3
Toxicity Assessment

The toxicity of some phthalates1 in animals has been known for decades, although few data are available on the toxicity of these chemicals in humans. Several human studies have reported associations of exposure of some phthalates with adverse reproductive outcomes and developmental effects similar to those in the rat. However, for the purposes of this chapter, reliance will be placed on the data obtained from animal studies. Species differences (mainly quantitative) in response will be referred to in the text with citation of human data when available. As noted in Chapter 1, the outcomes chosen for emphasis in this report are effects on the development of the male reproductive system. The reproductive developmental processes in rats are analogous to those in humans, and disruption of those processes in rats should be representative of what would occur in humans if the same processes are disrupted (reviewed in Foster 2005).

This chapter first discusses male sexual differentiation in mammals. That information serves merely to provide context for the discussion that follows; references to several reviews are provided for readers who would like further information. The results of early teratology studies are mentioned, and the reproductive effects of phthalates are then discussed. Aspects of the phthalate syndrome—its relationship to the hypothesized human testicular dysgenesis syndrome, structure-activity relationships, and mechanisms of action—are described next. Agents that produce effects on reproductive development similar to those of phthalates are noted. Although cancer is not the focus of this report, carcinogenic effects were the focus of much research on phthalates in past years, so the committee felt that the chapter would not be complete without a brief discussion of them. This chapter provides the context for the discussion on cumulative risk assessment and is not meant to be a comprehensive toxicity assessment or an exhaustive review of phthalate toxicity.

1

As stated in Chapter 1, the term phthalates used in this report refers to diesters of 1,2-benzenedicarboxylic acid, the o-phthalates.



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3 Toxicity Assessment The toxicity of some phthalates1 in animals has been known for decades, although few data are available on the toxicity of these chemicals in humans. Several human studies have reported associations of exposure of some phthal- ates with adverse reproductive outcomes and developmental effects similar to those in the rat. However, for the purposes of this chapter, reliance will be placed on the data obtained from animal studies. Species differences (mainly quantitative) in response will be referred to in the text with citation of human data when available. As noted in Chapter 1, the outcomes chosen for emphasis in this report are effects on the development of the male reproductive system. The reproductive developmental processes in rats are analogous to those in hu- mans, and disruption of those processes in rats should be representative of what would occur in humans if the same processes are disrupted (reviewed in Foster 2005). This chapter first discusses male sexual differentiation in mammals. That information serves merely to provide context for the discussion that follows; references to several reviews are provided for readers who would like further information. The results of early teratology studies are mentioned, and the re- productive effects of phthalates are then discussed. Aspects of the phthalate syn- drome—its relationship to the hypothesized human testicular dysgenesis syn- drome, structure-activity relationships, and mechanisms of action—are described next. Agents that produce effects on reproductive development similar to those of phthalates are noted. Although cancer is not the focus of this report, carcinogenic effects were the focus of much research on phthalates in past years, so the committee felt that the chapter would not be complete without a brief discussion of them. This chapter provides the context for the discussion on cu- mulative risk assessment and is not meant to be a comprehensive toxicity as- sessment or an exhaustive review of phthalate toxicity. 1 As stated in Chapter 1, the term phthalates used in this report refers to diesters of 1,2- benzenedicarboxylic acid, the o-phthalates. 39

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40 Phthalates and Cumulative Risk Assessment: The Tasks Ahead MALE SEXUAL DIFFERENTIATION IN MAMMALS Sexual differentiation in males follows complex interconnected pathways during embryo and fetal development that have been reviewed extensively else- where (see, for example, Capel 2000; Hughes 2001; Tilmann and Capel 2002; Brennan and Capel 2004). Critical to the development of male mammals is the development of the testis in embryonic life from a bipotential gonad (a tissue that could develop into a testis or an ovary). The “selection” is genetically controlled in most mammals by a gene on the Y chromosome. The sex-determining gene (sry in mice and SRY in humans) acts as a switch to control multiple downstream pathways that lead to the male phenotype. Male differentiation after gonad determination is exclusively hormone-dependent and requires the presence at the correct time and tissue location of specific concentrations of fetal testis hormones—Mullerian inhibiting substance (MIS), insulin-like factors, and androgens. Although a fe- male phenotype is produced independently of the presence of an ovary, the male phenotype depends greatly on development of the testis. Under the influence of hormones and cell products from the early testis, the Mullerian duct regresses, and the mesonephric duct (or Wolffian duct) gives rise to the epididymis and vas deferens. In the absence of MIS and testosterone, the Mullerian ductal system develops further into the oviduct, uterus, and upper vagina, and the Wolffian duct system regresses. Those early events occur before the establishment of a hypothalamic-pituitary-gonadal axis and depend on local control and production of hormones (that is, the process is gonadotropin-independent). Normal devel- opment and differentiation of the prostate from the urogenital sinus and of the external genitalia from the genital tubercle are also under androgen control. More recent studies of conditional knockout mice that have alterations of the luteinizing-hormone receptor have shown normal differentiation of the genitalia, although they are significantly smaller. Testis descent (see Figure 3-1) appears to require androgens and the hor- mone insulin-like factor 3 (insl3; Adham et al. 2000) to proceed normally. The testis in early fetal life is near the kidney and attached to the abdominal wall by the cranial suspensory ligament (CSL) and gubernaculum. The gubernaculum contracts, thickens, and develops a bulbous outgrowth; this results in the loca- tion of the testis in the lower abdomen (transabdominal descent). The CSL re- gresses through an androgen-dependent process. In the female, the CSL is re- tained with a thin gubernaculum to maintain ovarian position. Descent of the testes through the inguinal ring into the scrotum (inguinoscrotal descent) is un- der androgen control. Because the majority of studies discussed below were conducted in rats, it is helpful to compare the rat and human developmental periods for male sexual differentiation (see Figure 3-2). Production of fetal testosterone occurs over a broader window in humans (gestation weeks 8-37) than in rats (gestation days [GD] 15-21). The critical period for sexual differentiation in humans is late in

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41 Toxicity Assessment deferens FIGURE 3-1 Stages of testicular descent. Testicular descent in scrotal mammals (such as humans and rats) can be conveniently divided into two phases. The first is the transab- dominal phase in which the cranial suspensory ligament (CSL) disappears, and the tes- tes—located near the kidneys—move into the lower abdomen. The first phase is under the control of the hormone, insulin-like factor 3. The second phase is the inguinoscrotal phase in which the gubernaculum (Gub) develops further, and the testes move through the body wall (inguinal ring) into the developing scrotum. The second phase is under the control of androgen. Source: Klonisch et al. 2004. Reprinted with permission; copyright 2004, Developmental Biology. the first trimester of pregnancy, and differentiation is essentially complete by 16 weeks (Hiort and Holterhus 2000). The critical period in rats occurs in later ges- tation, as indicated by the production of testosterone in the latter part of the ges- tational period, and some sexual development occurs postnatally in rats. For example, descent of the testis into the scrotum occurs in gestation weeks 27-35 in humans and in the third postnatal week in rats. Generally, the early postnatal period in rats corresponds to the third trimester in humans. Given the above discussion, it is clear that normal differentiation of the male phenotype has specific requirements for fetal testicular hormones, includ- ing androgens, and therefore can be particularly sensitive to the action of envi- ronmental agents that can alter the endocrine milieu of the fetal testis during critical periods of development.

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42 Phthalates and Cumulative Risk Assessment: The Tasks Ahead FIGURE 3-2 Comparison of periods of male reproductive development in rat and hu- man. The shaded area under the curve indicates the changing testosterone concentrations in the fetal testis. Gestational weeks (humans) were measured from time of last menstrual period, so birth occurs at 40 weeks in this diagram. S.V., seminal vesicles. Source: Welsh et al. 2008. Reprinted with permission; copyright 2008, Clinical Investigation. EARLY TERATOLOGY FINDINGS The early studies that examined the potential for phthalate exposure to cause adverse effects on fetal development were standard teratology studies, in which pregnant animals were exposed during GD 6-15, and the offspring were examined just before birth, when the reproductive tract is immature. Generally, the concentration of a phthalate required to cause developmental toxicity in those studies was relatively high, and maternal toxicity was typically observed (NTP 2000, 2003a,b,c,d,e,f, 2006). Typical malformations observed included neural-tube defects, cleft palate, and skeletal abnormalities. On the basis of the early data, the National Toxicology Program (NTP) and its Center for the Evaluation of Risks to Human Reproduction (CERHR) (NTP 2000, 2003a,b,c,d,e,f, 2006) concluded that there was clear evidence of adverse devel- opmental effects in animals for BBP, DBP, DEHP, and DIDP and some evi- dence for DINP but only limited evidence for DHP and DOP. However, as dis-

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43 Toxicity Assessment cussed further below, the design of the standard teratology study was shown to be inadequate for detecting the spectrum of male reproductive effects that have now been reported because of their failure to include exposure during critical gestational windows. REPRODUCTIVE EFFECTS The testis was identified as a target organ in some of the first toxicity stud- ies undertaken with phthalates (see, for example, Gray et al. 1977). Although the effects in young adult animals were seen only at high doses in rat studies, it was obvious that testicular lesions could be produced with relatively short-term dos- ing models. Those lesions were the most severe manifestations of testicular tox- icity in that there was complete tubular atrophy. Initial experiments also indi- cated that there was an age sensitivity: pubertal animals had effects at doses lower than those in the corresponding studies in adult animals. Investigations of structure-activity relationships in the pubertal-rat model showed that the ester side-chain length of linear-chain phthalates needed to be four to six carbon atoms to produce testicular toxicity (Foster et al. 1980). Di-n- pentyl phthalate was the most potent in producing testicular toxicity. Phthalates of one to three carbons (methyl, ethyl, and n-propyl) did not produce testicular toxicity when given at a dose equimolar with DBP at 2 g/kg-d. Similarly, linear- chain phthalates of seven or eight carbons did not produce adverse effects. DEHP, which has eight carbons and a branched structure, had activity more similar to that of di-n-hexyl phthalate than to its linear isomer di-n-octyl phthal- ate. Investigation of the isomers of DBP indicated that the esters needed to be in the ortho configuration in that equimolar doses of the n-butyl esters in the meta- and para- positions were without effect in the pubertal-rat model (Foster et al. 1981a). Other studies with butyl phthalates indicated that the iso and sec esters were equivalent to the n-butyl but that the tert ester was without effect at equi- molar doses (Foster et al. 1981b). Detailed morphologic examination of the phthalate-induced testicular le- sions in pubertal rats (Foster et al. 1982; Creasy et al. 1983) and adult rats (Creasy et al. 1987) indicated that the Sertoli cell was the initial testicular target and that loss of support of the germ cells resulted in their rapid sloughing into the seminiferous tubular lumen, which resulted in a spermatogenic stage-specific lesion in adult animals. The effects of the various n-alkyl phthalates could be modeled with in vitro systems of mixed Sertoli and germ cell cultures (Gray and Beamand 1984), which demonstrated the same structure-activity relationships as that described for in vivo testicular toxicity. The in vitro Sertoli cell culture sys- tems also provided some insight into a potential mechanism of action for the pubertal model; effects on responsiveness of follicle-stimulating hormone were noted (Lloyd and Foster 1988; Heindel and Chapin 1989). Other in vitro studies of developing Sertoli cells and gonocytes taken from neonatal animals indicated

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44 Phthalates and Cumulative Risk Assessment: The Tasks Ahead that these cells showed an even greater sensitivity to phthalates than did the cells derived from pubertal animals; the increased sensitivity could be reproduced in neonatal rat pups (Li et al. 1998, 2000; Li and Kim 2003). The number of known environmental agents that produce adverse testicu- lar responses in male humans is not large, and although there may be differences in sensitivity based on dose, all of them have been shown to induce effects in rodents, especially the rat. Accordingly, most of the studies of effects of phthal- ates on male reproduction have been conducted in rodents, primarily rats. Gray et al. (1982) evaluated species differences in the induction of testicular toxicity of DBP and DEHP in the rat, mouse, guinea pig, and hamster. They found that the rat was the most sensitive, the guinea pig was broadly equivalent, the mouse was much less sensitive, and the hamster was resistant. The differences in tes- ticular toxicity were suggested to be due largely to pharmacokinetic differences. The results for the guinea pig were in stark contrast with the species differences observed in effects on the induction of hepatic growth and peroxisome prolifera- tion. The lower male reproductive toxicity observed for the mouse was consis- tent with the results of other studies of reproductive toxicants. For example, a number of the classic human testicular toxicants, such as 1,2-dibromo-3- chloropropane (Oakberg and Cummings 1984) and gossypol (Hahn et al. 1981; Kalla et al. 1990), do not seem to produce infertility or testicular toxicity in the mouse, so the rat is more commonly used as a model for male reproductive- toxicity studies. Although that does not imply that all agents known to produce injury in the rat would cause toxicity in humans, it does suggest that the rat is generally a good model of human male reproductive toxicity. The ability of specific phthalates to alter reproductive development in utero was first demonstrated by a multigeneration study of DBP in the rat by NTP (NTP 1991; Wine et al. 1997), although the critical nature of the effects was not immediately recognized. In that study (see Table 3-1), the highest dose of DBP (1% in the diet) produced few functional effects on the parental genera- tion; all the exposed males were able to sire litters, but decreases in litter size were noted. However, only one of 20 F1 males produced a litter at the same 1% dietary dose, and this indicated the importance of exposure during early life (gestation and lactation and up to puberty) as a contributing factor. The number of underdeveloped epididymides in F1 males and the presence of other rare re- productive tract malformations recorded at low incidence were also noteworthy. The adverse effects on the development of the reproductive system were not reported in the standard prenatal developmental toxicity studies. It was later discovered that the exposure period in the standard studies (from implantation to the closure of the hard palate, GD 6-15 in the rat) does not cover the critical de- velopmental window, now known to be GD 15-17 for phthalates (Carruthers and Foster 2005; see Figure 3-3). The U.S. Environmental Protection Agency (EPA 1998) has since extended the dosing period in its guidelines for prenatal devel- opmental toxicity testing to GD 6-20 (in the rat) to avoid some of the pitfalls inherent when agents that might affect the development of the reproductive

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45 Toxicity Assessment TABLE 3-1 Reproductive and Developmental Effects of DBP in the National Toxicology Program Reproductive Assessment by Continuous Breeding Study (1991) Effect Noted F0 Generation F1 Generation Decrease in fertility – + Decrease in litter size (of fertile animals) + + Decrease in testes weight (and – + histopathology) Decrease in pup weight + + Decrease in sperm count – + Cryptorchidism Not applicable + Male reproductive tract malformations Not applicable + (epididymide, external genitalia) Female reproductive tract weight (and – – histopathology) Estrus cyclicity – – Note: +, positive response; –, negative response. system are evaluated. However, there has been no change in the time of exami- nation of fetuses (usually just before term—around GD 21 in rats), so diagnosis of reproductive tract malformations remains problematic. It was only when the DBP multigeneration study was followed up with a more defined exposure pe- riod (Mylchreest et al. 1998, 1999) that the increased sensitivity of the fetus to DBP was described (Mylchreest et al. 2000). THE PHTHALATE SYNDROME OF EFFECTS ON MALE REPRODUCTIVE DEVELOPMENT Since the recognition of the critical importance of exposure during GD 15- 17, many studies have been conducted to determine the full spectrum of effects that can result from exposure to phthalates in utero. Studies have shown that male rats exposed to biologically active phthalates in utero during the period of sexual differentiation exhibit a number of reproductive tract abnormalities, which may include underdeveloped or absent reproductive organs, malformed external genitalia (hypospadias), undescended testes (cryptorchidism), decreased anogenital distance, retained nipples, and decreased sperm production (Myl- chreest et al. 1998, 1999; Gray et al. 2000). Studies evaluating DBP found that the fetal testes of phthalate-exposed males are characterized by seminiferous cords that contain multinucleated gonocytes (Barlow and Foster 2003; Hutchi- son et al. 2008). Phthalate exposure also results in regions of Leydig cell hyper- plasia. Barlow et al. (2004) showed that a small percentage of male offspring

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46 Phthalates and Cumulative Risk Assessment: The Tasks Ahead * Cryptorchid 100 10 Testis Epididymis * Hypospadias 10 * * Cleft prepuce 80 Animals with Malformation (%) 11 9 Phthalate syndrome 60 * * * 10 10 7 40 * * * 7 8 6 * * 6 20 * 5 * 5 4 2 2 1 1 0 0 250 500 750 Dose of DBP (mg/kg-d) * P < 0.05 (Fisher's exact) FIGURE 3-3 Effect of DBP given over 3 days on reproductive tract malformations. Pregnant Sprague-Dawley rats were given DBP on GD 15-17, critical window for induc- tion of phthalate syndrome, at 0, 250, 500, or 750 mg/kg-d by gavage in corn oil (5 mL/kg-d). Reproductive tract malformations were assessed in male offspring at postnatal day 100. Litters (10-12) were evaluated in each dose group; numbers of litters responding are indicated above bars. Control animals exhibited only cryptorchidism. Only when exposure occurred over GD 15-17 was the full suite of reproductive tract malformations that make up the phthalate syndrome observed. Other short-term (2-d) dosing regimens over GD 15-20 will produce specific reproductive malformations but not the full suite of malformations (Carruthers and Foster 2005). exposed to DBP in utero also develop Leydig cell adenomas as early as the age of 3 months. As discussed above, younger rodents are more sensitive to the ad- verse testicular effects of phthalates than older rodents. Pubertal and prepubertal rodents are more sensitive to the adverse effects of phthalates on the testes than adults (Foster et al. 1980; Sjoberg et al. 1986, 1988), and the fetal testes respond to phthalate concentrations that would be without effect in pubertal or adult animals (Gray et al. 2000; Mylchreest et al. 2000; Lehmann et al. 2004). Thus, the pubertal and prepubertal rat is sensitive, but the prenatal period is the most sensitive time for the testicular effects of phthalates.

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47 Toxicity Assessment Testicular Dysgenesis Syndrome Human males exhibit a high incidence of reproductive disorders. Crypt- orchidism and hypospadias are the most common male birth defects. In the United States, cryptorchidism affects 2-4% of male newborns (Barthold and Gonzalez 2003), and hypospadias occur in about one of 250 male newborns (Paulozzi et al. 1997).2 The incidence of male germ-cell cancers is thought to be on the rise (Skakkebæk et al. 2001), and studies suggest that semen quality has been decreasing (Carlsen et al. 1992; Swan et al. 2000). Testicular germ-cell cancers arise from abnormal fetal germ cells (Rajpert-De Meyts et al. 1998; Rorth et al. 2000), and disorders of sperm production may also arise during ges- tation (Sharpe and Franks 2002). The above disorders are risk factors for each other and share other pregnancy-related risk factors (Skakkebæk et al. 2001). On the basis of those observations, it has been hypothesized that they comprise a “testicular dysgenesis syndrome,” which arises in fetal life during reproductive system development because of disruption of critical gene programming in the fetal testis by either genetic or environmental factors (Skakkebæk et al. 2001; Sharpe and Skakkebæk 2008). The actions of phthalates on the developing re- productive tract of male rats exhibit excellent concordance with the end points of concern in the human male population that make up the testicular dysgenesis syndrome (see Table 3-2). However, there are no human data that directly link phthalate exposure with the hypothesized syndrome. TABLE 3-2 Comparison of Human Male Reproductive Effects of Concern with Effects of in Utero Phthalate Exposure in Rats Human Reproductive Effects with a Effects of in Utero Phthalate Possible in Utero Origin Exposure in Rats Infertility √ Decreased sperm count √ Cryptorchidism √ Reproductive tract malformations √ Hypospadias √ Testicular tumorsa √a a Testicular tumors in rats are Leydig-cell-derived, not germ-cell-derived as in humans. 2 There is some uncertainty in the rates reported, which depend on diagnostic criteria and on the time at which evaluation is conducted. Some subtle changes are not always noted, and newborns have a different incidence of cryptorchidism from infants at 6 months. Moreover, prospective studies with defined diagnostic criteria tend to provide better information than studies using registry data.

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48 Phthalates and Cumulative Risk Assessment: The Tasks Ahead Structure-Activity Relationships As discussed above, high-dose acute oral exposure to various n-alkyl phthalates induced testicular toxicity in pubertal rats and revealed differences in activity based on chemical structure (Foster et al. 1980). The studies indicated that only phthalates with chain lengths of four to six carbon atoms were capable of inducing testicular damage; di-n-pentyl phthalate yielded the most severe response. DEHP had toxicity that more closely resembled that induced by n- hexyl phthalate rather than that induced by its isomer di-n-octyl phthalate, which was without testicular toxicity. That observation indicated that branching of the ester side chain was also important. A similar structure-activity relationship has been demonstrated after in utero exposure (Gray et al. 2000). Phthalates with chain lengths of four to six carbons (dibutyl, butylbenzyl, dipentyl, and diethyl- hexyl) reduced fetal testicular testosterone and impaired male reproductive de- velopment, whereas phthalates with shorter or longer side chains (dimethyl, di- ethyl, and dioctyl) did not have an effect on male reproductive development (see Table 3-3).3 The developmentally toxic phthalates are indistinguishable in their effects on global gene expression in the fetal testis (Liu et al. 2005). The com- mon targeting of specific fetal testis genes by a select group of phthalates indi- cates common molecular mechanisms of action. Mechanism of Action The primary target of phthalates after in utero exposure is the fetal testis. One of the earliest phthalate-related fetal effects observed in rats was distur- bance of fetal testicular Leydig cell function or development (Parks et al. 2000; Shultz et al. 2001; Mylchreest et al. 2002; Fisher et al. 2003). That disturbance results in large aggregates of fetal Leydig cells (at GD 21) in the developing testis. The morphologic changes were preceded by a decrease in fetal testicular production of the androgen testosterone, which reached only 10% of control concentrations in some animals (Shultz et al. 2001; Lehmann et al. 2004; How- deshell et al. 2008). Androgen insufficiency at critical times in male reproduc- tive system development results in the failure of the Wolffian duct system to develop normally into the vas deferens, epididymis, and seminal vesicles (Bar- low and Foster 2003). Lower testosterone concentrations also affect the dihydro- testosterone (DHT)-induced development of the prostate and external genitalia (testosterone is converted to DHT by 5α-reductase). DHT is also responsible for the normal apoptosis of nipple anlagen4 in males, which results in the lack of 3 Although DIBP is strictly considered a phthalate with a chain length of three carbons, it produced toxicity similar to that of DBP. 4 Anlagen is defined as a precursor tissue.

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49 Toxicity Assessment TABLE 3-3 Effect of in Utero Phthalate Exposure on Male Rat Reproductive Outcomes Lowest Observed- Effect Level Phthalate Doses Effect (mg/kg-d) Phthalate Syndrome (mg/kg-d) Observed Reference DMP 750 Gray et al. 2000 – – DEP 750 Gray et al. 2000 – – DBP 0.1, 1.0, 10, 50 Reduced Lehmann et al. + 30, 50, 100, testosterone 2004 500 DIBP 100, 300, 300 Reduced Howdeshell et + 600, 900 testosterone al. 2008 BBP 50, 250, 750 250 Reduced Tyl et al. 2004 + anogenital distance Di-n- 25, 50, 100, 100 Reduced Howdeshell et + pentyl 200, 300, testosterone al. 2008 600, 900 DEHP 0.09-0.12, 14-23 Reduced NTP 2004 + 0.47-0.78, reproductive 1.4-2.4, 4.8- organ weight 7.9, 14-23, 46-77, 392- 592, 543- 775 DCHP 18, 90, 457 90 Reduced Hoshino et al. + anogenital 2005 distance DINP 750 750 Nipple Gray et al. 2000 + retention nipple development, and for the growth of the perineum to produce the normal male anogenital distance (AGD), about twice that of the female (Imperato- McGinley et al. 1985, 1986). Thus, the observed changes in androgen-dependent developmental landmarks are consistent with the lowered fetal concentrations of testosterone. Separately from effects on testosterone synthesis, in utero phthalate expo- sure disrupts seminiferous cord formation and germ-cell development and leads to the appearance of large multinucleated germ cells in late gestation (Myl- chreest et al. 2002; Barlow and Foster 2003; Kleymenova et al. 2005). The mul- tinucleated germ cells disappear postnatally. Germ-cell maturation is delayed in phthalate-exposed fetal testes. Postnatally, there is a delay in the resumption of germ-cell mitosis, and germ-cell number and presumably sperm count are re- duced (Sharpe 2008).

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57 Toxicity Assessment A recent study in mice, however, suggests that DEHP-induced hepatocar- cinogenesis occurs in the absence of PPARα expression. Ito et al. (2007) ex- posed wild-type and PPARα-null-type mice to 0.01% and 0.05% DEHP in the diet. The wild-type mice showed no statistically significant differences in hepa- tocarcinogenesis. However, a significant trend for an increase in total hepatic tumors was observed at 0.05% DEHP in PPARα-null-type mice compared with control PPARα-null-type mice. Although PPARα-null-type mice exhibit a high background incidence of hepatocarcinogenesis (Howroyd et al. 2004), statistical comparisons were made within the same groups; therefore, that fact should not have affected the reported results. Thus, the results suggest that DEHP might cause hepatic cancer in rodents through a mechanism that is independent of PPARα, as has been suggested by others (see, for example, Takashima et al. 2008). There is a known difference between rodents and humans in the ability of PPARα ligands to cause changes in the liver, including increases in cell growth and peroxisome proliferation (Peters et al. 2005), and it has been suggested that the hepatocarcinogenic effects of DEHP and DINP are unlikely to occur in hu- mans (Klaunig et al. 2003). More recent evidence supports that idea: mice that express human PPARα in the absence of mouse PPARα are refractory to the hepatocarcinogenic effects of PPARα ligands (Morimura et al. 2006). The lack of a hepatocarcinogenic effect of PPARα ligands in the “humanized” mouse model appears to be due to a species-specific differential regulation of a microRNA that regulates c-myc, an oncogene that is thought to be involved in cell proliferation (Shah et al. 2007). The differential regulation of this mi- croRNA might also explain the lack of changes in hepatic markers of cell prolif- eration observed in nonhuman primates exposed to DEHP or DINP (Rhodes et al. 1986; Pugh et al. 2000). However, whether exposure to PPARα ligands, such as phthalates, causes hepatic cancer in humans is unclear; further research is needed to answer this question definitively (Peters et al. 2005). In addition to hepatic cancer, some phthalates can cause tumors in other cell types. For example, a “tumor triad”—liver tumors, testicular Leydig cell tumors, and pancreatic acinar-cell tumors—has been described for some PPARα ligands, such as DEHP (Klaunig et al. 2003). BBP causes hepatic cancer and pancreatic acinar-cell tumors but not Leydig cell tumors (NTP 1997). It has been postulated that pancreatic acinar-cell tumors and Leydig cell tumors may also be mediated by PPARα (Klaunig et al. 2003). There are known species differences in response to PPARα ligands in the liver that appear to be mediated by differen- tial changes in gene expression that lead to differences in c-myc expression, and similar differences in PPARα-mediated events suggest that humans might not be susceptible to the nonhepatic tumors. However, further work is necessary to establish those putative PPARα-dependent mechanisms in the testicular Leydig cell tumors and the pancreatic acinar-cell tumors because the current evidence supporting those mechanisms is not strong (Klaunig et al. 2003). Thus, the non- hepatic tumors reported to occur after phthalate exposure in animal models may be mediated through mechanisms that are independent of PPARα.

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58 Phthalates and Cumulative Risk Assessment: The Tasks Ahead CONCLUSIONS In undertaking an examination of agents that produce a syndrome of de- velopmental response, such as the phthalate syndrome, it is normal to observe an increase in the appearance, severity, or frequency of the different malformations as the dose administered to the pregnant animal or fetus increases. Not all the animals would exhibit the full suite of malformations even at high doses, and at low doses only some of the specific effects may be manifested. It is the change in severity and frequency with respect to dose that is used to include specific agents in the characterization of specific developmental syndromes, such as the two syndromes described here (the phthalate and androgen-insufficiency syn- dromes). Other agents may, for example, interfere with AR action by the seques- tration of cofactors after binding to other nuclear receptors, such as the aryl hy- drocarbon receptor (AhR). However, none of the AhR ligands has been shown to elicit the full suite of adverse outcomes that have been described in connec- tion with more classical antiandrogens, and such agents have therefore not been included in the committee’s description of androgen insufficiency (see also Chapter 5). As noted previously and illustrated in Figure 3-5, the phthalate syndrome observed in rats has parallels with the hypothesized human testicular dysgenesis syndrome (Sharpe 2001; Fisher et al. 2003; Joensen et al. 2008; Schumacher et al. 2008; Sharpe and Skakkebæk 2008) and shows similarities to other known human genetic syndromes involving impaired androgen responsiveness in the sexual differentiation of the reproductive tract (for a review, see Hughes 2001). Humans, in common with all mammals, have a specific requirement for andro- gen for the normal differentiation of the male reproductive tract during fetal life. Androgen insufficiency is well described in humans with a focus on 5α- reductase deficiencies or alteration in AR structure and function (see reviews Brinkmann 2001; Sultan et al. 2002), and disorders of androgen action are the main cause of male pseudohermaphroditism and can result in a wide spectrum of under virilization in male offspring ranging from complete external feminization to male infertility. Thus, the pathways for the critical action of androgens during fetal life are highly conserved and operate in humans as they do in experimental animals. It is biologically plausible that adverse reproductive outcomes could occur if specific phthalates or mixtures of phthalates reach the developing hu- man fetus at the appropriate concentration and in the appropriate developmental window. REFERENCES Adham, I.M., J.M. Emmen, and W. Engel. 2000. The role of the testicular factor INSL3 in establishing the gonadal position. Mol. Cell Endocrinol. 160(1-2):11-16. Barlow, N.J., and P.M. Foster. 2003. Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to di(n-butyl) phthalate. Toxicol. Pathol. 31(4): 397-410.

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