The recognition that body weight is important in analyzing and understanding acquisition of puberty is strengthened by the work of Kennedy and Mitra ( 1963) and Carney et al. ( 1998) who showed that body weight and food intake are factors in the initiation of puberty in the rat, and by the work of Holsapple et al. ( 1998) who put groups of 26 timed mated SD rats on standard diets at 100% ( control), 70%, or 50% of historical control feed intake levels from gd 7 through weaning on pnd 21. Selected weanlings were continued on feed restriction until ten weeks ( with 100% feed from ten to 20 weeks of recovery) or until 20 weeks of age, with necropsy of all offspring at 20 weeks of age. Feed restriction resulted in reduced weight gains for dams and pups related to the degree of restriction. In both the 50% and 70% feed restriction groups, gestation length was significantly increased, and age at VP and PPS was also delayed ( by one day at 70% restriction and by six days at 50% restriction for both parameters). AGD at birth was significantly reduced in both sexes in the 50% restriction group, but AGD: body weight ratios were essentially identical across groups, indicating that smaller ( low body weight) pups had shorter AGDs and that the effects were proportional. The authors conclude that " these results show that certain reproductive and developmental endpoints are altered by feed restriction in the range relevant to common testing scenarios" ( Holsapple et al. 1998).

<IP_ADDRESS> Vaginal Patency in Females In females, acquisition of puberty is indicated by vaginal opening or patency ( VP), dependent on E2 and resulting from E2 dependent cornification of the vaginal seam. In control CD ® ( SD) rats in the authors' laboratory, the grand mean age at VP is 31.1 days ( based on 16 studies from 1996 to 2000). VP may be observed first as the appearance of a small " pin hole( s)" or perforations but is recorded as acquired when vaginal opening is complete. Vaginal threads across the vaginal opening may be temporary or persistent ( Wolf et al. 1999; Flaws et al. 1997).

Vaginal opening may be advanced by estrogenic compounds, and estrogen receptor modulators either advanced or delayed with various environmental chemicals ( see Review by Goldman et al. 2000). In a study of the effects of BPA on sexual development in two strains of rats, SD and Alderley Park ( AP), Tinwell et al. ( 2002) found strain related differences in VP. Vaginal opening was significantly delayed in AP rats and not SD rats exposed to BPA. There was no effect on age of first estrus ( an explanation of this endpoint follows). 44 <IP_ADDRESS> Age of First Estrus in Females On or within a few days of VP, the female exhibits her first estrus, so age at first estrus ( absolute age and/ or interval from VP to first estrus) is also useful. Late follicular growth of the first ovulatory cells is stimulated about the time of vaginal opening, although there is some variation in the initial release of oocytes. Following vaginal opening, daily vaginal smears are monitored to determine the age of first estrus and/ or first vaginal cycle. Irregular estrous cycles are often seen in the immediate postpubertal period ( Goldman et al. 2000).

<IP_ADDRESS> Preputial Separation in Males Acquisition of puberty in males is indicated by preputial separation ( PPS; balanopreputial separation) or separation of the foreskin of the penis from the glans. PPS is dependent on androgens. PPS is a process that leads to the cleavage of the epithelium through cornification, forming the squamous lining of the prepuce of the penis ( Goldman et al. 2000). As a sign of puberty and an essential prerequisite for further development of the ejaculatory process, PPS has been used as a reliable, noninvasive endpoint by which to monitor rodent pubertal development and perturbations of this process. This landmark of acquisition generally occurs during the peripubertal period ( pnd 36 55 or 60; Stoker et al. 2000). In control CD ® ( SD) rats in the authors' laboratory, the grand mean age at PPS is 41.9 days ( based on 16 studies from 1996 to 2000).

Estrogenic and anti androgenic compounds have been shown to delay PPS, while androgen receptor agonists accelerate PPS ( see review by Stoker et al. 2000). Tinwell et al. ( 2002) found that BPA had no effect on PPS in two strains of rats ( AP and SD) at a dose that delayed vaginal opening in female AP rats only. In AP rats, vaginal opening was at 33.8 ± 0.8 days in control animals, compared to 35.4 ± 0.6 days in rats exposed to 50 mg/ kg/ day BPA. Sensitivity to BPA was found to be not only strain related but sex related.

2.5.8 Estrous Cyclicity and Ovulation Rate in Postpubertal Females After the initial release of ova, female rats begin to exhibit four to five day estrous cycles, with accompanying changes in vaginal cytology and circulating hormones. The acquisition of estrous cyclicity results from shifts in the hypothalamic pituitary ovarian endocrine axis and is the culmination of the maturation of reproductive processes that began prenatally. As indicated above, irregular estrous cycles are more common in the first weeks after acquisition of puberty.

Ovulation rate is affected by the dose and ratio of FSH to LH, stage of the cycle, and age of the female. Large genetic differences in ovulation rate exist between strains of mice and in response to exogenous gonadotropins ( Spearow et al. 1999; Spearow and Barkley, 1999). 45 Ovulation rate ( the number of eggs ovulated per female) is not included in the endpoints discussed in this White Paper because the protocols of the proposed EDSP assays preclude measurement of ovulation rate in order to measure other relevant endpoints. Ovulation rate is based on the number of corpora lutea counted on the ovaries. These are postovulation ruptured follicles ( one per ovulated ovum) producing large amounts of P4 and lesser amounts of E2, to prepare the uterus for implantation of the conceptuses. However, maternal ovarian corpora lutea involute beginning at delivery of offspring ( with involution completed on or about pnd 4) to become corpora albicans, indistinguishable from corpora albicans from previous ovulation cycles. All of the studies in Tiers I and II that involve production of offspring require that the dams remain with their pups through lactation to weaning. The parental females are necropsied at the weaning of their litters on pnd 21, when the corpora lutea are no longer present on the ovaries. The inability to collect ovarian corpora lutea counts in these studies also precludes calculation of percent preimplantation loss:

What can be ascertained, and is therefore included in the list of endpoints to be discussed, is percent postimplantation loss, which is based on the number of uterine implantation sites ( i. e., the number of conceptuses implanted; these " nidation scars" persist at least 40 days after delivery) and the number of total pups delivered. Both of these parameters are present and recorded in the Tier I and II studies involving generation of offspring. The calculation for percent postimplantation loss is:

In studies comparing estrous cycles across rat strains, there were strain related differences in estrous cyclicity in response to food deprivation ( Tropp and Markus, 2001). Prior to food restriction, Brown Norway rats had irregular estrous cycle patterns while SD, LE, and F344 rats had regular estrous cycle patterns. By day 5 of food deprivation, 75% of F344 rats and 100% of Brown Norway rats stopped cycling and SD and LE rats were unaffected ( the animals' weights were reduced to 85% of ad libitum body weight). It is possible that SD and LE rats, which have generally larger body masses may have more energy store and therefore be less sensitive to changes in body weight. Another possibility is that sensitivity to food deprivation is higher in inbred versus outbred strains. However, given the fact that food deprivation schedules were adjusted to account for differences in initial body weight, it would seem unlikely that simple strain differences in body weight account for the results. These data suggest outbred strains selected for larger litter size are relatively resistant to the disruption of estrous cyclicity by dietary restriction. 46 In a study comparing estrous cycles in Lewis and F344 rats, by obtaining vaginal smears and quantitating E2, P, FSH, and LH levels at different phases of the cycle, Smith et al. ( 1994) reported that metestrus was significantly longer, while diestrus and estrus were significantly shorter in Lewis rats compared to F344 rats. Proestrus was similar in both strains. During estrus, E2 levels were significantly higher in Lewis compared to F344 rats, and P levels were significantly higher in all stages of the estrous cycle in Lewis compared to F344 rats. LH and FSH levels did not differ between strains at any stage of the estrous cycle. The authors suggest that elevated E2 and P levels may affect corticosterone levels which could affect hypothalamic pituitary adrenal axis responsiveness.

In response to endocrine disrupting chemicals, strain differences in the ovarian cycle have been reported. Cooper et al. ( 2000) reported that LE rats were more sensitive than SD rats to atrazine induced disruption of the ovarian cycle. In addition, Ando Lu et al. ( 1998) found that in aging Donryu rats, estrous cycle abnormalities ( e. g. persistent estrus) were more common than in F344 rats. Finally, in a study by Eldridge et al. ( 1994), atrazine administration to SD and F344 rats for up to 12 months produced changes in estrous cyclicity in SD rats ( increased the number of days of vaginal estrus), increased E2, decreased P, and increased incidence of mammary tumors in SD rats only, with no significant treatment related effects in F344 rats.

BPA, an environmental estrogen, has been found to stimulate Prl secretion in F344 but not SD rats ( Steinmetz et al. 1997). More recently, Long et al. ( 2000) found that BPA increased DNA synthesis and cell proliferation in the vaginal epithelium of F344 rats but not SD rats. Thus, the rat vagina, an estrogen target tissue, is more sensitive to the effect of BPA in a strain specific manner. Long et al. ( 2000) also showed that F344 and SD rats showed no difference in clearance of 3H BPA from the blood, concentration or affinity of estradiol receptor, or induction of early gene c fos in response to BPA. Since BPA increased vaginal cell proliferation and DNA synthesis in F344 but not in SD, these data show that strains differ in the intermediate effects of these xenoestrogens downstream of the ER.

Differences in estrous cyclicity have been reported in outbred strains of mice which have been selected for large litter size, high embryo survival, or small litter size ( Barkley and Bradford, 1981; DeLeon and Barkely, 1987). Selection for large litter size ( Line S1) and high embryo survival ( Line E) increased the regularity of estrous cycles, and selection for small litter size ( CN) dramatically decreased the regularity of estrous cycles. Therefore, the BN rat, F344 rat, or the CN mouse may provide better animal models than strains that have been bred for large litter size.

2.5.9 Andrology Depending on the age of the male rodent when sampled, the cauda epididymis (> 80 days old) or the entire epididymis ( 65 80 days old) is evaluated for total number of sperm per cauda or per gram cauda, motility and progressive motility ( as percent of total sperm examined. This evaluation must be done within two minutes of animal's demise 47 with microscope slide and buffer kept at 37 ° C). Percent malformed sperm should be examined usually manually by microscopic examination of 200 fixed and stained ( Eosin Y) sperm per male. In addition, one testis at necropsy should be frozen and subsequently homogenized in buffer and evaluated for homogenization resistant spermatid head counts ( SHC) to calculate daily sperm production ( DSP) and efficiency of DSP.

For andrological studies, epididymal sperm counts and testicular homogenizationresistant spermatid head counts are found to be good markers for altered spermatogenesis. Wilkinson et al. ( 2000) compared the outbred strains of Wistar and SD with the inbred strain Dark Agouti ( DA). While a small number of SD were used in this study, the DA rat has lower absolute and relative (% body weight) testes weight, and more variability in sperm counts but there was no significant difference in testicular histology, sperm count per gram of testis, or epididymal sperm count. There were also no differences in weights ( relative to body weight) of the epididymis, seminal vesicles, or ventral prostate or of testosterone values for whole blood. DA rats are deficient in CYP2D1 activity, and several P450 cytochromes may also be absent.

In the Tinwell et al.( 2002) study that found that the weak xenoestrogen BPA had no effect on PPS in two strains of rats ( AP and SD), they reported that 50 mg/ kg BPA decreased total sperm count and daily sperm count in AP rats but not in SD rats. Thus, there were strain related differences in the effects of BPA in rats.

Apostoli et al. ( 1998), in a review article on the toxicology of lead, stated that SD rats appeared to be relatively resistant to the toxicological effects of lead. However, in general, lead impaired spermatogenesis and decreased androgens in other rat strains ( e. g. Wistar and Charles Foster rats). Concentrations of blood lead > 40 : l/ dl were associated with decreased sperm counts, volume, motility, morphology and endocrine effects.

In mice, strain differences in andrological parameters have been observed. For example, CD 1 mice have been shown to be much greater than 16 fold more resistant than C57Bl/ 6J ( B6) or C17/ Jls strain mice to the inhibition of spermatogenesis by pubertal exposure to estradiol ( Spearow et al. 1999). Additional studies in Spearow's laboratory have confirmed these observations and have also shown CD 1 mice to be much more resistant to the inhibition of testes weight, elongated spermatids per seminiferous tubule crosssection and epididymal sperm counts than outbred wild derived Mus spretus/ RP/ Jls mice.

2.5.10 Organ Weights and Histopathology ° Reproductive ( including accessory sex organ weights). Reproductive organ weights should be obtained at adulthood and should include: ( a) ovaries and uterus for females and ( b) testes, epididymides ( total and separated into caput, corpus and cauda), prostate ( whole, and dorsolateral and ventral lobes separately; dissection may be postfixation), seminal 48 vesicles, coagulating glands, preputial glands, bulbourethral ( Cowper's) glands, and levator ani/ bulbocavernosus ( LABC) complex for males.

° Thyroid. Thyroid hormones ( T3 and T4) are necessary for normal growth, development, differentiation, and regulation of most organ systems ( Goldman et al. 2000; Stoker et al. 2000). Disruption of the feedback control of thyroid function may result in either a hypertrophic ( goiter) or hypotrophic thyroid, depending on the mechanism of disruption. These changes would be evident in the weight of the thyroid gland. Since the thyroid gland surrounds the trachea, the thyroid plus embedded trachea is fixed and the trachea dissected away post fixation. The thyroid can then be weighed with little or no damage to the organ for subsequent histopathology.

° Systemic ( liver, kidneys, brain, etc.). Systemic organ weights should be obtained at adulthood in both sexes and should include liver, kidneys, adrenal glands, pituitary, brain ( regions), etc. Comparison of the effect of the test compound on these organ weights ( absolute and relative) to effects on reproductive organ weights will provide a more complete characterization of toxicity and suggest whether observed toxicity is more or less targeted to the endocrine system.

° Absolute and relative to body weight ( and brain weight). Organ weights ( both reproductive and systemic) should be presented as absolute and relative to terminal body weight and brain weight. Relative organ weights will correct for effects on body weights ( i. e., systemic toxicity). Brain weight is generally considered more stable than body weight after exposure to exogenous compounds and provides a basis for determination if changes in organ weights are primary or secondary to altered body weights.

In a study by Putz et al. ( 2001), the estrogenic effects of neonatal exposure to ß estradiol 3 benzoate ( EB) were studied in two rat strains, SD and F344. Neonatal rats were injected with EB ( over a 7 log range of doses from 0.015 : g/ kg/ day to 15 mg/ kg/ day in SD and a 5 log range of doses from 0.15 : g/ kg/ day to 1.5 mg/ kg/ day in F344) on pnd 1, 3, and 5. While F344 were not examined on pnd 35, SD male rats on pnd 35, exhibited significant increases in absolute and relative testis and epididymis weights at the low dose, 0.015 : g/ kg/ day, and significant reductions at higher doses ( 1.5 and 15 mg/ kg/ day). Since hepatic testosterone hydroxylase activity was increased in the low dose animals, it may have advanced puberty, therefore resulting in increased organ weights. On pnd 90, in SD males exposed neonatally to the highest dose used in both strains ( 1.5 mg/ kg/ day), there were significant reductions in absolute and relative seminal vesicle, and coagulating gland weights, but not in testis or epididymis weights. SD rats on pnd 90 also showed an increase in testis and epididymis weights at the lowest dose ( at one order of magnitude lower than the increase observed on pnd 35). This dose was not tested in F344 rats. In F344 males, the reduction in male reproductive organ weights ( absolute and relative to body weight) at pnd 90 was greater at the highest dose ( 1.5 mg/ kg/ day) than in SD rats. Whereas relative testicular weights 49 were 44% of controls in F344 rats, they were 67% of controls in SD rats. Similarly, 1.5 mg/ kg EB reduced epididymal weights to 36% of controls in F344 versus 87% of controls in SD rats. Pnd 90 testis and epididymal weights were reduced much more by 1.5 mg/ kg EB in F344 than at a 10 fold higher dose ( 15 mg/ kg) in SD rats. Thus, SD rats were greater than 10 fold more resistant than F344 to the inhibition of testes weight by EB.

Strain related differences in absolute pituitary weights have been reported in ovariectomized rats exposed to E2 ( silastic implants) for 10 or 20 days ( Schechter et al. 1987). Pituitary weights were dramatically increased in F344 rats, with comparatively minimal effects in SD rats, and Prl levels were dramatically increased in F344 rats ($ 1000 fold), while only moderately increased in SD rats ( 100 fold). In addition, E2 implants in F344 strain rats produced a dramatic hyperplasia of anterior pituitary lactotropes, activation of phagocytic folliculo stellate cells ( FS), increase of cells positive for basic fibroblast growth factor, and reorganization of the blood supply from vessels in the adjacent meninges. Estradiol treated SD rats did not show comparable responses ( Schechter and Weiner 1991). Pituitary weights were also different across strains in ovariectomized rats exposed to E2 ( 10 mg s. c. pellet) for four weeks ( Yin et al. 2001). Control pituitary gland weights were the lowest in Brown Norway rats ( 4.4 ± 0.2 mg), and more than two fold higher in Wistar rats ( 13.0 ± 2.1 mg); F344 control pituitary weights were 7.5 ± 0.1 mg, and Donryu 10.7 ± 1.0 mg. After exposure for four weeks to E2, there was a significant > 3 fold increase in pituitary weights in F344 rats, a significant > 0.5 fold increase in pituiary weights in Brown Norway rats, and no difference in pituitary weights of Wistar and Donryu rats. The F344 strain was the most susceptible to estrogen induction of pituitary tumorigenesis, followed by Wistar and Brown Norway. The work of Schechter et al. and Yin et al. demonstrates that the pituitary gland of F344 rats is more sensitive to the effects of E2.

Differential effects of DES in particular rat strains have been demonstrated in studies by Gorski et al., who showed strain differences in estrogen dependent pituitary mass ( Edpm) and pituitary tumor growth ( Wendell et al. 1996; Wendell and Gorski 1997; Chun et al. 1998; Wendell et al. 2000). While F344 are highly susceptible to DESinduced pituitary growth/ tumors, Brown Norway ( BN) and SD rats are highly resistant. Following DES treatment, F344 strain rats and F344 x BN F2 rats with largest pituitary tumors showed a reduction in retinoblastoma susceptibility gene product ( pRb) ( Chun et al. 1998). QTL linkage analysis in a F344 x BN F2 mapped several additive and epistatic loci controlling Edpm, including susceptibility alleles from F344 and from BN ( Wendell and Gorski 1997). Through QTL mapping in a ( F344 x BN) x F344 backcross Wendell et. al. ( 2000) showed that several QTL including Edpm2 1, Edpm3, Edpm5, and Edpm9 2 all had significant effects on pituitary mass. While Edpm2 1 and Edpm9 2 primarily affected DNA content, Edpm5 primarily affected hemoglobin/ DNA ratio, and Edpm3 affected all of these traits equally ( Daun et al. 2000). These data defining genes controlling susceptibility to estrogenic agent induced tumors among genetically defined strains provide a powerful tool for understanding genetic differences in susceptibility to endocrine disruption by estrogenic agents. These data also have value as historic controls. Since these studies used genetically defined isogenic parental strains, they 50 can easily be repeated and enhance the identification of genes controling susceptibility to environmentally induced disease in humans as well.

In a review by Kacew et al. ( 1995), strain related differences in mammary tumorigenesis were summarized. SD rats are more susceptible to mammary tumorigenesis after exposures to 2 acetylaminofluorene, 1,4 bis( 4 fluorophenyl) 2 propynyl N cyclooctyl carbamate, and atrazine than were F344 rats. In addition, Wistar and SD rats were more susceptible than Copenhagen or LE rats to the effects of DMBA ( 7,12 dimethylbenz ( a) anthracene), while Wistar were more sensitive than LE to the effects of 2 acetylaminofluorene. Therefore, there is an inherent difference in mammary tissue sensitivity among rat strains. In males, the sensitivity of the tumorigenic response in the prostate of F344, ACI, Lewis, CD and Wistar rats to 3,2' dimethyl 4 aminobiphenyl ( DMAB) was ordered as follows: F344> ACI> Lewis> CD; the Wistar rats were insensitive ( Shirai et al. 1990).

Strain differences in susceptibility to the effect of chemicals on testis weight in mice have been reported. Oishi ( 1993) found that administration of di 2 ethylhexyl phthalate ( DEHP) ( 0, 0.1, 0.2, 0.4, and 0.8% in feed, for two weeks) to two strains of mice ( Jcl: ICR and CD 1) caused significant increases in absolute and relative liver weights in both strains at the highest doses and reduced testicular weights in CD 1 mice only ( at a dose as low at 0.2%). DEHP was associated with testicular atrophy in CD 1 mice only, at the doses administered.

In another report demonstrating strain differences in mice, Nagao et al. ( 2002) exposed male C57BL/ 6N and ICR mice to BPA at 0, 2, 20 or 200 : g/ kg/ day for various periods encompassing adulthood, the juvenile period ( just after weaning), and the embryo/ fetal period. Though BPA did not affect male reproductive organ weights during any dose/ exposure period, E2 ( 10 : g/ kg from pnd 27 to 48, as a positive control) produced significant decreases in absolute and relative testes, epididymides, and seminal vesicle weights ( as low as 55% of control values) compared to controls in C57BL/ 6N mice, while ICR mice were unaffected. Histopathology showed that 10 : g/ kg E2 was without effect on ICR males, while B6 males showed slight to severe effects on elongated spermatids, decreased epididymal sperm, and seminal vesicle atrophy. Thus, C57BL/ 6N mice were more sensitive than ICR mice to the effects of E2. These data are consistent with data presented by Spearow et al. ( 1999).

In male mice, strain related differences in susceptibility to endocrine disruption by endocrine active chemicals have been reported ( Spearow et al. 1999; Spearow et al. 2001). Mouse strains included B6 ( an inbred strain), CD 1 ( outbred, with larger litter size), C17/ Jls ( bred randomly, then inbred), and S15 ( bred for large litters, then inbred). In control mice, testicular weight ( absolute and relative to body weight) was higher in CD 1 and S15 strains selected for larger litter sizes. In juvenile male mice exposed for three weeks to E2 ( at 0, 2.5, 10, 20 or 40 : g in silastic implants), B6 and C17/ Jls were sensitive to E2, showing a maximal suppression of testis weight and spermatogenesis even at the lowest dose of E2 ( 2.5 : g), with no effect on testis weight or spermatogenesis in CD 1 or S15 at any dose up to 10 : g E2. Thus, Spearow et al. 51 ( 2001) demonstrated genetic differences in sensitivity to estrogen that may be related to breeding animals for high fecundity.

Additional studies exposing juvenile male mice from 3 to 7 weeks of age to 0, 0.625, 2.5, 10, 40 and 160 : g E2 in silastic implants showed a dramatic strain difference in susceptibility to endocrine disruption ( Spearow et al. 2002; Spearow et al. 2003). CD 1 mice were greater than 195 fold more resistant than B6 mice to the disruption by E2 of testes weight, number of elongated spermatids per tubule and Spermatogenic Index ( SI). CD 1 strain mice were also > 41 times more resistant than B6 strain mice to the inhibition by E2 of epididymal sperm counts, and were more resistant than outbred wild derived Mus spretus mice to the disruption of testes weight and spermatogenesis by E2.

In a separate experiment, immature B6 males, outbred CD 1, CD 1 derived inbred strains CD10 and CD3, and F1 crosses were implanted subcutaneously at 3 weeks of age with silastic implants containing 0, 2.5, or 40 : g E2 ( Spearow et al. 2003). Susceptibility to endocrine disruption by estrogenic agents ( SEDE) was evaluated 4 weeks later by determining testicular weight, histology and epididymal sperm counts. The effects of Strain, Dose of E2 and the Strain x E2 Dose interaction were all highly significant on testes weight ( TW), seminiferous tubule diameter, elongated spermatids per tubule, spermatogenic index and epididymal sperm counts ( P< 0.0003) ( Spearow et al. 2003). Resistance of mouse strains to disruption of testes weight by E2 ranked: B6 << CD3 < CD10 < CD 1. While CD10 x B6 F1 mice showed limited hybrid vigor or heterosis for resistance to the disruption of testes weight by E2, the CD10 x CD3 F1 showed a large amount of heterosis in this trait. The data suggest that susceptibility to the disruption of testes weight by estrogen is controlled by additively and non additively acting genes. Thus the observed > 16 fold to > 195 fold strain differences in susceptibility to the disruption of spermatogenesis and testes weight between strains questions the adequacy of the standard 10 fold within species safety factor if only genetically resistant strain( s) are used for toxicological safety testing.

2.5.11 Behavioral Assessments/ Clinical Observations Courtship and mating behaviors in both sexes, and maternal and neonatal behaviors involving nesting, pup retrieval, and nursing are also under the control of the endocrine system. Qualitative evaluation of these behaviors, as they affect viability and ability to thrive, provides another measure of possible endocrine disrupting activity of a test compound. Strain related differences in lordotic behavior have been reported. In LE rats exposed gestationally to 1,4,6 androstatriene 3,17 dione, high levels of lordotic behavior are observed in male adult offspring treated with estrogen and progesterone, while SD rats only showed slight effects ( Whalen et al. 1986). In an earlier study by Emery and Larsson ( 1979), Wistar males retained copulatory behavior longer than SD males following castration and systemic para chlorophenylalanine treatment ( which facilitates copulatory behavior). The castrated Wistar males also were more responsive to androgen replacement than SD males. In ovariectomized females, Wistar females were behaviorally more sensitive to estrogen than SD females. 52 2.5.12 Hormonal Controls The endocrine system ( also referred to as the hormone system) is made up of glands located throughout the body, hormones that are synthesized and secreted by the glands into the bloodstream, hormone carrier proteins ( e. g. steroid hormone binding proteins, globulin and albumin, " fetoprotein), receptors in the cell membranes, cytosol and nucleus of the cells of various target organs, and tissues that recognize and respond to the hormones. The function of the system is to regulate a wide range of biological processes, including control of blood sugar ( through the hormone insulin from the pancreas), growth and function of reproductive systems ( through the hormones T and estrogen and related components from the testes and ovaries), regulation of metabolism ( through the hormones cortisol from the adrenal glands and thyroxin from the thyroid gland), development of the brain and the rest of the nervous system ( estrogen and thyroid hormones), and development of an organism from conception through adulthood and old age. Normal functioning of the endocrine system, therefore, contributes to homeostasis ( the body's ability to maintain itself in the presence of external and internal changes) and to the body's ability to control and regulate reproduction, development, and/ or behavior. An endocrine system is found in nearly all animals, including mammals, nonmammalian vertebrates ( e. g., fish, amphibians, reptiles, and birds), and invertebrates ( e. g., snails, lobsters, insects, and other species). In humans, the system comprises more than 50 different hormones, and the complexity in other species appears to be comparable.

Puberty, the period in which sexual maturation occurs, begins in the hypothalamic pituitary gonadal ( HPG) axis and leads to the development of secondary sex characteristics and fertility in both males and females ( Stoker et al., 2000; Goldman et al., 2000). Within the hypothalamus, gonadotropin releasing hormone ( GnRH) from neurosecretory neurons act as the primary controller, whereas in the anterior lobe of the pituitary, gonadotropes, which secrete luteinizing hormone ( LH) and follicle stimulating hormone ( FSH), and lactrotropes, which secrete prolactin ( Prl), serve the controller function. The primary gonadotropin responsive elements in males are the Leydig and Sertoli cells in the testes, whereas in the female, the thecal and granulosa cells in the ovarian follicle respond.

° Hypothalamus ( GnRH). It is generally believed that the CNS is the trigger point for initiation of sexual maturation in the male and female rat ( Goldman et al., 2000; Stoker et al., 2000). GnRH is present in the fetal brain and slowly increases until the second postnatal week in females and the third postnatal week in males. At that point, GnRH increases steeply and remains elevated until puberty. At puberty, the GnRH neurons undergo a morphological change, developing spiny like processes that may be related to an increase in synapses on the cells. It has been shown that at puberty, the GnRH neurons become more responsive to neurotransmitter ( norepinephrine and dopamine) stimulation. GnRH is released in a pulsatile manner in both male and female animals, which induces a similar pattern of LH and FSH secretion from the 53 anterior pituitary. GnRH levels can be viewed as an indicator of initiation of sexual maturation.

° Pituitary ( FSH, LH, Prl, TSH). The gonadotropins FSH, LH, and Prl, secreted by the anterior pituitary, are essential in the process of sexual maturation. In the male, LH stimulates T secretion by direct action on the Leydig cells in the testis, and FSH binds to the Sertoli cells within the seminiferous tubules to aid spermatogenesis. FSH also increases the number of LH receptors in the testis, which in turn increases T production and testis growth. Increased prolactin is associated with growth of the prostate and seminal vesicle glands. In the female, FSH and LH act on the ovarian follicular granulosa and thecal cells, respectively, to stimulate production of E2, follicular/ oocyte maturation and ovulation. An increase in prolactin levels is essential in the acquisition of vaginal opening and the transition to sexual maturity. Thyroid stimulating hormone ( TSH) also from the anterior pituitary, is the trigger for the release of T3 and T4 from the thyroid gland ( though T3 is also produced locally in target organs) , and is essential in the regulation of thyroid activity ( see below).

Endocrine disrupting chemicals have been shown to alter levels of pituitary hormones, such as prolactin and LH. Cummings et al. ( 2000) dosed four strains of rats, either diurnally or nocturnally with atrazine ( 0, 50, 100, or 200 mg/ kg/ day on the first eight days of pregnancy). There were reductions in LH levels in Holtzman rats and LE rats but not in SD or F344 rats after diurnal dosing, and reductions in LH levels in LE and F344 at the highest dose ( 200 mg/ kg) after nocturnal dosing, with no effect on SD or Holtzman rats. There were strain related differences in control levels of LH. For example, serum LH levels were low in Holtzman and F344 rats and significantly higher in SD and LE rats. Control progesterone levels tended to be higher in F344 but the ranking of other strains differed according to time of collection. Thus basal levels of pituitary hormones may contribute to the sensitivity of certain strains to endocrine disrupting chemicals.

In a study by Cooper et al. ( 2000), 50 300 mg/ kg/ day of atrazine was administered to ovariectomized SD and LE rats for 1, 3, or 21 days, and surges of LH and Prl induced by estrogen were examined. After one or three doses of atrazine ( 300 mg/ kg), LH and Prl were suppressed in ovariectomized LE but not SD rats. After 21 doses, LH and Prl were suppressed in both rat strains in a dose dependent manner. Therefore, although a longer exposure resulted in similar effects in both strains, LE rats were more sensitive to shorter exposures of atrazine.

The differential effects of an environmental estrogen, bisphenol A ( BPA) were studied in female F344 and SD rats by Steinmetz et al. ( 1997). Basal levels of Prl were 40 and 25 ng/ ml in F344 and SD rats respectively. Within three days, E2 ( in silastic capsules inserted s. c.) significantly increased Prl levels 10 fold in F344 rats and only 3 fold in SD rats; while BPA significantly increased Prl levels 7 8 fold in F344 rats, with no effect on SD rats. Interestingly, E2 increased anterior pituitary weight in F344 rats, but not in SD rats, while BPA had no significant effect on pituitary weight in either strain. 54 While the authors speculated that genetic differences in estrogen receptors may be involved in strain related sensitivities, subsequent studies in uteri and vagina showed that F344 and SD rats differ in the intermediate effects of xenoestrogens downstream of the estrogen receptor ( Long et al. 2000).

° Gonads ( T, DHT, E2, P). Androgens are essential in the development of the male reproductive tract, as well as for feedback regulation of the hypothalamic pituitary axis, sex accessory organ development and maintenance, and spermatogenesis ( Goldman et al., 2000; Stoker et al., 2000). T and DHT are the two most active androgens. Testes descent and development; maturation of the epididymides, vas deferens, seminal vesicles, coagulating glands, prostate, Cowper's glands, levator ani/ bulbocavernosus, and other aspects of the male reproductive tract are dependent upon T, whereas DHT is responsible for male AGD, and external genitalia. DHT is key in the development and maintenance of the external genitalia, prostate, and urethra. E2 and progesterone ( P) serve similar developmental and maintenance functions in the female.

In four strains of rats, Holtzman, LE, SD and F344, dosed with atrazine ( 0, 50, 100 or 200 mg/ kg/ day, for the first eight days of pregnancy) one group diurnally and the other nocturnally, Holtzman rats were the only strain to show a significant postimplantation loss and decreased P levels ( Cummings et al., 2000). In the same study, serum E2 was increased only in SD rats dosed diurnally with the high dose of atrazine ( 200 mg/ kg). In control animals, there were strain related differences in both P and E2 levels. For example, serum P levels on day 9 controls were significantly higher in F344 and Holtzman rats than in SD or LE rats. At the same time, E2 levels in F344 rats were significantly lower than those of the other three strains. Thus, there are strain differences in control gonadal hormone levels, as well as in response to atrazine.

In 19.5 day old male rat fetuses, gestational exposure to TCDD ( 0, 0.5, 0.1, 0.5 or 1.0 : g/ kg) was associated with increases in prenatal T and pituitary LH production in Han/ Wistar but not LE rats ( Haavisto et. al., 2001). The lowered sensitivity of fetal LE rats may be associated with prenatal T levels which are only 15% of those in Han/ Wistar rats. Conversely, adult LE rats are 1000 times more sensitive to TCDD compared to Han/ Wistar rats ( Pohjanvirta et al., 1988). Interestingly, strain related differences in sensitivity to TCDD, are also age dependent. In the same report, gestational exposure to DES ( 100 : g/ kg in Han/ Wistar rats; 100, 200 or 300 : g/ kg in SD rats) significantly decreased prenatal T production in SD and Han/ Wistar male rats, thus the authors concluded that both TCDD and DES exposure in utero may interfere with the timing of the prenatal T surge.

° Thyroid ( T3, T4). TSH released from the pituitary gland stimulates the thyroid to secrete triiodothyronine, T3, and thyroxine, T4. T4 is more prevalent in the blood ( 98%) than is T3 ( 2%). T3 is predominantly produced locally in target tissues. Prenatally, maternal T4 is essential for normal offspring development. Thyroid hormones are well known to play essential roles in vertebrate 55 development ( Dussault and Ruel, 1981; Myant, 1971; Porterfield and Hendrich, 1993; Porterfield and Stein, 1994; Timiras and Nzekwe, 1989). Experimental work focused on the effects of thyroid hormone on brain development in the neonatal rat supports the concept of a " critical period" during which thyroid hormone must be present to avoid irreversible damage ( Timiras and Nzekwe, 1989). Though the duration of this critical period may be different for different thyroid hormone effects, the general view has developed that the period of maximal developmental sensitivity to thyroid hormone occurs during the lactational period in the rat ( Oppenheimer et al., 1994; Timiras and Nzekwe, 1989). Although thyroid hormone receptors are expressed in fetal rat brains ( Bradley et al., 1989; Strait et al., 1990) and thyroid hormone can exert effects on the fetal brain ( Escobar et al., 1990; Escobar et al., 1987; Escobar et al., 1988; Porterfield, 1994; Porterfield and Hendrich, 1992, 1993; Porterfield and Stein, 1994), the lactational period represents a stage of rapid expansion of the thyroid hormone receptors ( Perez Castillo et al., 1985) and an increase in the number of demonstrated effects of changed levels of thyroid hormone on brain development.

In a study of the effects of 2,3,7,8 Tetrachlorodibenzo p dioxin ( TCDD) on two strains of adult rats, LE, and Han/ Wistar, there were strain related differences in control and treated rats ( Pohjanvirta et al., 1989). Control T3 values in both strains were not significantly different, but T4 levels were about 1.2 times higher and TSH levels twice as high in Han/ Wistar compared to LE rats. Rats were injected once i. p. with 0, 5, 50, or 500 ( Han/ Wistar only) : g/ kg of TCDD, and tissues and hormones were collected at 1, 4, 8 and 16 days post treatment. TCDD decreased T4 levels slightly more in LE ( 59%) compared to Han/ Wistar rats ( 43%) after 4 days. After 16 days, T4 levels had returned to basal levels in the two highest dose groups in the Han/ Wistar rats, but in LE rats, T4 levels remained more than two fold lower than controls with no sign of recovery. By day 16, TCDD increased T3 levels in the two highest dose groups in the Han/ Wistar rats and had decreased T3 levels by one half in LE rats. Thus, LE rats exhibited a greater sensitivity to TCDD with respect to thyroid hormone levels. The greater sensitivity of LE rats to TCDD has been confirmed in more recent work by Pohjanvirta et al. ( 1999) who showed that LE rats are 1000 times more sensitive to the acute lethal effects of TCDD than are Han/ Wistar rats.

Another study compared the effects on levels of TSH, T3 and T4 after an endocrine challenge test [ with thyrotropin releasing hormone ( TRH), and TSH], on two strains of male rats, SD and F344 ( Fail et al., 1999). Both strains of rats responded to the challenge with increases in TSH levels. In F344 rats, there were significant increases in levels of T3 and T4, while in SD rats, there were only increases in T4.

Strain related differences in various hormone levels and organ weights were reported after exposure to the weak antiandrogen, p, p' DDE, by O'Connor et al. ( 1999). After exposure of adult male LE and CD rats for 15 days to p, p' DDE ( 0, 100, 200, 300 or 350 mg/ kg/ day in CD rats; 0, 200 or 300 mg/ kg/ day in LE rats), the following effects were reported. 56 CD LE 8 relative liver weight, 9absolute epididymis weight 8relative liver weight, 8absolute epididymis and relative accessory organ weight 8E2 ($ 200 mg/ kg/ day), no change in T, DHT 8E2 ( 300 mg/ kg/ day), T, DHT($ 200 mg/ kg/ day)

9FSH ($ 200 mg/ kg/ day) , no change in Prl, LH no change in FSH, Prl or LH 9T4 ($ 100 mg/ kg/ day), no change in TSH 8TSH and 9T4 ($ 200 m g/ kg/ day)

These data demonstrate strain sensitive differences in response to an endocrinedisrupting chemical. CD rats were much less sensitive to the effects of p, p' DDE than were LE rats.

There is also a report by Dhaher et. al, ( 2000) of intraspecies differences in estrogen receptor number and binding affinity between Balb/ c strain mice and a strain used as a systemic lupus erthematosus ( SLE) model, MRL/ MP lpr/ lpr ( Dhaher, Greenstein et al. 2000). The MRL mice showed significantly higher affinity for E2 binding ( using 3H moxestrol as ligand) than did the Balb/ c mice, which may be related to the exacerbation by E2 of SLE in the mouse model.

2.5.13 Uterine Weight Uterine weight is sensitive to estrogenic compounds. The increase in uterine weight after estrogenic compound exposure may be due to increased fluid uptake ( imbibition), increased cell size ( hypertrophy), and/ or increased cell number ( hyperplasia). Although the uterotrophic assay is being standardized and validated by an OECD/ EPA initiative, the measurement of uterine weight is a sensitive parameter for inclusion in many of the in vivo screens involving females. The uterotrophic assay was designed to identify chemicals that act as estrogen receptor agonists or antagonists, directly on the uterus in ovariectomized females ( since the HPG axis is not intact). An alternative is to use prepubertal intact females as the animal model. In this case, the test chemical may affect any point along the HPG axis, and the end organs such as the uterus, and the chemical may not be an ER agonist or antagonist since it does not require ER binding in the uterus. A description of processes and endpoints to evaluate chemical effects on uterine weight follows.

1. Ovariectomized adult females are exposed to test material for 3 5 days ( po, sc, etc.) and are evaluated for the following:

A. Increased uterine wet/ blotted weight, 6 to 24 ( hypertrophy and hyperplasia, respectively) hours after test dose; must be due to a uterine 57 estrogen receptor mediated response ( since gonad is missing, HPG axis is not intact); an estrogen receptor agonist will be detected.

B. Administration of authentic E2, the potent endogenous estrogen plus the test material; if uterine wet and blotted weights increased with E2, but to lesser extent from E2 plus test chemical, then test material is an estrogen antagonist.

2. Intact prepubertal females are exposed to test material for 3 days ( po, sc, etc.) prior to normal onset of puberty and are evaluated for the following:

A. Increased uterine wet/ blotted weight ( can occur through HPG axis since it is intact; if uterine weight is increased, the test material is an estrogenmimic or estrogen like ( not necessarily an estrogen receptor agonist).

B. Adminstration of E2 and test material for detection of anti estrogens ( need not be an estrogen receptor antagonist).

A three day uterotrophic assay for detecting the estrogenic activity of octylphenol, nonylphenol, methoxychlor, and bisphenol A in prepubertal LE rats was found to be the most accurate method of detecting estrogenic activity when compared to age of VO and estrous cyclicity ( Laws et al., 2000), and may provide a sensitive endpoint for detection of endocrine disrupting chemicals that act via estrogen receptor binding ( ovariectomized female), or via interaction with the HPG axis ( intact prepubertal female).

In studies performed in ovariectomized Wistar, Da/ Han, and SD rats, dosed two weeks after surgery with increasing doses of genistein ( 25, 50 and 100 mg/ kg/ day), p tert octylphenol ( 5, 50 and 200 mg/ kg/ day), bisphenol A ( 0, 5, 50 and 200 mg/ kg/ day), and as a positive control 100 : g/ kg EE, there was a strong stimulation by EE in uterine weight in the Wistar and Da/ Han rats and less pronounced response in the SD rats ( Diel et al., 2001). All strains showed comparable slight uterotrophic responses to 50 and 100 mg/ kg genistein and comparable moderate uterotrophic response to 200 mg/ kg p tert octylphenol. No doses of bisphenol A applied stimulated uterine wet weight in Wistar or Sprague Dawley rats, whereas in the Da/ Han rats a slight stimulation was detected in the highest dose ( 200 mg/ kg BW). These studies demonstrated a strain and chemical specific sensitivity in the uterotrophic assay with SD rats less sensitive than DA/ Han rats to EE and BPA. While Wistar rats were more sensitive than SD to EE, both Wistar and SD rats were resistant to BPA.

Similarly, EE ( 1,3,10 or 30 : g/ kg/ day), DES ( 0.5, 1.5, 5 and 15 : g/ kg/ day) and a weak phytoestrogen, coumestrol ( CE) ( 10, 35, 75 and 150 mg/ kg/ day), as positive controls, produced increases in uterine weight in SD and F344 rats ( McKim et al., 2001). However, in response to the chemical D4 ( 0,10,50,100, 250, 500 and 1000 mg/ kg/ day), the maximal uterine weight was increased 160% relative to control values in the SD rats and only 86% relative to control values in F344 rats. Thus, SD rats were more sensitive to the effects of octamethylcyclotetrasiloxane ( D4) in the uterotrophic assay. McKim et 58 al. ( 2001) suggested that the metabolism of D4 may be slower in SD versus F344 rats based on pharmacokinetic data.

In a study by Christian et al. ( 1998), the uterotrophic assay was compared across three rat strains, Wistar Chbb: THOM SPF, and Wistar CRL:( WI) BR, and SD ( Charles River). When administered DES, all three strains exhibited a positive uterine response, with a similar response in both Wistar strains and a slightly lower response in SD rats. Variability of responses was associated with background spontaneous incidences of abnormally high relative uterine weights possibly due to fluctuations in estrogen occurring between pnd 21 to 25. Due to differences in control mean uterine weights between rat strains ( i. e. means were lower in Wistar Chbb: THM SPF versus Wistar CRL and SD), criteria for biological outliers were different in Wistar Chbb: THOM SPF ($ 0.15%) versus Wistar CRL and SD rats ($ 0.20%). Christian et al. ( 1998) demonstrated the importance of historical control data in the determination of statistically significant effects in subsequent studies.

Sometimes genetic differences have no observable effect on an endocrine endpoint. A study by Odum et al., 1999a, investigated the effects of p Nonylphenol ( NP) ( 0 to 250 mg/ kg/ day orally for 3 or 11 days; and 0 to 7.12 mg/ kg/ day via mini pumps implanted s. c., for 11 days) in the uterotrophic assay in Alderley Park ( Wistar derived) and SD rats. Results were similar in both strains with a positive response to both DES ( 0.01 mg/ kg/ day) and NP ( 250 mg/ kg/ day), which were of the same magnitude as in previous studies performed in Noble rats ( Odum et al., 1999b). The uterotrophic effects of NP and DES were found to be independent of rat strain.

3.0 Interspecies Similarities and Differences in Endocrine Endpoints Few studies have been conducted in a single laboratory comparing the effects of endocrine disrupting chemicals in more than one strain within a species, and even fewer studies have been conducted in a single laboratory comparing the effects of endocrinedisrupting chemicals in more than one species. Therefore the criterion that only studies performed in the same laboratory across species would be included in this white paper could not be applied. One review paper which looked at many studies on many chemicals across many laboratories in mice versus rats was included as well as other studies comparing endocrine endpoints across species in response to endocrinedisrupting chemicals.

There are many reviews on species differences in reproductive and developmental toxicology studies. Among these is a comparison of reproductive organ weights, sperm parameters, and vaginal cytology from fifty 13 week studies involving 24 chemicals in seven different laboratories ( and four routes of exposure for the National Toxicology Program in B6C3F1 mice and F344 rats ( Morrissey et al., 1988). Considerable interlaboratory variability was demonstrated, but overall, it was concluded that there were no differences in types of sperm head abnormalities between control and treated rats and mice, and that testis, epididymis, and cauda epididymis weights and sperm motility were the most statistically powerful endpoints evaluated. Of all the 59 chemicals tested, only one, methylphenidate in the rat, produced an increase in abnormal sperm without effects on any other male endpoint. The agreement in results in these endocrine endpoints in response to reproductive toxicants between rats and mice was about 58%. A combination of confounding factors and species differences may have accounted for disparity in toxicological data.

Several reports have focused on differences in endocrine endpoints between rats and mice. In the uterotrophic assay, the estrogenic activity of parabens was assessed in B6D2F1 mice and Wistar rats ( Hossaini et al., 2000). The parabens tested were methyl, ethyl, propyl and butyl p hydroxybenzoate, and p hydroxybenzoic acid, which were administered either orally or subcutaneously for three days at doses up to 1000 mg/ kg/ day, and E2 ( 0.1 mg/ kg) was used as a positive control. In the mouse uterotrophic assay, there was no significant effect on uterus weight in doses up to 1000 mg/ kg/ day for all parabens. In the rat uterotrophic assay, 600 mg/ kg/ day of butyl paraben produced a positive response. Thus the estrogenic activity of parabens was found to be weak in rats and was not observed in mice.

Two separate studies reported species differences in the ovarian toxicity of reproductive toxicants. In one study ( Doerr et al., 1996), 1,3 butadiene epoxides ( butadiene monoepoxide, BMO; and butadiene diepoxide, BDE) were administered at doses up to 1.43 mmol/ kg/ day intraperitoneally for 30 days, to young SD rats and B6C3F1 mice. BMO was ovotoxic in mice, producing decreases in follicle counts and reproductive organ weights, with no effects in rats at the doses tested. BDE was ovotoxic in both rats and mice, with a greater sensitivity to BDE in mice, resulting in reductions in uterine and ovarian weights in mice at lower concentration than in rats. In addition, follicle counts were greatly reduced in mice at lower doses of BDE than in rats. The authors speculate that metabolic differences affecting the conversion of BMO to BDE may be responsible. In another study by Takizawa et al., 1985, intraovarian injection of increasing concentrations of benzo( a) pyrene, a polycyclic aromatic hydrocarbon, reduction in small oocytes occurred in a dose dependent fashion at doses ranging from 0.01 to 30 : g/ ovary in C57BL/ 6N and DBA/ 2N mice and 0.8 to 240 : g/ ovary in SD rats. Thus effects of benzo( a) pyrene on small oocyte number were present in both SD rats and two strains of mice.

Cadmium has also been found to induce ovarian toxicity in animals. In a study by Rehm and Waalkes ( 1988), the effects of cadmium were assessed in immature and mature female Syrian hamsters, four mouse strains ( BALB/ cAnNCr, DBA/ 2NCr, C57BL/ 6NCr, NFS/ NCr) and two rat strains [ F344 and Wistar Furth ( WF)] after a single injection ( sc) of a dose ranging from 20 to 47.5 : mol/ kg, and the reproductive tracts were examined by light microscopy. Syrian hamsters were the most sensitive to cadmium induced ovarian hemorrhagic necrosis, in particular, shortly before ovulation. In mice, only the DBA/ 2NCr strain showed significant cadmium induced ovarian hemorrhagic necrosis, and uterine lesions in any of the mouse strains were rare. Though rats showed dose and age dependent toxicity of the ovaries, uterus, cervix, and liver, cadmium induced uterine lesions only in mature F344 rats, not WF rats. Thus species and strain differences in cadmium induced reproductive toxicity were reported. 60 As female mice were more sensitive than female rats to the ovotoxic effects of 1,3 butadiene ( Doerr et a., 1996), male mice were found to be more sensitive to the reproductive effects of 1,3 butadiene, while the same doses produced no effects in rats. Anderson et al. ( 1998) compared the effects of exposure by inhalation to 1,3 butadiene ( up to 1250 ppm in rats and up to 130 ppm in mice) for 10 weeks prior to mating, in male CD 1 mice and male SD rats. Exposure in mice resulted in F1 abnormalities and increases in early deaths. None of these effects were observed at the same exposure concentrations in SD rats.

In an earlier study by Brinkworth, Anderson, and McLean ( 1992), dietary restriction in CD 1 mice was found to increase abnormal sperm which may have been related to a decrease in calories, and to decrease epididymal sperm counts, which may have been due to a lack of protein. In CD rats, dietary restriction only reduced epididymal sperm count. Thus dietary changes had different impacts on spermatogenesis in mice compared to rats.

A differential sensitivity to the developmental toxicity of BPA was reported by Morrissey et al. ( 1987) in CD 1 mice compared to CD rats. Mice and rats were dosed daily from gd 6 to 15, with 0 640 mg/ kg/ day BPA ( rats) and 0 1250 mg/ kg/ day BPA ( mice). Maternal toxicity ( as evidenced by significant decreases in maternal weight gain) occurred in rats at $ 160 mg/ kg/ day and at a much higher dose ( 1250 mg/ kg/ day) in mice. No fetal toxicity occurred at doses up to 640 mg/ kg/ day in rats, and in mice, at 1250 mg/ kg/ day, there were significant increases in the number of resorptions, and a decreases in average fetal body weight. Maternal mice were more sensitive to toxic doses of BPA than maternal rats, and fetal malformations were not observed in either rats or mice.

Species differences in endocrine receptor binding characteristics have been reported in in vitro experiments. Mathews et al. ( 2000) examined the ability of several natural and synthetic chemicals to compete with 3H E2 for binding to bacterially expressed estrogen receptor alpha D, E and F domains from five different species ( human, mouse, chicken, anole, and rainbow trout) fused to glutathione S transferase ( GST). While all of these fusion proteins displayed high affinity for E2 ( Kd of 0.3 to 0.9 nM), species differed in affinity of binding other estrogenic chemicals. With, for example, rainbow trout, ER fusion proteins showing much higher relative binding affinity for alpha zearalenol, Bisphenol A, octylphenol, o, p' DDT, Methoxychlor, p, p' DDT, o, p' DDE, p, p' DDE, alpha endosulfan and dieldrin than ERs from the human, mouse, chicken and green anole ( Matthews, Celius et al. 2000). Thus sequence variation in the ER ligand binding domain between strains and between species could be a major source of variation in EDSP assays, in evaluating compound which act via the ER.

4.0 Summary and Conclusions of Intraspecies and Interspecies Similarities and Differences in Endocrine Endpoints and Conclusions Endocrine mediated toxicity of chemicals varies among strains of animals of the same species and among different species. Endocrine endpoints vary in sensitivity to 61 chemicals across strains and species. The sensitivity to endocrine active chemicals is obviously dependent on the endpoint evaluated, the chemical administered, and the genotype of the animal model. Genetic variability which exists within outbred strains, between inbred and outbred strains as well as in the animal and human populations to which the results will be applied has the potential to confound the detection and interpretation of reproductive toxicity. The following table summarizes strain related similarities and differences in endocrine endpoints in many key research articles designed to investigate the effects of endocrine disruptors. The focus of the comparison is on rat strains, with some examples in mouse strains. 62 Table 2. Intraspecies Comparisons of Endocrine Endpoints Endocrine Endpoints Strains Chemical Similarities Differences Key References Uterine Weight Wistar, SD, Da/ Han Alderley Park, SD SD, F344 SD, F344 Wistar, SD SD, F344 Ethinyl Estradiol ( EE), BPA NP E2, BPA EE, DES and octamethylcyclot etrasiloxane ( D4)

DES E2,4 OH tamoxifen ( 4 OHT) Wistar and SD less sensitive to BPA than Da/ Han Results were similar in both strains with a positive response to NP ( 250 mg/ kg/ day), which were of the same magnitude as previous studies perform ed in Noble rats ( Odum et al., 1999b)

Both strains showed increased uterine weight in response to E2 EE, DES and D4 produced similar positive uterine response in SD and F344 rats.

SD and W istar( CRL) had similar control values for uterine weight; sim ilar uterotrophic response in both Wistar strains.

F344 and SD showed similar uterine weight responses to E2 SD less sensitive than Da/ Han to EE and BPA, W istar more sensitive than SD to EE.

250 m g/ kg NP resulted in slightly greater uterotrophic response in Alderlery Park ( 1.84 fold) than in SD ( 1.55 fold).

F344 more sensitive to BPA and E2 Maximal uterine response to D4 was two fold higher in SD than F344 rats. F344 m ore sensitive to EE.

Wistar( Chbb THOM SPF) had lower mean uterine weights in controls versus SD and Wistar( CRL), less variability in response to DES F344 more sensitive than SD to E2 induced uterine epithelial cell height. SD more sensitive than F344 to induction of uterine weight and epithelial cell height by 4 OHT 1Diel et al., 2001 2Odum et a., 1999a 3Steinmetz et al., 1998 4McKim et al., 2001 5Christian et al., 1998 6Bailey et al., 2002 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 63 Male and female sexual development ( AGD, PPS, VO) SD, LE SD, F344 p, p' DDE; flutamide $ E2 3 benzoate PPS same time in LE and SD controls; p, p' DDE had no effect on PPS or VO in either strain; flutamide decreased AGD and caused nipple retention and changes in male reproductive organ weights in both strains neonatal exposure to 1.5 mg/ kg/ day decreased male reproductive organ weights in both F344 and SD males ( pnd 90), and delayed PPS p, p' DDE produced significant decrease in AGD in male LE rats, not in SD; p, p' DDE produced nipple retention at a lower dose in SD than LE; VO earlier in LE controls versus SD controls At pnd 90, in rats exposed to 1.5 mg/ kg/ day, there were greater reductions in reproductive organ weights in F344 rats than in SD rats ( greater responsiveness in F344, higher sensitivity in SD) 7You et al., 1998 8Putz et al., 2001 Female reproductive tract SD, Alderley Park F344, SD LE, Holtzmann BPA BPA TCDD There was no effect on age of first estrus in either strain.

Metabolic clearance of BPA is same 1 : g/ kg on gd 15 produced malformations of F1 offspring female external genitalia and increased UVD in both strains. VO delayed in Alderley Park, but not in SD rats BPA increased DNA synthesis and cell proliferation in the vaginal epithelium of F344 rats but not of SD rats 9Tinwell et al., 2002 10Long et al., 2000 11Gray and Ostby, 1995 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 64 Male reproductive tract/ andrology F344, ACI, Lewis, SD, Wistar LE, SD LE, Wistar 3,2' dimethyl 4 amino biphenyl ( DMAB)

p, p' DDE Vinclozolin In both SD and LE rats, there were increases in liver weight, and in E2; decreases in T4; and no change in Prl or LH 200 mg/ kg/ day from gd 14 to pnd 3 produced malformations of m ale external genitalia, nipple retention lasting into adulthood, and increased inflammation of epididymides, prostate, seminal vesicles and coagulating glands in both strains. 12 mg/ kg produced transient retention of nipples/ areolae in preweanling males of both strains. Similar NOAEL in both strains, 12 and 6 mg/ kg bod wt. In Wistar and LE respectively. tumorigenic response in the prostate of F344, ACI, Lewis, CD and W istar rats to DMAB was more sensitive in F344> ACI> Lewis> CD and W istar rats were insensitive In SD rats there was a decrease in epididymis weight, no change in T, DHT, or TSH, and a decrease in FSH. In LE rats, there was an increase in epididymis weight, increases in T, DHT and TSH, and no change in FSH.

200 mg/ kg/ day from gd 14 to pnd 3 reduced maternal body weights and pup weights in Wistar but not LE. Chronic inflammation of urinary bladder, and testis atrophy were noted in LE and not Wistar. At 12 mg/ kg/ day, nipple/ areolae retention persisted into adulthood in LE, not in Wistar. Adult offspringLE also had reduced prostate, seminal vesicle, and coagulating gland weights; these effects were not seen in W istar adult offspring.. 12Shirai et al., 1990 13O'Connor et al., 1999 14Hellwig et al., 2000 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 65 AP, SD BPA No difference in PPS . 50 mg/ Kg BPA decreased total sperm count and daily sperm production in AP ( Wistar derived) rats but not SD. 15Tinwell et al., 2000 Wistar, SD and Dark Agouti ( DA)

SD versus other strains ( review)

F344, SD none lead Neonatal E2 3 Benzo ate ( EB) DA exhibited lower absolute testis weight than the other two strains, no difference in sperm count among the three strains.

SD more sensitive than other strains to testicular toxicity of lead.

F344 more sensitive and responsive than SD to reduction in TW, Epididymis, Seminal Vessicle and Coagulating gland weights. 16Wilkinson et al., 2000 17Apostoli et al., 1998 18Putz et al., 2001 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 66 Estrous cycle/ ovulation F344, LE, SD Donryu, F344 LE, SD SD, F344 diet ( feed restriction)

none atrazine atrazine Prior to food restriction, Brown Norway rats had irregular estrous cycle patterns while SD, LE and F344 rats had regular estrous cycle patterns. By day 5 of food restriction, 75% of F344 rats and 100% of Brown Norway rats stopped cycling and SD and LE rats were unaffected In aging Donryu rats, estrous cycle abnormalities ( e. g. persistent estrus) were more common than in F344 rats LE rats were more sensitive than SD rats to atrazine induced disruption of the ovarian cycle.

Atrazine administration to SD and F344 rats for up to 12 months produced changes in estrous cyclicity in SD rats ( increased the number of days of vaginal estrus), increased E2, decreased P, and increased incidence of mam mary tumors in SD rats only, with no effect in F344 rats. 19Tropp et al., 2001 20Ando Lu et al., 1998 21Cooper et al., 2000 22Eldridge et al. ( 1994) Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 67 Gonadal/ Pituitary Hormone levels Han/ Wistar, SD, LE SD, LE SD, F344 SD, LE, Holtzman, F344 TCDD, DES atrazine E2, BPA atrazine gestational exposure to DES ( 100 : g/ kg in Han/ Wistar rats; 100, 200 or 300 : g/ kg in SD rats) significantly decreased prenatal T production in SD and Han/ Wistar male rats After 21 doses of atrazine LH and Prl were suppressed in SD and LE rats. BPA had no effect on pituitary weight in either strain. In 19.5 day old male rat fetuses, gestational exposure to TCDD ( 0, 0.5, 0.1, 0.5 or 1.0 : g/ kg) was associated with increases in prenatal T and pituitary LH production in Han/ Wistar but not LE rats.

LH and Prl were suppressed in LE but not SD rats after 1 and 3 doses of atrazine.

After 3 days of E2 exposure, Prl was increased 10X in F344, only 3X in SD; increased pituitary weight in F344, not in SD. BPA increased Prl 7 8X in F344, no effect on SD rats.

Serum E2 increased by atrazine in SD rats, O nly Holtzman strain with decreased P levels. Control and treated levels of E2 much lower and non detectable in Dirunal F344 rats. Control levels of E2 higher nocturnally in LE rats. Diurnal atrazine reduced LH in Holtzman and LE but not in SD or F344. Nocturnal atrazine reduced LH in LE and F344 but not in SD or Holtzman. 23Haavisto et al., 2001 24Cooper et al., 2000 25Steinmetz et al., 1997 26Cummings et al, 2000 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 68 Thyroid hormone levels LE, Han/ Wistar SD, F344 TCDD TRH, TSH T3 levels in controls were comparable T4 and TSH levels were higher in Han/ Wistar controls; TCDD produced a greater reduction in T4 levels in LE than in Han/ Wistar rats.

Challenge with TSH and TRH increases levels of T3 and T4 in F344 rats, and increased T4 only in SD rats. 27Pohjanvirta et al., 1989 28Fail et al., 1999 Fertility/ reproductive parameters SD, LE, Holtzman, F344 SD, LE, F344 F344, SD Wistar Hannover, CD atrazine atrazine BDCM none no effect of atrazine on pre or post implantation loss in SD and LE rats full litter resorption at highest dose ( 200 mg/ kg/ day) in all three strains.

75 mg/ kg/ day significantly reduced body weights in both strains Increase in % preimplantation loss in F344; SD and LE not affected. Increase in % postimplantation loss only in Holtzman rats only.

At lower doses ( 50 and 100 mg/ kd/ day) pregnancy loss in F344, not in SD or LE 75 mg/ kg/ day BDCM produced 62% litter resorption in F344, no effect on SD Control W istar Hannover have lower pregnancy rates and litter sizes; higher % pre and postimplantation loss and resorptions versus control CD ® . 29Cummings et al., 2000 30Narotsk y et al., 2001 31Bielm eier et al., 2001 32Liberati et al., 2002 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 69 organ weights, histopath ology ( rats) SD, F344 Brown Norway, F344, W istar, Donryu F344, BN, SD F344, W F E2 E2 DES Cadmium No difference in pituitary weights of Wistar and Donryu rats, after E2 exposure. In ovariectomized rats exposed to E2 ( silastic implants) for 10 or 20 days, pituitary weights and Prl levels were dramatically increased in F344, minimal effects in SD rats.

In ovariectomized rats, control pituitary gland weights were the lowest in Brown Norway > W istar rats; After E2 treatment ( 10 mg s. c. pellet for 4 weeks), there was a significant > 3 fold increase in pituitary weights in F344 rats, a significant > 0.5 fold increase in pituitary weights in Brown Norway rats F344 sensitive to DES induced pituitary tumors; SD and BN resistant to effects of DES on pituitary gland.

Cadmium induced toxicity of the ovaries, uterus, cervix, and liver, cadmium induced uterine lesions in F344 not W F 33Schechter et al., 1987 34Yin et al., 2001 35Wendell et al., 1996, 1997, 1998, 2000; Chun et al., 1998 36Rehm and Waalkes, 1988 Table 2. ( continued)

Endocrine Endpoints Strains Chemical Similarities Differences Key References 70 Organ weights, histopathology spermatogenesis ( mice) Jcl: ICR and CD 1 mice C57BL/ 6N and ICR mice C57BL/ 6J ( B6), CD 1, C17/ Jls, and S15 BALB/ cAnNCrD BA/ 2NCr, C57BL/ 6NCr, NFS/ NCr di 2 ethylhexylphthalate ( DEHP)

BPA, E2 E2 Cadmium Increase in liver weight, both strains BPA did not affect m ale reproductive organ weights during any dose/ exposure period DEHP decreased testicular weight in CD 1 mice not Jcl: ICR E2 ( 10 : g/ kg from pnd 27 to 48, as a positive control) produced significant decreases in absolute and relative testes, epididymides, and sem inal vesicle weights compared to controls in C57BL/ 6N mice, while ICR m ice were unaffected.

B6 and C17/ Jls were sensitive to E2 showing a maximal suppression of testis weight and spermatogenesis even at the lowest dose of E2 ( 2.5 : g), with no effect on testis weight or spermatogenesis in CD 1 or S15 up to 10 : g E2.

cadmium induced ovarian hemorrhagic necrosis only observed in DBA/ 2NCr mice 37Oishi et al., 1993 38Nagao et al., 2002 39Spearow et al., 1999; 2001 40Rehm and Waalkes, 1988 1 40Footnotes are used for the identification of references in Table 3 71 Thus after conducting this literature review, strain related differences in effects on endocrine mediated endpoints in response to a wide variety of endocrine disrupting chemicals was obvious. The sensitivities of various strains to chemicals producing effects on various endocrine endpoints are summarized in Table 3. Since effects on many of the endpoints were also chemical specific, the chemicals are also included in Table 3.

Table 3 Summary of Agent and Endpoint Specific Intraspecies Differences Endocrine Endpoint Chemical Sensitive* Strains Less Sensitive/ Insensitive Strains References ( from Table 2)

Uterine Weight EE Wistar, Da/ Han SD 1 BPA Da/ Han Wistar, SD 1 NP AP> SD 2 EE, DES SD, F344 3 D4 SD F344 4 E2 SDïF344 6 tamoxifen SD F344 6 AGD p, p' DDE LE SD 7 flutamide SD, LE 7 Nipple retention p, p' DDE SD LE 7 flutamide SD, LE 7 vinclozolin LE > W istar 14 PPS E2 F344, SD 8 p, p' DDE SD, LE 7 VO p, p' DDE SD, LE 7 BPA AP SD 9 Male reproductive organ wts. flutamide LE, SD 7 E2 F344, SD 8 low dose E2 SD F344 8 vinclozolin LE Wistar 14 BPA C57BL/ 6N, ICR 38 E2 C57BL/ 6N 38 E2 B6, C17/ Jls ICR, CD 1, S15 39 DEHP CD 1 Jcl: ICR 37 Table 3. ( continued)

Endocrine Endpoint Chemical Sensitive* Strains Less Sensitive/ Insensitive Strains References ( from Table 2)

72 Estrous cycle/ ovulation feed restriction F344, BN SD, LE 18 atrazine LE SD 21 atrazine SD F344 22 Fertility/ gestational effects atrazine Holtzman F344, SD, LE 29 atrazine F344 SD, LE 30 BDCM F344 SD 31 Andrology BPA AP SD 15 lead SD 17 E2 B6, C17/ Jls CD 1, S15 39 Hormone Levels p, p'DDE SD ( FSH, E2, T4) LE ( FSH, Prl, LH) 13 Hormone Levels ( continued) p, p'DDE LE ( E2, T4, T, DHT, TSH) SD ( Prl, LH, T, DHT, TSH) 13 E2 SD ( Prl) F344 ( Prl) 22 TCDD Han/ W istar ( T, LH) LE ( T, LH) 23 atrazine LE ( LH, Prl) SD ( LH, Prl) 24 atrazine Holtzman ( P) SD ( E2, P) 26 E2 F344 ( Prl) SD ( Prl) 25 BPA F344 ( Prl) SD ( Prl) 25 TCDD LE ( T4) 27 TSH, TRH SD, F344 ( T4) SD ( T3) 28 TSH, TRH F344 ( T3) 28 Pituitary Weights E2 F344 SD 33 E2 F344> BN Wistar, Donryu 34 DES F344 SD, BN 35 Histopathology ( reproduct ive organs) BPA F344 ( females) SD ( females) 10 DMAB F344> ACI> Lewis> CD ( males) Wistar ( males) 12 vinclozolin LE ( males) Wistar ( males) 14 Table 3. ( continued)

Endocrine Endpoint Chemical Sensitive* Strains Less Sensitive/ Insensitive Strains References ( from Table 2)

73 cadmium F344 ( females) WF ( cadmium) 40 Histopathology ( continued) atrazine SD ( females) F344 ( females) 22 *" Sensitive" refers to a greater response, and may not reflect dose level Table 3 summarizes the intraspecies comparisons of the effects of endocrinedisrupting chemicals on endocrine endpoints in EDSP assays. In the uterotrophic assay, the SD rat is sensitive to many endocrine disruptors, except for EE and BPA in one study, in addition to many other strains. Less sensitive strains were F344 in response to D4, and Wistar in response to BPA. LE and SD rats were differentially sensitive to the effects of flutamide and p p' DDE on AGD and nipple retention. SD and F344 rats showed sensitivity to PPS in response to E2, while SD and LE were insensitive to p, p' DDE. AP rats were sensitive to BPA on VO, while SD and LE rats were less sensitive to BPA. Sensitivity to the effects on male reproductive organ weights was found in most rat strains and chemicals studies except for F344 ( low dose E2), and Wistar ( vinclozolin). Different strains of mice were differentially sensitive to the effects of E2 on male reproductive organ weights and andrology. Different strains of rats were differentially sensitive to the effects of BPA, atrazine, BDCM, p, p' DDE, TCDD, E2, lead, cadmium, DES, vinclozolin, and DMAB, on estrous cycle, fertility, pregnancy loss, andrology, hormone levels and histopathology of the reproductive organs. Pituitary weights were affected in F344 rats after exposure to DES and E2 while in other strains pituitary weights were unaffected. Overall, there was no one strain which was sensitive to all of the endocrine disrupting chemicals at most of the endocrine endpoints. Effects on endocrine endpoints were dependent on strain and chemical, and effects on strains were dependent on the endpoints and chemicals. There were no clear patterns indicating the optimal strain for detection of effects due to the majority of endocrineactive compounds tested. In addition, it was not clear that inbred strains or outbred strains would be the better choice in species/ strain selection. In selection of the appropriate species/ strain for EDSP assays, it may be important to consider the endocrine endpoints assessed and the test chemical employed. Obviously, it would be more thorough to conduct multi strain assays to increase to chances of detecting endocrine effects, but extremely difficult to determine which strains should be chosen considering the considerable variation across strains, even under the same experimental conditions ( which would minimize confounders). 74 The five example assays under consideration by the EDSP ( Table 1) were compared against strain sensitivities ( Table 3). In the one generation, two generation, and in utero lacational assays, a similar array of endpoints are assessed, from gestational indices, reproductive development, onset of puberty, estrous cyclicity, hormone levels, andrology, organ weights, and histopathology. Selecting a rat strain that is sensitive at the greatest number of endpoints is difficult, due to the different strain sensitivities observed with different chemicals. For example, if selecting one chemical, in this case, p, pNDDE, SD rats are sensitive at about half of the endpoints, and LE rats are sensitive at the other half of the endpoints. These data support the case for performing reproductive toxicity assays in more than one strain to maximize the probability of detecting an effect at an endocrine endpoint. Uterine weight, as assessed in the uterotrophic assay, which is currently in the proposed in utero lactational assay, is an endpoint which appears relatively sensitive across strains. In a review of uterotrophic assays performed in 19 different laboratories and two model systems ( ovariectomized and immature rats), the response to EE was robust, reproducible and sensitive across laboratories, regardless of differences in strain, diet, bedding, housing, and vehicle ( Kanno et al., 2001). In endpoints like fertility and gestational parameters, the SD rat appears less sensitive than the F344 rat to several chemicals, suggesting that the F344 rat may be a better strain for assessing the effects of chemicals at these endpoints. However, the F344 has a small litter size, thus reducing the number of animals available for multiple evaluations, and has a high incidence of spontaneous testicular tumors, which may confound potential effects on male reproductive organs. Both the adult male and pubertal male and female assays include assessments of hormone levels. Chemical effects on hormone levels were highly strain dependent, with no one strain insensitive to changes in every hormone level. With inclusion of enough different hormone level determinations, the possibility detecting a chemical induced change in one strain would be enhanced. Inclusion of an additional rat strain ( e. g. the LE rat) in the pre validation of EDSP assays may provide more information on the strain sensitivity of the various endpoints and assays, in addition to providing more flexibility to laboratories in selecting strains for performance of the assays.

Since interspecies comparisons performed under the same experimental conditions are few, and strain is obviously also a confounder in comparing speciesrelated differences in sensitivity to endocrine disrupting chemicals, conclusions about comparisons are limited. There are differences in the response of the rat and mouse reproductive tracts to various reproductive toxicants. In some cases, mice appear more sensitive and in other cases rats are more sensitive. In a comparison of reproductive organ weights, sperm parameters, and vaginal cytology from fifty 13 week studies involving 24 chemicals in seven different laboratories ( and four routes of exposure) for the National Toxicology Program in B6C3F1 mice and F344 rats ( Morrissey et al., 1988), there was considerable interlaboratory variability due to confounding factors ( such as different suppliers and environmental conditions) resulting in only a 58% agreement in endocrine endpoints ( i. e. either no response in either species, or at least one endpoint affected in both species) in response to reproductive toxicants. It is possible that EDSP assays involving only one species and strain, namely the SD rat, may not detect effects in endocrine endpoints that occur in mice ( and vice versa). However, a major question 75 that cannot be answered is which animal model will provide the most appropriate data on the ability of the test chemical to interact with the endocrine system, in order to predict the effects of endocrine active chemicals in humans, and/ or other species of concern. Since we cannot identify the most relevant or sensitive animal model with the existing data, because the sensitivity depends on the endpoint( s) chosen, the chemicals evaluated, the timing, duration and route of exposure, the dose levels, and is confounded by the varying genotype by environment interactions, the main issue is whether to use inbred versus outbred strains. Inbred strains are homogeneous at all loci, and have a limited range of responses ( less variability, but an effect may be missed), though using several genetically defined inbred strains in endocrine screens may provide a wider spectrum of responsivity. If selecting a single strain for endocrine screens, outbred strains have more genetic variability, exhibit a broader range of responsivity ( with a greater likelihood of detecting an effect), and may be more appropriate. 76 5.0 References Adham, I. M., Steding, G., Thamm, T., Bullesbach, E. E., Schwabe, C., Paprotta, I. And Engel, W. 2002. The overexpression of the insl3 in female mice causes descent of the ovaries. Mol. Endocrinol. 16, 244 262.

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