MALE SEXUAL DIFFERENTIATION IN MAMMALS

Sexual differentiation in males follows complex interconnected pathways during embryo and fetal development that have been reviewed extensively elsewhere (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 female 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 development 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 hormone 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 location of the testis in the lower abdomen (transabdominal descent). The CSL regresses through an androgen-dependent process. In the female, the CSL is retained with a thin gubernaculum to maintain ovarian position. Descent of the testes through the inguinal ring into the scrotum (inguinoscrotal descent) is under androgen control.

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 transabdominal phase in which the cranial suspensory ligament (CSL) disappears, and the testes—located (more...)

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 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 gestation, as indicated by the production of testosterone in the latter part of the gestational 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.

FIGURE 3-2

Comparison of periods of male reproductive development in rat and human. 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, (more...)

Given the above discussion, it is clear that normal differentiation of the male phenotype has specific requirements for fetal testicular hormones, including androgens, and therefore can be particularly sensitive to the action of environmental agents that can alter the endocrine milieu of the fetal testis during critical periods of development.

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 developmental effects in animals for BBP, DBP, DEHP, and DIDP and some evidence for DINP but only limited evidence for DHP and DOP. However, as dis 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 studies 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 dosing models. Those lesions were the most severe manifestations of testicular toxicity in that there was complete tubular atrophy. Initial experiments also indicated 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 phthalate. 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 equimolar doses (Foster et al. 1981b).

Detailed morphologic examination of the phthalate-induced testicular lesions 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 systems 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 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 testicular 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 phthalates 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 testicular 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 proliferation. The lower male reproductive toxicity observed for the mouse was consistent 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 generation; 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 reproductive 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 developmental 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 developmental 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 system are evaluated. However, there has been no change in the time of examination 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 period (Mylchreest et al. 1998, 1999) that the increased sensitivity of the fetus to DBP was described (Mylchreest et al. 2000).

TABLE 3-1

Reproductive and Developmental Effects of DBP in the National Toxicology Program Reproductive Assessment by Continuous Breeding Study (1991).

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 induction of phthalate syndrome, at 0, 250, 500, or 750 mg/kg-d by gavage in corn oil (5 mL/kg-d). Reproductive (more...)

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 (Mylchreest 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; Hutchison et al. 2008). Phthalate exposure also results in regions of Leydig cell hyperplasia. Barlow et al. (2004) showed that a small percentage of male offspring 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 adverse 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.

Testicular Dysgenesis Syndrome

Human males exhibit a high incidence of reproductive disorders. Cryptorchidism 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 gestation (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 reproductive 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.

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 diethylhexyl) reduced fetal testicular testosterone and impaired male reproductive development, whereas phthalates with shorter or longer side chains (dimethyl, diethyl, 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 common targeting of specific fetal testis genes by a select group of phthalates indicates common molecular mechanisms of action.

TABLE 3-3

Effect of in Utero Phthalate Exposure on Male Rat Reproductive Outcomes.

AGENTS THAT PRODUCE SIMILAR EFFECTS ON REPRODUCTIVE DEVELOPMENT

Although the spectrum of effects of some phthalates on male reproductive development in utero in rats is specific (the phthalate syndrome), a number of other types of agents can produce similar outcomes through a perturbation in androgen concentrations or androgen-receptor (AR) signaling. Indeed, in many of the reproductive tissues that require androgen for their normal development, it is unlikely that one can differentiate between a decreased concentration of the ligand (testosterone or DHT) and a blockade of the AR; the response or consequences would be identical, producing common adverse outcomes (see Figure 3-4).

FIGURE 3-4

Fetal androgen insufficiency and common adverse outcomes.

Although inhibition of insl3 appears unique to the effects of phthalates, some phthalates can reduce fetal testicular testosterone production. That property is shared by an array of agents that can produce “androgen insufficiency” in the developing fetus, which in turn can yield effects on male reproductive devel opment that would include many of the same malformations caused by phthalates. The processes that would be affected would include the development of the Wolffian duct into the epididymis, vas deferens, and seminal vesicle (predominantly under the control of testosterone) and the development of the urogenital sinus into the prostate and the genital tubercle, which develops into the penis (all of which are predominantly under DHT control). Effects on the length of the perineum (AGD) and apoptosis of the nipple anlagen in rats are also under DHT control. Indeed, the syndrome of androgen insufficiency could be considered a subset of the phthalate syndrome, with only the effects on insl3 and germ-cell development being different. Figure 3-5 shows the relationship between the phthalate syndrome and the androgen-insufficiency effects and compares the phthalate syndrome noted in rats with the hypothesized human testicular dysgenesis syndrome. There is a remarkable overlap in response between the phthalate syndrome and the hypothesized human testicular dysgenesis syndrome, except for responses for which rats are sexually dimorphic (retention of nipples) or that rodents do not exhibit (for example, rats do not develop testicular germ-cell cancer—the most common cancer in young men—but rather Leydig cell tumors of the testis, which are commonly noted both spontaneously and after exposure to biologically active phthalates). However, there are no human data that directly link the hypothesized human syndrome with phthalate exposure.

FIGURE 3-5

Relationship of phthalate syndrome in rats to that noted for agents that perturb androgen action to produce androgen insufficiency and to the hypothesized testicular dysgenesis syndrome in humans. End points in brackets are restricted to findings in experimental (more...)

The agents that can produce androgen insufficiency can be loosely grouped into three main classes: AR antagonist, mixed-function inhibitors, and 5α-reductase inhibitors. The spectrum of induced malformations is similar to that of phthalates, but the precise tissue sensitivity and therefore the most common malformations observed after in utero exposure to each group are different.

Comparison of Agents

Table 3-4 indicates the variety of predominant malformations associated with the different molecular mechanisms. The overall spectrum of induced malformations resulting from disturbances in androgen concentration is very similar to that resulting from disturbances in signaling. Although there might be quantitative differences in the individual malformations produced, depending on precise mechanisms or doses, the similarity in response of the androgen-dependent organs indicates that few independent pathways of response exist in relation to androgen disturbances. Thus, a developing prostate seems to respond in the same manner irrespective of the agent that lowers the concentration of a ligand, whether testosterone or DHT, or that blocks or alters signaling of the AR in the target tissue. Accordingly, the prostatic malformations induced by phthalates (which lower fetal testicular testosterone production), AR antagonists (such as flutamide and vinclozolin), mixed acting agents (such as prochloraz), and the 5α-reductase inhibitor finasteride are identical.

TABLE 3-4

Effects of Agents That Can Produce Androgen Insufficiency by Different Pharmacologic Activities or Mechanisms and the Most Common Resulting Malformation after in Utero Exposure of Pregnant Rats during Sexual Differentiation.

CANCER

This chapter has primarily addressed male reproductive effects of phthalates. However, much research on phthalate toxicity has focused on the carcinogenic effects observed in animal models. One of the best described carcinogenic effects of phthalates is hepatic cancer, although hepatic neoplasms are not observed in response to long-term exposure of all phthalates. Evidence from multiple reports (reviewed in NTP 2000) demonstrates that DEHP and DINP cause hepatic tumors in rats and mice (Table 3-5). Some phthalate monoesters—including MEHP, MINP, MBP, MBZP, MOP, and MIDP—can activate peroxisome-proliferator-activated receptor-α (PPARα), as demonstrated by Bility et al. (2004), who used an in vitro reporter assay. The ability of phthalate monoesters to activate PPARα increases with increasing chain length. Generally, the mouse PPARα can be activated by lower concentrations of the phthalate monoesters than can the human PPARα, and the response of the mouse PPARα is much greater than that of the human PPARα (Bility et al. 2004). Accordingly, DEHP and DINP are thought to cause hepatocarcinogenesis through their monoester metabolites at relatively high exposure because of ligand activation of PPARα, which is known to mediate hepatocarcinogenic effects in rodents (Peters et al. 1997; Hays et al. 2005).

TABLE 3-5

Summary of Hepatocarcinogenic Effects of Phthalates.

A recent study in mice, however, suggests that DEHP-induced hepatocarcinogenesis occurs in the absence of PPARα expression. Ito et al. (2007) exposed 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 hepatocarcinogenesis. 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 humans (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 microRNA might also explain the lack of changes in hepatic markers of cell proliferation 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 differential 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 nonhepatic tumors reported to occur after phthalate exposure in animal models may be mediated through mechanisms that are independent of PPARα.

CONCLUSIONS

In undertaking an examination of agents that produce a syndrome of developmental 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 syndromes). Other agents may, for example, interfere with AR action by the sequestration of cofactors after binding to other nuclear receptors, such as the aryl hydrocarbon receptor (AhR). However, none of the AhR ligands has been shown to elicit the full suite of adverse outcomes that have been described in connection 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 androgen 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 human fetus at the appropriate concentration and in the appropriate developmental window.

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Footnotes

1

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

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.

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.