OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION UNITED STATE S ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

RE : Meeting between Dow AgroScience s (DAS) and EPA to Discuss Lung Cholinesterase and Toxicity Adjustment Factor for Chlorpyrifos List of Documents 1. Meeting Summary Page 1 2. Meeting Sign in Sheet Page 2 3. DAS PowerPoint Presentation Page 3 Meeting Summary On Thursday , December 6 , 2012, EPA’s Office of Pesticide Programs met with DAS to discuss the use of lung cholinesterase inhibition as the endpoint for the inhalation risk assessment for chlorpyrifos . DAS discussed their point of view as presented in their PowerPoint presentation . EPA reiterated its position, which is presented in the chlorpyrifos preliminary human health risk assessment 1 (PHHRA) as well as the April 2012 Scientific Advisory Panel that addressed “ scientific issues concerning health effects of chlorpyrifos. ” 2 The toxicity adjustment factor was also discussed. EPA ’ s position is consistent with what appears in the PHHRA.

1 Available at http://www.regulations.gov/#!documentDetail;D=EPA - HQ - OPP - 2008 - 0850 - 0025 . 2 Documents available at http://www.epa.gov/scipoly/sap/meetings/2012/041012meeting.html .Name EPA and Registrant Meeting Chlorpyrifos December 6, 2012 Affiliation Phone Number "10 3**,.}o~** -I(( 11:13 EPA/Ht:O 7 03 3 0 cf '+13 5 

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### Michael Bartels, Cheryl Cleveland, Jon Hotchkiss, Daland Juberg December 6, 2012 

# Perspectives on Risk Assessment Elements

for Chlorpyrifos| 2 

## Outline

• Current Status/Assumptions • Existing Acute Inhalation Study for CPF • Lung ChEI as Endpoint? • Risk Assessment Implications • Use of TAF for Oxon • Data Development/Modeling Plans • Discussion| 3 

## Assumptions • Registration Review – bystander inhalation in 2011 preliminary HHA – Agency to revisit after new policies/procedures (based on 2009 SAP) are in place • EPA will translate fumigant methodology (focus on 24 hr TWA and PERFUM) for semi - volatiles • EPA is interested in DAS views on lung ChEI as a POD for acute inhalation risk assessment • DAS comment to EPA 6/30/11 assessment – consideration of ‘severity of effects’ supports reduction of UF DB 

### = 10X (LOAEL to NOAEL) to 3X for acute inhalation • DAS supporting EPA efforts through additional modeling/PBPK enhancements as recommended by 2011 SAP| 4 

## Dow AgroSciences Acute Inhalation Study and Context



# • Review of Existing 2010 Hotchkiss Study • Current Interpretations of Endpoints • Lung Endpoint Analysis • Literature Review 

# • Regulatory Implications of Lung Endpoints| 5



## CPF Aerosol Inhalation Study: Background
• Previous CPF inhalation studies conducted with CPF vapor > Saturated vapor concentration of CPF at 25 ° C is ~0.489 mg/m 3 > Absorbed dose from 6 hr exposure to saturated CPF vapor is ~99 μg/kg bw 

### • Nose - only inhalation of saturated CPF vapor does not result in detectable inhibition of blood or tissue ChE activity • DAS commissioned a nose - only CPF aerosol exposure study (Hotchkiss, 2010) to provide toxicokinetic data to extend CPF PBPK/PD model (Timchalk, 2002) for inhalation route of exposure > Nose - only inhalation of CPF aerosol necessary to achieve an absorbed dose sufficient to induce detectable ChE inhibition following a single 6 hr exposure > The particulate nature of the CPF aerosol exposure makes this study unique • Impact on regional dosimetry and uptake kinetics compared to a vapor study| 6 

## DAS Acute Inhalation Study (MRID 48139303)
• 6 h nose - only exposure to 0, 3.7, 12.9, 22.1, 53.5 mg/m 3 

### CPF aerosol > Highly respirable particulate aerosol (MMAD 1.8 – 1.9 μm) > Plasma, RBC, Brain, Lung ChEI and TK endpoints 

### • Study reported RBC ChEI NOEC at 3.7 mg/m 3 

### • Brain ChEI only at highest dose (53.5 mg/m 3 

### ) • Plasma and lung ChEI statistically identified • Analysis of the data (Table 5) suggests: > 3.7 mg/m 3 may be a NOEC for all 4 tissues > Statistically identified differences influenced by variability of control values • Inherent variability in the ChE assay as well as inter - and intra - group variability > Little to no evidence of a biological response to treatment in lung or plasma • Statistically identified differences reflect variability around control ChE levels > RBC is the most consistent and sensitive marker relative to D/R| 7 

## Interpretations of Endpoints by Time Frame for CPF Aerosol Study
• From 6 hr acute study, EPA named a 6 and 24 hr endpoint for CPF based on 3.7 mg/m 3 

### exposure level as LOAEL for lung and plasma. > 6 hr HEC = 2.48 mg/m3 (used for 2 hr – spray drift) > 24 hr HEC = 0.62 mg/m3 

### • The existing study has additional data with 2 and 4 hrs of exposure > Concentration and time dependence can be assessed as probit response for the % of control activity and suggests > 7.7 mg/m3 for RBC at 2 hour > 36 mg/m3 for lung at 2 hour 

### • Is lung an appropriate and valid endpoint?| 8 

## Use of Acute Inhalation Study for HEC (mg/m3)

2.48 0.6 2 0 2 6 24

time CXT policy 7.7 X 36 X| 9 Figure 9: Effect of CPF Exposure Concentration on Peak CPF and Oxon Blood Levels and the Magnitude of ChEI in Plasma, RBC, Lung and Brain| 10 

## Hotchkiss Study (Table 5 Inputs) Plasma and Lung Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m 3

• Little difference in tissue - specific (plasma and lung) ChE activity levels between control and 3.7 mg/m 3 - exposed groups • High control values contributed to statistical significance identified in plasma and lung| 11 

## Hotchkiss Study (Table 5 Inputs) Plasma and Lung Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m 3

• Little difference in tissue - specific (plasma and lung) ChE activity levels between control and 3.7 mg/m 3 - exposed groups • High control values contributed to statistical significance identified in plasma and lung| 12 

## Hotchkiss Study (Table 5 Inputs) RBC and Brain Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m 3

• Little difference in tissue - specific (RBC and brain) ChE activity levels between control and 3.7 mg/m 3 - exposed groups • Low control values contributed to statistical significance identified in RBC| 13 

## Hotchkiss Study (Table 5 Inputs) RBC and Brain Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m 3

• Little difference in tissue - specific (RBC and brain) ChE activity levels between control and 3.7 mg/m 3 - exposed groups • Low control values contributed to statistical significance identified in RBC| 14 

## Potential Confounding of Lung ChE by Plasma ChE

• Lung in DAS inhalation study not cleared of blood (by perfusion) • Contribution of blood ChE to lung ChE activity • Askar et al (2011) demonstrated importance of blood rinsing when evaluating tissue ChE • Given high correlation between plasma and lung ChE and that the lung tissues from DAS inhalation study were not rinsed of blood, lung ChE was likely influenced by blood perfusion • Lung ChE measurements likely reflect activity from 3 possible independent contributors (lung, RBC, plasma) Askar et al. 2011. Comparative analysis of cholinesterase activities in food animals using modified Ellman and Michel assays. Can. J. Vet. Res. 75:261 - 270.| 15 

## Impact of Lung ChE Activity on Biological Function/Physiology
• Acute toxicity/lethality > Central apnea, seizures, bronchoconstriction, bronchorrhea , etc. > Morbidity/mortality due to respiratory failure resulting from loss of central respiratory drive not direct peripheral cholinergic effects 

### • OPs linked to airway hyperreactivity /bronchoconstriction > Do not directly induce bronchoconstriction but can potentiate vagally induced bronchoconstriction > Increased airway reactivity due to OP - induced dysfunction of autoinhibitory M2 mucarinic receptors on parasympathetic nerves 

### • CPF inhibits M2 receptor function at doses that do not inhibit lung ChE • Good correlation of RBC ChE activity with target tissue (brain, lung, diaphragm) activity| 16 

## Literature Review

• Chemical Abstracts, Medline and CAB Abstracts were searched on the following: • Organophosphorus insecticides or chlorpyrifos • Airway, diaphragm, lung, bronchi*, pulmonary, respiratory • Asthma, lung function, airway hyperactivity or airway hyperreactivity , pulmonary uptake/metabolism or cholinesterase inhibition. • 144 citations reviewed for insight/input| 17 Marable et al 2007 – Differential sensitivity of blood, peripheral and central cholinesterases in beagle dogs following dietary exposure to chlorpyrifos . Reg. Tox . Pharmacol

. 47:240 - 248. • Beagle dogs exposed for 4 or 6 (main study) weeks to chlorpyrifos (0.3 to 2.0 mkd ) • Cholinesterase activity measured in plasma, RBC, brain, left atrium and ventricle, diaphragm, quadriceps, and nodose ganglia • Main study results: > RBC AChE inhibited at all dose levels in both sexes > Diaphragm, quadriceps, and nodose ganglia AChE was unaffected by treatment > Brain AChE decreased by 6% in high - dose groups – threshold effect • Conclusion – RBC AChE in more sensitive than brain or peripheral tissue AChE to inhibition by CPF and that protection of brain AChE would protect peripheral tissue AChE| 18 Nostrandt et al. 1997. The relationship of oral chlorpyrifos effects on behavior, cholinesterase inhibition, and muscarinic receptor density in rat. Pharmacol . Biochem . Behav . 58:15 - 23.



### • Adult male rats dosed with 0, 10, 30, 60, or 100 mkd CPF • Rats tested for FOB before testing and at 3.5 hr after testing • Tissue collection at 3.5 hr and 24 hr • Half brain, individual brain areas, retina, liver, heart, diaphragm, quadriceps, and blood (whole, plasma, RBC) • Differential tissue cholinesterase activity response • RBC more sensitive than diaphragm at both timepoints 

### • >60% brain ChE inhibition before neurobehavioral effects evident| 19 

## Lung ChEI Endpoints?

• Does EPA intend to require lung ChE a standard study or endpoint for cholinesterase - inhibiting pesticides? > Lung ChEI endpoint appears unique to CPF 

### • No existing EPA endpoints based on lung cholinesterase inhibition > Canvassed 27 OP and carbamate scoping documents and REDs > Of chemicals examined, 12 inhalation endpoints from inhalation studies and 15 had values based on oral studies 

### • For inhalation study derived endpoints most based on ChEI (many in tissue combinations) > 5 on brain (2 brain only) 6 on plasma, 8 were based on RBC (1 RBC only) > 2 endpoints other than ChEI . > In many cases, ChEI seen in multiple locations at the LOAEL.| 21 

## DAS Conclusions on Lung ChEI as POD

• RBC ChEI has historically and consistently been used as a reliable and conservative marker of exposure and POD • Acute inhalation study supports RBC is more dose - responsive to treatment than lung • Acute inhalation study lung ChEI measurements appear driven by control variability and influenced by RBC/plasma ChE activity • Oral studies support RBC as consistently more sensitive than peripheral (e.g., diaphragm) tissue ChEI • Therefore, RBC NOEC of 3.7 mg/m 3 

### serves as the most reliable and biologically responsive and conservative POD for acute inhalation risk assessment| 22



# Questions?| 23 

## Broader Questions for CPF Human Health Risk Assessment

• DAS concerned CPF bystander assessment may set multiple precedents > EPA implementing aspects of multiple policies on a case - by - case basis, without finalized policies. • There’s been SAP and public discussion but no clear resolution on bystander, spray drift and volatility policies > EFED scientists recently raised a significant number of basic research issues for use of flux data for semi - volatiles > When CPF case includes selection of: 1) models/algorithms, 2) defaults, 3) databases 4) percentiles and 5) timeframes, EPA is effectively setting policy through process which impacts the larger community • Data source for contiguous field size will be key input for PERFUM| 24 

## Human Health Risk Assessment within Registration Review
• In 2011, Agency preliminary bystander inhalation exposure assessment used available monitoring data > DAS provided an corrected Table 24 in which no ambient air concentration exceeded MOE of 300 (based on lung LOAEC) 

### • The field flux study was originally conducted for EPA’s ecological risk assessment > No inhalation study for oxon was part of DCI 

### • EPA is now interested in the Human Health Assessment > DAS proposes to conduct additional work to set an inhalation endpoint for oxon inhalation or to determine a TAF for inhalation route.| 25 

## Flux DER and the TAF as Input to HHRA

• Although flux of CPF and CPF oxon can be calculated separately, EFED used oral TAF of 12X for inhalation RPF between CPF and CPF oxon. > Use of TAF within DER buries untested underlying assumption of translation of the oral TAF for inhalation route 

### • Has HED reviewed the use of TAF for inhalation oxon in light given > An straight comparison of CPF and oxon endpoints is 7X • DAS proposed 1X based on CCA study and other related studies > DAS 2012 white paper on oxon vs CPF parent on peripheral tissue effects > SAP warnings about extrapolations of inhalation endpoint| 26 

## TAF and Routes

• A route - to - route extrapolation of TAF is questionable for CPF/and CPF oxon • DAS proposes to generate data to support establishment of an inhalation endpoint for oxon. This is consistent with 2009 SAP recommendations 

### .| 27 

## Considerations in TAF Assignment

• A route - to - route extrapolation of TAF is questionable for CPF/and CPF oxon • Before this is done, we need to determine the relative inhibition profile of Oxon across tissues, via the inhalation route, to understand which endpoint is most relevant for systemic effects • DAS has provided multiple pieces of information to aid in this: > October 2011 response which discussed brain as appropriate target tissue for oxon in oral route with no TAF for CPF to oxon > September 2012 DAS response, and following slides, on Oxon effects via drinking water 

### • An inhalation toxicity study with Oxon is also proposed for 2013| 28 

## Outline

• Background information • Route - comparison ChE study data, showing comparable ChE inhibition across blood and peripheral tissues • Comparative Cholinesterase Assay study data, showing preferential inhibition of RBC over brain • Update on PBPK model development • Prediction of Oxon tissue concentrations in Human • Proposal for Brain as relevant endpoint for TAF| 29 

## Background Information
• EPA is interested in improving the determination of any Toxicity Adjustment Factors that might be needed to derive POD values from 

### dietary Oxon exposures • Chlorpyrifos is unique in that multiple studies are available which provide data on ChE inhibition in peripheral tissues • In the 2010 DAS CCA study, Oxon was shown to elicit ChE inhibition in RBC, but not brain tissue > Oxon - based ChE inhibition in RBC tissue is also more pronounced from Oxon than from Chlorpyrifos 

### • To inform the relevance of Oxon - based RBC and Brain ChE inhibition 

### it is important to: > assess the impact of 1 st pass metabolism > utilize data from non - oral route ChE studies can inform the relevance of Oxon - based RBC and Brain ChE inhibition| 30 • Inhibition of ChE in Various Tissues of Rats following IV, IP, Dermal or Oral administration of CPF - Oxon (0.4, 12, 15 and 40 mg/kg, respectively; samples collected 4 hr post - dosing). Dose levels were selected to induce relatively similar degrees of ChE inhibition in plasma, RBCs, brain and diaphragm, oral dosing required the highest dose level. (data from Costa et al. 1990). Equal inhibition in the absence

of 1 st pass metabolism More inhibition of RBC due to presystemic hydrolysis of Oxon in blood

Brain effects more conserved across routes, due to systemic exposure| 31 Inhibition of ChE in Various Tissues of Rats following IP administration of CPF - oxon (30 mg/kg). Note - data from Carr et al. (2002).

Brain also representative of numerous peripheral tissues| 32 Dose - rate comparison of ChE inhibition in RBC and brain tissue of adult female SD rats (Marty et al. 2012) Dietary administration of CPF decreases systemic bioavailability (~ 7x) vs. gavage dosing (not shown) • Results in diminished brain effects, but more pronounced RBC inhibition • Trend consistent with high first pass metabolism of CPF • Supports rationale that RBC does not reflect systemic effects when appreciable presystemic metabolism occurs| 33 Test material comparison of ChE inhibition in RBC and brain tissue of adult female SD rats (Marty et al. 2012)

* 40 mg/kg Brain data from Costa et al., 1990 Substantially different inhibition profiles also seen for CPF & Oxon: • CPF systemically bioavailable, while no Oxon seen in blood • Oxon is significantly less toxic to peripheral tissues than CPF • This is due to extremely rapid, and complete presystemic hydrolysis of Oxon by ChE and other esterases| 34 Comparison of ChE inhibition in RBC and brain tissue of adult female SD rats administered CPF or CPF - oxon via oral gavage (Marty et al. 2012) 

### CPF CPF - Oxon

MOA for peripheral tissue effects dependent on systemic bioavailability of CPF or Oxon| 35 Sites of Presystemic Hydrolysis of Oxon 

#   To facilitate predictions of PK and PD from Oxon, the existing PBPK model for CPF has been modified: • incorporates Oxon - specific uptake rates • utilizes existing rates of presystemic metabolism of Oxon
Presystemic RBC inhibition also results in Oxon hydrolysis| 36 PBPK Model Validation: Predictions of ChE inhibition following single or repeat doses of CPF

- oxon to adult rats (Preliminary Model fits) CPF - Oxon - 0.5 mg/kg (single po ) CPF - Oxon - 0.5 mg/kg (repeat po )

Good correlation of model predictions with data| 37 PBPK Model: Predictions of CPF - Oxon tissue concentrations in adult human following a single oral dose of Oxon (1 μg/kg bw = 35 μg/L/day) ( Preliminary Data

)

• A high degree of presystemic hydrolysis of Oxon predicted • Non - detectable concentrations predicted in systemic blood and brain tissue| 38 PBPK Model: Predictions of CPF and Oxon - based ChE inhibition in Brain and RBC of adult human exposed via drinking water ( Preliminary Data

)

• Maximum predicted water concentrations result in biologically irrelevant ChE inhibition • Oxon - based inhibition in brain comparable to CPF| 39 

## Summary
• CPF and Oxon have unique pharmacokinetic profiles > CPF is primarily metabolized in the liver • but is systemically available above 0.1 mg/kg > Oxon is more labile, being rapidly hydrolyzed by numerous tissues, including Intestinal lumen, portal vein RBC & plasma esterases and liver • resulting in no systemically available Oxon > Increasing CPF metabolism, by dietary administration, also increases 1 st pass metabolism affording a more Oxon - like inhibition profile 

### • As a result, RBC is predictor of presystemic exposure to Oxon, vs. 

### systemic exposure to CPF • In contrast, brain sensitivity to Oxon effects has been shown to be comparable to numerous other peripheral tissues > Therefore, DAS proposes that the most appropriate endpoint for prediction of adverse effects from oral exposure to Oxon, relative to CPF, is inhibition of brain ChE activity 

### • Determination of the relevant endpoint for Oxon effects via other routes will be best made with empirical data| 40 

## Proposed 2013 Inhalation Studies

• Oxon - and Chlorpyrifos - induced ChE Inhibition with TK Endpoints > Oxon Pilot Study • 2 Oxon exposure concentrations + control • 6 hour nose - only inhalation exposure to aerosolized Oxon • Canulated rats for RBC/Plasma ChE inhibition time course • During and after exposure through 3 days • Collect urine for dose estimation > Oxon/ Chlorpyrifos Comparison Study • 3 Oxon + 3 equivalent Chlorpyrifos concentrations + Control • Serial sacrifice (during and after exposure through 3 days post exposure) • Blood oxon , chlorpyrifos , and TCP levels • RBC, Plasma, Lung, and Brain ChE activity to provide TK data • Urinary excretion to estimate dose| 41 

## 2013 PBPK Inhalation Model Plans
• Prediction of Oxon - and Chlorpyrifos - induced ChE Inhibition in Rats and Human > A LifeStage PBPK model has been developed and validated for prediction of CPF tissue dosimetry and biological effects from oral exposure route • presented to 2011 SAP • manuscript in sponsor review > LifeStage model currently expanded to include predictions from Oxon exposures via oral route (drinking water) • separate presentation of draft results, today > In 2013, model expanded to include predictions of PK and PD from inhalation route of exposure • model will be calibrated with existing oral route parameters • new lung - tissue metabolic rates will be determined for rat and human • lab work initiating 1Q2013 • TK and ChE inhibition data from 2010 CPF and 2013 Oxon inhalation studies will be used to validate the model • model completion planned for July 2013