25. Scientific and Technological Support on in vivo assays for the Agency's Endocrine Disrupter Screening and Testing program.

LE Gray, J Ostby, J Furr, R Cooper, T Stoker, S Laws, J Goldman, R Tyl.

1 2 3

Assign to Treatment

Dose Females (Adults - ~ 1 Month Post-Ovariectomy)

Necropsy ~ 6 hours after last am dose:

Collect serum for hormone analysisLavage

Body weight Weigh Uterus with and without fluid

Tier 1 Screening - Rat 3-Day Uterotropic Tier 1 Screening - Rat 3-Day Uterotropic Assay in Immature (21 Day Old) or Adult Assay in Immature (21 Day Old) or Adult (60 - 90 Day Old) Ovariectomized Females(60 - 90 Day Old) Ovariectomized Females

(am) Weigh,Dose andLavage

(am) Weigh,Dose andLavage

(am) Weigh,Dose andLavage

Uterotropic Assay Protocol

Required endpoints:

Growth

Uterine Wet and Dry Weights

Ovarian Weights (Immature Females)

Vaginal cytology (Adult Ovariectomized Females)

Optional measures

Uterine histology

Liver, kidney, pituitary and adrenal weights

Receptive Behavior - Lordosis quotient

Detects exogenous estrogenic compounds.

Effects of Xenoestrogens on Uterine Weight, Vaginal Cornification Effects of Xenoestrogens on Uterine Weight, Vaginal Cornification and Lordosis Behavior in Adult Ovariectomized Rats.and Lordosis Behavior in Adult Ovariectomized Rats.

INDUCTION OF ESTROGENIC EFFECTS INDUCTION OF ESTROGENIC EFFECTS

OILBPA sc200

BPB sc200PCPsc200

OC Tsc200N Psc/oral200

METsc200METSC 400

METOR AL200METOR AL400

E2D icofolsc200

DB Psc200DB Psc400

D B Poral1000

Lordosis Quotient

Vaginal Cytology (%C or N - 20%)

Uterine Weight ( - con)

PRIORITIZATION FOR SCREENINGBASED UPON QSAR, HPTS, VOLU ME, ETC

TIER 1 SCREENINGAbout 100 rats per chemical

(about 10,000 Rats/100 chemicals)

HOLD95%

NegativeNoTesting

90% (-)

10%(+)True + (5%)

False + (5%)(Type II Error)

(10 chemi cals per 100 screened)ReplicateTier 1

Repeat

False positives 5%

(about 50 rats per repeat

or 500 rats per 100 chemicals

screened)

Positives 5%Tier 2 Testing

(about 1, 500 rats per chemical ,

or 7,500 rats per 100 screened)

Including a "Tier 1 Repeat" step between T1S and T2T can reduce animal use in EDSP. Our Est imate of Animal Use in EDSP

(Assuming 5% of Chemicals are endocrine active)18,000 Rats per Hundred Chemicals Evaluated

42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

Assign to Treatment

Dose Males(~ 10 Days)

Necropsy:Serum for Hormone Analysis

Weigh: Glans Pe nisVentral Prostate

Seminal Ve sicles /Coagulating Glands (with/without fluid)Levator Ani/Bulbocavernosus Muscle

Cowper's Gla ndsLiver

AdrenalsKidneys

Tier 1 Screening Tier 1 Screening OECD Hershberger Assay ProtocolOECD Hershberger Assay Protocol

Castrate

Days of age

Androgen-Dependent Tissues Regress

Dose (54 - 63)

OECD Hershberger Assay Protocol

Required endpoints:Glans Penis WeightsVentral Prostate WeightsSeminal Vesicle/Coagulating Gland Weights with and without fluidLevator Ani/Bulbocavernosus Muscle WeightsCowper’s Glands WeightsLiver WeightAdrenals WeightKidneys Weight

Optional measures:Cardiac Blood for Serum Hormone AnalysisLiver, kidney and adrenal histology

Detects exogenous (anti)androgens which inhibit or stimulate growth of androgen-dependent tissues (Testosterone Propionate stimulates growth of androgen-dependent tissues after the regression of these tissues following castration).

A comparison of the dose-response curves for A comparison of the dose-response curves for TP sc in the Hershberger Assay and an in utero TP sc in the Hershberger Assay and an in utero

study (Gray et al.; Wolf et al.)study (Gray et al.; Wolf et al.)

TP dose sc mg/kg/d

0

20

40

60

80

100

Per

cent A

ffec

ted/E

ffec

t

Hershberger Developing Female

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Start Checking for VOLavage Daily after VO

Dose Female

Necropsy: T4 and TSH

Weigh: OvariesUterus (with/without fluid)

LiverKidneysPituitaryAdrenals

Histology: Uterus and Ovaries

Tier 1 Screening Tier 1 Screening Rat Pubertal Female AssayRat Pubertal Female Assay

DosingInitiated

Days of age

Assign to TRT

Immature (21 - 43 Days of Age) Intact Female Rat Protocol toEvaluate Pubertal Development and Thyroid Function

Required endpoints: Growth Age and weight at vaginal opening (VO)Vaginal cytology Serum thyroxine (T4) and thyroid-stimulating hormone (TSH)

Ovarian and uterine weights and histology Liver, kidney, pituitary and adrenal weights

Detects exogenous antithyroid or estrogenic compounds which alter pubertal development or thyroid function.

Daily oral (m g/kg/d) Methoxychlor or Octylphenol treatment Daily oral (m g/kg/d) Methoxychlor or Octylphenol treatment

induces pseudoprecocious puberty in the 21 day old female rat. induces pseudoprecocious puberty in the 21 day old female rat.

Data are expressed as the number of days that VO was accelerated Data are expressed as the number of days that VO was accelerated

(Gray and Ostby, 1998). (Gray and Ostby, 1998).

0 50 100 150 200

Dose of Chemical (mg/kg/d)

0

2

4

6

8

Days that VO is accelerated versus control

Octylphenol Methoxychlor

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Start Checking for

PPS

Dose Male

Necropsy:T4 and TSH

Weigh: Tes tesEpididymides

Ventral ProstateSeminal Vesicles/ Coagulating Gland (with/without fluid)

Leva tor Ani/Bulbocavennosus MuscleHistology: Testes , Epididymides and Thyroid

Alternative Tier 1 Screening Alternative Tier 1 Screening Rat Pubertal Male AssayRat Pubertal Male Assay

DosingInitiated

Days of age

Assignto TRT

Immature (23 - 54 Days of Age) Intact Male Rat Protocol to Evaluate Pubertal Development and Thyroid Function

Required EndpointsGrowth Age and body weight at preputial separationSerum thyroxin (T4) and thyroid-stimulating hormone (TSH)Thyroid histologySeminal vesicle plus coagulating gland weight (with/without fluid)Ventral prostate weightLevator ani/bulbocavernosus muscle weightTestis and epididymal weights and histology

Optional MeasuresSerum testosterone, estradiol, LH, prolactin and tri-iodothyronine

(T3)Liver, kidney, adrenal and pituitary weights and histologyex vivo testis and pituitary hormone productionHypothalamic neurotransmitter levels

Detects exogenous antithyroid, estrogenic, androgenic or antiandrogenic compounds and EDCs which alter pubertal development via follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH) or hypothalamic function.

Oral administration (21-55 days of age) of EDCs Oral administration (21-55 days of age) of EDCs delays Puberty (Preputial Separation) in the Male rat.delays Puberty (Preputial Separation) in the Male rat.

0 5 10 15 25 30 10038

40

42

44

46

48

50EPA RTI

Vinclozolin

40

45

50

55EPA 1EPA 2

EPA 3EPA 4

RTI

Methoxychlor

0 10 30 10040

42

44

46

48

50

52 H EA LTH Y EPA 1

R U N TS EPA 2

p,p' DDE

0 10 20 40404142434445464748

LINURON

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 230

1

BreedAssign to Treatments Birth

Dose Dam

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

1

PPS

Wean

Postnatal life in weeks

AGD

Areolas

Dose Dam Dose pups: Optional

Necropsy all offspring: (n= 50/sex/dose/8 litters)

Gestation (Days)

Alternative Tier 2 TestingAlternative Tier 2 TestingGeneral Transgenerational ProtocolGeneral Transgenerational Protocol

PO

F1

VO

Sexual Differentiation

Transgenerational Protocol

Required endpoints:Maternal Growth F1 Pup Growth and Viability (Count and Weigh Litter by Sex Weekly Until Weaning)Pups at Birth – Weigh Litters by Sex to Determine Litter Size, Average Bodyweight by Sex and Sex Ratio for Each LitterAnogenital Distance and Bodyweight (All F1 Pups - PND 2)Areolas and Position/Male and Weigh Litters by Sex (PND 13) Wean – Sacrifice Dams and Count Implantation Scars, House F1 Offspring in Unisexual Littermate Groups of 2 – 3 Pups/CagePuberty - Start Checking Females (PND 27) for Vaginal Opening and Record Age and Bodyweight at VO and Start Checking

Males (PND 37)for Preputial Separation and Record Age and Bodyweight at PPSMale Necropsy:

Serum for Hormone AnalysisShave and Check for Nipples and External MalformationsGlans Penis WeightVentral Prostate WeightSeminal Vesicles/Coagulating Glands (with/without fluid) WeightRight and Left Testes Weights and HistologyRight and Left Epididymal Weights and HistologyLevator Ani/Bulbocavernosus Muscle WeightPaired Cowper’s Glands WeightLiver WeightAdrenals WeightKidneys WeightNote any Internal Malformations (Particularly Androgen-Dependent Tissues)

Optional MeasuresMating and/or Play BehaviorSonication Resistant Testicular Sperm Head CountsCauda and Caput Epididymal Sperm CountsVentral Prostate, Seminal Vesicle/Coagulating Gland, Liver, Kidney, and Adrenal HistologyAR Immunohistochemistry (Androgen-Dependent Tissues such as Ventral Prostate)Ex-vivo Testicular Testosterone Production (hCG stimulated and unstimulated)

Detects exogenous estrogenic and (anti)androgenic compounds which permanently alter reproductive development and can be used to establish low and no observed effects levels.

Number of Areolas/Male at 13 Days of Age(Data are Litter Means)

0

2

4

6

8

1 0

1 2

Mean 0 9.1 6.5 1.1 0 0 0

SE 0 1 1 0.5 0 0 0

CON DEHP ** BBP ** DINP DEP DMP DOTP

Research Impact

EDC action can seriously alter reproductive development when administered during critical, sensitive life stages. In utero, perinatal and pubertal exposures to EDCs can produce severe effects on both male and female offspring, depending upon the mechanism of action. Some effects are not obvious until late in life or are latent and not expressed until after maturity or old age. The only protocols that can detect these types of effects are long-term transgenerational or multigenerational studies because they are the only protocols that 1) expose the animals during development and 2) evaluate the reproductive system in the offspring throughout life. Our studies typically dose the mother during gestation and lactation and observe the development of male and female rat offspring through maturity. The determination of the cellular and molecular mechanism of action of an EDC is critical to understanding its potential to affect other species, including humans. In order to elucidate mechanisms of action, we execute parallel in vitro or ex vivo studies with EDCs or their active metabolites. In addition, we are attempting to obtain data on fetal tissue levels of the active metabolites during the critical exposure period. Over the last few years we have examined a variety of chemicals with different mechanisms of endocrine action. We have studied estrogens (methoxychlor, zearalenone, and chlordecone), inhibitors of steroidogenesis (ketoconazole, fenarimol (inhibits aromatase), germ cell toxicants (busulfan and benzidine-based diazo dyes), phthalate esters (inhibit fetal testosterone production) and fungicides that are AR antagonists. We are now beginning to study environmental chemicals with androgenic activity. Over the last seven years, most of our work has been with environmental antiandrogens. In 1994 we published the first study that demonstrated that a pesticide altered male rat sexual differentiation by acting as an AR antagonist. This mechanism was confirmed using in vitro AR binding and transcriptional activation assays, and by examining AR-dependent ventral prostate gene expression (1997). In 1995 we were the first to report in Nature that the p,p’ DDE also was an AR antagonist. These studies have now been extended to several other original observations on pesticides like procymidone and linuron. Our work on linuron was the first to demonstrate that this herbicide induced reproductive tract malformations, and points to weaknesses in former and current Agency multigenerational test guidelines, as these malformations had been missed in several published developmental toxicology and multigenerational tests. In addition to AR antagonists, we, along with the laboratory of Dr P Foster at CIIT, have been studying the antiandrogenic effects of phthalate esters. We published the only report to date that demonstrates that diethyl hexyl phthalate (DEHP) inhibits masculinization of the male rat reproductive system by inhibiting fetal testosterone production. Currently, we are attempting to increase our ability to examine the cellular and molecular events associated with the effects of these chemicals on the fetal reproductive tract and testis. We also have collected tissues from some of these studies for chemical residue or mRNA analyses. For example, we have submitted fetal amniotic fluid and maternal urine samples from two phthalate ester studies to the Center for Disease Control (CDC) for determination of MEHP and MBP levels. We can then compare the dose to the fetus associated with adverse developmental effects in the rat and we can compare MEHP and MBP levels to those seen in humans. In a risk assessment, the Agency considers many different exposure scenarios besides intrauterine development. For example, EDC administration during puberty can alter this developmental process. Estrogens accelerate pseudoprecocious puberty in the female, while antiandrogens delay puberty in the male rat. In this regard, our pubertal studies on vinclozolin (1999) were the first demonstration that an antiandrogenic pesticide could delay the onset of pubertal landmarks in the male rat. These data were used by EPA to restrict uses of this chemical that were likely to result in childhood exposures to vinclozolin at “levels of concern”. We also found that linuron, di-n-butyl phthalate, diethyl hexyl phthalate and p,p’ DDE delay the onset of puberty in the male rat.

Products

Goldman, J.M., Laws, S.C., Balchak, S.K., Cooper, R.L., and Kavlock, R.J. Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid activity in the female rat. A focus on the EDSTAC recommendations. Crit Rev Toxicol. 30:135-196, 2000. Gray LE Jr, Ostby J, Sigmon R, Ferrell J, Rehnberg G, Linder R, Cooper R, Goldman J, Laskey J. The development of a protocol to assess reproductive effects of toxicants in the rat. Reprod Toxicol. 1988;2(3-4):281-7. Review. Gray LE Jr, Ostby JS, Ferrell JM, Sigmon ER, Goldman JM. Methoxychlor induces estrogen-like alterations of behavior and the reproductive tract in the female rat and hamster: effects on sex behavior, running wheel activity, and uterine morphology. Toxicol Appl Pharmacol. 1988 Dec;96(3):525-40. Gray LE Jr, Ostby J, Ferrell J, Rehnberg G, Linder R, Cooper R, Goldman J, Slott V, Laskey J. A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundam Appl Toxicol. 1989 Jan;12(1):92-108. Gray LE Jr, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulniers D, Wilson E, Zacharewski T, et al. (33 authors). Endocrine Screening Methods Workshop Report: Detection of Estrogenic and Androgenic Hormonal and Antihormonal Activity for Chemicals that act via Receptor or Steroidogenic Enzyme Mechanisms. Reproductive Toxicology 11(4):719-750, 1997. Gray LE Jr, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. J Toxicol Ind Health 15 (1-2):94-118, 1999. Gray LE Jr, Ostby J, Cooper RL, Kelce WR. The estrogenic and antiandrogenic pesticide methoxychlor alters the reproductive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats.Toxicol Ind Health 15(1-2):37-47, 1999. Gray LE Jr , Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DNR, Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L. Effects of environmental antiandrogens on reproductive development in experimental animals. Human Reprod Update. 7(3):248-64. 2001. Gray LE Jr, Lambright C, Parks L, Tyl R, Orlando E, Guillette L, Wolf C, Seely J, Chang TS, WilsonV, Hotchkiss A, Ostby J. (In press) Emerging issues related to endocrine disrupting chemicals and environmental androgens and antiandrogens. In: Handbook of Environmental Chemistry: Endocrine distuptors. (M Metzler Ed). 2001. Monosson E, Kelce WR, Lambright C, Ostby J, Gray LE Jr. Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol Ind Health 15(1-2):65-79, 1999. Stoker, TE, Parks, LG, Gray, LE, and Cooper, RL. Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations. Endocrine Disrupter Screening and Testing Advisory Committee. Crit Rev Toxicol. 30:197-252, 2000. Hertzberg VS, Lemasters GK, Hansen K, Zenick HM. Statistical issues in risk assessment of reproductive outcomes with chemical mixtures. Environ Health Perspect. 1991 Jan;90:171-5. Review.

Future Directions

EDSP ISSUES.Hershberger Assay. As the Lead Laboratory Ananalyze final Phase 1 OECD interlaboratory studies and draft report. Design Phase 2 and present proposal to OECD VMG for approval.Initiate Phase 2, analyze data and complete report and publish results after OECD approval.Publish background document on this Assay.