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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)
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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.
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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
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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?