Atrazine: Adverse Outcomes and 

Ral

ph L. Coo

per, Ph.D,

Atrazine: Adverse Outcomes and 

p p, ,

Chief, Endocrine Toxicology Bran

Toxicity Assessment Division, 

National Health and Environmental E

Research Laboratory,

ORD

U.S. EPA, 

RTP, NC 

July 2011
History of MOA/and Hazard Assessm

Research How did we get to LH as a ke

• Initial Concerns At i 

. How did we get to LH as

a ke

• Initial Concerns: Atrazine exposure cause

premature development of mammary gland tuthe female SD rat.

• 2000 SAP on Atrazine: Hazard and Dos

Response Assessment and Characterizagreed that atrazine-induced alterations in thof luteinizing hormone specifically the ovulatof luteinizing hormone, specifically the ovulat

of LH, was a key event in the development of mammary gland tumors.
The key events as described in the 2

IRED included:

• Hypothalamic effects resulting in changes in 

catecholamine function and regulation of the release of GnRH.

• Attenuation of the LH surge Attenuation of the LH surge.• Cessation of ovulation with the ensuing pers

release of estrogen.

• Increased prolactin release by the pituitary as 

secondary consequence resulting from the eestrogen levels estrogen levels.

• Prolactin and estrogen-induced proliferative 

in the mammaryg g g land leadin

g to tumori

gene
Relevance of cancer/noncancer 

• Reproductive aging is driven by brain in 

e e a ce o ca ce / o ca ce 

• Reproductive aging is driven by ovary in • Thus, the MoA for mammary gland tumor 

development is not relevant to humans.

• But, there are other LH-dependent advers

outcomes that have been identified in the outcomes that have been identified in the 

may be relevant.
Adverse Reproductive Effects in Ro

• Delayed puberty in male and female

• LOAEL M l 12 5 F l 30 /k /k

p

LOAEL, Male =12.5; Female 30 mg/kg/kg

• Ovarian cycle

• LOAEL = 75 mg/kg/day 21 days• LOAEL <40 /k h t LOAEL <40 mg/kg short-t 22 /k l term; ~22 mg/kg lon• Early reproductive senescence 3.65 mg/kg 6 

• Pregnancy maintenance

• FLR 50 /k GD 8 FLR 50 mg/kg GD 8-11.

• Prostatitis after lactational exposure.

• LOAEL 25 mg/kg (12 5 mg/kg twice a day) LOAEL, 25 mg/kg (12.5 mg/kg twice a day)

• Altered mammary gland development.
HPG Axis Perturbations: Summary of Tem

Dose Concordance

Axis Key Event Time Dose (NOEL/LOEL) Reference

Hypothalamus↓G RH l 4 da

ys N.A./25 m

g/k

g/da

y‡ Coo

per et a

↓G

nRH release

y gg y

p

4 days NA/50 mg/kg/day Foradori et 1 day N.A./300 mg/kg/day Cooper et a

1 day NA/100 mg/kg ↑ Goldman In

HPG Axis

PituitaryAttenuation of LH surge

3 days N.A./50 mg/kg/day Cooper et a4 days N.A./6.25 mg/kg/day Cooper et a4 days BMDL 2.56 Cooper et a

HPG Axis

4 days mg/kg/day 2011

5days NA/30 mg/kg/day McMullin et 26weeks 1.8/3.65 mg/kg/day Morseth et 

Ovary 1 day NA/300 mg/kg/day Cooper et aEstrous cyclicity disruption

1-day

N.A./300 mg/kg/day Cooper et a

4 days 25/75 mg/kg Cooper et a

2011

21 days N.A./75 mg/kg/day Cooper et a

26 weeks 1.8/3.65 mg/kg/day Morseth (19
2000 SAP on Atrazine

• “The reproductive developmental effects in rats ( delayed p

prostatitis) are consistent with a primary action of atrazine oaxis. In the case of delayed puberty, a suppression of GnRH would obviously delay increases in estradiol secretion by thcharacterize puberty in females This would lead to the obsecharacterize puberty in females. This would lead to the obse

delays in vaginal opening. In the male, similar delays in incrsecretion would lead to delays in T elevations that would redelayed preputial separation etc. Therefore, the proposed 

acti f d l t l ff t h id bl 

tion 

for 

developmen

t

al effec

ts s

hares considerable o

the proposed mode of action for carcinogenicity. Indeeneuroendocrine actions of atrazine are probably the domechanisms b

y p

 which atrazine exerts re

productive dev

effects. The same endocrine perturbations that induce tumappear to play a role in at least some reproductive developmeffects. 
Effect of Four Equimolar Doses of Atrazine 

Metabolites on the Pre-Ovulatory Surge of Proestrous Females Proestrous Females.

20 022.5

LH: Vehicle vs. DIA

V hi l

12.515.017.520.0

*

*

g/ml20

25

+

L)

Vehi

cle

12.5 mg/kg ATR

75 mg/kg ATR

2 55.07.5

10.0

LH ng

5

1015

+

+

**

LH (ng/m

1600 1800 2000

0.02.5

Hours

1600 1800 2000

0

Hours
MoA and Toxicity Pathway

LH is a key event leading to the disruption of hypothalamic gonadaand as a consequence altered reproductive performance and canc( t ) i th t

y y

(mammary tumors) in the rat. 

Organ 

System Molecular Cellular Tissue Organ 

T i it P th

Organ

System

Toxicity Pathway LH alteratio

Ch i l Receptor/Ligand

Gene 

activation Altered 

Toxicant

Cellular 

Responses

Organ

Responses

O

R

MacroMolecular

Interactions

Chemical

Properties

p g Interaction

DNA Binding

Protein Oxidation

Protein 

production

Altered 

signaling

physiology

Disrupted homeostasis

Altered tissue 

development/

De

Re

development/ function
Mammary Gland Developme

• Effect of gestational Atrazine exposure on mammar

tumor development was reviewed at the September 

a a y G a d e e op e 

• Atrazine stunted mammary gland growth thro

67. Rayner et al. (2005); Enoch et al. (2007).

• Currently Rayner and Enoch findings can not be rep

• Hovey et al. (2011) observed no differences in 

gland morphology in Long Evans rats exposeatrazine from GD 13-19.

• In response to the September 2010 SAP’s sug

ORD used both subjective and objective measeffort to compare and contrast the two approarats exposed GD 14-21 on PND 45 (dose range mg/kg/day) Davis et al. (2011).
Mammary Whole Mount Morphom

(Quantitative) Evaluations

2520

x 3 mmb.i.d.(Blocks 2 & 4)

(Quantitative) Evaluations

1510

5

ranches / 3 

0

x

0 1 5 20 100

11 10 9 10 6

of TEBs B

0 1 5 20 100

60504030

uct

Number o

0 1 5 20 100

302010

0

11 8 9 9

6

1510

a

5

nce (mm)

de - Most Distal Du

5

0

ATR Treatment (mg/kg/d)

Dista

Lymph Nod

0 1 5 20 100

11 9 10 10 6
Mammary Whole Mounts Subjective 

(Qualitative

)

Assessments

4 Treatment GD 14-21

Assessment PND 45

(

)

3

3.5

Atraz

Assessment PND 45

1

1 5

2

2.5

Scoremg/k

1

0 5

1

1.5

0

0.5

s.i.d. b.i.d.

Blocks 1 & 3 Blocks 2 & 4

Dosing Regimen

Blocks 1 &

3 Blocks

2

&

4
Summary

• The reproductive/developmental effects in rats (de

puberty ovarian cycling FLR) are consistent with puberty, ovarian cycling, FLR) are consistent with 

action of atrazine on the HPG axis, namely LH sec

• Alt ti i LH id th l t LOEL 

Alterations in serum LH provides the lowest LOEL

NOELs available and these serum hormone measua sentinel for two of the three adverse outcomes id

(puberty and ovarian function) (puberty and ovarian function).

• Although the proposed MoA for prostatitis is diffe

LOEL d NOEL f LH lt ti t ti LOELs and NOELs for LH alterations are protectiv

prostatitis as well.
One vs four-days of dosing on L

During the September 2010 SAP, the Panel raised the issue 

multiple atrazine exposure effects on the LH surge. In their r

A th SAP t d Agency the SAP commen

t

e

d:

“Data are clear in identifying that a greater-than-one pulse of exposatrazine is necessary for attenuation of the LH surge For example atrazine is necessary for attenuation of the LH surge. For example

doses (over 100 mg/kg) administered on the morning of proestrus characteristics of the LH surge occurring later the same day. Additiclearly demonstrate a once daily dose for 4 days and beginning on 

induce significant inhibition of the LH surge peak. In this instance, a response is observed. However, what is not clear is if less than 4, bthan 1 days’ exposure is sufficient to alter the LH surge. Further cothe matter it is not clear if a 4 the matter, it is not clear if

a

4-day exposure beginning on a differe

day exposure, beginning on

a differe

cycle, will result in changes in the LH surge similar to those when dbegins on the morning of proestrus. Understanding of the relatio

between duration of exposure and phase of the cycle will be k

translating rodent data to humans for risk assessment purpos
Role of estrogen and progesterone 

re

g g ulation of the LH sur

g

e

• Estrogen alone will induce sma

days later (E)

4 0

O V X - E strad io l Im p lan t / s c P ro g estero n e

• Estrogen for 3-4 days followed b

progesterone on day 4 will signincrease the amplitude of the LH (E+P).

3 02 0

E + P

P E

• The opposite order, P followed b

eliminate any E-dependent LH s

• Hypothesis: One dose of atrazi

administered on vaginal proestr

0

- 6 -4 -2 P eak +2 +4 +6

H o u r s fr o m P e a k

1 0 E o n ly

P + 

E on the morning the expected su

OVXed, E-primed female will incamount of LH as a result of the induced increase in progesteron

ou s o ea

al., 2009; Fraites et al. 2009).

• Atrazine dosing for 4 days will i

amplitude because of the daily rprogesterone.

• But we thought one dose had n
Effect of Atrazine on Ovariectomized

Estrogen-primed LH Surge

One-Day Exposure Three-Day Ex

• Long-Evans Rats• A th d ti f d i i th ff 

As the 

duration o

f

dosing increases, the effe

dose decreases. Cooper et al
One Dose of Atrazine increases peak heig

Area under the Curve. Area under the Curve.

40

10

*

/k

Sampling 

30

789

0

100

(11)(10)

mg/k

g

ml)

ml)

1400-1600-1800-2000h 

AUC

20

4

56

( )

*

m LH (ng/

 LH (ng/m

10

2

3

4

Serum

Serum

0

0 

-6 -4 -2 Peak +2 +4

01

Ti R l ti t P k At i ( Time 

R

elative 

to 

Pea

k Atrazine (m
Two Doses of Atrazine decreases peak h

and Area under the Curve

10

mg/

k

g Sampling 1400-1600-1800-2000h 

40

AUC

and Area under the Curve.

789

ng/ml)

0

100

(9)(9)

g/ g

g/ml) 30

35

4

56

erum LH (n( ) 152025

rum LH (ng

1

2

3

4

Se

5

1015

Ser

-4 -2 Peak +2 +4 +6

0

1

Time Relative to Peak

0

5

Atrazine (m

0 

Time Relative to Peak Atrazine (m
Four Doses of Atrazine decreases peak 

height and Area under the Curve height and Area under the Curve.

10

mg/kg

40

Sampling 1400-1600-1800-2000h 

AUC

789

0

100

(10)

(10)

mg/kg

30

35

ng/ml)

g/ml)

4

56

(10)

152025

p= 0.06

erum LH (

rum LH (ng

2

3

4

5

1015

*

Se

Ser

-4 -2 Peak +2 +4

0

1

0

5

0 

Time Relative to Peak Atrazine (mTime Relative to Peak Atrazine (m
Summary

• Responding to the September 2010 SAP’s comments rega

vs. multiple exposures, ORD scientists examined what cha

l if ld i t t f 

Summary

release, if any, would occur in response to one, two, or fo

ATR

• One dose of ATR significantly increased LH (in OVXed + 

demonstrating a clear effect on LH regulation in a manner with an effect of the increase in progesterone observed fo

at i d i i t ti trazine administration. T d wooses of ATR l d f ATR revealed no c

LH from control. Four doses led to the previously describin LH. Again, all of these changes are consistent with a re

the atrazine induced changes in adrenal progesterone the atrazine induced changes in adrenal progesterone.

• These findings speak only to the duration of dosing and 

LH Th d t i l d ff t f LH. They do not imply an adverse effect for i d 

increased 

OVXed E-primed rat. 
MoA and Toxicity Pathway

LH is a sensitive key event leading to the disruption of hypothalamfunction and as a consequence altered reproductive performance ( t ) i th t

y y

(mammary tumors) in the rat. 

Organ 

System Molecular Cellular Tissue Organ 

T i it P th

Organ

System

Toxicity Pathway LH alteratio

Ch i l Receptor/Ligand

Gene 

activation Altered 

Toxicant

Cellular 

Responses

Organ

Responses

O

R

MacroMolecular

Interactions

Chemical

Properties

p g Interaction

DNA Binding

Protein Oxidation

Protein 

production

Altered 

signaling

physiology

Disrupted homeostasis

Altered tissue 

development/

De

Re

development/ function
Thank You

ORD NHEERL TAD NTB OCSPP OPP HED

Thank You

ORD, NHEERL, TAD, NTB• Jerome Goldman, Ph. D• Susan Laws, Ph.D

OCSPP, OPP, HED• Elizabeth Mendez, • Vicki Dellarco, Ph. 

• Tammy Stoker, Ph. D. • Michael Narotsky, Ph. D• Melanie Fraites, Ph.D.

• Jack Fowle, Ph.D 

Melanie Fraites, Ph.D.

• Lori Davis, Ph.D.

• Angela Buckalew,

• Deborah Best Deborah Best• Ashley Murr• Michelle Hotchkiss

200

300

***

mL)

Female Studies. Eff t f At i DIA

100200 **

*

*

ACTH (pg/mEffec

t o

f Atrazine, DIA 

DACT for 4 days on pituitary adrenal activi

Vehicle

ATR 12.5

ATR 75

DIA 10

DIA 60.2

DACT 8.4

DACT 50.6

0

**

L)

D

D

20 **

L)

200400

*

**

**

terone (ng/mL 1015

* ***

*

terone (ng/mL

cle

2 5

75

10

0 2

8 4

0 6

0

200

Corticost

hicle

12.5

R 75

A 10

60.2

05

*

Progest

Vehicle

ATR 12.5

ATR 75

DIA 10

DIA 60.2

DACT 8.4

DACT 50.6

Vehic

ATR 12

ATR 7

DIA 1

DIA 60

DA
800

*** Female Studies. 

Eff t f At i DIA

400600

**

TH (pg/mL)Effec

t o

f Atrazine, DIA 

DACT for 1 day on pituitary adrenal activi

Vehicle ATR 75 DIA high DACT high

0

AC200

400

*** ***

+

mL)

2025 *** ***

ng/mL)

200

+

CORT (ng/m

5

1015

gesterone (n

Vehicle ATR 75 DIA high DACT high

0

Vehicle ATR 75 DIA hi

g

h DAC

05

Pro
Effect of Atra

the Ovulatory of LH in ProesFemales.

LH: Vehicle vs. DIA

25

e a es

20.022.5

Vehicle

12 5 mg/kg Atr

152025

+

g/mL) 10.0 12.5

15.017.5 *

*

ng/ml

12.5 mg/kg Atr

75 mg/kg Atr

5

10 +

** 

LH (ng

0 02.55.07.5

n

1600 1800 20

0

Hours

1600 1800 2000

0.0

Hours
Plasma Chlorortriazine Concentrations

(15 minutes post gavage) 1 Dose 75 mg/kg ATR 60.2 mg/kg DIA 50.6 m

ATR

+ 26.4 ± 4.0 (9)

DEA

+ 153.1 ± 17.8 (9)

(15 minutes post gavage)

DIA

+ 600.9 ± 50.5 (9) 4611.1 ± 359.0 (9)

DACT

+ 78.0 ± 10.5 (9) 271.9 ± 53.6 (9) 980.4 

4 Doses 12.5 mg/kg ATR 10 mg/kg DIA 8.4 mATR* 8.8 ± 1.2 (6)DEA* 63.8 ± 13.7 (6)

4 d 75 /k ATR 60 2 /k DIA 50 6 

DIA* 252.2 ± 51.7 (6) 1877.7 ± 253.9 (7)DACT** 747.5 ± 55.5 (2) 2088.3 ± 435.5 (3) 5977.0 

4

doses 75 mg/kg ATR 60.2 mg/kg DIA 50.6 m

ATR* 14 ± 2.1 (6)DEA* 101.7 ± 14.6 (6)DIA* 475.0 ± 62.2 (6) 3886.0 ± 356.0 (9)DACT** 4138.0 ± 1780.0 (3) 13136.0 ± 2401.0 (2) 12687.
Serum Hormones After a Single Dose 

Atrazine in the Male Rat

*

500 Control

5 mg/kg50 /k

*

ACTH

8 Progesterone

*

*

Atrazine in the Male Rat

*

*

*

*

*

*

200300400 50 mg/kg

100 mg/kg200 mg/kg

(pg/ml)

*

*

246

*

* *

*

(ng/ml)

*

0 5 15 30 60 180

0

100

0 5 15 30 6

02 

*

*

**

*

*

300400

/ml)

Corticosterone

68

10

Testosterone

/ml)

**

*

*

*

*

*

0 5 15 30 60 180

0

100200

(ng/

024

(ng/

0 5 15 30 60 180

Time (minutes)

0 5 15 30 6

0

Time (min.)
Serum Hormones After a Single Dose : 

DACT vs. ATR

ACTH

400500 Control

3.37 mg/kg

33.7 mg/kg

67.5 mg/kg

*

DACT 400

500

*

ACTH 

ATR 

100200300

67.5 mg/kg

135.0 mg/kg

* * * *

(pg/ml)

DACT

100200300

*

*

*

*

*

(pg/ml)

ATR

0

5 15 30 60

0

100 * * * *

0

5 15 30 

0

100

Corticosterone 

300400500

ml

DACT

*

*

**

*

300400

)

Corticosterone

ATR

100200300

*

*

*

*

*

ng/m

*

*

100200

(ng/ml)

0

0

5 15 30 60

Time (Minutes)

0

5 15 30 

0

Time (M
Atrazine Induces a Chemical Specif

Activation of HPA

*

500 

*

ACTH

Activation of HPA

*

*

*

*

*

200300400

(pg/ml)

• Control/DACT not 

affected,

• Dose response

0 5 15 30 60 

0

100

Time (minutes)

Dose response

• Not gastrointestinal 

distress (vagotomy/ measurement of

*

*

*

**

*

200300400

(ng/ml)

measurement of Corticosterone

gherlin)

• Activation with 

i t

*

*

0 5 15 30 60 

0

100

Time (minutes)

in

travenous 

administration.

Time (minutes)
Serum Corticosterone After a Sing

vs. Multiple Doses

• ATR DEA and DIA s

Corticosterone - 15 msingle dose 400

*

*

*

• ATR, DEA and DIA s

release of adrenal ste15 min. after a single 

100 male rat200300

CORT ng/ml male rat

Control ATR DIA DEA DACT

0

100

Dose Equimolar to 200 mg/kg ATR

400500

Control

DIA 4 mg/kg

*

*

* DIA 10 mg/kg

*

*

g/ml)

100200300

DIA 10 mg/kgDIA 40 mg/kgDIA 80 mg/kg

ATR 100 mg/kgDACT 67 5 mg/kg

*

*

CORT (ng

0

15 30

Post-Dosing Time (minutes)

DACT 67.5 mg/kg
Comparison of after 1 or 4-Days 

Exposure to Atrazine and metabolites

1000

One Dose Four Doses

1000

b

Exposure to Atrazine and metabolites

400600800

400

TH (pg/mL) 600 800

a

H (pg/mL)

ehicle

R 12.5

TR 75

DIA 10

A 60

2

0

200

b

b

AC

Vehicle ATR 75 DIA 60.2 DACT 50.6

0

ACT 200

Veh

ATR

ATR

DIA

DIA 6

600

b

g/mL)600

b

b

ng/mL)

300

a

a

rticosterone (n 200 400

icosterone (n

Vehicle

ATR 12.5

ATR 75

DIA 10

DIA 60

2

0

Cor

Vehicle ATR 75 DIA 60.2 DACT 50.6

Cort

0

i
20.0

22.5

Eff t f F E

Vehicle

12.5 mg/kg ATR

75 /k ATR

12.515.017.520.0

*

*

ng/ml

Effect of Four E

Doses of Atra

Metabolites on 

75 mg/kg ATR

2.55.07.5

10.0

LH n

Ovulatory Surg

Proestrous F

1600 1800 2000

0.0

5

Hours LH: Vehicle vs. DIA

25

LH: Vehicle vs. DACT

25

1520

+

g/mL) 15

2025

Vehicle8.4 mg/kg DACT50.6 mg/kg DACT

g/m L)

5

10 +

** 

LH (ng

5

10

LH (ng

1600 1800 20

0

Hours

1600 1800 2000

0

Hours