Session 3 : Reconsideration of two concerns previously raised by the HSRB in its June 2009 review of an intentional human dosing study with chlorpyrifos (Kisicki et al., 1999); additional pertinent information was made available by the sponsor related to these two concerns.

Background and context:

At its June 2009 meeting, the Human Studies Review Board considered several completed studies involving intentional human exposure to the organophosphate insecticide chlorpyrifos. In its review, the Board raised a number of scientific concerns about one of these studies: Kisicki J,Seip C, Combs M. 1999. A Rising Dose Toxicological Study to Determine the No-ObservableEffect-Levels (NOEL) For Erythrocyte Acetylcholinesterase (AChE) Inhibition and Cholinergic Signs and Symptoms of Chlorpyrifos at Three Dose Levels. Dated April 15-19, 1999. Unpublished study prepared by MDS Harris Laboratories under Project No. 21438 and Dow AgroSciences Study No. DR K-0044793-284. 578 p. MRID 44811002.

Two of the concerns about the Kisicki et al. (1999) study were considered to be major, and raised questions about whether the data arising from the study were scientifically valid. One concern was whether the urine samples were subjected to acid hydrolysis before processing and quantifying the chlorpyrifos metabolite trichloropyridinol. The second concern was why the absorption of chlorpyrifos was so much different compared to an earlier study of chlorpyrifos by Nolan et al. (1982) that was also reviewed at the June 2009 meeting. The relevant section of the Board's final report is appended below, with these two concerns highlighted yellow.

Following the June 2009 meeting, the study sponsor, Dow Agrosciences was asked by EPA to provide a written response to Agency regarding HSRB concerns. This response was not provided to the HSRB.

At the recent EPA Scientific Advisory Panel (SAP) meeting held in February 2011, data on chlorpyrifos from many sources were discussed, including the human intentional dosing data. The final report from the June 2009 HSRB was also provided to the SAP as part of the background materials. When the HSRB's concerns were discussed, Dow Agrosciences provided the SAP with its earlier response; this response was taken into consideration in the subsequent
SAP discussion.

The HSRB may have come to a different conclusion regarding these two issues - the analytical procedures used to analyze the urine samplesfor trichloropyridinol in Kisicki et al. (1999) and the difference in observed absorption between Kisicki et al. (1999) and Nolan et al. (1982) - had this information been provided to the Board prior to completion of the June 2009 final report. If this is the case, the HSRB should request that its conclusions and recommendations about the Kisicki et al. (1999) study be corrected in the public record.

The original HSRB report and Dow Agrosciences' response to the Agency are thus being provided to the HSRB to determine whether a correction to the public record is warranted.Charge questions:

1. Do the recommendations provided by the HSRB at its June 2009 meeting and in its subsequent report regarding the reliability of data on the urinary metabolite trichloropyridinol in the Kisicki et al. (1999) study change in light of the new information provided?

2. Do the recommendations provided by the HSRB at its June 2009 meeting and in its subsequent report regarding the level of absorption of chlorpyrifos in the Kisicki et al. (1999) study change in light of the new information provided?Extract from the Final Report of the June 2009 HSRB Meeting re: Kisicki et al. (1999)

Assessment of Completed Research Study MRID 44811002: Kisickiet al. (1999) A Rising Dose Toxicology Study to Determine the No-Observable Effect-Levels (NOEL) For Erythrocytes Acetylcholinesterase (AChE) Inhibition and Cholinergic Signs and Symptoms of Chlorpyrifos at Three Dose Levels.

Overview of the Study

This study was a double blind, randomized and placebo-controlled rising dose study designed to determine the no-observable effect level (NOEL) for inhibition of erythrocyte acetylcholinesterase in human volunteers. The dosages used in the study were 0.5, 1 and 2 mg/ kg, with the starting dose of 0.5 mg/kg based on the results of Nolan et al . (1982) discussed previously. In addition to assessing cholinesterase activity, blood and urine were collected and analyzed for chlorpyrifos and TCP to help define the pharmacokinetic properties in humans. Finally, paraoxonase status of each subject was determined (but will not be discussed here).

Chlorpyrifos was administered orally in a gelatin capsule with 0 (lactose), 0.5 and 1 mg chlorpyrifos/kg administered in Phase I of the study followed by a second phase that include 0 (control) and 2 mg/kg dosages. There were six male and six female participants for each dose level. Subjects were confined to the testing facility overnight prior to treatment through the first 48 hr after dosing. Additional samples were collected thereafter at 24 hr intervals through 168 hr (one week) post dosing. Blood, collected pre-dose (-10 and 0 hr) and at 2, 4, 8, 24, 36, 48, 72, 96, 120, 144 and 168 hr post treatment, was used for determining erythrocyte acetylcholinesterase activity and chlorpyrifos and TCP levels. Voided urine was collected at 12 hr intervals starting 48 hr prior to dosing and through the 168 hr dosing period (with the exception that immediately after dosing urine was collected at 0-6 and 6-12 hr).

Science

Charge to the Board

Are the blood and urine measurements of chlorpyrifos and/or TCP from the Kisicki et al. (1999) study reliable and appropriate for use in characterizing the results of epidemiological studies with chlorpyrifos?

Board Response to the Charge

HSRB Recommendation

Because of concerns about the analytic procedures' potential inability to control for the glucuronide-conjugated TCP, and apparent discrepancies in the absorption data when comparedwith the data from Nolan et al . (1982), the Board questioned the reliability and utility of the and urine measurements of chlorpyrifos and/or TCP from Kisicki et al. (1999) risk assessment

purposes.

HSRB Detailed Recommendations and Rationale

With respect to the general elements considered in the evaluation of the scientific reliability of the chlorpyrifos exposure data, this study provided clear documentation of the administered dose. Chlorpyrifos was added to a gelatin capsule normalized with lactose powder and the weight of the filled capsule was determined. There was one noted discrepancy that one participants in the 2 mg/kg dose group may have received a lower dose (1.63 mg/kg) based on conflicting data on body weight and administered dose data, but overall the documentation of the administered dose was satisfactory.

There was also generally good reliability with respect to sample collections. Participants remained in the testing facility for the first 48 hrs after dosing and 48 of the 60 subjects provided all periodic urine samples. Urine collections were judged to be complete based on creatinine excretion for 19 of the 60 subjects. One participant in the 2-mg/kg-dose group failed to return for sample collection and clinical monitoring after the initial 48 hr post-dosing period.

Chlorpyrifos and TCP were measured in blood and urine with GC/MS detection, and TCP was derivatized with N-methyl-(N-tertbutyldimethylsilyl)-trifloroacetamide. In general, these methods were similar to those described by Honeycutt and DeGeare with the exception that stable labels of chlorpyrifos and TCP (13C and/or 15N-labeled) were used as internal standards in the present analyses.

In reviewing this study, the HSRB identified several limitations or issues that may impact the overall reliability of the data. These included:

1. The amount of chlorpyrifos absorbed after oral administration in the present study was markedly lower than that reported by Nolan et al . (1982). A dose of 0.5 mg/kg was common to both studies, with Nolan et al. reporting approximately 70%, and
Kisicki et al. reporting approximately 35% absorbed. The rationale provided for this large difference was that chlorpyrifos was administered using lactose tablets and gelatin capsules in the Nolan et al. and Kisicki et al. studies, respectively. Kisicki et al. suggested that oral absorption is slowed by the dissolution of the gelatin capsule with a concurrent reduction in the total amount absorbed. Although the Board agreed that absorption of chlorpyrifos from the gelatin capsule might be slower than that from a lactose tablet, there was some skepticism that the difference in dosage form would yield the large discrepancy in total percent absorbed in the two studies.

2. In the methodological details provided for the analysis of urinary levels of TCP, there was no indication that urine samples were subjected to acid hydrolysis required toliberate the glucuronide conjugate of TCP, which is the major urinary metabolite of chlorpyrifos. This is an important distinction, as both Nolan et al. (1982) and Honeycutt and DeGeare (1993) indicated that urine samples were treated with concentrated sulfuric or hydrochloric acid and heated in order to hydrolyze the glucuronide conjugate and liberate TCP which is then derivatized (in the methods used by Honeycutt and DeGeare and Kisicki et al. ). In the analytical portion of the
Kisicki et al. (Brzak 2000), there is a clear designation that blood samples were treated with concentrated hydrochloric acid prior to extraction, but no similar details are provided for the analysis of the urine samples. Although it seems unlikely that this important (and necessary) step was omitted, the lack of explicit indication of this critical analytical step raises some doubt that it was done. If urine samples were not properly hydrolyzed prior to analysis, then the validity and reliability of these data are uncertain. Furthermore, since urinary TCP is used to estimate oral absorption of chlorpyrifos, it is possible that differences in sample handling may explain or at least contribute to the lower percent absorption reported by Kisicki et al

.

3. The total mass balance (total recovery of the administered chlorpyrifos) was not determined. Inclusion of total recovery could increase the confidence in the estimated percent of absorption in the study and possibly clarify whether there was an issue with the TCP analysis conducted by Kisicki et al

.

4. There were several questions regarding the designation and use of alternate participants in the study. The basis for participant replacement and verification of body weight and dose administered to the alternate participants were not adequately provided.

Because of concerns about the analytic procedures' potential inability to control for the glucuronide-conjugated TCP, and apparent discrepancies in the absorption data when compared
with the data from Nolan et al . (1982), the Board thus questioned the reliability and utility of the and urine measurements of chlorpyrifos and/or TCP from Kisicki et al. (1999) risk assessment

purposes.

Charge to the Board

Are the measurements of cholinesterase activity/inhibition from the Kisicki et al. (1999) study reliable?

Board Response to the Charge

HSRB Recommendation

The Board concluded that these measurements likely represent a reliable set of data, but cautioned the Agency about relying on data from the incomplete profile for the one responder atthe highest dose level. The Board also cautioned against relying on the statistical analyses as presented in the report.

HSRB Detailed Recommendations and Rationale

In this study, erythrocyte acetylcholinesterase activities were determined pre-dose (2 samples) and 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144 and 168 after dosing (Ellman method). Plasma cholinesterase activity was not measured. The results showed a relatively high degree of variability, and samples collected at 96 hr (Phase I; 0, 0.5 and 1 mg/kg) were judged to be low relative to all other treatment times. One participant in the 1-mg/kg dose group showed a 24.5% decrease in activity at the 96 hr time point. However, given the decreased activity observed in the entire group of samples collected at 96 hr along with the fact that peak inhibition would be expected to occur before 96 hr, this result was not considered to be a real finding. Only one participant in the 2-mg/kg-dose group showed a reduction in erythrocyte cholinesterase activity in the study, with a nadir (72% of her baseline level) observed 12 hr after treatment. However, this volunteer did not return for blood collections after the initial 48 hr period, resulting in an incomplete profile. This participant did not appear to show adverse clinical signs associated with cholinesterase inhibition. Based on the reduction in erythrocyte cholinesterase activity in this subject, the NOEL for enzyme inhibition was considered to be 1 mg/kg.

In reviewing this study, the Board identified several limitations or issues that may impact the overall reliability of the data. These included:

1. There was generally poor documentation provided for the grouping or batch processing of the blood samples. Given the recognized inter-day and inter-assay variability associated with the Ellman method, this is a critical weakness. The study records were noted to be insufficient to confirm that samples were batched correctly to allow the proper utilization of the unexposed control data to correct for day-to-day or batch-to-batch variations in the mean lab results. This limitation is particularly important to detect or to quantify accurately small changes within an exposed population.

2. The interpretation of the results is centered on the single individual who showed more than a 25% inhibition of erythrocyte cholinesterase activity at the 2-mg/kg-dose level. The conclusions regarding both the duration and magnitude of inhibition depend upon the inclusion of this participant, and the lack of follow-up in this participant beyond 48 hr thereby limits the interpretation of the results.

3. The reported statistical analyses of these data are highly problematic, and the results in Appendix 3 were judged to be clearly wrong. Specifically, it was noted that a univariate repeated measures ANOVA was performed (Kisicki et al . 1999, 25), and Appendix 3 summarizes the results of such analyses (Kisicki et al . 1999, 151-158, 184-188, 206-211). Although it is not possible to replicate these analyses, the reportsuggests that the variable being analyzed is the Normalized Percent of Baseline (Kisicki et al . 1999, 24). The report indicates that the Group Mean Square (Kisicki et al . 1999, 157) is 19.1207 while the Error Mean Square is 52,835,395,914.644. It is virtually impossible that these two values can be this different for real data. Furthermore, the corresponding Individual Hour Error Mean Squares from the corresponding mixed model analysis range from about 14.39 to 48.26. These are much more reasonable values. The report (Kisicki et al . 1999, 25) also indicates that mixed effects models were used where gender and treatment were considered to be fixed factors, and that time was considered to be a random continuous covariate. According to the mixed model analyses reported in Appendix 3, time is not being considered a random continuous covariate. Time is treated as a continuous covariate, but not as a random continuous covariate. Overall, the Board concluded that the statistical analyses are likely incorrect, and there should be some effort to perform correct analyses before relying on these data.

4. Plasma cholinesterase activity was not determined in the study. It is recognized that the erythrocyte cholinesterase activity is more biologically relevant for assessing potential adverse effect of chlorpyrifos, and as such, the lack of data on plasma cholinesterase is not a serious limitation. However, given the issues raised concerning sample handling, batch processing and statistical evaluations, inclusion of plasma cholinesterase may have helped to assess the overall reliability of the data.

The Board thus concluded that the measurements of erythrocyte acetylcholinesterase activities were generally reliable. However, Board members cautioned that the incomplete profile obtained from the only subject who showed inhibition at the 2 m/kg dose level limited the utility of the data. Moreover, the Board recommended that, although the data might be reliable, the statistical analyses in general and particularly for the data in Appendix 3 should be replicated prior to applying these results to any risk assessment or model development.