John C. O’Connor

DuPont Haskell Laboratory for Health and

Environmental Sciences

The 15-Day Intact Adult Male Assay As An Alternative Tier I Screening

Assay For Detecting Endocrine-Active Compounds

2

Outline

• Background

• Overview of the 15-day intact adult male assay– Study rationale

– Study design considerations

– Case study with flutamide, ketoconazole, & finasteride

• Path Forward

3

Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for

Identifying EACs

Recommended Screening Battery

Alternate Screening Battery No. 1

Alternate Screening Battery No. 2

In vitro assays In vitro assays In vitro assays ER binding/transactivation ER binding/transactivation ER binding/transactivation AR binding/transactivation AR binding/transactivation AR binding/transactivation Minced testis assay Placental Aromatase Placental Aromatase In vivo mammalian assays In vivo mammalian assays In vivo mammalian assays Uterotrophic assay (3-day) Uterotrophic assay (5-day) Uterotrophic assay (3-day) Hershberger Intact adult male Pubertal male Pubertal female

4

Desirable Attributes of a Screen

• Reliable (identifies known EACs for EAT activity)• Predictive (known EACs are identified for their mode

of action)• Sensitive (low false-negatives)• Quick (i.e., short-term)• Cost effective• Minimize animal usage

5

Study Design: 15-Day Intact Adult Male Assay• Model:

– 10-Week old intact male rats– n = 15/group– Control + 4 dose groups– 15-Day test (oral)

• Required Endpoints:– Organ weights - liver, testes, thyroid, epididymides, prostate, seminal ves., ASG unit– Histopathology - testis, epididymides, thyroid– Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH– Biochemical - preparation of hepatic microsomes

• Optional Endpoints (if warranted by other findings)– Histopathology – liver– Biochemical Assessment

• Hepatic UDP-glucuronyltransferase activity• Hepatic aromatase activity

– Others?

CNS

Hypothalamus

Anterior Pituitary

GnRH (+)

FSH (+)

Sertoli Cell Leydig Cell

Inhibin Testosterone

Aromatase

Estradiol

(-)

(-) (-)

(-)

Testis

LH (+)

X

X

X

X

DHT

X 5-Reductase

Target Peripheral Tissues

X

Hypothalamic-Pituitary-Testis Axis

7

Adult Male Assay: “Expected” Profile for Unknowns

Endocrine Activity

ASG Unit (% body weight)

Thyroid (% body weight)

Test.

(ng/ml)

E2

(pg/ml)

PRL

(ng/ml) LH & FSH

(ng/ml) TSH

(ng/ml) T4

(g/dl) Receptor Agonists Estrogen Receptor –/ – –/ –/ – – Androgen Receptor – – – Receptor Antagonists Estrogen Receptor – – – – – –/ – – Androgen Receptor –/ – –/ – – – Steroid Biosynthesis Inhibitors

Testosterone Biosynthesis – – – – – 5-Reductase – – – –/ – – Aromatase – – – –/ – – Thyroid-Active Compounds – –/ – –/ –

8

Adult Male Assay: Study Design Issues

• Oral dosing – most relevant route• Dose level selection

– Based on range-finder studies– Target ≤ 10% final body weight

• Based on dietary restriction studies– O’Connor et al., 2000 (Toxicol. Sci. 54: 338-354)

• Adult vs. immature animals– Immature are more sensitive to organ weight changes– Mature are more sensitive to hormonal changes– Mature have greater blood volume for hormonal assessment

• Duration – 2-week• Strain sensitivity differences

9

Final body

(% of control)

Testes

Epididymides Accessory sex

gland unit

Seminal vesicles

Prostate

Thyroid

Absolute Organ Weights (grams)

100 3.3 0.1 1.14 0.02 2.3 0.1 1.6 0.1 0.617 0.021 0.025 0.001

90 3.2 0.0 1.11 0.03 2.0 0.1 1.5 0.1 0.555 0.031 0.021 0.001*

85 3.3 0.1 1.08 0.02 1.8 0.1* 1.2 0.1* 0.529 0.034 0.019 0.001*

79 3.2 0.1 1.11 0.01 1.8 0.1* 1.3 0.1* 0.524 0.039 0.019 0.001*

74 3.2 0.1 1.06 0.02* 1.6 0.1* 1.1 0.1* 0.454 0.029* 0.019 0.001*

Relative Organ Weights (% body weight)

100 0.79 0.02 0.276 0.006 0.552 0.018 0.396 0.017 0.149 0.005 0.006 0.0003

90 0.86 0.01* 0.296 0.007* 0.548 0.020 0.394 0.016 0.149 0.008 0.006 0.0003

85 0.94 0.02* 0.308 0.005* 0.504 0.020 0.350 0.020 0.150 0.009 0.006 0.0003

79 0.97 0.02* 0.338 0.004* 0.561 0.036 0.411 0.026 0.160 0.012 0.005 0.0003

74 1.04 0.02* 0.344 0.006* 0.516 0.022 0.364 0.018 0.148 0.010 0.006 0.0003

Mean standard error. N = 15/group. * Significantly different (p < 0.05) from control by Dunnett’s Test.

Effect of Diet Restriction on Organ Weights in Sprague-Dawley Rats

10

Effect of Diet Restriction on Serum Hormones in Sprague-Dawley Rats

Final body

(% of control)

Testosterone

(ng/ml)

Estradiol (pg/ml)

Dihydro- testosterone

(pg/ml)

Prolactin (ng/ml)

Follicle stimulating hormone (ng/ml)

Luteinizing hormone (ng/ml)

100 3.5 0.5 11.1 1.4 162.3 25.4 17.9 2.9 13.1 0.7 4.4 0.3

90 3.9 0.6 11.9 1.2 175.3 19.6 11.8 1.5 14.9 0.9 5.2 0.4

85 3.7 0.8 12.9 1.2 176.3 32.6 16.5 2.3 13.4 0.6 4.8 0.3

79 1.6 0.4 # 12.8 1.6 81.3 14.0 # 9.9 1.4 # 13.9 0.6 5.1 0.3

74 0.9 0.3 # ND 60.6 12.9 # 10.1 2.1 # 12.8 0.7 5.1 0.3

Mean standard error. ND – not determined. N = 15/group. # Significantly different (p < 0.05) from control by Jonckheere’s test for trend.

11

Final body (% of control)

TSH

(ng/ml)

T3

(ng/dl)

T4

(g/dl)

100 17.3 1.3 80.7 4.0 4.3 0.2

90 17.0 1.8 79.9 3.6 4.0 0.2

85 16.7 1.5 68.1 3.7 # 3.6 0.2 #

79 14.1 1.1 70.5 3.7 # 3.2 0.2 #

74 10.8 1.5 # 60.8 2.8 # 3.1 0.2 #

Mean standard error. N = 15/group. # Significantly different (p < 0.05) from control by Jonckheere’s test for trend.

Effect of Diet Restriction on Thyroid Hormones in Sprague-Dawley Rats

12

Data Interpretation• High dose level should not exceed MTD, defined as <10% difference in

final body weight when compared to the control group

• Organ Weights– Absolute

• Testes, epididymis

– Relative (% final body weight)

• Liver, thyroid, prostate, seminal vesicles, ASG unit

• Histopathology - testis, epididymides, thyroid• Control + high dose unless effects are observed

• Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH

• Evaluate trends across dose response and overall pattern of hormonal effects

13

Testes ASG Sem Ves. Prostate0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Control Flutamide (10 mg/kg)

Weig

ht (%

Bo

dy W

eigh

t)*

*

Testes ASG Sem Ves. Prostate0.0

0.1

0.2

0.8

0.9

1.0

*

*

*

Wei

gh

t (%

Bo

dy W

eigh

t)Immature vs. Mature RatsImmature Mature

14

Testosterone LH0

2

4

6

8

10

12

14

16

18

20

ng/m

l

*

*

Testosterone LH0

2

4

6

8

10

12

14

16

18

20

ng/m

l

Control 10 mg/kg Flutamide

**

Immature vs. Mature RatsImmature Mature

15

Immature vs. Mature Comparison: Study Design• Goal: Compare responses of immature and mature rats to 2 positive

controls using identical study designs, and evaluate the intrinsic variability of serum hormone and organ weight measurements in mature and immature rats.

• Model:– Immature intact male rats (age 53 days at necropsy)– n = 15/group– 15-Day test (oral)– Control + 4 dose groups

• Vinclozolin (75 and 150 mg/kg/day)• Phenobarbital (5 and 25 mg/kg/day)

– Included feed restriction group (targeted to 10% body weight decrement from con.– Included two additional “control” groups (age 49 and 57 days at necropsy)

– Second experiment currently being conducted with PTU, flutamide, linuron, & DBP

16

0.0

0.1

0.2

0.3

0.4

0.5

0.6

***

*

*

Gra

ms

or %

Bod

y W

eigh

t

Control 75 mg/kg/day 150 mg/kg/day

Epididymis Sem. Ves. Prostate (grams) (%BW) (%BW)

*

Age – 53 Days

0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

Epididymis Sem. Ves. Prostate (grams) (%BW) (%BW)

Gram

s or % B

ody Weight

Organ Weights in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days

Age – 84 Days

Note: Organ weight alterations were more pronounced at 300 mg/kg/day in mature rats, and included microscopic alterations of the testes at 150 and 300 mg/kg/day.

17

Testosterone LH FSH0

3

6

9

12

15

18

21

24

27*

*

**

Seru

m C

onc. (n

g/ml)

Control 75 mg/kg/day 150 mg/kg/day

Testosterone LH FSH0

3

6

9

12

15

18

21

24

27

*

**

*

Ser

um

Con

c. (

ng/

ml)

Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days

Age – 53 Days Age – 84 Days

Note: Hormonal alterations were more pronounced at 300 mg/kg/day in mature rats.

18

Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Phenobarbital for 15 Days

Age – 53 Days Age – 84 Days

TSH T3 T40

10

20

30

40

50

60

70

80

90

*

*

Seru

m C

onc.

*

TSH T3 T40

10

20

30

40

50

60

70

80

90

*

*

*

*

Control 5 mg/kg/day 25 mg/kg/day

Seru

m C

onc.

Note: Thyroid weight was significantly increased at both dose levels in mature rats but was NOT affected in immature rats.

19

45 50 55 60 65 70 75 80 850

2

4

6

8

10

12

14

Tes

tost

eron

e (n

g/m

l)

Age (Days)

45 50 55 60 65 70 75 80 85

0

200

400

600

800

1000

Dih

ydro

test

oste

ron

e (p

g/m

l)

Age (Days)

45 50 55 60 65 70 75 80 85-2

0

2

4

6

8

10

12

14

16

18

Est

rad

iol (

pg/

ml)

Age (Days)

Correlation Between Age and Variability in Serum Hormone Concentrations in Rats

20

45 50 55 60 65 70 75 80 850

1

2

3

4

5

6

Lu

ten

izin

g H

orm

one

(ng/

ml)

Age (Days)

45 50 55 60 65 70 75 80 85

4

6

8

10

12

14

16

18

20

Fol

licl

e S

tim

ula

tin

g H

orm

one

(ng/

ml)

Age (Days)

45 50 55 60 65 70 75 80 85

0

10

20

30

40

50

60

70

80

Pro

lact

in (

ng/

ml)

Age (Days)

Correlation Between Age and Variability in Serum Hormone Concentrations in Rats

21

45 50 55 60 65 70 75 80 851

2

3

4

5

6

7

Th

yrox

ine

(g/

dl)

Age (Days)

45 50 55 60 65 70 75 80 850

5

10

15

20

25

30

35

40

45

Th

yroi

d S

tim

ula

tin

g H

orm

one

(ng/

ml)

Age (Days)

45 50 55 60 65 70 75 80 8530

40

50

60

70

80

90

100

110

120

Tri

iod

oth

yron

ine

(ng/

dl)

Age (Days)

Correlation Between Age and Variability in Serum Hormone Concentrations in Rats

22

Adult Male Assay: Thyroid Timecourse

1 Week 2 Week 4 Week0

25

50

75

100

125

*

UD

P-G

T (n

mol/m

g/m

in)

**

1 Week 2 Week 4 Week0.000

0.005

0.010

0.015

0.020

Control PB (100 mg/kg) PTU (1 mg/kg)

Th

yro

id W

eigh

t (%

bod

y w

eig

ht)

*

*

*

*

*

23

Adult Male Assay: Thyroid Timecourse

1 Week 2 Week 4 Week0

10

20

30

40

50

60

70

Control PB (100 mg/kg)l PTU (1 mg/kg)

*

*

*

*

TS

H (

ng/m

l)

1 Week 2 Week 4 Week0

1

2

3

4

T4 (m

g/d

l)

*

*

*

*

*

*

24

Detection of p,p’-DDE in the Adult Male Assay

• Weak AR antagonist

• Strain differences observed in 15-day intact male assay

• O’Connor et al., 1999 (Toxicol. Sci. 51: 44-53)

– CD rats – not clearly identified as an AR antagonist

– LE rats – identified as an AR antagonist

– Consistent with strain differences observed in studies of You et al., 1998 (Toxicol. Sci. 45, 162-173)

25

Dosage (mg/kg/day)

Final body

(grams)

Liver

(% body weight)

Testes

(grams)

Epididymides

(grams)

Accessory sex gland unit

(% body weight)

Seminal vesicles (% body weight)

Prostate

(% body weight)

Sprague-Dawley Rats

0 427 6 3.8 0.1 3.3 0.1 1.26 0.02 0.525 0.018 0.319 0.019 0.198 0.012

100 389 7 * 5.0 0.1 * 3.3 0.1 1.23 0.03 0.550 0.019 0.368 0.018 0.176 0.011

200 338 13 * 5.6 0.2 * 3.2 0.1 1.09 0.04 * 0.477 0.028 0.299 0.023 0.192 0.011

300 362 12 * 5.6 0.1 * 3.2 0.1 1.13 0.04 * 0.531 0.026 0.339 0.021 0.184 0.015

Long-Evans Rats

0 402 6 4.5 0.1 3.3 0.1 0.88 0.03 0.346 0.025 0.221 0.020 0.118 0.011

200 374 8 6.7 0.1 * 3.5 0.1 1.02 0.10 0.382 0.019 0.250 0.014 0.122 0.008

300 361 11 * 6.7 0.1 * 3.3 0.2 1.04 0.07 * 0.450 0.019 * 0.289 0.013 * 0.156 0.010 *

Mean standard error. * Significantly different (p < 0.05) from control by Dunnett’s Test.

Effect of p,p’-DDE on Organ Weights in the Adult Male Assay

26

Dosage (mg/kg/day)

Testosterone

(ng/ml)

Estradiol (pg/ml)

Dihydro- testosterone

(pg/ml)

Prolactin (ng/ml)

Follicle stimulating hormone (ng/ml)

Luteinizing hormone (ng/ml)

Sprague-Dawley Rats

0 3.0 0.8 4.4 0.9 51.5 14.4 10.3 1.7 14.5 0.6 2.9 0.2

100 2.5 0.5 5.4 0.9 51.3 9.1 13.6 2.4 15.1 0.7 3.3 0.3

200 0.8 0.4 11.7 1.3 # 33.6 10.0 11.7 1.8 10.9 0.8 # 2.7 0.3

300 2.0 0.5 9.7 1.0 # 62.5 12.2 9.3 0.9 11.9 0.7 # 2.9 0.2

Long Evans Rats

0 1.0 0.4 7.9 0.8 37.6 17.3 10.0 1.3 11.0 0.7 3.2 0.1

200 2.4 0.5 # 10.3 1.2 87.9 21.2 # 8.5 1.1 11.1 0.4 3.4 0.2

300 3.1 0.7 # 13.8 1.3 # 103.3 14.7 # 8.7 1.6 13.0 1.3 3.5 0.4

Mean standard error. # Significantly different (p < 0.05) from control by Jonckheere’s test for trend.

Effect of p,p’-DDE on Serum Hormone Levels in the Adult Male Assay

27

Dosage (mg/kg/day)

Thyroid stimulating hormone (ng/ml)

T3

(ng/dl)

T4

(g/dl)

Sprague-Dawley Rats

0 13.4 0.9 55.3 3.3 2.9 0.2

100 20.9 2.4 44.8 2.8 1.2 0.1 #

200 16.0 1.6 49.8 3.8 0.5 0.1 #

300 15.3 1.0 49.1 3.8 0.5 0.1 #

Long Evans Rats

0 15.7 1.1 74.2 2.8 4.5 0.3

200 35.1 4.3 # 73.0 3.0 2.1 0.1 #

300 28.7 2.6 # 71.5 2.9 1.6 0.1 #

Mean standard error. # Significantly different (p < 0.05) from control by Jonckheere’s test for trend

Effect of p,p’-DDE on Thyroid Hormone Levels in the Adult Male Assay

28

Case Study

With Flutamide, Ketoconazole,

& Finasteride

CNS

Hypothalamus

Anterior Pituitary

GnRH (+)

FSH (+)

Sertoli Cell Leydig Cell

Inhibin Testosterone

Aromatase

Estradiol

(-)

(-) (-)

(-)

Testis

LH (+)

X

X

X

X

DHT

X 5-Reductase

Target Peripheral Tissues

X

Hypothalamic-Pituitary-Testis Axis

30

Case Study: Study Design• Model:

– 10-Week old intact male rats

– n = 15/group

– 15-Day test (oral)

– Control + 4 dose groups

• Dose levels selected based on range-finder studies

– Flutamide (10 mg/kg/day; high dose)

– Ketoconazole (100 mg/kg/day; high dose)

– Finasteride (25 mg/kg/day; high dose)

• Measured Endpoints:– Organ weights - liver, testes, thyroid, epididymides, prostate, seminal ves., ASG unit

– Histopathology - testis, epididymides, thyroid

– Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH

31

Case Study: Organ Weights

Body ASG Unit Prostate0

20

40

60

80

100

**

*

*

*

*

Per

cen

t of

Con

trol

FLUT (20 mg/kg) KETO (100 mg/kg) FINA (25 mg/kg)

32

Test DHT LH0

50

100

150

200

250

300

350

400

FLUT (20 mg/kg) KETO (100 mg/kg) FINA (25 mg/kg)

*

*

*

* *

*

**

Per

cen

t of

Con

trol

Case Study: Serum Hormones

33

Effect Of Ketoconazole On Testosterone Biosynthesis From Isolated Rat Leydig Cells

0 0.1 0.15 0.2 0.5 1 5 100.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

PROGESTERONE

ng

/mL

0 0.1 0.15 0.2 0.5 1 5 100.0

0.5

1.0

1.5

2.0 17-OH-PROGESTERONE

0 0.1 0.15 0.2 0.5 1 5 100.0

0.2

0.4

0.6

0.8

1.0 ANDROSTENEDIONE

+hCG

-hCG

ng

/mL

Ketoconazole (uM)

0 0.1 0.15 0.2 0.5 1 5 100

1

2

3

4

5 TESTOSTERONE

Ketoconazole (uM)

34

Testosterone Biosynthesis (4 Pathway)① Cholesterol SCC enzyme (CP-450)

a. 20-hydroxylaseb. 22-hydroxylasec. 20,22-lyase

② 3-Hydroxysteroid Dehydrogenase ③ 4,5-Ketosteroid Isomerase ④ 17-Hydroxylase (CP-450) ⑤ C-17,20-Lyase (CP-450) ⑥ 17-Hydroxysteroid Dehydrogenase ⑦ 5-Reductase ⑧ Aromatase (CP-450)

Pregnenolone

Cholesterol

Progesterone

17-Hydroxyprogesterone

Androstenedione

Testosterone

1a

1b

1c

②③

④ Ketoconazole

⑤

⑥

5-Dihydroxytestosterone

⑦

⑧

⑧

Estrone

17-Estradiol

Leydig Cell

X

FinasterideXTarget peripheral tissuesX

Flutamide

35

Case Study: Flutamide, Ketoconazole, & FinasterideComparison of Organ Weight Data

Compound Testes

(g) Epid (g)

ASG (% BW)

Sem. Ves. (% BW)

Prostate (% BW)

Flutamide -

Ketoconazole -

Finasteride -

Statistically significant (p<0.05) decrease ( ) or no change (-) in endpoint response.

Cannot differentiate mode of action based on organ weight changes

36

Compound T

(ng/ml) DHT

(pg/ml) FSH

(ng/ml) LH

(ng/ml)

Flutamide Ketoconazole

Finasteride - - Statistically significant (p<0.05) increase ( ), decrease ( ), or no change (-) in endpoint response.

Mode of action can be determined based on hormonal changes

Case Study: Flutamide, Ketoconazole, & FinasterideComparison of Serum Hormone Data

Hypothalamic-Pituitary-Testis Axis

CNS

Hypothalamus

Anterior Pituitary

GnRH (+)

FSH (+)

Sertoli Cell Leydig Cell

Inhibin Testosterone

Aromatase

Estradiol

(-)

(-) (-)

(-)

Testis

LH (+)

X

X

DHT

X 5-Reductase

Target Peripheral Tissues

XAntiandrogens

(Flutamide)

Antiandrogens(Flutamide)

5-Reductase Inhibitors(Finasteride)

Steroid Inhibitors(Ketoconazole)

38

Intact Male EAC Endocrine Activity

+ 17-Estradiol Estrogen receptor agonist (full or potent)

+ Coumestrol Estrogen receptor agonist (weak or partial)

+ ICI-182,780 Estrogen receptor antagonist + Testosterone Androgen receptor agonist + Flutamide Androgen receptor antagonist (full or potent)

+ p,p’-DDE Androgen receptor antagonist (weak or partial) + Vinclozolin Androgen receptor antagonist (weak or partial) + Cyproterone Acetate Androgen receptor antagonist (weak or partial) + Linuron Androgen receptor antagonist (weak or partial) + Progesterone Progesterone receptor agonist

+ Mifepristone (RU486) Progesterone receptor antagonist Apomorphine D2 receptor agonist + Haloperidol D2 receptor antagonist

+ Reserpine Dopamine depletor (broad catecholamine depletion) + Phenobarbital Thyroid hormone excretion enhancer

+ Oxazepam Thyroid hormone excretion enhancer + Propylthiouracil Thyroid hormone synthesis inhibitor

+ Propylimidazole-2-thione (PTI) Thyroid hormone synthesis inhibitor

+ Finasteride 5-Reductase inhibitor + Ketoconazole Testosterone biosynthesis inhibitor

+ Di-n-butyl phthalate Steroid biosynthesis inhibitor + Anastrozole Aromatase inhibitor + Fadrozole Aromatase inhibitor + Ammonium Perfluorooctanoate Aromatase inducer

Compound was detected (+) or not detected (-) in the intact male assay.

EACs Examined in the Adult Male Assay

39

EACs Examined in the Pubertal Male/Female Assays

Pubertal Male Pubertal Female EAC Endocrine Activity + 17-Estradiol Estrogen receptor agonist (full or potent) + Ethinyl Estradiol Estrogen receptor agonist (full or potent) + Diethylstilbestrol Estrogen receptor agonist (full or potent) + Coumestrol Estrogen receptor agonist (weak or partial) Bisphenol A Estrogen receptor agonist (weak or partial) Butyl benzyl phthalate Estrogen receptor agonist (weak or partial) + Octylphenol Estrogen receptor agonist (weak or partial) + Nonylphenol Estrogen receptor agonist (weak or partial) + + Methoxychlor Proestrogen Testosterone Androgen receptor agonist + Flutamide Androgen receptor antagonist (full or potent) +# p,p’-DDE Androgen receptor antagonist (weak or partial) + Vinclozolin Androgen receptor antagonist (weak or partial) + Cyproterone Acetate Androgen receptor antagonist (weak or partial) + Dibutyl phthalate Antiandrogen (non-receptor mechanism) + + Bromocryptine D2 receptor agonist Haloperidol D2 receptor antagonist + Sulpiride D2 receptor antagonist + Phenobarbital Thyroid hormone excretion enhancer + + Propylthiouracil Thyroid hormone synthesis inhibitor + Finasteride 5-Reductase inhibitor + + Ketoconazole Testosterone biosynthesis inhibitor + Anastrozole Aromatase inhibitor + Fadrozole Aromatase inhibitor + Testolactone Aromatase inhibitor + + Atrazine Neuroendocrine mechanism

Compound was detected (+) or not detected (-) in the specified assay. # Detected as an AR antagonist in one of two experiments in CD rats.

40

Proposed Tier I Screening Battery

Tier I

UterotrophicAssay

Receptor Binding/

Transactivation

Intact Adult MaleAssay

Thyroid EffectsSteroid Biosynthesis

ER/AR AgonistsER/AR Antagonists

ER AgonistsER Antagonists

Agonist/Antagonist(ER, AR)

41

Advantages of Tier I Using Adult Male Assay

• Comprehensive mode-of-action screen– Capable of evaluating several different modes of action in a single assay

-- by measuring mechanistic endpoints (androgen, estrogen and thyroid agonists/antagonists; steroid hormone synthesis (aromatase & steroidogenesis)

– Tier I with intact male provides mode of action “profile” to focus direction of any further testing

– Reduces the number of animals needed for Tier I

• Intact endocrine system– Design allows integration of new endpoints if desired

• O’Connor et al., (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32: 521-549.

42

Completed Inter-Laboratory Studies Using the 15-Day Intact Adult Male Rat Assay Protocol

Laboratory EAC Endocrine Activity Result*RTI

(CRO)Methoxychlor ER agonist Positive1

RTI

(CRO)Linuron AR agonist Positive1

BASF

(Industry)Nonylphenol ER agonist Positive2

Syngenta

(Industry)Genestein ER agonist Negative2

Dow

(Industry)Flutamide AR antagonist Positive2

WIL

(CRO)Methyltestosterone AR agonist Positive2

*Data available though the EPA (1) or ACC (2)

43

Proposed Inter-Laboratory Studies Using the 15-Day Intact Male Rat Assay Protocol

Laboratories Chemicals Chemicals

A Ketoconazole*

(steroidogenesis inhibitor) Phenobarbital

(thyroid)

B Ketoconazole*

(steroidogenesis inhibitor) Linuron

(anti-androgen)

C Phenobarbital

(thyroid) Linuron

(anti-androgen) Leads

(Dupont & Dow) Negative Chemical (to be identified)

Ketoconazole* (steroidogenesis inhibitor)

*Compounds only evaluated by DuPont; all other compounds have been evaluated in 2 or more laboratories.

44

Rationale for Compound Selection

• The mode-of-action of the compounds focus on EAT– Linuron – weak anti-androgen

– Phenobarbital – weak thyroid-active agent

– Ketoconazole – steroid biosynthesis inhibitor

• All compounds have been previously run in the 15-day intact male assay

– Provide a base-line for expected results

– Will facilitate inter-laboratory comparisons of the transferability of the 15-day intact male assay

• All compounds have been previously evaluated in other potential Tier I screening assays (Hershberger, pubertal male and/or pubertal female assays)

– Will facilitate future comparisons among assays

45

Issues/Questions for the EDMVAC

• Is the protocol ready for inter-laboratory validation?– If not, what additional data is needed?

• Does the EDMVAC agree with the test material selection for inter-laboratory validation studies?