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-01-023.

• 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).

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

Uterine Weight E2 SD, F344Steinmetz et al., 1998

Uterine Weight E2 SD, F344Bailey et al., 2002

Uterine Weight EE Wistar, Da/Han SD Diel et al 2001

Uterine Weight NP AP SDOdum et al., 1999a

Female Tract Histopathology E2 F344 (females) SD (females)

Steinmetz et al., 1998

Female Tract Histopathology E2 F344 (females) SD (females)

Bailey et al 2002

Hormone Levels E2 F344 (Prl) SD (Prl)Steinmetz et al., 1997

Pituitary Weight E2 F344 SD Schechter et al., 1987

Pituitary Weight E2 F344>BN Wistar, Donryu Yin et al., 2001

Uterine Weight E2 BenzoateB6, C17/Jls, Mus spretus CD-1, CD10

Spearow et al., 2002

Fertility/ Gestational Loss E2 B6 CD-1

Spearow et al., 2003

Strain Variation in Susceptibility to E2 in Females

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.

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

PPS E2 F344, SD Putz et al., 2001

Male Reprod. Organ Wts. E2 F344 SD

Putz et al., 2001

Male Reprod. Organ Wts. E2 C57BL/6N ICR

Nagao et al., 2002

Male Reprod. Organ Wts. E2 B6, C17/Jls > S15 CD-1

Spearow et al., 1999, 2001

Andrology (Sperm Counts) E2 B6, C17/Jls CD-1

Spearow et al., 1999, 2001

Male Reprod. Organ Wts. DES F344 > Wistar-MS SD

Inano et al., 1996

Hormone Levels DESF344 > Wistar-MS (Testosterone)

SD (Testosterone)

Inano et al., 1996

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.

0

1

2

3

4

Wistar ControlWistar DES

F344 ControlF344 DES

SD ControlSD DES

Inano1996 data.txt

Testes Weight (g)

Strain x Treatment

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.

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

Uterine Weight BPA Da/Han Wistar, SD Diel et al 2001

Uterine Weight BPA F344 SD Steinmetz et al., 1998

Hormone Levels BPA F344 (Prl) SD (Prl)Steinmetz et al., 1997

Female Tract Histopathology BPA F344 (females) SD (females)

Long et al., 2000

Female Tract Histopathology BPA F344 (females) SD (females)

Bailey et al 2002

VO BPA AP SD Tinwell et al., 2002

Andrology (Sperm Counts) BPA AP SD

Tinwell et al., 2000

Uterine Weight NP AP SDOdum et al., 1999a

Male Reprod. Organ Wts. BPA C57BL/6N, ICR

Nagao et al., 2002

Strain Variation in Susceptibility to Bis Phenol A

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

Estrous cycle/ovulation Atrazine LE SD

Cooper et al., 2000

Fertility/gestational effects Atrazine Holtzman, F344 SD, LE

Cummings et al., 2000

Fertility/gestational effects Atrazine F344 SD, LE

Narotsky et al., 2001

Hormone Levels Atrazine LE (LH, Prl) SD (LH, Prl)Cooper et al., 2000

Hormone Levels Atrazine Holtzman (P) SD (E2, P)Cummings et al., 2000

Estrous cycle (Persistent Estrus) Atrazine SD F344

Eldridge et al., 1994

Mammary Gland Histopath. Female Atrazine SD (females) F344 (females)

Eldridge et al., 1994

Hormone Levels Atrazine SD (E2) F344 (E2)Eldridge et al., 1994

Uterine Weight tamoxifen SD F344Bailey et al., 2002

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

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

AGD flutamide SD, LE You et al., 1998Nipple retention flutamide SD, LE You et al., 1998Male Reprod. Organ Wts. flutamide LE, SD You et al., 1998AGD p,p’-DDE LE SD You et al., 1998Nipple retention p,p’-DDE SD LE You et al., 1998PPS p,p’-DDE SD, LE You et al., 1998VO p,p’-DDE SD, LE You et al., 1998

Hormone Levels p,p’DDE SD (FSH, E2, T4) LE (FSH, Prl, LH)O'Connor et al., 1999

Hormone Levels p,p’DDELE (E2, T4, T, DHT, TSH)

SD (Prl, LH, T, DHT, TSH)

O'Connor et al., 1999

Nipple retention vinclozolin LE > Wistar Hellwig et al., 2000

Male Reprod. Organ Wts. vinclozolin LE Wistar

Hellwig et al., 2000

Reprod. Organ Histopath. vinclozolin LE (males) Wistar (males)

Hellwig et al., 2000

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

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

Hormone Levels TCDD Han/Wistar (T, LH) LE (T, LH)Haavisto et al., 2001

Hormone Levels TCDD LE (T4) Han/WistarPohjanvirta et al., 1989

Hormone Levels TSH, TRH SD, F344 (T4)Fail et al., 1999

Hormone Levels TSH, TRH F344 (T3) SD (T3)Fail et al., 1999

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.

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

Litter Size UntreatedBN < DA < Lewis < F344 Wistar< LE = SD

Harlan Breeding Data

Estrous cyclefeed restriction F344, BN SD, LE

Tropp et al., 2001

Estrous cycle/ovulation Atrazine LE SD

Cooper et al., 2000

Fertility/ Gestational Loss Atrazine Holtzman, F344 SD, LE

Cummings et al., 2000

Fertility/ Gestational Loss Atrazine F344 SD, LE

Narotsky et al., 2001

Fertility/ Gestational Loss BDCM F344 SD

Bielmeier et al., 2001

Litter Size / Gestational Loss Untreated

CN- = CN1, Small litter size Strains < C17, B6 Normal Litter Size

Large Litter Size S15<CD-1

Spearow Breeding Records

Fertility/ Gestational Loss E2 B6 CD-1

Spearow et al., 2003

Strain Variation in Susceptibility to the Disruption of Estrous Cyclicity or Gestation

Endocrine Endpoint Chemical

Sensitive Strains

Less Sensitive Strains Reference

Reprod. Organ Histopath. DMAB

F344>ACI> Lewis>CD Wistar (males)

Shirai et al, 1990

Andrology (Sperm Counts) lead SD

Apostoli et al., 1998

Reprod. Organ Histopath. Female cadmium F344 WF (cadmium)

Rehm and Waalkes, 1988

Uterine Weight D4 SD F344McKim et al., 2001

Fertility/gestational effects BDCM F344 SD

Birlmeier et al., 2001

Male Reprod. Organ Wts. DEHP CD-1 Jcl:ICR

Oishi et al., 1993

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 Recommendation1) 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

F344 8.0 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 RatsOutbred 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

References Cited: Eklund, J. and G. E. Bradford (1977). "Genetic analysis of a strain of mice plateaued for

litter size." Genetics 85(3): 529-42.Inano, H., K. Suzuki, M. Onoda and K. Wakabayashi (1996). "Relationship between

induction of mammary tumors and change of testicular functions in male ratsfollowing gamma-ray irradiation and/or diethylstilbestrol." Carcinogenesis 17(2):355-60.

Li, D., T. Li, F. Wang, H. Tian and H. H. Samuels (2002). "Functional evidence forretinoid X receptor (RXR) as a nonsilent partner in the thyroid hormonereceptor/RXR heterodimer." Mol Cell Biol 22(16): 5782-92.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12138189

Putz, O., C. B. Schwartz, G. A. LeBlanc, R. L. Cooper and G. S. Prins (2001). "Neonatallow- and high-dose exposure to estradiol benzoate in the male rat: II. Effects on malepuberty and the reproductive tract." Biol Reprod 65(5): 1506-17.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11673268

Spearow, J. L. and M. Barkley (2001). "Reassessment of models used to test xenobioticsfor oestrogenic potency is overdue." Hum Reprod 16(5): 1027-9.http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/referer?http://humrep.oupjournals.org/cgi/content/abstract/16/5/1027

Spearow, J. L., D. R. Morris, U. Wong, R. Altafi, T. Stearns, K. J. Mogel, M. R.Sanford, S. W. Eteiwi and M. Barkley (2002). Genetic Control of Susceptibility toEndocrine Disruption of Testicular Development and Spermatogenesis by Estradiol.2002 Annual Meeting of the Endocrine Society, San Francisco, CA.

Spearow, J. L., D. R. Morris, U. Wong, A. Hawatky, E. Burden, L. Martinez, B. M.Lang, H. Ordonez and M. Barkley (2003). Genetic Variation Between Strains ofMice in Susceptibility to the Disruption by Estradiol, of Pubertal ReproductiveDevelopment, Spermatogenesis, Fetal Survival and Gestation. 36th Annual Meetingof the Society for the Study of Reproduction, Cincinnati, Ohio.