1

Dan Campbell

Team Lead, Herbicides

Regulatory Affairs

NAFTA Herbicide

(336) 632-7627 (phone)

(336) 632-5688 (fax)

Syngenta Crop Protection, 

LLC

P.O. Box 18300

Greensboro, NC 27419-8300

www.syngenta.com

FedEx

January 13, 2011

Melanie Biscoe

U.S. Environmental Protection Agency

One Potomac Yard, Room S-4900

2777 South Crystal Drive

Arlington, VA 22202-4501

Dear Ms. Biscoe,

As a follow up to our December 15, 2010 meeting and the December 17, 2010 release of the 

September FIFRA Scientific Advisory Panel (SAP) meeting, Syngenta Crop Protection, LLC. is 

providing details of our development of a Physiologically Based Pharmacokinetic (PBPK) model, 

including the planned functionality/utility, development plans and timing. We are also providing a 

description of the plans and timing of the studies we are conducting that were recommended by the

September 2010 SAP. 

Key Syngenta studies that are designed to address EPA’s and the SAP’s goal of using a benchmark 

dose based upon the LH surge suppression are as follows:

1. PBPK Data and Model Development for the Rat

2. Scaling Rodent Model to Non-Human Primates and Humans

3. External to Internal Dose Conversion using the PBPK model for Humans

4. Benchmark Dose Determination (BMD)

Dose Response and BMD Calculation

Effects on the LH Surge vs. Effects on Fertility

Strain Sensitivity Differences

Bolus vs. Distributed Dose Administration

5. Life Stage Sensitivity and the Relevance to Uncertainty Factors

6. Probabilistic Risk Assessment Based Upon Internal Dose and the BMD.

7. Drinking Water Monitoring 
PBPK Data and Model Development

The SAP of September 2010 recommended that better plasma data be used to provide “... tight 

definitions of the PK profiles of the individual compounds (parent compound and analytes)...”1

.

The panel also stated that “This information combined with good estimates of plasma partition 

coefficients should allow the development of physiologically based pharmacokinetic models that can 

be used to extrapolate from rodents to other species, including humans.”2

 

Syngenta is in the process of enhancing the rodent model developed by McMullin et.al (2007) by using 

the data from the recent and ongoing pharmacokinetic studies carried out in vitro and in vivo. The 

rodent model will be scaled to rhesus monkey and humans, as described in Attachment 1 and 2.

The plasma/tissue partition coefficient study is complete and will soon be submitted for publication 

(Tremblay et al. 2011) and a new study on absorption through a gut monolayer cell culture is being 

initiated.

Benchmark Dose Determination:

Syngenta has ongoing studies summarized in Attachment 3 that are relevant to following areas.

Dose Response and the Calculation of BMD

The SAP recommended that “With regard to demonstration of a dose response of inhibition of the 

LH surge to atrazine exposure, several replicates of the dose response curve, ideally in each strain 

of rat tested, would help to validate the precision of the BMR determination.”3

 The SAP also 

recommended additional approaches to analyze the Cooper 2010 data as well as previous Syngenta 

LH studies4

. 

Effects on the LH Surge vs. Effects on Fertility

The SAP expressed the need to correlate the degree of LH suppression caused by atrazine dosing 

to the potential for effects on fertility. The SAP stated, “...while attenuation of the LH surge over 

one estrous cycle represents a sensitive biological response, a causal association between the 

observed modest, yet statistically significant, changes in LH surge peak and adverse fertility 

measures has not been made.” 5

 and that the “...biological significance and degree of adversity of 

the (LH) effect used to derive the benchmark value remains to be addressed robustly”6

 

Strain Sensitivity Differences:

Concerning the comparative strain data of Sprague-Dawley and Long Evans rats, the SAP 

recommends that “...data from these two strains should not be combined for the determination of 

a BMR. Their differences should be assessed, acknowledged and taken into consideration, but 

 1 Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting held September 14 - 17, 2010 on the ReEvaluation of the Human Health Effects of Atrazine: Review of Non-cancer Effects and Drinking Water 

Monitoring Frequency, December 9, 2010, 

http://www.epa.gov/scipoly/sap/meetings/2010/september/091410minutes.pdf, page 10 2 Ibid, page 48 3 Ibid, page 56 4 Ibid, page 54

5 Ibid, page 52 6 Ibid, page 53
their data should be analyzed separately”7

. The study being conducted will allow for a side-byside comparison of the BMD in both strains. 

Distributed Dose Administration Compared to Bolus Dose Administration

The SAP recommended that data generated from dietary administration should be considered, 

since it “... provides a smoother plateau, is less stressful and disruptive than gavage, and is more 

realistic representation of human exposure through drinking water, depending on the presence of 

food in the digestive tract.”8

 Towards this end, Syngenta has generated comparative (dietary vs 

gavage) PK data, and has ongoing studies generating LH dose - response data (peak and areaunder-the-curve) comparing the two routes of administration. 

Potential for Life-Stage Sensitivity and the Relevance to Uncertainty Factors

The SAP expressed the need to determine the potential for sensitivity to LH suppression across the 

reproductive lifespan. The Panel states, “Specific data which examine long-term diminution of the LH 

surge over the reproductive lifespan and resultant effects on fertility have not been studied with 

atrazine... Studies specifically addressing the effects of atrazine during all key phases of the 

reproductive lifespan are lacking.”9

The Panel stated these data “...need to be taken into account when, for example, selecting the 

appropriate uncertainty factors, along with the adequacy of the toxicokinetic and toxicodynamic data 

that will inform the inter- and intra-species extrapolations.”10

New Syngenta work relevant to these questions appears in Attachment 4.

Risk Assessment

The use of the new atrazine PBPK model in risk assessment is summarized in Attachment 5. Briefly, 

the PBPK model will be used as a tool for converting external doses resulting from the consumption of 

chlorotriazine residues in drinking water to internal dose metrics. Subjects will be run iteratively in a 

Monte Carlo simulation using CARES or an Excel-based tool that has been previously submitted to 

EPA (Sielken 2006). An acute risk assessment will be conducted probabilistically according to EPA 

guidance for such analysis. All tools, documentation and results will be provided to EPA.

Other Syngenta Research Pertinent to Risk Assessment

Syngenta has two additional ongoing activities that are pertinent to the final use of the PBPK model in 

the risk assessment simulation. These are described in Attachment 6 and 7.

Drinking Water Monitoring Program in 2011

Syngenta plans to conduct a pilot monitoring program for the collection of daily water samples from 

community water systems (CWS) during the atrazine use season. The data are intended to augment the 

daily data currently available, for use in model validation and development and to respond to the 

September SAP comments. These data are important for the use in developing methodologies, as stated 

 7 Ibid, page 56

8 Ibid, page 48

9 Ibid, page 56-57 10 Ibid, page 53
by the SAP, “...it is important to have true daily data when developing a methodology...”11 

Additionally, the Panel stated that autosamplers should be used, since, “(a)utosamplers can better 

capture transient events that happen during periods when personnel are not available to do the 

sampling.”12

The number and location of the CWS representing different water body types are currently being 

determined, and it is anticipated that monitoring will begin this spring and preliminary information 

will be available in the summer of 2011. Additional statistical analyses of the impact of different

sampling frequencies in different surface water body types (e.g. reservoirs, small and large streams) 

are ongoing.

We look forward to discussing the information from the ongoing studies described in the attachments

during our February meeting. We appreciate the hard work of the EPA scientist on the atrazine data 

assessments. Please call me at (336) 632-7627 if you have any questions on this letter.

Sincerely,

Dan Campbell

Regulatory Team Lead, Herbicides

Syngenta Crop Protection, LLC.

 11 Ibid, page 62 12 Ibid, page 60
Attachment 1:

Syngenta Studies and Projects Relevant to PBPK Modeling

Syngenta Study 0033836 (WIL Study 639081) has characterized the pharmacokinetics of atrazine and 

its chlorometabolites in the young, adult female Sprague-Dawley rat when atrazine was administered 

as a bolus dose (gavage) or in feed over a 24 hour dosing period at doses of 3, 10 or 50 mg/kg/day over 

4 days.

Syngenta Study 0040036 and (WIL Study 639084) has characterized the rate of atrazine metabolism to 

its chlorometabolites in hepatocytes obtained from young adult female rats and humans at 

concentrations equivalent to plasma concentrations expected to be achieved in vivo.

Hamner Institute PBPK Models for rat, rhesus monkey and human.

Relevance to EPA’s Review:

• Will Study 639081 will be used to revise the McMullin PBPK rodent model (McMullin et al.

2007) to more accurately predict the internal dose of atrazine and its chlorometabolites 

following a bolus or distributed dose.

• Will Study 639084 provides key data that permits the PBPK rodent model to be scaled to 

humans.

• The PBPK model will provide an improved method for calculating internal dose metrics for 

atrazine and chlorotriazine metabolites in rodents, primate and human (Attachment 2).

• Predictions from the rodent PBPK models can be validated with plasma, urine and tissue 

concentrations measured in rats and primates in other previously submitted studies, for 

example the studies by Hui et al.(1996 a,b,c) and Davidson (1988).

New Study Submission Dates:

WIL Studies 639081, 639084: January 31, 2011

Hamner Institute PBPK Models: Provide to EPA at meeting February 10 or 11, 2011 (or 

another date convenient for EPA)
Attachment 2:

Specification for the Chlorotriazine PBPK Model at the Hamner Institute

1) The code for the model will be written using the ACSLX software program.

2) The model will be based upon the PK dataset collected following atrazine (ATZ) administration by 

gavage (in 5% methylcellulose and Tween 80), or in feed to the young adult (13-week) female 

Sprague-Dawley (SD) rats (WIL Study 639081; See Attachment 1 for study design).

3) The atrazine absorption rate constant will be derived from in vivo (C14 atrazine/total chlorotriazine 

[TCT] data) and ex vivo (Caco2 cells) data (Study yet to be initiated).

4) The model will be developed based upon the in vitro and in vivo rat data and parameterized for 

mouse, rhesus monkey and human (females) so as to permit tissue/organ concentration of atrazine 

and its chlorometabolites in these species.

5) Model compartments will include but not be limited to the following tissues and organs: plasma, 

liver, brain (hypothalamus – inside the blood-brain barrier and pituitary (outside the blood brain 

barrier), adrenals, ovary, testes, mammary tissue, urine, feces.

6) The model will be capable of receiving input doses for atrazine, deisopropyl atrazine (DIA), 

deethylatrazine (DEA), diaminochlorotriazine (DACT) at a temporal resolution of 5 minutes. In 

the routine use of the model, it is expected that input will be hourly and exposure will be modeled 

for up to 7 days of intermittent exposure.

7) The model will be capable of predicting the tissue compartment concentrations of ATZ, DIA, 

DEA, DACT and TTR (Total Triazine Residue) with a temporal resolution of 5 minutes. In the 

routine use of the model, it is expected that modeled compartment concentration will be generated 

hourly for up to 7 days of intermittent exposure.

8) TTR will be calculated proportionally based upon the relationship between TTC and TTR in 

rodent, primate and man as reported in Syngenta studies.

9) One model output should be the area under the plasma concentration curve for atrazine and DEA, 

DIA and for TCT. The duration of time over which the calculation is determined should be 

capable of being specified as an input variable. Ideally, the AUC metric should be capable of 

advancing in temporal steps of 1 hour duration integrated over a period from 1 hour up to 7 days of 

intermittent exposure.

10) The rate of conversion of atrazine to DEA, DEI and DACT will be estimated from the rat and 

human in vitro hepatocyte study (WIL Study 639084).
11) The model will be validated using the results from additional ongoing studies where atrazine was 

administered to young adult female SD and Long Evans rats (WIL Study 639091). Predicted 

plasma and tissue concentrations will be compared to measured plasma and tissue concentrations.

12) Predicted plasma and tissue concentrations based upon female SD rats will be compared to 

measured plasma and tissue concentrations measured in female Long Evans rats.

13) The model will be parameterized for young adult CD-1 mice. Plasma and tissue concentration 

prediction will be compared to measured concentrations (Ross et al, 2009).

14) The model will be parameterized for young adult rhesus monkey. Plasma and tissue concentrations 

will be predicted and compared to measured concentrations (Hui et al, 1996a, b).

15) The model will be parameterized for humans. Plasma and urine concentrations will be predicted 

by the model and compared to measured concentrations (Davidson, 1988).

16) Probabilistic risk assessments will be conducted by Sielken and Associates using the human PBPK 

model to calculate internal dose as a function of external dose. 
Attachment 3:

Syngenta Studies Relevant to Benchmark Dose Determinations

Syngenta Study Number 0033837 (WIL Study Number 639080) characterizes the dose-response 

relationship for the effect of 4-day exposures to atrazine by gavage or in diet on the LH surge of intact 

female Sprague-Dawley rat. 

Relevance to EPA’s Current Review

• This study provides on the differences in the LH response when the dose is distributed over the 

day compared to a single bolus dose.

• Distributed dosing is more relevant to human exposure scenarios.

• BMD’s can be calculated from these data.

Report Submission Date: April 29, 2011

Syngenta Study Number 0052067 (WIL Study Number 639089) compares the effect of 4 days 

exposure to atrazine by gavage or in the diet on the LH surge of intact female Sprague-Dawley rats to 

the effect on the LH surge in intact female Long-Evans rats. 

Relevance to EPA’s Review: 

• This study will provide additional information on potential sensitivity differences between the 

Long Evans rat compared to the Sprague-Dawley rat.

• BMD’s can be calculated for the bolus dose versus the distributed dose scenarios.

Protocol Provided: October 22, 2010

Report Submission Date: April 29, 2011.

Syngenta Study Number 0052068 (WIL Study Number 639090) compares the effect of 4 days of 

atrazine on the reproductive performance of intact female Sprague-Dawley and Long-Evans rats. 

Atrazine was administered by gavage at doses that suppress the LH surge or at equivalent doses in diet

that have no effect on the LH surge. 

Relevance to EPA’s Review:

• This study shows that NOELs and BMD’s calculated from functional outcome measures are 

more than 10 fold greater than NOELs/BMD’s based upon a biomarker (LH) for such effects.

• This study reduces uncertainty about whether an EPA standard based upon LH is protective.

Protocol Submitted to EPA: October 22, 2010

Report Submission Date: April 29, 2011.
Attachment 4:

New Syngenta Studies Relevant to Uncertainty Factors

Syngenta study 0023168 (WIL Study 639060) has shown that peri-pubertal female Sprague-Dawley 

rats are less sensitive to the effect of atrazine on the LH surge than are young adult 13- week old 

females. 

Longer duration studies have already been submitted by Syngenta.

Relevance to EPA’s Review:

• The results from Will Study 639060 will provide information on relevance of the 3X FQPA 

uncertainty factor currently used to calculate the RfD for atrazine. 

• The allometric scaling of dose from rat to human in PBPK models typically reduces the 

standard 10 fold interspecies scaling factor to approximately 3 fold.

Report Submission Dates: 

WIL Study 639060 January 29, 2011

Hamner Institute PBPK Models February 18, 2011.
Attachment 5:

Syngenta Projects Relevant to Risk Assessment 

Inputs to Probablistic Risk Assessment Models

Chemographic Profiles: Syngenta will use chemographic profiles for different durations of atrazine 

and its chlorotriazine metabolites in a subset of CWSs. 

Water Intake: Two surveys of water intake (CSFII and Bayer CropScience Drinking Water 

Consumption Survey (DWCS)) will be provided with the risk assessment tool. CSFII does not contain 

information on the timing of direct water consumption within a day, whereas the DWCS does.

Body Weight: Will be either derived from the water intake survey in the case of CSFII or derived 

from user-specified distributions (e.g., EPA Exposure Factors Handbook).

Ingested Dose: The ingested dose of atrazine and its chlorometabolites for individuals will be 

calculated as a product of concentration and volume of water consumed divided by body weight. 

Benchmark Dose: BMDs, BMDLs, and the distribution of BMDs will be derived from dose-effect 

data using the appropriate data sets (LH suppression, Functional Effect), appropriate animal models

(e.g. Long-Evans, Sprague-Dawley), and appropriate durations (4 days, 90 days, 6 months) of atrazine 

or DACT administered as either a bolus or distributed dose.

Outputs from the Human PBPK Model

Internal Plasma Concentration

• Internal plasma concentration of atrazine, DEA, DIA, DACT will be calculated using the 

human PBPK model at time steps driven by the frequency of measurement of water 

consumption.

• A suitable dose metric (e.g. peak concentration, AUC, AUC above a baseline) of each analyte 

or combination thereof, depending on the biological response of interest.

 Tissue Concentrations

• Tissues concentrations can be calculated by the PBPK model.

 Monte Carlo Simulation

• A Monte Carlo simulation will be conducted using either CARES (Drinking Water Minute 

Module (DWMM)) or an Excel based simulation model (Sielken et al. 2006) for appropriate 

age, ethnic, region, and season)

• Input files for each simulation and the random seed number will be provided.

• Output from the simulation will be tabled and graphed as cumulative probability density 

functions for each CWS evaluated.

• Distributions and 99th or some other suitable percentiles for intakes, response levels, 

equivalent distributed vs. bolus doses, and MOEs will be calculated for analysis.

• Sensitivity analyses will be conducted.

 Integrated PBPK / Simulation tool provided by: April 31, 2011
Attachment 6

Syngenta Studies Relevant to Defining the Critical Dose Duration

Syngenta study 0023168 (WIL Study 639060) showed that 4 days of high doses of atrazine treatment 

of approximately 13-week old female Sprague-Dawley suppresses the LH surge whereas continuous 

dosing from conception until 13 weeks of age has no effect suggesting that an adaptive process has 

been triggered.

Syngenta project (Foradori 2009) has shown that the effect of atrazine on the LH surge was reduced 

approximately 50% 2 days after the termination of treatment and the LH surge had returned to normal 

4-days after termination of dosing.

Syngenta study 0052070 (WIL Study 639091) is designed to evaluate the number of doses of atrazine 

(4, 8 or 14) are required before the LH surge response disappears. 

Syngenta plans to initiate a new study that will determine the minimum number of days of treatment 

needed to produce an effect on the LH surge in the female Sprague-Dawley and Long Evans rat.

Report Submission Dates: 

WIL Study 639060: January 31, 2011

WIL Study 639091: April 29, 2011
Attachment 7

Syngenta Studies Relevant to Defining the Critical Toxiphore/Dose Metric

Syngenta study 0033836 (WIL Study 639081) has shown that atrazine, deethylatrazine (DEA), or 

deisopropylatrazine (DIA) do not appear to persist long enough in plasma to mediate the effect on the 

LH surge. In contrast the plasma concentration of diaminochlorotriazine (DACT) persists for a long 

enough duration to be the “candidate toxiphore” following gavage treatment with atrazine.

A new study is planned to determine if 4 days of dosing with atrazine, DACT, or DACT’s glutathione 

conjugate have effects on the LH surge. 

Report Dates: 

WIL Studies 639081: Submission January 31, 2011

New WIL Study Protocol: February 15, 2011

Final Report: TBD
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