1 D an 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

F ed E x

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 develop ment 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 an d 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 sup pression are as follows :

1. PBPK Data and Model D evelopment for the Rat

2. Scaling Rodent Model to N on

- H uman P rimates and H uman s

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. E ffects on F ertility
Strain Sensitivity Differences

Bolus vs. Distributed Dose Administration

5. Life Stage S ensitivity and the R elevance to Uncertainty Factors

6. Probabilistic Risk A ssessment Based Upon Internal Dose and the BMD.

7. Drinking Water MonitoringPBPK Data and Model Development

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

definiti ons 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 pharm acokinetic 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 o n going pharmacokinetic studies carried out in v itro 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 stu dy on absorption through a gut monolayer cell culture is being

initiated.

Benchmark Dose Determination :

Syngenta has ongoing studies summarized in Attac hment 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 r eplicates 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 studies 4

.

Effects on the LH Surge vs. Effects on Fertility The SAP expressed the need to correlate the degree of LH suppre ssion caused by atrazine dosing to the potential for effec ts on fertility. T he 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 statist ically 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

St

rain 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 Re

E valuation of the Human Health Effects of Atrazine: Review of Non

- cancer Effects and Drinking Water

M onitoring 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 53the ir data should be analyzed separately” 7 . The study being conducted will allow for a si de

- by

side comparison of the BMD in both strains.

Distributed Dose Administration Co mpared to Bolus Dose Administration The SAP recommended that data generated from dietary a dministration 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 tra ct.” 8 Towards this end, Syngenta has generated comparative (dietary vs gavage) PK data, and has ongoing studies generating LH dose

- response data (peak and area

under

- 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 k ey 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 wi ll inform the inter

- and intra

- species extrapolations.” 10

New Syngenta work relevant to these question s appear s 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 con verting 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 a n 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 Resear ch Pertinent to Risk Assessment

Syngenta has t wo 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 s ystems (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 commen ts

. These data are important for the us e in developing methodologies, as stated

7 Ibid, page 56

8 Ibid, page 48

9 Ibid, page 56

- 57

10 I bid, page 53by 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 t hat 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

. Additiona l statistica l analyses of the impact of different

samp ling 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 t he 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 Stud ies 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 exp ected 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 atr azine 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 metri cs for

a trazine 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 , f or example the studies by Hui et al. ( 1996 a,b,c ) and Davidso n ( 1988 )

.

New S tudy Submission Dates:

WIL Studie s 639081, 639084 : January 31, 201 1

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 (WI L Study 639081; See Attachment 1 for study design)

.

3) The atrazine absorption rate constant will be derived from in vivo (C 14 atrazine/total chlorotriazine
[TCT] data ) and ex vivo (Caco2 cells) data (Study yet to be initiated).

4) The model will be developed bas ed 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 d ays 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 t hat 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) On e 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 metri c 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 hep atocyte 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) . Predi cted
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 t issue 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 (Davi dson, 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 th e 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.

**•** Distrib uted 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 r educes 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 Re levant to Uncertainty Factors

Syngenta study 002316 8 (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 current ly 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 approximate ly 3 fold

.

Report Submission Date s : 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 durati ons of atrazine

an d its chlorotriazine metabolites in a subset of CWSs.

Water Intake: Two surveys of water intake (CSFII and Bayer CropScience Dri nking Wate r 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 int ake 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 individual s 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 set s (LH suppression, Functional Effect)

, appropriate animal model s ( e.g. Long

- Evans, Sprague

- Da wley) , and appropriate duration s (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 biolog ical 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 simul ation 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

function s for each CWS evaluated.

**•** Distributions and 99 th or some other suitable percentiles for intakes, response levels, equivalent distributed vs. bolus doses, and MOEs will be calculated for analysis.

**•** Sensitivity analyse s will be conducted.

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

## Attac h ment 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

d osing 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 termin ation 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 disappe ars.

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 Date s : WIL Stu dy 639060: January 31 , 2011 WIL Study 639091 : April 29, 2011

## Attachme nt 7 Syngenta Studies Re levant 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 effe ct on the LH surge. In contrast the plasma concentration of diaminochlorotriazine (DACT) persist s for a long enough duration to be the “candidate toxiphore” following gavag e treatment with atrazine

.

A new study is planned to determine if 4 days of dosing with atrazine, DACT, or DACT ’ s glutat h ione conjugate have effect s on the LH surge

.

Report Date s : WIL Studies 639081 : S ubmission January 31, 2011 New WIL Study Protocol: February 15, 2011 Final Report: TBDReferences

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