1 of 69 June 26, 2006 EPA HSRB 06 01 George Gray, Ph. D. Science Advisor Office of the Science Advisor 1200 Pennsylvania Avenue, NW Washington, DC 20460 Subject: April 4 6, 2006 EPA Human Studies Review Board Meeting Report Dear Dr. Gray:

The United States Environmental Protection Agency ( EPA or Agency) requested the Human Studies Review Board ( HSRB) to provide advice on Agency scientific and ethics reviews of completed human studies concerning the following pesticide active ingredients: aldicarb, amitraz, azinphos methyl, dichlorovos ( DDVP), ethephon, methomyl, oxamyl, and sodium cyanide. The studies reviewed included both studies on which the Agency proposed to rely in actions under the pesticide laws and studies that the Agency had decided not to use in its risk assessments, either for ethical or scientific reasons. The enclosed HSRB report addresses the Board's response to EPA charge questions for the Board's consideration at its April 4 6, 2006 meeting.

The HSRB was extremely impressed with the high quality scientific and ethical review brought before the Board. A summary of the Board's conclusions on the scientific and ethical considerations of the human toxicity studies for the eight pesticides under review are provided below.

Aldicarb Scientific considerations   The cholinesterase data from the aldicarb human study were reliable for use in the aldicarb single chemical, aggregate risk assessment.

  The cholinesterase data from the aldicarb human toxicity study were reliable for use in the cumulative risk assessment for N methyl carbamates. 2 of 69 Ethical considerations   The aldicarb human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted), nor that information provided to participants seriously impaired their informed consent.

Methomyl Scientific Considerations   The methomyl human study could be appropriately applied to the inter species risk factor for methomyl and for use in the cumulative risk assessment of N methyl carbamates.

Ethical Considerations   The methomyl human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted), nor that information provided to participants seriously impaired their informed consent.

Oxamyl Scientific Considerations   Although the Board had some questions about the Agency's conclusions regarding lack of sex difference and of the difference between brain and RBC ChE based on only one species, the Board supported the Agency's conclusion that there were no study deficiencies identified that would have affected the outcome or conclusions of this study.

  Considering the high quality of the design and the conduct of the study, the Board agreed that this intentional human dosing study of oxamyl was sufficiently robust to be used for reducing the 10x inter species ( i. e. animal to human) uncertainty factor in the cumulative risk assessment for the N methyl carbamates. 3 of 69 Ethical Considerations   The oxamyl human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear or convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

Azinphos Methyl Scientific Considerations   Data from the 28 day repeat oral dose study of azinphos methyl should not be used in developing a point of departure for extrapolation of risk to workers exposed to azinphosmethyl via the dermal and inhalation routes.

  Data from the 28 day repeat oral dose study of azinphos methyl cannot be used to inform the inter species factor in the cumulative risk assessment.

Ethical Considerations   The AZM human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

DDVP Scientific Considerations   The DDVP repeat dose human toxicity study was sufficiently robust for developing a point of departure for estimating dermal, incidental oral, and inhalation risk from exposure to DDVP in the single chemical risk assessment.

  The DDVP repeat dose human toxicity study should not be used to support reducing the default 10X inter species factor in the cumulative risk assessment of the organophosphate pesticides. 4 of 69   The HSRB concluded that the other DDVP human toxicity studies available for the Board's consideration should not be used for determining a reduction in the 10X uncertainty factor to derive reference dose values for DDVP based on animal toxicity endpoints.

Ethical Considerations   The DDVP repeat dose oral human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

Ethephon Scientific Considerations   The Board concluded that the scientific quality of either the ethephon 28 day oral toxicity study or the ethephon 16 day human oral toxicity study was not adequate on its own. The 16 day study can be used to inform the ethephon 28 day human oral toxicity study. The Board approved the 28 day study for use in EPA risk assessments, emphasizing that the dose level administered is almost certainly not the lowest dose at which adverse effects are likely to be observed. However, its use in lieu of the animal studies will result in greater protection for exposed human populations.

Ethical Considerations   Both the 28 and 16 day oral human toxicity studies failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted), nor that information provided to participants seriously impaired their informed consent.

Sodium Cyanide Scientific Considerations 5 of 69   Data from the amygdalin trial could be used for establishing a point of departure in the acute dietary risk assessment for sodium cyanide.

Ethical Considerations   The sodium cyanide human oral toxicity study appeared to meet the specific ethical standards prevalent at the time the research was conducted   There was no evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained   There was no evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

Amitraz Scientific Considerations   The results from the single oral dose study were informed by the human metabolism study such that the single oral dose study was appropriate for developing a point of departure for acute dietary risk.

  The combined results from the single oral dose study and the human metabolism study were not appropriate for developing a point of departure for chronic dietary risk, shortterm oral exposure, or inhalation risk.

  The majority of the Board concluded that the human dermal study was not appropriate for developing a point of departure for dermal exposures of various durations.

Ethical Considerations   The amitraz acute oral and dermal human toxicity studies failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however   There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and   There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted), nor that information provided to participants seriously impaired their informed consent.

In conclusion, the EPA HSRB appreciated the opportunity to advise the Agency on the scientific and ethical aspects of human subjects and looks forward to future opportunities to continue advising the Agency in this endeavor. 6 of 69 Sincerely,

Celia Fisher, Ph. D. Chair EPA Human Studies Review Board 7 of 69 NOTICE This report has been written as part of the activities of the EPA Human Studies Review Board, a Federal advisory committee providing advice, information and recommendations on issues related to scientific and ethical aspects of human subjects research. This report has not been reviewed for approval by the Agency and, hence, the contents of this report do not necessarily represent the view and policies of the Environmental Protection Agency, nor of other agencies in the Executive Branch of the Federal government, nor does mention of trade names or commercial product constitute a recommendation for use. Further information about the EPA Human Studies Review Board can be obtained from its website at http:// www. epa. gov/ osa/ hsrb/. Interested persons are invited to contact Paul Lewis, Designated Federal Officer, via e mail at lewis. paul@ epa. gov.

In preparing this document, the Board carefully considered all information provided and presented by the Agency presenters, as well as information presented by public commenters. This document addresses the information provided and presented within the structure of the charge by the Agency. 8 of 69 United States Environmental Protection Agency Human Studies Review Board Chair Celia B. Fisher, Ph. D. Marie Ward Doty Professor of Psychology, Director, Center for Ethics Education, Fordham University, Department of Psychology, Bronx, NY Vice Chair William S. Brimijoin, Ph. D., Chair and Professor, Molecular Pharmacology and Experimental Therapeutics, Mayo Foundation, Rochester, MN Members David C. Bellinger, Ph. D., Professor of Neurology, Harvard Medical School Professor in the Department of Environmental Health, Harvard School of Public Health Children's Hospital, Boston, MA Alicia Carriquiry, Ph. D., Professor, Department of Statistics, Iowa State University Snedecor Hall, Ames, IA *

Gary L. Chadwick, PharmD, MPH, CIP, Associate Provost, Director, Office for Human Subjects Protection, University of Rochester, Rochester, NY Janice Chambers, Ph. D., D. A. B. T., William L. Giles Distinguished Professor, Director, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Wise Center, Mississippi State, MS Richard Fenske, Ph. D., MPH, Professor, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle WA Susan S. Fish, PharmD, MPH, Professor, Biostatistics & Epidemiology, Boston University School of Public Health, Co Director, MA in Clinical Investigation Boston University School of Medicine, Boston, MA Suzanne C. Fitzpatrick, Ph. D., DABT, Senior Science Policy Analyst, Office of the Commissioner, Office of Science and Health Coordination, U. S. Food and Drug Administration, Rockville, MD Kannan Krishnan, Ph. D., Professor, Département de santé environnementale et santé au travail, Faculté de medicine, Université de Montréal, Montréal, Canada KyungMann Kim, Ph. D., CCRP, Professor & Associate Chair, Department of Biostatistics & Medical Informatics, School of Medicine and Public Health, University of Wisconsin Madison, Madison, WI 9 of 69 Michael D. Lebowitz, Ph. D., FCCP, Professor of Public Health & Medicine. University of Arizona, Tucson, AZ Lois D. Lehman Mckeeman, Ph. D., Distinguished Research Fellow, Discovery Toxicology, Bristol Myers Squibb Company, Princeton, NJ Jerry A. Menikoff, M. D., Associate Professor of Law, Ethics & Medicine, Director of the Institute for Bioethics, Law and Public Policy, University of Kansas Medical Center, Kansas City, KS Robert Nelson, M. D., Ph. D., Associate Professor of Anesthesiology and Critical Care, Department of Anesthesiology and Critical Care, University of Pennsylvania School of Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104 Sean M. Philpott, Ph. D., Research Scientist, David Axelrod Institute, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY Human Studies Review Board Staff Paul I. Lewis, Ph. D., Designated Federal Officer, United States Environmental Protection Agency, Washington, DC * Not in attendance at April 4 6, 2006 Public Meeting 10 of 69 TABLE OF CONTENTS INTRODUCTION.............................................................................................................................................. 11 REVIEW PROCESS.......................................................................................................................................... 12 CHARGE TO THE BOARD AND BOARD RESPONSE................................................................................. 13 1. ALDICARB.................................................................................................................................................... 13 2. METHOMYL ................................................................................................................................................. 19 3. OXAMYL ...................................................................................................................................................... 25 4. AZINPHOS METHYL...................................................................................................................................... 32 5. DDVP......................................................................................................................................................... 38 6. ETHEPHON ................................................................................................................................................... 47 7. HYDROGEN CYANIDE................................................................................................................................... 54 8. AMITRAZ...................................................................................................................................................... 58 11 of 69 INTRODUCTION On February 6, 2006 ( 71 Federal Register 6137), EPA published its final rule for protection of subjects in human research. This new rule included creating an independent Human Studies Review Board ( HSRB) to provide advice, information and recommendations related to the scientific and ethical issues of such research.

The Food Quality Protection Act ( 1996) mandated that the Agency reassess pesticide tolerances ( i. e., the legal limit of pesticides in food) by August, 2006. The Agency is in the final stages of reassessing all tolerances and has elected for the inaugural April 4 6, 2006 HSRB meeting to focus on those pesticides which are subject to the August, 2006 statutory deadline for tolerance reassessment and which have a human toxicity study being considered for incorporation in quantitative human health risk assessment. The HSRB reviewed toxicity studies involving intentional exposure of human subjects to eight pesticide active ingredients: aldicarb, amitraz, azinphos methyl ( AZM), ethephon, dichlorvos ( DDVP), methomyl, oxamyl, and sodium cyanide. Three of these pesticides are members of the N methyl carbamate ( NMC) common mechanism group ( aldicarb, methomyl, oxamyl); two are members of the organophosphorus pesticides ( OP) common mechanism group ( AZM, DDVP). The three remaining pesticides ( amitraz, ethephon, sodium cyanide) each have toxicity profiles that differ from the other chemicals.

The Agency prepared a variety of documents which provided background information. These included the proposed and final Human Studies Rule, report from the National Academy of Sciences regarding human studies, EPA, Office of Pesticide Programs' ( OPP's) policy regarding the use of cholinesterase data in human health risk assessments, and a slide presentation providing a broad overview of the OPP's human health risk assessments. The Agency used the framework provided in Emanuel ( 2000) as the basis for its ethics reviews. The Agency described the approach for performing scientific review of human studies and for incorporating human toxicity data into risk assessment.

For each of the human studies under consideration, the Agency provided the complete study report as submitted to the Agency. For each chemical, the Agency developed a review of the ethical conduct of the study. Each ethics review identified any deficiencies noted in the conduct of the specific study compared to both current ethical standards and the ethical standards prevailing at the time the research was performed. The Agency intentionally deferred making a final determination of whether an individual study satisfied the ethical standards for acceptability in 40 CFR sections 26.1704   26.1706, pending the advice of the Board.

For most studies, the Agency develops documents, called Data Evaluation Records ( DERs), containing a scientific review of the study. DERs contain summaries of the study design, methods and results, describe potential deficiencies, and provide conclusions about the usefulness of the study in risk assessment. In addition to the DERs, the Agency prepared for each chemical a " Weight of Evidence" ( WOE) memorandum that discusses the differences and similarities between the human and animal toxic responses to each chemical and characterizes the usefulness of the human toxicity studies for human health risk assessment. The WOE memos 12 of 69 express the Agency's most current scientific conclusions on which the Agency solicited comments from the Board.

The HSRB reviewed both studies on which the Agency proposes to rely on actions under the pesticide laws and studies that the Agency has decided not to use in its risk assessments, either for scientific reasons or because they do not meet the standards in EPA's final human studies rule. The Agency asked the HSRB to advise the Agency on a range of scientific and ethics issues regarding how the studies should be assessed against the provisions in 40 CFR sections 26.1701   26.1704 of EPA's final human studies rule. The Board anticipates having a series of meetings to review pesticide human studies relevant to pending re registration, tolerance reassessment and new registration decisions, in accordance with the provisions of the final rule. This report transmits the HSRB's comments and recommendations from its inaugural April 4 6, 2006 meeting.

REVIEW PROCESS On April 4 6, 2006 the Board had a public face to face meeting in Arlington, Virginia. Advance notice of the meeting was published in the Federal Register " Human Studies Review Board: Notice of Public Meeting and Proposed Candidates for Membership to the Board ( 71 FR 46, 12194). At the public meeting, following welcoming remarks from Agency officials, Celia B. Fisher, HRSB Chair, proposed a set of scientific and ethics criteria consistent with the language of 71 Federal Register 24, 6137 to guide Board evaluation of each protocol. The Chair's scientific criteria asked the Board to consider the following two questions: ( 1) Did the research design and implementation meet scientific standards and ( 2) Did the data generated by the protocol have implications for the Agency's Weight of the Evidence ( WOE) review and when applicable aspects of the risk assessment? The Chair's ethics criteria asked the Board to consider three questions: ( 1) Did the study fail to fully meet specific ethical standards prevalent at the time the research was conducted; ( 2) Was the conduct of the study fundamentally unethical ( i. e., specifically was there clear and convincing evidence that the research was intended to seriously harm participants or failed to obtain informed consent); and ( 3) Was the conduct of the study significantly deficient relative to the ethical standards prevailing at the time ( i. e., was there clear and convincing evidence that identified deficiencies could have resulted in serious harm based on knowledge available at the time the study was conducted or the information provided to participants could seriously impair informed consent). The Board then heard presentations from EPA on the following topics: ( 1) introduction ( summary of EPA protections for subjects of human research, science and ethics considerations of human subjects research); ( 2) carbamate pesticides ( science and ethics of aldicarb, methomyl and oxamyl human studies); ( 3) organophosphate pesticides ( science and ethics of azinphos methyl and DDVP human studies); ( 4) other pesticides ( science and ethics of ethephon, sodium cyanide and amitraz human studies). The Board also heard oral public comments from the following individuals:

Carbamate pesticides Mr. Angus Cameron representing BCG Europe Neil Carmichael, Ph. D. representing Bayer Crop Science Jennifer Sass, Ph. D. representing Natural Resources Defense Council 13 of 69 Organophosphate Pesticides Monty Eberhart, Ph. D. and Mr. Dan Van Geothen representing Bayer Crop Science and Mahktesheim Agan Robert Levine, Ph. D. representing Amvac Mr. Ian Chart representing Amvac Laura Plunkett, Ph. D. representing Amvac Thomas Starr, Ph. D. representing Amvac Jennifer Sass, Ph. D. representing Natural Resources Defense Council Ms. Shelley Davis representing the Farmworker Justice Fund Other Pesticides Neil Carmichael, Ph. D. representing Bayer Crop Science Following Agency presentations and public comments, the Board deliberated on the charge questions. For their deliberations, the Board considered the materials presented at the meeting, written public comments and Agency background documents on each individual pesticide ( i. e., pesticide human study, Agency data evaluation record ( DER) of the pesticide human study, weight of evidence review, risk assessment and ethics review).

CHARGE TO THE BOARD AND BOARD RESPONSE 1. Aldicarb Charge to the Board Aldicarb is a N methyl carbamate ( NMC) pesticide whose primary toxic effect is neurotoxicity caused by the inhibition of the enzyme acetylcholinesterase, via carbamylation followed by rapid recovery. Aldicarb can, at sufficiently high doses, lead to a variety of clinical signs. The Agency is conducting an acute, aggregate ( single chemical, multi route) risk assessment of aldicarb. In addition, aldicarb is a member of the N methyl carbamate common mechanism group and is thus included in the cumulative ( multi chemical, multi route) risk assessment for the NMCs.

1.1 Scientific considerations The Agency's " Weight of the Evidence" ( WOE) document and Data Evaluation Records ( DERs) for aldicarb described the study design and results of the aldicarb acute oral, human toxicity study. The WOE document also discussed the Agency's conclusions regarding the usefulness of the human study in the acute, aggregate, single chemical risk assessment and in the cumulative risk assessment for the NMCs. Regarding the aldicarb human study, the Agency concluded that the study was sufficiently robust for reducing the inter species ( i. e., animal to human) uncertainty factor in the aggregate and the cumulative risk assessments. 14 of 69 The Board was asked to comment on the scientific evidence that supports whether the aldicarb human study was sufficiently robust for reducing the inter species ( i. e., animal to human) uncertainty factor in: a. single chemical, aggregate risk assessment and b. cumulative risk assessment.

Board Response to the Charge Brief Overview of the Study The study ( Wyld et al. 1992) consisted of a double blind, placebo controlled, single oral dose of aldicarb in orange juice, taken during the course of a meal. A variety of both subjective and objective observations were accumulated, including red blood cell ( RBC) cholinesterase activities, blood pressure, and respiratory parameters among others. Several dose levels were investigated and several time points were also investigated.

Critique of Study The strengths of the study were: it was designed with multiple doses so a dose response relationship could be studied; it was double blind; a large number of parameters that are relevant to anticholinesterase were studied ( including both subjective data such as headache and 1several objective physiological measures such as blood pressure, pulse, pulmonary function and saliva production); there were exclusion criteria; there were frequent measurements of cholinesterase over a 1 day period in addition to pre treatment measurements; the observations of clinical signs were made by trained observers; both sexes were studied ( although there were fewer doses and individuals in the female group); and the cholinesterase depression was both dose and timedependent in both males and females.

The weaknesses of the study were: there were typically only 4 8 individuals per dose; there were fewer female subjects and they were only tested at the middle dose levels; there were a large number of reported signs across all groups, including the placebo, making interpretation difficult; the cholinesterase methodology may have not assayed all of the cholinesterase in RBCs; and the first time point of cholinesterase measurement may have been after the time of peak cholinesterase inhibition.

The Board also noted that, with respect to statistical analysis, the study was likely to be under powered. With the large number of endpoints and the relatively large between subject variances in almost all endpoints, the number of subjects was probably insufficient to guarantee adequate statistical power. In addition, no adjustments were made for multiple comparisons. Since each endpoint was analyzed separately and because many comparisons were carried out across genders, time points, doses, etc., the overall confidence level is probably much below the nominal 95%. It was also noted that the univariate statistical models that were fitted to the data account for the repeated measures structure of the design, but do not account for the multivariate structure in the measurements. Because endpoints were measured on the same subjects, one could expect meaningful correlations between the various endpoints that could contain additional information about the treatment effects and the effects of covariates. Consequences of not using 15 of 69 multivariate methods on this data set include a decrease in the power of statistical tests and erosion of confidence levels that occur when many models are fitted to many endpoints independently as was done in this study. These considerations throw some doubt on some of the study results that suggest no statistically significant treatment effects.

Nevertheless, while it is clear that the statistical power of the study was low, the data do show a very clear and predictable dose and time dependency in the RBC cholinesterase data. The response of the cholinesterase data is what would be expected of a transient carbamate anticholinesterase. Additionally, it would be expected that blood cholinesterase would be the most sensitive endpoint. RBC cholinesterase is well recognized as a sensitive biomarker of exposure to anticholinesterases. Its inhibition is not responsible for clinical signs of anticholinesterase toxicity, so no cause and effect relationship should be expected between RBC cholinesterase inhibition and changes in any of the physiological parameters measured. Therefore, it is also reasonable that inhibition of the RBC cholinesterase would occur whereas the other parameters related to clinical signs, such as salivation or blood pressure, would not. Lastly, the occurrence of numerous clinical signs in the placebo group gives credence to the conclusion that the RBC cholinesterase inhibition was a treatment related effect whereas clinical signs were not.

The Board concluded that the results of the study could be used in the WOE analysis to determine a NOEL for RBC cholinesterase and clinical signs in males and that the RBC cholinesterase demonstrated a dose dependent and time dependent pattern of inhibition in both males and females.

HSRB Consensus and Rationale The Board concluded that the cholinesterase data from the aldicarb human study were reliable for use in the aldicarb single chemical, aggregate risk assessment.

The Agency Data Evaluation Report for Aldicarb ( Sette 1992) suggested that the NOAEL and LOAEL were based upon sweating in males. While sweating is a possible clinical sign resulting from cholinesterase inhibition, the responses in the subjects were not consistently doserelated Although sweating is an objective endpoint, the WOE document indicates that the RBC cholinesterase inhibition would be the more appropriate objective endpoint. There are additional reasons why there is more confidence in the cholinesterase data. First, the blood cholinesterase data, despite their weaknesses, are the most consistent responses to aldicarb exposure, because they follow time and dose dependent patterns, as would be expected of a transient anticholinesterase. Second, the cholinesterase data, though probably based on incomplete fractions of the entire RBC cholinesterase population due to the nature of the analysis method, would still be expected to be internally consistent within this study between samples of treated and placebo individuals, and therefore the inhibition levels can probably be believed. There was a dose and time to recovery related relationship to cholinesterase inhibition in both sexes, so the cholinesterase data are also consistent between the sexes.

The Board concluded that the cholinesterase data from the aldicarb human toxicity study were reliable for use in the cumulative risk assessment for N methyl carbamates. Aldicarb, 16 of 69 being an N methyl carbamate, belongs to this common mechanism group which acts via acetylcholinesterase inhibition. Therefore, the endpoint in the cumulative risk assessment must be cholinesterase inhibition. Moreover, the dose response data from the human study appeared such that BMD and BMDL can be calculated. The HSRB concluded that the aldicarb human study appears to be a scientifically valid study, suitable for use in both the aggregate risk assessment and the cumulative risk assessment.

While the Board concluded that the cholinesterase data could be reliably used in the cumulative risk assessment, it did note some limitations. The animal data were not supplied so the HSRB cannot address the accuracy of the corresponding animal data. Because of the rapid reactivation of carbamylated cholinesterase, it is unclear whether accurate cholinesterase inhibition values were obtained in the human study or that peak inhibition was measured because the earliest time point measured was 1 hour post dose. The WOE document stated that the human and rat cholinesterase inhibition were comparable at the 0.05 mg/ kg dose level; a question arises as to whether this comparison was made at the same point in the inhibition/ recovery patterns of both species. However, it should be possible to make extrapolations of the possible peak from animal data on the time course pattern of cholinesterase inhibition and recovery, and the Agency is urged to make certain that BMD and BMDL comparisons between animals and humans are being made on comparable times in the inhibition/ recovery pattern.

1.2. Ethical considerations Charge to the Board a. The Agency requests that the Board provide comment on the following:

In light of the ethics committee's instruction that the lay summary be " greatly expanded," and the fact that the materials used to obtain informed consent listed a limited range of symptoms of carbamate toxicity ( excluding some reported as adverse effects in the study), included multiple references to the test material as a drug, and failed to identify dose levels to be administered to male subjects, whether, the materials used to obtain informed consent should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted Whether the absence from the protocol of discussion of the potential risks to subjects or benefits to society of conducting the proposed research ( as required by the 1989 Declaration of Helsinki, Principle # 4, with which the research asserted compliance) should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted;

b. The Agency asks that the Board provide comment on the following, taking into account all that is known about the ethical conduct of this study:

OPP's conclusion that there was not clear and convincing evidence that the conduct of the research was fundamentally unethical. 17 of 69 Whether there is clear and convincing evidence that the conduct of the study was significantly deficient relative to the ethical standards prevailing when the study was conducted.

Board Response to the Charge Study Overview The study was completed on March 11, 1992. The study sponsor was located in France and the performing laboratory was located in Scotland. The protocol documents specifically stated that the research was conducted in compliance with the Principles of Good Clinical Practice as promulgated by the EMEA CPMP in May 1990, and the Declaration of Helsinki ( in either the 1983 or 1989 version, as cited in two different portions of the study documents). Although the May 1990 document on good clinical practice was the precursor for the subsequent 1997 document promulgated by the International Conference on Harmonization, the earlier document did not include any specification of the contents of the informed consent form. However, the supporting and supplementary study documents claimed that the research ethics committee provided independent review and oversight of the research functioned according to the standards of the FDA regulations found in 21 CFR 56. These regulations cross reference the informed consent requirements found in 21 CFR 50 Both FDA regulations were effective on January 27, 1981. As such, the research ethics committee should have applied the FDA regulations from nearly a decade before the conduct of this particular research protocol. It should be noted that the sponsor subsequently claimed that the study also was performed according to the requirements of 40 CFR 26, based on a retrospective analysis published in 2003.

Critique of Study The Board concurred with the Agency on the factual observations of the strengths and weaknesses of the study, as detailed in Carley ( 2006a). However, further comments are warranted on ( 1) the documentation and process of informed consent, and ( 2) the minimization of risk with respect to the study design.

It is clear that the written documentation for informed consent is not up to the standards found in the above regulations. The Volunteer Information that was given to research subjects as an appendix to the signed informed consent document did not have an adequate discussion of the risks of the research. At the request of the research ethics committee ( dated December 24, 1991), the Volunteer Information included the possibility that administration of the test product could result in " gut motility effects ( abdominal pains), effect on eye pupil size and muscle weakness"). In addition to the concerns that the risk information was insufficient in the written consent documentation, selected subjects were enrolled a second time. This was not discussed or in the protocol or the informed consent documentation and therefore raises doubts to the Board about the adequacy of the consent process. In effect, the re dosing of selected subjects suggests that the investigator was unblinded to group assignment, reducing the supposedly double blind study to a single blind study and raising doubts ( unanswered by the documentation) about the sufficiency of informed consent. In particular, the re use of selected subjects suggests that some study participants were randomized to receive placebo versus active compound while other 18 of 69 subjects were not. As a result, the burden of risks was unlikely to have been distributed evenly among all study participants.

There were several observations that raised concerns about the extent to which the study design minimized risk to research subjects. First, the dosing schedule did not adhere to a strict dose escalation design. As a result, it is possible that subjects dosed at higher levels would experience significant adverse events that could have been anticipated in a more traditional dose escalation design. The Board recognized that the public comments by Bayer CropScience presented during the public comment period at the HSRB meeting suggested that there was previous human testing data available to indicate that such risks would be unlikely. Second, the stopping rule based on a 70% inhibition of cholinesterase activity is fairly liberal ( i. e. not protective), given that 70% inhibition would likely result in significant adverse effects. Although no subject in the research actually experienced this level of cholinesterase reduction, the selection of such a stopping rule increases the risk to subjects. Finally, the letter to the subjects' general practitioner does not contain any information about the actual study and the product being administered other than the name of the compound. As such, this minimizes the extent to which the opinion of the general practitioner could protect the research subject.

HSRB Consensus and Rationale The Board concluded that:

a) The aldicarb human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted.

b) There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained.

c) There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted), nor that information provided to participants seriously impaired their informed consent.

Informed Consent: While informed consent was obtained, the informed consent documents did not include adequate discussion of the research risks as documented by Carley ( 2006a) and did not fully meet ethical standards prevalent at the time the study was conducted. There was a suggestion that additional aspects of risk may have been discussed in the Volunteer Information. Although the Board recognized that the documentation would not meet today's standards, the lack of documentation does not in and of itself demonstrate a significant deficiency that would have seriously impaired the participants' ability to provide informed consent.

Participant risk: The Board concurred that the stopping rule based on 70% reduction in cholinesterase activity raised the possibility of exposing subjects to inappropriate risk. However, the Board acknowledged that data existed at the time to suggest that the doses used in the study 19 of 69 would likely not achieve this level. Thus, there was no evidence that serious harm was intended nor that serious harm could have resulted from the implementation of the study.

The Board concluded that there was no obvious reason why the Agency cannot rely on the results of this study, as appropriate under current pesticide laws, given the absence of clear and convincing evidence that the research was fundamentally unethical or significantly deficient relative to the ethical standards prevailing when the study was conducted.

2. Methomyl Charge to the Board Methomyl is a member of the N methyl carbamate ( NMC) common mechanism group based on its ability to inhibit acetylcholinesterase via carbamylation. The Agency had previously completed the acute, aggregate ( single chemical, multi route) risk assessment of methomyl. At the present time, the Agency is considering the use of the methomyl acute oral, human toxicity study to inform the inter species uncertainty factor used in the cumulative risk assessment of the NMCs.

Scientific considerations The Agency's WOE document and DER for methomyl describe the study design and results of the methomyl acute oral human study. The WOE document also discusses the Agency's conclusions regarding the usefulness of the human study in the cumulative risk assessment for the NMCs. For methomyl, the Agency had concluded that the human toxicity study supports a 10X inter species uncertainty factor for methomyl in the cumulative risk assessment of the NMCs.

Please comment on the scientific evidence that supports this conclusion.

Board Response to the Charge Study Overview A study with methomyl was conducted in human subjects using a double blind, placebocontrolled single ascending dose design ( McFarlane et al. 1998). The defined goal of the study was to determine an acute no adverse effect level ( NOAEL) based on inhibition of plasma and RBC cholinesterase, and in the original protocol, the doses were projected to be 0.1, 0.3, 0.5, 0.75, 1 and 1.5 mg/ kg. Methomyl was administered as a bolus dose in a capsule immediately ( about 5 minutes) following a meal, and post dose blood sampling commenced at 15 minutes after dosing. Collection of blood samples for analysis of plasma and RBC acetylcholinesterase activity was continued for 24 hours after dosing. Plasma and RBC acetylcholinesterase activities were compared to a baseline measurement, determined from two predose samples collected 16 hours and 30 minutes prior to dosing, for each subject. The results were compared to the placebo group. In addition, numerous physiological parameters including ECG, heart rate, pulse, blood pressure, body temperature, clinical chemistry and hematology parameters, determination of 20 of 69 pupil size and salivation were assessed during the entire 24 hour period after dosing. The criterion for not escalating to the next dose was inhibition of RBC cholinesterase exceeding 40%.

Critique of Study The scientific validity of this study was determined by its comparative strengths and weaknesses. The strengths of this study were determined by the comprehensive analysis of cholinesterase inhibition that was obtained including:

  The design and results established a clear dose response and time dependent relationship for inhibition of RBC cholinesterase. This was based on the execution of three dose levels in the ascending dose paradigm. The dosages evaluated were 0.1, 0.2 and 0.3 mg/ kg.   The analysis of the temporal pattern of RBC cholinesterase inhibition was comprehensive and complete. Specifically, blood samples were collected at 15 minutes after dosing, with 15 minute sampling intervals for the first 2 hours, followed by sample collection for evaluation at 2, 3, 4, 6, 8, 12 and 24 hours after dosing. This time course established the peak of cholinesterase inhibition and assured recovery of enzyme activity in all subjects.   The inclusion of clinical endpoints associated with increased cholinergic stimulation as additional endpoints provided additional relevant measures of potential adverse effects during the course of the 24 hour study.

Several weaknesses of the study were noted, and these weaknesses are also based on aspects relating to its design and conduct and include:

  All subjects were male, with no data obtained from females   The group size ( 5/ dose level) was small   The establishment of the baseline RBC cholinesterase level from which all subjects were compared was the mean of 2 determinations, which, given the general pattern of variation, reduces the confidence in the calculation of percent inhibition of the enzyme.

One additional issue concerning the design and execution of this study was the initial proposed dose escalation which ranged from 0.1 to 1.5 mg/ kg ( 0.1, 0.3, 0.5, 0.75, 1, and 1.5 mg/ kg) and the criterion not to escalate the dose when inhibition of RBC cholinesterase exceeded 40%. There was one instance in which this criterion was not adhered to. In the first session of the study, one subject was dosed with placebo and the other received methomyl at 0.1 mg/ kg. The subject receiving methomyl showed evidence of RBC cholinesterase inhibition exceeding 40% ( approximately 43%), but this change was determined at 8 hours after dosing. The study author concluded that, based on the general knowledge and understanding of the rapidity of enzyme inhibition observed with the carbamates, this finding was likely to be a spurious result. In review of the data for RBC cholinesterase inhibition for this session, it was also noted that the placebo treated subject in this session showed a 20% decrease in baseline enzyme activity, thereby suggesting a possible, albeit unconfirmed, issue regarding the accuracy and reliability of the analysis of enzyme activity in these samples. Therefore, the sponsor's conclusion that the effect observed in the subject dosed with 0.1 mg/ kg methomyl was most likely to be a spurious 21 of 69 result was considered scientifically reasonable and justifiable, and the decision to escalate the dose in the next session was not in violation of the protocol.

In the second session of the study, the lead subject dosed with methomyl at 0.3 mg/ kg also showed a greater than 40% inhibition of RBC cholinesterase. Consequently, the next session completed the five subjects to be used in this group but did not escalate the dose further as per the study protocol. Therefore, the highest dosage evaluated in the study was 0.3 mg/ kg, which was much lower than the original intended escalation up to 1.5 mg/ kg. To establish a better dose response relationship, the study was unblinded, and a dosage of 0.2 mg/ kg was added to the protocol after which the study was re blinded and completed.

HSRB Consensus and Rationale The Board concluded that the methomyl human study could be appropriately applied to the inter species risk factor for methomyl and for use in the cumulative risk assessment of N methyl carbamates.

The HSRB concluded that data from the study in human subjects exposed to methomyl provided a meaningful assessment of the dose response relationship and time course for inhibition of RBC cholinesterase in human subjects. The strengths of the study design and conduct outweigh the indicated weaknesses, and the results are scientifically valid. Therefore, the data can be used to inform the assessment of interspecies uncertainty factors for methomyl.

Ethical considerations Charge to the Board 2.2.1. The Agency requested that the Board provide comment on the following:

  Whether the investigators' decision to administer a dose to additional subjects in session 3, when one subject receiving that dose in session 2 displayed RBC ChEI greater than 40%, a response that triggered the protocol's anti escalation provision, should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted;

  Whether the timing of the investigators' report to the ethics committee of the adverse effects observed in one subject during session 2 should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted;

  Whether the failure of the investigators to request approval from the ethics committee for certain amendments to the approved protocol, as required by the protocol, when the changes were administrative and had no effect on the safety of the subjects should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and 22 of 69   Whether the absence from the protocol of discussion of the potential risks to subjects or benefits to society of conducting the proposed research ( as required by the Declaration of Helsinki, Principle # 5) should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and 2.2.2. The Agency asked that the Board provide comment on the following, taking into account all that is known about the ethical conduct of this study:

  OPP's conclusion that there was no clear and convincing evidence that the conduct of the research was fundamentally unethical.

  Whether there was clear and convincing evidence that the conduct of the study was significantly deficient relative to the ethical standards prevailing when the study was conducted,

Board Response to the Charge Brief Overview of the Study The study was performed by a commercial research organization, Inveresk Clinical Research of Edinburgh, Scotland and sponsored by the Haskell Laboratory for Toxicology and Industrial Medicine, E. I. du Pont de Nemours and Company, of Newark, Delaware. The protocol documents specifically state that the research was conducted in compliance with the Principles of Good Clinical Practice promulgated by the International Conference on Harmonization ( CMPM/ ICH/ 135/ 95), and the 1996 version of the Declaration of Helsinki.

Critique of Study The Board concurred with the Agency's factual observations of the strengths and weaknesses of the study, as detailed in Carley ( 2006b). However, further comments are warranted on certain events that took place during the conduct of the study.

1. Determination That Certain Data Were Spurious In session one of the study, one of the subjects exhibited a greater than 40% inhibition in RBC cholinesterase activity at one point, without any signs or symptoms. According to the protocol, such inhibition of cholinesterase was a criterion for not escalating to the next dose level. Because this occurred at 8 hours post dose, study personnel considered this to be a spurious result and went ahead with escalation to the next dose without consulting with the ethics review board ( It is somewhat unclear when the results of the assay became available; the observation about this result is described as being noted " retrospectively at the time of reporting, when there was no sample available for retesting."). If this determination of spuriousness had been incorrect, then giving the following subject an escalated dose might have placed that subject at considerable risk. 23 of 69 The Board concluded that the determination of spuriousness was reasonable at the time, given the late timing of the change in cholinesterase levels, and the similar change observed in cholinesterase levels for a subject in session one who received placebo. Nonetheless, the Board determined that a far more appropriate action would have been for the researchers to have first consulted with the ethics review committee about repeating that 0.1 mg/ kg dose before proceeding with dose escalation. Moreover, the report of the study results stated that " laboratory tests showing abnormal values for any subject were repeated as often as deemed necessary by the clinical investigator until the test values returned to accepted limits or until an explanation other than compound effect was given." In spite of this, it does not appear that any additional tests were performed; study participants only received scheduled laboratory tests at the 12 hour and 24 hour points. Although there was a possibility that these events might have placed subjects at an increased risk of harm, the Board determined that there was not clear and convincing evidence that this could have resulted in serious harm to subjects, particularly given the information known about the compound being tested and the other protections in place as part of the protocol.

2. Decision to Proceed with Third Session In session two of the study, one subject who received an escalated dose of 0.3 mg/ kg exhibited a greater than 40% inhibition in RBC cholinesterase, together with self reported clinical symptoms ( i. e. headache). As noted above, the protocol indicated that dose escalation would not occur if any subject experienced such a level of inhibition. The protocol was followed in this regard: additional subjects were tested at the 0.3 mg/ kg dose, but not at any higher doses. However, the protocol also stated that the study would stop if a subject both had greater than 40% inhibition and also demonstrated signs or symptoms of carbamate toxicity ( one of which might be considered to be a headache). Although the Board was concerned that this decision to continue the study and to test additional subjects at the 0.3 mg/ kg dose was made without consulting the ethics review committee, it determined that the provisions of the protocol were sufficiently unclear as applied to these facts ( particularly regarding what constitute signs and symptoms of toxicity); the actions of the investigators were thus consistent with the protocol as written. In addition, given the protections that were in place as part of the protocol, and the information then known about the compound being studied, the Board concluded that there was not clear and convincing evidence that this decision presented a risk of substantial harm to the subjects. Nonetheless, the Board concluded that a more appropriate course of action would have been for the researchers to have consulted with the ethics review board before proceeding with the study. This conclusion and the desirability of having taken similar actions after the apparently " spurious" result in session one is underscored by public comments presented to the Board that the observation of acetylcholinesterase depression at 0.3 mg/ kg was " surprising." There is a danger in explaining away unexpected findings based on prior expectations, and ethics review boards should play an important role in preventing investigators from falling prey to such expectations.

3. Amendment of the Protocol Following the testing of the subjects at the 0.3 mg/ kg level as part of session three, the protocol was amended to allow additional subjects to be tested at the 0.2 mg/ kg level. This 24 of 69 change was approved by the chair of the ethics review committee, consistent with procedures for the committee which allowed the chair to approve amendments that did not increase the risk level ( i. e., did not involve testing subjects at a dose level above those approved by the convened board). Although the Board did not conclude this was a formal violation of then existing ethical standards, some members of the Board were concerned that the ethics committee chair's actions were inappropriate and that the protocol change should have been reviewed by the full committee because additional subjects were being exposed to a study compound beyond those contemplated by the unamended protocol.

HSRB Consensus and Rationale The Board concluded that:

a. The methomyl human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however b. There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and.

c. There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted), nor that information provided to participants seriously impaired their informed consent.

( 1) The Board concluded that the decision to administer a dose to additional subjects in session three could not be determined to be significantly deficient relative to the ethical standards prevailing when the study was conducted because, as discussed above, that decision could be interpreted as consistent with the terms of the protocol and was not likely to expose the subjects to additional serious harm.

( 2) The Board concluded that the timing of the investigators' report to the ethics committee regarding what happened to the subject who experienced cholinesterase inhibition during session two should not be considered significantly deficient relative to the ethical standards prevailing when the study was conducted, given the subject's minimal symptoms and the fact that RBC cholinesterase inhibition by itself is not considered an adverse event.

( 3) The Board determined that the failure of the investigators to request approval from the ethics committee for certain amendments to the approved protocol, while deficient relative to the ethical standards prevailing at the time the study was conducted, should not be considered significantly deficient, given that these changes were purely administrative.

( 4) The Board determined that the absence from the protocol of discussion of the potential risks to subjects or benefits to society of conducting the proposed research ( as required by the Declaration of Helsinki, Principle # 5) should not be considered significantly deficient relative to the ethical standards prevailing when the study was conducted. The potential risks and 25 of 69 benefits are described in the research subject information sheet/ informed consent document. While not in the protocol itself, they are nonetheless included in the materials provided to the ethics committee ( and to the potential research subject). Thus, the ethics committee and the potential research subjects had the opportunity to weigh the risks and benefits.

The Board concluded that there was no obvious reason why the Agency cannot rely on the results of this study, as appropriate under current pesticide laws, given the absence of clear and convincing evidence that the research was fundamentally unethical or significantly deficient relative to the ethical standards prevailing when the study was conducted.

3. Oxamyl Charge to the Board Similar to aldicarb and methomyl, oxamyl is a member of the N methyl carbamate ( NMC) common mechanism group based on its ability to inhibit acetylcholinesterase via carbamylation and is thus included in the NMC cumulative risk assessment. The Agency had previously completed the acute, aggregate ( single chemical, multi route) risk assessment of oxamyl. The Agency is now considering the use of the oxamyl acute oral, human toxicity study to inform the inter species uncertainty factor in the cumulative risk assessment of the NMCs.

3.1. Scientific considerations The Agency's WOE document and DER for oxamyl described the study design and results of the oxamyl acute oral human toxicity study. The WOE document also discussed the Agency's conclusions regarding the usefulness of the human study in the cumulative risk assessment for the NMCs. For oxamyl, the Agency had concluded that the human toxicity study was sufficiently robust for reducing the 10X inter species ( i. e., animal to human) uncertainty factor in the cumulative risk assessment.

Please comment on the scientific evidence that supports this conclusion.

Board Response to the Charge Study Overview The stated primary objective of this randomized, double blind, ascending oral dose study with oxamyl ( McFarlane and Freestone 1999a) was to estimate the no observable adverse effect level ( NOAEL) for oxamyl in humans after oral administration.

The study used 40 healthy male subjects, aged 19 39. Each was given a single oral dose of oxamyl in a gelatin capsule at doses of 0 ( placebo), 0.005, 0.015, 0.03, 0.06, 0.09 or 0.15 mg/ kg BW. With the exception of dose sessions 1 ( 2 placebos), 2 ( 1 placebo and 1 at 0.005), 7 ( 1 placebo and 4 at 0.09), 8 ( 1 placebo and 1 0.15), and 9 ( 1 placebo and 4 at 0.15), all other dose sessions had 1 placebo, 4 at current dose, and 1 at the next higher dose. Plasma and RBC cholinesterase activity were assayed at screening and 2 days, 16 hours and 30 minutes prior to 26 of 69 dosing and at 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 12, and 24 hours post dose and 7 days post dose. Other clinical parameters indicative of carbamate exposure, such as urinalysis, pupillometry, saliva increase etc. were also measured.

No treatment related effects for ECG, heart rate, pulse, blood pressure, respiratory rate, body temperature, hematology, clinical chemistry, urinalysis, or pupillometry were recorded.

At 0.09 mg/ kg b. w., 7 12% plasma and RBC ChE inhibition was observed with three of five volunteers exhibiting greater than 20% plasma ChE inhibition. Therefore, 0.06 mg/ kg b. w. was considered the NOAEL. The Agency generated BMD and BMDL estimates of 0.083 mg/ kg and 0.069 mg/ kg, respectively, based on the RBC ChE data from the study.

Critique of the Study The scientific validity of this study was analyzed by weighing its strengths and weaknesses in order to determine if the study was sufficiently robust to be of use in the Agency's risk assessment for oxamyl.

Appropriate endpoints for this study were selected. Acetyl cholinesterase inhibition in the nervous system was viewed as a key molecular event in the mechanism of toxicity of carbamate compounds. Measurements of acetyl cholinesterase inhibition in both RBCs and brain are considered to be biomarkers of exposure for these compounds. In its policy on the use of data on cholinesterase inhibition for risk assessments of organophosphorus and carbamate pesticides ( USEPA, 2000), the Agency stated that it would treat cholinesterase inhibition in the blood as a surrogate for effects on the peripheral nervous system in animals and for effects on the peripheral and central nervous system in humans.

The animal data for oxamyl were not available to the Board to allow it to make an independent judgment as to whether the methods used to determine acetyl cholinesterase inhibition in the animal studies and the human study were comparable. According to EPA, however, the methods used in the animal studies are comparable to that in the human study.

Previous animal data was used to predict that the starting dose 0.005 mg/ kg would have no detectable effect on humans. Oral administration of the compound is the most predictive mode of administration since exposure to residues would come from food consumption. There was no justification for the sample size of 4 for oxamyl for each dose session.

Forty healthy males of similar age and weight were used in this study. A weakness of this study was that no female subjects were used in this study and there was no indication that any human female data exists from other studies. However, the Agency advised the Board that, based upon its evaluation of the animal data, no sex differences would be expected.

The specific schedule for dosing was described in the protocol. It was an ascending double blind, placebo controlled study. Dose escalation did not occur if there were clinically significant symptoms/ signs of toxicity. Stopping rules were equal to or greater than 40% inhibition of RBC cholinesterase at a single time point or 25% at two consecutive time points. If 27 of 69 there were associated symptoms/ signs of carbamate toxicity, no further dose escalation would occur.

A repeated measures analysis of variance was used to analyze the percentage change from baseline in RBC and plasma cholinesterase activity, pupillometry and saliva collection. At each time point of measurements, a test for linear trend with dose was performed using a linear contrast. If the test for linear trend was significant at the 5% significance level, the pairwise comparisons at each timepoint were not adjusted for multiple comparisons. Otherwise, a Bonferroni adjustment was applied to the pairwise test at the 0.83% significance level.

At 0.09 mg/ kg b. w., 7 12% plasma and RBC ChE inhibition was observed with three of five volunteers exhibiting greater than 20% plasma ChE inhibition. Therefore, 0.06 mg/ kg b. w. was considered the NOAEL. The Agency generated BMD and BMDL estimates of 0.083 mg/ kg and 0.069 mg/ kg, respectively, based on the RBC ChE data from the study The Board highlighted the strengths of the study as follows:

( a) The study design, including both the increasing dose levels and the time point of observations/ measurements, was very rich in information content to allow evaluation of doseresponse and estimation of the NOAEL and the dose escalation in a step wise fashion was scientifically sound considering the uncertainties in the risk.

( b) The study had a very clearly defined dose escalation scheme with properly justified stopping rules. The schedule of observation and measurements were frequent enough to give sufficient information on the time course effect of oxamyl on subjects.

( c) The statistical methods used for analysis, including the adjustment for multiple comparisons between placebo and active doses when there was no statistical significance in the linear trend test, were quite adequate for the study.

The Board highlighted the weaknesses of the study as follows:

( a) There appeared to be no specific justification for the sample size of four for oxamyl for each dose session and no traditional statistical test of effect size regarding hypotheses. Because the study was exploratory versus confirmatory, the lack of justification was not critical. Nevertheless it would have been helpful to know what effect size could be detected between the placebo and the active dose groups given the sample size of the study.

( b) Because of lack of justification for the sample size, the lack of statistical significance in the observed difference between placebo and active doses could not be interpreted appropriately. Specifically the statement in the Agency Data Evaluation Report ( Moyer and Milznez 2001) that " RBC cholinesterase activity was statistically similar to placebo at all time points in the 0.005, 0.015, 0.03, and 0.06 mg/ kg dose groups" was not justified as the study was not designed to test for statistical similarity. 28 of 69 ( c) The Agency concluded that " there is no indication from the available rat data for either oxamyl or methomyl to suggest a sex difference." The animal data were not supplied to the Board and therefore the Board could not independently verify this conclusion. However the Board was not uncomfortable with accepting EPA's conclusions on this matter.

The Agency also concluded that " Given the similarity between brain and RBC ChE in rats, the Agency is not aware of any biological or physiological reason that human brain ChE would be more sensitive than the rat brain." Again without seeing the animal data, the Board could not verify this conclusion about rats, but the conclusion about human brain and RBC cholinesterase was reasonable scientifically.

HSRB Consensus and Rationale Although the Board had some questions about the Agency's conclusions regarding lack of sex difference and of the difference between brain and RBC ChE based on only one species, the Board supported the Agency's conclusion that there were no study deficiencies identified that would have affected the outcome or conclusions of this study.

Considering the high quality of the design and the conduct of the study, the Board agreed that this intentional human dosing study of oxamyl was sufficiently robust to be used for reducing the 10x inter species ( i. e. animal to human) uncertainty factor in the cumulative risk assessment for the N methyl carbamates.

3.2. Ethical considerations Charge to the Board a. The Agency requested that the Board provide comment on the following:

  Whether inclusion in the protocol submitted to the ethics committee of a factually inaccurate statement regarding unavailability of data on accidental or incidental exposure to oxamyl should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted;

  Whether the absence from the protocol of any discussion of the potential risks to subjects or benefits to society of conducting the proposed research ( as required by the Declaration of Helsinki, Principle # 5) should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and b. The Agency asks that the Board provide comment on the following, taking into account all that is known about the ethical conduct of [ this/ each] study:

  OPP's conclusion that there was not clear and convincing evidence that the conduct of the research was fundamentally unethical. 29 of 69   Whether there was clear and convincing evidence that the conduct of the study was significantly deficient relative to the ethical standards prevailing when the study was conducted.

Board Response to the Charge Study Overview The study was performed by a contract research organization, Inveresk Clinical Research of Edinburgh, Scotland and sponsored by the Haskell Laboratory for Toxicology and Industrial Medicine, E. I. du Pont de Nemours and Company, of Newark, Delaware. The protocol documents specifically state that the research was conducted in compliance with the Principles of Good Clinical Practice promulgated by the International Conference on Harmonization ( CMPM/ ICH/ 135/ 95), and the 1996 version of the Declaration of Helsinki. Comments provided by du Pont Crop Protection ( Dupont 2006), also assert compliance with the 1996 version of the Declaration of Helsinki.

Critique of Study The Board concurred with the factual observations of the strengths and weaknesses of the study, as detailed in Carley 2006c). However, further comments are warranted regarding: 1) whether the exclusion of data on accidental or incidental exposure to oxamyl meets the legal definition of research misconduct prevalent at the time the study was conducted; 2) whether the escalating dose oral exposure protocol used was designed to minimize risks to study participants; and 3) whether the documentation and process of study subject enrollment was sufficient to meet prevailing standards of voluntary informed consent.

1) Exclusion of Available Data on Accidental or Incidental Exposure Page 15 of the original study protocol provided to the independent ethics review committee established by Inveresk Clinical Research included the following statement: " No data on accidental or incidental exposure of people to oxamyl is available" ( McFarlane and Freestone 1999). This statement is false. Prior to the start of the Randomised Double Blind Ascending Oral Dose Study with Oxamyl in late 1998, Du Pont submitted eleven adverse event reports involving exposure of humans to oxamyl for inclusion in the EPA's Office of Pesticide Programs ( OPP) Incident Data System ( IDS). Between 1982 and 1994, the State of California Department of Food and Agriculture's Pesticide Illness Surveillance Program recorded an additional 61 incidents, including 30 cases involving occupational exposure to oxamyl alone. In comments provided to the EPA on March 31, 2006, Du Pont Crop Protection stated that " most of the accidents or incidents reported in these databases lack confirmation of the product involved or the amount of product involved in the exposure. Also, some reports lack information on symptoms or details of the exposure. Therefore, none of this data was useful for designing the study." ( DuPont 2006). This latter statement may be true, but the discounting of the relevance of adverse event data from accidental or incidental exposures is ill advised and borders on the unprofessional. This may have been an appropriate decision but the data, evaluation and assessment should have been discussed in the protocol and the consent document. Furthermore, 30 of 69 the statement in the original study protocol remains untrue. The question before the Board, therefore, was whether or not this misstatement met the legal definition of research misconduct.

The Agency has accepted the federal policy on research misconduct ( Classification No.: 3120.5; Approval Date: 03/ 18/ 2003; Review Date: 03/ 18/ 2006), as defined in 42 CFR 93.103:

Research misconduct means fabrication, falsification, or plagiarism in proposing, performing, or reviewing research, or in reporting research results.

( a) Fabrication is making up data or results and recording or reporting them.

( b) Falsification is manipulating research materials, equipment, or processes, or changing or omitting data or results such that the research is not accurately represented in the research record.

( c) Plagiarism is the appropriation of another person's ideas, processes, results, or words without giving appropriate credit.

( d) Research misconduct does not include honest error or differences of opinion.

Although the failure to include the incidental exposure data may meet this definition of falsification, a finding of research misconduct made under 42 CFR 93.104 requires that: 1) there be a significant departure from accepted practices of the relevant research community; 2) the misconduct be committed intentionally, knowingly, or recklessly; and 3) the allegation be proven by a preponderance of the evidence. Under the standards outlined in 42 CFR 93.103 104, given the limitations in the studies cited by Du Pont, and lack of evidence that the absence of information was intentional, knowing, or reckless, it is the opinion of the Board that this omission does not meet the evidentiary threshold necessary for a finding of research misconduct based on falsification.

2) Minimization of Risks to Study Participants Reasonable exposure levels appear to have been chosen using available animal data. Although human exposure data from the EPA OPP IDS and California's Pesticide Illness Surveillance Program also may have been useful in designing the study so as to minimize risk to research subjects, there was no clear and convincing evidence of willful or blatant disregard for participant safety. Adherence to a strict dose escalation protocol, with a stopping rule based on 40% inhibition of RBC cholinesterase, reduced the likelihood that study subjects would experience unanticipated adverse clinical events. Based on the knowledge available to investigators at the time the study was conducted, this double blind ascending dose oral exposure trial appears to have been designed to adequately minimize potential risks to study participants.

3) Voluntary Informed Consent 31 of 69 It was clear that the written documentation for informed consent failed to meet the standards outlined in Principles of Good Clinical Practice ( CMPM/ ICH/ 135/ 95) and the 1996 version of the Declaration of Helsinki.

The information sheet that was given to research subjects as an appendix to the signed informed consent document lacked adequate discussion of the potential risks of participating in the research study. Several examples of what Board members consider inadequate discussion of potential risks are illustrated below:

Data on incidents involving accidental oxamyl exposure were available through both the EPA's OPP IDS and the California Pesticide Illness Surveillance Program, as previously discussed. These data included reports of adverse clinical events, including detailed reports of systemic effects, eye effects and dermatological effects resulting from exposure to oxamyl. These data were not provided to study participants.

The background information provided to study participants enrolling in both the original and amended study included the statement that " results from this study would further confirm that the use of oxamyl for crop protection would not pose an unreasonable risk to human health." ( McFarlane and Freestone 1999). Although the informed consent document clearly stated that this is the first study involving intentional exposure of human subjects, particularly given the incident data discussed above, this statement as written might have inappropriately downplayed concerns that study participants might have with regard to participation.

Study participants enrolling in the amended study, designed to look for clinical or biological outcomes associated with exposure to 0.15 mg/ kg body weight of oxamyl, were informed that " no significant side effects [ were] noticed in any of [ the previous lower] dose levels." ( McFarlane and Freestone 1999). Previous trial participants receiving 0.09 mg/ kg body weight of oxamyl exhibited 4 24% and 12 22% RBC and plasma cholinesterase inhibition. The study investigators concluded that these observed levels of cholinesterase inhibition did not qualify as adverse events because of the variability in the data, but they also state that the level of inhibition observed in study participants receiving 0.09 mg/ kg body weight of oxamyl achieved statistical significance. Because the consent form described cholinesterase inhibition as a side effect of oxamyl exposure, it can be argued that this information should have been provided to subjects participating in the 0.15 mg/ kg dose study.

The letter to the subjects' general practitioner contained no information about the actual study and the product being administered other than the name of the compound. It thus minimized the extent to which the opinion of the general practitioner could protect the research subject.

In hindsight, it is clear that the documentation provided failed to rigorously meet the standards of voluntary informed consent applicable to studies conducted in Scotland in 1998 1999. However, there was no clear and convincing evidence that these deficiencies knowingly and seriously impaired the informed consent process.

HSRB Consensus and Rationale 32 of 69 The Board concluded that:

a) The oxamyl human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted, however b) There was no clear or convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained, and c) There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

Although the study failed to meet the specific ethical standards prevalent at the time the research was conducted, including those cited in the study protocol itself, the Board concurred with the assessment of the Agency that there was no clear and convincing evidence that the conduct of the research was fundamentally unethical in that it was not intended to harm subjects nor failed entirely to obtain informed consent.

It is believed that potential risks to study participants were inadequately addressed in the documents provided to research participants at enrollment, thus impairing the informed consent process. Nevertheless, the Board believed that there was no clear and convincing evidence to suggest that the study investigators failed to obtain voluntary informed consent from all research participants.

The Board was of the opinion that the deficiencies noted above are unlikely to have resulted in serious harm to study participants, based on the knowledge available to the investigators at the time.

The Board concluded that there is no obvious reason why the Agency cannot rely on the results of this study, as appropriate under current pesticide laws, given the absence of clear and convincing evidence that the research was fundamentally unethical or significantly deficient relative to the ethical standards prevailing when the study was conducted.

4. Azinphos Methyl Charge to the Board Azinphos methyl ( AZM) is an organophosphate pesticide ( OP). Consistent with other OPs, AZM elicits neurotoxicity through the inhibition of the enzyme, acetylcholinesterase, via phosphorylation of the active site. At sufficiently high doses, exposure to AZM can lead to a variety of clinical signs. The Agency is developing an assessment to estimate risk to workers from exposure to AZM. In addition, AZM is a member of the OP common mechanism group and is thus included in the cumulative risk assessment for the OPs. 33 of 69 Scientific considerations The Agency's WOE document and DER for AZM describe the study design and results of the AZM repeat dose, oral, human toxicity study. The WOE document also discussed the Agency's conclusions regarding the usefulness of the human study in the worker risk assessment and in the cumulative risk assessment for the OPs. For AZM, the Agency has concluded that the human toxicity study was appropriate for developing a point of departure for extrapolation of risk to workers exposed to AZM via the dermal and inhalation routes. For the cumulative risk assessment, the Agency has determined that because no cholinesterase inhibition was seen in the human toxicity study, it was not possible to evaluate whether steady state had been reached in humans at 28 days of exposure. Thus, the Agency had concluded that the AZM repeat dose, oral, toxicity study was not sufficiently robust for informing the inter species factor in the cumulative risk assessment of the OPs.

Please comment on the scientific evidence that supports the conclusions for:

a. the use of the human toxicity study to develop a point of departure for extrapolation of risk to workers in the worker risk assessment and b. the determination that the human toxicity study cannot be used to inform the interspecies factor in the cumulative risk assessment.

Board Response to the Charge Brief Overview of the Study In a repeat dose oral human toxicity study, conducted in 1999 by the Inveresk Laboratory ( McFarlane & Freestone, 1999b), four males were dosed with a placebo ( lactose) and eight males were dosed with 0.25 mg/ kg/ day of AZM for 28 consecutive days. The sole dose of 0.25 mg/ kg was chosen based on the results of a subchronic rat study and an earlier study with human subjects that indicated this would be a NOAEL for inhibition of plasma and RBC cholinesterase. Previous human studies were conducted in 1972 and 1974 and regulatory agencies were reluctant to rely on the data " due to deficiencies in documentation, specifically the availability of raw data and the lack of GLP compliance."

Dosing was orally by capsule and the subjects were housed in a clinic throughout the dosing period. The subjects were monitored for reactions and samples of blood were taken at predose ( eight times) and daily throughout the dosing period. On some days, a blood sample was taken both prior to administering the dose and four hours later. Both plasma cholinesterase ( ChE) and RBC acetylcholinesterase ( AChE) were measured. In addition, hematology, clinical chemistry, and urinalysis were evaluated as well as blood pressure and electrocardiogram ( ECG). There were no reactions to treatment and blood pressure and ECG were not affected. Eight of the 12 volunteers were noted to have symptoms of a viral infection and some were treated with paracetamol. The postdosing group mean data were compared three ways in an effort to see if AZM caused inhibition of either plasma ChE or RBC AChE. None of the comparisons indicated inhibition of either measure. Also, samples taken four hours after dosing did not indicate 34 of 69 inhibition. It was concluded that a dose of 0.25 mg/ kg/ day for 28 days did not result in either clinical symptoms or inhibition of either plasma ChE or RBC AChE in human males, so that this dose could be considered a NOAEL.

Critique of the Study One strength of the study was its design as a randomized, double blind study including a placebo group. The clear inclusion and exclusion criteria for subject selection appeared to have been chosen to reduce the likelihood of confounding factors. The subjects were in good health, were not using prescription or illicit drugs, were non smokers, and were not agricultural workers or pesticide applicators who might have experienced OP exposure within the preceding month. Subjects resided in the clinic from the day before study start to 24 hours after the last dose was administered, received a standardized diet, and were under medical and nursing supervision throughout.

The assessment of critical outcomes was comprehensive, involving a general clinical exam, measurement of systolic and diastolic blood pressure, the administration of an ECG, and various blood and urine studies. Multiple measurements were made of plasma ChE and RBC AChE, namely on 8 pre dose days then daily 4 hours after dosing. Moreover, on 12 post dosing days, two blood samples were taken, one before the dose and one 4 hours afterwards.

Possible group differences in plasma ChE and RBC AChE were evaluated in several ways, both in grouped and individual data: a) Comparison of the day to day ChE activities within the AZM group to the same day values in the control group b) Comparison of same day predosing ChE activities with ChE activities 4 hours after dosing c) Comparison of the day to day ChE activities values within the AZM group to predosing baseline value.

Several weaknesses were identified in the study. First, the subjects were all male, however there have been no consistent sex differences in sensitivity with regard to inhibition of plasma or RBC cholinesterase in animal studies ( at higher dose levels that elicit more serious toxicity, there are gender related differences in responses). The sample size was not large, involving only 12 adult males ( four in the placebo group, eight in the AZM group), raising a concern that the statistical power might have been inadequate to detect a modest degree of inhibition in the AZM group.

The standard deviations of the ChE levels were large, as great as 20% for plasma ChE and 15% for RBC ChE, reducing confidence in the consistency of these endpoints.

A variety of symptoms were reported over the course of the study, with the majority occurring in subjects administered AZM. The conclusion drawn in the Agency's Data Evaluation Record ( Doherty 1999) that none of the symptoms likely reflected effects of AZM was not verifiable, nor was it convincing. 35 of 69 Certain aspects of the statistical analyses are problematic. For the analyses of percentage change each day from baseline ( predosing) plasma and RBC cholinesterase activity in the subjects receiving AZM, the choice of repeated measures ANOVA as the statistical approach was appropriate given the structure of the data. This would have allowed each subject to be used as his own control, but this does not appear to be the way the analyses were done. The pooled baseline RBC and plasma ChE values were used as covariates rather than individual subject values. This essentially ignores the value of the individual levels in increasing statistical power by taking account of pre dosing inter individual differences in ChE levels. The other two types of analyses conducted were not limited by this deficiency, but the analysis of within subject change from baseline is potentially the most sensitive analysis in this study.

In proposing to use the results of this study for dermal and inhalation risk assessment, the Agency applied a dermal absorption factor of 42% based on a rat study and an inhalation absorption factor of 100%. Bayer Crop Science commented that data are available supporting a lower estimate of human dermal absorption ( 21.5%) but these data were not available for evaluation by the Board. Another concern was whether the effects of a single oral dose administered daily provide a reasonable estimate of the effects of the continuous dosing via all routes of exposure that would be experienced by agricultural workers.

The major concern regarding this human study was it was designed to demonstrate a NOAEL. No LOAEL could be established to validate the NOAEL because only one dose was used. The fact that inhibition was not observed at the single dose used raised concern about sensitivity of the methods used. The Agency was reassured by the laboratory's observation of plasma and RBC cholinesterase inhibition in other studies of carbamates and organophosphates, including aldicarb, methomyl, and oxamyl. Nevertheless, it is not possible to be sure if the absence of an effect in this study was because the methods used were, for some reason, not up to the standards of those used in the other studies, and therefore less sensitive.

HSRB Consensus and Rationale The Board concluded that:

a. Data from the 28 day repeat oral dose study of AZM should not be used in developing a point of departure for extrapolation of risk to workers exposed to AZM via the dermal and inhalation routes.

b. Data from the 28 day repeat oral dose study of AZM cannot be used to inform the inter species factor in the cumulative risk assessment.

Despite the study's strengths, the HSRB had only limited confidence in the inferences that can be drawn from this study, in large part because only one AZM dose was used. In order to use these data, one must assume that the lab would have detected inhibition had it been present at the single dose administered. The conclusions drawn could be correct, but this cannot be determined with certainty. The conclusion that this study is not appropriate for developing a point of departure for either the worker risk assessment or the cumulative risk assessment is 36 of 69 consistent with the recommendation of the NAS committee ( NAS 2001) that the Agency should not consider data from NOEL only studies.

Ethical considerations Charge to the Board a. The Agency requests that the Board provide comment on the following:

Whether the informed consent materials   which refer to " the company" and " supervising doctor" without further identification, and contain no discussion of who would benefit from the research   should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and,

Whether the absence from the protocol of any discussion of the potential risks to subjects or benefits to society of conducting the proposed research ( as required by the 1996 Declaration of Helsinki, Principle # 5, with which the research asserted compliance) should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and b. The Agency asks that the Board provide comment on the following, taking into account all that is known about the ethical conduct of [ this/ each] study:

OPP's conclusion that there was not clear and convincing evidence that the conduct of the research was fundamentally unethical.

Whether there was clear and convincing evidence that the conduct of the study was significantly deficient relative to the ethical standards prevailing when the study was conducted.

Brief Overview of the Study As noted above, the study was conducted in 1999 by a contract research organization, Inveresk Clinical Research of Edinburgh, Scotland, at its Elphinstone Research Centre in Tranent. The study sponsor was the Bayer Corporation, Agricultural Division, of Stilwell, Kansas. The protocol documents specifically state that the research was conducted in compliance with the guideline for Good Clinical Practice, and in accordance with the guidelines of the Declaration of Helsinki, 1964, as amended through 1996.

Critique of Study The Board concurred with the factual observations of the strengths and weaknesses of the study, as detailed in Carley ( 2006d). However, further comments are warranted with regard to whether the documentation and process of study subject enrollment was sufficient to meet prevailing standards of voluntary informed consent. 37 of 69 It is clear that the written documentation for informed consent failed to meet fully the standards contained in the 1996 version of the Declaration of Helsinki.

In particular, the information sheet that was given to research subjects as an appendix to the signed informed consent document contained the following statement under the subheading Aim: " The aim of the study is to establish a recommended daily intake following long term dietary exposure and to determine the most appropriate way of assessing workers exposure." By using the phrase " recommended daily intake," this statement would appear to suggest that it is actually desirable to consume a daily minimum amount of the study compound. This sentence is thus misleading. However, other portions of the information sheet made it clear that the study compound was a pesticide, and that consuming it is not good for people, thus leading the Board to conclude that this one sentence by itself does not provide clear and convincing evidence that the consent process as a whole was significantly deficient relative to the ethical standards prevailing at the time the study was conducted.

HSRB Consensus and Rationale The Board concluded that:

a) The azinphos methyl human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted.

b) There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained.

c) There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

The Board determined that the informed consent materials   which refer to " the company" and " supervising doctor", without further identification, and which contain no discussion of who would benefit from the research   did not fully comply with the ethical standards at the time. However, the Board did not find clear and convincing evidence that the level of non compliance should be considered significantly deficient relative to those thenprevailing ethical standards. With regard to the use of the terms " the company" and " supervising doctor," this type of language, though highly undesirable, is unfortunately not uncommon, even at the present. With regard to the benefit issue, the Board determined that the consent materials, while far from ideal, did in fact somewhat address this issue. They indicate, for example, that the study may lead to further information about the risk that this compound poses to workers and consumers.

The Board determined that the absence from the protocol of discussion of the potential risks to subjects or benefits to society of conducting the proposed research ( as required by the Declaration of Helsinki, Principle # 5) should not be considered significantly deficient relative to the ethical standards prevailing when the study was conducted. The potential risks and benefits 38 of 69 are described in the research subject information sheet/ informed consent document. While not in the protocol itself, they are nonetheless included in the materials provided to the ethics committee ( and to the potential research subject). Thus the ethics committee and the potential research subjects had the opportunity to weigh the risks and benefits.

The Board concluded that there was no obvious reason why the Agency cannot rely on the results of this study, as appropriate under current pesticide laws, given the absence of clear and convincing evidence that the research was fundamentally unethical or significantly deficient relative to the ethical standards prevailing when the study was conducted.

5. DDVP Charge to the Board Like AZM, DDVP is an organophosphate pesticide ( OP) which elicits neurotoxicity through the inhibition of acetylcholinesterase, via phosphorylation of the active site. The Agency is conducting an aggregate ( single chemical, multi route, multi duration) risk assessment of DDVP. In addition, DDVP is a member of the OP common mechanism group and is thus included in the cumulative ( multi chemical, multi route) risk assessment for the OPs.

5.1. Scientific considerations a. The Agency's WOE document and DER for DDVP described the study design and results of the DDVP repeat dose oral human study. The WOE document also discussed the Agency's conclusions regarding the usefulness of this study in the aggregate risk assessment and in the cumulative risk assessment for the OPs. For the single chemical risk assessment, the Agency had concluded that the human study was sufficiently robust for developing a point of departure for estimating dermal, incidental oral, and inhalation risk from exposure to DDVP in the single chemical risk assessment. For the cumulative risk assessment, the Agency had determined that results of the DDVP multi dose human toxicity study do not support reducing the default 10X inter species factor in the cumulative risk assessment of the OPs.

Please comment on the scientific evidence that supports the conclusions for:

a) the Agency's conclusions for use of the human study for developing a point of departure for estimating risk in the single chemical, aggregate risk assessment and b) the Agency's determination that the human study cannot be used to reduce the interspecies factor in the cumulative risk assessment.

Board Response to the Charge Study Overview A single blind, randomized placebo controlled oral study was conducted with DDVP in which six healthy male volunteers were administered a daily dose of DDVP at approximately 39 of 69 0.1mg/ kg/ day and three volunteers were administered a placebo ( Gledhill 1997). DDVP was dissolved in corn oil and administered daily in a gelatin capsule to fasted subjects ( subjects did not eat after midnight prior to each morning dose) for a total of 21 days. Blood samples were collected for analysis of red blood cell ( RBC) cholinesterase activity. For each subject, the baseline level of RBC cholinesterase activity was established from seven pre dose samples, and a single blood sample was collected approximately 24 hours after dosing ( just prior to dosing on the following day) on days 1, 2, 4, 7, 9, 11, 14, 16 and 18 of the study. No blood samples were collected on the last three days of the study ( days 19 21). After cessation of dosing, subjects returned approximately 1 week later ( on or about day 25) for a follow up analysis of RBC cholinesterase activity. The criterion for withdrawal from the study was inhibition of RBC cholinesterase activity of 20% or more on a given day's analysis followed by a further decrease in activity in the next successive sample. A linear mixed model was fitted to the cholinesterase activity and significant differences were found within subjects and also between groups ( treatment versus placebo subjects) for pre dose and post dose cholinesterase activity.

Critique of Study The scientific validity of the repeated dose study for DDVP was determined by the relative strengths and weakness of the design and conduct of the study.

The strengths of the study were considered to be as follows:   The repeat dosing paradigm affords the opportunity to more critically evaluate the sustained nature of RBC cholinesterase inhibition associated with OPs.   The analysis of RBC cholinesterase activity is fairly robust, given the assessment of predose baseline values on 7 separate occasions. The baseline values were fairly consistent both within and between subjects ( 16,000   21,000 IU).   A low, but statistically significant effect was observed in RBC cholinesterase inhibition.

There are, however, numerous weaknesses of the study that are relevant to the utility of these data in establishing a point of departure for use in single chemical, aggregate and cumulative risk assessments for DDVP. These weaknesses included:

  A single dosage was used, preventing establishment of dose response relationships.

  The sample size was small and included only male volunteers. In this regard, it was not clear whether the study was properly powered, given that no sample size calculations seem to have been used in order to arrive at the number of volunteers used in the study.

  RBC cholinesterase inhibition was determined only at 24 hours post dosing. Although organophosphates are known to show prolonged inhibition of cholinesterase activity, it was not clear whether the peak of RBC cholinesterase was established.

  The sponsor did not include the analysis of plasma cholinesterase measurements in the study. The reason for this omission was not clear, and it was noted that virtually every toxicological and exposure assessment study that has focused on cholinesterase inhibition due to organphosphorus insecticides includes measurement of both plasma ( serum) 40 of 69 cholinesterase and RBC cholinesterase. The combination of these measurements has been used routinely for risk assessments by numerous regulatory agencies, including the EPA, as well as by clinicians who diagnose pesticide poisonings. Current worker surveillance programs in some states ( California and Washington) require measurement of both plasma and erythrocyte cholinesterase, and both measurements are used in combination to initiate workplace investigations and remove workers from pesticide related job activities until enzyme activities return to baseline.

  Sampling was incomplete because the last day of analysis of enzyme inhibition was day 18 but dosing continued until day 21. It is not clear whether any additional decrease in enzyme activity would have been observed on days 19 21. In the sampling schedule outlined above, the maximum time gap between samples during the dosing period ( days 1 21) was three days ( between days 11 and 14). Logically then, an additional sample would have been taken on day 21 or 22, the time in the study when maximum effects might be observed. In this regard, two subjects presented with inhibition values of 22 and 23%, exceeding the 20% cut off for subsequent analysis and consideration for withdrawal on day 18 of the study. The lack of additional measurements in these subjects during the remainder of the dosing period was scientifically inadequate and ethically troublesome.

  It is neither clear nor convincing that steady state inhibition of RBC cholinesterase activity was achieved in the study. The data suggest that the level of enzyme inhibition was still increasing, particularly in the time period from 11 18 days, with a difference of 16% in activity on Day 18 compared to pre dose values. Dosing was stopped on Day 21, with no further analysis until approximately 4 days after cessation of dosing. Although the difference in activity between day 18 post dose and the average of the pre dose measurements was deemed to be " not biologically significant" by the study sponsor, the possibility that RBC cholinesterase inhibition may have reached or exceeded 20% by the end of the study cannot be excluded.

  There was a lack of appropriate follow up in all subjects in this study. As noted earlier, two subjects presented with inhibition values of 22 and 23%, exceeding the 20% cut off for subsequent analysis and consideration for withdrawal on Day 18. However, there were no additional analyses of RBC cholinesterase activity in these individuals on days 19 21 of dosing. Additionally, a blood sample was collected within 4 to 10 days following completion of the 21 day dosing period for all subjects. The mean inhibition of the enzyme observed at seven days following cessation of dosing was 17%, suggesting little if any recovery of enzyme at this time, but these data were not analyzed for statistical significance. Furthermore, one subject ended his participation in the study ( day 25) with RBC cholinesterase inhibition greater than 20%. In all cases, there was no additional follow up of the subjects after the single post dose analysis of cholinesterase activity.

HSRB Consensus and Rationale The Board concluded that: 41 of 69 a) The DDVP repeat dose human toxicity study was sufficiently robust for developing a point of departure for estimating dermal, incidental oral, and inhalation risk from exposure to DDVP in the single chemical risk assessment.

In specific response to the Agency's charge question, the HSRB concluded that there were numerous technical limitations to the data obtained in the study. In addition to the weaknesses of the study design and execution outlined above, the HSRB concluded that the evaluation of only a single dose level of DDVP along with the omission of plasma cholinesterase measurements greatly limit the value of this study in terms of producing new knowledge that can be used in the regulatory process and in medical diagnosis. Furthermore, study investigators have an obligation to design a study that provides appropriate oversight of subjects until indications of the effects of the administered dose are no longer present, and the Board considered that the design of this study which continued intentional dosing without collection of blood samples for cholinesterase analysis was not defensible scientifically. Following considerable discussion, the HSRB concluded that the dosage evaluated in the repeat dosing human study can be used as a LOAEL for the single chemical aggregate risk assessment for DDVP. Although a study using a single dose level is not ideal for establishing a LOAEL, there was general consensus that RBC cholinesterase is a well characterized endpoint for compounds that inhibit acetylcholinesterase activity and therefore, because the decreased activity in RBC cholinesterase activity observed in this study was at or near the limit of what could be distinguished from baseline values, it was unlikely that a lower dose would produce a measurable effect in RBC cholinesterase activity. However, the HSRB strongly recommended that, in most cases, studies in human subjects designed to define the NOAEL or LOAEL for a compound should include more than a single dose level.

b) Results of the DDVP repeat dose human toxicity study should not be used to support reducing the default 10X inter species factor in the cumulative risk assessment of the organophosphate pesticides.

Although the data obtained in this study can be used to establish a point of departure for the single chemical aggregate risk assessment, the consensus of the HSRB was that the scientific limitations of the study design do not justify its use in the cumulative risk assessment. In particular, the lack of sample collection through to the completion of dosing, the lack of a clear demonstration that steady state inhibition of RBC cholinesterase inhibition had been achieved, and the lack of any dose response data limit the overall utility of the human data. Accordingly, the HSRB recommended that the default interspecies uncertainty factor should be applied for the cumulative risk assessment for DDVP.

Charge to the Board b. The Agency has concluded that other human studies made available to the Board do not provide sufficient scientifically sound information to warrant any reduction in the 10X interspecies uncertainty factor used to derive reference dose values for DDVP based on animal toxicity endpoints. 42 of 69 Please comment on the scientific evidence that supports these conclusions.

Board Response to the Charge HSRB Consensus and Rationale The HSRB concluded that the other DDVP human toxicity studies available for the Board's consideration should not be used for determining a reduction in the 10X uncertainty factor to derive reference dose values for DDVP based on animal toxicity endpoints.

The Board's deliberations for this question focused on the acceptability or not of these human studies for the Agency's intended purposes, rather than on the evaluation of the magnitude of the interspecies uncertainty factor. Since the results of animal studies or benchmark dose modeling were not provided to the Board, the review was restricted to the evaluation of the acceptability of the human studies for the intended purposes. The Board concluded these other human studies did not warrant use in determining the inter species uncertainty factor because of policy considerations ( e. g., exposures involving infants, children, pregnant women) or due to uncertainty associated with the study itself ( e. g., inappropriate duration, questionable route, exposure data of limited utility, lack of time course data on critical effect). Board concerns regarding these other human studies are provided in Table 1. 43 of 69 Table 1. Evaluation of Specific DDVP Human Studies Provided to the HSRB Reference Observations/ Concerns Gledhill Acute toxicity study Insufficient time course measurements of cholinesterase Cavagna ( 1970) Conducted in newborns Slomka ( 1981) Exposure through plastic bead ( polyvinyl resin) formulation; rate of absorption not known; data provided as ranges Cervoni ( 1969) Efficacy trial; measurement of cholinesterase inhibition not the main focus; no assurance of consistent methods of measurement of inhibition Leary ( 1974) Exposure via insecticide strips; possibility children were involved, but composition of families not given; inhalation route   caveat of dose calculations; no information on consent of subjects Smith ( 1972) Performed in altitude chamber; short term inhalation study; no information on consent of subjects Cavagna ( 1969) Exposure via insecticide strips included pregnant women, infants and children in a hospital Pena Chavarria ( 1969) Exposure of patients ( most of them anemic, treated for parasite infection); not designed to identify NOAEL or LOAEL Stein ( 1966) Insufficient information on the time course of cholinesterase inhibition; doses not quantitated Funckes ( 1963) Study population included children, some below six years; time course evaluation on inhibition insufficient Witter ( 1961) Simulated aircraft cabin; one hour exposures; time course information lacking Gratz ( 1963) Inadequate information to evaluate dose and NOAEL; inadequate information about how many people exposed at each concentration; single application based exposures 5.2. Ethical considerations Charge to the Board a. The Agency requested that the Board provide comment on the following: 44 of 69 Whether references to the test material as a drug and other statements that could indicate the study constituted medical research, that appear in the materials used to obtain informed consent should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted;

Whether the administration of the test material for three additional days without monitoring subjects' cholinesterase levels following the detection of cholinesterase inhibition greater than 20 % in some subjects should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and Whether the lack of medical surveillance of subjects, following the termination of dosing, to establish the subjects' cholinesterase levels returned to normal should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted; and b. The Agency asked that the Board provide comment on the following, taking into account all that is known about the ethical conduct of the Gledhill repeated dose study:

OPP's conclusion that there was not clear and convincing evidence that the conduct of the research was fundamentally unethical; and Whether there is clear and convincing evidence that the conduct of the Gledhill repeat dose study was significantly deficient relative to the ethical standards prevailing when the study was conducted.

Board Response to the Charge Brief Overview of the Study As noted above, the study was conducted in 1996 by the Zeneca Central Toxicology Laboratory, Cheshire, United Kingdom. The study sponsor was the Amvac Chemical Corporation. The protocol documents specifically state that the research was conducted in compliance with the guideline of the Declaration of Helsinki, 1964, as amended through 1989.

Critique of Study The Board concurred with the Agency's factual observations of the strengths and weaknesses of the study, as detailed in Carley ( 2006e). However, further comments are warranted with regard to: 1) whether the provisions in the protocol for monitoring the subjects adequately protected them, and 2) whether the documentation and process of study subject enrollment was sufficient to meet prevailing standards of voluntary informed consent.

1. Monitoring of Subjects While Receiving Study Compound The protocol permitted subjects to continue to be administered the study compound for three days following the detection of RBC cholinesterase inhibition of greater than 20%. Given 45 of 69 that study subjects were not monitored during most of the study ( and were not full time residents at the study laboratory), it was possible that they might develop clinically significant symptoms during this period and no one would be available to detect that and to treat them. The Board concluded that this aspect of the study design was inadequate. However, given that there had been a prior similar study at triple the dose used in this study, and that study had demonstrated minimal symptoms, the Board could not conclude that there was clear and convincing evidence that this element of the study design could have resulted in serious harm based on the knowledge available to the investigators at the time.

2. Monitoring of Subjects at End of Study The protocol also provided that, at the end of the study, inhibition levels would not be retested for periods as long as seven days even though some subjects had RBC cholinesterase inhibition of greater than 20%. During this period of time, due to the long acting nature of the study compound, subjects' cholinesterase inhibition levels could have been increasing, and thus symptoms might have developed and, theoretically have become serious at a time when they were being infrequently monitored. The Board determined that this aspect of the study design was inadequate. However, as noted above, given that there had been a prior similar study at triple the dose used in this study, and that study had demonstrated minimal symptoms, the Board could not conclude that there was clear and convincing evidence that this element of the study design could have resulted in serious harm based on the knowledge available to the investigators at the time.

The inadequate monitoring during and at the end of the study undermines the scientific value of the study. The fact that the research subjects were unsupervised during the study, and the ascertainment of adverse event data was through self report, renders the data establishing a NOAEL suspect. The risk of a " false negative" conclusion is quite high. In addition, the failure to achieve a steady state in the inhibition of acetyl cholinesterase activity may render these data inadequate as well, and of limited utility. This fact, also noted in the Board's scientific review, can negatively influence the prospective benefit to risk ratio.

3. Informed Consent It was clear that the written documentation for informed consent failed to fully meet the standards outlined of the 1989 version of the Declaration of Helsinki.

In particular, the information sheet that was given to research subjects as an appendix to the signed informed consent document under the subheading Possible Adverse Events noted that if " any symptoms possibly due to enzyme blocking did occur, they could rapidly be reversed by a specific antidote." Although that statement is a correct statement of the scientific issue, it failed to take into account the fact that this study did not involve subjects remaining under 24 hour supervision. For most of the time, the subjects would not be under any supervision. Given that enzyme inhibition could build up over time due to the long acting nature of the compound being studied, symptoms could likely occur at a time when there was no one available to administer the antidote. Telling the subjects that an antidote could rapidly reverse symptoms without explaining 46 of 69 that there were circumstances in which that antidote would not be readily available was not an adequate way to inform subjects of the true nature of this risk.

In hindsight, it is clear that the documentation provided failed to rigorously meet the standards of voluntary informed consent applicable to studies conducted in the United Kingdom in 1996. However, there was not clear and convincing evidence that these deficiencies knowingly and seriously impaired the informed consent process.

HSRB Consensus and Rationale The Board concluded that:

a) The DDVP repeat dose oral human toxicity study failed to fully meet the specific ethical standards prevalent at the time the research was conducted.

b) There was no clear and convincing evidence that the research was fundamentally unethical intended to seriously harm participants or that informed consent was not obtained.

c) There was no clear and convincing evidence of significant deficiencies in the ethical procedures that could have resulted in serious harm ( based on the knowledge available at the time the study was conducted) nor that information provided to participants seriously impaired their informed consent.

The Board determined that there was not clear and convincing evidence that references to the test material as a drug and other statements that could indicate the study constituted medical research, that appear in the materials used to obtain informed consent should be considered significantly deficient relative to the ethical standards prevailing when the study was conducted. These statements are highly undesirable, and should not be used as part of modern day practice in writing such consent materials. Nonetheless, it was unfortunately not uncommon for such wording to have been used in consent materials in the past. In addition, other wording in the consent materials clearly advised subjects that this was a study involving consuming an insecticide. The Board, however, specifically rejects the arguments of the study sponsor that it was appropriate to use the " drug" terminology because the consent materials were generic materials used for a variety of studies. It also rejects the study sponsor's argument that because the test material is sometimes used as a drug, it was appropriate to use that terminology in this study even though this study in no way related to its use as a drug.

For the reasons discussed above, the Board determined that there was not clear and convincing evidence that the administration of the test material for three additional days without monitoring subjects' cholinesterase levels following the detection of cholinesterase inhibition greater than 20 % in some subjects could have led to serious harm. There the study should not be considered significantly deficient relative to the ethical standards prevailing when the study was conducted.

For the reasons discussed above, the Board determined that there was not clear and convincing evidence that following the termination of dosing, the lack of medical surveillance to 47 of 69 establish the subjects' cholinesterase levels returned to normal could have led to serious harm. Therefore the study should not be considered significantly deficient relative to the ethical standards prevailing when the study was conducted.

The Board concluded that the Agency can rely on the results of this study, as appropriate under current pesticide laws, given the absence of clear and convincing evidence that the research was fundamentally unethical or significantly deficient relative to the ethical standards prevailing when the study was conducted.

6. Ethephon Charge to the Board Ethephon is an organophosphorus compound that, upon absorption into plants, forms ethylene gas which is an important component of the plant hormone complex. The Agency is conducting an aggregate ( single chemical, multi route) risk assessment of ethephon.

Scientific consideration The Agency's WOE document and DERs for ethephon describe the study design and results of the ethephon repeat dose, oral, human toxicity studies. The WOE document also discusses the Agency's conclusions regarding the usefulness of the human studies in the aggregate, single chemical risk assessment. The Agency has concluded that the 28 day human study is sufficiently robust to establish a point of departure for extrapolating acute and chronic dietary risk.

Please comment on the scientific evidence that supports this conclusion.

Board Response to the Charge Brief Overview of the Study According to the Agency's weight of evidence document ( WOE) ( Kent 2006) , " ethephon is an organophosphorus compound that, upon absorption into plants, forms ethylene gas which is an important component of the plant hormone complex. The production of ethylene by plants occurs naturally as crops mature but it can be slow during periods of unfavorable weather. Ethylene generated from ethephon is absorbed by the plant tissues and moderates the growth process. It is not similar in structure to other organophosphate pesticides. Ethephon registrants have submitted many toxicity studies to support the continued pesticidal use of the chemical, and among these studies are two involving direct dosing of human subjects that OPP is considering using to establish endpoints to assess risk from exposure to ethephon. The WOE document compares the strengths and weaknesses of the human and animal toxicity studies and discusses how the human studies fit in with the animal studies, i. e., are the human data consistent with animal data in terms of types of effects and effect levels or are there notable differences between animals and humans." 48 of 69 According to the WOE document, " as a strong acid, ethephon is corrosive to the skin and eyes ( Category I). In acute and subchronic neurotoxicity studies in rats, there were signs of neurotoxicity at doses of 500 mg/ kg and above ( acute) and 300 mg/ kg ( subchronic). In dogs there were no clinical signs of neurotoxicity evident at 75 mg/ kg/ day although plasma and RBC cholinesterase were inhibited in animals at doses as low as 7.5 mg/ kg/ day."

There were two human studies under consideration, both involving direct dosing of human subjects and are repeated dose oral studies, one for 28 days duration ( Reese 1972) and the other for 16 days ( Weir 1977).

28 Day Oral Toxicity Study In a non guideline oral toxicity human study ( Reese 1972), ethephon was administered by capsule in a powdered formulation ( 10% a. i. weight/ weight) of hydrated silica and starch to human adults ( 5/ sex) at an average dose of 1.8 mg a. i./ kg/ day or placebo 0 mg/ kg/ day ( 3/ sex) for 28 consecutive days. Administration of the test material or the placebo was given orally by capsule divided into three daily doses. Each subject received 2 capsules postprandially for the first two dosing periods; the third dose was given at the end of the work day in order to simulate as closely as possible ingestion of the material as a crop residue. The capsules were dispensed daily ( 5 days/ week) by an assistant who observed the subjects while they took each capsule and recorded solicited comments related to the ingestion of the material during the first 8 hours. The subjects and the assistant did not know which capsules were placebo or the test material. Each Friday, the subjects received a supply of capsules to be taken during the two day weekend. All subjects were monitored regularly for adverse effects. Hematology measurements, clinical chemistry and urinalysis were conducted initially and on Days 7, 14, 21, 28 and 2 weeks following the last administration. ChE ( plasma and RBC) determinations were conducted using the   pH/ hr method ( modified Michel method) initially, and on Days 1, 2, 7, 14, 28 and 2 weeks after the last administration.