Reviewers Presentation on US Environmental Protection Agency " White Paper on Species/Stock/Strain on Endocrine Disruptor Assays" Reviewed by but not in complete concurrence Jimmy L. Spearow, Ph.D. Associate Research Geneticist University of California at Davis Work conducted for Battelle Memorial Institute under EPA Prime Contract 68-W

The US EPA is planning to test a multitude of chemicals for endocrine activity and their ability to disrupt reproductive development and function using in vivo EDSP screening assays. While Sprague-Dawley derived CD rats, and CD-1 mice have been most frequently used for such reproductive toxicology testing, these strains were previously selected mainly for large litter size for over 80 generations. Selection for large litter size results in correlated responses in reproductive endocrine traits, especially decreased sensitivity to estrogen. Strain differences have also been observed in susceptibility to many toxicants. Raises concern that use of a single highly prolific outbred strain in the EDSP could underestimate effects on sensitive genotypes (Spearow et al., 1999; 2001).

Strain Variation in Susceptibility to E2 in Females

While uterine weight responses to E2 were similar between SD and F344 rats, SD rats were less sensitive than F344 rats to the effects of E2 on increasing uterine epithelial cell height, pituitary weight and serum prolactin. SD rats were less sensitive than other strains to EE and NP. F344 rats were consistently as sensitive, and for most female reproductive responses more sensitive than SD rats to E2. Females large litter size selected CD-1 and CD10 mice were also less sensitive than B6, C17 and wild-derived Mus spretus strain mice to the effects of E2

Except for effects on PPS, large litter size selected strains including SD rats and CD-1 strain mice are much more resistant than F344 rats and B6 strain mice to the inhibitory effects of estrogens on male reproductive organ weights, sperm counts, and/or testosterone levels. Strain Variation in Susceptibility to E2 in Males

*In contrast to some of the assertions of the 7/25 version of the White Paper, available scientific data shows that: F344 rats are greater than 10-fold more sensitive than SD rats to the inhibitory effects of E2 on reproductive organ weights Putz et al., (2001). Data from Table 3 of the white paper has been corrected accordingly. Litter size of high prolificacy selected strains does not decline to the "original" population mean following relaxation of selection (Eklund and Bradford, 1977). Relaxed selection in SD derived CD IGS strain rats is thus unlikely to reverse resistance to EDC resulting from long-term selection for high prolificacy and return this strain to the sensitivity of the “original” population.

The Data of Inano et al., (1996) show that SD rats were less responsive, than Wistar-MS, and F344 rats to the inhibition of testis weight, seminal vesicle weight, prostate weight and serum testosterone by DES implants (P<0.01) Effect of exposure to DES from 3 to 15 months of age * Data of Inano et al., 1996 was requested but not included in the 7/25 version of the white paper.

From Spearow et al., 1999 Evidence for Strain Differences in Susceptibility of males to estrogenic agents includes: Large Litter Size Selected CD-1 mice are much less sensitive than B6 and C17 strain mice to inhibition of testes weight by pubertal exposure to E2 (Spearow et al., 1999). Recent studies show that CD-1 mice are less sensitive than wild-derived Mus spretus mice, and >195-fold less sensitive than B6 strain mice to disruption of testes weight by E2 (Spearow et al., 2003).

Effects of pubertal exposure from 3 to 7 weeks of age to E2 on Testicular Histopathology CD-1 0µg E2 Implants B6 0µg E2 Implants Normal spermatogenesis in CD-1 and B6 strain mice in response to pubertal exposure to 0 µg E2 Implants.

Effects of pubertal exposure from 3 to 7 weeks of age to E2 on Testicular Histopathology. CD-1 40µg E2 Implants B6 40µg E2 Implants CD-1 strain mice are much less sensitive than B6 strain mice to the complete disruption of spermatogenesis by E2. Increasing doses of E2 had minimal effects in CD-1, while B6 strain mice showed loss of elongated spermatids and atrophy of seminiferous tubules, with some tubules showing a Sertoli cell only phenotype.

From Spearow et al., 1999 Large Litter Size Selected CD-1 mice are much less sensitive than C57BL/6J (B6) and C17 strain mice to inhibition of spermatogenesis by pubertal exposure to E2 (Spearow et al., 1999).

SD rats are consistently less sensitive than DA/Han, Alderley Park and especially F344 rats to the effects of BPA on many phenotypes including Uterine Weight, Uterine Histopathology, Puberty, and Sperm counts. Strain Variation in Susceptibility to Bis Phenol A

SD and in some cases LE rats were less sensitive than some other strains to the effects of atrazine on estrous cyclicity, gestational loses, as well as LH, Prl and/or Progesterone levels. But, SD is more sensitive than F344 to tamoxifen and effects of long term (> 3 month exposure) to atrazine on persistent estrous, elevated E2 and mammary tumors. Strain Variation in Susceptibility to Atrazine & Tamoxifen

A very limited number of strains, all of which are outbred, have been compared for reproductive responses to androgen disruptors. Data on inbred strains is needed, the existing data do not show a consistent strain difference, except LE is more sensitive than Wistar rats. Strain Variation in Susceptibility to Androgen Disruptors

Strain Variation in Susceptibility to Thyroid Hormone Disruptors TCDD has multiple targets and multiple adverse effects. Male Turku Long-Evans rats are greater than 300-fold more resistant than Han/Wistar rats to the lethal effects of TCDD. LE rats were more sensitive than Han/Wistar rats to the inhibition of T4 by TCDD. In utero exposure to TCDD increased Testosterone (T) in Han/Wistar but not in LE rat testes. Strain differences in effects of RxR ligands are unknown.

Large litter size selected strains including SD and LE rats, as well as, CD-1 mice were less sensitive than F344 and BN rats & B6 mice, respectively, to the disruption of estrous cycles and/or gestation by feed restriction, atrazine, BDCM and/or E2. The robust gestation of large litter size selected strains is associated with decreased sensitive to the disruption of pregnancy. Strain Variation in Susceptibility to the Disruption of Estrous Cyclicity or Gestation

The pattern of strain differences in susceptibility to endocrine disruption by other EDC is not as clear as for estrogenic agents, and likely depends on genetic mechanisms by which each EDC acts F344 rats are sensitive to several EDC, except for being about 50% less responsive to D4 than SD rats. Additional strains need to be compared for EDC with other mechanisms of action. Strain Variation in Susceptibility to Other Diverse Chemicals

Partial Recommendation 1) SD rats were much less sensitive than other strains, and especially F344 strain rats to the disruption of: Most male and female reproductive phenotypes by estrogen; All male and female reproductive phenotypes by BPA; Gestation by diverse EDC; and, Several phenotypes by short term exposure to atrazine. Thus, SD rats should be avoided and F344 rat preferred to provide a more sensitive and reproducible model for such endocrine exposures. 2) However, F344 is less sensitive for several endpoints to partial estrogen agonists/ antagonists such as tamoxifen, long term atrazine and agents with other mechanisms of action such as D4. 3) The sensitivity of F344 to androgen, thyroid hormone and/ RXR disruptors is largely unknown. Thus, multiple, diverse strains in addition to F344 are required to insure that at least one strain in the EDSP is sensitive to the EDC being tested.

Conclusions: 1)Strains of rats and/or strains of mice differ significantly in susceptibility to the disruption of reproductive development and function by: Estrogenic agents, including E2, DES, Bisphenol A, & NP; Partial E2 agonists/antagonists including Atrazine & Tamoxifen; Androgen disruptors, including p,p'-DDE & Vinclozolin; Thyroid hormone disruptors, including TCDD; EDC with other mechanisms of action including, DMAB, & Cadmium; EDC which disrupt gestation including E2, BDCM, & Atrazine. 2)While few studies have conducted extensive dose response curves, the magnitude of Strain Differences in Susceptibility to EDC range from a few percent to over 195-fold. 3)There was no one strain most sensitive to all EDC, or all phenotypic endpoints. 4)There was no one strain most sensitive to all androgen disruptors, or all "other" EDC.

Conclusions: 5)Strain differences in susceptibility to endocrine disruption were clearest for estrogenic agents and for the disruption of gestation. 6)Large litter size selected SD rats were less sensitive than F344, and in some cases other strains of rats, to the effects of E2 on most endpoints in males, and females. Large litter size selected CD-1 mice were also less sensitive than B6 strain mice to the effects of E2 on most endpoints in males, and females. 7)Large litter size selected SD rats were also consistently less sensitive than F344 strain rats to the effects of BPA on both male and female endpoints. 8)Reproductively robust, large litter size selected strains including SD and LE rats, as well as, CD-1 mice were less sensitive than F344 and BN rats & B6 mice, respectively, to the disruption of estrous cycles and/or gestation by several EDC. 9)Thus, SD strain rats should be avoided and F344 rats preferred as one of the strains needed to provide a more sensitive and reproducible model for detection of estrogenic EDC.

Summary 10) The use of isogenic strains rather than outbred strains in the EDSP will provide more precise and reproducible bioassays for detecting endocrine disrupting activities. 11) Given the finding of many Strain x Endocrine disrupting agent interactions, and no single optimal strain for detecting all endocrine agents, conducting EDSP assays with multiple strains on different isogenic genetic backgrounds would better insure that all the animals tested are not resistant to the endocrine disrupting chemical (EDC) being analyzed. Otherwise, the use of a single strain, especially one which is genetically resistant to certain EDC in the EDSP, risks underestimating effects of EDC on sensitive genotypes.

*Confounders, Ligands, and Potential Genotype x Environment interactions that need consideration in EDSP assays: RxR ligands disrupting Thyroid Hormone action: Thyroid hormone Receptor (TR) / Retinoid X receptor (RxR) heterodimer binds to Thyroid hormone Response Element (TRE) to regulate gene transcription. Since the RxR is not a silent partner (Li, et al 2003), the EDSP needs to consider both potential TR and RxR ligands in in vitro and in vivo screens for Thyroid Hormone disruptors. Potential for strain differences in interactions of thyroid and reproductive hormones on the induction of hepatic Cyp, Epoxide hydrolase and other enzymes involved in xenobiotic metabolism. Using a no/low rather than a high phytoestrogen diet (PMI 5002) since: a) varieties of Soybeans with much lower and much higher phytoestrogens are being developed, risking the uniformity of EDSP diets and, b) variation in phytoestrogen levels is likely to confound the detection of weak estrogenic agents with beneficial as well as detrimental effects.

Reference Data: Some Commercially Available Inbred strains of rats Inbred (Harlan Data) Rat Strain Approx. Litter Size Coat color Genetic origin ACI Agouti Black Closely related to COP (E2 sensitive) BN 4.5 Non Agouti Brown Wild Copenhagen (COP) Brown Hooded Curtis and Dunning at Columbia DA 6.0 Agouti F Albino From Curtis at Columbia Lewis 7.5 Albino Wistar derived PVG 4.5 Black hooded Other Wistar Inbreds ~5 Albino Wistar Wistar Furth 5.5 Albino Wistar Note that use of strains with different coat colors in the EDSP will eliminate potential strain mix-ups.

Reference Data: Some Commercially Available Outbred strains of Rats Outbred Strain Approx. Litter Size Coat color Genetic origin Previous Selection (Harlan Data) History SD (HSD) 10.5 Albino Wild x Wistar derived * LLSS SD (CD) (CRL) Albino Wild x Wistar derived Extensive LLSS Long Evans (CRL) Black hooded Wild x Wistar derived Extensive LLSS Long Evans (HSD) 10.8 Black hooded Wild x Wistar derived LLSS (Blue Spruce) Wistar 9.5 Albino Wistar (CRL) LLSS Wistar- Hannover 9.5 Albino Wistar *LLSS = Historic Large Litter Size Selection either by former or current commercial breeder/ supplier. Charles River Laboratories (CRL) selected CD, LE and Wistar outbred stocks mainly for large litter size with more limited selection for increased vigor from the acquisition of the stocks until the early 1990s. Note after 1991 most stocks were no longer selected for large litter size due to concerns raised. Yet these stocks are the products of long term selection mainly for large litter size. Harlan Sprague Dawley = HSD

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