Evaluation of a Male Reproductive Toxicant

11.03 Evaluation of a Male Reproductive Toxicant P S Coder, E D Sloter, D G Stump, and M D Nemec, WIL Research Laboratories, LLC, Ashland, OH, USA C J Bowman, Pfizer Global Research and Development, Groton, CT, USA ª 2010 Elsevier Ltd. All rights reserved.

11.03.1 11.03.2 11.03.2.1 11.03.2.2 11.03.2.3 11.03.2.4 11.03.2.5 11.03.2.6 11.03.3 11.03.3.1 11.03.3.1.1 11.03.3.1.2 11.03.3.1.3 11.03.3.2 11.03.3.2.1 11.03.3.2.2 11.03.4 11.03.5 References

Introduction Study Designs – Strategies and/or Considerations Regulatory Guidelines Study Design Considerations The ICH Study Designs The Multigeneration Reproduction Studies Screening Studies Timing of Reproductive Toxicity Studies Endpoints and Methodologies Evaluation of Effects on the Adult Male Functional spermatogenic assessments Macroscopic and microscopic evaluation Functional assessment of fertility Evaluation of Effects on the Developing Male Effects on male development Male-mediated effects on development Data Interpretation Conclusions

Abbreviations AGD ALH BCF CASA ECHA EDS EDSP FSH GD H&E HESI HPT ICH ILSI IND

anogenital distance amplitude of lateral head displacement beat cross frequency computer-assisted sperm analysis European Chemicals Agency ethane 1,2-dimethane sulfonate Endocrine Disruptor Screening Program Follicle stimulating hormone Gestation day hematoxylin and easin Health and Environmental Sciences Institute Hypothalamic-pituitary-testicular axis International Conference on Harmonization International Life Sciences Institute Investigational New Drug

LH LIN NTP OECD PAS PAS-H PDE4D4 PRL RACB STR US EPA US FDA VAP VCL VSL

62 63 63 67 68 69 70 71 72 72 72 74 78 79 79 80 81 83 83

luteinizing hormone linearity National Toxicology Program Organization of Economic Cooperation and Development periodic acid-Schiff Periodic acid-Schiff-hematoxylim phosphodiesterase type 4 variant 4 prolactin reproductive assessment by continuous breeding straightness of trajectory United States Environmental Protection Agency United States Food and Drug Administration average path velocity curvilinear velocity straightline velocity

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62 Male Reproductive Toxicology: Strategies for Evaluation

11.03.1 Introduction

widely associated with adverse effects on fetal development, the effects of paternal exposure are less obvious. Over the past three decades, evidence for paternally mediated effects on reproduction and development has slowly accumulated. One of the secondary objectives of this chapter is to provide an overview of potential sites and mechanisms of action of toxicants that have been shown to affect male reproduction. The life cycle of a male is presented in Figure 1. Schematically, the stages from conception to sexual maturity and conception of the next generation to in utero development in the female are presented. Nonclinical toxicity studies defined by different regulatory agencies attempt to evaluate consequences of exposure of all stages of development from conception to sexual maturity. It is often necessary to allow exposure and observations to be continued from one generation to the next in order to identify immediate and/or latent effects. In Figure 1, various endpoints evaluated during nonclinical toxicity studies are presented in context of the timing of the assessment with reference to the male life cycle.

Male reproductive toxicology is the study of the potential adverse effects of an agent on the structure and function of organs and organ systems involved in male reproduction. For the purposes of this chapter, a toxicant is defined as a xenobiotic of chemical or biological origin, including an investigational new drug. Male reproductive toxicology includes scientific techniques that range from general systemic toxicity testing and functional effects on reproductive organs of the male to mechanistic and molecular approaches that might be conducted in isolated cells. The primary objective of this chapter is to review historical and current trends in the evaluation of a toxicant in the context of the assessment of male reproductive toxicity. The importance of assessing male reproductive toxicity stems from the likelihood of downstream effects on fertility, fecundity as well as possible transmission of exposure-related effects to the fetus consequent to paternal exposure. While maternal exposure and placental transfer of toxicants have been

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Figure 1 The reproductive life cycle of a male from conception to sexual maturity and conception of the next generation to in utero development. Standard endpoints evaluated in nonclinical toxicity studies are shown.

Evaluation of a Male Reproductive Toxicant

The primary goal of the male reproductive system is to produce viable functional sperm that can lead to the production of viable offspring following conception. Male reproduction and the process of spermatogenesis have been shown to be influenced by a variety of toxicants. Adverse effects of toxicant exposure can be noted on a number of sites including the hypothalamic–pituitary–testicular axis (HPT) axis (hormonal interaction between the hypothalamus, the pituitary, and the testis), various cell types within the testes, the posttesticular ductal system (efferent ducts, epididymis, and vas deferens), the accessory sex glands, the external genitalia, and the seminal fluid or the sperm itself. Toxicants that influence the HPT axis usually manifest their effects by altering the release or function of pituitary gonadotropins or neurotransmitters. Further downstream, exposure to the vast majority of toxicants leads to loss of germ cells by apoptosis. Toxicants such as ethane 1,2-dimethane sulfonate primarily cause apoptotic loss of Leydig cells (Yang et al. 2006) and antiandrogens such as flutamide and vinclozolin have been shown to inhibit androgen-receptor function (O’Connor et al. 2002a). In both instances, however, the secondary consequence is apoptotic loss of germ cells due to a lack of testosterone. Alterations in Sertoli cell structure and function due to alterations in the blood–testis barrier or the disruption of Sertoli cell protein secretion also result in apoptotic loss of germ cells due to reductions in Sertoli cell supportive capacity. Reduction in germ cell number due to apoptosis or reduction in the number of spermatids released from the seminiferous epithelium could potentially result in downstream effects on male fertility due to a reduction in the number of mature sperm. However, it is the extent of injury which ultimately determines the final outcome. Primary indications of injury to male reproduction may be noted as alterations in organ weights and/or size or as subtle changes in tissue histopathology. Table 1 lists mechanistic targets sensitive to toxicants that produce adverse effects on male reproduction. While it is not the intent of this chapter to provide an overarching review of each and every class of known male reproductive toxicants, attempts have been made to encompass a wide variety of toxicants that affect the same mechanistic target.

11.03.2 Study Designs – Strategies and/or Considerations Assessment of a toxicant, in the context of assessment of male fertility, usually begins early in the toxicity testing paradigm during the conduct of acute and

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repeat-dose (subchronic toxicity) studies. Usually conducted in rodent as well as nonrodent species, each study involves detailed histopathologic evaluation of organs and organ systems, including the male reproductive organs and accessory sex glands. Acute and subchronic studies, for a drug candidate, must be completed before a company can file an Investigational New Drug (IND) application with the United States Food and Drug Administration (US FDA) to allow for the commencement of human clinical trials (Eaton and Klaassen 2001). According to the current ICH (International Conference on Harmonization) guidelines M3(R2), human male subjects may be included in phase I and phase II clinical trials, without having conducted male fertility studies but an assessment of male fertility should be included prior to large scale or long duration clinical trials (e.g., phase III). The basis for this allowance is that the repeat-dose studies are expected to allow for identification of pathologic alterations in the testes and/or accessory sex glands. 11.03.2.1

Regulatory Guidelines

At present, regulatory guidelines for the assessment of reproduction and development can be divided into three broad categories – those intended for pharmaceuticals (US FDA 1994, 1996; ICH 2005), for industrial and agricultural chemicals (OECD 1983, 1995, 1996, 2001a,b; US EPA 1998a,b,c,d) and for food additives and ingredients (US FDA 1982, 2000). The detailed history of the progression of regulatory guidelines for developmental and reproductive toxicity assessment has been reviewed extensively in previously published manuscripts (Collins 2006; Holson et al. 2006). In recent months, international discussions of a proposed addendum to ICH guideline S6 have included details on how to detect effects on reproduction for biotechnology-derived pharmaceuticals (biotherapeutics); particularly when the only pharmacologically relevant model is the nonhuman primate (Buckley et al. 2008; Martin et al. 2009). The problems associated with evaluating functional effects on fertility (e.g., mating trial) in nonhuman primates are so significant that alternative methods are typically employed. These alternative strategies include addition of reproduction-specific endpoints to chronic monkey studies (Oneda et al. 2009; Weinbauer et al. 2008), use of surrogate test articles in rodents (Martin et al. 2009; Treacy 2000), or even validation of alternative models of fertility such as the guinea pig (Wehner et al. 2009).

Table 1 Testicular toxicants and mechanistic targets Site of action

Mechanistic target

Drug/chemical

Drug/chemical class

References

Hypothalamic–pituitary axis

Synthesis/secretion of prolactin Synthesis/secretion of LH

Cimetidine Ranitidine Cadmium DBCP Cadmium Cetrorelix DBCP Acyline EDS Cadmium Bromocriptine Bromocriptine Chlorambucil Bromocriptine

Histamine (H2) antagonist Histamine (H2) antagonist Transition metal Nematocide Transition metal GnRH antagonist Nematocide GnRH antagonist Alkylating agent Transition metal Dopamine agonist Dopamine agonist Nitrogen mustard alkylating agent Dopamine agonist

Delitala et al. (1979) Delitala et al. (1982) Lafuente et al. (2001) Kaplanski et al. (1991) Lafuente et al. (2001) Pareek et al. (2007) Kaplanski et al. (1991) Pareek et al. (2007) Laskey et al. (1994), Yang et al. (2006) Gunnarsson et al. (2007) Pakarinen et al. (1994) Pakarinen et al. (1994) Delic et al. (1986) Pakarinen et al. (1994)

Flutamide Vinclozolin Cadmium TCDD Cyclophosphamide Cimetidine Cadmium Cisplatin

Antiandrogen Antiandrogen Transition metal Halogenated organic compound Nitrogen mustard alkylating agent Histamine (H2) antagonist Transition metal Alkylating agent

MEHP Cisplatin Colchicine 2,5-Hexanedione MEHP TCDD Cisplatin 2,5-Hexanedione MEHP Cadmium 1,3-Dinitrobenzene MEHP

Plasticizer metabolite Alkylating agent Plant alkaloid Metabolite of n-hexane Metabolite of DEHP Halogenated organic compound Alkylating agent Metabolite of n-hexane Metabolite of DEHP Transition metal Environmental pollutant Metabolite of DEHP

O’Conner et al. (2002a) O’Conner et al. (2002a); Kelce et al. (1998) Haouem et al. (2008); Sen Gupta et al. (2004) Choi et al. (2008) Elangovan et al. (2006) Sasso-Cerri and Cerri (2008) Janecki et al. (1992) Gotoh et al. (1990), Kopf-Maier (1992), Pogach et al. (1989) Grasso et al. (1993) Huang et al. (1990), Pogach et al. (1989) Allard et al. (1993) Hall et al. (1991), Johnson et al. (1991) Richburg and Boekelheide (1996) Lai et al. (2005) Huang et al. (1990), Nambu et al. (1995) Boekelheide (1988) Richburg and Boekelheide (1996) Wong et al. (2004) Foster (1989) Giammona et al. (2002)

Synthesis/secretion of FSH

Testis–Leydig cells

Cell death LH receptor expression LH receptor function PRL receptor expression/ function Androgen receptor antagonist Testosterone synthesis/ secretion

Testis–Sertoli cells

Testis–Sertoli cells

Cell death Blood–testis barrier

FSH receptor function Synthesis/secretion of ABP Microtubule/vimentin structure Sertoli cell secretions (MIS, lactate, transferrin, etc.) Sertoli cell function

Paracrine signaling

Testis–germ cells

Spermatogonial cell death

Differentiating germ cell death (Various stages)

Testis–germ cells

Differentiating cell death (Various stages)

Spermatogenic arrest Spermiogenesis

Accessory sex organs

Vascular bed or blood flow Enzyme activity; secretions Smooth muscle contractility Secretion into seminal fluid

Cisplatin Doxorubicin Bleomycin Etoposide DBCP Chlorambucil Methoxyacetic acid MEHP Doxorubicin 2,5-Hexanedione Cyclophosphamide Cetrorelix Acyline Cisplatin DBCP TCDD Lead Ethylnitrosourea Cadmium Gentamicin Neomycin Boric acid Deoxynivalenol BCA EDS Ritanserin Ketanserin TOCP Sertraline Paroxetine Methadone Heroin Thalidomide Cyclophosphamide Penicillin

Alkylating agent Anthracycline antibiotic Glycopeptide antibiotic Type II topoisomerase inhibitor Nematocide Nitrogen mustard alkylating agent Metabolite of EGME

Sawhney et al. (2005), Zhang et al. (2001) Hou et al. (2005), Shinoda et al. (1999) Russell et al. (2000b) Russell et al. (2000) Meistrich et al. (2003) Delic et al. (1986) Brinkworth et al. (1995)

Metabolite of DEHP Anthracycline antibiotic Metabolite of n-hexane Nitrogen mustard alkylating agent GnRH antagonist GnRH antagonist Alkylating agent

Richburg and Boekelheide (1996) Shinoda et al. (1999) Blanchard et al. (1996) Velez de la Calle et al. (1989) Pareek et al. (2007) Pareek et al. (2007) Sawhney et al. (2005), Seaman et al. (2003), Zhang et al. (2001) Meistrich et al. (2003) Choi et al. (2008) Adhikari et al. (2001) Rodriguez et al. (1983) Sen Gupta et al. (2004) Schlegel et al. (1991) Schlegel et al. (1991) Ku et al. (1993), Linder et al. (1990) Sprando et al. (2005) Tully et al. (2005)

Nematocide Halogenated organic compound Heavy metal Alkylating agent Transition metal Aminoglycoside antibiotic Aminoglycoside antibiotic Antiseptic, insecticide Mycotoxin Drinking water disinfection byproduct Alkylating agent 5HT antagonist 5HT antagonist Organophosphate SSRI SSRI Opioid analgesic Opioid analgesic Immunomodulator Nitrogen mustard alkylating agent -Lactam antibiotic

Yang et al. (2006) Collin et al. (1996) Collin et al. (1996) Somkuti et al. (1987a,b) Ozyavuz et al. (2004) Kesim et al. (2004) Ragni et al. (1985, 1988) Swanson et al. (1978a) Ragni et al. (1985, 1988) Lutwak-Mann et al. (1967); Teo et al. (2001) Hales et al. (1986) Green and Green (1985) (Continued )

Table 1

(Continued)

Site of action

Mechanistic target

Drug/chemical

Drug/chemical class

References

Accessory sex organs

Secretion into seminal fluid

Sperm

Sperm motility

Cocaine Tetracycline Clindamycin Aspirin Ciprofloxacin Phenytoin Boric acid Erythromycin EGEE Morphine TOCP Neomycin Nitrofurantoin Dicloxallin Cyclophosphamide Ethylnitrosourea Cimetidine Cadmium Boric acid TCDD DBCP TOCP Cyclophosphamide Vinyl acetate Deoxynivalenol Amitraz Ampicillin Novobiocin Erythromycin Heroin Morphine TOCP Deoxynivalenol Vinyl acetate Methadone Ethylnitrosourea

Dopamine reuptake inhibitor Tetracycline antibiotic Antibiotic Analgesic Quinolone antibiotic Antiepileptic Antiseptic, insecticide Macrolide antibiotic Antifreeze Opioid analgesic Organophosphate Aminoglycoside antibiotic Antibiotic -Lactam antibiotic Nitrogen mustard alkylating agent Alkylating agent Histamine (H2) antagonist Transition metal Antiseptic, insecticide Halogenated organic compound Nematocide Organophosphate Nitrogen mustard alkylating agent Industrial chemical Mycotoxin Pesticide -Lactam antibiotic Aminocoumarin antibiotic Macrolide antibiotic Opioid analgesic Opioid analgesic Organophosphate Mycotoxin Industrial chemical Opioid analgesic Alkylating agent

Yazigi et al. (1991) Eriksson and Baker (1967) Klemmt and Scialli (2005) Kershaw et al. (1987) Naber et al. (1993) Swanson et al. (1978b) Linder et al. (1990) Schlegel et al. (1991) Wang et al. (2006) Singer et al. (1986) Somkuti et al. (1987a,b) Schlegel et al. (1991) Schlegel et al. (1991) Schlegel et al. (1991) Elangovan et al. (2006) Ficsor et al. (1984) Van Thiel et al. (1979) Haouem et al. (2008) Yoshizaki et al. (1999) Choi et al. (2008) Kaplanski et al. (1991) Somkuti et al. (1987a,b) Elangovan et al. (2006) La¨hdetie (1988) Sprando et al. (2005) Al-Thani et al. (2003) Schlegel et al. (1991) Schlegel et al. (1991) Schlegel et al. (1991) Ragni et al. (1985, 1988) Singer et al. (1986) Somkuti et al. (1987a) Sprando et al. (2005) La¨hdetie (1988) Ragni et al. (1985, 1988) Ficsor et al. (1984), Panda et al. (1988)

Sperm count

Sperm fertilizing capacity Sperm viability Sperm

Sperm abnormalities

Sperm enzyme secretions

5HT, serotonin; DBCP, dibromochloropropane; EGEE, ethylene glycol monoethyl ether; EGME, ethylene glycol monomethyl ether; EDS, ethane 1,2-dimethane sulphonate; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; MEHP, monoethylhexyl phthalate; PRL, prolactin; ABP, androgen-binding protein; TCDD, 2,3,7,8-tetrachlorodibenzop-dioxin; TOCP, tri-o-cresyl phosphate; SSRI, selective serotonin reuptake inhibitor.

Evaluation of a Male Reproductive Toxicant

11.03.2.2 Study Design Considerations In a contract research organization, one often encounters questions that might come instinctively to a study director that is actively running male fertility studies, but that may pose challenges for general toxicologists or someone new to this field. What are the most important aspects of study design? How do I know how long I need to treat males with my compound? What criteria do you use for dose selection? Do we need to do sperm analysis? Do I have to evaluate all reproductive tissues microscopically? If we have no effect on pregnancy rate, do we still need to evaluate sperm parameters? Dose selection is perhaps the most important criteria during the design of a male fertility study. Doses that do not produce any signs of overt toxicity may be unacceptable to the regulatory agencies. On the contrary, doses that are too high may result in excessive moribundity or mortality resulting in an inadequate number of progeny being available for evaluation. Acute and repeat-dose toxicity studies provide useful insight into the effects of a xenobiotic on moribundity, mortality, and target organ toxicity. Furthermore, evaluation of organ weights and microscopic examination of various tissues provide useful information on systemic toxicity and aid in dose selection for fertility studies. In the absence of overt systemic toxicity, hematology and clinical chemistry data should be evaluated for determination of subtle toxicant-induced alterations. In the event that no evidence of systemic or target organ toxicity is available at the time of dose selection, the S5(R2) ICH Harmonized Tripartite Guidelines (ICH 2005) suggest that a limit dose of 1000 mg kg1 day may be adequate for reproductive toxicity studies if systemic exposure can be demonstrated. It is considered futile to increase the dosage beyond this limit if a test compound is inadequately absorbed and systemic exposure cannot be demonstrated. The duration of dose administration prior to cohabitation of males and females is another important consideration. For the majority of pharmaceutical agents, short-term exposure is expected. Thus, in general, only a 2- to 4-week precohabitation treatment period is recommended for males. This decision is further influenced by the fact that the majority of male reproductive toxicants are known to affect meiotic spermatocytes and postmeiotic spermatids, and thus direct effects on spermatogenesis, mating behavior, and fertility are likely to be detected following 2–4 weeks of treatment. For

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pharmaceutical compounds that are expected to have more long-lasting effects such as cytotoxic compounds intended for oncology indications or for compounds that are intended for use in maintenance programs for lifestyle diseases, a longer precohabitation treatment, of up to 10 weeks, may be desirable. A 10-week precohabitation treatment period is also recommended for chemicals where exposure might be expected to occur over a prolonged period of time (Takayama et al. 1995). Assessment of sperm quality parameters (sperm number, motility, and morphology) is a recommended endpoint in fertility and reproduction studies as suggested by the FDA, the United States Environmental Protection Agency (US EPA) as well as by the Organization of Economic Cooperation and Development (OECD). Furthermore, while the presence or absence of sperm, the rate of cell division in the germinal epithelium, and the sperm production rate can be assessed in multiple ways, there are no substitutes for evaluating sperm motility and morphology. In addition, with the use of computerassisted sperm analysis (CASA) one can even provide insight into progressive motility of sperm in a given sample (addressed in detail in Section 11.03.3.1.1). Since fertility is dependent not only upon a normal sperm count, but also upon functionally normal and motile sperm, assessment of sperm quality parameters makes the microscopic examination of reproductive organs somewhat redundant. Often by this stage in the development program, histopathology data are available from previous repeat-dose toxicity studies. It is prudent, however, to preserve all reproductive organs for future possible histopathologic examination. The second important question regarding sperm quality assessments is whether or not to conduct these assessments if no effects are noted on mating, fertility, and conception indices (addressed in detail in Section 11.03.2.3) or on litter sizes in mated females. It is important to remember the species differences that exist between nonclinical animal models and humans. In the historical control database for reproduction studies generated in the authors’ laboratory, daily mean sperm production rate for male rats is 13.4 million sperm per gram of testis. Humans, however, have been reported to have a daily sperm production rate that is only 25–35% of most species including rats and nonhuman primates (Johnson et al. 1992; Sharpe 1994). Furthermore, it has been demonstrated that in comparison with other species, human males produce fewer sperm relative to the number of sperm

68 Male Reproductive Toxicology: Strategies for Evaluation

required for fertility (Amann 1981; Working 1988). Thus, substantial reductions in sperm count or quality would be needed to adversely influence rat fertility (Klinefelter et al. 1994). However, it would be important to be able to detect a 5–10% reduction in sperm production in a rat, as these changes could be predictive of devastating consequences in humans. The number of animals assigned to the study, per group, per endpoint is another frequently considered aspect of study design. While meeting the minimum regulatory requirement, one should account for background variability in mating, fertility, and copulation indices and pregnancy rates. One should also consider that the minimum number of animals needed to provide adequate statistical power may be more than the minimum recommended number. Compound class effects should be discussed and utilized in study design, where possible, to allow for inclusion of endpoints that might provide insights into the toxicity of the compound, as well as adequate elucidation of compound-related effects. One should consider the benefits of assigning females that are cycling normally and are receptive to mating, to ensure that female-only effects do not produce bias in the dataset. Testes of males should at least be palpated to ensure that no abnormalities can be identified before assignment to study. One should be reminded that industry standards often vary vastly and are technologically more advanced than the required battery of tests required by any of the regulatory agencies. While regulatory guidance documents introduced in the early 1900s have evolved over the past several decades, use of more modern endpoints and newer technologies is often implemented in the industry prior to regulatory demand. Technologies such as CASA, endocrine endpoints (e.g., hormones) and blood and tissue dosimetry are just a few endpoints that have been included over the years. Contrary to conducting reproductive toxicity studies according to the ‘guideline minimum design,’ the authors’ laboratory frequently attempts to design studies that allow for maximum characterization/detection of toxicity as well as provide a greater probability of detecting rare event findings. Specific examples of this include full sperm assessment (motility, morphology, concentration, and production number), maintenance of >1 pup per sex per litter for F1 generation developmental landmarks, and incorporation of other biomarkers of exposure/effect based on available data from previous studies or other compounds in that chemical/pharmacological class.

11.03.2.3

The ICH Study Designs

The ICH 4.1.1 study allows for the determination of functional effects on male libido, mating, fertility, and sperm quality (motility, count, and morphology); endpoints that are not evaluated in acute and subchronic studies which typically include only histological examination of the male reproductive organs. Conceptually, the ICH 4.1.1 study involves treatment of adult sexually mature males and females prior to cohabitation to allow for manifestation of toxicant-related effects on spermatogenesis and/or ovulation, libido, and mating behavior. Typically, a 2- to 4-week precohabitation treatment period is recommended for males following which they are cohabited with naive or treated females and indices of reproductive performance are recorded. Mating is confirmed by evidence of a copulatory plug or by a vaginal lavage for sperm. Male mating index is defined as the percentage of the number of males with confirmed evidence of mating versus the total number of males that were cohabited. The male copulation index is defined as the percentage of males that sired a litter versus the total number of males that showed positive evidence of mating. The male fertility index is defined as the percentage of males that resulted in a successful pregnancy versus the total number of males that were cohabited. In addition, precoital intervals, defined as the number of days from the initiation of cohabitation until there is positive evidence of mating, are recorded. Females are generally euthanized in midpregnancy and intrauterine data are recorded. Males are euthanized following euthanasia of the females, and reproductive organ weights and spermatogenesis (number, morphology, and motility) are evaluated. The preferred and recommended species for an ICH 4.1.1 study is the rat provided that an appropriate pharmacologic or toxicologic response is elicited. The advantages associated with the use of rats for fertility and reproduction studies include cost-effectiveness, high fertility, short gestation period, large litter sizes, and the availability of extensive historical control data. However, the rat has also been shown to be an inappropriate model system due to its high susceptibility to dopamine agonists and an increased incidence of Leydig cell tumors (Holson et al. 2006). Where the rat is an inappropriate model system, or when an appropriate pharmacologic response cannot be elicited in the rat (for biologics such as monoclonal antibodies), other species such as

Evaluation of a Male Reproductive Toxicant

mouse, rabbit, dog, or the nonhuman primate may be used instead. When direct effects are expected in either sex, researchers often resort to conducting independent male and female fertility assessment studies in compliance with the ICH 4.1.1 guidelines. One or the other sex is treated, animals are mated with untreated naive animals of the opposite sex and mating, and copulation and conception indices are evaluated. Occasionally, the ICH 4.1.1 study is combined with the embryo/fetal development study, especially if no effects are expected on the process of implantation. Males (and females) are exposed as described above and exposure of the female continues, similar to a traditional ICH 4.1.3 developmental toxicity study, through closure of the hard palate (gestation day 17). It should be noted however that these two ICHdefined standard study designs fail to detect the effects on the reproductive organs in a male fetus. The critical window of development of the male reproductive tract lies between gestation days 16–18 (Carruthers and Foster 2005) and cessation of dosing at closure of the hard palate (GD 17) does not adequately encompass that critical window of exposure. In the ICH testing paradigm, only the ICH 4.1.2 (the pre- and postnatal development) study includes exposure from gestation to lactation (gestation day 6 to lactation day 20) and is thus ideal for identification of developmental effects in the male fetus. In this design, fetuses are exposed in utero during the critical window of male reproductive organ development between gestation days 16–18 and nursing. Once maternal treatment/exposure is stopped at weaning, F1 postnatal growth, development, and reproductive function are evaluated postexposure. This design is

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intended to identify hazards associated with latent/ permanent effects of in utero and lactational exposure. Some of the obvious differences in this multigenerational evaluation compared to the more traditional multigenerational studies described below are that the F1 generation is not directly exposed, and the mating and fertility assessment is testing for latent effects on reproduction whereas direct, functional effects on reproduction are more appropriately evaluated in the fertility design (ICH 4.1.1).

11.03.2.4 The Multigeneration Reproduction Studies In general, multigeneration studies are designed to assess the effects of long-term exposure to ambient low levels of industrial chemicals in the workplace, or to low-level pesticides or food additives in the diet (Holson et al. 2006). Specifically, these studies encompass the reproductive life cycle (F1) from preconception, in utero, peri- and postnatal development, puberty, sexual maturity, gestation and onward to the next generation as illustrated in Figure 1. Currently, multigeneration studies are required by the FDA (for food ingredients) and by EPA and OECD (for chemicals) OECD 2001b; US EPA 1998b; US FDA 2000. Overall, the three agencies have proposed very similar study designs that have been discussed extensively (Holson et al. 2006). Minor differences between the three study designs are presented in Table 2. The recommended species for these studies is the rat, although it may not always be the preferred model (e.g., based on mode of action). The multigenerational study is a labor-intensive study that is

Table 2 Differences across multigeneration study designs

Estrous evaluation

Breeding procedures

Sperm assessment Histopathology

Organ weights at weaning Optional assessments

US EPA (1998b)

US FDA (2000)

OECD (2001b)

Minimum 3 weeks prior to mating and throughout cohabitation No repairing of animals that show no evidence of mating All control and high-dose animals evaluated 10 Animals per sex per group that were selected for mating evaluated Two per sex per litter

Minimum 3 weeks prior to mating and throughout cohabitation No repairing of animals that show no evidence of mating

Period not specified; evaluated prior to mating and optional during cohabitation Females with no evidence of mating repaired with successful males 10 Males per group evaluated

Triggered 10 Animals per sex per group that were selected for mating evaluated One per sex per litter Extra options, functional tests, teratology, immunotoxicity

All control and high-dose animals evaluated One per sex per litter

70 Male Reproductive Toxicology: Strategies for Evaluation

not all inclusive of other potential developmental effects and is most often conducted in the absence of internal exposure/metabolism data. In recent years there have been many attempts to find suitable alternatives to conducting two-generation reproduction studies as a requirement for the types of agents described above. One alternative study design that appears to have scientific merit is the F1-extended one-generation reproduction study within the life stages testing paradigm proposed by the Agricultural Chemical Safety Assessment Technical Committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) (Cooper et al. 2006). Variations of this extended one-generation reproduction study have been discussed for a number of years as a possible alternative (Aoyama and Suzuki 2003; Janer et al. 2007), but one of the primary drawbacks appears to be implementation and experience with this assay to prove that it can fulfill the needs of risk assessment. Although there are many options to the extended one-generation study, the version described by Cooper et al. (2006) includes dosing and evaluation of three F1 animals per sex per litter postweaning to adulthood, robust evaluations of developmental landmarks (e.g., anogenital distance, nipple/areola retention, vaginal patency, balanopreputial separation), satellite toxicokinetic evaluation (for measurement of internal exposure), developmental neurotoxicity and immunotoxicity evaluations, and real-time data interpretation to trigger additional evaluations such as mating trials or prenatal developmental toxicity. Another multigenerational study design is reproductive assessment by continuous breeding (RACB). This unique study design was developed by the National Toxicology Program (NTP) more than 25 years ago for a similar purpose as the designs described earlier: to identify potential hazards to male and/or female reproduction and characterize those that cause toxicity through dose–response relationships (Chapin and Sloane 1996, 1997). Although the study design itself has evolved over the years, the robust functional assessment of mating and fertility has been maintained; specifically, mating pairs are kept together for the purposes of producing multiple litters (typically 4–5). Following postnatal development of pups produced from the last mating, these pups are used in another round of continuous breeding. In addition, exposed rats could be mated with naive rats of the opposite sex to isolate sex-specific effects on reproduction. Over the years it was recognized that these functional assessments were not always the most sensitive endpoint representing perturbation of the reproductive system,

so other critical endpoints (e.g., sperm assessment, estrous cyclicity, reproductive histopathology) have been incorporated as part of the standard design. Although the conduct of the RACB protocol has been largely limited to the NTP, it has generated a large, public database of reproductive toxicity data that has served as a useful tool over the years for risk assessment of specific chemicals as well as a greater understanding of rodent reproduction as a research toxicology model. 11.03.2.5

Screening Studies

The rodent dominant lethal assay (US EPA 1998c) was designed to detect mutagenic effects on male spermatogenesis that are detrimental to embryo survival, that is, embryo/fetal death is measured as an indicator of reproductive toxicity. Males are exposed to the test article for a short duration of 1–5 consecutive days followed by sequential mating trials that correspond to various stages of spermatogenesis, with the number of mating periods scheduled corresponding to at least one spermatogenesis cycle. Females are terminated in midpregnancy and examined for the number of implantation sites, the number of live/ dead conceptuses, and the number of corpora lutea. These numbers are used to determine decreases, if any, in the number of live conceptuses following treatment. While other causes cannot be completely ruled out, dominant lethality, or dead conceptuses, are generally considered to be the result of paternally derived chromosomal damage. In recent years, the use of the dominant lethal assay has seen a slow decline, primarily due to attempts to reduce animal usage and also because the ICH 4.1.1 has been accepted as a more robust and comprehensive approach not only to detecting effects on progeny survival, but also in elucidating effects on male reproductive organ weights, histopathology, and spermatogenic endpoints following a male exposure period that covers the length of a spermatogenic cycle. The design of the male fertility study also often better mimics real-life human exposure scenarios for pharmaceuticals. In addition to the more definitive studies described earlier (Sections 11.03.2.3 and 11.03.2.4), there are several relatively smaller study types focused on screening for possible endocrine, reproductive, or developmental effects. Ideally, screens would be used prior to investing the resources on larger definitive studies, but they are also often conducted to further characterize suspected modes of action based on data generated from apical endpoints

Evaluation of a Male Reproductive Toxicant

on definitive studies. General in vivo reproductive screens include the OECD 421 and 422 guideline studies and the in utero/lactation protocol (Tyl and George 2005). The OECD 421 reproductive screen is very limited, both in sample size and scope (e.g., no sperm assessments or histopathology on male reproductive organs). The OECD 422 screen is a 28-day repeat-dose study, combined with a mating and delivery segment prior to termination, and includes anatomical and clinical pathology. The in utero/lactation protocol is a more recent evolution stemming from the need for screening endocrine-active substances. Although data generated with this design are limited, it appears to be a more robust screening design in comparison to the OECD 421 and 422 designs, since animals are evaluated at least until weaning with the option to directly dose the F1 generation postweaning. Several more targeted screens are also available that have been shown to have substantial predictive power for effects on male reproductive function, as well as an increased sensitivity to detection. These include three bioassays currently being evaluated as part of the Endocrine Disruptor Screening Program (EDSP) by the US EPA for screening of toxicants for suspected endocrine activity in males (Hershberger, male pubertal, and 15-day intact adult male assays). The Hershberger assay is a historically well-established protocol (Gray et al. 2005, 2006; Hershberger et al. 1953) that has received much attention and has been undergone robust interlaboratory validation exercises for the development of a draft guideline and an optimized protocol (OECD 2008; Owens et al. 2006, 2007). This assay is designed to evaluate whether an agent is androgenic or antiandrogenic and utilizes weights of androgen-sensitive male reproductive organs. The male pubertal assay encompasses a repeat-dosing protocol that includes exposure from weaning to sexual maturity (1 month). Developmental landmarks (balanopreputial separation), reproductive hormones, organ weights, and histopathology of male reproductive organs as well as the thyroid gland are evaluated (Stoker et al. 2000). The strength of this design lies in the different endpoints which when evaluated together allow for detection of chemicals with antithyroid, androgenic, or antiandrogenic activity. Toxicants that alter pubertal development through their effect on gonadotropin and/or prolactin release and/or function or hypothalamic function could potentially be detected through this assay. The assessment of developmental landmarks combined with hormone assessments and histopathology make for a robust, yet labor-intensive

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screening assay. The 15-day intact adult male assay was developed a number of years ago and has also a robust history in evaluating many endocrine-active agents (O’Connor et al. 2002b). The strength of this bioassay lies in the measurement of several different hormones to create a fingerprint of responses that can lead to the prediction of modes of action when combined with an assessment of male reproductive organ weights. 11.03.2.6 Studies

Timing of Reproductive Toxicity

The FDA requires that fertility studies in males be conducted even in the absence of any observable effects on reproductive organs in the early repeatdose toxicity studies as these general toxicity studies do not allow for the assessment of functional fertility. Possible exceptions to this functional fertility assessment include biotherapeutics as discussed in Section 11.03.2.1. Early indications of injury to male reproduction may be noted in the development process during subchronic toxicity studies in the form of testicular and accessory organ weight changes and during subsequent histopathologic examination of the organs of the male reproductive tract. The recently updated ICH guideline M3(R2) provides basic guidance regarding the timeline of male reproduction and fertility studies with respect to the conduct of this resolution includes the requirement of standard histopathological examination of the testis and ovary in a repeated-does toxicity study (generally rodent) of at least 2 weeks in duration as this was found to be at least as sensitive as fertility studies to detection of adverse effects in male reproduction (ICH 2005; Sakai et al. 2000). Since exposure to most chemical entities and food additives is expected to occur at low levels, over an extended period of time, and human exposures are often ill-defined, the EPA, OECD, and FDA (food additives) do not have any stringent requirements regarding the timing of male and female fertility studies for chemicals. Instead, the quantity of manufactured materials usually triggers the assessment of reproduction and fertility. REACH is a new European Community regulation on chemicals and their safe use. It addresses the registration, evaluation, authorization, and restriction of chemical substances. The new law entered into force on 1 June 2007. The aim of REACH is to improve the protection of human health and the environment through earlier identification of the intrinsic properties of chemical substances. The REACH regulation gives greater responsibility to industry to manage the risks from chemicals and to

72 Male Reproductive Toxicology: Strategies for Evaluation

provide safety information on the substances. Manufacturers and importers will be required to gather information on the properties of their chemical substances, which will allow for their safe handling, and to register the information in a central database run by the European Chemicals Agency (ECHA). The regulation also calls for the progressive substitution of the most dangerous chemicals when suitable alternatives have been identified.

11.03.3 Endpoints and Methodologies Various in vivo and in vitro methodologies have been utilized to answer questions regarding the effects of a toxicant on male and/or female fertility and reproductive performance. In general, the nonregulatory in vitro approaches such as the androgen/estrogen receptor binding assays have been primarily used as screening tools or for the elucidation of mechanism of toxicant action. The reader is recommended to approach the wide variety of available literature on in vitro methodologies. The following sections present in vivo approaches that are widely accepted by the regulatory agencies for the conduct of male fertility and reproductive performance assessments. 11.03.3.1 Evaluation of Effects on the Adult Male 11.03.3.1.1 Functional spermatogenic assessments

The rat is the most common animal model evaluated in male reproductive toxicity studies due to similarities between rats and humans in underlying reproductive biology and responsiveness to reproductive toxicants. Consequently, the reproductive historical control databases for rats are typically more robust in most laboratories than for other animal models. The mouse, rabbit, dog, and nonhuman primate are alternative species used in fertility studies; however, these species have limited utility as they generally have lower fertility rates and numbers of live offspring compared to the rat. In addition, the dog and nonhuman primate are more expensive than rats. It should be noted that while the non-human primate (Cynomolgus sp.) is considered sexually mature, defined by complete histological maturation of the male reproductive tract by around 4 years of age, successful breeder males are often closer to 6 years of age (Meyer et al. 2006). The rabbit, dog, and

nonhuman primate, however, offer the distinct advantage of longitudinal-based evaluations of sperm parameters from ejaculates collected before, during, and after the treatment period; the optimal age for sperm assessment in various laboratory animals is presented in Table 3. The most commonly evaluated spermatogenic endpoints include sperm count (millions per gram of testicular or epididymal tissue), motility (percentage motile), and morphology (percentage morphologically normal). The sperm production rate (Blazak et al. 1985) is often calculated from sperm count data as follows: Sperm production rate ¼ Number of sperm per gram of testis=R

where R is the rate of turnover of the germinal epithelium in days, which in the male rat is 6.1 days. In rodents, the number of mature epididymal sperm generally correlates with the number of spermatogenic stem cells (Meistrich 1982), but a similar correlation has not been established in other species. No spermatogenic endpoint has emerged as a definitive indicator of male reproductive health, and evidence indicates that regulation of male fertility is multifactorial. Therefore, these spermatogenic parameters should be evaluated in conjunction with any available reproductive organ weight, histopathology, and fertility data to best assess male reproductive effects. The power to detect a drug-related effect in a study depends upon a number of factors, including the variability of the endpoint being assessed, the sensitivity of the method, and the sample size. Because animal models such as rodents and rabbits have a much greater reserve of sperm than humans, fertility indices are generally considered to be a relatively insensitive measure of reproductive health in rodents compared to sperm parameters. As a result, Table 3 Recommended minimum age for sperm assessment in various species Species

Age (min-)

Rat Mouse Rabbit Dog Monkey (cynomolgus) Human

12 weeks 10 weeks 6 months 10 months 6 years 18 years



Sperm can be obtained at younger ages but social/legal restrictions may apply.

Evaluation of a Male Reproductive Toxicant

the regulatory guidelines recommend that at least 16 litters per group be evaluated to adequately assess the effects on fertility. It is generally recommended that all males in a standard fertility study (e.g., at least 20 males) be evaluated for sperm motility, which is the minimum number of animals required to detect an approximate 10–15% decrease in the percentage of motile sperm (Seed et al. 1996; Working and Hurtt 1987). The same holds true for assessing effects on the percentage of morphologically normal sperm. For sperm enumeration, typically at least 12 males are required to attain sufficient power for detecting an approximate 10% decrease in sperm number; however, evaluation of all males in a group is the most recommended approach to maximize the power to detect a change in this endpoint. Collection of sperm samples for evaluation can occur at various locations along the male reproductive tract. Samples for sperm enumeration can be obtained from the testis, and samples for both sperm enumeration and motility can be obtained from the epididymis (cauda, caput, or entire organ), vas deferens, and, except for rodents, the ejaculate. No statistical differences were reported by Seed et al. (1996) in the motility or velocity values of sperm obtained from the cauda epididymis and vas deferens. Sperm samples obtained from the distal portion of the cauda epididymis and vas deferens are considered to have better sperm recovery and motility than proximal segments of these same organs (Slott et al. 1991). There is also some evidence that sperm motility parameters from different locations along the male reproductive tract (e.g., proximal vs distal cauda) may exhibit different sensitivities to toxic insult (Klinefelter et al. 1991). The absence of sperm can reflect a disruption in the activity of the seminiferous epithelium or, in the case of epididymal sperm, a posttesticular tubular obstruction. Sperm count in rodents typically refers to determination of the number of homogenizationresistant spermatids in late maturation stages of spermiogenesis. Mature spermatid nuclear DNA is highly condensed and resistant to damage from homogenization, while less mature round spermatids and other cell types can be destroyed by homogenization. Fresh or frozen homogenates prepared from the testis and/ or cauda epididymis can be used to estimate spermatid number. Sperm samples can also be obtained from an ejaculate in animal species such as the rabbit, dog, or primate. Sperm can easily be enumerated using replicate counting on a hemacytometer; however, the advent of CASA systems for sperm analysis, such as

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Hamilton-Thorne TOX IVOS system, has made enumeration of sperm much more efficient and less labor-intensive. Sperm motility is best assessed on sperm samples obtained through a diffusion method. Aspiration methods can cause shear force damage to sperm and affect motility. In the diffusion method, an incision is made in the epididymis, and the organ is placed into a specific volume of buffer. Most physiological buffered saline solutions at physiological pH have proven adequate for motility analysis over a short duration, although buffers do vary in their ability to support sperm survival for longer periods of time. Sperm (motile and immotile) are allowed to diffuse into the buffer for a period of time until adequately dispersed for analysis. Because sperm motility can be affected by temperature extremes, motility determinations are performed under constant temperature (generally approximately 34–37  C). Temperatures above 37  C can decrease sperm motility, and temperatures above 40  C can rapidly immobilize sperm. Sperm movement characteristics such as velocity and trajectory can vary tremendously within the same sample. Visual methods of evaluating sperm motility are inaccurate and highly subjective, making interlaboratory and interscorer comparisons problematic. Because of the laborious and subjective nature of assessing sperm motility, many research and toxicology laboratories have employed the use of CASA for assessing sperm kinematic parameters (Figure 2). With CASA, computerized images of sperm are recorded for a short duration, and the location of each sperm head within a microscope field is analyzed from frame to frame to generate a sperm track that is categorized based on specific movement criteria. The majority of sperm movement data generated by CASA is not reported in a standard male fertility study. The endpoint of interest in FDA guideline studies is percent motility – a motility score based on an estimate of the percentage of motile sperm within a sample, defined as follows: Percent motile sperm ¼

Number of motile sperm Total number of sperm counted  100

A sperm is defined as motile by CASA if the average path velocity (Figure 2) exceeds a user-defined threshold. Average path velocity, however, is not always predictive of the fertilizing capacity of the sperm. For example, recent data in humans have shown that the reduction in average path velocity

74 Male Reproductive Toxicology: Strategies for Evaluation

Average path velocity (VAP; µm s–1) Beat cross frequency (BCF; beats s–1)

Lateral head amplitude (ALH; µm)

Curvilinear velocity (VCL; µm s–1) Straight line velocity (VSL; µm s–1)

Figure 2 Sperm kinematic parameters measured by CASA. Straightline velocity (VSL) is the time-averaged velocity of a sperm head along the straight line between its first and last detected positions. Curvilinear velocity (VCL) is the average velocity measured over the actual point-to-point track followed by the cell. The average path velocity (VAP) measures the sperm head along its spatial average trajectory (i.e., smoothed version of VCL). Amplitude of lateral head displacement (ALH) is the maximum lateral displacement of a sperm head about its spatial average trajectory (i.e., track width). Beat cross frequency (BCF) is the timeaveraged rate at which the curvilinear sperm track crosses its average path trajectory. Calculated parameters include linearity (LIN), which is the linearity of the curvilinear trajectory calculated as VSL/VCL  100, and straightness of trajectory (STR), which is the linearity of the sperm average path calculated as VSL/VAP  100. The elongation ratio of minor to major axis of each sperm nucleus (ELO) and the size of each sperm nucleus (SIZ) in square microns can also be measured.

exhibited by the sperm of older men is not due to sperm swimming slower, but due to the increased curvature of the sperm path (Sloter et al. 2006). Therefore, the forward progress, and hence the fertilizing ability, of sperm can be hampered by excessive curving, even though sperm velocity is normal. Only sperm that exhibit a certain minimum velocity and forward progression are capable of fertilization. Progressive motility by CASA is defined as the percentage of sperm exceeding the user-defined thresholds for average path velocity (VAP) and straightness (STR) of trajectory (Figure 2). The EPA guidelines require that a measure of progressive motility accompany the percent total motility score. The data obtained using CASA is only as good as the user-defined setup parameters. Careful validation and testing must be performed to ensure that the settings are optimized for each animal species. For rodents, sperm sampling, processing, and analysis conditions should be optimized within each laboratory such that the percentage of motile sperm in untreated control animals is at least 70% (Working 1988). Common technical errors that result in poor sperm motility and count data include the following: clumping, uneven distribution of sperm, improper dilution, large amounts of debris or air-bubbles on the slide, uneven illumination of the sperm, incorrect chamber depth, out-of-focus microscope fields, or identifying nonswimming sperm (caused by vibrations, Brownian motion, or improper loading of the chamber) as motile. Sperm morphology is assessed in many different ways and depends primarily on the classification of sperm characteristics in the head, midpiece, and tail.

Generally, sperm samples used for motility analysis are also used for morphology assessment by light microscopy through a wet mount technique (Linder et al. 1992) or a modification of it. Abnormal forms of sperm are broadly classified into categories such as double heads, double tails, microcephalic or megacephalic, or normal-shaped head separated from flagellum. Because rats tend to have very low background levels of morphologically abnormal sperm, it is recommended that statistical comparisons across groups be based on the total number of morphologically normal and abnormal sperm in order to maximize power to detect treatment-related differences. A number of drugs (e.g., cyclophosphamide, thalidomide, and morphine) have been found to enter semen in varying concentrations depending on their chemical properties (lipid solubility and molecular weight). Seminal fluid consists mainly of secretions from the accessory sex glands and the epididymis. Xenobiotics may enter the seminal fluid through the prostate and seminal vesicles or cross the blood–testis barrier. The presence of xenobiotics in semen may result in female exposure through the reproductive tract, and there have been suggestions in the literature that exposure of the female through the semen may lead to adverse effects on the conceptus (see Section 11.03.3.2.2). 11.03.3.1.2 evaluation

Macroscopic and microscopic

At the time of necropsy, reproductive organ weights are recorded on all reproductive and fertility studies. Routinely weighed organs, in the authors’ laboratory,

Evaluation of a Male Reproductive Toxicant

include the brain, pituitary, testes, epididymides, prostate, and seminal vesicles. Organ weights are usually reported as absolute weights as well as relative to brain and body weights. Excessive toxicity, manifested in terms of reduced body weights or excessive malnutrition, may result in a concomitant reduction in some organ weights. Thus, organ weights relative to body weights provide a means of normalizing the data. Since brain weights in adult mammals are rarely affected even in severely affected animals, organ-to-brain-weight data are presented. Significant reductions in reproductive organ weights, in particular those of the testes and epididymides, may be indicative of reduced germ cell numbers within the testis and/or reduced number of sperm in the epididymis. Slight to moderate reduction in germ cell or sperm numbers may be nonadverse in rodents with animals still showing normal mating behavior and fertility. On the other hand, substantial increases in organ weights may be indicative of pathologic conditions such as edema, inflammation, or hyperplasia. Organ weights may also be increased in response to dramatic changes in the hormonal milieu. Increased reproductive organ weights in fetal or neonatal animals may be indicative of alterations in development. For example, hyperthyroidism during fetal or early neonatal life results in a greater than 150% increase in Sertoli cell number (Hess et al. 1993) and a corresponding increase in testis weight. Steps following the recording of organ weight include preparation of tissues for histopathology. Routinely evaluated tissues in the male include the testes, epididymides, prostate, seminal vesicles, coagulating glands, and the pituitary. While tissue preparation is important for each individual tissue type, this step is probably one of the most critical steps in the assessment of testicular histopathology due to the tendency of the seminiferous tubules to be easily damaged during tissue preparation. Thus, this section focuses primarily on the testes. The four main variables in tissue preparation are method of fixation, choice of fixative, choice of embedding media, and choice of histological stains (Russell et al. 1990). The most commonly used method of testicular fixation is immersion fixation; the tissue is simply dropped in the fixative. Tissue fixation: The size of the testes may prove to be a limiting factor in allowing access to the fixative and has been known to create artifacts (shrinkage, wrinkling, etc.) due to uneven or slow fixation. Shrinkage artifacts are seen increasingly within the intertubular interstitial area and closer to the periphery of the testis

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along the tunica albuginea. This process of immersion fixation may be accelerated by puncturing or nicking the tunica with a needle in several places to allow access to the fixative. It is also a common practice to allow the tissue to immersion fix for several hours, before creating several thick slices that can continue to soak in fixative before being prepared for embedding. While immersion fixation is adequate for light microscopic examination of the testis, it provides inadequate clarity and fixation of subcellular organelles and is thus an inappropriate method of fixation of tissues intended for ultrastructural examination. In smaller species such as the rat and mouse, vascular perfusion preceded by a heparinized wash to clear blood vessels is routinely used (Hess 1990; Sprando 1990; Xu and Xu 2001). In larger species such as the dog, it is impractical to use vascular fixation due to the volume of fixative required to infuse the whole animal, and perfusion through the testicular artery has been perfected by some investigators (Johnson et al. 1996; Lanning et al. 2002). Choice of fixative and embedding media: Buffered formalin has historically been the fixative of choice for both immersion and vascular perfusion of the testis. However, when combined with paraffin embedding, formalin results in exaggerated shrinkage artifacts which can be averted by embedding of formalinfixed testes in methacrylate (Chapin et al. 1984). Thus, both the choice of fixative and embedding media determine the degree of preservation and integrity of the tissue. Other factors influencing the choice of fixative and embedding media include the transparency, firmness, and the ability to section thinly. Embedding media such as methacrylate and epoxy are often used for applications requiring semithin (<2 mm) sectioning. Due to the far greater resolution afforded by these sections, perfusion with glutaraldehyde or glutaraldehyde variations is the recommended method of tissue fixation. Paraffin embedding is perhaps the most commonly used embedding technique that is compatible with a large variety of stains and allows sectioning as thin as 3 mm (routinely 5–10 mm). Bouin’s fixative is often considered to be the best fixative for the testis if the tissue is to be embedded in paraffin (Russell et al. 1990) and is also the fixative of choice in the authors’ laboratory. Modified Davidson’s fixative provides definite advantages over Bouin’s in that it is more convenient to use while the picric acid in Bouin’s is an irritant, is allergenic, potentially explosive, mutagenic, and possibly carcinogenic (Lanning et al. 2002; Latendresse et al. 2002). In comparison, tissues fixed

76 Male Reproductive Toxicology: Strategies for Evaluation

with Modified Davidson’s offer comparable resolution and relatively little shrinkage artifacts. Choice of histological stains: Standard histopathology examination of the testis uses hematoxylin and eosin staining, which provides gross nuclear and cytoplasmic staining. However, detailed histopathologic evaluation of the testis requires stains that draw attention to specific structures and cellular associations within the seminiferous epithelium. Periodic acid-Schiff’s (PAS) staining allows for the identification of polysaccharides and complex carbohydrate macromolecules by selectively oxidizing glucose residues to create aldehydes that can react with Schiff’s reagent to form a purple-magenta-colored complex. Within the testis, carbohydrates such as glycogen are typically found in the basal laminae and in the developing acrosome of differentiating spermatids. Differential staining patterns identify the various stages of spermatid differentiation revealing cellular associations at various stages in spermatogenesis. The additional use of hematoxylin as a light counter stain emphasizes nuclear structures within the tissue. Thus, PAS-hematoxylin (PAS-H) staining aids in identifying early disturbances in spermatogenesis in studies of short duration. For studies of durations longer than 90 days, subtle lesions noted as part of the early degeneration of spermatogenesis are rarely detected (Lanning et al. 2002), and carbohydrate staining with PAS-H does not provide any additional information over the standardly used hematoxylin and eosin (H & E) staining. Testicular histopathology is often considered to be a sensitive indicator of effects on spermatogenesis. Testicular tissue should be examined with prior knowledge of testis structure, the process of spermatogenesis, and the classification of spermatogenesis. The testis consists primarily of two main structural and functional compartments – the seminiferous tubules and the interstitial tissue. Sertoli cells support and sustain germ cells within the seminiferous epithelium and provide a specialized microenvironment by the secretion of hormonal and nutritive factors for the sustenance and viability of germ cells to enable cell division, maturation, and differentiation (Clermont 1993). Each Sertoli cell establishes contact with numerous germ cells at different stages of maturation (Staub et al. 2000). Sertoli cells divide rapidly during the late prenatal period and, in rodents, cease to divide by the second week of postnatal life. Since each Sertoli cell can support only a limited number of germ cells, the final number of Sertoli cells within the testis directly correlates with

sperm production rates and the number of spermatogonia in almost all species (Johnson et al. 1996). Spermatogenesis staging refers to the identification of cell-specific associations between developing and differentiating germ cells within a cross section of a single seminiferous tubule (Russell et al. 1990). It utilizes cellspecific markers such as the shape of the cell and the nucleus, the presence or absence of stage-specific subcellular structures, the development of the acrosome, and the shape of the nucleus of the elongating spermatids. In the authors’ laboratory, Bouin’s fixed, paraffinembedded, PAS-H-stained cross sections are most commonly used for testicular histopathology as well as staging. Standard H & E staining is conducted to obtain information regarding the overall state of the testis, the extent of cell loss, atrophy, presence or absence of a luminal space, condition of interstitial cells, and/or excessive vacuolation within the seminiferous tubules or within the interstitium. Staging of spermatogenesis, and the transient nature of some of the stages, makes for relatively subjective observations. Often findings may be non-stage-related, of varying severity and may require a concentrated effort to define and delineate the nature of the findings. It is often recommended that the same individual examine an entire study, and that the same individual define the criteria used to identify and grade the severity of observations before peer-review. Current regulatory guidelines require histopathologic evaluation of the male and female reproductive organs, either during the conduct of studies assessing effects of the toxicant on reproduction and fertility or during the long-term repeat-dose toxicity studies. The quantitative histopathologic assessment of spermatogenesis and spermatogenesis stages, however, is not an absolute requirement. While an understanding of spermatogenesis stages and the spermatogenic processes is essential in understanding and identifying toxicantrelated alterations in testicular histology, the knowledge of stage-specific insults is not considered necessary or relevant from a regulatory perspective, as long as information is obtained regarding any possible or putative functional deficits. On the contrary, spermatogenesis staging is frequently used in an academic setting to determine the first insult or the first visible effect on spermatogenesis. Furthermore, staging is beneficial in determining early disturbances, before any indications of functional deficits can be detected. In studies of duration longer than 90 days, where functional deficits begin to be clearly evident, the architecture of the seminiferous tubule may be too vastly altered to be staged.

Evaluation of a Male Reproductive Toxicant

Toxicant-induced alterations in testicular histopathology: Predominantly, there are three main cell types within the testis whose integrity is important to the maintenance of functional spermatogenesis. Two of these, the Sertoli cells and the sperm-producing germ cells, are contained within the main structural and functional compartment of the testis: the seminiferous tubules. The Leydig cells produce testosterone and are situated outside the seminiferous tubules in the interstitial compartment. Several different classes of toxicants (Table 1) have been shown to have direct toxic effects on each individual cell type. In the testis, the hallmark histopathologic signs of Sertoli cell injury are noted within the seminiferous tubules and include excessive vacuolation of the Sertoli cell cytoplasm, presence of giant multinucleated germ cells, missing germ cell layers, increased germ cell degeneration (apoptosis or necrosis), abnormal development of germ cells, apical sloughing and shedding of cellular material, primarily degenerating germ cells into the tubular lumen, reduced or failed spermiogenesis, and decreased seminiferous tubule fluid secretion. The size of the tubular lumen and the thickness of the seminiferous epithelium provide key information regarding the health of the Sertoli cells. Sertoli cells are relatively large elongated cells that extend from the tubular margin to the lumen. Each cell forms tight junctions with adjacent Sertoli cells, forming the blood–testis barrier that regulates the flow of nutrients and growth factors to cells with the blood–testis barrier (Petersen and Soder 2006). Each Sertoli cell contains an intricate system of cytoskeletal filaments (microtubules, actin and vimentin filaments) that aid in maintaining cell structure and polarity, and have also been implicated in the anchoring of germ cells to the Sertoli cell and in the translocation of differentiating germ cells along the length of the cell toward the lumen (Hess and Franca 2004). Toxicants such as cadmium (Janecki et al. 1992) and cisplatin (Pogach et al. 1989) compromise the integrity of the blood–testis barrier and chemicals such as colchicine (Allard et al. 1993), 2,5hexanedione (Hall et al. 1991; Johnson et al. 1991), and the phthalate esters (Richburg and Boekelheide 1996) cause cytoskeletal collapse due to effects on the microtubules and microfilaments, leading to collapse of cell structure resulting in an increased luminal size and consequently a thin-looking seminiferous epithelium. Recently, Sertoli cell apoptosis, in conjunction with cytoskeletal disarray, has been demonstrated using ultrastructural examination of rodent testis following exposure to cimetidine

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(Sasso-Cerri and Cerri 2008). This is the first report of Sertoli cell apoptosis in situ in response to any kind of physical or chemical insult. Toxicant-induced functional insufficiency is the most commonly reported sign of Sertoli cell injury. Since one of the primary functions of Sertoli cells is to provide endocrine and paracrine support for germ cell development and differentiation, toxicant-related alterations in Sertoli cell function also result in alterations in spermatogenesis and consequently reduced fertility. Reduced secretion of support factors such as androgen-binding protein (Huang et al. 1990; Pogach, et al. 1989), lactate, and transferrin (Huang et al. 1990; Lai et al. 2005; Nambu et al. 1995) and reduced Sertoli cell receptor function (Grasso et al. 1993) all lead to reduced germ cell supportive capacity and the consequent loss of developing germ cells. Signs of injury include missing germ cell subpopulations, Sertoli cell vacuolation, and general reduced cellularity of the seminiferous epithelium. Testicular germ cells are probably the first cell type to undergo apoptosis in response to toxicant injury. Different toxicants affect different cell types within the testis with the meiotic spermatocytes being the most commonly affected cell type. The prolonged meiotic prophase probably exacerbates the sensitivity of spermatocytes to a wide variety of chemicals, with the primary response being apoptotic cell death, based on the activation of a variety of apoptotic pathways. The nitrogen mustard, cyclophosphamide, has been shown to impair the G2/MI transition in pachytene spermatocytes due to DNA alkylation-related double-strand breaks, which eventually lead to initiation of apoptosis (Aguilar-Mahecha et al. 2005). Other alkylating agents such as cisplatin (Sawhney et al. 2005; Seaman et al. 2003; Zhang et al. 2001) and ethylnitrosourea (Rodriguez et al. 1983) have also been shown to cause increased apoptosis in actively dividing spermatocytes. Sertoli cell-only syndrome or the complete lack of noticeable meiotic spermatocytes and postmeiotic spermatids is most notably observed immediately following injury to toxicants affecting actively dividing and differentiating germ cells. Absence of specific cell stages could be indicative of increased death of a specific cell type, death of cells as they reach a certain stage of differentiation, or a failure of previous cell stages to differentiate. While this acute loss of germ cells can result in temporary infertility, the testis has the ability to repopulate itself with mature germ cells, provided that the stem cell population remains unharmed (Sawhney et al. 2005). Permanent

78 Male Reproductive Toxicology: Strategies for Evaluation

infertility and irreversible Sertoli cell-only syndrome are generally believed to be a consequence of apoptotic loss of stem spermatogonia (Sawhney et al. 2005) and the duration of azoospermia is believed to be related to the number of stem cells killed (Meistrich et al. 1989). Spermatogonial cell loss has been reported after exposure to antibiotics such as doxorubicin and bleomycin (Hou et al. 2005; Russell et al. 2000b; Shinoda et al. 1999), alkylating agents such as cisplatin and chlorambucil (Delic et al. 1986; Sawhney et al. 2005; Seaman et al. 2003; Zhang et al. 2001), nematocides such as dibromochloropropane (Meistrich et al. 2003), and DNA topoisomerase inhibitors such as etoposide (Russell et al. 2000a). Histopathologic alterations in other germ cell subtypes can be indicative of downstream effects on fertility and fecundity. The presence of elongated spermatids just below the surface of the lumen, with tails extended into the tubular lumen, is an identifying feature for spermatogenesis stages VII and/or VIII. At this point, the elongated spermatids are ready for release into the lumen from where they are translocated into the epididymis for further maturation. Presence of elongated spermatids, without any visible signs of tails extending into the lumen, might be an early sign of reduced motility. In a late stage VIII tubule in a rat seminiferous epithelium, these spermatids are usually accompanied by darkly staining residual cytoplasmic bodies. Presence of spermatids beyond stage VIII, when they are expected to have been released into the lumen, for example, stages X–XII in the rat, may be indicative of failure or slowdown in the process of spermiogenesis (spermatid release) which may lead to reduced epididymal sperm counts. Leydig cells reside in the interstitial compartment of the mammalian testes and are the primary source of testosterone. Leydig cell function is extensively regulated under the influence of hypothalamic, pituitary, and testicular hormones. The secretion and binding of the pituitary gonadotropin, luteinizing hormone (LH), to Leydig cells stimulates the production of enzymes responsible for the secretion of testosterone. Prolactin (PRL), also secreted by the anterior pituitary, has a stimulatory effect on Leydig cell steroidogenesis. Using microscopy and labeling techniques in mice, Faria et al. (2003) demonstrated that cellular death by apoptosis did not occur in Leydig cells in situ during the neonatal, peripubertal, puberty, or adult periods. However, these cells do have the capacity to induce and activate apoptotic pathways as has been demonstrated

using the synthetic glucocorticoid drug, methylprednisolone (Morris et al. 1997) and EDS (ethane 1,2dimethane sulfonate), an alkylating agent (Laskey et al. 1994; Morris et al. 1997; Yang et al. 2006). EDS, probably the most well-documented xenobiotic leading to apoptotic loss of Leydig cells, is frequently used in the laboratory as an investigative tool; the progressive loss of testosterone has been shown to cause a time-dependent increase in germ cell apoptosis (Woolveridge et al. 1999). Functional insufficiency of Leydig cells, noted in the inhibition of testosterone synthesis, for example, following exposure to the transition metal, cadmium (Haouem et al. 2008; Sen Gupta et al. 2004) or cyclophosphamide (Elangovan et al. 2006), is the more commonly noted response following toxicant exposure. Alterations in the expression and function of receptors for LH, FSH, PRL, or testosterone have also been noted following exposure to the dopamine antagonist bromocriptine (Pakarinen et al. 1994) or the antiandrogens, flutamide and vinclozolin (Kelce et al. 1998; O’Connor et al. 2002a). A detailed description of spermatogenic histopathology and spermatogenesis staging is beyond the scope of this chapter; however, for detailed morphological descriptions with light and electron micrographs, the reader is directed to the book by Russell et al. (1990). 11.03.3.1.3 of fertility

Functional assessment

The methodologies and endpoints used to detect functional alterations to male reproduction following exposure to test agents are very simple, yet powerful, apical tests. The most basic and powerful tests are those that evaluate the proportion of females that become pregnant (fertility index) and the changes in litter size (fecundity). Complementary functional evaluations may include sexual behavior (e.g., lordosis), precoital interval (time to evidence of mating), mating index (proportion of males with evidence of mating), and copulation index (proportion of males siring a litter from those with evidence of mating). There is also evidence in the peer-review literature that maleonly exposure can induce changes in litter size (preand/or postnatal mortality), fetal morphology, sex ratio, and growth retardation (see Section 11.03.3.2.2). It is well documented that relative to the methodologies described earlier (sperm assessment and microscopic examination) these functional endpoints of fertility/fecundity alone are relatively insensitive for detecting effects on male reproduction. However,

Evaluation of a Male Reproductive Toxicant

the most prudent course of evaluation couples functional assessments with microscopic evaluation and sperm assessment, as this suite of information has been shown to have the best predictive power of how a particular test agent may affect human reproduction (Ulbrich and Palmer 1995). As described earlier, the rat is the most common animal model for nonclinical evaluation of potential effects on reproduction. For the functional assessments this is most certainly the case with the mouse used less frequently followed by the rabbit and in some cases an abbreviated evaluation may be conducted with the dog, or with biotherapeutics, the guinea pig or now-human primate as described in Section 11.03.2.1. One of the key differences between the rats and the humans is that humans have a much lower sperm production rate (per gram testicular parenchyma) than rats. In addition, human sperm have a relatively high percentage of abnormal and nonmotile sperm, and the rat has a tremendous reserve of sperm stored in the caudal epididymis relative to the human. These biological features in the rat are the primary reason that the ability to detect a functional effect of a test agent on fertility is often less sensitive than specific evaluations of sperm (quality, quantity, or motility) and testicular histopathology. It has been demonstrated in surgically altered rats that a 90% reduction in the number of sperm available for ejaculation had no effect on breeding performance of males (Aafjes et al. 1980). Other investigators have corroborated that sperm output in rats or mice must decrease by >90% before a reduction in fertility is observed (Gray et al. 1992; Meistrich 1982; Robaire et al. 1984). There are also toxicological data that nitrobenzene-induced reduction of morphologically normal, motile spermatozoa produced per day by 80% only had a modest effect on breeding performance (Blazak et al. 1985). Evaluation of relationships among reproductive endpoints in the NTP database of reproductive toxicity tests has demonstrated a strong correlation between reduced epididymal sperm count (as small as 20% reduction) and reduced fertility/litter size over many studies in mice (Chapin et al. 1997). The authors surmised that this apparent difference with individual rodent studies described earlier was that the NTP dataset evaluated this correlation at a population level rather than in individual animals/ compounds, concluding that small reductions in sperm count might be expected to translate into fewer offspring in that population and should be considered adverse (Chapin et al. 1997).

79

Although these largely apical endpoints are sometimes considered to be insensitive, the approach used to design experiments can use these endpoints to answer very specific questions to address possible modes of action. One example is the reproductive assessment by continuous breeding (RACB) design used by the NTP where male rats go through repeated mating trials. Another example is to use serial weekly mating following a single or shortterm repeated dose and then evaluate the reproductive outcome (implantation sites or litter size). In both cases, it is possible to use the results of a continuous breeding protocol over time or the weekly mating trials to back calculate what stage of spermatogenesis was affected by treatment (e.g., late spermatocyte insult would manifest approximately 5 weeks following exposure) based on the known kinetics of spermatogenesis. It is not unusual for a test agent to elicit specific changes in male reproductive parameters (e.g., microscopic examination or sperm assessment) in the absence of a functional change in fertility; however, the most accurate safety/risk assessment of a test agent on male reproduction will include a functional assessment of mating, fertility, and fecundity in mature males following an appropriate exposure period. It is the synthesis of results following collection of all these endpoints that allow for the most appropriate interpretation. 11.03.3.2 Evaluation of Effects on the Developing Male In addition to direct reproductive effects on the adult male, the two other considerations are the ability of an agent to affect the developing male (e.g., in utero through puberty) and the ability of an agent to elicit toxicity through male-mediated effects on development (paternal origins of disruption of development/ survival of the next generation). 11.03.3.2.1

Effects on male development The male rat reproductive tract and secondary sex structures develop late in gestation. Specifically, fetal testicular testosterone peaks around day 17–19 of gestation in the rat triggering the subsequent development of the Wolffian ducts, followed by postnatal/ pubertal development and growth of the secondary sex structures (specifically descent of the testes and balanopreputial separation). Prins et al. (2008) demonstrated that prenatal exposure to estradiol during the critical window of prostate development

80 Male Reproductive Toxicology: Strategies for Evaluation

resulted in hypermethylation of the gene for phosphodiesterase type 4 variant 4 (PDE4D4), resulting in a higher incidence of neoplastic lesions in the prostate of the progeny. In standard pharmaceutical testing guidelines, any disruption of male reproductive tract development would likely not be detected in standard teratology studies that stop dosing around the time of closure of the hard palate (GD 17), rather multigenerational studies (see Section 11.03.2.4) are typically where this type of insult would manifest. A difficulty in assessing effects on male development (e.g., undescended testes, hypospadias, delayed balanopreputial separation, mating/fertility) is the latency between toxic insult and detection of functional deficits (Figure 1). Since these types of effects do not always penetrate the whole litter, when 1 pup per sex per litter is selected for the next generation following weaning, there is a random chance of selecting either responders or nonresponders, thus biasing results or reducing the sensitivity of identifying an effect on male development. In recent years, additional structural endpoints have been characterized and evaluated as markers for disruption of male development, specifically anogenital distance (AGD) and nipple/areolae retention (Gallavan 1999; Imperato-McGinley et al. 1986). These two endpoints can be measured preweaning while there is still robust litter representation prior to selection for the next generation and again as adults, if appropriate. Both of these endpoints are sensitive to reproductive hormones (dihydrotestosterone) in the normal male, androgenic growth of the peritoneum between the anus and the genital tubercle (AGD) and androgen-induced regression of the nipple/areolae. In addition to being very sensitive to alterations in male reproductive hormone status, both have been shown to be predictive to more severe structural malformations in rats such as hypospadias, undescended testes, and agenesis of male reproductive organs (Bowman et al. 2003; McIntyre et al. 2001). With regard to effects on human male development, hypospadias and cryptorchidism (undescended/ ectopic testes) are two of the most common male reproductive disorders in children. Decreased AGD in male babies with phthalate exposure has been confirmed (Swan et al. 2005) in a similar manner as would have been predicted from rat studies with phthalates (Mylchreest et al. 2000). In addition, the testicular dysgenesis hypothesis (Sharpe and Skakkebaek 2008) describes how hormone-active agents in the environment may be putative for the apparent increase in reproductive developmental abnormalities (decreased

sperm counts and increases in hypospadias, cryptorchidism, and male organ cancers). 11.03.3.2.2 Male-mediated effects on development

It is well established that exposure of the pregnant female to toxic chemicals can produce developmental as well as functional deficits in the developing fetus. Gestational and lactational exposure to various chemicals has been shown to cause fetal death, structural malformations, growth deficits as well as postnatal functional deficits. Exposure of the male and the downstream consequences on the offspring are less understood. Although several agents have been shown to adversely affect pregnancy outcome, contradictory reports exist in the literature and the exact role of paternal exposure remains undetermined. Commonly reported effects of paternal exposure include fetal/neonatal death (e.g., lead, X-rays), preand postimplantation loss (e.g., vincristine, ethanol, X-rays), growth retardation (e.g., ethanol), behavioral deficits (e.g., opiates, heavy metals, ethanol), and/or malformations (Agent Orange, 1,3-butadiene, ethanol, ethylnitrosourea). Table 4 provides a list of chemicals that have been implicated in causing adverse reproductive outcomes and developmental defects in the offspring following paternal exposure. In addition epidemiological studies in humans have demonstrated increased risks for congenital malformations, especially anencephaly following paternal exposure to agricultural chemicals (Lacasan˜a et al. 2006; Regidor et al. 2004,), organic solvents (Hooiveld et al. 2006), and electromagnetic fields (Blaasaas et al. 2002). Agricultural chemicals have also been implicated in an increased risk of fetal death (Regidor et al. 2004). Various xenobiotic agents have been implicated in causing damage to mammalian DNA. Intuitively, one would surmise that DNA damage in germ line cells could potentially cause adverse reproductive and developmental outcomes. The perfect example lies in the effects of ionizing radiation that has been shown to cause DNA double-strand breaks in germ cells. While most cells in the mammalian system have the ability to repair DNA, progressive loss of DNA damage repair capabilities has been demonstrated in postmeiotic mouse male germ cells (Marchetti and Wyrobek 2008). Thus, male germs cells are highly sensitive to DNA-damaging agents such as ionizing radiation, reactive oxygen species, and also chemical and environmental mutagens, and reduced DNA repair is expected to invoke the risk of paternally transmitted heritable damage (Tremellen 2008).

Evaluation of a Male Reproductive Toxicant

81

Table 4 Male-mediated developmental effects Drug/chemical Thyroxine Agent Orange X-rays Vincristine Morphine

SA

RI

+ +

RBW

CM

ND

DD

BD

+ +

+ + + + +

+ +

Ethylnitrosourea Ethanol 1,3-Butadiene Cyclophosphamide

RLS

+

Methadone Mercury Lead

PPL

+ +

+

+

+

+

+ +

+ +

+

+

+

+

+

+

+

Ethylene dibromide

+

+

+

+

References Bakke et al. (1976) Aschengrau and Monson (1990) Dobrzynska and Czajka (2005) Dobrzynska et al. (2005) Cicero et al. (1995), Lowery et al. (1990), Nelson et al. (1996) Joffe et al. (1976), Lowery et al. (1990), Nelson et al. (1996) Cordier (2008) Cordier (2008), Lindbohm et al. (1991), Min et al. (1996), Nelson et al. (1996), Stowe and Goyer (1971) Nagao and Fujikawa (1996) Abel and Lee (1988); Abel and Moore (1987); Bielawski and Abel (1997) Anderson et al. (1996) Lowery et al. (1990); Nelson et al. (1996) Lowery et al. (1990); Nelson et al. (1996)

SA, spontaneous abortions; RI, reduced number of implantations; PL, pre- and postimplantation loss; RLS, reduced litter size; RBW, reduced birth weights; CM, congenital malformations; ND, neonatal death; DD, developmental delay; BD, behavioral deficits.

An increased risk of DNA damage has also been noted for individuals with high levels of oxidative stress due to endogenous free radical generation (Tremellen 2008), lifestyle exposure to cigarette smoke (Ji et al. 1997), and ethanol (Maneesh et al. 2006). Increased oxidative stress has also been shown to damage sperm membranes, reduce motility, and also reduce the ability of a sperm to fertilize an egg (Tremellen 2008). Lifestyle exposures to cigarette smoke have also been shown to cause decreased motility and increased sperm aneuploidy and disomy (Robbins et al. 2005; Rubes et al. 1998). Chronic low-dose treatment to chemotherapeutic agents such as cyclophosphamide has also been shown to result in increased pre- and postimplantation loss (Hales et al. 2005a,b), congenital malformations (anasarca, hydrocephaly, growth retardation) (Anderson and Brinkworth 2001; Hales et al. 2005a), and a variety of tumors (Anderson and Brinkworth 2001). Furthermore, exposure to cyclophosphamide has also been implicated in induction of changes in expression profiles of DNA repair genes during preimplantation embryo development. Harrouk et al. (2000) demonstrated altered expression of transcripts for proteins involved in various DNA repair pathways in one- and eight-cell rat embryos following exposure of male rats invoking the possibility of transmittable modifications in sperm DNA.

Epigenetic modification of DNA refers to alterations in DNA methylation patterns or even chromatin remodeling, leading to changes in gene expression. It is a heritable phenomenon that is induced without changes in the primary DNA sequence (ReamonBuettner and Borlak 2007). Alteration in gene expression during fetal development has the potential of severe consequences on fetal morphogenesis, growth retardation, and even survival. It has been suggested that individual cell types in the developing embryo have individual epigenetic signatures that reflect the ultimate phenotype of each cell. Alterations in normal epigenetic imprints are suspected to have significant effects on the pre and postnatal organisms (Nafee et al. 2008). An exhaustive discussion of the role of male-mediated effects on development is outside the scope of this chapter; the reader is directed to the book edited by Robaire and Hales (2003).

11.03.4 Data Interpretation One of the primary objectives of a reproductive toxicity study, whether conducted as a male-only study or as a combined male/female study, is to quantitate reproductive outcome. Reduced pregnancy rates or reduced mating and/or copulation indices are initial signals of an adverse functional effect. In addition,

82 Male Reproductive Toxicology: Strategies for Evaluation

male reproductive effects may also be manifested as reduced litter sizes and increased embryo lethality upon examination of the females. At that juncture, this combination of findings presents the investigator with a pivotal decision point: whether or not the males should be re-paired with naive females and if further evaluation of functional effects is warranted. If the decision is made to proceed with the male necropsy at this stage, the next set of data (gross findings, sperm motility, morphology, and count and reproductive organ weights) may offer initial clues into possible effects on male reproductive function. These data represent the next decision point, which may trigger histopathologic examination of reproductive organs. Prior knowledge of histopathologic lesions noted during a repeated-dose toxicity study may offer guidance into this decision-making process. Histopathologic evaluation on a fertility study would ensure that the same animals that show functional deficits are evaluated for structural abnormalities that may explain the loss of function. On the contrary, the absence of functional deficits with a compound known to cause a lesion may trigger histopathologic evaluation on a fertility study. The investigators should bear in mind that fertility studies temporally separate functional assessments from terminal sperm assessments and organ weight evaluations. It is important to consider that a complete absence of systemic effects may trigger questions regarding the absorption and overall systemic exposure. It is thus beneficial to obtain exposure data where no prior data exist or to compare systemic effects with data from studies where exposure has been demonstrated. Once all these data have been obtained, the question remains how does one interpret the data? The reader must appreciate that the approach must be holistic. It is important to assess the entire spectrum of effects, or lack thereof, instead of evaluating each individual endpoint in isolation (weight of evidence). Knowledge of class effects or anticipated sequelae may help to elucidate the putative etiology of any observations. Holson et al. (2006) developed a ranked scheme of typical reproductive endpoints. The following is a modified list of male reproductive endpoints based on descending sensitivity: viable litter size/live birth index, assessment of sperm quality, weight/morphology (macroscopic and microscopic) of reproductive organs, mating/fertility indices and reproductive outcome, sexual behavior, sex ratio in progeny, and prenatal mortality.

A common example of confounding effects that influence the interpretation of reproductive toxicity data is a change in secondary sex organs correlated with reduced body mass. There are several possible explanations for the alterations that should be considered. One example is a direct effect on the reproductive process due to an antiandrogen (O’Connor et al. 2002a). Another possible explanation is declining body weights since these accessory sex structures are typically not spared under these conditions (O’Connor et al. 2002a). Feed restriction studies are often used as a surrogate for evaluating the possible impact of reduced body weights. Using feed restricted rats, Chapin et al. (1993a) demonstrated that prostate and seminal vesicle weights were reduced concomitant with reduced terminal body weights. Feed restriction of up to 30% had no impact on testicular and epididymal weights, fertility, and sperm count. These results were corroborated in a companion feed restriction study conducted in mice (Chapin et al. 1993b). More recently, Rehm et al. (2008) also demonstrated that seminal vesicle, ventral prostrate, and epididymal weights were reduced concomitantly with body weight. Plasma testosterone concentrations were also demonstrated to be lower in feed-restricted rats. In addition, degeneration of stage VII pachytene spermatocytes was also noted in feed-restricted rats. Interestingly, the duration of lower food intake and the initial age of lower food intake were shown to have differential consequences. Alternatively, systemic toxicity induced by chemical agents can produce a broader spectrum of effects than those observed with simple feed restriction. Interpretation of effects on male reproduction is usually based upon the assumption that a threshold of effects exists and that a greater severity or a broader complement of effects may be elicited with an increase in dosage levels. It is thus important to assess the entire spectrum of effects, or lack thereof, instead of evaluating each individual endpoint in isolation. A flat dose–response may indicate saturation of absorption or metabolic pathways, and it may be necessary then to further characterize the dose–response by assessing lower dose levels. For pharmaceutical compounds intended for acute or short-term exposure, any observed morphological or functional deficits may trigger the need to evaluate reversibility of effects in a subsequent study. One should consider the possibility of an atypical control group if all treated groups are similarly affected with respect to

Evaluation of a Male Reproductive Toxicant

the concurrent control and the control values lie outside of historical control ranges. For the most part, the underlying biology and responses to toxicants between humans and rodents are believed to be comparable (Holson et al. 2006). However, interspecies variation in responsiveness may arise due to physiological, hormonal, or metabolic differences between humans and rodents (Working 1988). Several examples have been cited in the literature of compounds that do not produce the same toxic metabolite or the ultimate toxicant may be more rapidly biotransformed in one species as opposed to others.

11.03.5 Conclusions Assessment of the potential of an agent to produce male reproductive toxicity requires an in-depth evaluation of the entire spectrum of effects noted during repeated-dose as well as reproductive toxicity studies. Study-specific data should be evaluated in the context of concurrent as well as historical control data. In addition, compound class effects should be taken into consideration. Assessment of individual endpoints should be avoided and the interpretation should be drawn from the overall weight of the evidence. The most sensitive endpoint of toxicity determines the no-observed-adverse-effect level, which is used for the purposes of human risk assessment. Compounds of the highest level of concern for reproduction would be those that cause reproductive toxicity below a threshold dose where generalized systemic effects are observed. In contrast, estimation of reproductive risk for agents that elicit reproductive toxicity concomitantly with other measures of systemic toxicity is less clear and further studies may be required for proper characterization of reproductive risk.

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