OFFICE OF CHEMICAL SAFETY

AND POLLUTION PREVENTION

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

RE: Meeting between Dow AgroSciences (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 

assessment1 (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 

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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 UFDB = 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/m3

> 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/m3 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/m3

• Brain ChEI only at highest dose (53.5 mg/m3)

• Plasma and lung ChEI statistically identified 

• Analysis of the data (Table 5) suggests:

> 3.7 mg/m3 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/m3 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

Inhaled Dose (mg CPF inhaled/kg bw)

[Exposure Concentration (mg CPF/m3)]

1.04 3.62 6.21 15.02 Peak Blood Concentration (ng/g blood)

0.1

1

10

100

Percent Control ChE Activity

(2 hr Post-Exposure)

0

40

80

120

160

CPF

Oxon

RBC

Plasma

Lung

Brain

[3.7] [12.9] [22.1] [53.5]

< LLQ

100%

of Control
| 10

Hotchkiss Study (Table 5 Inputs)

Plasma and Lung Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m3

• Little difference in tissue-specific (plasma and lung) ChE activity levels between control and 

3.7 mg/m3-exposed groups

• High control values contributed to statistical significance identified in plasma and lung Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour

0 mg/m3 0 mg/m3

1412 167 3357 1417 1306 3136 1390 3841 2294 2435 3127 4258 2993 2993 2710 2463 3304 3311

2521 2329 2830 2144 1549 1379 2062 2723 2131 3184 2788 6406 3551 3530 4166 2979 2760 2583

1521 2646 2177 2392 1534 2514 2160 3652 3537 3297 3516 3466 5226 2915 2618 3381 3078 3381

1675 2201 3421 1443 3225 2704 2327 2219 2015 3487 2936 4222 3113 2746 3586 3191 2929

3569 2757 2944 1671 3413 2782 2402 2816 2572 4102 3071 4420 5014 2866 2781 3092 2632 3000

2124 4398 2101 2364 2361 2473 3189 2178 2085 3664 3205 3947 3756 3374 2654 2512 3827 3318

Mean 2137.0 2416.3 2805.0 1905.2 2231.3 2498.0 2255.0 2904.8 2439.0 Mean 3361.5 3107.2 4453.2 3942.2 3070.7 2985.8 3002.2 3132.0 3087.0

Std. Dev. 814.5 1357.2 564.6 449.7 917.5 597.2 582.0 703.5 573.5 Std. Dev. 556.7 248.8 1013.6 956.4 310.0 662.6 452.5 426.0 308.7

3.7 mg/m3 3.7 mg/m3

1534 1145 1048 1354 1531 266 1464 1322 1365 3005 1924 2072 2481 2129 1846 2927 3069 3217

2653 1688 1733 1704 1540 1744 1279 2189 2047 2249 2841 2411 2157 2772 2609 2475 2906 2567

1368 1604 779 1095 2247 1726 1322 2409 2921 2567 3259 2122 2941 3062 2532 6524 2913 2680

2275 1632 1043 1405 1564 1716 2828 1984 2285 2433 1980 1937 2539 2164 2009 2567 2948 2779

1714 1567 2119 1076 1882 1831 1423 1609 1921 2249 2433 2608 1980 1867 3090 5111 2736 L A

2738 3615 2051 1596 1212 2291 2953 2418 1844 2433 2149 2934 2249 2030 2312 3309 2948 2948

Mean 2047.0 1875.2 1462.2 1371.7 1662.7 1595.7 1878.2 1988.5 2063.8 Mean 2489.3 2431.0 2347.3 2391.2 2337.3 2399.7 3818.8 2920.0 2838.2

Std. Dev. 588.5 874.4 577.1 255.4 356.3 687.1 788.0 444.9 517.9 Std. Dev. 280.6 528.0 377.1 339.5 469.7 448.3 1638.0 107.6 253.8

Plasma Cholinesterase Activity (U/L) Lung Cholinesterase Activity (U/L)

Exposure Post-Exposure Exposure Post-Exposure
| 11

Hotchkiss Study (Table 5 Inputs)

Plasma and Lung Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m3

Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour

0 mg/m3 0 mg/m3

1412 167 3357 1417 1306 3136 1390 3841 2294 2435 3127 4258 2993 2993 2710 2463 3304 3311

2521 2329 2830 2144 1549 1379 2062 2723 2131 3184 2788 6406 3551 3530 4166 2979 2760 2583

1521 2646 2177 2392 1534 2514 2160 3652 3537 3297 3516 3466 5226 2915 2618 3381 3078 3381

1675 2201 3421 1443 3225 2704 2327 2219 2015 3487 2936 4222 3113 2746 3586 3191 2929

3569 2757 2944 1671 3413 2782 2402 2816 2572 4102 3071 4420 5014 2866 2781 3092 2632 3000

2124 4398 2101 2364 2361 2473 3189 2178 2085 3664 3205 3947 3756 3374 2654 2512 3827 3318

Mean 2137.0 2416.3 2805.0 1905.2 2231.3 2498.0 2255.0 2904.8 2439.0 Mean 3361.5 3107.2 4453.2 3942.2 3070.7 2985.8 3002.2 3132.0 3087.0

Std. Dev. 814.5 1357.2 564.6 449.7 917.5 597.2 582.0 703.5 573.5 Std. Dev. 556.7 248.8 1013.6 956.4 310.0 662.6 452.5 426.0 308.7

3.7 mg/m3 3.7 mg/m3

1534 1145 1048 1354 1531 266 1464 1322 1365 3005 1924 2072 2481 2129 1846 2927 3069 3217

2653 1688 1733 1704 1540 1744 1279 2189 2047 2249 2841 2411 2157 2772 2609 2475 2906 2567

1368 1604 779 1095 2247 1726 1322 2409 2921 2567 3259 2122 2941 3062 2532 6524 2913 2680

2275 1632 1043 1405 1564 1716 2828 1984 2285 2433 1980 1937 2539 2164 2009 2567 2948 2779

1714 1567 2119 1076 1882 1831 1423 1609 1921 2249 2433 2608 1980 1867 3090 5111 2736 L A

2738 3615 2051 1596 1212 2291 2953 2418 1844 2433 2149 2934 2249 2030 2312 3309 2948 2948

Mean 2047.0 1875.2 1462.2 1371.7 1662.7 1595.7 1878.2 1988.5 2063.8 Mean 2489.3 2431.0 2347.3 2391.2 2337.3 2399.7 3818.8 2920.0 2838.2

Std. Dev. 588.5 874.4 577.1 255.4 356.3 687.1 788.0 444.9 517.9 Std. Dev. 280.6 528.0 377.1 339.5 469.7 448.3 1638.0 107.6 253.8

Plasma Cholinesterase Activity (U/L) Lung Cholinesterase Activity (U/L)

Exposure Post-Exposure Exposure Post-Exposure

• Little difference in tissue-specific (plasma and lung) ChE activity levels between control and 

3.7 mg/m3-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/m3

• Little difference in tissue-specific (RBC and brain) ChE activity levels between control and 

3.7 mg/m3-exposed groups

• Low control values contributed to statistical significance identified in RBC Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour

0 mg/m3 0 mg/m3

4696 2420 3310 3366 2080 4766 5034 4610 3070 52644 49154 43380 46192 60424 45762 56679 55294 40080

5798 3126 2716 2604 2094 4074 4526 4200 3564 53301 52129 50906 50256 52502 52432 50814 53118 51379

5020 3324 2858 3678 1910 4624 4666 4738 2758 51118 52326 33176 52552 51507 53605 60311 44801 49252

4610 3212 2576 2646 2956 3338 5190 4936 2576 53867 47888 49520 55026 49359 50715 32370 52475 52743

5076 3366 2504 3056 2532 4314 4242 4314 3748 48863 53012 55330 50362 54029 49323 52051 49429 46737

4454 2900 2618 3112 2504 3946 5288 5302 2208 51811 52997 47309 52892 49379 46383 53987 49584 50255

Mean 4942.3 3058.0 2763.7 3077.0 2346.0 4177.0 4824.3 4683.3 2987.3 Mean 51934.0 51251.0 46603.5 51213.3 52866.7 49703.3 51035.3 50783.5 48407.7

Std. Dev. 482.8 353.8 294.5 413.7 388.8 516.4 411.3 406.2 591.1 Std. Dev. 1801.2 2181.0 7672.8 3033.2 4121.0 3174.8 9757.8 3680.2 4558.3

3.7 mg/m3 3.7 mg/m3

5288 3238 4412 4388 2156 4204 3342 2226 2326 53688 51469 50614 53730 51703 49765 63864 51109 48459

4722 3606 3804 4234 2778 3640 3484 3032 2566 87105 51419 40898 48657 51236 50529 53653 52401 47406

4554 3268 2678 4276 2254 4558 3710 3526 2692 50684 52727 47829 50522 51491 53695 74922 56210 52959

5048 3252 2948 4474 2028 3102 3342 2918 2990 72485 47475 50614 54275 51327 48133 56147 50261 49957

4554 3436 4628 4388 2282 4008 3102 3342 2692 51455 52528 51575 43745 50310 53073 51328 50091 52918

5118 2914 4304 4840 2834 4248 3484 3796 3598 47250 52705 51688 50515 49123 47222 55907 54140 47780

Mean 4880.7 3285.7 3795.7 4433.3 2388.7 3960.0 3410.7 3140.0 2810.7 Mean 60444.5 51387.2 48869.7 50240.7 50865.0 50402.8 59303.5 52368.7 49913.2

Std. Dev. 312.7 231.1 812.3 217.1 335.7 517.9 202.5 550.7 441.6 Std. Dev. 15821.4 2007.3 4146.8 3829.0 978.5 2594.6 8738.1 2412.9 2500.4

RBC Cholinesterase Activity (U/L) Brain Cholinesterase Activity (U/L)

Exposure Post-Exposure Exposure Post-Exposure
| 13

Hotchkiss Study (Table 5 Inputs)

RBC and Brain Cholinesterase Activity: Individual animal data – Control and 3.7 mg/m3

Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour Exposure 2 hour 4 hour 6 hour 2 hour 6 hour 12 hour 24 hour 48 hour 72 hour

0 mg/m3 0 mg/m3

4696 2420 3310 3366 2080 4766 5034 4610 3070 52644 49154 43380 46192 60424 45762 56679 55294 40080

5798 3126 2716 2604 2094 4074 4526 4200 3564 53301 52129 50906 50256 52502 52432 50814 53118 51379

5020 3324 2858 3678 1910 4624 4666 4738 2758 51118 52326 33176 52552 51507 53605 60311 44801 49252

4610 3212 2576 2646 2956 3338 5190 4936 2576 53867 47888 49520 55026 49359 50715 32370 52475 52743

5076 3366 2504 3056 2532 4314 4242 4314 3748 48863 53012 55330 50362 54029 49323 52051 49429 46737

4454 2900 2618 3112 2504 3946 5288 5302 2208 51811 52997 47309 52892 49379 46383 53987 49584 50255

Mean 4942.3 3058.0 2763.7 3077.0 2346.0 4177.0 4824.3 4683.3 2987.3 Mean 51934.0 51251.0 46603.5 51213.3 52866.7 49703.3 51035.3 50783.5 48407.7

Std. Dev. 482.8 353.8 294.5 413.7 388.8 516.4 411.3 406.2 591.1 Std. Dev. 1801.2 2181.0 7672.8 3033.2 4121.0 3174.8 9757.8 3680.2 4558.3

3.7 mg/m3 3.7 mg/m3

5288 3238 4412 4388 2156 4204 3342 2226 2326 53688 51469 50614 53730 51703 49765 63864 51109 48459

4722 3606 3804 4234 2778 3640 3484 3032 2566 87105 51419 40898 48657 51236 50529 53653 52401 47406

4554 3268 2678 4276 2254 4558 3710 3526 2692 50684 52727 47829 50522 51491 53695 74922 56210 52959

5048 3252 2948 4474 2028 3102 3342 2918 2990 72485 47475 50614 54275 51327 48133 56147 50261 49957

4554 3436 4628 4388 2282 4008 3102 3342 2692 51455 52528 51575 43745 50310 53073 51328 50091 52918

5118 2914 4304 4840 2834 4248 3484 3796 3598 47250 52705 51688 50515 49123 47222 55907 54140 47780

Mean 4880.7 3285.7 3795.7 4433.3 2388.7 3960.0 3410.7 3140.0 2810.7 Mean 60444.5 51387.2 48869.7 50240.7 50865.0 50402.8 59303.5 52368.7 49913.2

Std. Dev. 312.7 231.1 812.3 217.1 335.7 517.9 202.5 550.7 441.6 Std. Dev. 15821.4 2007.3 4146.8 3829.0 978.5 2594.6 8738.1 2412.9 2500.4

RBC Cholinesterase Activity (U/L) Brain Cholinesterase Activity (U/L)

Exposure Post-Exposure Exposure Post-Exposure

• Little difference in tissue-specific (RBC and brain) ChE activity levels between control and 

3.7 mg/m3-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/m3 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

Entity Oral Route Inhalation Route

Chlorpyrifos 

(parent)

aPAD = 0.0036 mg/kg 

based on BMDL of 0.36 mg/kg for RBC

HEC of 2.48 mg/m3 

from 6hr study

Chlorpyrifos oxon 

(metabolite)

aPAD – 0.0005 mg/kg

based on BMDL of 0.05 mg/kg for RBC 

Unknown 

TAF 7.2X (ratio of aPADs)

12X (EPA model assessment)

unnecessary if first pass metabolism and 

brain ChE considered for oxon

Unknown 

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 1st 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).

Dose Route

Cholinesterase Activity (% Control)

IV Dermal IP Oral

0

20

40

60

80

100

Plasma

RBC

Brain

Diaphragm Equal inhibition in the absence 

of 1st 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).

Sampling Time (hr post-dosing)

2 hr 24 hr

0

20

40

60

80

100

Medulla-Pons

Corpus Striatum

Cerebral Cortex

Hippocampus

Skeletal Muscle

Lung

Heart

Brain also representative of numerous peripheral tissues
| 32

CPF Oral- Gavage

Dose (mg/kg)

Cholinesterase Activity (% Control)

0.01 0.1 1 10 100

0

50

100

RBC

Brain

Dose-rate comparison of ChE inhibition in RBC and brain tissue of adult female SD 

rats (Marty et al. 2012)

CPF Oral- Diet

Dose (mg/kg)

Cholinesterase Activity (% Control)

0.01 0.1 1 10 100

0

50

100

RBC

Brain

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 

CPF Oral- Gavage

Dose (mg/kg)

Cholinesterase Activity (% Control)

0.01 0.1 1 10 100

0

50

100

RBC

Brain

Oxon Oral- Gavage

Dose (mg/kg)

Cholinesterase Activity (% Control)

0.01 0.1 1 10 100

0

50

100

RBC

Brain

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

Key Event 1 Key Event 2 Key Event 3

Dose (mg/kg)

Cholineric 

effects in GI 

tissue

Inhibition of RBC 

Cholinesterase 

Activity

Inhibition of Brain 

Cholinesterase 

Activity

0.05 - - -

0.1 - - -

0.5 - - -

2 - + -

5-Pilot - ++ -

10 - +++ ++

10-Pilot - +++ ++

20-Pilot + * ++++ +++

* GI effects data in adult Male Long-Evan rats, from Moser et al. (1995)

Scale for ChE inhibition (% of control): ≥90% = "-", 75-90% = "+", 40-75% = "++", 

 5-40% = "+++", <5% = "++++"

Key Event 1 Key Event 2 Key Event 3

Dose (mg/kg)

Cholineric 

effects in GI 

tissue

Inhibition of RBC 

Cholinesterase 

Activity

Inhibition of Brain 

Cholinesterase 

Activity

0.01 - - -

0.05 - - -

0.1 - - -

0.5 - ++ -

1 - +++ -

1-Pilot - +++ -

5-Pilot + ^ ++++ -

10-Pilot + ^ ++++ -

^ Effect seen (mucoid feces), in only 1 of 3 study animals at this dose level

Scale for ChE inhibition (% of control): ≥90% = "-", 75-90% = "+", 40-75% = "++", 

 5-40% = "+++", <5% = "++++"

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)

0 10 20

0

50

100

Brain

RBC

Plasma

Time (hr)

Cholinesterase Activity (% Control)

0 50 100 150 200

0

50

100

Time (hr)

Cholinesterase Activity (% Control)

RBC

Brain

Plasma

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)

Intestine

Time (hr) Oxon Concentraion (uM)

0 2 4 6 8

110- 0 8

110- 0 7

110- 0 6

110- 0 5

110- 0 4

110- 0 3

110- 0 2

110- 0 1

1100 0

 Plasma (systemic)

Time Oxon Concentraion (uM)

0 2 4 6 8

110- 0 8

110- 0 7

110- 0 6

110- 0 5

110- 0 4

110- 0 3

110- 0 2

110- 0 1

1100 0

Liver

Time Oxon Concentraion (uM)

0 2 4 6 8

110- 0 8

110- 0 7

110- 0 6

110- 0 5

110- 0 4

110- 0 3

110- 0 2

110- 0 1

1100 0

Brain

Time Oxon Concentraion (uM)

0 2 4 6 8

110- 0 8

110- 0 7

110- 0 6

110- 0 5

110- 0 4

110- 0 3

110- 0 2

110- 0 1

1100 0

Initial water concentration

Limit of Quantitation

• 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 1st

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