Response to EPA (John Doherty) re: TCP hydrolysis and 

analysis methods and differences in oral absorption of 

chlorpyrifos between human volunteer studies 

Michael J. Bartels, Kathy A. Brzak and Shakil A. Saghir 

Toxicology Laboratory 

The Dow Chemical Company 

Midland, MI 48674

INTRODUCTION 

The Health Effects Division (HED) of EPA’s Office of Pesticide 

Programs recently brought several human volunteer studies involving 

chlorpyrifos to the EPA Human Studies Review Board (HSRB) for 

scientific and ethical review. Based on discussion at the HSRB 

meeting, EPA on July 13, 2009 requested additional information 

regarding conduct of chemical analyses and oral absorption results in 

two of the studies. Dow AgroSciences (DAS) is providing information 

in response to this request. 

ANALYTICAL PROCEDURES FOR TCP 

John Doherty of the HED, in an email inquiry to Dow Chemical of 

September 16, 2009, posed the following question: ”The issue is that 

the HSRB raised concerns that the Nolan (~70%) and Kisicki (~30%) 

studies have such a large recovery difference for TCP indicating 

more was absorbed in the Nolan study. One of the HSRB panel 

members challenged the methodology used to detect TCP in blood 

and urine. Previously, Ken Racke addressed the issue in blood 

indicating that no hydrolysis was necessary since based on previous 

work with rat blood, nearly all TCP in blood is unconjugated. The 

assumption that TCP in human blood was also unconjugated has 

been made as a defense for not attempting to hydrolyze the blood to 

free conjugated TCP. This is still an assumption that at least one 

person here is challenging. The HSRB appears to consider the Nolan 

study more reliable than the Kisicki study with respect to TCP 

analysis in the urine. For the Kisicki study the Board has "concerns 

about the analytical procedures' potential inability to control for the 

1 
glucuronide-conjugated TCP, and apparent discrepancies in the 

absorption data when compared with the data from Nolan et. al 

(1982), the Board questioned the reliability and utility of the blood and 

urine measurements of chlorpyrifos and/or TCP from Kisicki et al 

(1999) for risk assessment purposes." The Board also implied that 

the difference in absorption is related to differences in the 

methodology used to detect TCP. 

I went over both the Brzak original supplement to the Kisicki study 

(page 16) and the response the Ken Racke sent previously (July 24, 

2009, you are the second author) but cannot find where it specifically 

states that the sulfuric acid hydrolysis step was included in the 

preparation of the urine for TCP analysis. If the acid hydrolysis step 

was the same for both Nolan and Kisicki and it is the established 

method to free conjugated TCP, then we can eliminate that the 

difference in apparent absorption between Nolan and Kisicki is due to 

differences in sample preparation. Therefore if you can provide 

written documentation that there was the appropriate acid step in the 

preparation of the urine for TCP analysis and any other information 

that will resolve the reliability issue for TCP analysis in the Kisicki 

study, then the HSRB should reconsider its concerns. The 

documentation should include the amount of acid, temperature and 

time allowed for hydrolysis." 

To clearly address this question, a summary of the analytical 

methods used for analysis of total 3,5,6-trichloropyridinol (TCP), has 

been prepared (Table 1). This table summarizes the study, species, 

hydrolysis, extraction, derivatization and separation/detection 

methods for Nolan 1982 and Kisicki 1999 human volunteer studies. 

For comparision, the analogous TCP methods employed on two rat 

metabolism or pharmacokinetic studies are included. 

TCP in Urine: As shown by Nolan et al. in the Dow Chemical 1987 

rat metabolism study, 14C-chlorpyrifos-derived metabolites are 

excreted primarily in the urine of rats, accounting for 84-92% of the 

administered dose. The major metabolites found were free TCP and 

the glucuronide and sulfate conjugates of TCP. The authors showed 

that a 1 hr hydrolysis of the urine, at 80 degrees C, with a 1:10 

acidification with 12M HCl was sufficient to completely hydrolyze the 

two conjugates of TCP back to the free TCP metabolite. The TCP 

2 
analytical methods employed for analysis of urine samples from both 

the Nolan 1982 and Kisicki 1999 human volunteer studies utilized 

hydrolysis methods of at least this acid strength and hydrolysis 

temperature and duration. In the Nolan 1982 study, urine samples 

were hydrolyzed with 2:10 (acid:urine) ratio of 18M H2SO4, at 90 

degrees C for 1 hr. In the Kisicki 1999 study, the urine samples were 

analyzed for total TCP by Dr. Steven Summer of Battelle Laboratory 

(Columbus, Ohio). The acid hydrolysis method employed in this 

study was the same as in the original Nolan 1987 rat metabolism 

study (1:10, 12M HCl:urine @ 80 degC x 1 hr). Summer also verified 

that these conditions were sufficient for complete hydrolysis of the 

conjugated TCP in the Kisicki study urine samples by evaluating 

hydrolysis of numerous samples at 3 hr and 1hr. The extended 

hydrolysis time afforded no increase in total TCP found (<2% 

change). 

TCP in Blood: As shown by Mendrala et al. in the Dow Chemical 

1997 rat pharmacokinetic study, free TCP accounted for virtually all of 

the radioactivity in blood of rats administered either 5 or 100 mg/kg of 

14C-chlorpyrifos. Based on these results, the analysis of total TCP in 

the Nolan 1982 and Kisicki 1999 human volunteer studies did not 

require any acid hydrolysis step, as the TCP in human blood samples 

is assumed to be non-conjugated (see more complete discussion of 

this in Dow AgroSciences comments of July 24, 2009 (Racke 2009)). 

Some acid was added to the blood samples in both human volunteer 

studies, to protonate the analyte prior to SPE cleanup and/or 

subsequent extraction. 

Overall Extraction, Derivatization, Analysis Conditions: All of the 

quantitative analytical methods for TCP in blood or urine from the rat 

or human pharmacokinetic studies were fairly comparable. Following 

acidification (with subsequent hydrolysis for urine), the samples were 

extracted into an organic solvent. Note that the samples from the 

Nolan 1982 human volunteer study employed an intial solid-phase 

extraction (SPE) column cleanup step, as the GC detection method of 

Electron-Capture (ECD) is not as specific as the detection method 

employed in the later studies (GC/MS). However, once in an 

extraction solvent, the TCP in the sample extracts was derivatized 

with a silylating reagent to enhance volatility of this analyte. The 

derivatized TCP was then separated on a GC column and quantitated 

via either ECD or mass spectral detection. The reference for the 

3 
methods employed in any of the human or rat studies is listed in the 

Comments column of Table 1. Note that the recovery of TCP from 

any of the listed methods was quite high, ranging from 86 to 114%. 

More specifically, the methods used for analysis of urinary TCP from 

the two human volunteer studies was quite high, at 97 and 99%. 

Note that the method for urine analysis in the Kisicki study was a Dow 

AgroSciences Report by E. Olberding (1998), which is included as an 

Appendix in the TCP analysis report of Summer 1999. 

ORAL ABSORPTION OF CHLORPYRIFOS 

John Doherty of the HED, in an email inquiry to Dow Chemical of 

September 16, 2009, also posed a second question: ”If the acid step 

was included and total TCP was detected in the urine in the Kisicki 

study, then the difference in apparent absorption needs to be 

explained. In both cases either the pill or the capsule have to be 

dissolved in the stomach before the chlorpyrifos can pass the pyloric 

valve to enter the g-i tract to be absorbed. I discussed this with 

another toxicologist, particularly involved with metabolism and PK, 

and he agrees that there may be some differences in absorption but 

not such a large difference as seen with the Nolan and Kisicki 

studies. Therefore, HED is requesting that DOW provide additional 

support for the assumption that the differences in apparent absorption 

is related to differences in dosing method (pill vs. capsule). It is also 

suggested that any other explanations that can explain the large 

difference in apparent absorption between the Nolan and Kisicki 

studies be provided especially to remove concerns that differences in 

analytical procedure were responsible." 

As discussed above, the analysis of TCP in blood and urine from the 

Nolan 1982 and Kisicki 1999 studies used comparable methods for 

analysis of the total TCP present in either matrix. An adequate acidhydrolysis step was also employed in the analysis of urine samples 

from the Kisicki 1999 study samples. As a result, the difference in 

oral absorption between the two studies is most probably a result of 

test material formulation differences. 

4 
In the study of Nolan et al. (1982), the chlorpyrifos test material was 

dissolved in methylene chloride and the resulting solution applied to a 

lactose tablet. Following evaporation of methylene chloride carrier, 

the tablet was taken orally with water by the study volunteers. In 

contrast, the chlorpyfos test material used in the Kisicki 1999 study 

was formulated by weighing in crystalline test material into an empty 

capsule and filling the remainder of the capsule with lactose powder. 

It would be expected that the chlorpyrifos added as a solution to 

lactose would afford dried test material of a substantially smaller 

particle size and/or larger surface area than the corresponding 

crystalline product. Numerous authors have shown that altering 

either property of drugs that are poorly water soluble in water can 

substantially enhance oral absorption in animals or humans. Garner 

et al. (2002) showed that micronizing diosmin from a mean particle 

size of 36.5 μm to 1.79 μm resulting in an increase in oral absorption 

in human volunteers from 33% to 58%. Evans et al. (2006) have 

also shown a 4-7 fold increase in systemic bioavailability of 

ketoconazole in dogs upon nanostructuring to increase drug surface 

area via rapid freezing. Elder et al (2006) have shown subtantial 

increases in systemic bioavailability of danazol following reducing 

drug particle size via a controlled precipitation technique. Finally, Liu 

(2008) has also discussed the fact that oral absorption generally 

increases with reduced particle size, since for most poorly soluble 

compounds, dissolution into the GI fluids is the rate-limiting step in 

oral uptake. In fact, this author describes that the oral absorption of 

the antifungal agent griseofulvin was found to increase 2.5 fold when 

the surface area was increased about six-fold. This increase in 

absorption is quite consistent with the 2.2-fold increase in absorption 

seen for chlorpyrifos, when adminstered as a solution applied to a 

lactose tablet (then dried) vs. the crystalline solid (70% from Nolan 

1982 vs. 32% from Kisicki 1999). 

Based on these examples, and the known differences in test material 

formulation techniques between the Nolan and Kisicki human 

volunteer studies, it is expected that the lower oral bioavailability 

measured in the 1999 Kisicki study was due to a larger mean 

chlorpyrifos particle size (affording a smaller net surface area/mg test 

material) than that of the formulation employed by Nolan et al (1982). 

5 
References 

Brzak, K. A. (2000). A rising dose toxicology study to determine the 

no-observable-effect-levels (NOEL) for erythrocyte 

acetylcholinesterase (AChE) inhibition and cholinergic signs and 

symptoms of chlorpyrifos at three dose levels- Part B. Report of 

the Dow Chemical Company (DECO HET K-044793-284B), 

Midland, Michigan. (MRID 45144101). [Chemical analysis of 

blood samples from Kisicki et al., 1999]. 

Elder E.J., J.E. Hitt, T.L. Rogers, C.J. Tucker, S.A. Saghir, S. 

Svenson, and J.C. Evans (2006). Particle Engineering of Poorly 

Water Soluble Drugs by Controlled Precipitation, Chapter 19. In, 

Polymeric Drug Delivery Volume II: Polymeric Matrices and Drug 

Particle Engineering, (Svenson, S., Ed.), ACS Symposium Series, 

Vol. 924, Oxford University Press, Cary, NC, pp292-304. 

Evans, J.C. B.D. Scherzer, C.D. Tocco, G.B. Kupperblatt, J.N. 

Becker, D.L. Wilson, S.A. Saghir, and E.J. Elder (2006). 

Preparation of Nanostructured Particles of Poorly Water Soluble 

Drugs via a Novel Ultra-Rapid Freezing Technology, Chapter 21. 

In, Polymeric Drug Delivery Volume II: Polymeric Matrices and 

Drug Particle Engineering, (Svenson, S., Ed.), ACS Symposium 

Series, Vol. 924, Oxford University Press, Cary, NC, pp 320-328. 

Garner, R C, J V Garner, S Gregory, M Whattam, A Calam, and D 

Leong. 2002. Comparison of the absorption of micronized (Daflon 

500 mg) and nonmicronized 14C-diosmin tablets after oral 

administration to healthy volunteers by accelerator mass 

spectrometry and liquid scintillation counting. Journal of 

Pharmaceutical Sciences 91, 32-40. 

Kisicki, J. C., Seip, C. W., and Combs, M. L. (1999). A rising dose 

toxicology study to determine the no-observable-effect-levels 

(NOEL) for erythrocyte acetylcholinesterase (AChE) inhibition and 

cholinergic signs and symptoms of chlorpyrifos at three dose 

levels. Report of MDS Harris (Project# 21438), Lincoln, 

Nebraska. Sponsored by Dow AgroSciences, Indianapolis, 

Indiana (MRID 44811002 & MRID 45144101). 

6 
7 

Liu, Rong. 2008. Water-Insoluble Drug Formulation. CRC Press, 

January 18, p. 84. 

McKellar, R.L. (1979). Determination of Chlorpyrifos and 3,5,6-

Trichloro-2-Pyridinol in Whole Blood and Urine by Gas 

Chromatography. Report of the DowElanco Company, 

Indianapolis, Indiana (Report number ACR-79.9). 

Mendrala, A. L., and Brzak, K. A. (1998). Chlorpyrifos: Part A – 

Concentration-Time course of chlorpyrifos and chlorpyrifos-oxon 

in blood. Report of Toxicology & Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan 

(MRID 44648102). 

Nolan. R. J.; Rick, D. L.; Freshner, N. L.; et al. (1984). Chlorpyrifos: 

Pharmacokinetics in Human Volunteers Following Single Oral and 

Dermal Doses. Toxicology and Applied Pharmacology. 73, 8-15 

(MRID 00124144). 

Nolan, R. J., Dryzga, M. D., Landenberger, B. D., and Kastl, P. E. 

(1987). Chlorpyrifos: Tissue distribution and metabolism of orally 

administered 14C-labeled chlorpyrifos in Fischer 344 rats. Report 

of Toxicology & Environmental Research and Consulting, The 

Dow Chemical Company, Midland, Michigan MRID 40458901). 

Olberding, E. L. (1998). Determination of Residues of 3,5,6-

Trichloropyridinol in Urine by Capillary Gas Chromatography with 

Mass Selective Detection. Report of Dow AgroSciences LLC 

(Report number GRM-97.04.R1). 

Racke, K.D., Bartels, M.J., Rick, D.L., Brzak, K.A., and Mattsson, J.L. 

(2009). Response to EPA Query Regarding Chemical and 

Statistical Analyses of Human Volunteer Studies. Report of Dow 

AgroSciences LLC (Report number KDR07242009). 

Summer, S. J. 1999. A rising dose toxicology study to determine the 

no-observable-effect-levels (NOEL) for erythrocyte 

acetylcholinesterase (AChE) inhibition and cholinergic signs and 

symptoms of chlorpyrifos at three dose levels. Report of Battelle 

Testing Laboratory, Columbus, Ohio (Project Number N101121A). 

[Chemical analysis of urine samples from Kisicki et al., 1999]. 
Table 1. Summary of TCP analytical methods employed in human volunteer studies. 

8 

Hydrolysis Extraction Derivatization Derivatization Derivatization Analysis

Study Species Matrix Conditions Solvent Solvent Reagent Conditions Conditions 

Nolan, 1987Dow report HET-K-044793-(76)

Rat Urine 1:10 (12M HCl:urine)

80 degC x 1 hr

None None None None HPLC- 14C 

detection

Mendrala, 1997Dow report DECO HET T2.02-191-000-003A

Rat Blood None (HCl added to 

protonate TCP prior to extraction)

Ethyl acetate Toluene MTBSTFA 

silylating reagent

60 degC x 1hr NCI-GC/MS 

Nolan, 1982Dow report HET K-044793-061

Human volunteer

Blood None (HCl added to 

protonate TCP prior to extraction)

C18-SPE 

cleanup, then benzene extraction

Benzene BSA silylating 

reagent

None listedderivatization assumed to be instantaneous

GC/ECD 

Brzak, 2000 Dow report DECO HET K-044763-284B

Human volunteer (samples from Kisicki 1999 study)

Blood None (HCl added to 

protonate TCP prior to extraction)

Ethyl acetate Toluene MTBSTFA 

silylating reagent

60 degC x 1hr NCI-GC/MS 

Nolan, 1982Dow report HET K-044793-061

Human volunteer

Urine 2:10 (18M 

H2SO4:urine)

90 degC x 1 hr

C18-SPE 

cleanup, then benzene extraction

Benzene BSA silylating 

reagent

None listedderivatization assumed to be instantaneous

GC/ECD 

Summer, 1999 Battelle report N101121A

Human volunteer (samples from Kisicki 1999 

Urine 1:10 (12M HCl:urine)

80 degC x 1 hr Authors also investigated 3hr 

1-chlorobutane 1-chlorobutane MTBSTFA 

silylating reagent

60 degC x 1hr EI-GC/MS 

study) hydrolysis time