Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 1 of 36 EPA HSRB 06 02 1 2 George Gray, Ph. D. 3 Science Advisor 4 Office of the Science Advisor 5 1200 Pennsylvania Avenue, NW 6 Washington, DC 20460 7 8 Subject: May 2 3, 2006 EPA Human Studies Review Board Meeting Report 9 10 Dear Dr. Gray: 11 12 The United States Environmental Protection Agency ( EPA or Agency) requested the 13 Human Studies Review Board ( HSRB) to review scientific and ethics reviews of chromium, 14 carbofuran and methyl isothiocyanate. The enclosed HSRB report addresses the Board's 15 response to EPA charge questions for the Board's consideration at its May 2 3, 2006 meeting. 16 17 A summary of the Board's conclusions on the scientific and ethical considerations of the 18 human toxicity studies for the three pesticides are provided below. 19 20 Chromium 21 22 Scientific Considerations 23   The Board concluded that the 1994 Nethercott et al. dermal sensitization study was 24 sufficiently sound, from a scientific perspective, to be used to estimate a safe level of 25 dermal exposure to hexavalent chromium. 26 27   The study was properly designed, well conducted, and employed appropriate scientific 28 and clinical methods to determine a minimum elicitation threshold for dermal 29 sensitization due to hexavalent chromium. The MET10 reported in the study provided a 30 reasonable point of departure for risk assessment. 31 32 Ethical Considerations 33   The HSRB concluded there was insufficient information to determine whether the study 34 failed to fully meet specific ethical standards prevalent at the time the research was 35 conducted. 36 37   The Board concurred with the assessment of the Agency that there was no clear and 38 convincing evidence that the conduct of the research was fundamentally unethical in that 39 the deficiencies did not result in serious harm, nor seriously impair the informed consent 40 of the research subjects and; 41 42   The Board determined that there was not clear and convincing evidence that the conduct 43 of the study was significantly deficient relative to the ethical standards prevailing when 44 the study was conducted. 45 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 2 of 36 Carbofuran 1 2 Scientific Considerations 3 4   The HSRB concluded that there were numerous technical issues regarding the conduct of 5 the oral and dermal studies with carbofuran and that overall, the weakness of the studies 6 far outweigh the strengths. Accordingly, the HSRB did not recommend any of the oral or 7 dermal studies conducted with carbofuran in human subjects for the single chemical 8 assessment or in informing the interspecies uncertainty factor for the cumulative 9 assessment. 10 11 Ethical Considerations 12   For the oral study, there was no evidence that the study failed to fully meet specific 13 ethical standards prevalent at the time the research was conducted. 14 15   For the oral human toxicity study, there was no clear and convincing evidence of 16 significant deficiencies in the ethical procedures that could have resulted in serious harm 17 ( based on the knowledge available at the time the study was conducted) nor that 18 information provided to participants seriously impaired their informed consent. 19 20   For the oral study there was no clear and convincing evidence that the research was 21 fundamentally unethical ( e. g., intended to seriously harm participants or that informed 22 consent was not obtained). 23 24   The HSRB found deficiencies in both dermal human toxicity studies relative to specific 25 ethical standards prevalent at the time the study was conducted. 26 27   For both dermal human toxicity studies, there was clear and convincing evidence of 28 significant deficiencies in the ethical procedures for minimizing risk that could have 29 resulted in serious harm ( based on the knowledge available at the time the study was 30 conducted). The first dermal toxicity study was significantly deficient given the delay in 31 the administration of atropine to more than one subject experiencing the signs and 32 symptoms of carbamate toxicity. The second dermal toxicity study was considered 33 significantly deficient in that the lack of information provided about the results from the 34 initial dermal toxicity study seriously impaired their informed consent. 35 36   However, for both dermal human toxicity studies, there was no clear and convincing 37 evidence that the research was fundamentally unethical ( e. g., intended to seriously harm 38 participants or that informed consent was not obtained). 39 40 41 Methyl Isothiocyanate 42 43 Scientific Considerations 44   The Board concluded that air concentrations of methyl isothiocyanate sufficient to 45 produce eye irritation would lead to a conservative and prudent point of departure for 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 3 of 36 inhalation risk ( i. e., eyes were a sensitive endpoint in relation to the respiratory system). 1 The Board reached it decision based on eye irritation LOAELs are often lower than 2 respiratory irritation LOAELs for irritant gases. While the use of eye irritation data as a 3 surrogate for respiratory data is reasonable in this situation, one must be cautious as only 4 appropriate controlled human studies of the respiratory system can provide a final and 5 definitive respiratory point of departure, if ever determined. 6 7 Ethical Considerations 8   The HSRB determined there were minor deficiencies in the ethical procedures relative to 9 those prevalent at the time, however: 10 11   There was no clear and convincing evidence that the conduct of the research was 12 fundamentally unethical ( e. g., the research was intended to seriously harm participants or 13 failed to obtain informed consent) and; 14 15   There was no clear and convincing evidence that the conduct of the study was 16 significantly deficient relative to the ethical standards prevailing when the study was 17 conducted. 18 19 The Board also provided commentary of its scientific criteria for review of human dosing 20 studies. The Board's criteria encompassed the following: ( 1) justification; ( 2) dose selection; ( 3) 21 endpoint selection; ( 4) participants; ( 5) method; and ( 6) statistical analyses. In addition, the 22 Board established criteria for evaluating the utility of single dose level studies. 23 24 In conclusion, the EPA HSRB appreciated the opportunity to advise the Agency on the 25 scientific and ethical aspects of human subjects research and looks forward to future 26 opportunities to continue advising the Agency in this endeavor. 27 28 29 Sincerely, 30 31 32 33 Celia Fisher, Ph. D. Chair 34 EPA Human Studies Review Board 35 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 4 of 36 NOTICE 1 2 This report has been written as part of the activities of the EPA Human Studies Review 3 Board, a Federal advisory committee providing advice, information and recommendations on 4 issues related to scientific and ethical aspects of human subjects research. This report has not 5 been reviewed for approval by the Agency and, hence, the contents of this report do not 6 necessarily represent the view and policies of the Environmental Protection Agency, nor of other 7 agencies in the Executive Branch of the Federal government, nor does mention of trade names or 8 commercial product constitute a recommendation for use. Further information about the EPA 9 Human Studies Review Board can be obtained from its website at http:// www. epa. gov/ osa/ hsrb/. 10 Interested persons are invited to contact Paul Lewis, Designated Federal Officer, via e mail at 11 lewis. paul@ epa. gov. 12 13 In preparing this document, the Board carefully considered all information provided and 14 presented by the Agency presenters, as well as information presented by public commenters. 15 This document addresses the information provided and presented within the structure of the 16 charge by the Agency. 17 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 5 of 36 United States Environmental Protection Agency Human Studies Review Board 1 2 Chair 3 4 Celia B. Fisher, Ph. D. Marie Ward Doty Professor of Psychology, Director, Center for Ethics 5 Education, Fordham University, Department of Psychology, Bronx, NY 6 7 Vice Chair 8 9 William S. Brimijoin, Ph. D., Chair and Professor, Molecular Pharmacology and Experimental 10 Therapeutics, Mayo Foundation, Rochester, MN * ** 11 12 Members 13 14 David C. Bellinger, Ph. D., Professor of Neurology, Harvard Medical School 15 Professor in the Department of Environmental Health, Harvard School of Public Health 16 Children's Hospital, Boston, MA 17 18 Alicia Carriquiry, Ph. D., Professor, Department of Statistics, Iowa State University 19 Snedecor Hall, Ames, IA 20 21 Gary L. Chadwick, PharmD, MPH, CIP, Associate Provost, Director, Office for Human Subjects 22 Protection, University of Rochester, Rochester, NY 23 24 Janice Chambers, Ph. D., D. A. B. T., William L. Giles Distinguished Professor, Director, Center 25 for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State 26 University, Wise Center, Mississippi State, MS * 27 28 Richard Fenske, Ph. D., MPH, Professor, Department of Environmental and Occupational Health 29 Sciences, University of Washington, Seattle WA 30 31 Susan S. Fish, PharmD, MPH, Professor, Biostatistics & Epidemiology, Boston University 32 School of Public Health, Co Director, MA in Clinical Investigation 33 Boston University School of Medicine, Boston, MA 34 35 Suzanne C. Fitzpatrick, Ph. D., DABT, Senior Science Policy Analyst, Office of the 36 Commissioner, Office of Science and Health Coordination, U. S. Food and Drug Administration, 37 Rockville, MD 38 39 Kannan Krishnan, Ph. D., Professor, Département de santé environnementale et santé au travail, 40 Faculté de medicine, Université de Montréal, Montréal, Canada 41 42 KyungMann Kim, Ph. D., CCRP, Professor & Associate Chair, Department of Biostatistics & 43 Medical Informatics, School of Medicine and Public Health, University of Wisconsin Madison, 44 Madison, WI ** 45 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 6 of 36 Michael D. Lebowitz, Ph. D., FCCP, Professor of Public Health & Medicine. University of 1 Arizona, Tucson, AZ 2 3 Lois D. Lehman Mckeeman, Ph. D., Distinguished Research Fellow, Discovery Toxicology, 4 Bristol Myers Squibb Company, Princeton, NJ 5 6 Jerry A. Menikoff, M. D., Associate Professor of Law, Ethics & Medicine, Director of the 7 Institute for Bioethics, Law and Public Policy, University of Kansas Medical Center, 8 Kansas City, KS 9 10 Robert Nelson, M. D., Ph. D., Associate Professor of Anesthesiology and Critical Care, 11 Department of Anesthesiology and Critical Care, University of Pennsylvania School of 12 Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104 13 14 Sean M. Philpott, Ph. D., Research Scientist, David Axelrod Institute, Wadsworth Center for 15 Laboratories and Research, New York State Department of Health, Albany, NY 16 17 Human Studies Review Board Staff 18 19 Paul I. Lewis, Ph. D., Designated Federal Officer, United States Environmental Protection 20 Agency, Washington, DC 21 22 * Recused from carbofuran discussion and deliberation 23 ** Not in attendance at May 2 3, 2006 Public Meeting 24 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 7 of 36 TABLE OF CONTENTS 1 2 INTRODUCTION...................................................................................................................... 8 3 REVIEW PROCESS................................................................................................................... 9 4 CHARGE TO THE BOARD AND BOARD RESPONSE......................................................... 10 5 1. Chromium......................................................................................................................... 10 6 2. Carbofuran ....................................................................................................................... 16 7 3. Methyl Isothiocyanate ....................................................................................................... 26 8 COMMENTARY ON SCIENTIFIC STANDARDS FOR HUMAN DOSING STUDIES......... 33 9 REFERENCES......................................................................................................................... 35 10 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 8 of 36 INTRODUCTION 1 2 On May 2 3, 2006, the United States Environmental Protection Agency's ( EPA or 3 Agency) Human Studies Review Board ( HSRB) met to review scientific and ethical issues 4 concerning human toxicity studies involving two pesticide active ingredients, carbofuran and 5 methyl isothiocyanate ( MITC), and chromium, a constituent of wood preservative products 6 ( wood preservatives are regulated as pesticides under the Federal Insecticide, Fungicide, and 7 Rodenticide Act). 8 9 The Pesticide Registration Improvement Act ( PRIA) requires that EPA complete its 10 decision making process on certain types of applications to register a pesticide product within 11 specified amounts of time after receiving the application for registration. In addition, PRIA 12 established deadlines for EPA to complete " reregistration" of pesticide active ingredients that are 13 contained in pesticide products initially registered before 1984. Reregistration involves the 14 systematic reexamination of older pesticides, applying contemporary scientific and regulatory 15 standards. When a pesticide active ingredient is approved for use on food, EPA combines 16 reregistration with the tolerance reassessment process mandated by the Food Quality Protection 17 Act of 1996 ( FQPA). 18 19 Both MITC and carbofuran are undergoing reevaluation in the reregistration process. 20 EPA is considering the human health risks of chromium both in its reregistration program and as 21 part of its review of an application for registration pending under FIFRA and PRIA. 22 23 For each of the human studies under consideration, the Agency provided the Board with 24 the complete study report and any supplements available to the Agency. Each of these studies 25 was assigned a unique identifier, the Master Record Identifier or MRID, which the EPA, Office 26 of Pesticide Programs ( OPP) uses to manage documents in its archive. When a company submits 27 multiple documents pertaining to a single study, each document is assigned a unique MRID as it 28 is received and catalogued. Thus a study with several supplements, such as the MITC study 29 discussed at the meeting, may be associated with several MRIDs. 30 31 For each study, the Agency had provided a review of the ethical conduct of the study. 32 Each ethics review identified any deficiencies noted in the conduct of the specific study 33 compared to both current ethical standards and the ethical standards prevailing at the time the 34 research was performed. EPA has intentionally deferred making a final determination of 35 whether an individual study satisfies the ethical standards for acceptability in 40 CFR sections 36 26.1704   26.1706, pending the advice of the Board. 37 38 For most studies, the Agency develops documents, called Data Evaluation Records 39 ( DERs), containing a scientific review of the study; the Board was provided with one or more 40 DERs for carbofuran and MITC. DERs contain summaries of the study design, methods and 41 results, describe potential deficiencies, and provides conclusions about the usefulness of the 42 study in risk assessment. 43 44 In addition to the DERs, the Agency had prepared a Weight of Evidence ( WOE) 45 memorandum for carbofuran and MITC discussing the differences and similarities between the 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 9 of 36 human and animal responses to each chemical and characterizing the usefulness of the human 1 toxicity studies for human health risk assessment. The WOE memos expressed the Agency's 2 current scientific conclusions on which the Agency was soliciting the Board's comments. To 3 maintain the historical record of review, the Agency may, in some cases, include a DER for a 4 study that expressed scientific conclusions differing from those in the WOE document. 5 6 For chromium, the Agency provided a set of documents which contained similar 7 information to DERs and WOEs, but which had a slightly different format and presentation, due 8 to the procedural history of the EPA's review of this chemical. As noted above, chromium is a 9 constituent in wood preservative products. The Agency has concern about the potential for 10 chromium to elicit an allergic response in sensitized individuals who come in contact with 11 residues remaining in products made from wood that has been treated with chromium containing 12 wood preservatives. To assess the risk of potential dermal exposure, the Agency reviewed, 13 among other information, a study involving intentional exposure of sensitized subjects to 14 different levels of chromium, ( Nethercott et al. 1994). This assessment was one of the first 15 assessments of this kind performed by the Agency, and it raised significant scientific issues. 16 Accordingly, the Agency prepared a background document for its independent, peer review 17 advisory committee, the FIFRA Scientific Advisory Panel ( SAP). The SAP is a federally 18 chartered advisory committee of scientific experts who provide advice to the Agency on 19 pesticides and pesticide related issues as to their impact on human health and the environment of 20 regulatory actions. The Agency provided a copy of the materials given to the SAP for its review, 21 as well as a copy of the SAP's final report. After receiving the SAP's recommendations, the 22 Agency sought review and comment from other Agency scientists through the steering 23 committee of the Agency's internal Science Policy Council ( SPC) to ensure consistency across 24 programs in the approach to regulating substances that are skin sensitizers. Using the advice of 25 the SAP and the steering committee of the SPC, the Agency developed a memorandum 26 describing how it intended to use the results of the Nethercott study to derive a sensitization 27 Reference Dose. 28 29 The HSRB has reviewed studies on which the Agency proposes to rely in actions under 30 the pesticide laws and studies that the Agency has decided not to use in its risk assessments, 31 either for scientific reasons or because they do not meet the standards in EPA's final human 32 studies rule, 40 CFR Part 26. The Agency asked the HSRB to advise the Agency on a range of 33 scientific and ethics issues and on how the studies should be assessed against the provisions in 40 34 CFR sections 26.1701   26.1704 of EPA's final human studies rule. This report transmits the 35 HSRB's comments and recommendations from its May 2 3, 2006 meeting. 36 REVIEW PROCESS 37 38 On May 2 3, 2006 the Board had a public face to face meeting in Arlington, Virginia. 39 Advance notice of the meeting was published in the Federal Register " Human Studies Review 40 Board: Notice of Public Meeting ( 71 Federal Register 19725). At the public meeting, following 41 welcoming remarks from Agency officials, Celia B. Fisher, HRSB Chair, proposed a set of 42 scientific and ethics criteria consistent with the language of 71 Federal Register 6137 to guide 43 Board evaluation of each protocol. The Chair's scientific criteria asked the Board to consider the 44 following two questions: ( 1) did the research design and implementation meet scientific 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 10 of 36 standards and ( 2) Did the data generated by the study have implications for the Agency's Weight 1 of the Evidence ( WOE) review and when applicable aspects of the risk assessment? The Chair's 2 ethics criteria asked the Board to consider three questions: ( 1) did the study fail to fully meet 3 specific ethical standards prevalent at the time the research was conducted; ( 2) was the conduct 4 of the study fundamentally unethical ( i. e., specifically was there clear and convincing evidence 5 that the research was intended to seriously harm participants or failed to obtain informed 6 consent); and ( 3) was the conduct of the study significantly deficient relative to the ethical 7 standards prevailing at the time ( i. e., was there clear and convincing evidence that identified 8 deficiencies identified could have resulted in serious harm based on knowledge available at the 9 time the study was conducted or the information provided to participants could seriously impair 10 informed consent). The Board then heard presentations from the Agency on the following 11 topics: science and ethics of chromium human studies, science and ethics of carbofuran human 12 studies and science and ethics of methyl isothiocyanate human studies. The Board heard oral 13 public comments from the following individuals: 14 15 Chromium 16 Jennifer Sass, Ph. D. representing the Natural Resources Defense Council 17 18 Carbofuran 19 Donald Carson, Ph. D. and Ms. Jane McCarty on behalf of FMC Corporation 20 Jennifer Sass, Ph. D. representing the Natural Resources Defense Council 21 22 In addition, the Board received written public comments from CRLA Foundation, FMC 23 Corporation, and the Natural Resources Defense Council. Following Agency presentations and 24 public comments, the Board deliberated on the charge questions. For their deliberations, the 25 Board considered the materials presented at the meeting, written public comments and Agency 26 background documents on each individual pesticide ( i. e., pesticide human study, Agency data 27 evaluation record ( DER) of the pesticide human study, weight of evidence review, risk 28 assessment and ethics review). 29 CHARGE TO THE BOARD AND BOARD RESPONSE 30 31 1. Chromium 32 33 Charge to the Board 34 35 Hexavalent chromium is a component of a pesticide product intended to be used as a 36 wood preservative. Members of the general public may experience dermal exposure to residues 37 of hexavalent chromium remaining on wood treated with a wood preservative. Because 38 chromium has caused allergic contact dermatitis ( ACD) in occupational settings, 39 EPA has determined that it should assess the potential for ACD in the general public resulting 40 from the use of wood preservatives containing chromium. 41 42 In a meeting of the FIFRA Scientific Advisory Panel ( SAP) in May 2004, EPA obtained 43 independent peer review of scientific issues related to the assessment of the potential dermal risk 44 resulting from exposure to chromium. See www. epa. gov/ scipoly/ sap/ 2004/ final. doc The Agency 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 11 of 36 had carefully considered the report of the SAP, as well as the advice of EPA scientists through 1 the steering committee of the Agency's Science Policy Council. Taking all of this into account, 2 EPA had derived a " sensitization reference dose" ( RfD) based on the 10% Minimum Elicitation 3 Threshold ( MET 10) and use of a 10 fold uncertainty factor for potential variability within the 4 human population and other uncertainties. See ADTC Memorandum, " Hexavalent Chromium  5 Finalization of Issues related to Quantitation of Dermal Risk from exposure to treated wood 6 containing hexavalent chromium," August 31, 2004. 7 8 Scientific considerations 9 10 EPA had identified a study performed with subjects who had documented sensitivity to 11 chromium ( Nethercott, et al., 1994). The study was conducted to identify a level of exposure to 12 chromium below which dermal exposure did not appear to elicit an ACD response. Regarding 13 the Nethercott human study, the Agency had concluded that the study contains information 14 sufficient for assessing human risk resulting from potential dermal exposure. 15 16 Please comment on whether the Nethercott study was sufficiently sound, from a scientific 17 perspective, to be used to estimate a safe level of dermal exposure to hexavalent chromium. 18 19 Board Response to the Charge 20 21 Introduction 22 23 Hexavalent chromium ( CrVI) is known to cause allergic contact dermatitis ( ACD). CrVI 24 is a component of a pesticide product used as a wood preservative, and members of the general 25 public may be exposed through contact with treated wood. ACD is a delayed, immunologically 26 mediated, inflammatory skin disease consisting of various degrees of erythema, edema, and 27 vesiculation. ACD is typically characterized by two phases, termed induction and elicitation. 28 Induction occurs when there was an exposure of sufficient magnitude and/ or duration to activate 29 specific immune mechanisms resulting in the acquisition of sensitization, while elicitation occurs 30 from a subsequent exposure to the same chemical allergen. In general, the amount of allergen 31 exposure needed to produce induction is greater than that needed to produce elicitation in 32 previously sensitized individuals. Thus, the study of elicitation can provide an appropriate 33 critical endpoint for risk assessments. One approach to estimate an acceptable area dose for 34 protection against elicitation is the determination of a minimum elicitation threshold, or MET. 35 The concept behind the MET is that there was an elicitation threshold below which no 36 sensitization reaction is expected. 37 38 The EPA FIFRA Scientific Advisory Panel met in May 2004 to review human and 39 animal studies related to CrVI ( SAP, 2004). In August, 2004 the Agency's Antimicrobials 40 Division Toxicity Endpoint Selection Committee issued a memorandum that summarized its 41 assessment of dermal risk from CrVI ( ADTC, 2004). The Agency identified a study performed 42 with human subjects who had documented sensitivity to chromium ( Nethercott et al., 1994). The 43 study was conducted to identify a level of exposure to chromium below which dermal exposure 44 did not appear to elicit an ACD response. Regarding the Nethercott et al. study, the Agency had 45 concluded that the study contained information sufficient for assessing human risk resulting from 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 12 of 36 potential dermal exposure. The Agency had asked the HSRB to comment on whether this study 1 was sufficiently sound, from a scientific perspective, to be used to estimate a safe level of dermal 2 exposure to hexavalent chromium. 3 4 Critique of the 1994 Nethercott et al. Study 5 6 The purpose of the study was to determine the MET as mass of allergen per skin surface 7 area for CrVI by a patch testing technique. The study also included response to CRIII, but these 8 data were not discussed here. Five concentration levels of CrVI ( 4.4, 0.88, 0.18, 0.088, 0.018 9 µ g/ cm2) was used in the patch test, and " TRUE Test" patches were manufactured specifically for 10 use in the study to reduce the variability inherent in earlier patch preparation methods. The 11 highest concentration ( 4.4 µ g/ cm2) were used as a screening concentration to identify those who 12 were sensitized to CrVI. This first round of testing involved 102 volunteers ( 78 men and 24 13 women) previously shown to be sensitive to developing allergic contact dermatitis ( ACD) in 14 response to an allergen. CrVI elicited ACD in 54 ( 39 men and 15 women) of these 102 subjects. 15 Two lower concentrations ( 0.018 and 0.088 µ g/ cm2) were tested in these 54 volunteers in round 16 two. Those who had no ACD response during round two were tested with the next two higher 17 concentrations ( 0.18 and 0.88 µ g/ cm2) in round three. These concentrations were chosen to 18 provide a maximal ACD response. The study was double blind as to concentration, and each 19 concentration was matched with a control ( placebo) concentration within each volunteer. Patch 20 concentrations were validated analytically and found to be within Contract Laboratory Procedure 21 criteria for acceptability. The serial escalation of patch concentration level permitted the authors 22 to determine a dose response relationship and to calculate MET values. The authors calculated a 23 10% minimum elicitation threshold ( MET10) of 0.089 µ g/ cm2. 24 25 This study had a number of strengths. It involved both sexes, the study concentrations 26 were selected carefully based on previous studies, and the investigators determined a priori what 27 sample size and dosing group size were needed to establish statistical accuracy for the MET10. 28 Many elements of the experimental protocol ( e. g., employment of the control patch, serial 29 increase of the concentration until manifestation of ACD, double blinding of patch concentration 30 levels) were thoughtfully developed. The study was designed in accordance with current 31 scientific standards to address a clearly defined research question, included representative study 32 populations for the endpoint in question, and met requirements for adequate statistical power. It 33 appears to have been conducted in accordance with recognized good clinical practices, including 34 appropriate monitoring for safety. Finally, the study authors reported the design, conduct and 35 analysis very comprehensively. 36 37 There are several questions that can be raised regarding the scientific validity of this 38 study. First, the authors developed a cumulative response curve that included subjects who did 39 not respond to any of the doses presented in rounds two and three. These subjects were assigned 40 a minimum elicitation threshold value of 4.4 µ g/ cm2, although none were tested at doses between 41 0.88 and 4.4 µ g/ cm2. The assignment of this MET value appeared arbitrary, and potentially 42 distorts the shape of the cumulative response curve. However, this use of the high MET value 43 does not affect the calculation of the MET10, and so it was of no consequence to the study's 44 primary conclusion. Second, a recent study by Hansen et al. ( 2003) reported a MET10 of 0.03 45 ug/ cm2 for 18 subjects, a value substantially lower than that reported by Nethercott et al. 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 13 of 36 However, these two studies differed with respect to the reading scale employed. The reading of 1 the tests in the Nethercott et al. study followed rules adopted for the diagnostic patch test; that is 2 to say, the definition of a positive reaction was the appearance of erythema infiltration and 3 papules. This approach was consistent with current international clinical standards. For the 4 Hansen et al. study, the investigators used the same reading scale for diagnostic patch testing, but 5 for definition of thresholds they used any degree of reaction, including erythematous and 6 follicular reactions. The logic for this approach was that at very low concentrations irritation was 7 not an issue, so that the question of threshold was not a diagnostic decision. This more sensitive 8 reading approach, which at present was considered experimental, accounts for the difference in 9 MET10 values reported in these two studies. Third, Nethercott et al. ( 1994) used patches that 10 covered a very small area of skin ( 0.81 cm2). Workers, and presumably members of the public, 11 would typically be exposed over a much larger skin surface area than that used in this study. In 12 their article Nethercott et al. discussed the potential importance of patch surface area, and 13 described an additional experiment with four of the study subjects who had exhibited MET 14 values at 0.88 µ g/ cm2. In this experiment five patches were used for each subject, and the 15 exposure level of CrVI was reduced to 0.18 µ g/ cm2 for each patch. The data that resulted from 16 this experiment were not presented, but the authors stated that " sub MET concentrations of CrVI 17 applied over a larger skin surface area did not elicit the positive responses seen when the MET 18 concentration was applied in the standard patch." Current evidence indicated that the dose per 19 unit area was the most important parameter for studies of this kind. But there was no doubt that if 20 an extended area was exposed, such as the full arm, there may be an effect from absorption of an 21 ACD producing compound. This type of exposure could lead to a systemic contact dermatitis 22 reaction with spreading of the dermatitis to a vesicular palmar eczema, and eventually flexural 23 eczema. Such systemic spreads are well known in relation to major contact dermatitis reactions, 24 as occur in occupational exposures. The Nethercott et al. study, where relatively small skin 25 surface areas were exposed, does not exclude that such effects could happen if larger areas were 26 exposed. 27 28 HSRB Consensus and Rationale 29 30 The Board concluded that the 1994 Nethercott et al. dermal sensitization study was 31 sufficiently sound, from a scientific perspective, to be used to estimate a safe level of dermal 32 exposure to hexavalent chromium. 33 34 The 1994 Nethercott et al. study was properly designed, well conducted, and employed 35 appropriate scientific and clinical methods to determine a minimum elicitation threshold for 36 dermal sensitization due to hexavalent chromium. The MET10 reported in the study provided a 37 reasonable point of departure for risk assessment. 38 39 Charge to the Board 40 41 Ethical considerations 42 43 The Agency requested that the Board provide comment on the following: 44 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 14 of 36 a. Is there clear and convincing evidence that the conduct of the Nethercott study was 1 fundamentally unethical? 2 3 b. Is there clear and convincing evidence that the conduct of the study was significantly 4 deficient relative to the ethical standards prevailing at the time the research was conducted? 5 6 Board Response to the Charge 7 8 Brief Overview of the Study 9 10 A previously published study involving dermal exposure of 102 healthy volunteers to 11 increasing doses of CrVI was evaluated, hereinafter referred to as Nethercott et al. 1994. he study 12 sponsor was unknown, but is likely to be either the Chem Risk Division of McLaren/ Hart 13 Environmental Engineering, Alameda CA, or a client of McLaren/ Hart. The study was 14 conducted in 1992 at five U. S. and one Canadian academic institution: the Cleveland Clinic 15 Foundation ( Cleveland, OH), Johns Hopkins University ( Baltimore, MD), Pennsylvania State 16 University ( Hershey, PA), Stanford University ( Palo Alto, CA), the University of British 17 Columbia ( Vancouver, BC), and the University of Louisville ( Louisville, KY). The study was 18 conducted after the promulgation of federal protections for the protection of human participants 19 in research ( i. e. Common Rule) ( § 45CFR46; adopted by the EPA in 1991 and published at 20 § 40CFR26), so the regulatory requirements of the Common Rule were applicable. Furthermore, 21 all five US academic institutions participating had a valid Multiple Project Assurance of 22 Compliance with U. S. Department of Health and Human Services ( DHHS) Regulations for 23 Protection of Human Research Subjects at the time the study was performed. The University of 24 British Columbia, in contrast, held a Cooperative Project Assurance at that time, allowing its 25 participation in DHHS recognized research programs and documenting the University of British 26 Columbia's commitment to the protection of human research subjects in accordance with 27 § 45CFR46. 28 29 Critique of Study 30 31 The Board concurred with the factual observations of the strengths and weaknesses of the 32 study, as detailed in USEPA ( 2006a). However, further comments were raised regarding: 1) 33 whether the documentation and process of study subject enrollment was sufficient to meet 34 prevailing standards of voluntary informed consent and 2) whether the repeat high dose oral 35 exposure protocols used were designed to minimize risks to study participants. 36 37 1) Voluntary Informed Consent 38 39 The Common Rule provides a comprehensive framework for initial and continuing 40 review of research involving human subjects. In order to ensure that a study like Nethercott et al. 41 was performed ethically, the Common Rule requires that: 1) people who participate as subjects 42 in research are selected equitably and give informed and voluntary written consent; and 2) 43 research involving human subjects be reviewed and approved by an independent oversight group 44 such as an Institutional Review Board ( IRB). As published, however, the Nethercott et al. study 45 did not contain sufficient information for the Board to adequately determine whether or not the 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 15 of 36 informed consent process used to enrolled study participants met the standards outlined in 1 § 45CFR46. All that is known about the informed consent process is that " all volunteers provided 2 their doctors with written consent to participate in the study" ( Nethercott et al. 1994). 3 4 Given the paucity of documentation, the Board concluded there was no evidence that the 5 voluntary informed consent process used failed to meet the regulatory and ethical standards 6 applicable to research conducted in the United States and Canada in 1992. All six academic 7 institutions participating in this study had an assurance of compliance with DHHS Regulations 8 for Protection of Human Research Subjects at the time, requiring independent review of the 9 research protocol and consent documents by IRBs. These review boards were expected to 10 approve a study involving human subjects only if: 1) the risks to subjects were minimized by 11 using procedures which were consistent with sound research design and which do not 12 unnecessarily expose subjects to risk; and 2) the risks to subjects were reasonable in relation to 13 anticipated benefits to subjects, if any, and the importance of the knowledge that may reasonably 14 be expected to result ( see, e. g., § 45CFR46.111). The HSRB believed that it was unlikely that all 15 six of these IRBs would overlook deficiencies in the consent process that would seriously impair 16 the voluntary informed consent of the research subjects. 17 18 2) Minimization of Risks to Study Participants 19 20 The Nethercott et al. study employed a three step exposure protocol. Initially, 102 21 volunteers were screened for hexavalent chromium sensitivity by dermal exposure using a 22 chromium concentration equivalent to the standard dose used in patch testing for skin allergies 23 ( 4.4 µ g Cr( VI)/ cm2). Pregnant women, individuals receiving immunosuppressive or steroid 24 medications, and patients with recent or concurrent dermatological conditions were excluded 25 from study participation. 54 chromium sensitive subjects were identified by Nethercott et al. 26 These chromium sensitive subjects then participated in up to two rounds of additional testing. In 27 the first round, subjects were exposed to 0.018 and 0.088 µ g CrVI/ cm2 using a skin patch 28 approach. Five subjects developed allergic contact dermatitis to one or both of these lower doses; 29 these subjects were excluded from further testing. Subjects who failed to respond to either the 30 0.018 or 0.088 µ g dose, however, were subsequently exposed to ten fold higher doses ( 0.18 or 31 0.88 µ g Cr( VI)/ cm2. 27 subjects developed allergic contact dermatitis to one or both of these 32 higher doses. 33 34 In sensitized individuals, chromium exposure elicits an allergic contact dermatitis similar 35 to a poison oak or poison ivy rash. The result typically is an itching, red rash with bumps or 36 blisters; these transient symptoms usually are mild and can be treated with calamine lotion and 37 hydrocortisone cream. The use of patch testing, even when it knowingly results in allergic 38 contact dermatitis, thus meets the generally accepted definition of minimal risk. Furthermore, Dr. 39 Torkil Menne, a consultant to the HSRB, commented that most studies designed to determine the 40 minimum elicitation threshold to a dermal sensitizing agent like chromium have used a single 41 step protocol in which study subjects were exposed to the entire range of dermal concentrations 42 in a single round of testing. The study exclusion criteria and the use of a three step exposure 43 protocol, involving initial screening of subjects for chromium sensitivity followed by additional 44 rounds of testing, using doses significantly smaller than those routinely employed for allergy 45 testing and excluding reactive study participants from further exposure, seems designed 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 16 of 36 specifically to minimize the risk of serious harm to research participants. Thus, the Board 1 believed that there was not was clear and convincing evidence that these studies could have 2 resulted in serious harm based on the knowledge available to the investigators at the time. 3 4 HSRB Consensus and Rationale 5 6 The Board concurred with the assessment of the Agency that there was no clear and 7 convincing evidence that the conduct of the research was fundamentally unethical in that the 8 deficiencies did not result in serious harm, nor seriously impair the informed consent of the 9 research subjects. 10 11 The Board determined that there was no clear and convincing evidence that the conduct 12 of the study was significantly deficient relative to the ethical standards prevailing when the study 13 was conducted. 14 15 The Board based these two determinations on its conclusion that this study appeared to 16 have not deviated significantly from the ethical standards prevailing when the study was 17 conducted. However, this conclusion was based, in part, on a process that was hampered by a 18 lack of supporting documentation concerning independent ethical review by the study 19 investigators' home institutions. The Board strongly recommended that for all studies submitted 20 to the HSRB, the Agency make a good faith effort to obtain such documentation in the future. 21 22 2. Carbofuran 23 24 Charge to the Board 25 26 Carbofuran is an N methyl carbamate ( NMC) pesticide whose primary toxic effect is 27 neurotoxicity caused by the inhibition of the enzyme, acetylcholinesterase, via carbamylation 28 followed by rapid recovery. Carbofuran can, at sufficiently high doses, lead to a variety of 29 clinical signs. The Agency is conducting acute, aggregate ( single chemical, multi route) and 30 worker risk assessments of carbofuran. In addition, carbofuran is a member of the N methyl 31 carbamate common mechanism group and is thus included in the cumulative ( multi chemical, 32 multi route) risk assessment for the NMCs. 33 34 Scientific considerations: 35 36 The Agency's WOE document and DERs for carbofuran described the study design and 37 results of a carbofuran human oral study and two human dermal toxicity studies. The WOE 38 document also discusses the Agency's conclusions that these studies were useful in establishing 39 points of departure, both oral and dermal, for the single chemical assessment and in informing 40 the interspecies uncertainty factor for the cumulative assessment. 41 42 Please comment on the scientific evidence that supports these conclusions. 43 44 Board Response to the Charge 45 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 17 of 36 Study Overview 1 2 Three separate studies ( one oral, two dermal) were carried out with carbofuran in human 3 subjects. The study details are described separately below. 4 5 Overview of Oral Study 6 7 The oral study conducted with carbofuran was carried out in nine healthy male volunteers 8 using an ascending dose schedule and single doses of 0.05, 0.1 and 0.25 mg/ kg ( 1976). The goal 9 of this study was to determine the threshold for toxicity following a single oral dose. Initially, the 10 study was conducted in an open design ( subject and investigator knew that carbofuran was 11 ingested) until a dose level produced symptoms determined to be intolerable ( described below). 12 Once the intolerable dose was achieved ( 0.25 mg/ kg), the study was completed in a randomized, 13 double blind manner. Carbofuran was administered as a single dose in a capsule immediately 14 following breakfast, after which subjects remained under observation for 24 hours. Blood 15 samples were collected for analysis of plasma and RBC cholinesterase activity at 0.5, 1, 2, 3, 6 16 and 24 hours after dosing. The baseline level of RBC cholinesterase activity was established 17 from a predose sample collected immediately prior to dosing. For each subject, additional 18 physiological parameters including ECG, blood pressure, pupil size and accommodation and the 19 Fukuda step test were collected, and subjects were monitored continuously for additional 20 symptoms of toxicity, including sweating, salivation, headaches and nausea and vomiting 21 throughout the 24 hour post dosing period. A complete clinical chemistry profile was performed 22 predose and at 24 hours. The next highest dose was not initiated until data from the 24 hour 23 post treatment period were evaluated. Plasma and RBC cholinesterase levels were determined 24 using a modification of the Ellman colorimetric method with propionylthiocholine as substrate. 25 Subjects were allowed to smoke during the 24 hour sample collection period. 26 27 After administration of the 0.05 mg/ kg dose ( 2 subjects), no symptoms were noted and 28 RBC cholinesterase activity was decreased by 11 or 22% from baseline ( plasma cholinesterase 29 was decreased by 32 and 36%, respectively). Accordingly, the dose was escalated to 0.1 mg/ kg 30 ( 2 subjects). In this leg of the study, one subject exhibited an abnormal vestibular mechanism 31 prior to dosing and showed further deterioration after exposure to carbofuran. This subject also 32 showed changes in cardiovascular parameters including sinus bradycardia and sinus arrhythmia. 33 Two subjects presented with mild symptoms including headache ( 1 subject) or lightheadedness 34 ( the other subject). RBC cholinesterase activity decreased 33 and 31%, respectively, whereas 35 plasma cholinesterase activity was more variable ( decreased 56 and 35%, respectively). Based 36 on these results, the dose was escalated to 0.25 mg/ kg ( 2 subjects) where marked symptoms, 37 including drowsiness, nausea, vomiting, headache, salivation, and sinus bradycardia were noted. 38 Accordingly, this dose level was considered to have achieved the level of intolerable symptoms, 39 and an additional 2 subjects were exposed to this level along with one control subject in a double 40 blinded manner. At this dose level, RBC cholinesterase inhibition ranged from 46 63% and 41 plasma cholinesterase inhibition ranged from 33 100%. 42 43 Overview of Dermal Studies 44 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 18 of 36 The dermal studies conducted with carbofuran ( 1977 and 1978) involved application of 1 the compound to the backs of subjects for 4 hours. The two studies were similar in design, but 2 differed with respect to the commercial formulations tested and the mass applied per unit area of 3 skin. 4 5 The 1977 dermal study ( i. e., first dermal study) was carried out as a single, ascending 6 dose study and was designed to determine the threshold for toxicity under conditions of normal 7 and elevated temperatures. Carbofuran was provided in labeled capsules containing 75.4% 8 carbamate powder or placebo. This powder was applied to the backs of each subject over an area 9 described by a paper template and was then mixed with either water, an artificial sweat medium, 10 or normal saline to insure adhesion. Under normal temperature conditions ( approximately 70 ° F 11 and 35% humidity), the doses evaluated were 2, 4, 8 and 32 mg/ kg ( 2 subjects per dose level), 12 whereas under elevated temperature conditions ( approximately 90 ° F and 68 89% humidity), the 13 doses evaluated were 0.5, 1 and 2 mg/ kg ( 2 subjects per dose level). A control group ( 2 subjects) 14 was included in the high temperature leg of this study. For the high temperature conditions, 15 subjects were also made to exercise by riding a stationary bicycle ( 5 minutes of exercise 16 followed by 15 minutes of rest) throughout the entire 4 hour exposure period. The parameters 17 outlined above under the overview of the oral study were performed on all subjects in this study. 18 19 Under normal temperature conditions, no symptoms were noted at any dose level, and 20 changes in RBC and plasma cholinesterase were variable. RBC cholinesterase inhibition did not 21 exceed 24% ( observed at 32 mg/ kg). Plasma cholinesterase activity was highly variable, with a 22 maximal inhibition of 33% noted at the 4 mg/ kg dose, whereas only 0 or 2 % inhibition was 23 reported in the 2 subjects dosed with 32 mg/ kg. 24 25 Under conditions of high temperature and humidity, symptoms were observed in the two 26 subjects dosed at 2 mg carbofuran/ kg. One subject at this level exhibited severe symptoms 27 ( including hazy vision, vomiting, defecation with muscle cramps and chills) and required 28 atropine ( at 3 separate times) to ameliorate symptoms. Maximal inhibition of RBC 29 cholinesterase activity at this dose level was 45 and 65% in the 2 subjects ( 4 hours), whereas 30 plasma cholinesterase inhibition was maximal at 24 hours ( 12 and 16 %, respectively). 31 32 The 1978 dermal study ( i. e., second dermal study) was carried out as a single, ascending 33 dose study and was conducted under conditions of elevated temperature and humidity as 34 described above. The carbofuran used in this study was a formulation containing 44% active 35 ingredient and was applied at a concentration of approximately 0.5 mg/ cm2 using a 50% dilution 36 of the formulation. The doses evaluated were 0.5, 1, 2 and 4 mg/ kg ( 2 subjects per dose level). 37 There was no control group. The same parameters outlined above under the overview of the oral 38 study were performed on all subjects in this study. 39 40 One subject dosed at 0.5 mg/ kg reported nausea after treatment and the other subject 41 noted burning at the application site. In contrast, neither subject dosed with 1 or 2 mg/ kg 42 experienced any symptoms. A dose of 4 mg/ kg resulted in symptoms of nausea, dizziness and 43 weakness in both subjects, and atropine was administered to these subjects. Inhibition of RBC 44 cholinesterase activity showed some evidence of dose dependence but was variable, ranging 45 from 22 and 7% to 61 and 49% in the 2 subjects treated with 0.5 and 4 mg/ kg, respectively. 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 19 of 36 Plasma cholinesterase levels were highly variable, with 33 and 46% inhibition observed at 0.5 1 mg/ kg and 6 and 9% at 4 mg/ kg, respectively. 2 3 Critique of the Oral and Dermal Studies Conducted with Carbofuran 4 5 In the three studies described above, the major strength of the work was that the 6 experimental design included the evaluation of at least three dose levels from which dose 7 response relationships could be evaluated. Furthermore, the study outcomes were generally 8 consistent with fundamental principles of xenobiotic disposition including observations that 9 exposure from the oral route likely exceeded that from the dermal route ( reflected by the 10 observation of toxicity at much lower oral doses) and that dermal exposure was increased in an 11 environment of increased temperature and humidity. However, in evaluating all of the studies, 12 numerous weaknesses were noted by the HSRB. These weaknesses included: 13 14 1) There was no justification or rationale for the selection of doses used in any of the 15 three studies. 16 17 2) The sample size was very small ( typically two subjects per dose or condition) with few 18 or no controls ( no more than two control subjects in any study). Such a design prevented 19 evaluation of statistical significance for any parameter measured in the studies. 20 21 3) The values obtained for RBC and plasma cholinesterase levels were highly variable. 22 Factors that contributed to this variability included the small sample size, the inclusion of only a 23 single baseline sample collected immediately prior to dosing used to compare all post dosing 24 samples, the small number of control subjects, and an uncommon method for analytical 25 determination of cholinesterase activities. The contribution of potential laboratory error cannot 26 be ruled out. 27 28 4) Plasma cholinesterase levels were highly variable in all studies so as to preclude any 29 useful interpretation. In general, plasma cholinesterase levels were not consistent with changes 30 in RBC cholinesterase activities. 31 32 5) One subject who presented with abnormal vestibular mechanisms in the pre dose 33 evaluation was used in the oral study and showed serious symptoms after treatment. 34 35 6) Subjects were allowed to smoke during the study period. 36 37 While the oral and dermal studies shared these common weaknesses, there were also 38 serious limitations regarding the application of carbofuran in the conduct of the dermal studies. 39 In particular, it is known that dermal absorption is influenced by the concentration of compound 40 applied per unit surface area of skin, and it was clear that the studies were extremely different in 41 this regard. For example, as shown in the Table 1 below in the first dermal study ( high 42 temperature/ humidity), the mass loading range was 6,000 to 12,000 µ g carbofuran/ cm2. These 43 extremely high loading levels were not appropriate for evaluating potential dermal absorption 44 from occupational or environmental exposure to carbofuran. In the first dermal study, the 45 greatest skin surface area treated in the normal temperature leg of this study was 40 cm2; a mass 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 20 of 36 of 3,264 mg was applied to this area, equivalent to a loading of 81,600 µ g carbofuran/ cm2. In 1 contrast, mass loading was controlled to achieve approximately 500 µ g carbofuran/ cm2 at all 2 dose levels in the second dermal study. 3 4 5 Table 1. Calculation Of Loading Levels For Carbofuran For Subjects In The First Dermal Study 6 ( High temperature/ humidity conditions) 7 8 Subject Dose Body Wt Mass Template Loading ( mg/ kg) ( kg) ( mg) ( cm2) ( ug/ cm2)

1 0 63 0 2 0 65 0 3 0.5 72 36 6 6,000 4 0.5 66 33 5.72 5,769 5 1 74 74 8.55 8,655 6 1 64 64 7.94 8,060 7 2 74 148 12.16 12,171 8 2 78 156 12.49 12,490 9 A primary deficiency of the first dermal study was that it did not provide a realistic 10 worker exposure scenario; that is, the exposures of the subjects in these experiments did not 11 correspond to exposures likely to be seen among workers. Large amounts of carbofuran ( up to 12 3,000 mg) were applied to a relatively small skin surface area ( 6 40 cm2) in the experiments, 13 whereas we typically see much larger skin surface areas exposed to smaller amounts among 14 workers ( e. g., 1 10 µ g/ cm2). For example, the hands, a skin surface commonly exposed to 15 pesticides, have a total surface area of 990 cm2 ( EPA Exposure Factors Handbook, 1997). 16 Dermal dosing studies require careful consideration of three factors: mass applied to the skin, 17 surface area treated, and the duration of exposure. Therefore, the skin loadings and skin surface 18 areas exposed in both carbofuran dermal studies were not appropriate for determination of a 19 NOAEL or a LOAEL for risk assessment purposes. 20 21 HSRB Consensus and Rationale 22 23 The EPA concluded that the oral and dermal studies conducted with carbofuran in human 24 subjects were useful in establishing points of departure, both oral and dermal, for the single 25 chemical assessment and in informing the interspecies uncertainty factor for the cumulative 26 assessment. 27 28 However, while these studies were informative, the HSRB concluded that there were 29 numerous technical issues regarding the conduct of the oral and dermal studies with carbofuran 30 and that overall, the weakness of the studies far outweigh the strengths. The weaknesses 31 included the small sample size, the lack of control subjects, the highly variable results for RBC 32 cholinesterase activity and the inappropriate application methods used in the dermal studies. 33 Accordingly, the HSRB did not recommend any of the oral or dermal studies conducted with 34 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 21 of 36 carbofuran in human subjects for the single chemical assessment or in informing the interspecies 1 uncertainty factor for the cumulative assessment. 2 3 Additional Considerations: Potential For The Carbofuran Human Studies Data 4 5 The Board provided additional analysis in response to the Agency's charge to the Board 6 concerning the potential for the data in human subjects for carbofuran to be applied to: ( 1) the 7 calculation of a benchmark dose ( BMD10) and identification of the BMD10L ( lower confidence 8 limit); ( 2) the identification of a NOAEL or LOAEL for effects or ( 3) the comparison to other 9 species for possible adjustments to uncertainty factor for the cumulative assessment. 10 11 The HSRB provided the following additional perspective relative to the Agency's 12 question: 13 14 The utility of the human studies with carbofuran was limited by the very small sample 15 size used in all of the studies. The Agency proposed to use the RBC cholinesterase data for 16 determination of the BMD10L. However, under conditions where the group size was only two, it 17 would be imperative to have highly accurate, valid, reliable and consistent measures of RBC 18 cholinesterase activity in both control and carbofuran treated subjects. This rigor was simply not 19 achieved in the human studies. Rather, RBC cholinesterase activities were compared to a single 20 baseline value, were highly variable across subjects, including controls, and did not show any 21 consistency with plasma cholinesterase levels. As such, although a BMD10L could be calculated, 22 the magnitude of the error in the derived values would preclude a reliable, meaningful 23 assessment. Therefore, the HSRB reiterated its recommendation that the human data should not 24 be used for calculation of the BMD10. 25 26 In a similar manner, the small sample size, compounded by the lack of consistent changes 27 in cholinesterase activities in all studies, the inappropriate methods used for dermal application 28 of the compound in the dermal studies and the inclusion of at least one subject who presented 29 with abnormal vestibular function in a pre dose assessments limited the general utility of the 30 data. Collectively, the weaknesses in the carbofuran human studies conduct and outcomes cast 31 doubt on the utility of the data for identifying a NOAEL or LOAEL or for comparing across 32 species in consideration of the interspecies uncertainty factor for the cumulative risk assessment. 33 Thus the HSRB recommended that the human data should not be used for these evaluations. 34 35 Charge to the Board 36 37 Ethical Considerations 38 39 The Agency requested that the Board provide comment on the following: 40 41 Oral Toxicity Study: 42 Is there clear and convincing evidence that the conduct of the human oral study 43 conducted with carbofuran was fundamentally unethical? 44 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 22 of 36 Is there clear and convincing evidence that the conduct of the oral study was significantly 1 deficient relative to the ethical standards prevailing at the time the research was conducted? 2 3 Dermal Toxicity Studies: 4 Is there clear and convincing evidence that the conduct of either of the human dermal 5 studies conducted with carbofuran was fundamentally unethical? 6 7 Is there clear and convincing evidence that the conduct of the dermal studies was 8 significantly deficient relative to the ethical standards prevailing at the time the research was 9 conducted? 10 11 Board Response to the Charge 12 13 Study Overview 14 15 There were three studies involving either oral or dermal administration of carbofuran: an 16 oral toxicity study performed in 1976 ( IRB Review dated March 31, 1976; Final Report dated 17 September 17, 1976); a dermal toxicity study performed in late 1976 and early 1977 ( IRB 18 Review dated August 25, 1976; Final Report dated March 18, 1977); and a second dermal 19 toxicity study conducted in late 1977 ( REC Review date unknown; Final Report dated February 20 15, 1978). 21 22 The location for the research was the Quincy Research Center in Kansas City, Missouri. 23 All three studies were under the direction of a single principal investigator, John D. Arnold, MD. 24 The research appeared to have been performed under contract to the Midwest Research Institute, 25 also located in Kansas City. The responsible institutional review board was the Community 26 Review Committee, Inc., again located in Kansas City. The research sponsor was FMC 27 Corporation, located in Philadelphia, Pennsylvania with the manufacturing facility apparently 28 located in Middleport, New York. 29 30 No ethical or regulatory standards were mentioned in any of the study documents. Given 31 the dates of the research studies, Section 12 of FIFRA applied to the research. In addition, the 32 1975 version of the Declaration of Helsinki was available at the time. 33 34 Critique of Studies 35 36 The following comments apply to all three studies. 37 38 1) The fact that these studies have never been published should not be used as the sole 39 criterion to determine whether the purpose of the research was to obtain generalizable 40 knowledge. Publication is neither a necessary nor sufficient criterion of whether or not the 41 research was designed to allow for either a descriptive or causal inference. 42 43 2) The risks were minimized by the study design ( setting aside the actual conduct), 44 assuming that there was a valid scientific purpose in escalating the dose until achieving a " lowest 45 observable adverse effect level" ( LOAEL). Examples of the procedures that were incorporated to 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 23 of 36 minimize risk included the presence of a supervising physician who was readily available for 24 1 hours after dosing, confinement of the subjects for 24 hours, abstinence from alcohol during the 2 study, the exclusion of other drugs within two weeks of performing the study, the availability 3 and administration of atropine ( discussed further below), and a delay in dose escalation ( at least 4 in the oral toxicity study) until the 24 hour clinical data was available. In addition, subjects only 5 received the active compound once during each research study. 6 7 3) Measurements of RBC cholinesterase inhibition should serve as an adequate surrogate 8 measure of toxicity, obviating the need to induce clinical signs and symptoms of cholinergic 9 toxicity. The question however in judging these three studies was whether this standard was 10 either appreciated or applicable in 1976 and 1977. The fact that the research was designed to 11 cause clinical signs and symptoms of cholinergic toxicity as the study endpoint does not, in and 12 of itself, establish that the interests of the subjects did not prevail over other interests. The 13 Common Rule allows for the balancing of the risks of research against the knowledge that may 14 reasonably be obtained. The central question then was whether the risks were reasonable, not 15 whether the research was designed to elicit clinical toxicity. 16 17 4) With respect to informed consent, the list of signs and symptoms of cholinergic 18 toxicity found in the consent documents was fairly complete. The consent documents were fairly 19 straightforward about the fact that the testing involved a pesticide and that the research would be 20 of no benefit to the subject. The freedom to withdraw was emphasized, along with the fact that 21 additional testing to ensure the safety of subjects would be requested by the supervising 22 physician. In spite of these strengths, the consent documents failed to provide a description of 23 the study design ( i. e., dose escalation) and the anticipated endpoint of clinical toxicity. The 24 phrase " we do not expect any serious complications" is clearly open to interpretation. Some 25 would and some would not consider the clinical signs and symptoms of cholinergic stimulation 26 " serious." Regardless, the phrase does introduce a framing of these stated risks as " non serious." 27 Given the research design, the consent documents would have been improved if they had been 28 explicit about the dose escalation, the place of the specific subject within that dose escalation, 29 and the fact that someone would eventually have a 100% chance of experiencing clinical 30 toxicity. Although these changes are an admirable standard going forward, the consent 31 documents used for the oral and first dermal toxicity study met ( and some might argue exceeded) 32 the standards prevalent in 1976 and 1977. However, as discussed below, the consent document 33 for the second dermal toxicity study was seriously deficient. 34 35 The Board had specific comments about the conduct of each of the studies that can be 36 addressed under the general topic of the reasonableness of the risks ( and the efforts to reduce 37 those risks) that the subjects experienced in the conduct of this research. 38 39 1) Was it appropriate to expose additional subjects, in the oral toxicity study, to a dose 40 which had already been shown to cause clinical toxicity if the scientific purpose was to establish 41 a LOAEL? Given the criticism of attempting to determine a " no observable adverse effect level" 42 ( NOAEL) using a small sample size, the design chosen in these three studies to elicit a LOAEL 43 may be more reliable. However the small sample size, when combined with the variability and 44 unreliability of the RBC cholinesterase measurements, undermine confidence that the study was 45 designed to establish the real LOAEL. The repeat administration of the test substance absent 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 24 of 36 dose escalation was used in other cholinesterase inhibitor studies, but the endpoint driving the 1 decision to not escalate dosing was the more sensitive endpoint of the degree of RBC 2 cholinesterase inhibition. 3 4 2) There was documentation ( in a letter dated October 26, 1976) of the decision to start at 5 the 2.0 mg/ kg dose in the low temperature and low humidity phase of the first dermal toxicity 6 study. Although the responsible IRB was not consulted ( for which there were no procedural 7 guidelines in 1976), was the decision to bypass the 16 mg/ kg dose in favor of a 32 mg/ kg dose in 8 the low temperature and humidity phase of the dermal toxicity study reasonable? If the dose 9 response relationship based on the percent RBC acetyl cholinesterase inhibition was linear, yet 10 the onset of clinical signs and symptoms reflects a threshold response, this decision could have 11 placed the subjects given the higher dose at greater risk even though, in retrospect, the 32 mg/ kg 12 dose was well tolerated. 13 14 3) The administration of atropine as an antidote to cholinergic toxicity may have been 15 delayed for one or more of the subjects in the high temperature and high humidity phase of both 16 dermal toxicity studies. Although mention was made of written instructions for the 17 administration of atropine, these instructions were not included in the submitted documentation. 18 The question then was whether there could be any justification for the delay in the administration 19 of atropine. Two possible justifications might be: ( 1) the signs and symptoms were from non 20 muscarinic cholinergic receptors and thus would not be responsive to atropine ( which was not 21 the case); or ( 2) the supervising physician was concerned that any resulting tachycardia or other 22 side effects from the administration of atropine would be of greater risk ( highly unlikely). After 23 considerable reflection, the Board could find no scientific or clinical reason to delay the 24 administration of atropine. 25 26 4) Study participants were not fully informed of the risks of the study. It should have 27 been clear to study investigators that the escalating dose design used was likely to result in 28 serious harm to some research subjects. For example, several participants who received a 2.0 29 mg/ kg dose of carbofuran during the high temperature and high humidity phase of the first 30 dermal toxicity study exhibited clear clinical signs and symptoms of carbamate poisoning, 31 requiring administration of atropine as an antidote. Plasma and red cell cholinesterase inhibition 32 data also was obtained from these individuals, with participants demonstrating 46% and 65% 33 peak red cell inhibition respectively. In the subsequent second dermal toxicity study, however, 34 the data from the first dermal toxicity study were not used either to develop clear stopping 35 criteria or to modify the dosing protocol, thus exposing study participants to an unacceptable 36 level of risk. The two participants in the high temperature and high humidity second dermal 37 toxicity study who received a 2.0 mg/ kg dose of carbofuran did not exhibit any clinical 38 symptoms of carbamate poisoning. These individuals did, however, exhibit peak red cell 39 cholinesterase inhibition of 40% and 42% respectively, similar to the level of inhibition seen in 40 one of the symptomatic participants in the first dermal toxicity study. These data suggest that the 41 LOAEL for carbofuran was at or near 2.0 mg/ kg. Nevertheless, the decision was made to expose 42 two subjects to a dose of 4.0 mg/ kg carbofuran, once again resulting in severe clinical symptoms 43 indicative of carbamate poisoning and requiring administration of atropine as an antidote. In light 44 of the clinical and biomarker data obtained from the first dermal toxicity study, it should have 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 25 of 36 been obvious to study investigators that exposure of additional research subjects to a dose of 4.0 1 mg/ kg carbofuran was likely to have resulted in serious harm to these two study participants. 2 3 This conclusion, coupled with the observation that the consent documents from the 4 second dermal toxicity study explicitly stated study investigators "[ did] not expect a serious 5 complications" raises serious questions about the informed consent process. At least some study 6 participants were likely to experience clinical signs indicative of carbamate toxicity. To imply 7 otherwise in the informed consent documents suggests that the consent process was severely 8 flawed. Study participants were denied access to information that might have influenced their 9 decision to voluntarily enroll in the second dermal toxicity study. 10 11 HSRB Consensus and Rationale 12 13 Oral Toxicity Study 14 15 For the oral study, there was no evidence that the study failed to fully meet specific 16 ethical standards prevalent at the time the research was conducted. 17 18 There was no clear and convincing evidence that the research was fundamentally 19 unethical ( e. g., intended to seriously harm participants or that informed consent was not 20 obtained). 21 22 There was no clear and convincing evidence of significant deficiencies in the ethical 23 procedures that could have resulted in serious harm ( based on the knowledge available at the 24 time the study was conducted) nor that information provided to participants seriously impaired 25 their informed consent. 26 27 Dermal Toxicity Studies 28 29 The HSRB found deficiencies in both dermal human toxicity studies relative to specific 30 ethical standards prevalent at the time the study was conducted. 31 32 The majority of the Board concluded there was no clear and convincing evidence that the 33 research was fundamentally unethical ( e. g., intended to seriously harm participants or that 34 informed consent was not obtained). In light of the results obtained from the first dermal toxicity 35 study, one Board member concluded that the second dermal toxicity study was fundamentally 36 unethical in design. The Board member believed that this study was neither designed to 37 minimize the risk of serious harm to participants nor to ensure an adequate informed consent. 38 39 For both dermal toxicity studies, there was clear and convincing evidence of significant 40 deficiencies in the ethical procedures for minimizing risk that could have resulted in serious 41 harm ( based on the knowledge available at the time the study was conducted). The first dermal 42 toxicity study was significantly deficient given the delay in the administration of atropine to 43 more than one subject experiencing the signs and symptoms of carbamate toxicity. The second 44 dermal toxicity study was considered significantly deficient by a majority of Board members in 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 26 of 36 that the lack of information provided about the results from the initial dermal toxicity study 1 seriously impaired their informed consent. 2 3 4 3. Methyl Isothiocyanate ( MITC) 5 6 Charge to the Board 7 8 MITC is an irritating compound that has a limited animal database for toxicity via 9 inhalation, the key route of exposure. MITC can be used as a pesticide directly to treat wood 10 poles, but the major pathway of exposure to MITC is from degradation of several fumigant 11 pesticides ( i. e., metam sodium, metam potassium, and dazomet). Due to its volatility, MITC has 12 the potential to move off site, which can result in exposure to bystanders near treated areas and, 13 through ambient air, to people far away from treated areas. Use of the soil fumigants also results 14 in exposure to those handling the pesticides or working in treated fields. 15 16 Scientific considerations 17 18 The Agency's WOE document and DER for MITC describe the study design and results 19 of the MITC odor threshold and eye irritation human studies. The WOE document also discusses 20 the Agency's conclusions that the eye irritation study is useful for the assessment of potential 21 effects on bystanders and workers from exposures to MITC during acute ( 1 day) intervals. The 22 Agency had concluded that the odor threshold study is less useful than the eye irritation study for 23 assessing the human health effects of MITC, since the odor detection threshold for humans is 24 higher than the level that causes eye irritation. The Agency had decided, however, to use the 25 results of the eye irritation study for assessing the inhalation exposure of MITC. 26 27 Please comment on the scientific evidence that supports this conclusion. 28 29 Board Response to the Charge 30 31 Introduction 32 33 MITC is the primary and key degradate of these fumigant pesticides ( i. e., metam sodium, 34 metam potassium and dazomet). As a gas injected into soil, it can kill soil borne pests, such as 35 insects, microorganisms, weeds, and nematodes. The fumigant dissipates from the soil in a few 36 days to a couple of weeks. 37 38 According to the EPA Weight of Evidence ( WOE) document ( USEPA 2006b) " The 39 mode of toxic action for MITC is not known at this time. MITC is primarily an irritating 40 compound that produces non specific systemic effects in oral toxicity studies such as changes in 41 body weight, food consumption, and hematological parameters. Following air exposures to 42 MITC, consistent effects are observed in rats and humans. For example, clinical signs and 43 pathological changes of the respiratory tract consistent with an irritant have been observed in 44 laboratory studies in rat. Humans exposed to MITC complain of symptoms such as itchy and 45 burning eyes, rash and burning skin, nausea, scratchy throat, salivation, coughing, and shortness 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 27 of 36 of breath. In acute toxicity testing with animals, MITC is considered Acute Toxicity Category I 1 ( corrosive) for skin and eye irritation." Since the animal studies were either long term inhalation 2 or oral studies, they were not considered for a point of departure and therefore would be less 3 protective of human health. Thus, the Board recommended the eye irritation LOAL as a point of 4 departure. 5 6 Brief Overview of Study 7 8 The EPA WOE extracted a description of the odor and eye irritation study ( Russell and 9 Rush 1996) directly from the Risk Characterization Document for MITC of the Department of 10 Pesticide Regulation, Cal EPA ( July 25, 2003, pp 53 59), which was considered accurate and 11 quoted herein. 12 13 " In order to determine the NOEL for human eye irritation produced by MITC vapors, as 14 well as its odor threshold, human volunteers were exposed to air concentrations of MITC in a 15 laboratory setting ( Russell and Rush, 1996). The study specifically focused on assessing these 16 parameters at different times of exposure. An olfactometer was used which permitted the 17 operator to dispense the test material through a manifold system. The test material could thus be 18 diluted over a 100 fold concentration range. The material was dispensed by diffusion from a 19 glass vessel which could be maintained at any temperature ± 0.1 ° C over a range of 30 to 70 ° C. A 20 Total Hydrocarbon Analyzer ( THA) was used to monitor the flow of test material during the 21 exposure period. In addition, carbon tube samples were drawn once the system was equilibrated 22 prior to exposure, and at the end of the exposure. The test material was desorbed from the carbon 23 and analyzed by gas chromatography. Every effort was undertaken to minimize the reaction of 24 the test material with the tubing and other equipment used in the delivery system". 25 26 " In the olfactory threshold study, 33 individuals ( 16 males, 17 females) with a mean age 27 of 25 years ( range, 18 to 34 years) were tested. They were exposed to three positive control 28 odorants, pyridine, acetic acid, and n butyl alcohol as well as to MITC. The technician chose the 29 odorant and concentration level. The odorant was dispensed in double blind fashion from one of 30 three presentation ports. The subject was responsible for identifying from which of the 31 presentation ports the odorant was dispersed. A 30 second rest period between exposures was 32 permitted in order to allow the subject to recover prior to the next exposure. The operator tested 33 each subject over the range of concentrations for each odorant until he was assured that the 34 threshold had been adequately ascertained. A standard procedure was employed in order to make 35 this determination." 36 37 " In the NOEL determination for eye irritation, the olfactometer was modified by 38 attaching goggles to the presentation line. This permitted the test material to be directed only to 39 the eyes. Five parameters were used to ascertain an irritation response: 1. the subjects' subjective 40 estimation of irritation ( using the " Likert" scale); 2. photographs of the subjects' eyes prior to 41 and after exposure; 3. blink rate as measured by electromyography; 4. effect upon visual acuity; 42 5. tear production. Both a positive control ( acetic acid) and a negative control ( air) were 43 employed. Baseline responses for each of the assessment parameters were determined under pre 44 exposure conditions (" zero time controls") and upon exposure to the negative control (" air only 45 controls") for the prescribed period. A positive irritation response was based on three criteria: 1. 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 28 of 36 the average response must be quantitatively greater than the pre exposure response; 2. the 1 average response must be greater than pre exposure and greater than could be expected 2 statistically from individual to individual differences within the group; 3. the average treated 3 response must be greater than the air only group's response and greater than could be expected 4 from individual differences observed within the group. Seventy individuals ( 38 males, 32 5 females) with a mean age of 32 years ( range, 18 67 years; median age, 28 years) were exposed to 6 air, MITC, and/ or acetic acid. Between 9 and 16 subjects were examined under each dose/ time 7 period combination. Three exposure periods, 14 minutes, 4 hours and 8 hours were used. In the 8 eight hour test, subjective responses, blink rates and tearing were assessed at 0, 1.5, 3, 3.5, 6 and 9 8 hours ( tearing was not measured at 3.5 hours). Two 15 minute rest breaks and a 30 minute 10 lunch break were permitted during the 8 hour period. In the four hour test, these same parameters 11 were assessed at 0, 1, 2, 3 and 4 hours ( tearing was not measured at 0, 2 and 3 hours). In the 14 12 minute exposure protocol, subjective responses and blink rates were measured at 0, 1, 4 and 14 13 minutes after the start of exposure. Tearing was measured at 14 minutes only. Visual acuity and 14 ocular morphology were assessed at the beginning and end of each exposure period. All analyses 15 were performed in a double blind manner." T tests were used to compare responses at each 16 computed concentration level for each time period to both air control results and zero time 17 results. Both were significant and positive but responses to the control substance were not as 18 dramatic. 19 20 Critique of the Study 21 22 Introduction 23 24 Table 2 shows what the investigators called the NOEL, which the Agency's Data 25 Evaluation Record and WOE call the NOAEL and LOEL respectively ( EPA's RfC methodology 26 document included eye, nasal, and throat irritation in its list of adverse effects). 27 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 29 of 36 1 Table 2. Summary Of MITC Eye Irritation Effects From Human Subjects 2 3 Exposure time NOAEL ( ppm) LOAEL ( ppm) Source of observed Effect 1 minute 3.3   4 minutes 0.6 1.9 Subjective eye irritation 14 minutes 0.6 1.9 Subjective eye irritation 1 hour 0.23 a 0.8 Subjective eye irritation 1.5 hours 0.22 a

2 hours 0.23 a 0.8 Subjective eye irritation and blink rate 3 hours 0.23 a 0.8 Subjective eye irritation and blink rate 3.5 hours 0.22 a

4 hours 0.23 a 0.8 Subjective eye irritation 6 hours 0.22 a

8 hours 0.22 a

a The slightly different values obtained at the low dose NOAEL level ( 0.22 and 0.23 ppm) reflected 4 the fact that they were derived from tests performed on different days. 5 6 7 As the WOE stated " Exposure to 0.8 ppm ( 800 ppb) MITC resulted in a statistically 8 significant positive response based on averaging the subjective assessments by the subjects using 9 the Likert scale methodology. As many as 8 out of 9 subjects showed a positive response at 1 10 and 2 hours, the first two time points examined [ and also at 3 & 4 hours]. Shorter exposures to 11 0.6 ppm did not result in statistically significant Likert scale changes, though 1 of 9 individuals 12 appeared to respond at 4 and 14 minutes. Exposure to 1.9 ppm or 3.3 ppm MITC for 4 or 14 13 minutes resulted in positive subjective responses at 4 and 14 minutes. At 1 minute of exposure, 14 levels as high as 3.3 ppm did not evoke a statistically significant positive response." 15 16 " Mean blink rate determinations at 0.8 ppm were statistically significantly increased at 17 the 2 and 3 hour time points compared both to air only and zero time controls. Statistical 18 significance was not achieved at 1 and 4 hours, though a positive response was indicated in 19 several individuals. The blink response to 0.6 ppm and 1.9 ppm at 1, 4 and 14 minutes did not 20 show a positive response. At 3.3 ppm, statistical significance was achieved at 4 and 14 minutes. " 21 The Board agreed with the Agency's conclusion that " A strong suggestion of a response was also 22 present at 1 minute, though it was not statistically significant." In addition, the subjective ( Likert 23 scale) responses were the most sensitive and most variable. The eye blink rate was the next most 24 sensitive. The other tests were not as sensitive and usually were not significant. 25 26 The Board agreed with the Agency conclusions as noted in their DER: 27 28 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 30 of 36 "° For a one minute exposure, the NOAEL for eye irritation is 3.3 ppm due to a lack of response 1 in any parameter tested." 2 3 "° For exposures 4 14 minutes, the NOAEL for eye irritation is 0.6 ppm based on responses on 4 the Likert subjective scale at 1.9 ppm. " 5 6 "° For exposures of 1 8 hours, based on the statistically significant subjective ( Likert scale) 7 responses at 0.8 ppm MITC at 1 4 hours and the statistically significant eyeblink responses at 2 8 and 3 hours, 0.22 ppm was designated as the NOAEL for this study. The NOAEL for eye 9 irritation was consistent for the 1 8 hour measurements. It is reasonable to assume that exposures 10 up to 24 hours would likely yield a similar response." 11 12 Finally, in terms of the olfactory threshold study, the Board agreed with the Agency's 13 conclusion that " The observed odor threshold for MITC ranged from 0.2 to 8 ppm with a 14 geometric mean of 1.7 ppm." 15 16 Strengths of the study 17 18 The studies were well designed, equipped, carefully controlled and performed by 19 experienced investigators at a respected institute. The lowest concentration tested was the largest 20 sample size. Exclusion criteria were appropriate: abnormal irritation, contacts, frequent 21 headaches, recent asthma attacks, and pregnancy. 22 23 Weaknesses of the study 24 25 The eye irritation studies did not have a sufficient number of subjects in each of the 26 experiments and phases. In addition there was no information on the susceptibility status of 27 individuals tested nor information on within subject variation. Another shortcoming is that eye 28 irritation does not predict dermal nor respiratory effects. Thus, there may be lower NOAELs for 29 these latter effects. 30 31 Two tailed t tests were used to compare the responses of subjects receiving different 32 doses of MITC despite the presence of substantial skew in the data of some groups, with some 33 standard deviations exceeding the corresponding means. This was most common among the 34 subjects receiving the lower doses, an issue of particular concern insofar as the goal of the study 35 was to identify a NOAEL. A nonparametric test would have been a more appropriate choice. In 36 addition, because responses were measured repeatedly on the same subjects over time, a 37 statistical approach that took this into account would also have been more appropriate than the 38 series of independent t tests that were carried out. 39 40 The investigators were rather rigid in their approach to the interpretation of p values. For 41 instance, a group difference for which the p value was 0.052 was not considered evidence of an 42 effect. On the other hand, the investigators clearly stated their criteria for interpretation and 43 applied these rules consistently. Moreover, inspection of the tables indicated that the conclusions 44 reached would not have differed even if a somewhat more liberal criterion of statistical 45 significance had been applied. 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 31 of 36 1 This issue does raise a more general concern relating to the size of the study sample. The 2 investigators provided no rationale for the sample size that was used nor power calculations, 3 despite the important influence that sample size has on whether a group difference reaches some 4 level of statistical significance. The inclusion of a small number of additional subjects in the 5 different groups could well have caused some of the borderline p values to fall to a level that 6 would have met the investigators' criteria for significance and, potentially, change the inferences 7 drawn, as demonstrable by re calculations of significance. Thus it is important that one could be 8 confident that the sample size was adequate for the assessment of the study hypotheses. Ideally, 9 the investigators should have begun by specifying the magnitude of the response that they 10 consider meaningful and want to be able to detect, should it exist ( e. g., a 50% increase in the 11 response, a doubling of the response, etc). Then, after making additional assumptions, they 12 could calculate the number of subjects that would be necessary. As stated, this was not done. 13 14 HSRB Consensus and Rationale 15 16 The Board concluded that air concentrations of methyl isothiocyanate sufficient to 17 produce eye irritation would lead to a conservative and prudent point of departure for inhalation 18 risk ( i. e., eyes were a sensitive endpoint in relation to the respiratory system). The Board 19 reached its decision based on eye irritation LOAELs are often lower than respiratory irritation 20 LOAELs for irritant gases ( WHO 1979ab, NRC 1986; WHO/ EURO 1986). While the use of eye 21 irritation date as a surrogate for respiratory data is reasonable in this situation, one must be 22 cautious as only appropriate controlled human studies of the respiratory system can provide a 23 final and definitive respiratory point of departure, if ever determined ( NAS 1975). 24 25 Charge to the Board 26 27 Ethical considerations 28 29 The Agency requests that the Board provide comment on the following: 30 31 a. Is there clear and convincing evidence that the conduct of the human eye irritation 32 study with MITC was fundamentally unethical? 33 34 b. Is there clear and convincing evidence that the conduct of this study was significantly 35 deficient relative to the ethical standards prevailing at the time the research was conducted? 36 37 Board Response to the Charge 38 39 Brief Overview of the Study 40 41 The human eye irritation study was conducted in 1993 through 1995. The study was 42 performed in Davis, California by researchers at the Sensory Testing Laboratory, School of 43 Medicine, University of California, Davis, together with the Western Research Center of Zeneca 44 Ag Products, Richmond, California. The study sponsor was the Metam Sodium Task Force 45 ( representing chemical manufacturers), whose mailing address is in care of Zeneca Ag Products 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 32 of 36 of Wilmington, Delaware. The documents provided by the sponsor specifically state that the 1 research was conducted in compliance with the Declaration of Helsinki ( presumably the 1989 2 version, though no date is specified) and the Human Subject's Bill of Rights ( a provision of 3 California law). The study was reviewed and approved by the Human Subjects Review 4 Committee at the University of California, Davis, an institution which held a Multiple Project 5 Assurance with the U. S. Department of Health and Human Services. The documentation 6 provided by that Committee indicated that it reviewed this study pursuant to the standards of the 7 Common Rule ( 45 C. F. R. Part 46, Subpart A) and determined it to be in compliance with that 8 Rule. 9 10 The Board's comments only relate to the human eye irritation study, and not to the 11 human odor threshold study conducted by the same group of investigators. Consistent with the 12 charge presented to the Board by the EPA, the Board made no comments with regard to the 13 human odor threshold study. 14 15 Critique of Study 16 17 The Board concurred with the factual observations of the strengths and weaknesses of the 18 human eye irritation study, as detailed in USEPA ( 2006c). The Board concurred with the 19 Agency's conclusion that although there were deficiencies with regard to the applicable ethical 20 standards prevailing at the time this study was conducted, those deficiencies were relatively 21 minor. In addition to the deficiencies specified in USEPA ( 2006c), the Board wanted to comment 22 on two additional aspects of the study: 23 24 1. The Human Subjects Review Committee asked the investigators to add a provision to 25 the protocol and the consent form indicating that " if significant irritation is experienced, no 26 higher dose will be administered." The revised protocol never provided any specific criteria for 27 determining how it would be determined whether a subject was experiencing significant 28 irritation. It was appropriate that such stopping rules be relatively specific, if possible. 29 30 2. The original protocol for the eye irritation study involved exposing subjects to MITC 31 for a series of two minute periods, with twenty minute breaks between each exposure. In the 32 study investigator's memorandum to the IRB dated August 17, 1994, requesting renewal of the 33 protocol, the investigator indicated that he had apparently finished conducting at least part of the 34 study as initially described, and that it was " going well without any ill effects." He submitted a 35 protocol amendment so that he might study the effects of longer exposure to MITC ( up to eight 36 hours at a time). In the document submitted to the EPA describing the results of this series of 37 studies, however, no data were provided as to the results of the short term study. On page 26 of 38 the submitted documents, which outlines when subjects were exposed to this agent and for what 39 periods of time, there was mention only of the 8 hour, 4 hour, and 14 minute exposure periods. 40 The tables accompanying the report only gave details of the results of those longer exposure 41 periods. Since the longer exposures were premised on the favorable results from the short term 42 exposures, it would have been appropriate for the report to have also included details relating to 43 the results from the short term ( two minute) trials. The absence of such details makes it difficult 44 to determine any ethical irregularities that might have been revealed by such additional 45 information. 46 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 33 of 36 1 HSRB Consensus and Rationale 2 3 The Board concluded that: 4 5 There was no clear and convincing evidence that the conduct of the research was 6 fundamentally unethical ( e. g., the research was intended to seriously harm participants or failed 7 to obtain informed consent). 8 9 There was no clear and convincing evidence that the conduct of the study was 10 significantly deficient relative to the ethical standards prevailing when the study was conducted. 11 12 The Board based these two determinations on its conclusion that this study, based on the 13 evidence presented, appeared to have had only relatively minor deviations from the ethical 14 standards prevailing when the study was conducted. 15 16 COMMENTARY ON SCIENTIFIC STANDARDS FOR HUMAN DOSING STUDIES 17 18 The Chair asked the Board to articulate the set of scientific standards that has and will 19 continue to guide Board decision making for human dosing studies. Following Board 20 deliberation, scientific standards for human dosing studies in general and for single dose studies 21 in particular were adopted. 22 23 Scientific Standards for Human Dosing Studies 24 25 1. Justification 26 27   Is the scientific question worthwhile? 28   Are human subjects necessary to answer the question? 29   Is potential risk serious or irreversible? 30 31 2. Dose Selection 32 33   Sufficient to test the question? ( single dose in most cases is not sufficient to determine 34 NOAEL and LOAEL) 35   Based on appropriate data ( e. g. preclinical; previous studies) 36 37 3. Endpoint Selection 38 39   Consistent with the aim of the study? 40   Appropriate to answer questions about human responses ( e. g., sensitivity, accuracy, validity, 41 replicability)? 42   Measured accurately and reliably with good quality assurance? 43   Participants 44   Characteristics generalizable to question asked? 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 34 of 36   Appropriate inclusion/ exclusion criteria? 1   Are measurements taken at appropriate times to answer the study question? 2 3 4. Method 4 5   Is the sample size sufficient? 6   Is selection of control and experimental groups appropriate? 7   Is the staging of dose intervals, dose amounts, and type of exposure sufficient to answer the 8 question? 9   Is there quality assurance for observations, instruments and data? 10 11 5. Statistical Analyses 12 13   Can data be statistically analyzed? 14   Is the statistical method appropriate to answer the question? 15 16 Scientific Standards for Single Dose Level Study 17 18 Board definition of single dose level study  individual study that uses one dose level irrespective 19 of the number of subjects, frequency of dosing or inclusion of a control or placebo. 20 21 1. In general, single dose level studies have limited utility 22 23   Such studies cannot be used in isolation to establish a NOAEL or LOAEL 24   In rare instances they may have utility if interpreted within the context of one or more 25 supplementary studies that provide information at other dose levels under analogous 26 conditions. 27 28 2. Single dose level studies may be able to answer a very focused question 29 30   However in such instances its utility will depend upon the robustness of study design, the 31 rationale for the study and whether the study design was consistent with the rationale. 32   Evaluation of robustness will include questions of: control, relevant endpoints, evidence that 33 measures can identify an adverse effect or detect a change, use of a surrogate marker that is 34 quantifiable and recognized as an established function of the compound and other criteria for 35 scientific validity. 36 37 3. A single dose level study may have utility if it provides evidence of adverse effects observed 38 at lower levels than other studies have indicated. 39 40 41 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 35 of 36 REFERENCES 1 2 3 ADTC. 2004. 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National Academy 18 Press, 1986. http:// darwin. nap. edu/ books/ 0309037301/ html/ 13. html 19 20 Russell MJ and Rush TI. ( 1996) Methyl Isothiocyanate: Determination of human olfactory 21 detection threshold and human no observable effect level for eye irritation. Sensory Testing 22 Laboratory, University of California at Davis. Report No. RR 96 049B. September 10, 1996 23 MRID 44400401. 24 25 SAP. 2004. Transmittal of Minutes of the FIFRA Scientific Advisory Panel Meeting Held May 26 4 6, 2004: A Consultation On Dermal Sensitization Issues For Exposures To Pesticides. July 1. 27 28 United States Environmental Protection Agency. Data Evaluation Review. Methyl 29 Isothiocyanate. Special Study; Human Eye Irritation and Odor Threshold. 30 31 USEPA. 2006a. EPA's Initial Ethical Review of Hexavalent Chromium Human Sensitization 32 Study. April 11, 2006. 33 34 USEPA. 2006b. United States Environmental Protection Agency. Weight of Evidence 35 Discussion for Methyl Isothiocyanate. April 13, 2006. 36 37 USEPA. 2006c. EPA's Initial Ethical Review of MITC Human Odor Threshold and Eye 38 Irritation Studies, dated April 13, 2006. 39 40 WHO 1986. EHO/ EURO EH 13. 41 42 WHO 1979a. EHC 7. Environmental Health Criteria 7: Photochemical oxidants. World Health 43 Organization, 1979. http:// www. inchem. org/ documents/ ehc/ ehc/ ehc007. htm 44 45 Proposed Final Draft v. 1 Dated June 14, 2006; Do Not Cite or Quote 36 of 36 WHO 1979b . EHC 8 Environmental Health Criteria 8: SULFUR OXIDES AND 1 SUSPENDED PARTICULATE MATTER. World Health Organization, 1979. 2 http:// www. inchem. org/ documents/ ehc/ ehc/ ehc008. htm 3