Atrazine: Updates to the Do

Response Assessment wiResponse Assessment wi

Implications for Water 

Response Assessment wi

Implications for Water 

Monitoring Frequency

P t ti t S i Ad i P Presen

t

ation 

to 

S

cience Ad

visory 

Pan

July 26-29, 2011

Ch R d i Ph D Chester 

R

o

d

riguez, Ph.D.

Health Effects Division

USEPA Office of Pesticide Program
Outline

• Re-cap of September 2010 SAP meeting: interna

of exposure

• Analysis of additional animal pharmacokinetic stu

radiolabeled atrazine 

• Human pharmacokinetic study with atrazine• Pharmacokinetic modeling approach

• One-compartment linear model compartment linear model• Model evaluation

• Understanding of pharmacokinetics, internal dos

Understanding of pharmacokinetics, internal dos

LH attenuation

• Estimating the human intake dose rate through A

of water chemographs

• Apply methodologies to case studies
Human Health Risk Assess

A

trazin

e

Human Health Risk Assess

A

ta e r zin

e

• Previous risk assessment to support t

• Based on a NOAEL/LOAEL approach• Critical stud

y yy : 6 month dietar

y stud

y

• Key event: Luteinizing Hormone (LH) Att

• September 2010 SAP meeting 

• More sophisticated approaches introduc

• Benchmark dose modeling• Internal measures of exposure 
Internal Dose for LH Atten

Atrazine

External Dose

Pharmacokinetics

Body Inta

Internal Dosimetry 

(Atrazine & Metaboli

“The dose at the target site, the

internal dose, is the ultimate determinant of risk.” 

National Research Council,1994.

Mode of Action Key E

(LH Attenuation)

National Research Council,1994.

(LH Attenuation)

Observed Toxicity

(e.g., ovarian cyclicity dis

developmental delay
Internal Measure of Exposure 

Attenuation in the Rat Attenuation Attenuation in the Rat

• Why an “Internal Measure of Exposure”?

•

Atrazine is short-lived in the bod

yp y due 

primaril

y to 

• Metabolites also active in attenuating LH

• Area under the plasma concentration-time curve 

it l f n

ternal measure o

f exposure

Single dose effect

l)Cooper et al. 2000 25

Repeated daily dosing over 4 days

Cooper et al. 201

Serum LH (ng/m

5

10152025

utinizing Hormone

Serum (ng/ml)

time (hr) 0 1 3 6 1 3 6

0 50 100 200 300

Atrazine Dose (mg/kg bw)

1 3 6 1 3 6 1 3 6 Vehicle 1.56 3.12 6.25 12.5 25 0

5

Atrazine (mg/kg-d)

 Leu

• Duration is critical parameter for LH attenuation in 
AUC = Levels x Duratio

(“how much x for how Lon

• AUC selected was for plasma triazines based oradiolabeled atrazine studies

( 

radiolabeled atrazine studies

• Lack of detailed pharmacokinetic information foand its metabolites

• R di l b l d t i t di i t il bl R

adi

ola

b

ele

d a

trazine s

t

udies in ra

ts avail

able 

• Radiolabel studies generally achieve a high demass balance Cl

*

H

*

*

N N

N

H

Cl

*

H

*

*

N N

N

H

Cl

N

N

CH

3

H

C

3

H

CH

2CH

3 CH

N

N

N

CH

3

H

C

3

H

CH

2CH

3 CH

Metabolically Stable Radiolabeled Triazine Ring
Rationale for Study Select

Thede 1987 Radiolabeled Atrazine Pharmacokine• Selected to learn about the temporal relationship beatrazine exposure and LH attenuation in rats• Similar to the 4-day LH attenuation study:

• Used intact young female rats • Involved repeated once daily dosing with atrazin

Involved repeated once daily dosing with atrazin

days• Covered a wide range of atrazine doses (1- 100 

• Repeated plasma measurements including elimina
Plasma Profile from Thed

8

1 mg/kg bw ATRA3 mg/kg bw ATRA

7 mg/kg bw ATRA10 mg/kg bw ATRA

es (mg/L)

46

Plasma Triazine

0 24 48 72 96 120 144 168 192 216 240 264 288

02

Radiolabeled time (hrs)P

start of dosing pseudo steady state dosing stopped

• Repeated dosing with atrazine leads to plasma accof triazines to a plateau

/pseudo steady state level by 

day Plateau/pseudo steady state:

• Plasma levels sta

y within a s

pecific ran

ge as lo

y p g

dosing continues at the same level and frequenc
Pseudo-Steady State Co

Steady State Co

• Hypothetical: repeated dosing for 3 different duratioto the same to the same pseudo steady state plasma levels steady state plasma levels

600

Hypothetical

400

Pseudo steady state

Levels

0 200 400 600 

0

200 4 days 14 days 6 months

time (hr)

Plasma L

• As long as dosing continues at the same level and fplasma levels will remain within the same range

time (hr)

plasma levels will remain within the same range.
Critical LH Attenuation Stu

Different Durations

• The bioaccumulative profile of plasma triazines is cwith the similar LOAELs for LH attenuation across d

Different Durations

durations of atrazine exposure

Study Duration LOAEL (mg/kg bw/d

atrazine)

Cooper et al. 2010 4 days 3 12 .

Morseth 1996 6 months 3.65

• Proposed a daily steady state AUC for internal dose 

response analysis
Linear Pharmacokinetic Be

Thede 1987 Study

• Other features noted in the behavior of plasm

Thede 1987 Study

Linear Pharmacokinetic Behavior

1500 1 mg/kg bw/da

AUC for plasma triazines

scales directly with atrazine dose Linear elimination 

750

100012501500 y= 13.0x - 8.57

R2 = 0.994

udo-daily

y state AUC

mg/L-h) -2.0 -1.8

-1.6

ma triazines)

0 20 40 60 80 100

0

250500

ATRA dose (m

g/k

g/day)

pse

steady

(m

240 252 264 276 

-2.4-2.2

time (hrs)

Ln (plasm

( g g y)
Linear Elimination Kin

Change in plasma concentration Eliminatio

Rate of elimination 

ΔCp

Δ

t = kel * Cp dCp dt

=

=

Integrated form: LnCp = LnCp – kel*

0

Rate of elimination: • Linear expression with kel as slope Linear expression with kel as slope• Proceeds at a rate that is independent of plasma • Rate determined solely by kel (a constant)

C t t l h lf lif (i t l 2/k l) 

•

Cons

tan

t plasma 

h

alf

-life (i.e., 

t1/2 = ln2/k

el) - pre

for extrapolation across dose levels
Plasma Elimination Pha

Analysis: Thede 1987 St

8

1 mg/kg bw ATRA3 mg/kg bw ATRA

7 mg/kg bw ATRA10 mg/kg bw ATRA

nes (mg/L) 

Analysis: Analysis: Thede 1987 St

246

ed Plasma Triazin 

0 24 48 72 96 120 144 168 192 216 240 264 288

0

start of dosing pseudo steady state dosing stopped

Radiolabel time (hrs)e

Th d 1987 start of dosing pseudo steady state dosing stopped

Atrazine Dose Elimination rate constant Plasma half-life

Thede 1987

(mg/kg bw/day) (kel, h

r-1) (hrs)

1 0.010 72.6 (3.0 da3 0.015 47.2 (2.0 day7 0.013 52.9 (2.2 day

10 0.014 50.3 (2.1 day
Outline

• Re-cap of September 2010 SAP meeting: interna

of exposure

• Analysis of additional animal pharmacokinetic stu

radiolabeled atrazine 

• Human pharmacokinetic study with atrazine• Pharmacokinetic modeling approach

• One-compartment linear model compartment linear model• Model evaluation

• Understanding of pharmacokinetics, internal dos

Understanding of pharmacokinetics, internal dos

LH attenuation

• Estimating the human intake dose rate through A

analysis of water chemographs

• Apply methodologies to case studies
Addressing Limitations o

Thede 1987 Study

Limitations of the Thede 1987 Pharmacokinetic S

• Only 2 animals per dose group

Thede 1987 Study

Only

2 animals per dose group

• Elimination phase estimated with only 3 data points single animalPlasma elimination analysis from other rat studies with radiolabeled atrazine:• P l au

et l a

l. 1993 l t lti f 1993: plasma measuremen

ts resulting 

fr

single oral gavage doses of 1 or 100 mg/kg bw radioatrazine administered to groups of 3 male rats

• Simoneaux 1985: plasma measurements at varioupost-dosing resulting from oral gavage doses of 0.4 mg/kg bw radiolabeled atrazine given to 36 male rats mg/kg bw radiolabeled atrazine given to 36 male rats 

time point) for 7 days
Additional Rat Studies with Rad

Atrazine

Single dose of 1 mg/kg bw 14C- atrazine m)Single dose of 100 mg/kg bw 14C-

m)

• Paul et al. 1993 Study

Atrazine

0.40.6

Paul et al. 1993

ed Equivalents (ppm 10152025 

eled equivalents (ppm

0 48 96 144 192 240 288 336

0.00.2

time (hr)

Plasma radiolabele

0 24 48 72 96 120 144 168 192 216 24

05

10

time (hr)

Plasma radiolabe

Single dose of 1 mg/kg bw 14C-atrazine

-2

0

Slope = - 0.013 ± 0.002

R2 valents) (ppm)

= 0 74

Single dose of 100 mg/kg bw 14C-atrazine

4 Slope = - 0.014 ± 0.001

R2 = 0 94

ents) (ppm)

-8-6-4

2

Paul et al. 1993

R 0.74

asma radiolabeled equi 48 96 144 192 240 288 336 384 -2

02

R = 0.94

Paul et 

time (hr)

ma radiolabeled equival

0 48 96 144 192 240 288 336

time (hr)

ln (Pla -4

ln (Plasm
Additional Rat Studies with Rad

Atrazine

Repeated dosing with 0 4 mg/kg bw 14C Atrazine Repeated dosing with 4 0 mg/kg bw 14C 

Atrazine

Simoneaux 1985

Repeated dosing with

0.4 mg/kg bw 14

C-Atrazine

0.060.080.10

equivalents (ppm)

Repeated dosing with

4.0 mg/kg bw 14

C-

1.01.5

equivalents (ppm)

0 2 4 6 8 10 12 14 16 18

0.000.020.04

Simoneaux 1985

time (days)

Plasma radiolabeled e

0 2 4 6 8 10 12 14 16 

0.00.5 

time (days)

Plasma radiolabeled e time (days) time (days)

Elimination Phase Analysis: 0.4 mg/kg bw/day 14C-atrazine

s

1 ) (ppm)

Elimination Phase Analysis: 4.0 mg/kg bw/day 14C

1 ts) (ppm)

10 12 14 16 18

-4-3-2-1

01

Simoneaux 1985

 time (days)

diolabeled equivalents 10 12 14 16 1

3

-2-1

0

 time (days)

adiolabeled equivalent

-6-5

slope = -0.236 ± 0.031 day-1

= -0.010 ± 0.001 hr-1

Ln (Plasma rad -4

-3

slope = -0.258 ± 0.019 day-1 = -0.011 ± 0.008 hr-1

Ln (Plasma ra
Consistent Plasma Elimination 

Constants Across Studies Constants Across Studies

• A very consistent elimination rate constant acdifferent studies and oral doses of radiolabeled 

Atrazine Dose (mg/kg bw/day)

Elimination rate 

constant (kel, hr-1)

Plasma half-life

(hrs)

S

0.4 0.010 72.6 (3.0 days) Simone

1 0.013 53.3 (2.2 days) Paul e

1 0.010 72.6 ( y) 3.0 da

y

s) Thed

3 0.015 47.2 (2.0 days) Thed4 0.011 63.0 (2.6 days) Simone7 0 013 . 52 9 (2 2 52.9 (2.2 days) Thed

Thed

10 0.014 50.3 (2.1 days) Thed

100 0.014 50.3 (2.1 days) Paul e
Pharmacokinetic Studies with Radiol

Atrazine in Non Atrazine in Non-Human Primates (Mo

Human Primates (Mo

• Hui et al. 2011: rhesus monkeys dosed via oral gav1,, g 10 or 100 m

g of radiolabeled atrazine

• Plasma was sampled for radioactivity as various timpost-dosing

Most important observations

• AUC scaled linearly with a

• Linear elimination kinetics

14C-Atrazine

(mg)

AUC

0

→

∞

((mg/L)*hr)

kel

(hr-1)

Plasma Half-life

(hr)

1 1.61

± 0.35

0.04

± 0.03 20 

±

8

10 13.42

± 3 58

0.06

± 0 03 16 

±

9

±

3.58

±

0.03

100 151

± 24

0.03

± 0.00 24 

± 1

Hui et al. 20
Outline

• Re-cap of September 2010 SAP meeting: interna

of exposure

• Analysis of additional animal pharmacokinetic stu

radiolabeled atrazine 

• Human pharmacokinetic study with atrazine• Pharmacokinetic modeling approach

• One-compartment linear model compartment linear model• Model evaluation

• Understanding of pharmacokinetics, internal dos

Understanding of pharmacokinetics, internal dos

LH attenuation

• Estimating the human intake dose rate through A

of water chemographs

• Apply methodologies to case studies
Human Pharmacokinetic S

• Human information very limited

• Davidson 1985 re

port: 

• 6 human volunteers received a single oral dose of at(0.1 mg/kg bw)• Whole blood from a single subject analyzed for atraz

Whole blood from

a single subject analyzed for atraz

chlorotriazine metabolites DEA, DIA, and DACT• Urine from all subjects analyzed for atrazine, DEA, D

DACT

Results:• Only DIA and DACT detected in whole blood• O l DEA DIA d DACT d t t d i i O

nly DEA, DIA, an

d DACT

d

e

tec

t

e

d in urine

• Chlorotriazines detected exhibited linear elimination kine

when analyzed in blood (DIA & DACT) and urine (DEA, D• Lack of mass balance: DEA, DIA, and DACT accounted only 14.5% of the administered atrazine dose 
Summary of Human Pharmaco

Study

Human Pharmacokinetic Parameters 

Whole Blood

Chlorotriazine tmax 

(hr)

Linear Elimination 

Rate Constant (kel, hr-1)

Elimina

Lif

Atrazine ND ND DEA ND ND DIA

≤

2 0.25 2

DACT ~ 5 0.039 1

Urine 

At i raz ne N/A ND NDEA N/A 0.29 2.4 DIA N/A 0.30 2.3 DACT N/A 0.060 11.5 

ND = not detectedN/A = not applicable
Summary of Pharmacokin

Information

• Use of radiolabeled plasma equivalents includeand ll f it t b lit d all o

f its me

t

a

b

olites

• AUC as internal measure of exposure accounts duration 

– im

portant for LH attenuation

• Plasma triazines (atrazine and its metabolites) upon repeated dosing with atrazine to reach a 

l t / d t d t t l l b th 4th

pla

teau

/pseu

do s

tea

dy s

t

a

te level by the 

4 d 

th

day 

LH attenuation also observed by 4th day of once 

dosing• Proposed internal dose metric: daily steady staplasma triazines

Li b h i f l t i i i t t f 

• Linear 

b

e

havior o

f plasma 

t

riazines consis

ten

t

f

across doses and species
New Pharmacokinetic Inform

• An in vivo pharmacokinetic study in rats recently suthe Registrant• A new PBPK modeling effort based on this new data

A new PBPK modeling effort based on this new data

submitted by the Registrant in collaboration with the Institutes

•The Agency has not completed a thorough review a

evaluation of a new PBPK modeling effort
Outline

• Re-cap of September 2010 SAP meeting: interna

of exposure

• Analysis of additional animal pharmacokinetic stu

radiolabeled atrazine 

• Human pharmacokinetic study with atrazine• Pharmacokinetic modeling approach

• One-compartment linear model compartment linear model• Model evaluation

• Understanding of pharmacokinetics, internal dos

Understanding of pharmacokinetics, internal dos

LH attenuation

• Estimating the human intake dose rate through A

analysis of water chemographs

• Apply methodologies to case studies
Pharmacokinetic Model

• Estimate of internal dose metric for LH attenuation • Extrapolate of internal dose for LH attenuation to di

Extrapolate of internal dose for LH attenuation to di

species (humans), life-stages, exposure conditions, e• Relate the human ingested dose through drinking w

h l l l th t b d t th human plasma levels th

a

t can 

be compare

d

to the ra

point of departure level for LH attenuation

Ideal Approach: A Physiologically Based Pharmacoki

Model

In the absence of a PBPK model, the Agency is consother options for informing pharmacokinetics, internawater monitoringwater monitoring
Proposed Interim Pharmacoki

Modeling Approach Modeling Approach

A one compartment linear model

Plasma Triazines

(Cp, mg/L) kel (hr‐1) Triazines eliminated Oral dose of Atrazine

(mg)

Fast/complete

absorption

Vdss(L)

• Linear behavior: consistent feature of plasma triazindifferent studies doses and species different studies, doses, and species

• AUC scales directly with atrazine dose• Elimination is linear (constant plasma eliminatio

• C i t t ith i ff t b Ti h lk t l 1

Consis

ten

t with previous effor

ts 

by Timc

h

alk e

t al. 1

McMullin et al. 2007 and Hui et al. 2011 in modeling 

chlorotriazine equivalents

• Modeling overall behavior of all plasma triazines res

atrazine exposure
Proposed Interim Pharmacokin

Modeling Approach Modeling Approach

A one-compartment linear model

Plasma Triazines

(Cp, mg/L) kel (hr‐1) Triazines eliminated Oral dose of Atrazine

(mg)

Fast/complete

absorption

Vdss(L)

• Only two parameters are needed 

• volume of distribution (Vd)• kel

• Vd and kel at steady state (Vdss)• Vdss and kel estimated independently from single d

l dt i P l t l 1993

plasma 

d

a

ta in 

Paul e

t a

l. 1993
Evaluation of One Evaluation of One-Compart

Compart

Linear Model

Plasma Triazines

(Cp, mg/L) kel (hr‐1) Triazines eliminated Oral dose of Atrazine

Fast/complete

absorption

V (L)

Linear Model

(mg)

Vdss(L)

Vdss = 6.55 L/kg bw kel = 0.013 hr-1

Analytical solution to a one Analytical solution to

a one

-compartment linear model: compartment linear model:

Dose rate (mg/kg bw/day)

Vdss (L/kg bw)

* kel (hr‐1) * 24hr/day Cpss (mg/L)

=

Expression relates daily dose rate of atrazine to steady state plasma levels (

Can the model be used to predict plasma triazine levels reported inthe Thede 1987 study?

animal 1 animal 2

Atrazine dose rate (mg/kg/day) Cpss (mg/L) Cpss (mg/L)

1 0.56 0.623 1.88 2.16

Model Predicte

0.491.47

7 3.84 3.45

10 5.11 4.8350 22.80 25.61

100 53.10 56.84

3.434.89

24.4748.93
Outline

• Re-cap of September 2010 SAP meeting: interna

of exposure

• Analysis of additional animal pharmacokinetic stu

radiolabeled atrazine 

• Human pharmacokinetic study with atrazine• Pharmacokinetic modeling approach

• One-compartment linear model compartment linear model• Model evaluation

• Understanding of pharmacokinetics, internal dos

Understanding of pharmacokinetics, internal dos

LH attenuation

• Estimating the human intake dose rate through A

analysis of water chemographs

• Summary and next steps
Integrating Internal Dosimetry a

Attenuation in Rats Attenuation in Rats

Atrazine

O l

d ( /d )

IN

One-compartment linearmodel expression for 

d l

Oral gavage

dose (mg/day)

100% absorption

NPUT

Absorbed Dose (mg/day)

(Dose rate) rat

a

d

u

lt rats:

AUC =

Plasma triazines (Cp, mg/L)

Body volume of distribution (Vd, L)

clearance 

kel (hr‐1)

OUTP

Continued exposure

Vdss rat * kel rat

AUC

= OUTP

Continued exposure

Steady state

(Vdss, L)

bioaccumulation

Steady state plasma triazines (Cpss, mg/L)

Daily steady state AUC ((mg/L)*hr)

Duration
Relating Animal to Human Sit

Atrazine

Animal Studies

Atrazin

Human Sit

Orally administered at

constant dose level &

frequency

Drinking wat

variable dose 

and freque

Pharmacokinetics Pharmacok

and freque

Pharmacokinetics

Internal Dosimetry 

(Atrazine & Metabolites) 

Pharmacok

Internal Do

(Atrazine & M

LH attenuationLH attenuation
Human Internal Dosimetry Ap

Water Consumption

(L/day)

Chlorotriazinesin water

(μg/L)

One-compartment linearmodel with allometricallyscaled parameters for adult

Absorbed dose (mg)

INPUT 100% absorption

(Dose rate) human

scaled parameters for adult

humans:

AUC

=

cl

k

Continued exposure

Body volume of distribution (Vd, L) *

Plasma triazine concentration (Cp, mg/L)

Vdss human * kel humanAllometricall

y scaled 

AUC O

BioaccumulationEquivalenttime to steady state

(Vdss, L)

y

from rat values Averagedaily plasma concentration (Cpave, mg/L

duration

Human average daily AUC ((mg/L)

* hr) 

Human average daily AUC ((mg/L) hr)
Outline

• Re-cap of September 2010 SAP meeting: interna

of exposure

• Analysis of additional animal pharmacokinetic stu

radiolabeled atrazine 

• Human pharmacokinetic study with atrazine• Pharmacokinetic modeling approach

• One-compartment linear model compartment linear model• Model evaluation

• Understanding of pharmacokinetics, internal dos

Understanding of pharmacokinetics, internal dos

LH attenuation

• Estimating the human intake dose rate through A

analysis of water chemographs

• Apply methodologies to case studies
Estimating the Human Dos

• Variable dose rate and frequency

Hypothetical WaterChemograph HypotheticalWaterChemograph

304050

e Levels

0

1020

Triazine

Critical duration Time

• At the April 2010 SAP meeting, the Panel recommended ...an integrated (or daily average) internal measure along witan integrated (or daily average) internal measure along wit

drinking water exposure monitoring approach based on the the concentration-time curve (AUC)...”• Integrated (or daily average) internal measure

= plasma A

Integrated (or daily average) internal measure plasma A

• Proposed approach for water exposure = water chemogra(AUCwater

)
Estimating the Human Dos

e Levels

A 

Hypothetical Water Chemograph

304050

Levels

time

Chlorotriazine

Critical duration

0

102030

 Time

Triazine L Critical duration

divided into trapezoidsas dictated by the sampling fe e r

equ

ency

Area of trapezoid

= average levels

*duration

AUCwater for given duration = Area sum of all trapezoid

Area of trapezoid average levels duration

[atrazine]ave (mg/L)

= AUCwater ((mg/L)*hr) for given duration

# days

* 24 hrs

(4)

Expression 4 gives a time-weighted average level of atrazine in watealong with water consumption information results in a human dose r
Estimating Human Equivalent 

AUCs From Water Chemogr

Hypothetical Water Chemograph

304050

Levels

AUCs From Water Chemogr

Chemogr

Critical duration

0

102030

 Time

Triazine L

[atrazine]ave (ug/L) = AUCwater ((mg/L)*hr) for given duration

# days * 24 hrs

[triazine water levels]ave (μg/L) * water consumptio

kel (hr-1) * Vdss (L)

= Human average daily plasma AUC

Allometrically scaled from rat values 
Summary

• AUC: reasonable internal dose metric since durimportant for LH attenuation important for LH attenuation• Accumulation of plasma triazines to pseudo ste

consistent with temp y orality of LH attenuation

• Linear pharmacokinetics of plasma triazines acdifferent doses, studies, and species (including hsupport the use of a one-compartment linear mod• Extrapolation to adult humans possible with allscaled parameters (Vd and kel) scaled parameters (Vd and kel)• Human dose rate estimated through AUC analychemographs• Apply methodology to case studies