### 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 hum an volunteer studies involving chlorpyrifos to the EPA Human Studies Review Board (HSRB) for

scientific and ethical review. Bas ed on discussion at the HSRB meeting, EPA on July 13, 2009 requested additional information regarding conduct of chemical analys es and oral absorption results in two of the studies. Dow AgroSciences (DAS) is providing information in response to this request.

ANALYTICAL PROC EDURES FOR TCP

John Doherty of the HED, in an email inquiry to Dow Chemical of September 16, 2009, posed t he following question: ” The issue is that

the HSRB raised concerns that th e Nolan (~70%) and Kisicki (~30%) studies have such a large recovery difference for TCP indicating

more was absorbed in the Nolan st udy. One of the HSRB panel members challenged the methodolo gy used to detect TCP in blood and urine. Previously, Ken Ra cke addressed the issue in blood indicating that no hydrolysis was necessary since based on previous work with rat blood, nearly all TC P in blood is unconjugated. The assumption that TCP in human bl ood was also unconjugated has been made as a defense for not attemp ting to hydrolyze the blood to free conjugated TCP. This is still an assumption that at least one person here is challenging. The HS RB 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

1glucuronide-conjugated TCP, and apparent discrepancies in the absorption data when compared with the data from Nolan et. al (1982), the Board questioned the re liability 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 analysi s. If the acid hydrolysis step

was the same for both Nolan and Kisicki and it is the established method to free conjugated TCP, t hen we can eliminate that the difference in apparent absorption bet ween Nolan and Kisicki is due to differences in sample preparatio n. Therefore if you can provide

written documentation that there was the appropriate acid step in the preparation of the urine for TCP anal ysis and any other information that will resolve the reliability is sue 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, deriva tization and separation/detection

methods for Nolan 1982 and Kisicki 1999 human volunteer studies. For comparision, the analogous TC P 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, 14 C-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 conjug ates of TCP. The authors showed that a 1 hr hydrolysis of the uri ne, at 80 degrees C, with a 1:10

acidification with 12M HCl was suffic ient to completely hydrolyze the two conjugates of TCP back to t he free TCP metabolite. The TCP

2analytical methods employed for anal ysis 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 t he Nolan 1982 study, urine samples were hydrolyzed with 2:10 (acid:urine) ratio of 18M H 2 SO 4 , at 90 degrees C for 1 hr. In the Kisicki 1999 study, the urine samples were analyzed for total TCP by Dr. St even Summer of Battelle Laboratory (Columbus, Ohio). The acid hydro lysis 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 sufficien t 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 TC P accounted for virtually all of the radioactivity in blood of rats ad ministered either 5 or 100 mg/kg of

14 C-chlorpyrifos. Based on these resu lts, 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 (s ee more complete discussion of this in Dow AgroSciences comments of July 24, 2009 (Racke 2009)).

Some acid was added to the bloo d samples in both human volunteer studies, to protonate the anal yte 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 w ere fairly comparable. Following acidification (with subsequent hyd rolysis 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 sp ecific as the detection method employed in the later studies (GC/MS). However, once in an extraction solvent, the TCP in t he sample extracts was derivatized with a silylating reagent to enhance vo latility of this analyte. The derivatized TCP was then separated on a GC column and quantitated via either ECD or mass spectral det ection. The reference for the

3methods employed in any of the hum an 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 analysi s 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 th e 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, particularl y 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 requesti ng that DOW provide additional support for the assumption that the differences in apparent absorption is related to differences in dosing met hod (pill vs. capsule). It is also suggested that any other explana tions that can explain the large difference in apparent absorptio n 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 stud ies 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.

4In the study of Nolan et al. (1982), the chlorpyrifos test material was dissolved in methylene chloride and th e resulting solution applied to a lactose tablet. Following evaporat ion of methylene chloride carrier, the tablet was taken orally with water by the study volunteers. In contrast, the chlorpyfos test mat erial 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 chlo rpyrifos added as a solution to

lactose would afford dried test ma terial of a substantially smaller particle size and/or larger surf ace area than the corresponding crystalline product. Numerous auth ors have shown that altering either property of drugs that are poorly water soluble in water can substantially enhance o ral absorption in anim als or humans. Garner et al. (2002) showed that micronizi ng diosmin from a mean particle

size of 36.5 μ m to 1.79 μ m 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 nanostruc turing to increase drug surface area via rapid freezing. Elder et al (2006) have shown subtantial increases in systemic bioavailabili ty of danazol following reducing drug particle size via a controlled prec ipitation technique. Finally, Liu (2008) has also discussed the fa ct 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 descr ibes that the oral absorption of the antifungal agent griseofulvin wa s found to increase 2.5 fold when

the surface area was increased abou t six-fold. This increase in absorption is quite consistent with the 2.2-fold increase in absorption seen for chlorpyrifos, when adminst ered as a solution applied to a lactose tablet (then dried) vs. t he crystalline solid (70% from Nolan 1982 vs. 32% from Kisicki 1999).

Based on these examples, and the kn own differences in test material formulation techniques between the Nolan and Kisicki human volunteer studies, it is expected th at 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).

5References

Brzak, K. A. (2000). A rising dose to xicology study to determine the no-observable-effect-levels (NOEL) for erythrocyte acetylcholinesterase (AChE) inhi bition 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 Vo lume II: Polymeric Matrices and Drug Particle Engineering, (Sv enson, 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 ab sorption of micronized (Daflon 500 mg) and nonmicronized 14 C-diosmin tablets after oral administration to healthy volunteers by accelerator mass spectrometry and liquid scint illation 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 acetylcholin esterase (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).

67 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 (Repo rt number ACR-79.9).

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

Concentration-Time course of ch lorpyrifos and chlorpyrifos-oxon in blood. Report of Toxicolo gy & 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 Volunt eers 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 chlorpyrifo s in Fischer 344 rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midl and, 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. R eport 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) inhi bition and cholinergic signs and symptoms of chlorpyrifos at three dose levels. Report of Battelle Testing Laboratory, Columbus, Oh io (Project Number N101121A). [Chemical analysis of urine samples from Kisicki et al., 1999].Table 1. Summary of TCP analytical methodsemployed in human volunteer studies.8HydrolysisExtraction Derivatization Derivatization Derivatization AnalysisStudySpecies Matrix ConditionsSolvent Solvent Reagent Conditions Conditions CommentsNolan, 1987Dow reportHET-K-044793-(76)RatUrine 1:10 (12M HCl:urine)80 degC x 1 hrNoneNoneNone NoneHPLC- 14Cdetection84-92% of dose recovered in urine,as free TCP, glucuronide and sulfateconjugates.hydrolysis for 80 degC x 1hrcompletely cleaved both glucuronideand sulfate conjugatesMendrala, 1997Dow reportDECO HET T2.02-191-000-003ARatBlood None (HCl added toprotonate TCP prior toextraction)Ethyl acetate Toluene MTBSTFAsilylatingreagent60 degC x 1hr NCI-GC/MSFree TCP accounted for virtually all oftest material-derived radioactivityfound in blood(at 5 and 100 mg/kg).Original TCP method: Brzak et al.(1998), J. Anal. Tox., 22: 203-210.Method recovery of TCP from Blood was96-114%.Nolan, 1982Dow reportHET K-044793-061HumanvolunteerBlood None (HCl added toprotonate TCP prior toextraction)C18-SPEcleanup, thenbenzeneextractionBenzene BSA silylatingreagentNone listedderivatizationassumed to beinstantaneousGC/ECDOriginal TCP method: McKellar (1979),Dow report ACR 79.9 B 600-219-79.Method recovery of TCP from Blood andUrine was 86% and 97%, respectively.Brzak, 2000Dow report DECOHET K-044763-284BHumanvolunteer(samples fromKisicki 1999study)Blood None (HCl added toprotonate TCP prior toextraction)Ethyl acetate Toluene MTBSTFAsilylatingreagent60 degC x 1hr NCI-GC/MSOriginal TCP method: Brzak et al.(1998), J. Anal. Tox., 22: 203-210.Method recovery of TCP from Blood was96-114%.Nolan, 1982Dow reportHET K-044793-061HumanvolunteerUrine 2:10 (18MH2SO4:urine)90 degC x 1 hrC18-SPEcleanup, thenbenzeneextractionBenzene BSA silylatingreagentNone listedderivatizationassumed to beinstantaneousGC/ECDOriginal TCP method: McKellar (1979),Dow report ACR 79.9 B 600-219-79.Method recovery of TCP from Blood andUrine was 86% and 97%, respectively.Summer, 1999Battelle reportN101121AHumanvolunteer(samples fromKisicki 1999Urine 1:10 (12M HCl:urine)80 degC x 1 hrAuthors alsoinvestigated 3hr1-chlorobutane 1-chlorobutane MTBSTFAsilylatingreagent60 degC x 1hr EI-GC/MSOriginal TCP method: Olberding (1998),Dow AgroSciences report GRM-97.04.R1(appended to Summer 1999 Battellereport).study)hydrolysis timeMethod recovery of TCP from Urine was99%.Study results show no increase inurine TCP levels with 3hr hydrolysistime vs. 1 hr hyd