Chapter 20. Toxic Responses of the Reproductive System

• The gonads possess a dual function: an endocrine function involving the secretion of sex hormones and a nonendocrine function relating to the production of germ cells (gametogenesis).
• Gametogenic and secretory functions of either the ovary or testes are dependent on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary.
• The blood–testis barrier between the lumen of an interstitial capillary and the lumen of a seminiferous tubule impedes or prevents the free exchange of chemicals/drugs between the blood and the fluid inside the seminiferous tubules.
• Xenobiotics can act directly on the hypothalamus and the adenohypophysis, leading to alterations in the secretion of hypothalamic-releasing hormones and/or gonadotropins.
• Steroid hormone biosynthesis can occur in several endocrine organs including the adrenal cortex, ovary, and the testes.
• Female reproductive processes of oogenesis, ovulation, the development of sexual receptivity, coitus, gamete and zygote transport, fertilization, and implantation of the conceptus may be sites of xenobiotic interference.
• Xenobiotics may influence male reproductive organ structure, spermatogenesis, androgen hormone secretion, and accessory organ function.

Chemicals can adversely affect reproduction in males and females. Recent trends in human fertility point to the potential for declines in normal human reproduction and suggest that exposure to environmental chemicals and drugs may contribute to these declines. The reproductive cycle is outlined in Figure 20–1.

Numerous complex processes are orchestrated in a precise and sequential order for optimal performance at different stages of the life cycle of animals and humans. Following fertilization of an egg by a sperm, the resulting zygote must be transported along the oviduct while maturing into an early embryo. This embryo must then implant in the uterus successfully, differentiate, produce a placenta, and undergo normal embryogenesis and fetal development.

Acquisition of sexual maturity involves the generation of gametes by the gonads. For parental animals, once their reproductive life span has finished, the process of reproductive senescence then occurs. These processes all involve complex interplay between tissues and cells, under hormonal control that provides the critical signals and precise timing of these events. All these processes can be targets for the action of specific agents that can disturb events leading to adverse effects on reproduction, such that the normal production of viable offspring cannot occur.

During the seventh week of human gestation, the male and female morphological characteristics begin to develop. Gonadal differentiation depends on signals from the Y chromosome, which contains the genes necessary to induce testicular morphogenesis. One of these signals is the SRY gene, which is the sex-determining region on the short arm of the Y chromosome and acts as a “switch” to initiate transcription of other genes that contribute to testicular organogenesis. In the absence of the SRY protein, the gonad remains indifferent for a short period of time before differentiating into an ovary.

Interstitial Leydig cells produce the male sex hormone testosterone, which induces masculine differentiation of the Wolffian duct and external genitalia. Figure 20–2 provides a diagrammatic representation of sexual differentiation in the human male. In rodent and human species, fetal testicular androgen production is necessary for proper testicular development, normal male sexual differentiation, and differentiation of the Wolffian ducts into the epididymides, vasa deferentia, and seminal vesicles.

Androgens derived from the Leydig interstitial cells stimulate the mesonephric (or Wolffian) ducts to form the male genital ducts, while Sertoli cells produce Müllerian-inhibiting substance (or anti-Müllerian hormone), which suppresses development of the paramesonephric (Müllerian) ducts, or female genital ducts.

In the humans, the external genitalia are indistinguishable until the 9th week of gestation, and not fully differentiated until the 12th week of development. Development of the external genitalia coincides with gonadal differentiation. Fetal testicular androgens are responsible for the induction of masculinization of the androgynous external genitalia. Thus, male, but not female, reproductive tract development is totally hormonally dependent and inherently more susceptible to endocrine disruption.

The mammalian oocyte (Figure 20–3) begins meiosis during fetal development but arrests partway through meiosis I and does not complete the first division until ovulation; the second division is completed only if the egg is fertilized. In the males, meiosis begins at puberty and is a continuous process, with spermatocytes progressing from prophase to the meiotic second division in little more than a week. This difference in strategy has implications for the action of toxicants and critical time periods when these cells may be vulnerable to attack.

Late in gestation and at birth, male rats display longer anogenital distances (AGD) than do female rats with neonatal male AGD being more than twice as long as females. There are homologous sex differences in humans. In many mammalian species, including humans and rats, males of the species engage in more aggressive play than do females. Both AGD and behavior can be altered by exposure to hormonal and antihormonal agents.

During the infantile period of development, emergence of the nipple buds and areolae in females as well as maturation of the hypothalamic–pituitary axis occurs. Emergence of the nipple buds is prevented in males by prenatal androgen-induced atrophy of the nipple anlagen. Prenatal androgen-treated females may display reproductive tract malformations (retained male tissues or vaginal agenesis).

Puberty is initiated by activation of the hypothalamic–pituitary–gonadal (HPG) and hypothalamic–pituitary–adrenal (HPA) axes (see Figure 20–4). At the onset, the HPG axis releases gonadotropin-releasing hormone (GnRH) pulses with increasing frequency and amplitude that induces complimentary pulsatile secretions of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary. In turn, LH and FSH stimulate the gonads inducing gonadarche characterized by the onset of gonadal hormone production. In the females, secretion of androgens from theca cells and estradiol from granulosa cells of maturing follicles prior to ovulation is followed by secretion of progesterone from the corpus luteum after ovulation. In the males, LH stimulates testicular synthesis and secretion of androgens and insulin-like peptide 3 hormone from the Leydig cells of males.

Premature thelarche and premature adrenarche are often referred to as pseudoprecocious puberty when the full spectrum of pubertal changes does not occur. Premature thelarche in girls and gynecomastia in boys result from direct exposure to estrogen-containing personal care and “natural” products. Untoward consequences of these conditions may occur with prolonged exposure, including shortened stature due to effects of estrogens on the growth plates of the long bones and sexual–social behavior that is inappropriate for the chronological age of the child. Concerns have also been expressed that premature thelarche may enhance the likelihood of developing diseases like breast cancer and endometriosis.

The association of pubertal alterations with environmental exposure to persistent halogenated organic chemicals such as polychlorinated biphenyls (PCBs), brominated flame retardants, dioxin, hexachlorobenzene, endosulfan, and heavy metals also has been studied but a consensus about the causative role of these chemicals in altering puberty has not been achieved.

Rodent Models of Puberty

Rodents are important animal models in the study of the effects of toxicants on puberty. In the laboratory rats, the standard landmarks of puberty are the age of preputial separation (PPS) in the males, and the ages of vaginal opening (VO) and first estrus in females.

Onset of pubertal landmarks in rats can be altered after acute in utero and/or lactational exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), busulfan, androgens, and endocrine-disrupting chemicals (EDCs). Also, onset of pubertal landmarks in rats is delayed after peripubertal exposures to antiandrogenic chemicals. Throughout puberty and into adulthood, the sex accessory glands and other androgen-dependent tissues (i.e., muscles and nervous system) continue to depend on testosterone and 5α-dihydrotestosterone for maturation and maintenance of function.

Hypothalamo-pituitary–Gonadal Axis

FSH and LH are glycoproteins synthesized and released from the pituitary gland. Hypothalamic neuroendocrine neurons secrete specific releasing or release-inhibiting factors into the hypophyseal portal system, which carries them to the adenohypophysis, where they act to stimulate or inhibit the release of anterior pituitary hormones. GnRH acts on gonadotropic cells, thereby stimulating the release of FSH and LH.

The neuroendocrine neurons have nerve terminals containing monoamines (norepinephrine, dopamine, and serotonin) that impinge on them. Reserpine, chlorpromazine, and monoamine oxidase (MAO) inhibitors modify the content or actions of brain monoamines that affect gonadotropin production.

In the females (Figure 20–5), LH acts on thecal cells of the ovary to induce steroidogenesis, particularly the production of progesterone and androgens that are transferred to the granulosa cells that can be stimulated by FSH to produce estradiol. These steroids provide feedback on the hypothalamus and pituitary to regulate gonadotropin production.

Similarly in the males (Figure 20–6), FSH acts primarily on the Sertoli cells, but it also appears to stimulate the mitotic activity of spermatogonia. LH stimulates steroidogenesis in the interstitial Leydig cells. A defect in the function of the testis (in the production of spermatozoa or testosterone) will tend to be reflected in increased levels of FSH and LH in serum because of the lack of the negative feedback effect of testicular hormones.

The HPG feedback system is a very delicately modulated hormonal process. Toxicants that alter the hepatic and/or renal biotransformation of endogenous sex steroid may be expected to interfere with the pituitary feedback system.

Ovarian Function

Oogenesis

About 400,000 follicles are present at birth in each human ovary. After birth, many undergo atresia, and those that survive are continuously reduced in number. Any chemical that damages the oocytes will accelerate the depletion of the pool and can lead to reduced fertility in females. About one-half of the numbers of oocytes present at birth remain at puberty; the number is reduced to about 25,000 by 30 years of age. About 400 primary follicles will yield mature ova during a woman's reproductive life span. During the approximately three decades of fecundity, follicles in various stages of growth can always be found. After menopause, follicles are no longer present in the ovary.

Although ovarian weight does not fluctuate during the estrous cycle, ovarian weight and histology can provide very useful information about the effects of toxicants on the female reproductive system. Ovarian weight can be reduced by either depletion of oocytes or disruption of the HPG axis. Toxicants induce various ovarian lesions, including polyovular follicles, oocyte depletion, interstitial cell hyperplasia, corpora albanicans, and an absence of corpora lutea.

Case Study: Busulfan

Busulfan is an alkylating agent used to treat several diseases in humans, including chronic myelogenous leukemia, certain myeloproliferative disorders such as severe thrombocytosis, and polycythemia vera. Busulfan causes ovarian failure and prevents or delays the onset of puberty in girls.

In rodents, administration of busulfan specifically inhibits germ cell development. Offspring display permanent reproductive and CNS alterations. The most severely affected females do not display estrous cycles or spontaneous sexual behavior as a consequence of this exposure. Even though the gonads of both sexes are affected at similar dosage levels, fertility and gonadal hormone production are much more easily disrupted in female than male offspring, because the steroid-producing cells in the ovary fail to differentiate in the absence of the oocyte. A diagrammatic representation of the sites of actions of female reproductive toxicants is presented in Figure 20–7.

Ovarian Cycle

The cyclic release of pituitary gonadotropins involving the secretion of ovarian progesterone and estrogen is depicted in Figure 20–8. These female sex steroids determine ovulation and prepare the female accessory sex organs to receive the male sperm. This axis can be disrupted, resulting in infertility at any level of the endocrine system. For example, chemicals that block the LH surge transiently can prevent or delay ovulation, resulting in infertility or lower fecundity due to delayed fertilization of ova.

The blood–testis barrier between the lumen of an interstitial capillary and the lumen of a seminiferous tubule impedes or prevents the free exchange of chemicals/drugs between the blood and the fluid inside the seminiferous tubules.

Targets for Toxicity

For an adult male, there are numerous potential targets for the action of chemicals on the system (Figure 20–9). Dopamine analogs and estrogens interfere with gonadotropin production and release, thereby affecting spermatogenesis. However, perturbing the homeostasis of nutrients can lead to direct effects on spermatogenesis and subsequent issues with fertility. Similarly, chemicals that have direct effects on the liver (e.g., CCl4) can disturb the normal metabolism of sex steroids leading to changes in clearance (predominantly of glucuronide and sulfate conjugates of hydroxytestosterones in the male), indirectly affecting the HPG axis and exerting effects on male repro-duction.

The testis also has a finely tuned circulatory system in mammals, termed the pampiniformplexus, designed to shunt the arterial venous blood supply and aid in scrotal cooling. Some chemicals (e.g., cadmium) can actually target this structure and the testicular circulatory system to induce ischemic shock to the testes, resulting in injury and reduced fertility.

Testicular Structure and Spermatogenesis

Spermatogenesis is an extremely ordered process in the rat. The spermatogonia have populations that act as the stem cells for the seminiferous tubules and a proportion of these cells then undergo a series of mitotic divisions to increase numbers and move into meiotic prophase, and are then committed to becoming spermatozoa, which are released into the lumen of the seminiferous tubule.

Different biochemical events can go on during the different stages and indeed this can provide clues as to potential modes of action of chemicals that produce stage-specific lesions. Such occurrences do occur regularly with certain phthalate esters, glycol ethers, and antiandrogenic agents.

Once sperms are released into the seminiferous tubule lumen and proceed to the epididymis, they can also be the target of toxicant action. Chlorosugars and epichlorohydrin have both been shown to inhibit energy metabolism in sperm, preventing them from functioning normally. The number of environmental chemicals that produce adverse responses in human males is not large. All of these have been shown to induce effects in rodents and especially the rat, although there may be differences in sensitivity based on dose.

Posttesticular Processes

During the movement along the epididymal tubule, fluid is removed by active transport and this stage of the process is one that can be interfered with by toxicants, resulting in an inappropriate environment for normal sperm development.

Erection and Ejaculation

Little is known concerning the effects of chemicals on erection or ejaculation. Pesticides, particularly the organophosphates, are known to affect neuroendocrine processes involved in erection and ejaculation. Many drugs act on the autonomic nervous system and affect potency. Impotence, the failure to obtain or sustain an erection, is rarely of endocrine origin; more often, the cause is psychological.

Case Studies for Effects on the Male

m-Dinitrobenzene

m-Dinitrobenzene (m-DNB) has been extensively studied for its ability to produce a rapid deleterious effect on the rat testis. Testicular weight remained reduced for many weeks after the treatment period with significant dose-related effects on fertility (measured by pregnancy rate and implantation success).

Ethylene Glycol Monomethyl Ether (EGME)

EGME has been shown to produce testicular toxicity in a wide variety of species, including nonmammals. A number of studies have described Sertoli cell vacuoles, swollen germ cell mitochondria, and a breakdown of the membrane between the Sertoli cell and the pachytene spermatocyte. This is followed within hours by the death of (probably those) pachytene spermatocytes.

EGME is metabolized to active intermediates methoxyacetaldehyde and methoxyacetic acid (MAA). Treating animals with MAA produces identical testicular lesions as that of the parent compound. The lesion is not characteristic of a low-androgen testicular lesion, and reduced accessory sex organ weights are not a prominent feature associated with the early testicular pathology.

Because the transition from early to midpregnancy in the rat requires hormones from the feto-placental unit, if implantation or uterine decidualization is blocked by a chemical, then the female would resume her estrous cycles and the corpora lutea would regress. Chemicals that induce whole-litter loss at mid- to late pregnancy may cause abortions in some of the females, whereas others fail to deliver and appear pregnant for an unusually long period of time.

Many abortifacients induce pregnancy loss by reducing progesterone levels in the rat. Generally, reducing midpregnancy progesterone levels by half or more is sufficient to terminate pregnancy.

Perinatal exposure to toxicants with estrogenic activity can defeminize the HPG axis such that the female rats are acyclic and infertile, whereas less affected females display the “delayed anovulatory syndrome” and become anovulatory and acyclic at an early age.

In males, a decrease in androgen is noted in around 20 percent of fit 60-year-old men, but the value of androgen supplementation is not clear with regard to reproductive senescence.

Currently, the potential effects of EDCs on human health and the proven effects of EDCs on wildlife are a major focus among the scientific community. It has been suggested that in utero exposure to environmental estrogens, antiandrogens, or chemicals like phthalates or 2,3,7,8-TCDD could be responsible for the reported 50 percent decline in sperm counts in some areas and the apparent increase in cryptorchid testes, testicular cancer, and hypospadias.

Phthalate exposures have been associated with reduced AGD in boys and lower testosterone levels in men. In females, exposure to EDCs during development could contribute to earlier age of puberty and to increased incidences of endometriosis and breast cancer. Besides pesticides and other toxic substances in the environment, many compounds that are phytosterols, estrogens, antibiotics, beta-blockers, antiepileptics, and lipid-regulating agents have significant endocrine-disrupting activity and are capable of inducing reproductive toxicity.

In the area of wildlife toxicology and ecosystem health, it is apparent that clear-cut cause and effect relationships exist between exposure to EDCs and adverse effects in several vertebrate classes from fish to mammals.

Known Effects of EDCs in Humans and Animals

The list of chemicals that are known to affect humans, domestic animals, and/or wildlife via functional developmental toxicity or endocrine mechanisms includes 2,3,7,8-TCDD, PCBs and polychlorinated dibenzofurans (PCDFs), methylmercury, ethinylestradiol, alkylphenols, plant sterols, fungal estrogens, androgens, chlordecone, DBCP, dichlorodiphenyltrichloroethane (DDT), and other organochlorine compounds. In addition to these xenobiotics, over 30 different drugs taken during pregnancy have been found to alter human development as a consequence of endocrine disruption. These drugs are not limited to estrogens, like diethylstilbestrol (DES). EDCs are known to alter human development via several mechanisms besides the estrogen receptor (ER), including binding to retinoic acid (RAR and RXR) receptors, and inhibiting synthesis of steroidogenic enzymes or thyroid hormones. Findings on the effects of background levels of PCBs on the neurobehavioral development of the child have contributed to the concerns about the effects of EDCs on human health via alteration of hormone function.

Human Sexual Differentiation

Exposure to hormonally active chemicals during sex differentiation can produce pseudohermaphroditism. Androgenic drugs like danazol and methyltestosterone can masculinize human females (i.e., “female pseudohermaphroditism”). The drug aminoglutethimide, which alters steroid hormone synthesis in a manner identical to many fungicides, also masculinizes human females following in utero exposure.

Transplacental exposure of the developing fetus to DES causes clear cell adenocarcinoma of the vagina, as well as gross structural abnormalities of the cervix, uterus, and fallopian tube. These DES-exposed women are more likely to have an adverse pregnancy outcome, including spontaneous abortions, ectopic pregnancies, and premature delivery. Some of the pathological effects that develop in males following fetal DES exposure appear to result from an inhibition of androgen action or synthesis (underdevelopment or absence of the vas deferens, epididymis, and seminal vesicles) and anti-Müllerian duct factor (persistence of the Müllerian ducts).

Known Effects of Plant and Fungal Products in Animals and Humans

Although most naturally occurring environmental estrogens are relatively inactive, the phytoestrogen miroestrol is almost as potent as estradiol in vitro and even more potent than estradiol when administered orally. In addition, many plant estrogens occur in such high concentrations that they induce reproductive alterations in domestic animals. “Clover disease,” which is characterized by dystocia, prolapse of the uterus, and infertility, is observed in sheep that graze on highly estrogenic clover pastures. Permanent infertility can be produced in ewes by much lower amounts of estrogen over a longer time period than are needed to produce “clover disease.”

Known Effects of Organochlorine Compounds in Humans

Several pesticides and toxic substances have been shown to alter human reproductive function. An accidental high-dose in utero exposure to PCBs and PCDFs has been associated with reproductive alterations in boys, increased stillbirths, low birth weights, malformations, and IQ and behavioral deficits. In addition to the effects associated with this inadvertent exposure, subtle adverse effects were seen in infants and children exposed to relatively low levels of PCBs and PCDFs.

One metabolite of DDT (mitotane, o,p′-DDD) was found to alter adrenal function with sufficient potency to be used as a drug to treat adrenal steroid hypersecretion associated with adrenal tumors. In addition, lower doses of mitotane restored menstruation in women with spanomenorrhea associated with hypertrichosis.

Occupational Exposures

Occupational exposure to pesticides and other toxic substances (i.e., chlordecone and DBCP) in the workplace has been associated with reduced fertility, lowered sperm counts, and/or endocrine alterations in male workers. Workers exposed to high levels of chlordecone, an estrogenic and neurotoxic organochlorine pesticide, displayed intoxication, severe neurotoxicity, and abnormal testicular function. Male workers involved in the manufacture of 4,4′-diaminostilbene-2,2′-disulfonic acid (DAS), a key ingredient in the synthesis of dyes and fluorescent whitening agents, had lower serum testosterone levels and reduced libido as compared with control workers. Thus, it is surprising that occupational exposures to potential EDCs at effective concentrations have not been entirely eliminated from the workplace.

Environmental Androgens

Androgenic activity has been detected in several complex environmental mixtures. Pulp and paper mill effluents (PME) include a chemical mixture that binds androgen receptors (AR) and induces androgen-dependent gene expression in vitro. This mode of action is consistent with the masculinized female mosquitofish (Gambusia holbrooki) collected from contaminated sites. Male-biased sex ratios of fish embryos have been reported in broods of eelpout (Zoarces viviparus) in the vicinity of a large kraft pulp mill on the Swedish Baltic coast, suggesting that masculinizing compounds in the effluent were affecting gonadal differentiation and skewing sex ratios. Effluents from beef-cattle concentrated animal feeding operations have been shown to display androgenicity.

Environmental Antiandrogens

Fungicides

Vinclozolin and procymidone are two members of the dicarboximide fungicide class that act as AR antagonists. These pesticides, or their metabolites, competitively inhibit the binding of androgens to AR, leading to an inhibition of androgen-dependent gene expression.

Administration of vinclozolin during sexual differentiation demasculinizes and feminizes the male rat offspring such that treated males display female-like AGD at birth, retained nipples, hypospadias, suprainguinal ectopic testes, a blind vaginal pouch, and small to absent sex accessory glands.

Procymidone induces shortening of the AGD in male pups, and older males display retained nipples, hypospadias, cryptorchidism, cleft phallus, a vaginal pouch, and reduced sex accessory gland size. Fibrosis, cellular infiltration, and epithelial hyperplasia are noted in the dorsolateral and ventral prostatic and seminal vesicular tissues in adult offspring.

Linuron (Herbicide)

This herbicide binds rat and human AR and inhibits DHT–hAR-induced gene expression in vitro. In utero linuron exposure produces male rats displaying epididymal and testicular abnormalities. Also, fetal testosterone production is significantly reduced in linuron-treated fetal males.

Phthalates (Plasticizers)

In utero, some phthalate esters alter the development of the male rat reproductive tract at relatively low dosages. Prenatal exposures to DBP, benzyl-butyl phthalate (BBP), di-isononyl phthalate (DINP), and diethylhexylphthalate (DEHP) cause a syndrome of effects, including underdevelopment and agenesis of the epididymis and other androgen-dependent tissues and testicular abnormalities. The phthalates are unique in their ability to induce agenesis of the gubernacular cords, a tissue whose development is dependent on the peptide hormone insulin-like peptide 3.

Environmental Estrogens

Methoxychlor is an estrogenic pesticide that produces estrogen-like effects. The active metabolites activate estrogen-dependent gene expression in vitro and in vivo in the female rats, thereby stimulating a uterotropic response, accelerating VO and inducing constant estrus, and reducing infertility. In the ovariectomized female rats, methoxychlor also induces estrogen-dependent reproductive and nonreproductive behaviors, including female sex behaviors, running wheel activity, and food consumption.

When given to the dam during pregnancy and lactation, both male and female offspring are affected. Females display irregular estrous cycles and reduced fecundity. Male fertility is unaffected at doses up to 200 mg/kg per day.

Ethinylestradiol is a synthetic derivative of estradiol that is in almost all modern formulations of combined oral contraceptive pills. This drug is found in many aquatic systems contaminated by sewage effluents, originating principally from human excretion. Thus, ethinylestradiol plays a major role in causing widespread endocrine disruption in wild populations of fish species and other lower vertebrate species.

EDC Screening Programs

The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) proposed a tiered screening (tier 1) and testing (tier 2) strategy for EDCs. The recommended screening battery was designed to detect alterations of HPG function; estrogen, androgen, and thyroid hormone synthesis; and AR- and ER-mediated effects in mammals and other taxa.

In Vivo Mammalian Assays

EDSTAC recommended the laboratory rat as the species of choice for the endocrine screening and testing assays. The EDSTAC proposed three short-term in vivo mammalian assays for the tier 1 screening battery: the uterotropic, Hershberger, and pubertal female rat assays.

Uterotropic Assay

Estrogen agonists and antagonists are detected in a 3-day uterotropic assay using subcutaneous administration of the test compound. The selected uterotropic assays for estrogens and antiestrogens use either the intact juvenile or the castrated ovariectomized adult/juvenile female rat.

Hershberger Assay

The second in vivo assay in tier 1, the Hersh-berger assay, detects antiandrogenic activity simply by weighing androgen-dependent tissues in the castrated male rat. In this assay, weights of the ventral prostate, Cowper's glands, seminal vesicle (with coagulating glands and fluids), glans penis, and levator ani/bulbocavernosus muscles are measured after 10 days of oral treatment with the test compound. This assay is very sensitive for detection of androgens and antiandrogens.

Pubertal Female Rat Assay

The third in vivo mammalian/rat assay in the screening battery is the pubertal female rat assay. Weanling female rats are dosed daily by gavage for 21 days while the age at VO (puberty) is monitored. The females are necropsied at about 42 days of age. This assay detects alterations in thyroid hormone status, HPG function, inhibition of steroidogenesis, estrogens, and antiestrogens, and has been found to be highly reproducible and very sensitive to certain endocrine activities including estrogenicity, inhibition of steroidogenesis, and antithyroid activity.

Screens and Multigeneration Studies

Significant attention has focused on the development of “screens” for reproductive toxicity. The screens currently employed have been developed to prioritize chemicals for more comprehensive testing.

The most comprehensive assessment of reproductive toxicity would be provided by a protocol that exposes the animal model throughout the reproductive cycle (see Figure 20–1) and assesses multiple endpoints at different life stages during this continuous exposure. The protocol and guideline coming closest to this ideal is the multigeneration reproduction study (Figure 20–10) used for the assessment of chemicals, pesticides, and some food additives. Multigeneration studies normally encompass detailed measurements of reproductive performance (number of pregnant females from number of pairs mated, number of females producing a litter, litter size, and number of live pups with their birth weights and sex). Measurement of growth and analysis of the reproductive organs in the F0 parental generation is conducted. Similar measurements to those undertaken for the F0 are made on the F1 parents, and the offspring are examined at birth (and sexually dimorphic endpoints may be collected such as AGD), at weaning, and at puberty (particularly the assessment of VO and time of first estrus in females and belanopreputial separation in males) in addition to the adult measurements of reproductive performance, organ weights, histology, etc.

Testing for Endocrine-Disrupting Chemicals

In the tiered screening and testing approach, only chemicals that display positive reproducible responses in tier 1 screening (T1S) would continue evaluation in full-life cycle or multigenerational tests. In tier 2 testing (T2T), issues of dose–response, relevance of the route of exposure, sensitive life stages, and adversity are resolved.

Data should be summarized in a manner that clearly delineates the proportion of animals that are affected. In teratology studies, data are typically presented and analyzed in this manner, indicating the number of malformed/number observed on an individual and litter basis, whereas multigenerational studies are frequently presented and analyzed differently, even when clear teratogenic and other developmental responses are noted after birth. Multigenerational protocols are used in T2T because only these protocols expose the animals during all critical stages of development and examine reproductive function of offspring after they mature.

Although the EPA multigenerational test provides for a comprehensive evaluation of the F0 or parental generation, too few F1 animals (offspring with developmental exposure) are examined after maturity to detect anything but the most profound reproductive teratogens. F0 animals within a dose group typically respond in a similar fashion to the chemical exposure; however, the response to toxicants in utero can vary greatly even within a litter with only a few animals displaying severe reproductive malformations in the lower dosage groups.

“Transgenerational” protocols typically use fewer litters (7 to 10 per dose group) but examine all of the animals in each litter. These protocols actually use fewer animals but provide enhanced statistical power to detect reproductive effects in the F1 generation. The life-long exposure of both males and females in the F1 generation, which allows one to detect effects induced in utero, during lactation, or from direct exposure after puberty, can confound the identification of when the effect was induced (i.e., during adulthood versus development) or of which sex was affected.

Some EDCs disrupt pregnancy by altering maternal ovarian hormone production in F0 dams at dosage levels that appear to be without direct effect on the offspring. In such cases, the standard EPA multigenerational protocol with minor enhancements would be recommended, or a transgenerational protocol with exposure continued after weaning. The transgenerational or in utero lactational protocols fill a gap in the testing program for EDCs that should be used only on a case-by-case basis.

Testing Pharmaceuticals

In the case of pharmaceuticals, it is rare for multigeneration studies to be conducted, because it is not common for all the population to use a specific drug and exposure to the drug is over many different life stages, and not necessarily chronic. Typically three specific studies are undertaken:

1. A study of fertility and early embryonic development (see Figure 20–11). Parental adults are exposed to the test chemical for 2 weeks (females) or 4 weeks (males) prior to breeding and then during breeding. Females then continue their exposure through to implantation. Males can be necropsied for the endpoints noted above for the multigeneration studies after pregnancy has been confirmed, and for the pregnant females, necropsy takes place any time after midgestation. Reproductive and target organs are weighed and examined histologically, sperm parameters are assessed in males, and the uterine implantation sites and ovarian corpora lutea are counted in females, as well as live and dead embryos.

2. A study of effects on pre- and postnatal development including maternal function (see Figure 20–12). In this study, pregnant females are exposed from implantation until weaning of their offspring (usually PND 21 in the rat). After cessation of exposure, selected offspring (one male and one female per litter) are raised to adulthood and then mated to assess reproductive competence. These animals are observed for maturation and growth (but are not exposed). Puberty indices, as employed in the multigeneration study, are measured. In addition, sensory function, reflexes, motor activity, learning, and memory are also evaluated.

3. A study of embryo–fetal development (see Figure 20–13). This study tests for enhanced toxicity relative to that noted in pregnant females and, unlike the previous two studies, is normally conducted in two species (typically the rat and rabbit). Exposure occurs between implantation and closure of the hard palate and females are killed just prior to parturition. At necropsy, dams are observed for any affected organs and corpora lutea are counted. Live and dead fetuses are counted and examined for external, visceral, and skeletal abnormalities.

One of the following three summary risk conclusions would be applied to the drug label: (1) the drug is not anticipated to produce reproductive and/or developmental effects above the background incidence for humans when used in accordance with the dosing information on the product label; (2) the drug may increase the incidence of adverse reproductive and/or developmental events; or (3) the drug is expected to increase the incidence of adverse reproductive and/or developmental effects in humans when used according to the product label.

An examination of the reproductive cycle in a comparison of these three most likely options for FDA studies indicates an obvious gap in the exposure regime for the complete reproductive cycle, namely exposure of weanlings through puberty to adulthood. This exposure period has become of increasing interest to many companies developing drugs for specific administration to infants and juveniles, and “bridging-type” protocols have been developed to specifically address toxicity that may occur after exposure during this specific life stage.

There are a number of general points that the investigator should note in any estimation of potential reproductive toxicity:

• Adequacy of experimental design and conduct. Was there sufficient statistical power in the evaluation(s)?
• Occurrence of common versus rare reproductive deficits. Biological versus statistical significance.
• Use of historical control data to place concurrent control data into perspective and to estimate population background incidence of various reproductive parameters and deficits.
• Known structure–activity relationships for inducing reproductive toxicity.
• Concordance of reproductive endpoints. Did a decrease in litter size relate to ovarian histology and changes in vaginal cytology?
• Did the reproductive deficits become more severe with increases in dose? Did histological changes at one dose level become decrements in litter size and then reductions in fertility at higher dose levels in any generation?
• Did the reproductive deficits increase in prevalence (more individuals and/or more litters) with dose level in any generation?
• Special care should be taken for decrements in reproductive parameters noted in the F1 generation (and potentially later generations) that were not seen in the F0 generation, which may suggest developmental, as well as reproductive, toxicity. Likewise, findings in an F1 generation animal may (or may not) be reproduced in F2 offspring. For example, effects in the F1 generation on reproductive parameters may have resulted in the selection out of sensitive animals in the population, thus not producing F2 offspring for subsequent evaluation.