Bratt, S.J. Jackson, and C.R. Elcombe. 1986. Comparative pharmacokinetics and subacute toxicity of di(2-ethylhexyl) phthalate in rats and marmosets: Extrapolation of effects in rodents to man. Environ. Health Perspect. 65:299-308. Rothenbacher, K.P., R. Kimmel, S. Hildebrand, F.W. Schmahl, and P.C. Dartsch. 1998. Nephrotoxic effects of di(2-ethylhexyl) phthalate hydrolysis products on cultured kidney cells. Hum. Exp. Toxicol. 17:336-342. Ruddick, J.A., D.C. Villeneuve, I. Chu, E. Nestmann, and D. Miles. 1981. An assessment of the teratogenicity in the rat and the mutagenicity in Salmonella of mono-2-ethylhexylphthalate . Bull. Environ. Contam. Toxicol. 27:181-6. Schmid, P., and C. Schlatter.1985. Excretion and metabolism of di(2-ethyl-hexyl) phthalate in man. Xenobiotica 15 :251 -6.

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To protect space crews from contaminants in potable and hygiene water, NASA requested that the National Research Council (NRC) provide guidance on how to develop water exposure guidelines and subsequently review NASA’s development of exposure guidelines for specific chemicals.

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