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The Effect of the Type of Respondent on Risk Estimates of Pesticide Exposure in a Non-Hodgkin's Lymphoma Case-Control Study Geary W. Olsen, DVM, PhD * Kenneth M. Bodner, MSPH ABSTRACT. There has been considerable controversy among epidemiologists whether proxy interviews provide agricultural pesticide use data comparable to that obtained from direct informants, who are assumed to be generally more knowledgeable about their use of pesticides. The purpose of this analysis was to determine whether odds ratios for pesticide use, in particular 2,4-dichlorophenoxyacetic acid (2,4-D), varied by the type of respondent in a resurvey of 310 subjects in the National Cancer Institute's Iowa/Minnesota Non-Hodgkin's lymphoma case-control study. Pesticides were grouped according to crop insecticides, animal insecticides, and herbicides (with and without 2,4-D), as well as 2,4-D itself. Using logistic regression, only animal insecticides had consistent risk estimates between the proxy and direct informants for the three categories of frequency of use employed in the analysis (1-4 days/yr., 5-9 days/yr. and 10+ days/yr.). Significant (p < 0.05) interaction terms (respondent type by each frequency of use category) were observed for crop insecticides at 1-4 days/yr., for herbicides including 2,4-D at 10+ days/yr. and for herbicides excluding 2,4-D at 10+ days/yr. use. For 2,4-D use, the proxy-derived odds ratio was 2.5 (95% Cl 0.8-8.0) for the highest frequency of use (10+ days/yr.) compared to a direct informant-derived odds ratio of 0.7 (95% Cl 0.3-1.9) (interaction term p = 0.08). The results from this analysis, as well as other published data, suggest that the type of respondent may act as an effect modifier in the association between cancer and pesticide exposure. We agree with others who have recommended that agricultural pesticide use risk estimates derived from casecontrol study data should be analyzed and presented separately by the type of respondent. (Article copies available from The Haworth Document Delivery Service: 1-800-342-9678.) KEYWORDS. Cancer, epidemiological methods, herbicide, insecticide, non-Hodgkin's lymphoma, pesticide, 2,4-dichlorophenoxy-acetic acid, 2,4-D Geary W. Olsen and Kenneth M. Bodner are affiliated with the Department of Epidemiology, Health and Environmental Sciences, The Dow Chemical Company, Midland, MI. Address correspondence to: Dr. Geary W. Olsen, 3M Occupational Medicine, 3M Center, Building 220-3W-05, P.O. Box 33220, St. Paul, MN 55133-3220. Journal of Agromedicine, Vol. 3( 1 )1996 1996 by The Haworth Press, Inc. All rights reserved. Chapter 1 Rationale for the Phenoxy Herbicide Benefits Assessment ORVINC. BURNSIDE1 Table of,,Contents WHY THE ASSESSMENT WAS UNDERTAKEN During the past several years a National Agricultural Pesticide Impact Assessment Program (NAPIAP) Task Force has conducted a biologic and economic assessment of benefits from the use of phenoxy herbicides in the United States. Phenoxy herbicides, such as 2,4-D, have provided economical, selective, postemergence control o f broadleaf weeds in grass crops and noncropland for the past live decades (8, 12, 13). A major use o f 2,4-D today is in combination with other herbicides because it economically enhances the weed control spectrum o f many herbicide mixtures. The herbicide 2,4-D is registered for use on 65 crops in the United States and other phenoxy herbicides are registered on 25 crops, and these herbicides also have numerous noncropland uses. The chemical, 2,4-D, was first registered with the United States Patent Office for use as a herbicide in 1945 (12). Like other pesticides registered prior to November 1984, the phenoxy herbicides must be re-registered with the Environmental Protection Agency (EPA) by 1997 or all present registrations will be lost. This benefits assessment o f 2,4-D and other phenoxy herbicides encompasses all their cropland and noncropland uses in the United States, along with their biologic and economic benefits. This report will provide EPA personnel and others with benefits information about the phenoxy herbicides that will be useful in deciding whether this class o f herbicides should be re-registered. Also, we are presenting a historical account o f the development of phenoxy herbicides in the United States, and the impact that these herbicides have had on weed management technology and the development o f the discipline of weed science. 1 Prof., Dep. Agron. and Plant Genet., Univ. Minnesota, St. Paul, MN 55108. ENVIRONMENTAL AND TOXICOLOGICAL INFORMATION Environmental concerns and risk data development for 2,4-D re-registration was completed by the Industry Risk Assessment Task Force II in time to meet the December 1995 EPA deadline. Other Industry Task Forces have conducted risk research, as specified by EPA, for re-registration o f other phenoxy herbicides used in the United States. A synopsis o f environmental and toxicology information with the phenoxy herbicides is presented here, but in Chapter 3, epidemiological and toxicological information is presented in greater detail. Based on dissipation studies, the acid, salt, and ester formulations o f 2,4-D readily break down in the soil with an average half-life of 4 to 10 days (1). The herbicide 2,4-D can persist in detectable amounts in the environment for 1 to 4 weeks after application. Movement in soil is usually less than 6 inches downward from the site of application. In sandy soils with low organic matter content in California, 2,4-D has leached only 12 to 18 inches even after large amounts o f water were applied (1 ). Thus, groundwater contamination with 2,4-D across the United States has remained very low (2), and new phenoxy herbicide management practices should further decrease the potential for groundwater cortfamination (4). Phenoxy herbicides decompose primarily by microbial activity, and degradation Page 2 of 6 increases with increased soil pH, moisture, organic matter, and temperature. Little genetically based weed resistance development has been observed despite the extensive use o f the phenoxy herbicides during the past five decades (7). Dissipation of 2,4-D is rapid in surface water and trace amounts are only occasionally detected (2), even though some ester and salt formulations o f 2,4-D are registered and used to control aquatic weeds in lakes and ponds. The Health Advisory Level for 2,4-D that was established by EPA for potable water is 70 ppb (3). EPA has rated 2,4-D as "practically nontoxic" for both warm water and cold water fish, and monitoring studies indicate no evidence of bioaccumulation in fish or the environment (1). EPA has classified 2,4-D's bird-dietary-toxicity and honeybee-toxicity as "practically nontoxic" (1). Because there is little residue from phenoxy herbicides in the air, soil, or water and because residues in treated vegetation decline rapidly, exposure o f wildlife to phenoxy herbicides has been low. The major effect phenoxy herbicides have on wildlife is habitat modification, because of reduction o f broadleaf vegetation, but these effects may even be beneficial to wildlife (6). The phenoxy herbicides are low in toxicity to humans and animals (1, 9). No scientifically documented human health risks, either acute or chronic, exist from the approved uses o f the phenoxy herbicides. Acute toxicity to humans, based on oral, dermal, ocular, or inhalation administration, may vary with the 2,4-D formulation. However, phenoxy herbicides have been used widely by numerous individuals (9) from home owners to farmers and ranchers, and even with significant exposure, humans have shown essentially no acute toxicity (11). In the late 1970's and the 1980's, questions were raised about the chronic effects to humans from repeated exposure to the phenoxy herbicides because o f the occurrence o f three rare forms o f cancer (10). A comprehensive study of the scientific evidence relating to the safety of 2,4-D was published in 1992 (11). This report stated that: "The case-control epidemiological studies that have been the source of the cancer risk hypothesis are inconclusive. Problems in assessing exposure based on patients' memories make these studies difficult to interpret. Cohort studies o f 2,4-D-exposed workers do not generally support the specific hypothesis that 2,4-D causes cancer. Taken together, the epidemiological studies provide, at best, only weak evidence o f an association between 2,4-D and the risk of cancer." The report further states: "Historical exposures to 2,4-D by user groups, particularly farmers, forestry workers and commercial applicators, would be higher than those sustained under present rigorous standards for application which involve the use of protective clothing and other measures to reduce exposure. Proposed label changes indicate that in the future exposures will be even further reduced. Viewed in this context, the available data indicate that the potential public health impact of 2,4-D, including the risk o f human cancer, was negligible in the past and would be expected to be even smaller in the present and future." Another Advisory Panel convened by the EPA to evaluate toxicology studies on 2,4-D since the prior report in 1992 (5) stated: "The Committee concludes that the data are not sufficient to find that there is a cause and effect relationship between the exposure to 2,4-D and NHL (non-Hodgkins lymphoma)." Thus, the risk to htlrfians from exposure to the phenoxy herbicides has been considered negligible. Therefore, a benefits assessment of the phenoxy herbicides in the United States, which is the objective o f this report, would be very useful considering the limited evidence of any apparent adverse human health or environmental risks from the use o f these herbicides. H O W THE ASSESSMENT WAS CONDUCTED An organizational meeisjg on NoveipJ)er 4, 1992 w'as attended by Orvin Burnside, Ronald Davis, Page 3 of 6 Leonard Gianessi, Dennis Kopp, Craig Osteen, and Nancy Ragsdale. We decided to conduct a biologic and economical benefits assessment of phenoxy herbicide use in the United States with Burnside as the Task Force chair. A Task Force o f eminent weed scientists and an economist would be selected by Burnside to prepare a benefits assessment o f all uses o f the phenoxy herbicides. The first meeting o f the Task Force on January 13, 1993 was attended by Rodney Bovey, Orvin Burnside, Herman Delvo, Clyde Elmore, Leonard Gianessi, Larry Hammond, Ellery la k e , Dennis Kopp, Carole Lembi, John Nalewaja, Michael Newton, and James Parochetti. We then selected an established weed scientist from each state, who we contacted by telephone before sending them generic questionnaires (Appendix 1) for each registered phenoxy herbicide use in their state. Our subsequent meetings were generally held in conjunction with the Weed Science Society o f America annual meetings to reduce travel expenses, because most Task Force members normally attended those meetings. At the February 9,1993 meeting, Philip Szmedra became a part of the Task Force to replace Herman Delvo as our economist Additional meetings were held February 7, 1994, May 3, 1995, and February 7, 1996 to update Task Force members, agree on solutions to concerns needing attention, and answer questions that did arise. 4 Questionnaires (Appendix 1) were sent out during early 1993 to our selected weed scientist in each state plus Puerto Rico, and they were requested to respond for each registered phenoxy herbicide use in each cropland or noncropland situation. I f the weed scientist did not feel qualified to provide information about some phenoxy herbicide use in their state, they were instructed to contact an individual, or individuals, with the best knowledge o f the needed inforand they were requested to respond for each registered phenoxy herbicide use in each cropland or noncropland situation. I f the weed scientist did not feel qualified to provide information about some phenoxy herbicide use in their state, they were instructed to contact an individual, or individuals, with the best knowledge o f the needed information. Separate questionnaires were furnished for each EPA registered use of Questionnaires were returned to Herman Delvo and he worked with Leonard Gianessi in recording and summarizing the data which Philip Szmedra then analyzed. Missing data from states caused us to alert the appropriate expert on our Task Force who in turn contacted the state weed scientists in question. I f no data were received on some specific use in any state, our Task Force expert then provided such data based on results from surrounding states or from personal knowledge o f the situation. Thus, we either received data from all states or we extrapolated to 100% o f a specific cropland or noncropland use in the United States. We compared estimated use o f phenoxy herbicides in the various cropland or noncropland areas with the actual sale o f phenoxy herbicides for the 1992 use year. If there was a discrepancy between the two amounts, the Task Force member responsible for that cropland or noncropland use made appropriate contacts and revisions to make these two estimates agree within reasonable limits: Efforts were made throughout the data analysis process to avoid double counting phenoxy herbicides uses or to avoid omissions o f use areas. Actual data analysis procedures are discussed in Chapter 4. The return rate for these phenoxy herbicides questionnaires (over 90%) was among the highest received by NAPIAP assessments. We believe such high returns were due to picking reliable respondents, contacting them for their concurrence before the questionnaires were sent, and following up by letters and telephone calls. Also, respondents were interested in retaining the use o f the phenoxy herbicides in the United States. ApDITIONAL INFORMATION ABOUT THIS REPORT Page 4 of 6 Each chapter in this report is intended to "stand alone," having its own Abstract, Literature Cited, and so forth. The various chapters are written by a recognized expert in that area o f phenoxy herbicide use, economics, epidemiology, or toxicology. For phenoxy herbicides and other herbicides that are derived from an acid, use rates are conventionally expressed in terms o f weight o f acid equivalent (a.e.) per unit area. For all other herbicides, use rates are conventionally expressed in terms o f weight o f active ingredient (a.i.) per unit area. This is how herbicide use rates are expressed in this report, but to reduce repetition we have dropped the use o f a.e and a.i., and we just express rate in pounds o f herbicide broadcast over one acre, which we abbreviate lb/A. Other abbreviations or acronyms used in this report are given in Appendix 2. We have used common names for plants and herbicides, to avoid cluttering the text with the Latin names o f plants and the chemical names o f herbicides included in this report. However, we have provided this information in Appendices 3 ,4 , and 5 for those who want more detailed information. Crops mentioned in this report are listed by common name in Appendix 3. The term crops is used in a broad sense, and it includes, for example, such things as trees in forests and orchards, and grasses in turf and rangeland. Weeds mentioned in this report are listed by common name in Appendix 4. The term weeds is also used in a broad sense, and it includes, for example, such things as woody species and plant species that would be considered crops, if they were growing where they were wanted. Herbicides mentioned in this report are listed by common name in Appendix 5. There, in addition to the chemical names, we have included some proprietary names (i.e., registered trade names, registered trademark names, and brand names). And finally, Appendix 6 is a glossary of terms used in weed science, most o f which have been used in this report, ACKNOWLEDGEMENTS Dennis Kopp, administrative advisor to this Task Force, and NSncy Ragsdale, Director o f the United States Department of^Agriculture NABIAP organization, were most helpful and encouraging during the entire assessment effort. They had the needed information, data, resources, advise, and counsel when and wheredt was needed. It was both an educational and enjoyable experience to work with such dedicated and capable individuals. Task Force members provided yeoman efforts when it was needed to complete this assessment in a timely manner. We appreciated their capable efforts in this "labor-of-love" as they all donated their time and effort to assuring the continued use o f these important phenoxy herbicides. Our selected state contacts were most generous with their time and talents as they met our many deadlines for the data that they were most capable o f providing. The assessment results are credible because we selected those most knowledgeable and actively engaged in the areas surveyed in each state plus Puerto Rico. We could not have done this assessment without them so they should accept our sincere thanks for a job well done. Thpse individuals are listed in the front pages o f this assessment under "Respondents to Our Questionnaires." Herman Delvo sent out the questionnaires, Leonard Gianessi compiled the survey data, Philip Page 5 of 6 Szmedra analyzed the results, and team members added the expertise that explained and summarized the results received. Also, there were many reviews o f the data and manuscripts as it went through the editorial process, which added to the creditability and value o f this phenoxy herbicide assessment. Finally, we want to thank our capable typist, Patricia Kessler, who typed the various chapters and made sure that we followed basic rules o f composition and editorial format. Eric Ristau produced our computer generated pie chart figures. Providing uniformity among the various chapters and readability was an objective that we strived to achieve. Also, we want to thank the publishers at Richtman Printing Companies, 301 NP Avenue, Fargo, ND 58107 who took our material and produced the finished product. We believe that we have produced an accurate and useful assessment o f the biologic and economic benefits of the phenoxy herbicides in the Unherbicides in the United States during 1992. This publication is the collaborative effort o f many individuals knowledgeable about the phenoxy herbicides, and it is dedicated to all individuals who have worked for the continued use o f these efficacious and economical herbicides. Table of Contents LITERATURE CITED 1. Ahrens, W. H., ed. 1994. Herbicide Handbook. Weed Science Society o f Amrica, Champaign. IL. 352 p. 2. Anonymous. 1986. National Water Quality Inventory, 1986 Report to Congress. Office o f Drinking Water, U.S. Environmental Protection Agency, Washington. EXt. EPA-44014-87-008. 185 P- 3. Anonymous. 1990. Drinking Water Regulations and Health Advisories. Office of Drinking Water, U.S. Environmental Protection Agency, Washington, D.C. 4. Anonymous. 1993. Agricultural Research to Protect Water Quality. Conf. Proc., Soil and Water Cons. Soc., Ankeny, IA. 755 p. 5. Anonymous. 1994. An SAB Report: Assessment o f Potential 2,4-D Carcinogenicity, Review of the Epidemiological and Other Data on Potential Carcinogenicity o f 2,4-D by the SAB/SAP Joint Committee. U.S. Environmental Protection Agency, Washington, D.C. Science Advisory Board, Report EPA-SAB-EHC-94-005. 6. Bramble, W. C. and W. R. Bums. 1974. A long-term ecological study of game food and cover on a sprayed utility right-of-way. Purdue University, Lafayette, IN. Bull. No. 918. 16 p. 7. Duke, S. O., ed. 1995. Herbicide-Resistant Crops: Agricultural. Environmental, Economic, Regulatory, and Technical Aspects. Lewis Publishers, CRC Press, Boca Raton, FL. 416 p. 8. Gianessi, L. P. 1993. The Use o f Phenoxy Herbicides in U.S. Agriculture. Resources o f the Future, Washington, b.C . 3 p. 9. Harris. S. A., K. R. Solomon, and G. R. Stevensort. 1992. Exposure o f home owners and bystanders to 2.4-dIHlordphehoxycetiC (2,4-D). J. Environ. Sci. Health. B27(l):23-28. 10. Linet, M. S. 1986. ftV W : AgfifclilkiHl Hefbicide Use and Risk o f Lymphoma and Soft Tissue Page 6 of 6 Sarcoma, and Other Epidemiologic Studies. Examining the Association Between the Herbicide 2,4-f), Herbicides in General, Farming and Malignant Lymphoma. John Hopkins School o f Public Health, Baltimore, MI). *, 11. M unjo, I. ., Q. L. Carlo, J. C. Op*, K. G. Sund, R.Ivf. Wilson, E. KennepoM, B. S. Lynch, M. fahinske, ap<^ N*X. Lee. 1992. Comprehensive, integrated review apd evaluation o f the scientific vidence relating to the safety of the herbicide 2,4-D. J.' Amer. Cptl. Toxicol. 1 (5):559^64. 12. Page, D. L. 1995. The reregistration of2,4-D ; where in th w o r l^ - a n d the environment --are We? p. 2-12. in Industry Tftsk Force IFbj 2,4 -ti R'se^rhJata. DowEj^n^, Indianapol^N . ' * - ` ! s f i l Pokomy, ^ 1941. New compounds; Amer. Ghem. Soe. 63:1768. ' --. '; Phenoxy Herbicides Chapter 2 Page 1 of 15 Chapter 2 The History of 2,4-D and Its Impact on Development of the Discipline of Weed Science in the United States ORVIN C. BURNSIDE1 Table of Contents A bstract. Research in the early 1940's on 2,4-D in the United States and MCPA in England, their release after World War II. and their rapid acceptance by farmers put selective weed control in the "public spotlight" worldwide. Phenoxy herbicides have provided economical, selective, postemergence control of broadleaf weeds in grass crops and noncropland for the past five decades. Weed science technology has had a major impact during this herbicide era in increasing crop yields and reducing labor requirements for controlling weeds. Farmers rely heavily on 2,4-D and subsequently developed herbicides as a major component of their weed management program. Because the phenoxy herbicides were so effective, weed scientists initially concentrated their research and educational efforts on chemical control o f weeds. This trend continues today and has impeded research on alternate weed management technology. Herbicide development will continue to be the focus o f chemical company personnel because o f the profit incentive. Consequently, publicly-funded weed scientists will need to emphasize preventive, cultural, mechanical, biological, and integrated control technologies so they can provide growers and land users with more balanced systems of weed management. 'Prof., Dep. Agron. and Plant Genet., Univ. Minnesota, St. Paul, MN 55108. INTRODUCTION Weeds are the number one pest problem in crop production because they reduce crop yields and increase production costs (3, 4, 9). Herbicides are the most widely used class o f pesticides in the United States because they are effective and economical in controlling weeds, and because weeds are the most important pest in crop production. In addition, herbicides reduce manpower and horsepower requirements and erosion-producing tillage in crop production. No exact figure has been placed on the economical benefits provided to world agriculture by the introduction o f the phenoxy herbicides, which started the herbicide revolution in developed countries, but the estimated value to humankind from reduced labor requirements alone is in the billions of dollars annually. Sale of phenoxy herbicides in the United States was about $171 million in 1992, but the loss o f these herbicides would increase alternative herbicide and non-chemical weed control costs by $947 million annually (10, 16). The phenoxy herbicides still represent over 10% o f herbicide poundage used in the United States, and they are the most widely used family o f herbicides worldwide. Our NAPIAP report will provide EPA personnel information about the phenoxy herbicides that will be useful in their deciding whether this class o f herbicides should be re-registered for agricultural crops, forestry, pastureland, rangeland, roadsides, turfgrass, aquatics, and other noncropland uses in the United States ( 10). The objectives o f this chapter are: (a) to trace the discovery and development o f 2,4-D, and (b) to discuss the impact o f 2,4-D and subsequent herbicides on the development o f the discipline of weed Phenoxy Herbicides Chapter 2 Page 2 of 15 science. DISCOVERY AND D EV ELO PM EN T O F 2,4 D The phenoxy herbicides, and 2,4-D in particular, ushered in the chemical weed control revolution in the mid 1940's. Even 51 years after its introduction, 2,4-D continues to be the most commonly and widely used herbicide worldwide. This discussion encompasses the numerous research and development pieces that completed the 2,4-D "puzzle" and its subsequent extensive utilization as a selective herbicide (Table 1). No one can give credit to all the personnel involved in the basic, applied, developmental, and marketing activities that brought 2,4-D use to fruition; however, that should not prevent an attempt at recognizing some major developmental and utilizational milestones. Table 1 is a chronological listing of some major advances in the history of 2,4-D development in the United States and worldwide, and when and where these developments occurred. Table 1. Initial research on selective herbicides. The original research on selective herbicides was carried out by Bolley in the United States, Bonnett in France, and Schultz in Germany around 1900 (20). These researchers independently found that solutions of copper salts and other inorganic compounds would selectively control broadleaf weeds in cereals. Bolley, a plant pathologist, wrote in 1908 (6) that "When the farming public has accepted this method (selective weed control) of attacking weeds . .. the gain to the country at large will be much larger in monetary consideration than that which has been afforded by any other single piece of investigation applied to field work in agriculture." Bolley continued with, "If, therefore, this method o f attacking weeds by means of chemical spray is one-quarter or one-half as successful in general operation as the writer is willing to vouch for, the money returns to the spring wheat growing states must far exceed the hopes of the most optimistic." This prophecy by Bolley regarding new methods o f weed management actually came to fruition with the advent of the phenoxy herbicides in the mid-1940 era (32). Basic research th at led to 2,4-D. Botanists have long been intrigued with plant shoot and root gtowth and the mechanisms causing plants to respond to stimuli. Darwin from England in 1880 (33) reported that plants bend towards the light and that some influence from the tip of oat coleoptiles caused this bending or phototropic response. Boysen-Jensen from Denmark in 1911 (33) reported that when an oat coleoptile that was exposed to directional light was excised and reattached to unexposed oat tips, the bending still occurred. He concluded that the influence from coleoptile tips was thus chemical not physical. Went from the Netherlands in 1926 (33) collected the chemical from oat coleoptile tips and found it to be an active plant growth regulator. He developed a quantitative measurement of the regulator and thus stimulated considerable research in this area. Kogl and Haagen-Smit from the Netherlands in 1934 (33) reported the isolation of indoleacetic acid (IAA) from plants and human urine and identified it as the principal naturally occurring hormone (later called an auxin) in plants. When humans eat fresh vegetables they injest the hormone IAA (chemically very similar to the more stable 2,4-D) and excrete it in their urine. Because IAA was unstable outside o f plants, researchers began synthesizing and investigating the effect of IAA derivatives and homologs on plant growth activity. Zimmerman and Wilcoxson from the United States in 1935 (37) reported the discovery of phenylacetic acid and naphthyl acetic acid, which affected plants by preventing premature fruit drop, inducing rooting, accelerating fruit ripening, and causing seedless tomatoes. Pokomy from the United States in 1940 (29) synthesized 2,4-D and 2,4,5-T while looking in vain for a fungicide. In June 1941, Pokomy described the chemical synthesis of 2,4-D (29). In 1942 Zimmerman and Hitchcock (36) reported that 2,4-D was 300 times more potent than indolebutyric acid, the major plant growth regulator at that time, for inducing seedless Phenoxy Herbicides Chapter 2 Page 3 o f 15 tomatoes. ' * ' 'i Monochloroacetic acid and 2,4-dichlorophenol were used to make 2,4-D (29). Subsequently, salts were made by adding the appropriate amine or inorganic hydroxide to the acid (1). Esters were synthesized by reacting 2,4-D with the appropriate alcohols. The discovery o f 2,4-D could be considered more fortuitous than systematic, but one must recognize that discovery begets further discovery, and the process seems to gain momentum and often culminates in numerous and diverse scientific advances. W ent stated that, "When I worked 25 years ago with the growth hormone (2,4-D), I had many wild ideas about what it might do once it was available in large quantities, but I never dreamed that it would lead to the development o f weed killers. This is an excellent example, how fundamental research may lead to the solution o f very practical problems." (2). Therefore, basic research on plant growth regulators during the 1930's and earlier (33) facilitated the development o f selective phenoxy herbicides in the 1940's. Thus, the discipline o f weed science evolved from the field o f economic botany or more specifically the study o f plant growth regulators. M ilitary interest ill 2,4-D. Interest in 2,4-D within the scientific community seemed to lose momentum during World War II when both United States and England scientists initiated secret biological warfare research on plant growth regulators with the objective o f destroying enemy crops. Kraus at the University o f Chicago had observed since 1936 that certain growth regulators were phytotoxic (28). Kraus was aware o f the inadequacy o f existing herbicides, and in 1941 he was first to propose that growth regulators might work as herbicides, because they oftenwhen both United States and England scientists initiated secret biological warfare research on plant growth regulators with the objective o f destroying enemy crops. Kraus at the University o f Chicago had observed since 1936 that certain growth regulators were phytotoxic (28). Kraus was aware of the inadequacy o f existing herbicides, and in 1941 he was first to propose that growth regulators might work as herbicides, because they often killed test plants. In late 1941, Kraus and other prominent scientists convinced Secretary o f War, H. L. Stimson, o f the potential dangers In November 1942, the United States Army began developing Camp Detrick (later renamed Fort Detrick) in Frederick, Maryland, as the center for research and testing of chemicals for biological warfare with special emphasis on crop destroying chemicals. In March 1943, the United States Army paid the University o f Chicago $3500 for herbicide research completed by Kraus (28). In January 1944, research at Camp Detrick was accelerated on crop destroying herbicides. W. B. Ennis, Jr. was one of a small group o f military scientists assigned to Camp Detrick, and he provided the following information regarding research by the military with the phenoxy herbicides2. 2Ennis, W. B., Jr. 1995. Personal communication from one o f the military scientistsassigned to Camp Detrick, Maryland, to O. C. Burnside, Dep. Agron. and Plant Genet., Univ. Minnesota, St. Paul, MN. While laboratory facilities were under construction at Camp Detrick, E. J. Kraus at the University o f Chicago and J. W. Mitchell with the USDA at Beltsville, Maryland, were supported by contract to continue studies of the herbicidal effects o f plant growth regulators. Somewhat later, a synthesis program was commenced under contract with A. S. Newman at Ohio State University. In 1944, the biological warfare effort at Camp Detrick was stepped up by the Special Projects Division o f the Chemical Warfare Service, U.S. Army under a heavy cloak of secrecy. Herbicide research with crop destruction as the objective was vigorously pursued under the direction o f A.G. Norman by a team of about a dozen scientists drawn from other assignments in the services. Most were recent Ph.D. graduate students or graduates in agronomy or botany. Phenoxy Herbicides Chapter 2 Page 4 o f 15 The research program was broadly conceived to throw light on the nature o f plant responses,,p la n t1 and herbicide specificities, environmental effects, effective rates o f application, spray volumes and droplet size, co-agents and carriers, etc. Although attention was soon centered on halogen-substituted aiyloxy acids, particularly the phenoxyacetics, many new compounds and derivatives were synthesized and screened for growth regulating properties. Sufficient amounts o f the more active compounds, such as 2,4-D, 2,4,5-T and others, were procured for field trials, which included aerial application. Attention was also given to forest defoliants for the purpose o f reducing cover o f enemy defense positions in the expected assault on Japan. All the research conducted at Camp Detrick was kept under military secrecy until the end o f World War H. Then the scientists were free to publish results o f their research. The entire June 1946 (Vol. 107) issue o f the Botanical Gazette consisted o f papers from Camp Detrick scientists. These papers reported well-designed experimental observations likely to be o f general interest. Muchadditional information was obtained, but not all in the rigorous experimentaldetail justifying journal publication. Additional papers appeared later in the Agronomy Journal, American Journal o f Botany, Science, Weeds, and other journals. The research at Camp Detrick formed a major part of the beginning of modem weed science and represented a significant part ofthe foundation knowledge on weed control with herbicides. Among the accomplishments o f the Camp Detrick scientists were the development of methods for evaluating over 1000 chemical compounds fortheir herbicidal properties, determining histological effects o f 2,4-D and 2,4,5-T, cytological effects o f propham, demonstration that weeds can becontrolled with ultra low-volume sprays, investigating theimportance o f carriers for 2,4-D (solvents, surfactants, granules,etc.), defining the selective action of sprays on broadleaf plants,identifying the herbicidal effects o f soil and water applications, anddetermining the dosages - required. The herbicide 2,4-D was patented by American Chemical Paint Co. with licensing provisions for production by Dow Chemical Co. and other companies. Commercialization followed in the mid to late 1940's. After the war most o f the scientists in the program returned to civilian life, some to universities to complete graduate programs and later to embark on careers that stemmed from their involvement in the Camp Detrick program. The Camp Detrick program, however, was not terminated. After a few months o f reduced activity it was converted to a civilian staffed operation administered by the Civil Service. Security was reduced, yet certain aspects remained classified. A new staff was slowly assembled, including some o f the wartime group. The program was less specifically targeted than previously and broadened to encompass various types o f growth responses in plants. Synthesis and screeningwere continued and improved procedures for assessingherbicidal activity were devised. Camp Detrick research wassupplemented by contracts with universities, and research was initiated on abscission and the gibberellins. Leaf abscission and the testing o f defoliants for woody species were pursued during 1961-72 under the direction o f C. E. Minarik, which led to their military use that became so controversial in Vietnam operations. All biocidal research activities were terminated in 1972, following adeclaration by President R. M. Nixon that the United States would nolonger conduct biological warfare research or develop biologicalweapons. The herbicide/defoliant research at Camp Detrick was then closed and its personnel dispersed. Some of the plant researchlaboratories and facilities were taken over by USDA-ARS scientists and personnel for research relevant to their ongoing programs." Phenoxy Herbicides Chapter 2 Page 5 o f 15 The development of organic herbicides was greatly facilitated during World War H because of their military potential as biological warfare weapons (28). Throughout the war, United States Army research on the phenoxy herbicides was done in secrecy until January 3,1946 when Secretary o f War, R. P. Patterson, announced that more than 1000 different chemical agents had been tested on living plants. In May 1946, G. W. Merck (28), Director o f the Civilian War Research Service, declared that, "...only the rapid ending o f the war prevented field trials in an active theater that would, without injury to human or animal life, affect the growing crops and make them useless." Thus, herbicides were not used in chemical warfare until November 21, 1962 when the United States and South Vietnam military personnel sprayed 2,4,5-T plus 2,4-D (Agent Orange) in Vietnam to defoliate trees along military routes to reduce sniper activity (7,11). Several Vietnam veterans in my subsequent weed science classes praised the use o f Agent Orange because o f the safety it provided from sniper fire for our military personnel. Antiwar protestors seized on the military's use o f Agent Orange in their successful campaign to discredit this unpopular war. In the 1970's, following cessation o f military hostilities, spokespersons for Vietnam veterans alleged that health problems and deaths of veterans were related to Agent Orange exposure in the military, and these allegations persisted. Thus, the public announcement on March 15,1979 by EPA Administrator, W. D. Ruckelshaus, of an Emergency Suspension Order and Threat to Cancel further use o f 2,4,5-T and silvex in the United States was strongly propelled by political pressure and not based upon scientifically developed risk assessment data. Unfortunately, on January 2, 1985 agriculturalists and foresters lost all registered uses o f 2,4,5-T and silvex, effective and widely used woody plbiological warfare weapons (28). Throughout the war, United States Army research on the phenoxy herbicides was done in secrecy until January 3, 1946 when Secretary o f War, R. P. Patterson, announced that more than 1000 different chemical agents had been tested on living plants. In May 1946, G. W. Merck (28), Director of Commercial development of 2,4-D. Public plant scientists in the United States (22, 36) and England (5, 30) continued to do limited research with 2,4-D during World War Et. Research in England stressed the development of MCPA, a herbicide similar to 2,4-D. MCPA was favored in England because o f a plentiful supply o f cresol extracted from coal and used to make MCPA versus a plentiful supply o f phenol from oil refineries in the United States which was used to make 2,4-D. In 1942, Zimmerman and Hitchcock (36) reported that the phenoxyacetic acids could induce seedless tomatoes and that they were potent synthetic plant hormones. Kraus and Mitchell tested 2,4-D at the University o f Chicago and Beltsville, Maryland, but, because of the war, did not publish their results until 1944 (28). In June 1944, Mitchell and Hamner with the United States Department o f Agriculture (USDA), Bureau of Plant Industry at Beltsville, Maryland, made the first public announcement of using 2,4-D as a herbicide that exhibited differential weed kill (22). Hamner then left the USDA and joined the Agricultural Experiment Station at Geneva, New York. In August 1944, Hamner and Tukey (17) caused considerable public interest when they reported that within 10 days after spraying field bindweed with 2,4-D, the weeds died. English researchers had worked with MCPA, 2,4-D, and other plant growth regulators during the early 1940's but delayed publishing their results until after World W arff (5, 30). Marth and Mitchell in 1944 (22) sprayed 2,4-D on a lawn infested with dandelions at Beltsville, Maryland, and achieved selective broadleaf weed control, with no injury to the lawn grasses. Mitchell et al. (24) then conducted additional studies on a golf course in August and September 1944 and in October 1944 reported selective broadleaf weed control in turfgrass. Dayis in 1945 (12), with the United States Golf Association, was the first person to direct a developmental program that would Phenoxy Herbicides Chapter 2 p age 6 o f 15 result in practical selective weed control with 2,4-D in turfgrass and lawns. Davis even sprayed 2,4-D on turfgrass of the National Capital Park Service including the White House lawn. In 1945, workers at various state agricultural experiment stations began the first extensive field testing o f 2,4-D (28). Also, the popular abbreviation, 2,4-D, first appeared in the literature in 1945. When the second annual North Central Weed Control Conference (NCWCC) workers met November 26 to 28,1945 in St. Paul, Minnesota, Timmons indicated that data were reported from 30 cooperators with 140 experiments conducted in the United States and 36 experiments conducted in Canada (31). Kephart with the USDA chaired a panel discussion on new chemicals for weed control, and plant injury problems due to phenoxy herbicide drift were first reported (19). Experiments in the late 1940's proved that Kraus had been right in 1941 when he first envisaged the use o f plant growth regulators as herbicides (28). Military secrecy that covered the Camp Detrick research did not extend to all herbicide applications that scientists discovered. Thus, the original patent o f 2,4-D and related compounds (U.S. Patent Number 2,322,761) was as plant growth regulators by John F. Lontz and assigned to E.I. du Pont de Nemours and Company dated June 29, 1943 (28). Franklin D. Jones with the American Chemical Paint Company (ACPC) filed on March 20, 1944 and received use patent 2,390,941 in December 1945 for 2,4-D as a herbicide3. In 1944, Mitchell paid $12.50 to ACPC and Davis paid $78.00 to Sherwin-Williams Company for a pound of 2,4-D, but the price dropped to less than $3.00 per pound in 1945 and $0.50 per pound in 1950 (12, 28). In June 1945, ACPC marketed 2,4-D under the brand name Weedone, which was the first selective, systemic herbicide produced and sold on a commercial scale. In 1945, Weedone sold so poorly that many dealers wanted to return their unsold stocks, but appropriate advertising in Better Homes (now Better Homes and Gardens) and the Reader's Digest stimulated enough sales for ACPC so that in 1946 150,000 acres were treated.3 However, one must not overlook the tremendous impact o f research by public weed scientists across the United States and Canada during 1945 and thereafter, which generated immediate farmer interest in selective weed control (12,19,31). 3Jones, F. D. 1964. Personal communication, from the individual who patented 2,4-D as a herbicide, to C. J. Willard, Dep. Agron., Ohio State Univ., Columbus, OH who was then editor o f the journal W eals.) The Hamner and Tukey article about field bindweed control (17) precipitated such interest among regulatory personnel, farmers, and home owners in 2,4-D that manufacturers rushed to market the herbicide. Witman wrote 4 that the agricultural chemical industry in the UnitedStates had geared upchemical industry in the UnitedStates had geared up to merchandise 2,4-D w h en ,11. . . the industry was thunderstruck on December 11, 1945 when U.S. Patent 2,390,941, on herbicides containing chlorophenoxyacetic acids, was issued to Franklin D. Jones and assigned to American Chemical Paint Company. However, cool-headed businessmen were able to negotiate licensing o f the Jones Patent for a very small royalty fee for anyone who sought to make and sell chlorophenoxyacetic acid herbicides, and the 1946 season went forward to a spectacular success for Sherwin-Williams Company lawyers filed a lawsuit against ACPC's patent o f 2,4-D as aherbicide and the following judgement was rendered;5 "Under this settlement, the Government and the Public are given a free license to make and use, but not to sell,compositions covered by the patents involved in file litigation. In addition, the same free license is given to theGovemment and the Public under the three other Jones' patents. Americansell,compositions covered by the patents involved in the litigation. In addition, the same free license is given to theGovemment and the Public under the three rnenoxy nermciaes tmapter l Page 7 of 15 other Jones' patents. American Chemical PaintCompany, Ambler, Pa., will, upon request grant licenses to any responsible party desiring tomanufacture and sell the patented compositions. The royalty to be charged has been fixed at amaximum o f 2% of the net sale and price o f patented compositions, with a minimum amountroyalty o f $250 and a maximum total royalty for the lives of the patents of $10,000." Obviously, 4 Witman, E. D. 1978. Personal communication from a product development specialistwith PPG industries. Inc. to J. H. Dawson. Res. Center. Washington Agric. Stn., Prosser.WA who was then editor of the WSSA Newsletter. 5 Press release. 1947. Announcement of the settlement on October 24, 1947 of Sherwin-Williams' litigation against the American Chemical Paint Company patent o f 2,4-D as aherbicide. USDA officials also recognized the importance o f 2,4-D and ordered human toxicity studies in 1945 and all o f these proved negative. Mitchell et al. (25) reported that treating pastures with twice the normal use rates o f 2.4-D produced no toxic effects in sheep and cows grazing on them, and feeding a cow 5 1/2 grams of pure 2,4-D per day for 3 months produced no ill effects to the cow or her calf fed entirely on milk from that cow. Kraus even announced that he had personally eaten one-half gram of 2,4-D per day for 3 weeks with absolutely no ill effects (19). Several 2,4-D esters with low volatility were patented (U.S. Patent Numbers 2,523,227 and 2.523.228) in 1950 by W.H. Mullison and assigned to Dow Chemical Company. The butoxyethyl ester of 2,4-D with low volatility was patented (U.S. Patent Number 2,543,397) in 1951 by W. W. Allen and assigned to Union Carbide. The first year o f widespread testing and sale o f 2,4-D in the United States was 1945 and 917,000 pounds were produced (28). Production rose to 5,466,000 pounds in 1946 and 14, 36. and 54 million pounds in 1950, 1960, and 1964, respectively. At present, the annual production o f 2,4-D for use in the United States is about 47 million pounds (10, 16), so 2,4-D is still one o f the more widely used herbicides in the United States and also worldwide. This continued demand for 2,4-D shows that it has markedly advanced humankind's utopian dream o f lifting the hoe from the farmer's hand and reducing much of the drudgery associated with crop production. Table of Contents DEVELOPMENT OF THE WEED SCIENCE DISCIPLINE Weed science is one o f the younger academic disciplines in agriculture, first receiving significant recognition in the United States in the mid-1940's because o f the introduction o f the phenoxy herbicides (9, 20). Research in the early 1940's on 2,4-D in the United States and MCPA in England, their release after World War II, and their rapid acceptance stimulated selective weed control research and education worldwide. Extensive research on mechanical and cultural weed management before 1945 should have brought recognition to the discipline o f weed science, but these efforts were generally identified as part o f agronomic practices or crop production systems. Leighty (21), for example, wrote in the 1938 Yearbook of Agriculture that, "Weeds, aside from the noxious species, are not a serious problem on a well-organized diversified farm. Indeed, there is little excuse for such a farm to be weedy." And he goes on to say, "In some kinds o f one-crop farming, weeds, while plentiful, do not prevent the production of good yields." Furthermore, even though the major benefit o f tillage is weed control, many farmers tended to identify cultivation benefits as coming from tillage per se. Thus, many early agricultural scientists did not give adequate recognition to weeds and the Phenoxy Herbicides Chapter 2 Page 8 o f 15 importance o f their control until the advent of the phenoxy herbicides (9,18). Weed control results from 2,4-D and subsequent herbicides were so spectacular that weed workers concentrated their efforts on chemical control o f weeds and this shift impeded research and education on alternative weed management technology (9, 34, 35). Also, this shift in weed research, development, education, and marketing efforts over the past five decades has impacted all areas of weed science. The objectives o f this section are: (a) to enumerate the economic importance o f weeds and the extent o f herbicide use, and (b) to discuss the impact o f 2,4-D on the development of the herbicide industry, regulatory agencies, farmers, and publicly-funded weed scientists. Monetary losses from weeds. In the early 1960's, USDA scientists estimated the relative proportion o f total losses in productivity o f United States agriculture from insect pests to be 10%, soil erosion 13%, livestock diseases 17%, plant pathogens 26%, and weeds 34% (3). In addition, farmer costs were about three times higher for controlling weeds than for controlling plant pathogens and insect pests combined. In 1971, after substantial use of pesticides for controlling pests (various biological organisms that reduce crop yields or quality) in agriculture, USDA scientists estimated annual United States losses to crop pests as follows: nematodes 3%, plant pathogens 27%, insect pests 28%, and weeds 42% of total pest losses in crop production (4). These relative loss comparisons probably have not changed greatly in the past two decades. Annual weed losses (mainly reduced crop yields and increased costs o f control) in the United States were estimated to be over $20 billion in 19936. Thus, weeds remain an important impediment to crop production because they are omnipresent, they reduce crop yields, and weed control often constitutes the major cost o f producing a crop even though we have eliminated much of our reliance on manual weed control. It must be recognized, however, that crop losses from weeds, costs o f control, and crop production costs in general would be much greater if farmers were forced to revert to crop management methods used prior to 1945. Herbicide costs and usage. In 1991, pesticides were a $26.8 billion industry worldwide with sales totaling $6.4 billion in the United States or 24% o f worldwide pesticide sales (23). Herbicides represented about 65% o f total pesticides used in the United States in 1990 and 85% o f pesticides used on cropland (13). Herbicide use on crops in the United States grew from 115 million pounds in 1966 to a peak o f 500 million pounds in 1982 when over 90% o f the acreage o f major crops was treated with one or more herbicides (15). Since 1982, total pounds o f herbicides used has decreased about 10% because o f the introduction of compounds that are active at lower rates, reductions in use rates o f older herbicides, and government sponsored acreage diversion programs (9). The phenoxy herbicides still represent about 10% of total herbicide usage in the United States. 6 Bridges, D. C. 1993. Personal communication from the chairman o f the Weed Science Society o f America, Weed Loss Committee, to O. C. Burnside, Dep. Agron. and Plant Genet., Univ. Minnesota, St. Paul, MN.) Impact of 2,4-D on the herbicide industry. The phenoxy herbicides stimulated chemical industry involvement in herbicide development because industry personnel recognized the weed control need in agriculture and noncropland and the profit potential o f this technology. The spectacular results with 2,4-D stimulated an industry-wide search for additional herbicides, which ushered in the herbicide era that has lasted for five decades. Total numbers o f hedevelopment because industry personnel recognized the weed control need in agriculture and noncropland and the profit potential of this technology. The spectacular results with 2,4-D stimulated an industry-wide search for additional Phenoxy Herbicides Chapter 2 Page 9 o f 15 herbicides, which ushered in the herbicide era that has lasted for five decades. Total numbers of herbicides available in the United States as listed by Timmons (32) or in Weed Science Society o f America journals (9) were 14 in 1940, 25 in 1950, 61 in 1960, 131 in 1970, 156 in 1980, and 145 in 1990. Thus, industiy rapidly expanded personnel in herbicide development, manufacture, and sales in order to exploit the market potential o f herbicides that increased rapidly from 1945 until about 1982 when herbicides use peaked in the United States (15). Since 1982 there has been increased competition among chemical companies because the herbicide market in the United States had matured and profits were decreasing as companies competed for the same market share rather than exploiting mainly untapped markets. T7 projects that this consolidation may continue until only six full-service pesticide companies (synthesis of pesticides on through to sales) will remain worldwide. 7Carpenter, W. D. 1995. Personal communication from the retired Vice President andGeneral Manager, New Products Division, Monsanto Company, St. Louis, MO to O. C. Burnside, Dep. Agron. and Plant Genetics, Univ. Minnesota, St. Paul, MN.) Impact of 2,4-D on the regulatory agencies. The phenoxy herbicides were vigorously promoted by personnel o f various weed control regulatory agencies that were already in place when 2,4-D was first sold in 1945. For example, the Connecticut legislators enacted in 1726 a European barberry control law which was the initial United States program to aid in the control o f black stem rust o f wheat, but a more effective barberry control law was enacted in 1917 that encompassed 13 North Central and Western States (32). Federal and states' Ribes spp. Quarantine and Eradication Laws aimed at the control o f white pine blister rust were initiated during 1 9 1 2 to l9 1 9 in 2 6 states. In 1914 Virginia passed a law requiring the eradication o f red cedar as an aid in the control o f apple rust, and similar legislation was also enacted in four other states. Numerous States and Canadian Provinces enacted Seed Laws beginning in 1821 in Connecticut and Noxious Weed Control Laws beginning in 1872 in Minnesota, and these regulatory personnel immediately recognized the importance o f 2,4-D in carrying out their legislative mandates (32). However, 2,4-D did not turn out to be the panacea weed control herbicide that it was advertised to be, and thus no noxious weeds were eradicated (2). Laws aimed at the prevention or spread of undesirable weeds such as Seed laws have been moderately effective in reducing the spread o f weeds, Weed Control Laws more often than not have been ineffective, and Eradication Laws have been ineffective because eradication o f a weedy species from a large area has proven to be an impossible objective (8). Thus, the impact o f regulatory efforts in weed management and financial support of these regulatory programs has eroded over the past several decades. However, regulation o f pesticides by the EPA and similar state agencies has increased over the past several decades. Impact of 2,4-D on the farmer. The phenoxy herbicides raised weed management to a higher level, and producers accepted this new technology rapidly and with enthusiasm, because they were able to achieve better weed control with less labor and expense (20, 31,32). Phenoxy herbicides provided effective, economical, and selective broadleaf weed control in small grains, com, sorghum, pastureland, rangeland, and turfgrass. I remember when farmers commonly lost crops to weeds such as quackgrass, Canada thistle, or wild mustard. Now such a farmer would be considered a poor steward of the land and would be the topic of conversation at the local coffee shop. I also remember having to get off the tractor and remove wild mustard plants, or they would clog my father's small grain binder. Then, 2,4-D was introduced, and we were all amazed at its ability to selectively control wild mustard in small grains at a very Phenoxy Herbicides Chapter 2 Page 10 o f 15 economical cost (see picture on the cover page). Farmers began expecting selective herbicides for numerous other cropping systems, and the agricultural chemical industry responded positively because o f the profit potential. Herbicides such as 2,4-D and MCPA established the concept of postemergence, selective weed control; chloramben and atrazine established preemergence, selective weed control; and EPTC and trifluralin established preplant-soil-incorporated, selective weed control. Examples o f changes in herbicide technology over the past five decades are inorganic to organic herbicides, nonselective to selective herbicides, postemergence to soil-applied (some incorporated) herbicides and now back again, mechanical to more chemical control of weeds, single herbicides to mixtures and even multiple applications, formulated herbicides used alone to utilizing various herbicide additives, and crops bred or transformed for tolerance to specific herbicides rather than herbicides being screened for crop tolerance (9). The American farmer relies heavily on herbicides as the major method of weed control, and herbicides allow them to farm more and more acres (9,14,35). Herbicide use and farm equipment development has resulted in substantial decreases in number o f farmers and farm laborers required for crop production in the United States (26). Adequate alternative methods o f weed control are not currently available in the event o f future restrictions on herbicide usage that may result because o f public concern about herbicides in ground and surface water, toxicity to humans, chronic health effects, food safety, and wildlife mortality (9). Operators o f large acreage farms will continue to demand more and better selective herbicides; whereas, an increasing group o f organic and smaller acreage farmers are looking for alternative and more economical weed management methods (34, 35). Impact of 2,4-D on publicly supported weed scientists. The phenoxy herbicides had such an impact on weed technology that most publicly-supported weed scientists became engrossed in chemical weed control research and education endeavors (9, 34). Thus, weed control methods have changed because weed scientists slighted or abandoned alternative weed management technology. We have witnessed five decades in which most o f the weed science research in the United States has had some connection with herbicides. However, this herbicide era has markedly advanced our ability to manage weeds in cropland and noncropland, and greatly reduced labor requirements and crop losses in agricultural production. University and USDA-Agricultural Research Service (ARS) administrators began hiring weed scientists after the advent of 2,4-D, but employment numbers never reflected the fact that weeds were a greater production hazard than either plant diseases or insect pests (2,9,27). Possibly administrators rationalized that the herbicide industry would support or supply many o f the weed science research and educational needs. For example, federal funding for pest management research at universities in fiscal year 1991 was $61 million for Entomology, $50 million for Plant Pathology, $9 million for Nematology, and $6 million for Weed Science (27). In 1995, Ogg (27) contacted the business managers of the Entomological Society o f America to learn that they had 8100 members and 6075 were publicly-funded, the American Phytopathological Society had 5000 members and 4250 were publicly-funded, and the Weed Science Society o f America had 2200 members and 550 were publicly-funded. Such public-funding and hiring practices forced the few publicly-funded weed scientists to conduct research and education activities in the areas where they could make the most rapid progress and receive industry support, i.e., herbicide technology. Is it any wonder that recent assessments of weed management technology (9 ,1 4 ,3 4 ,3 5 ) have shown that farmers place an overwhelming emphasis on chemical weed control technology? If public weed Phenoxy Herbicides Chapter 2 Page 11 o f 15 scientists do not conduct preventive, mechanical, cultural, biological, and integrated weed management research, it will not be done. We coverwhelming emphasis on chemical weed control technology? If public weed scientists do not conduct preventive, mechanical, cultural, biological, and integrated weed management research, it will not be done. We cannot expect to entice large numbers o f chemical company weed scientists into researching alternative weed control technology. They must research, develop, and sell marketable products that return a profit or there will be no herbicide industry (9). M ost alternative weed management methods do not have the profit potential to merit private-sect FUTURE TRENDS IN WEED SCIENCE One can predict that weed science will continue to show modest growth in public research, education, and herbicide regulation areas, as well as in the area of private consulting; however, the number of weed scientists within the chemical industry will decrease because o f continuing consolidation o f pesticide companies (9). Academic employment opportunities should increase for those trained in the areas o f weed biology, ecology, and management Weed control activities will move from the largely chemical approach to systems involving greater use o f preventive, cultural, mechanical, biological, and integrated weed management activities. Herbicides will continue to be important in weed management, but they will be used more selectively and judiciously. The use o f postemergence herbicides may increase as they will be used to "back up" alternative weed control methods so that fanners do not experience crop yield loss while initiating the use of new weed management technology. However, public weed scientists must redirect their activities after five decades o f largely herbicide-focused research and undertake a major change in weed research objectives to develop basic information about weeds and problem solving research using integrated weed management systems (9,34,35). If there is one thing that I have learned from 35 years o f weed research, it is that there is no panacea weed control method. Thus, one must emphasize an integrated approach to weed management, as certain weed species have survived all individual weed control methods that humankind has so far devised. Greater use of on-farm research may aid in developing the needed weed management technology. Public weed scientists have exciting years ahead as they emphasize research and education directed toward weed biology and life histories, weed seedbanks, seed and bud dormancy, cover and smother crops, rotation o f crops with different life cycles, weed-competitive crops, biological and ecological weed management, postemergence herbicide technology, precision herbicide applications that vaiy rates or herbicides based on soil type and climate and weed species, environmentally benign herbicides or mycoherbicides, spray equipment activated by the presence o f weeds, interference modeling of crop-weed systems, reduced-tillage crop production systems, and the destruction or inactivation of weed propagules to develop integrated, sustainable, and environmentally safe weed management methods. We should not abandon herbicides, but we can use them more judiciously (9). We need to conserve natural resources such as soil and fossil fuels while increasing the productivity, profitability, and competitiveness o f agriculture in the United States. Also, a major challenge for us in the future is to optimize weed management systems to meet or exceed the water quality and environmental impact standards demanded by the public. Implementing alternative weed control methods, not more monitoring programs, is the way to solve problems o f pesticide contamination o f water or the environment. To accomplish these objectives, an increase in public funding for the development of weed science as a discipline must occur because development of alternative weed management methods will not attract substantial private funding. Phenoxy Herbicides Chapter 2 Page 12 of 15 Now weed scientists must settle down and attend to research and educational endeavors that have been put on the "back burner." while continuing to pursue the judicious use of herbicides. The future will be just as exciting as the past, as weeds do not give up their turf easily. Table of Contents LITERATURE CITED 1. Ahrens. W. H., ed. 1994. Herbicide Handbook. Weed Sci. Soc. America, 1508 West Univ. Ave., Champaign, IL. 352 p. 2. Andersen, R. N. 1991. The North Central Weed Control Conference: Origin and Evolution. North Central Weed Sci. Soc., Champaign, IL. 206 p. 3. Anonymous. 1965. Losses in Agriculture. U.S. Dep. Agric., Washington, D.C. Handbook No. 291. 120 p. 4. Anonymous. 1971. The Economics of Agricultural Pest Control. USDA Pesticide Review, Econ. and Stat., Washington, D.C. Report No. 14. 60 p. 5. Blackman, G. E. 1945. A comparison o f certain plant growth substances with other selective herbicides. Nature 155:500-501. 6. Bolley, H. L. 1908. Weed control by means o f chemical sprays. North Dakota Agric. Exp. Stn. Bull., Fargo, ND. 80:541-574. 7. Bovey, R. W. and A. L. Young. 1980. Military uses o f herbicides, pp. 371-403. m Bovey. R.W. and A.L. Young, eds. The Science of 2,4,5-T and Associated Phenoxy Herbicides. John Wiley and Sons, New York. NY. 8. Burnside, O. C. 1970. Progress and potential for nonherbicidal control o f weeds through preventive weed control, pp. 464-483. in Holstun, J.T., ed. FAO International Conf. on Weed Control, Weed Sci. Soc. America, Champaign, IL. 9. Burnside, O. C. 1993. Weed science -- the step child. Weed Technol. 7:515-518. 10. Burnside, O. C., R. W. Bovey, C. L. Elmore, E. L. Knake, C. A. Lembi, J. D. Nalewaja, M. Newton, and P. Szmedra. 1996. Biologic and economic assessment of benefits from use o f phenoxy herbicides in the United States. WSSA Abstr. 36:36. 11. Davis, D. E. 1979. Herbicides in peace and war. BioScience 29(2):84, 91-94. 12. Davis, F. F. 1945. What's been observed in the tests o f 2,4-D on weeds. Golfdom 19:11- 17. 13. Delvo, H. W., compiler. 1990. Agricultural Resources - Inputs Situation and Outlook Report. Resour. Technol. Div., Econ. Res. Serv., U.S. Dep. Agric., Washington, D.C. AR-17. 59 p. 14. Duke, S. O. 1992. Weed science -- the need and the reality. Phytoparasitica 20:183-186. 15. Gianessi, L. P. 1992. U.S. Pesticide Use Trends: 1966-1989. Quality o f the Environment Division, Resources for the Future, Washington, D.C. 55 p. Phenoxy Herbicides Chapter 2 Page 13 o f 15 16. Hammond, L. E. 1995. N ew perspectives on an essential product: 2,4-D. Down to Earth. 50(2): 1-5. 17. Hamner, C. L. and H. B. Tukey. 1944. The herbicidal action o f 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid on bindweed. Science 100:154-155. 18. Holm, L. 1975. The role o f weeds in world food production. Proc. North Central Weed Control Conf. 32:18-24. 19. Kephart, L. W. 1945. Panel discussion on new chemicals for weed control. Proc. North Central Weed Control Conf. 2:68-75. 20. Klingman, G. C., F. M. Ashton, and L. J. Noordhoff. 1982. Weed Science: Principles and Practices. John Wiley & Sons, New York, NY. 449 p. 21. Leighty, C. E. 1938. Crop rotation, pp. 406-430. m Knight, H.G., ed. Soils and Men, 1938 Yearbook o f Agric. U.S. Dep. Agric., Washington, D.C. 22. Marth, P. C. and J. W. Mitchell. 1944. 2,4-Dichlorophenoxyacetic acid as a differential herbicide. Bot. Gaz. 106:224-232. 23. McDougall, J. and M. Phillips. 1992. Agrochemical Overview 1991. County Natwest Woodmac, Kintore House, 74-77 Queen St., Edinburgh, Scotland. 5 p. 24. Mitchell, J. W., F. F. Davis, and P. G. Marth. 1944. Turf weed control with plant growth regulators. Golfdom 18:34-36. 25. Mitchell, J. W., R. E. Hodgson, and C. F. Gaetzens. 1946. Tolerance o f farm animals to food containing 2,4-dichlorophenoxyacetic acid. J. An. Sci. 5(2):226-232. 26. Nalewaja, J. D. 1974. Energy requirements for various weed control practices. Proc. North Central Weed Control Conf. 29:19-23. 27. Ogg, A. G. 1995. Expanding the Weed Science Society o f America beyond weed science. Weed Technol. 9:406-408. 28. Peterson, G. E. 1967. The discovery and development o f 2,4-D. Agric. Hist. 41:243- 253. 29. Pokomy, R. 1941. Some chlorophenoxyacetic acids. J. Amer. Chem. Soc. 63:1768. 30. Slade, R. C., W. G. Templeman, and W. A. Sexton. 1945. Plant growth substances as selective weedkillers. Nature 155:497-498. 31. Timmons, F. L. 1945. Tuesday morning session opening remarks. Proc. North Central Weed Control Conf. 2:16-19. 32. Timmons, F. L. 1970. A history o f weed control in the United States and Canada. Weed Sci. 18:294-307. 33. Went, F. W. and K. V. Thimann. 1937. Phytohormones. The Macmillan Co., New York, NY. 294 P- 34. Wyse, D. L. 1992. Future o f weed science research. Weed Technol. 6:162-165. Phenoxy Herbicides Chapter 2 Page 14 o f 15 ,35. Zimdahl, R. L. 1991. Weed Science --A Plea for Thought. U.S. Dep. Agric., Coop. State Res. Serv., Washington, D.C. Symposium Preprint, 34 p. 36. Zimmerman, P. W. and A. E. Hitchcock. 1942. Substituted phenoxy and benzoic acid growth substances and the relation o f structure to physiological activity. Contr. Boyce Thompson Inst. 12:321-344. 37. Zimmerman,- P. W. and F. Wilcoxson. 1935. Several chemical growth substances which cause initiation o f roots and other responses in plants. Contr. Boyce Thompson Inst. 7:209- 229. Table 1. Chronology of selected developments in the history o f phenoxy herbicides (generally 2,4-D) mainly in the United States but also worldwide.a Year 1880 1908 1911 1926 1934 1935 1940 1941 1942 1942 1942 1943 1944 1944 1944 1945 1945 1945 1945 1945 Individuals or groups Development Darwin Studied tropisms in plants (33). Bolley, Bonnet, and Schultz Selectively controlled broadleaf weeds in small grains with copper salts (20). Boysen-Jensen Documented chemical control o f tropisms in plants (33). Went Measured quantitatively a natural plant growth regulator (33). Kgl and Haagen-Smit Identified IAA as the natural plant growth regulator (33). Zimmerman and Wilcoxson Discovered PAA and NAA to be plant growth regulators (36). Pokomy Synthesized 2,4-D and 2,4,5-T (29). Kraus Proposed that growth regulators might work as herbicides (28). Zimmerman and Hitchcock Found 2,4-D to be the most active o f the plant growth regulators (36). Stimson Initiated biological warfare research in the U.S. with crop killing herbicides (28). U.S. Army Established Camp Detrick for biological warfare research (28). Lontz, DuPont Co. Patented 2,4-D as a plant growth regulator (28). Mitchell and Hamner Made the first public announcement that 2,4-D was a selective herbicide (22, 28). Marth and Mitchell Selectively controlled dandelions in turfgrass with 2,4-D (22). Hamner and Tukey Selectively controlled field bindweed with 2,4-D (17). NCWCC scientists Used the name "2,4-D" in the literature for the first time (28). ACP Co. Were the first to market 2,4-D as a selective herbicide (28). Davis Used 2,4-D first for commercial control of weeds in turfgrass (12). NCWCC scientists Conducted extensive research with 2,4-D as a selective herbicide (19,31). Mitchell, Hodgson, and Gaetzens Reported that animal toxicity studies with 2,4-D were negative (25). Phenoxy Herbicides Chapter 2 Page 15 o f 15 1945 Kraus. Ate 1/2 gram o f 2,4-D per day for 3 weeks with no ill effects (19). 1945 1945 Kephart Jones, ACP Co. Chaired a NCWCC symposium on 2,4-D in which drift damage was reported (19). Patented 2,4-D as a selective herbicide (28). 1946 Patterson Reported that the U.S. Army had screened over 1000 chemicals for their crop killing potential (28). 1946 1947 U.S. Army scientists Sherwin-Williams Co. Reported on their herbicide research at Camp Dietrick, filling the entire June issue o f the Botanical Gazette 128). Completed litigation against ACP's 2,4-D patent (28). 1950 1951 Mullison, Dow Co. Patented several low volatile 2,4-D esters (28). Allen, Union Carbide Co. Patented butoxyethyl ester o f 2,4-D (28). 1962 U.S. Army Sprayed Agent Orange as a defoliant in Vietnam (7,11). 1970 USDA-ERS Estimated that restricting phenoxy herbicide use would cost U.S. farmers $290 million annually (4). 1979 Ruckelshaus, EPA Suspended all uses o f 2,4,5-T and silvex in the U.S. (7,11). 1985 U.S. District Court, Washington, DC Banned all use o f 2,4,5-T and silvex in the U.S. (7,11). 1988 Agriculture Canada Reported that banning phenoxy herbicides would cost Canadian growers $420 to $488 million annually (16). 1990 EPA Established the Health Advisory Level o f 70 ppb o f 2,4-D in potable water (16). 1992, 1994 EPA Received reports from Advisory Committees o f toxicologists indicating that 2,4-D was not a carcinogen (16). 1995 Industry Task Force II Submitted the 2,4-D risk assessment re-registration data requested by EPA (16). 1996 NAPIAP Presented a progress report of the phenoxy herbicide benefits assessment (10). Abbreviations used were 2,4-D [(2,4-dichlorophenoxy)acetic acid], IAA (indoleacetic acid), PAA (phenylacetic acid), NAA (naphthylacetic acid), 2,4,5-T [(2,4,5-trichlorophenoxy)acetic acid], NCWCC (North Central Weed Control Conference), ACP (American Chemical Paint), USDA (United States Department o f Agriculture), ERS (Economic Research Service), ppb (parts per billion), EPA (Environmental Protection Agency), and NAPIAP (National Agricultural Pesticide Impact Assessment Program)* Page 1 o f 24 Chapter 3 Risk Assessment of Phenoxy Herbicides: An Overview of the Epidemiology and Toxicology Data REBECCA A. JOHNSON and ELIZABETH V. WATTENBERG1 'Assist. Profs., Div. Environ. & Occup. Health, School Public Health, Univ. Minnesota, Minneapolis, MN 55455. Table of Contents A bstract. Despite the societal benefits of phenoxy herbicides, results from a series o f epidemiologic studies have raised questions about possible human health risks associated with their use. However, the results from these studies have been inconsistent and may have been due to errors in the reporting o f pesticide exposures. Recent methodologic research indicates that pesticide exposures may be reported inaccurately to such a degree that it may not be possible to interpret the results from epidemiologic studies where these data have been provided by study participants. Therefore, the evidence from such epidemiologic studies should not be given much weight when determining whether there is a causal association between cancer and phenoxy herbicides in general, or between cancer and 2,4-D specifically. Thus, the only conclusion that can be drawn from the previously conducted epidemiologic studies is that the data are insufficient to assess the carcinogenicity of phenoxy herbicides, or 2,4-D, in humans. Recent toxicologic research indicates that 2,4-D alone is not carcinogenic and suggests no known mechanism by which it could be carcinogenic. Rarely are chemicals carcinogenic in humans, but not in animals. Furthermore, a review o f noncarcinogenic effects suggests that individuals who are exposed to 0.01 mg/kg/day o f 2,4-D over their lifetimes would not experience an appreciable risk o f toxic effects. Based upon data from exposure studies, the general public is exposed to levels o f 2,4-D that are below this reference dose; therefore, the general public should not be at risk of adverse health effects due to 2,4-D exposure. Occupational exposures, however, may exceed the reference dose. To reduce risk, workers should use appropriate protective equipment. INTRODUCTION The societal benefits associated with use of phenoxy herbicides have been described in other chapters. Despite these benefits, possible risks also must be considered in the re-registration of phenoxy herbicides. In this context, risk is defined as the likelihood that some adverse effect to humans will occur (69). In general, pesticides may be thought to pose a certain amount o f risk because: (a) most pesticides are synthetic chemicals, which are deliberately released into the environment; (b) pesticides are specifically designed to harm or kill living things, and they have the potential to be toxic to nontarget organisms, including humans; and (c) many humans may be exposed to pesticides, either occupationally, or from nonoccupational sources (47, 69). However, the degree o f risk or lack thereof varies with the specific pesticide, as well as with the frequency, intensity, and duration of exposure (69). Initially, much o f the concern about pesticides was directed at adverse ecological effects. During the last few decades, however, the focus of concern has shifted from the environment to human health Page 2 o f 24 (69), The purpose o f this chapter is to review the epidemiology and toxicology data relating to phenoxy herbicides, or more specifically, 2,4-D. E PID EM IO LO G Y Epidemiology background. Epidemiology is the study o f the occurrence of, and factors related to, states o f health and disease in human populations. The potential for pesticide exposures to cause cancer has been the foremost human health effect o f concern; therefore, this section o f the review will focus on those epidemiologic studies that have evaluated phenoxy hrbicides in relation to cancer. In the past decade, a number o f reviews of the epidemiologic data relating to pesticides and cancer have been published (2 ,4 ,5 ,7 , 8, 10,11, 1 2 ,1 6 ,2 0 ,3 5 ,3 8 ,4 2 ,4 8 ,4 9 ,5 5 , 62). Thus, the summary o f the epidemiologic literature presented here is, in part, drawn from these reviews. Furthermore, this review is not intended to be comprehensive, but instead, it includes select articles that reflect on the cancer risks associated with human exposure to 2,4-D or other phenoxy herbicides. In addition, the key mthodologie issues surrounding these epidemiologic studies are discussed, as well as the results of epidemiologic studies designed to investigate these issues. S tudy designs an d risk estim ates. In the late 1970's, the first analytic epidemiologic investigations o f phenoxy herbicides and cancer were begun. Prior to that time, only descriptive studies had been done and these typically provide less useful information. The analytic epidemiologic studies conducted have examined either specific types o f cancer (i.e., case-control studies) or specific exposed populations (i.e., cohort studies). In these case-control studies, people who have a specific cancer (i.e., cases) and people who do not have the cancer (i.e., controls) are identified. Then, information about prior exposure to a suspected carcinogen is obtained for both the cases and controls. The odds of exposure among the cases is compared to the odds o f exposure among the controls, and this risk estimate is called the odds ratio (OR). The way to interpret the value o f the OR is as follows: (a) if the OR equals 1.0, the cases and controls have been exposed equally; (b) if the OR is less than 1.0, the controls have been exposed more than the cases; and (c) if the OR is greater than 1.0, the cases have been exposed more than the controls. If exposure is more common among the cases than the controls, the exposure may be associated with the occurrence of the cancer. In a cohort study, groups o f individuals with and without exposure are followed over time. The incidence o f disease in the exposed group is compared to the incidence o f disease in the unexposed group and this risk estimate is called the relative risk (RR). The way to interpret the value o f the RR is as follows: (a) if the RR equals 1.0, the incidence o f disease is equal among the exposed and unexposed; (b) if the RR is less than 1.0, the incidence o f disease is higher for the unexposed than for the exposed; and (c) if the RR is greater than 1.0, the incidence o f disease is higher for the exposed than for the unexposed. I f the incidence o f disease is more common among exposed persons than among those who are unexposed, the exposure may be associated with the occurrence o f the disease. In addition, the OR and RR usually are provided with an interval that shows the variability in the risk estimate and specifies the probability that the interval includes the true risk. This interval is called the confidence interval (Cl) and it is used to help determine whether the risk estimate is meaningfully different from 1.0. The risk estimate is said to be significantly different from 1.0 if the Cl does not include 1.0. Summaries o f the case-control and cohort studies are presented separately below. The case-control studies are discussed first because findings from the early case-control studies prompted much o f the epidemiologic research on phenoxy herbicides, including 2,4-D. Page 3 o f 24 In the summaries and tables, reference is made to the following exposures: "pesticides," "herbicides," "phenoxy herbicides," and "2,4-D." These terms are presented as such to accurately reflect how the questions were asked in the epidemiologic studies. For example, in a particular study, participants may have been asked, "Did you ever use herbicides?" and "Did you ever use 2,4-D?" Case-control studies. The majority o f case-control studies have focused on the following types of cancer: (a) soft-tissue sarcomas (STS), (b) Hodgkin's disease (HD), and (c) non-Hodgkin's lymphoma (NHL). In Sweden, a clinical observation (28) o f a number o f patients with STS and prior exposure to phenoxy herbicides led to the first case-control study (30). In this case-control study in northern Sweden, a fivefold increase in risk for STS (OR = 5.3; 95% Cl = 2.4 to 11.5) was observed for exposure to phenoxy herbicides. However, 2,4,5-T was frequently used by those who were exposed, and this phenoxy herbicide has been found to contain 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) as a contaminant. Another case-control study subsequently was undertaken in southern Sweden to try to confirm these findings (25). In southern Sweden, commonly used phenoxy herbicides included 2,4-D, MCPA, and others thought to be free of 2,3,7,8-TCDD. The risk for STS associated with exposure to any phenoxy herbicides found in this second Swedish study was nearly sevenfold (OR = 6.8; 95% Cl = 2.6 to 17.3). A fourfold risk (OR = 4.2; Cl not provided) was reported for exposure to phenoxy herbicides not contaminated by 2,3,7,8-TCDD. Around the same time, a number o f patients with malignant lymphoma and previous exposure to phenoxy herbicides was reported in Sweden (29). Once again, a case-control study was performed to see if there was a relation between this type o f cancer and exposure to these herbicides (32). The results of this case-control study of malignant lymphoma showed almost a fivefold increase in risk (OR = 4.8; 95% Cl = 2.9 to 8.1) with exposure to phenoxy herbicides. Separate results were not provided for HD and NHL, but were reported to be similar. Due to the substantial risks observed in the early Swedish studies and the potential for widespread exposure to phenoxy herbicides, a number of case-control interview studies of STS, HD, and NHL followed in Sweden, New Zealand, Italy, United States, and Australia (21, 31,33, 34, 53, 54, 56, 58, 59, 60, 61, 65, 70, 71, 74). The basic design features and results o f these studies are summarized in Table 1 and are not discussed further. Despite the strong associations observed by Swedish researchers, almost all o f the other studies conducted elsewhere have failed to confirm these findings. In Kansas, Hoar et al. (34) found a significant association with NHL and use o f phenoxy herbicides and 2,4-D. In addition, the risk o f NHL significantly increased with the number of days o f herbicide exposure per year. OR's o f 1.4, 1.6, 2.6, and 6.0 were found for using herbicides for 1 to 5, 6 to 10, 11 to 20, and more than 20 days per year, respectively. Frequency o f use data were not collected for specific pesticides such as 2,4-D. In Nebraska, this same researcher and colleagues (74) observed a nonsignificant elevation in risk for NHL attributable to 2,4-D. In addition, an association was suggested between increasing days of 2,4-D exposure per year and risk o f NHL. C ohort studies. Cohort studies that have examined cancer risk and phenoxy herbicides have included those persons potentially exposed during manufacturing or application. STS, HD, and NHL are relatively rare cancers and are believed to occur many years after initial exposure to a carcinogen; therefore, most of the cohort studies conducted to date have not had either a sufficiently large cohort or an adequate number of years of follow-up to be informative. The relevant cohort studies (14, 15, 24, 43, 44, 45, 52, 66, 67, 68) are summarized in Table 2 and are not described further. The findings from the cohort studies do not support those of the Swedish case-control studies. Page 4 of 24 Methodologic issues. Epidemiology is fraught with inconsistent studies; however, it is unusual for risk estimates to vary as greatly as those observed in the case-control studies o f STS, HD, and NHL and phenoxy herbicides. Several papers (6, 7 ,8 ,9 ,1 2 ,1 3 , 50) have been published about methodologic issues that may explain the inconsistent case-control study findings. Error in the measurement of exposure has been suggested as one o f the possibleexplanations and continues to be the primary methodologic issue o f concern (9). Overview of exposure measurement concepts. Error in the measurement o f exposure, also called exposure misclassification, is the difference between the measured exposure and the true exposure. Although exposure misclassification occurs to some extent in almost all epidemiologic studies, it is particularly problematic because it leads to bias in the risk estimate. This type of bias is called misclassification or information bias. Differential misclassification occurs when the exposure measurement error differs according to disease status; nondifferential misclassification is independent o f disease status. Nondifferential misclassification usually results in an underestimate o f the true risk. Differential misclassification can either underestimate or overestimate the risk and can make it impossible to draw any conclusions from the study results. Case-response or recall bias is a form o f differential misclassification of exposure in which cases report exposures differently than controls. Compared to controls, cases may better recall or falsely report exposures in an attempt to identify what may have caused their disease (3). Information about the validity and reliability o f measured exposure data is needed to determine the extent and direction o f exposure misclassification. Validity refers to the accuracy o f a measure (i.e., whether it correctly measures the true exposure) and reliability refers to the reproducibility o f a measure (i.e., whether it consistently provides the same results on two or more occasions). Long-term reliability refers to the reproducibility obtained when the measurements are made a long time apart and short-term reliability refers to the reproducibility obtained when the measurements are made a short time apart Reliability is only important for what it reveals about validity. If an exposure measure is not reliable, it cannot be valid. A reliable exposure measure, however, does not imply a valid measure. Therefore, it is preferable to evaluate the validity o f an exposure measure. In order to measure the validity of an exposure measure, however, the true exposure must be known. A measure o f the true exposure usually is not available or is not practical to use; otherwise, this perfect measure would be used instead o f the less accurate one. As a result, the "validity" o f an exposure measure often refers to how the measure under study compares with a measure that is deemed to be the most accurate (i.e., a "gold standard") (3). Percentages o f agreement often are calculated to assess the reliability of an exposure measure. Sensitivity and specificity often are calculated to assess the validity o f an exposure measure. Sensitivity is calculated as the percentage o f truly exposed persons who report that they have been exposed. Specificity is calculated as the percentage o f truly unexposed persons who report that they have not been exposed. Ideally, a valid exposure measure will have both high sensitivity and high specificity. A number o f questions have been raised regarding the exposure assessment procedures in epidemiologic studies of pesticides and cancer. STS, HD, NHL, and other cancers o f interest occur infrequently and are characterized by long cancer induction periods following exposure. Consequently, studies o f these cancers most often have used a case-control design to alleviate the need for large numbers o f subjects and a long follow-up period. Therefore, information about past pesticide exposures usually has been obtained by interviewing subjects, or when necessary, proxy-respondents. The accuracy o f these pesticide exposure measurements is dependent on the Page 5 of 24 memory or recall o f the individuals being interviewed. The ability to recall details related to pesticide use may decrease with time; thus, the accuracy o f data regarding early uses o f pesticides may be most suspect. Unfortunately, these data may be the most relevant with regard to initiation o f cancer (6). Recall is hindered by the fact that subjects exposed to pesticides almost always have used a number o f different pesticides during their lifetimes, and the available and recommended pesticides have changed over the years (8). Recall also is difficult because most subjects use pesticides relatively infrequently, often only seasonally (8, 12, 13). Proxy-respondents (e.g., spouses, siblings, children, and parents) are interviewed on behalf o f subjects when the subjects themselves are unable to be interviewed due to death, illness, dementia, or some other reason. In case-control studies of pesticides and cancers, the cancers being studied often have high death rates; thus, by necessity, proxy-respondents must be used. However, the proxy-respondents may never have known which pesticides the subjects used. It is commonly believed this lack of knowledge would cause proxy-respondents' recall o f pesticide use to be less than studysubjects' recall (8, 9), but it also is possible that proxy-respondents may be prone to case-response bias. In the Nebraska study, the risk of NHL associated with 2,4-D was higher among subjects with proxy-respondents than among self-respondents (74). For proxy-respondents, OR's o f 2.2, 2.2, and 2.4 were found for handling 2,4-D for 1 to 5, 6 to 20, and more than 20 days per year, respectively. For self-respondents, the corresponding OR's were 1.0, 1.6, and 1.4. Similarly, in a case-control study of NHL and leukemia conducted in Iowa and Minnesota (18, 21), the proxy-respondent OR's for use o f 2,4-D for 1 to 4, 5 to 9, and more than 10 days per year were 0.8, 1.0, and 2.5, respectively (51). The self-respondent OR's for the same categories o f use were 0.5, 0.2, and 0.7. Thus, in the case-control studies in Nebraska and in Iowa-Minnesota, there was evidence that case-response bias may have occurred. Mthodologie studies. The issues noted above illustrate the difficulties associated with retrospectively obtaining information about pesticide exposures and the need to assess the reliability and validity o f reported use of pesticides. Given the complexity o f pesticide exposures and the controversy surrounding the inconsistent findings from case-control studies o f pesticides and cancer, it is remarkable how little mthodologie research has been conducted in this area (17, 19, 30, 34, 39, 40, 70). Such research may not have been undertaken because it has been assumed that pesticide exposure misclassification always would reduce estimates o f risk (i.e., that the misclassification is nondifferential) or because of the resources required. The few studies that have been done are summarized in Table 3 and described briefly below. In the first case-control study o f STS and pesticides conducted in Sweden, 50 employers were mailed a questionnaire to verify employment and use o f phenoxy herbicides or chlorophenols reported by study subjects (30). Only a small proportion (40%) o f the employers responded. Due to a lack of records regarding exposures to these chemicals, responses received mainly were based on employers' recall. The authors stated that the results obtained were uncertain and difficult to interpret. Despite these limitations, the authors concluded that the reported use of phenoxy herbicides probably was reliable because a high degree of agreement was observed for use of chlorophenols in sawmills or the pulp industry. The measure used to assess agreement and the actual agreement observed were not provided. In addition, possible differences in agreement for cases and controls were not evaluated. A similar approach was used in the case-control study o f STS and NHL in Washington state (70). In Page 6 of 24 this study, supervisors and coworkers were contacted by telephone to corroborate self-reported exposures to specific phenoxy herbicides, chlorophenols, or other chemicals for those jobs with potential exposures to these chemicals. The authors indicated that confirmation o f subjects' responses was provided in essentially all instances in which the supervisors or coworkers were successfully reached. No response rate was given, but a greater number o f supervisors and coworkers could be reached for recent occupations. In addition, the majority o f verification contacts attempted (approximately 80%) were for recent occupations. The authors also stated that there were no significant differences in agreement for cases and controls. No agreement percentages were provided. In the case-control study o f malignant lymphoma and STS conducted in Kansas, pesticide suppliers were interviewed on behalf o f 110 subjects with farming experience to see whether they could confirm the pesticide use reported by the fanners (34). For these interviews, the suppliers relied on their memories or written records, or both (6,12). Overall agreement between the farmers' and suppliers' responses regarding use o f pesticides was only about 50% (6). When only the previous 10 years were considered, agreement between suppliers and subjects was higher. Suppliers typically reported less pesticide use than the farmers (34). For all subjects, the percentage o f agreement for herbicides was approximately 60%. Similar percentages were found for cases and controls (12,13, 34). Agreement was considerably lower for specific years o f exposure to herbicides (12,13). For use o f 2,4-D, agreement was 83% for NHL cases and 74% for controls (6). The number o f cases and controls who reported using herbicides was multiplied by the percentage o f verified herbicide use and the OR for NHL was recalculated. The recalculated OR was 1.8, which was higher than the OR o f 1.6 calculated from only the interview responses (34). A recalculation o f the OR was not performed for 2,4-D. The authors attributed the lack of agreement between the two sources to the large number o f suppliers fanners could have used, the closing o f suppliers, the lack o f historical records about pesticide purchases at many suppliers, the long period o f time during which farmers had purchased pesticides, and the number o f pesticides purchased (6). In Iowa, 95 Iowa male controls from the Iowa-Minnesota case-control study o f NHL and leukemia (18,21) were reinterviewed about selected agricultural pesticides that the fanner previously had reported using, and these responses were compared with responses provided by proxy-respondents (19). The percentages o f agreement for 2,4-D use before and after 1960 were 97 and 95%, respectively. In each time period, proxy-respondents reported slightly more subjects as exposed to 2,4-D than the subjects reported themselves. Agreement for the usual number o f days herbicides were used per year was 52%. Similarly, the agreement percentages relating to the average annual frequency o f 2,4-D use before 1960 was 48%, while after 1960 it was 56%. Because the study included only controls, no assessment could be made of possible reporting differences for cases and controls. The authors concluded that agreement between the responses o f subjects and proxies was excellent and that data from proxy-respondents will be sufficient for epidemiologic purposes, assuming that differential recall is minimal. In another study, 270 male cancer cases were initially interviewed for a multicenter case-control study o f STS, HD, NHL, and other cancers (17). For the cases who died during the 4-year study interval, proxy-respondents also were interviewed. Accuracy o f data from proxy-respondents was assessed by calculating theother cancers (17). For the cases who died during the 4-year study interval, proxy-respondents also were interviewed. Accuracy o f data from proxy-respondents was assessed by calculating the sensitivity and specificity (i.e., data from the self-respondents were assumed to be the "gold standard"). Agreement between self- and proxy-respondents for pesticide and herbicide exposure data was low (sensitivities ranged from 14 to 51% and specificities ranged from 95 to 100%). The sensitivity for herbicides used infarming was approximately 45%, and the specificity was Page 7 of 24 approximately 95%. For herbicideproducts containing 2,4-D, the sensitivity was 35% and the specificity was 100%. Becausethe study included only cases, no assessment could be made o f possible reporting differen As part o f a large mthodologie study done by one o f us (39), proxy-respondents wereinterviewed for 328 Minnesota subjects from the Iowa-Minnesota case-control study of NHLand leukemia (18, 21) who died or became incompetent since the initial interview (40). Asquestions increased in detail, agreement percentages decreased. Agreement percentagesranged from 48 to 91% for specific pesticides, with the majority between 60 and 80%. Among the NHL cases, leukemia cases, and controls, the agreement percentages for allherbicides were 79, 70, and 71%, respectively. For these same groups o f subjects, theagreement percentages for 2,4-D were 75, 61, and 64%, respectively. Exposuremisclassifications had varying effects on the OR's and 95% Cl's. Generally, OR's calculatedfrom proxy respondent data were less than those from self-respondent data; however, severalexceptions occurred. For example, proxy-respondents had an OR for leukemia o f 2.4 (95%CI = 0.9 to 6.0) for herbicides; whereas, the corresponding OR for self-respondents was 1.1(95% Cl = 0.5 to 2.3). The leukemia OR for 2,4-D was 2.6 (95% Cl = 1.1 to 6.3) forproxy-respondents and 1.0 (95% Cl = 0.4 to 2.1) for self-respondents. However, for NHL,OR's for self- and proxy-respondents were more similar for ever using 2,4-D or anyherbicides. Due to the presence of nondifferential and differential misclassification, a numberof the self- and proxy-respondent OR's were markedly different from each other. Thus, thefindings from this component indicated that pesticide data provided by proxy-respondents willnot necessarily result in the same estimate o f risk or lead to the same conclusion as dataprovided by self-respondents. Another component of this large mthodologie study (39) examined the long-termreliability o f reported pesticide information by reinterviewing, 7 to 10 years later, 389 self-respondents and 369 proxy-respondents from Minnesota who participated in the earlier Iowa-Minnesota case-control study o f NHL and leukemia (18, 21). Percentages o f agreement for first and second interview responses were higher for self-respondents than for proxy-respondents. For 2,4-D, agreement percentages ranged from 78 to 81% for self-respondents and from 59 to 73% for proxy-respondents. OR's and 95% Cl's, calculated using data from the first and second interviews, were variable for both types of respondents. The disparities in the percentages o f subjects exposed as reported in the first and second interviews had varying effects on the OR's. For leukemia, the self-respondent OR's did not differ much by interview; whereas, for NHL, there were some differences in the self-respondent OR's by interview. There were a number of differences in the proxy-respondent OR's by interview for both leukemia and NHL. The findings from this component showed that self- and proxy-respondents may not provide reliable information about past pesticide exposures due to nondifferential and differential exposure misclassification. The final components o f the large mthodologie study (39) were to evaluate the short-term reliability and validity o f reported pesticide exposure data. Short-term reliability was assessed by interviewing a sample o f 157 farmers twice, 3 to 10 months apart. For 42 o f the farmers who participated in the initial interview, validity was assessed by comparing the interview responses to data about 2,039 pesticide purchases collected from 14 local suppliers. The percentage o f agreement was used to compare first and second interview responses provided by the farmers. The agreement o f the farmers' responses in the first interview with the suppliers' records was evaluated by calculating the percentage o f agreement, sensitivity, and specificity. The information from the suppliers was assumed to be the "gold standard". More general pesticide exposure data (i.e., ever use) were reported reliably; whereas, more definitive Page 8 of 24 pesticide exposure data (i.e., years o f use, frequency o f use, and duration o f use) were reported less reliably. Agreement for ever use of any herbicides was 94%, For 2,4-D, the agreement percentage was 80%. Agreement percentages for 2,4-D for the average number o f days used per year and the average number o f hours used per day were 71 and 72%, respectively. As observed in this component o f the study, the inability o f respondents to reliably recall specific details o f pesticide use is important because many o f the positive associations between phenoxy herbicides and NHL reported to date have occurred among subgroups defined by frequency o f exposure. The validity o f reported pesticide data was poor. The validity o f pesticide use data is more critical than the reliability, because data may be repeatable, but inaccurate. For most pesticides, low values of sensitivity compared with those of specificity indicated that underreporting was a greater problem than overreporting; however, 2,4-D was a notable exception to this pattern. The overall agreement percentage for the farmers and suppliers was 81% for herbicides. In addition, the sensitivity was 89% and the specificity was 20%. For 2,4-D, the agreement percentage was 60%, the sensitivity was 63%, and the specificity was 53%. Findings from this component of the study demonstrated the potential for exposure misclassification and resulting errors in risk estimates for studies in which pesticide data are obtained by interview. Epidemiology conclusion. In addition to the published reviews o f the epidemiologic literature, a number of expert panels have weighed the evidence regarding the carcinogenicity of phenoxy herbicides, or 2,4-D alone. In 1987, a panel convened by the Canadian Centre for Toxicology (20) concluded that, "... there is limited evidence o f carcinogenicity in man from exposure to phenoxy herbicides. In terms o f exposure to 2,4-D specifically, the evidence must still be regarded as inadequate to classify it as a carcinogen." The International Agency for Research on Cancer Working Group reached the same conclusion for phenoxy herbicides (64). No distinction was made for 2,4-D. In 1989, a panel convened by the Harvard School o f Public Health Center for Risk Analysis (35) concluded that, "Although a cause-effect relationship is far from being established, the epidemiological evidence for an association between use o f 2,4-D and non-Hodgkin's lymphoma is suggestive and requires further investigation. There is little evidence o f an association between use o f 2,4-D and soft-tissue sarcoma or Hodgkin's disease, and no evidence o f an association between 2,4-D use and any other form o f cancer." Another panel sponsored by the Industry Task Force n on 2,4-D Research D ata (49) concluded, "... the epidemiological studies provide, at best, only weak evidence o f an association between 2,4-D and the risk o f cancer." Finally, in 1993, a Special Joint Committee o f the Science Advisory Board and the Scientific Advisory Panel o f the EPA (64) judged that, "... at this time, the data are not sufficient to conclude that there is a cause and effect relationship between the exposure to 2,4-D and NHL." In weighing the evidence, one must consider that the results o f the epidemiologic studiesare only as good as the data on which they are based. In particular, OR's can be affected by differences in exposure misclassification for cases and controls. Thus, the reliability andvalidity o f reported pesticide exposure data, and the comparability of such data obtained fromself- and proxy-respondents have been explored in mthodologie studies. Only one mthodologie study, however, has demonstrated the effect of pesticide exposuremisclassification on the OR (39). Furthermore, this is the only study to systematicallyexamine the validity of pesticide exposure data, as well as to evaluate the short- and long-termreliability o f such data. The combined results o f this study demonstrated the potential for bothdifferential and nondifferential exposure misclassification and the potential for resulting errorsin risk estimates for studies in which pesticide data are obtained by interview. In particular,there was Page 9 of 24 substantial evidence regarding the poor quality o f reporting exposures to 2,4-D. As a result, findings from the previously conducted case-control studies should not begiven much weight when determining whether there is a causal association between phenoxyherbicides, or 2,4-D, and cancer. At this point in time, the only conclusion that can be drawnfrom the epidemiologic studies is that the data are insufficient to assess the carcinogenicity ofphenoxy herbicides, or 2,4-D. in humans. Table of Contents TOXICOLOGY Toxicology background. The chemical structure o f 2,4-D allows it to mimic the plant hormone, indoleacetic acid (49). Because of it's structural similarity to this plant hormone, absorption o f 2,4-D through roots and leaves can result in altered plant metabolism, abnormal growth, and plant death. The results from a series of epidemiologic studies, described in the preceding section, raised questions about the safety o f 2,4-D for humans (49). A number of toxicologic studies have been conducted in response to these safety concerns, and to fulfill the EPA requirements for re-registration o f 2,4-D (36, 49). In 1992, the manufacturers of 2,4-D commissioned an extensive review of these studies, which was published in the Journal of the American College of Toxicology (49). This report was reviewed by a panel of nonindustry experts in toxicology and epidemiology. In addition, the Science Advisory Board o f the EPA has issued a report that reviews the epidemiologic and toxicologic data on the potential carcinogenicity of 2,4-D (64). Data related to the carcinogenicity o f 2,4-D also was reviewed by an expert panel convened by the Harvard School of Public Health (35). This section of our review presents a summary and update o f toxicologic studies relating to the health risk assessment o f 2,4-D, drawn mainly from the reviews already mentioned, and from summaries of the data collected for submission to the EPA for the reregistration o f 2,4-D (36). The results from some o f the key toxicologic studies are presented in Table 4, Environm ental fate. In 1989, the World Health Organization published a review and evaluation of the environmental effects of 2.4-D (73). The general conclusion from this review was that 2,4-D does not persist in the soil because it is rapidly broken down and detoxified by light and by microorganisms in the soil (73). Because most organisms rapidly excrete 2,4-D, this herbicide would not be expected to accumulate or remain stored in the tissues o f animals (73). C ontam inants. The purity o f technical grade 2,4-D can range from less than 90% to 99% (72). The forms o f 2,4-D most commonly used in forestry and agriculture are the alkali salts, amine salts, or esters (73). Technical grade formulations of 2,4-D may contain solvents or "wetting agents" that help the herbicide stick to and cover the plants (73). The presence o f other types o f impurities depends on the purity of the starting material, the procedure used to manufacture the specific form of 2.4-D, and the storage conditions (72). Reactions that take place at high pH and high temperatures tend to increase the formation of polychlorinated dibenzo-p-dioxins (PCDD's), some o f which are toxic (72). A study done by researchers at Agriculture Canada in the early 1980's tested samples o f 2,4-D for the presence of PCDD's (23). The levels o f PCDD's differed depending on the form o f 2,4-D. Out o f 11 samples of 2,4-D acid analyzed, none had levels o f PCDD's above the detection limit o f 1 ppb. By contrast, eight out of 26 samples of 2,4-D amine salts had detectable PCDD levels, which ranged from 5 to 587 ppb. Page 10 o f 24 The samples o f 2,4-D esters had the most widespread PCDD contamination. PCDD's were detected in 20 out of 21 samples o f 2,4-D esters, with levels ranging from 35 ppb to 23.8 ppm. Another study, however, detected 6.8 ppb 2,3,7,8-TCDD in a German formulation o f 2,4-D (27). The Canadian Government limits PCDD contamination o f 2,4-D formulations to 10 ppb (49). In the United States, the technical product of each manufacturer is analyzed for the presence o f PCDD's, and the health risk posed by the contaminants is evaluated on a case-by-case basis.2 Nitrosamines, which can be carcinogenic, have been detected in amine formulations o f 2,4-D (72). Nitrosamines may form when 2.4-D amine salts are stored along with nitrates inmetal containers. Nitrates have been added to metal containers to prevent corrosion. Thechange in packaging o f 2,4-D from metal containers to plastic or epoxy-lined containers wouldbe expected to eliminate the formation o f nitrosamines (49). Few studies rigorously address the toxicology o f complex mixtures. Therefore, the effectof solvents, additives, or impurities on the toxicology of 2,4-D remains largely unknown. Onestudy found, however, that when rats were fed 2,4-D for 13 weeks, technical grade 2,4-D acid(97.3% pure) was no more toxic than purified 2,4-D acid (26). Estim ated exposure levels. The highest levels o f exposure to 2,4-D occur in occupationalsettings. The amount of 2,4-D absorbed from occupational exposures will vary depending onthe type o f work and on the type o f safety measures used. It has been estimated that, withoutprotective gear, commercial applicators may be exposed to 2 to 160 g o f 2,4-D/kg/day and forestry workers may be exposed to 0.86 to 129 g of 2,4-D/kg/day (49). Workers whospray herbicides from a backpack may receive some o f the highest doses of 2,4-D. It has beenestimated that workers using backpack sprayers and wearing protective clothing had intemaldoses ranging from 30 to 244 g/kg/day (49). The doses received by workers involved inaerial spraying have been estimated to range from 0.53 to 8.54 g/kg/day (49). One studyestimated that farmers who do not wear protective gear received an average dose o f 5.78 g/kg o f 2,4-D during eachspray operation. A variety of surveys indicate that commercial foods and public water supplies do notpresent a significant source o f exposure of 2,4-D to the general public (72). Duringapplication periods, the general population in herbicide use areas usually is not exposed to doses o f 2,4-D greater than 2 g/kg/day (72). Likewise, the dose o f 2,4-D received by home gardeners has been estimated to be less than 2 g/kg/day (49). 2 Personal communication. 1995. Stephen Funk, Chemistry Branch, EPA, Washington, D.C. A bsorption. Approximately 90% of exposure o f agricultural workers to 2,4-D occurs through skin contact. The observation that 2,4-D can be detected in the urine 4 hours after dermal exposure suggests that 2,4-D is rapidly absorbed through the skin, and also rapidly excreted (49). Absorption of 2,4-D is slower through the skin than through ingestion. In addition, skin can serve as a reservoir for 2,4-D (49). The amount o f 2,4-D absorbed through the skin depends on a number of factors, including the chemical form of 2,4-D, the condition o f the skin, the site of dermal contact on the body, and the solvent in which 2,4-D is dissolved. For example, one study found that, when applied to the forehead of humans, approximately 58% of the applied dose of 2,4-D dimethylamine was absorbed in contrast to 6% o f the applied dose o f 2,4-D isooctyl ester (49). The interpretation o f data from toxicity studies conducted in animals is difficult because the efficiency of absorption of 2,4-D varies across animal species. For example, whereas 2,4-D dimethylamine is absorbed relatively efficiently when applied to the foreheads o f humans, only 20% o f a dose of 2,4-D dimethylamine was Page i l of 24 absorbed when it was applied to the shaved backs o f rats over the course o f 7 days (49). Exposure to 2,4-D through inhalation may occur in some occupational settings. Inhalation has been estim ated to account for approxim ately 2% o f exposure for applicators who spray 2,4-D (35). Inhalation may be a significant route o f exposure for workers involved in the m anufacturing o f 2,4-D. U nfortunately, there is little research data on the absorption o f 2,4-D through inhalation (49). Absorption o f 2,4-D appears to be rapid and complete from the gastrointestinal tracts o f humans and experimental animals (49,72). A lthough ingestion is usually a relatively m inor route o f exposure, m ost toxicity testing o f 2,4-D has been conducted by oral exposure. Because 2,4-D is absorbed more efficiently through the gastrointestinal tract than through the skin, 2,4-D should be more toxic when ingested than when applied to the skin. Therefore, using oral studies o f 2,4-D to test toxicity may add some m argin o f safety when these data are used to predict risk from exposure to 2,4-D through skin contact. Distribution through the body. Following absorption, 2,4-D is distributed throughout the body (49). Studies conducted in a variety o f species show that, after oral dosing, 2,4-D is found in the liver, kidney, lung, and to a lesser extent in the brain (49). The distribution o f 2,4-D through the human body appears to be sim ilar to that in test species. W hen organs were examined following fatal poisonings, the highest levels o f 2,4-D were found in kidney and liver, w ith lower levels found in brain, muscle, and heart (72). It also has been reported that up to approximately 17% o f a dose o f 2,4-D can cross the placenta in pregnant m ammals (72). Transport o f 2,4-D into tissues and across the blood-brain barrier probably occurs by a cellular transport system, such as the organic acid transporter (49). Because 2,4-D is w ater soluble it is excreted in the urine, and does not tend to be stored or to accum ulate in tissues in contrast to fat-soluble compounds (49). Binding o f 2,4-D to plasm a proteins can occur and may affect distribution (72). W hen 2,4-D is bound to the plasm a proteins, it cannot reach tissues where it m ight cause damage. High doses o f 2,4-D can saturate or use up all o f the plasm a protein binding sites, which could result in a dramatic rise in the concentration o f "free" 2,4-D. Free 2,4-D may be excreted. A t very high doses o f 2,4-D, however, the rate o f excretion m ay slow down, and therefore the concentration o f 2,4-D that can reach tissues in the body may increase and therefore cause toxicity. Exposure to high doses o f other compounds that compete w ith 2,4-D for binding to plasm a proteins may also cause in a rise in the concentration o f free 2,4-D. Excretion. A ll species studied, including humans, excrete 2,4-D mainly in the urine (72). The rate o f excretion depends on the dose o f 2,4-D. Excretion o f 2,4-D is through the organic acid transport system in the kidney (49). Low doses o f 2,4-D are excreted more rapidly than high doses, which can saturate this active kidney transport system. This phenomenon was demonstrated in a pharm acokinetic study o f 2,4-D in rats, designed to measure the rate at which 2,4-D is absorbed and excreted (26). In this study, rats were given single oral doses o f 14C labeled 2,4-D at 10,25,50,100, or 150 mg/kg. The concentration o f labeled 2,4-D in the plasm a and the urine was m easured 6 hours later. The rats given 100 and 150 mg/kg 2,4-D exhibited a dramatic increase in the plasm a concentration o f labeled 2,4-D compared w ith rats given the low er doses. The rats in the two high-dose groups also exhibited a corresponding decrease in urinary excretion o f labeled 2,4-D. M unro et al. (49) point out that the doses o f 2,4-D that m ost hum ans are exposed to should be below doses that saturate active kidney transport. Therefore, they suggested that toxicologic results from animals treated w ith high doses o f 2,4-D that saturate renal transport should be interpreted with caution because they may not be relevant to typical human exposures (49). Page 12 o f 24 M etabolism . M any compounds are broken down or transform ed by specific enzymes present in alm ost all tissues, but present in especially high concentrations in the liver. Although this type o f metabolism can detoxify chemicals, sometimes these enzymes transform a compound into a chemical that is even more toxic than the original one. Studies indicate that 2,4-D is not extensively m etabolized in humans or other mammals, and furtherm ore 2,4-D does not appear to be transformed into a more toxic compound by m etabolic enzymes (49). In one study, five m ale humans were given an oral dose o f 5 m g/kg 2,4-D acid (57). This study reported that 2,4-D was efficiently absorbed and then eliminated. H alf o f the initial dose o f 2,4-D was elim inated by 11.6 hours. Approximately 95% o f the administered dose o f 2,4-D was found eventually in the urine. Depending on die individual, from 0 to 12% o f the 2,4-D found in the urine was in the form o f conjugates, compounds that have undergone a specific detoxification reaction. Conjugates containing 2,4-D also have been identified in the urine o f rats and pigs following oral exposure to 2,4-D. M ore toxic or reactive form s o f 2,4-D, as a result o f metabolism, have not been identified (49). A cute toxicity. Acute toxicity usually refers to toxicity that occurs w ithin a day or a few days after short-term , high dose exposure. Acute toxicity studies usually are intended to reflect a situation that would result in obvious poisoning. A common indicator o f acute toxicity is the LD50, the dose that kills 50% o f the test animals w ithin a specified tim e interval. The lower the LD50, the m ore toxic the compound. The LD50 for 2,4-D varies depending on the form o f the compound, the route o f exposure (i.e., oral, skin, or inhalation), and the species to w hich it is administered. An early study determined an oral LD50 o f 100 mg/kg in dogs, which appear to be the most sensitive species (72). A more recent study compared the oral LD50's o f various forms o f technical grade 2,4-D in rats (26). The test m aterials included: 2,4-D acid (95.0% pure), 2,4-D isoctyl ester (94.0% pure), 2,4-D dim ethylam ine salt (67.9% pure), 2,4-D isobutyl ester (96.1% pure), 2,4-D sodium salt (90.8% pure), 2,4-D butoxyethanol ester (97.1% pure), and 2,4-D butyl ester (98.6% pure). The LD50's ranged from 533 m g/kg for 2,4-D isobutyl ester in female rats (424 m g/kg 2,4-D acid equivalent) to 1090 mg/kg 2,4-D dimethylamine salt in male rats (619 mg/kg 2,4-D acid equivalent). Results from this study also indicate that acute exposure to 2,4-D through the skin is less toxic than acute oral exposure. The single dose dermal LD50's for all o f the test m aterials was greater than 2000 mg/kg in rabbits. These results indicate that the acute dermal toxicity o f 2,4-D is low. The acutely toxic dose o f 2,4-D in humans is difficult to determine. D eath has resulted from ingestion o f 80 m g/kg 2,4-D dimethylamine (72). An earlier report suggested that doses o f 2,4-D as high as 36 mg/kg m ay not cause acute oral toxicity (72). In any case, workers should be able to avoid accidental exposure to acutely poisonous doses o f 2,4-D through the use o f proper occupational safety procedures. Subchronic to x id ty . Subchronic toxicity studies typically involve treating animals for approximately 3 months, and usually are intended to reflect a short-term exposure to a chemical. These studies usually report the highest experimental dose that does not cause toxicity, w hich is called the no-observed-adverse-effect-level (NOAEL). The NOAEL is an indication o f a safe dose. These studies also usually report the low est experimental dose that causes toxicity, which is called the low est-observed-adverse-effect-level (LOAEL). Although m ost subchronic studies o f 2,4-D have been oral studies, one subchronic study investigated the effects o f dermal exposure to 2,4-D on both skin irritation and systemic (overall) toxicity (36). The results from this study support the conclusion from the acute study thintended to reflect a short-term exposure to a chemical. These studies usually report the highest experim ental dose that does not cause toxicity, which is called the no-observed-adverse-effect-level (NOAEL). The NOAEL is an indication o f a safe dose. These studies also usually report the low est experimental dose that causes toxicity, which is called the Page 13 o f 24 low est-observed-adverse-effect-level (LOAEL). Although m ost subchronic studies o f 2,4-D have been oral studies, one subchronic study investigated the effects o f dermal exposure to 2,4-D on both skin irritation and systemic (overall) toxicity (36). The results from this study support the conclusion from the acute study that dermal exposure to 2,4-D has low systemic toxicity. This study also dem onstrated that the doses that The 2 ethylhexyl ester and the dimethylamine salt o f 2,4-D were significantly more irritating to the skin than was 2,4-D acid. The highest dose o f 2,4-D acid, 1000 mg/kg/day, did not cause dermal irritation. These results indicate a NOAEL for this effect o f 1000 mg/kg/day, but a LOAEL could not be determined. By contrast the LOAEL for dermal irritation for 2,4-D ethylhexyl ester was 162.5 mg/kg/day w ith a NOAEL o f 16.3 mg/kg/day. The results for 2,4-D dimethylamine salt were similar, yielding a LOAEL for dermal irritation o f 180.1 mg/kg/day w ith a NOAEL o f 18 mg/kg/day. These results indicate that 2,4-D ethylhexyl ester and 2,4-D dimethylamine induce local effects on the skin at m uch lower doses than those required to cause systemic toxicity. A subchronic oral study (26) conducted in rats compared the toxicity o f purified 2,4-D to technical grade 2,4-D (97.3% pure). The rats were fed either purified or technical grade 2,4-D acid for 13 weeks at doses o f 0, 1 5 ,6 0 ,1 0 0 , or 150 mg/kg/day. No overt signs o f toxicity or behavioral changes were noted during daily observations. M icroscopic examination o f nervous system tissue revealed no treatm ent-related effects. Significant treatm ent-related effects o f other tissues included altered levels o f the thyroid hormone tetraiodothyronine (T4), increased liver weights, and kidney lesions. Females w ere more sensitive than m ales when effects o f 2,4-D acid on body weight, levels o f serum T4, and the liver w ere examined. Both males and females appeared to be more sensitive to purified 2,4-D acid than to the technical grade. The LOAEL for decreased body weight gain in females fed purified 2,4-D was 60 m g/kg/day, w ith a corresponding NOAEL o f 15 mg/kg/day. The LOAEL for m ales w as 100 mg/kg/day, w ith a NOAEL o f 60 mg/kg/day. The LOAEL for decreased body weight gain for anim als fed technical grade 2,4-D was 150 mg/kg/day for both males and females, w ith a corresponding NOAEL o f 100 mg/kg/day. B oth purified and technical grade 2,4-D appeared to cause a decrease in serum T4 levels in females, but not in males. The LOAEL for purified 2,4-D for was 60 m g/kg/day, and the NOAEL 15 mg/kg/day. The LOAEL for technical grade 2,4-D was 100 m g/kg/day, w ith a NOAEL o f 60 mg/kg/day. M icroscopic exam ination o f thyroid tissue revealed no treatm ent-related changes. Purified 2,4-D acid caused an increase in relative liver weights in both males and females. The LOAEL for females for 2,4-D was 60 mg/kg/day, w ith a NOAEL o f 15 mg/kg/day. The LOAEL for males was 150 mg/kg/day, with a NOAEL o f 100 mg/kg/day. Technical grade 2,4-D caused an increase in relative liver weights in females, but not males. The LOAEL for females fed technical grade m aterial was 150 mg/kg/day, w ith a NOAEL o f 100 mg/kg/day. M icroscopic examination o f liver tissue revealed minor, nonspecific effects primarily in the 100 and 150 mg/kg/day dose groups fed either purified or technical grade 2,4-D acid. In this study, the kidney appeared to be the m ost sensitive target organ. In contrast to the other effects o f 2,4-D acid, m ales were more sensitive to kidney effects than females. A significant increase in relative kidney w eight was observed in all dose groups o f males fed either purified 2,4-D or technical grade 2,4-D. The LOAEL for increased kidney weight was 15 mg/kg/day* but no NOAEL could be determined. The LOAEL for females fed purified 2,4-D was 60 mg/kg/day, w ith a NOAEL o f 15 mg/kg/day. The LOAEL for technical grade 2,4-D in this study was 150 mg/kg/day, w ith a NOAEL o f 100 mg/kg/day. M icroscopic exam ination o f kidney tissue also revealed treatm ent-related changes in males and females. Changes in kidney tissue were more extensive at 60 mg/kg/day and higher doses w here the authors predicted that 2,4-D would saturate active kidney transport. This Page 14 o f 24 study indicates a LOAEL o f 15 mg/kg/day for oral subchronic exposure in rats based on effects on the kidney. A NOAEL could not be determined from this study. M ore recent rodent studies indicate a NOAEL o f 15 mg/kg/day for subchronic ingestion o f 2,4-D.3 These studies, completed in 1991, examined the toxic effects o f 1 ,1 5 ,1 0 0 , or 300 mg/kg/day 2,4-D on rats or mice. The LOAEL for rats was 100 mg/kg/day based on histopathology, changes in body weight, and clinical pathology. The LOAEL in m ice was 100 mg/kg/day based on decreased levels o f glucose and T4, and increased kidney weights. The NOAEL for both rats and mice was 15 mg/kg/day. Dogs appear to be more sensitive to the subchronic oral toxicity o f 2,4-D toxicity than rodents (22). One dog study compared the toxicity o f 2,4-D acid (96.7% pure), 2,4-D dimethylamine salt (66.7% pure), and 2,4-D 2-ethylhexyl ester (95.1% pure). Beagles w ere fed 2,4-D for 13 weeks at doses o f 0, 1.0,3.75, and 7.5 mg/kg/day. The 2,4-D acid also was given at an additional dose o f 0.5 mg/kg/day for 13 weeks. No effects on hematology (blood), urinalysis, or ophthalmology were observed. All three forms o f 2,4-D had significant effects on body weight gain, some parameters o f clinical chemistry, and testes weight. Although all three form s o f 2,4-D caused a significant decrease in body weight gain, 2,4-D acid and 2.4- D 2-ethylhexyl ester appeared to be more potent than 2,4-D dimethylamine salt. The LOAEL for 2.4- D acid and 2,4-D 2-ethylhexyl ester was 3.75 m g/kg/day, and the NOAEL was 1 mg/kg/day. The LOAEL for 2,4-D dimethylamine salt was 7.5 m g/kg/day with a NOAEL o f 3.75 mg/kg/day. Dose-related effects were noted for 5 o f the 17 clinical chemistry param eters examined. These included an increase in blood urea nitrogen (BUN), creatinine, and alanine aminotransferase; a decrease in alkaline phosphatase; and, in dogs treated w ith 2,4-D dimethylamine salt, an increase in aspartate aminotransferase. These results indicate a LOAEL o f 1 mg/kg/day for 2,4-D dimethylamine salt and 2,4-D 2-ethylhexyl ester for changes in clinical chemistry. A NOAEL could not be determined. In the case o f 2,4-D acid, the study established a LOAEL o f 1 mg/kg/day for changes in alanine aminotransferase and creatinine, w ith a corresponding NOAEL o f 0.5 mg/kg/day; and a LOAEL o f 3.75 mg/kg/day for changes in BUN, with a NOAEL o f 1 mg/kg/day. The authors state, however, that they did not consider these changes in clinical chemistry significant because they did not observe any treatm ent related m icroscopic changes in the kidney. In addition, progression in these effects was not observed in a chronic study o f 2,4-D discussed below (22). M icroscopic examination did reveal changes in the liver. The authors derived a LOAEL o f 7.5 mg/kg/day and a NOAEL o f 3.75 mg/kg/day for all three formulations based on liver effects. Treatment w ith either o f the three form ulations resulted in a significant decrease in testes weight. The LOAEL for the effect o f 2,4-D acid on testes w eight was 3.75 mg/kg/day w ith a NOAEL o f 1 mg/kg/day. The LOAEL for the 2,4-D amine and the 2,4-D ester was reported as 7.5 m g/kg/day w ith a NOAEL o f 3.75 mg/kg/day. The authors did not observe significant treatm ent-related effects on the w eights o f other organs. The authors concluded that these studies indicate a overall NOAEL o f 1 mg/kg/day for subchronic ingestion or any o f the three form s o f 2,4-D. These data w ere reviewed by the EPA Office o f Pesticide Program s, w hich concurred w ith the authors conclusion.3 Collectively, these studies indicate a NOAEL o f 1 mg/kg/day for subchronic ingestion based on dogs as the most sensitive species. 3 Personal communication. 1995. Jess Rowland, Office Pesticide Programs, EPA, W ashington, D.C. Page, 15 o f 24 Chronic toxicity. The results from oral chronic (i.e., long-term ) studies conducted on rats and dogs are consistent w ith the results from the subchronic studies (22,36). First, these studies indicate that dogs are more sensitive to chronic toxicity o f 2,4-D than rodents. Second, the chronic dog study also supports a NOAEL o f 1 mg/kg/day for 2,4-D (22). A chronic ingestion toxicity study o f 2,4-D acid in dogs was designed based on the results from the subchronic study discussed previously (22). The authors only studied 2,4-D acid, based on their conclusion that the subchronic toxicity o f 2,4-D acid is sim ilar to that o f 2,4-D dimethylamine salt and 2.4- D 2-ethylhexyl ester. Beagles w ere fed diets containing 0 ,1 .0 , 5.0, or 7.5 mg/kg/day 2,4-D acid (96,7% pure) for a year. Consistent with the findings from the subchronic study, the authors detected no significant alterations in hematology or urinalysis. In addition, microscopic examination revealed no treatm ent-related effects on bone marrow, lymph nodes, or spleen. Treatment-related effects included decreased body weights, changes in clinical chemistry, and effects on the kidney and liver. Treatment with 2,4-D resulted in a decreased body weight gain for females, but not m ales. The LOAEL for females was 7.5 m g/kg/day, w ith a NOAEL o f 5 mg/kg/day. The NOAEL for m ales was 7.5 m g/kg/day, but a LOAEL could not be determined. Changes in some o f the clinical chemistry parameters were noted in both males and females. Dose-related changes in clinical chemistry included decreased glucose levels, and increases in BUN, creatinine, cholesterol, and alanine aminotransferase. The LOAEL for these effects was 7.5 mg/kg/day for both m ales and females, w ith a NOAEL o f 5 mg/kg/day. M icroscopic examination revealed effects on the liver and kidney in both males and females in the 5 and 7.5 mg/kg/day dose groups. These results indicate a LOAEL o f 5 mg/kg/day and a NOAEL o f 1 mg/kg/day based on the liver and kidney effects. These data w ere review ed by the EPA Office o f Pesticide Programs, which concurred w ith the NOAEL o f 1 m g/kg/day.3 The results o f chronic rodent studies are sim ilar to those o f the dog study; A 2-year chronic oral study conducted in rats, completed in 1984, supports the NOAEL derived from the chronic dog study.3 The LOAEL from this study was 5 mg/kg/day, w ith a NOAEL o f 1 mg/kg/day based on kidney effects. M ore recent rodent studies indicate that dogs are more sensitive to 2,4-D than rats. A chronic study conducted on rats indicates a LOAEL o f 75 mg/kg/day for 2,4-D acid based on decreases in body weights, w ith a NOAEL o f 5 mg/kg/day (36). This study is discussed later in our "Carcinogenicity" section. A chronic oral mouse study, also discussed under "Carcinogenicity," indicates a LOAEL o f 150 mg/kg/day based on renal effects and a NOAEL o f 5 m g/kg/day (36). Collectively, these studies suggest an overall NOAEL o f 1 mg/kg/day for chronic exposure to 2,4-D, based on the observation that dogs are the m ost sensitive species and that liver and kidney are the target organs. The NOAEL from the dog study is used to calculate the reference dose (RfD) for 2.4- D currently used by the EPA Office o f Pesticide Programs. The RfD is an estim ate o f the dose that humans, including sensitive subpopulations, could be exposed to daily, throughout their lifetime w ithout appreciable risk o f toxic effects. The calculation o f the RfD is discussed in the "Toxicology conclusion" section at the end o f this chapter. Neurotoxicity. Some case reports have suggested an association between exposure to 2,4-D and the developm ent o f nervous system effects ranging from peripheral polyneuropathy and reduced nerve conduction velocity to depression, anxiety, and other symptoms o f post-traum atic stress syndrome in Vietnam veterans (49). In test animals, doses o f 2,4-D above 100 mg/kg can cause m yotonia o f the skeletal m uscle (49), and oral doses above 150 mg/kg o f 2,4-D can damage the blood-brain barrier (46). Toxicologic studies in rats and rabbits indicate, however, that neurotoxic effects o f 2,4-D do not Page 16 o f 24 occur below doses that saturate the kidney transport system. Therefore, neurotoxic effects would only be expected to occur at high doses o f 2,4-D. A short-term study conducted in rats investigated the neurotoxic effects o f skin contact w ith 2,4-D (46). In this study, all four limbs o f m ale rats were treated w ith a 12% or 24% solution o f 2,4-D dimethylamine. The authors noted that when 2,4-D dimethylamine is used as an herbicidal spray it usually is diluted to final concentration o f 0.5 to 2% . The rats w ere treated for 2 hours per day, 5 days a week. Treatm ent w ith 24% 2,4-D dimethylamine was stopped after 2 weeks because the animals developed severe derm atitis. Treatment with 12% 2,4-D dimethylamine was conducted for 3 weeks as planned, inasmuch as the animals exhibited only minimal skin change. The levels o f 2,4-D m easured in plasm a w ere 323 g/m l after 2 weeks o f treatm ent w ith a 24% solution, and 66.5 g/ml after 3 weeks o f treatm ent w ith a 12% solution. The dramatic increase in plasm a levels o f 2,4-D at the higher dose was attributed to increased absorption through the damaged skin. Derm al treatm ent w ith 2,4-D dimethylamine resulted in significantly decreased body weights and increased kidney weights, but no treatm ent-related effects on nervous system tissue (46). Decreased body weights were observed in animals treated with either 12% or 24% 2,4-D dimethylamine. Rats treated for 3 weeks w ith a 12% solution also exhibited an increase in kidney weights. Histology did not reveal any treatm ent-related effects on the kidney, suggesting that the increase in kidney weight may be an adaptive response to the active excretion o f 2,4-D. One measure o f neurotoxicity, grip strength, was increased after 3 weeks o f treatm ent w ith a 12% solution. This result is consistent w ith other reports that indicate that 2,4-D can increase forelimb and hindlimb grip strength. The significance o f the increased grip strength o f the rats is not clear. Histology did not reveal any treatm ent-related lesions in central and peripheral nervous system tissues. This study indicates that short-term exposure to 2,4-D does not cause observable pathological effects on the nervous system. To address the effects o f prolonged exposure on the nervous system , the Industry Task Force II on 2,4-D Research D ata conducted a 1-year oral neurotoxicity study on rats (36). Rats w ere fed diets providing 0 ,5 , 75, or 150 mg/kg/day o f 2,4-D acid. The rats were evaluated by a functional observational battery (a series o f observational tests to assess neurotoxicity), forelimb and hindlimb grip perform ance, landing foot splay, and an autom ated test o f motor activity. Evaluation was conducted before exposure, and after 3, 6, 9, and 12 months o f exposure. A t the end o f 12 months, central and peripheral nervous system tissues from the control and high dose group underwent microscopic examination. A summary o f the study results reported that the only significant neurological effects occurred in the high dose group. Retinal degeneration was noted in fem ales treated w ith 150 mg/kg/day. Forelimb grip perform ance, norm alized for body weight, was significantly increased in both males and females treated w ith 150 mg/kg/day. Systemic effects occurred at low er doses. Significantly decreased body weights were observed in the 75 and 150 mg/kg/day dose groups. The effect on body weight is consistent w ith the results o f the rat subchronic study conducted by Gorzinski et al. (26). These results indicate a LOAEL o f 150 mg/kg/day and a NOAEL o f 75 m g/kg/day for neurotoxicity. This study also indicates a LOAEL o f 75 mg/kg/day for a decrease in body weights, with a corresponding NOAEL o f 5 mg/kg/day. Reproductive and developmental toxicity. To examine the effects o f 2,4-D on reproduction, the Industry Task Force II on 2,4-D Research D ata conducted a two generation study in rats (36,49). The rats w ere adm inistered 0 ,5 ,2 0 , or 80 mg/kg/day 2,4-D acid as a single dose from days 6 through 15 o f gestation. The 80 mg/kg/day dose o f 2,4-D caused excessive m aternal toxicity, as indicated by Page 17 o f 24 reduced maternal body weights and food consumption. The high dose group also exhibited decreased length of gestation, and reduced live-litter size. In addition to the maternal toxicity observed in the high dose group, decreased pup weight was observed in the 20 mg/kg/day group. The LOAEL for reproductive effects was determined to be 20 mg/kg/day based on the decreased pup weight. The NOAEL for reproductive effects was 5 mg/kg/day. Studies conducted in rats and rabbits indicate that 2,4-D does not cause birth defects or effect development (36). In one study, female Fischer 344 rats were administered 2,4-D acid via stomach tube once a day from days 6 through 15 o f gestation. The dose range in this study was 0, 8, 25, or 75 mg/kg/day. No teratogenic or embryotoxic effects were observed at these doses. There was a decrease in maternal body weight gain, however, in the 75 mg/kg/day dose group. This study indicated a LOAEL o f 75 mg/kg/day for maternal toxicity based on effects on body weight gain, with a corresponding NOAEL of 25 mg/kg/day. * Similar results were obtained from a study that investigated the potential developmental toxicity o f 2,4-D triisopropanolamine, 2,4-D isopropylamine, and 2,4-D butoxyethyl ester (36). In this study, rabbits were administered 0, 10, 30, or 75 mg/kg/day acid equivalents o f 2,4-D on days 7 through 19 o f gestation. No treatment-related effects on embryo-fetotoxicity or teratogenicity were observed at any o f the doses. Maternal toxicity was noted at 30 and 75 mg/kg/day for all three forms o f 2,4-D. Maternal toxicity was indicated by a number o f effects including decreased body weight gains, myotonia, and urine discoloration. This study indicates a NOAEL for maternal toxicity of 10 mg/kg/day, and a LOAEL o f 30 mg/kg/day for all three forms of 2,4-D. These results indicate that rabbits are more sensitive to the general toxic effects o f these forms o f 2,4-D than to the general toxic effects o f 2.4-D acid. C arcinogenicity. The potential carcinogenicity o f 2,4-D has been under review for over a decade. In August, 1980, the EPA requested that 2,4-D be tested for carcinogenicity in rats and mice (64). In 1987, the EPA classified 2,4-D under Group "D", "not classifiable as to human carcinogenicity," pending additional data (64). As previously discussed in the "Epidemiology conclusion" section, the Harvard School of Public Health convened a panel o f 14 scientists (35) to, "...examine the weight o f evidence on the potential carcinogenicity of 2,4-D." More recently, a public advisory group to the EPA, the Joint Committee o f the Science Advisory Board and the Scientific Advisory Panel (SAB/SAP), met in 1993 to review the results from epidemiologic studies, carcinogen bioassays, and other relevant data concerning the potential carcinogenicity o f 2,4-D (64). Both the SAB/SAP and the panel convened by the Harvard School o f Public Health concluded that results from the rodent carcinogen bioassays available at the times o f the reviews provided weak evidence that 2,4-D is carcinogenic (35, 64). The SAB/SAP evaluated data from a variety o f mutagenicity studies and concluded that 2,4-D does not appear to be mutagenic (64). The Ames test, mouse micronucleus assay, and unscheduled DNA synthesis assay were uniformly negative for various forms o f 2,4-D. The SAB/SAP found that other types o f mutagenesis assays that sometimes yielded positive results, such as tests o f cytogenetics in human and animal lymphocytes, had significant experimental deficiencies. For example, 2,4-D was positive in some tests, but lacked a dose-response. Other reports o f positive results did not specify the source and purity o f the 2,4-D. These types o f problems raised questions about the validity o f the positive results. The SAB/SAP concluded that the available data suggests that 2,4-D is not genotoxic. The report from this committee also noted that although positive results would have strengthened the Page 18 0124 case that 2,4-D is carcinogenic, negative results do not necessarily mean that 2,4-D is not carcinogenic. The SAB/SAP and the panel convened by the H arvard School o f Public H ealth evaluated data from the same tw o rodent carcinogenicity studies. A 2-year feeding study conducted in B6C3F1 mice was completed in 1987. The mice were fed 0 ,1 ,1 5 , or 45 mg/kg/day 2,4-D (35, 49). The study reported no excess tumors in males or fem ales in any o f the dose groups (35). A second 2-year feeding study conducted in Fisher 344 rats was completed in 1986 (35,49). The rats w ere fed the same doses o f 2,4-D as the mice (35). The dose range was chosen on the basis o f a 13-week subchronic study, which indicated that 60 mg/kg/day o f 2,4-D produces kidney damage. The results from this study indicated no statistically significant increase in tum ors in female rats. In male rats, however, there appeared to be a statistically significant increase in the occurrence o f a type o f brain tum or, astrocytom as, in the high dose group. One astrocytom a was identified among the 60 controls, none w ere observed in the 1 and 5 mg/kg/day dose groups, two w ere observed among the 58 rats in the 15 m g/kg/day group, and five were identified among the 60 rats fed 45 mg/kg/day. The incidence o f astrocytom as in the m ale rats was considered w eak evidence o f carcinogenicity by criteria used to evaluate potent neurocarcinogens such as methyl nitrosourea (3 5 ,4 1 ,4 9 ). For example, inbred rodents have a variable rate o f spontaneous brain tum or incidence. Although there is not as m uch inform ation on the F344 rats as other inbred rodents, it has been argued that the increase in astrocytom a incidence in the 2,4-D study is w ithin the range o f incidences o f spontaneous brain tum or developm ent. In addition, rats treated w ith 2,4-D did not appear to have signs o f brain toxicity, preneoplastic lesions, or early neoplastic proliferations. On the other hand, the EPA Health Effects Division Carcinogenicity Peer Review Panel questioned w hether this study used an adequate dose range. They specifically questioned w hether the highest dose used in the study was a maximum tolerated dose. The maximum tolerated dose is the highest dose that w ill not decrease the lifetim e o f the animal due to causes other than carcinogenicity. This dose typically causes an approximately 10% decrease in body weight. The maximum tolerated dose is usually the highest dose used in carcinogen bioassays. To establish w hether the incidence o f astroycytom as was treatm ent-related, EPA personnel requested that additional rodent carcinogenicity studies be conducted at higher doses o f 2,4-D (35, 64). Two additional rodent carcinogenicity studies w ith 2,4-D w ere completed in M arch o f 1995. One study was conducted in Fisher 344 rats. In this study, male and female rats were administered 0, 5, 75, or 150 mg/kg/day 2,4-D for either 12 months (lF isher 344 rats. In this study, male and female rats were adm inistered 0, 5 ,7 5 , or 150 mg/kg/day 2,4-D for either 12 months (15 animals/sex/dose) or 24 months (50 anim als/sex/dose) (37). No treatm ent-related i1 mice. The mice were fed 1 ,5 ,1 5 0 , or 300 mg/kg/day for up to 24 months. The m id and high doses were excessively toxic to males. Therefore, the male mice were term inated at 1 year, and the study was continued w ith only the females. N o treatm ent-related increase in tum ors in fem ale m ice were noted up through 2 years. These recent rodent carcinogenicity studies support the suggestion that 2,4-D alone is not carcinogenic. Toxicology conclusion. C arcinogenicity. Recent rodent carcinogenicity assays indicate that treatm ent w ith 2,4-D alone does not cause an increase in tum or development. In addition to being negative in standard carcinogenicity bioassays, 2,4-D also was negative in a rat liver model for tum or prom oters (1). These results indicate that 2,4-D is not carcinogenic alone, and furtherm ore is not synergistic w ith known mutagenic Page 19 o f 24 carcinogens. N oncarcinogenic effects. Studies of noncancer endpoints indicate that 2,4-D is not a developmental toxicant, and that 2,4-D does not cause neurotoxic effects below doses that saturate kidney transport. Dogs appear to be more sensitive to 2,4-D than rats with respect to general toxicity. This may be due to species differences in the capacity for excretion o f 2,4-D. The goal of most risk assessments for noncarcinogenic effects is the calculation o f an RfD. An RfD is an estimate of the reference dose that humans, including sensitive subpopulations, could be exposed to daily, throughout their lifetime without appreciable risk of toxic effects. An RfD is calculated by dividing a NOAEL by an uncertainty factor. The NOAEL is usually obtained from the study with the most sensitive species and most sensitive target organ. The uncertainty factor is calculated by assigning numbers ranging from 1 to 10 to account for specific sources of uncertainty when using data from animal studies to estimate a safe dose for humans. RfD (mg/kg/day) = NOAEL (mg/kg/dav) Uncertainty factor Because dogs appear to be the most sensitive species to 2,4-D, the RfD will be calculated using the NOAEL from the chronic ingestion study conducted on dogs (22). The NOAEL from that study was 1 mg/kg/day based on the liver and kidney effects. Using conventional risk assessment methods, an RfD for 2,4-D would be derived by dividing the NOAEL o f 1 mg/kg/day by an uncertainty factor o f 100. The uncertainty factor o f 100 is calculated by multiplying an uncertainty factor o f 10 to account for interspecies extrapolation by another uncertainty factor o f 10 to account for sensitive subpopulations. This would yield an oral RfD o f 0.01 mg/kg/day. This RfD is consistent with the RfD for 2,4-D currently listed on the EPA's Integrated Risk Information System (63). This RfD should also be adequate for skin exposure, because absorption o f 2,4-D via ingestion is very efficient and more rapid than the absorption of 2,4-D through the skin. SUMMARY Collectively, the epidemiologic and toxicologic data show that 2,4-D is not likely to be carcinogenic in humans unless it is acting through an unknown mechanism that is not evident in animals. Based upon the calculated RfD and data from exposure studies, the general public should not experience toxic effects from exposure to 2,4-D. Because workers involved in the manufacture or application of 2,4-D may be exposed to levels above the RfD, appropriate protective equipment always should be used. Table of Contents LITERATURE CITED 1. Abdellatif, A. G., V. Preat, J. Vamecq, R. Nilsson, and M. Roberfroid. 1990. Peroxisome proliferation and modulation of rat liver carcinogenesis by2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, periluorooctanoic acidand nafenopin. Carcinogenesis. 11:1899-1902. 2. American Medical Association Council on Scientific Affairs. 1988. Cancer risk o f pesticides in agricultural workers. JAMA. 260:959-966. 3. Armstrong, B. K., E. White, and R. Saracci. 1992. Principles o f ExposureMeasurement in Epidemiology. Monographs in Epidemiology and Statistics, Vol. 21. Oxford Univ. Press, New York, NY. Page 20 of 24 4. A xelson, 0 . 1987. Pesticides and cancer risks in agriculture. M ed. Oncol.Tumor Pharmacother. 4:207-217. 5. Blair, A ., H. M alker, K. P. Cantor, L. Burm eister, and K. W iklund. 1985. Cancer among farmers: a review. Scand. J. W ork Environ. H ealth. 11:397-407. 6. B lair, A ., S.H. Zahm, K.P. Cantor, and P.A. Stewart. 1989. Estim ating exposure to pesticides in epidem iological studies o f cancer, p. 38-46. in W ang, R. G. M. C. A. Franklin, R . C. Honeycutt, and J. C. Reinert, eds. Biological M onitoring for Pesticide Exposure: M easurement, Estim ation, and Risk Reduction. American Chem. Soc., W ashington, DC. 7. Blair, A ., O. Axelson, C. Franklin, O. E. Paynter, N. Pearce, J. Stevenson, E. T ro sk o ji. Vainio, J. W oods, and S. H. Zahm. 1990. 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Pearce, N . E., A. H. Sm ith, J. K. Howard, R. A. Sheppard, H. J. Giles, and C. A. Teague. 1986. Non-Hodgkin's lymphoma and exposure to phenoxyherbicides, chlorophenols, fencing work, and m eat works employment: a case-control study. Br. J. Ind. Med. 43:75-83. 54. Pearce, N. E., R. A. Sheppard, A. H. Smith, and C. A. Teague. 1987. Non-Hodgkin's lymphoma and farming: an expanded case-control study. Br. J.Cancer. 39:155-161. 55. Pearce, N. and J. S. Reif. 1990. Epidemiologic studies o f cancer in agriculturalworkers. Am. J. Ind. M ed. 18:133-148. 56. Persson, B., A-M . Dahlander, M. Fredriksson, H. N. Brage, C-G. Ohlson, and O. Axelson. 1989. M alignant lymphomas and occupational exposures. Br. J. Ind. M ed. 46:516-520. 57. Sauerhoff, M. W ., W. H. Braun, G. E. Blau, and P. J. Gehring. 1977. The fate o f 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man. Toxicology. 8:3-11. 58. Smith, A. H ., D. O. Fisher, H. J. Giles, and N. Pearce. 1983. The New Zealand soft tissue sarcom a case-control study: interview findings concerning phenoxyacetic acid exposure. Chemosphere. 12:565-571. 59. Smith, A. H ., N, E. Pearce, D. O. Fisher, H. J. Giles, C. A. Teague, and J. K.Howard. 1984. Soft tissue sarcom a and exposure to phenoxyherbicides andchlorophenols in N ew Zealand. J. Natl. Cancer Inst. 73:1111-1117. 60. Smith, A. H. and N. E. Pearce. 1986. Update on soft tissue sarcom a and phenoxyherbicides in New Zealand. Chemosphere. 15:1795-1798. 61. Smith, J. G. and A. J. Christophers. 1992. Phenoxyherbicides and chlorophenols: acase control study on soft tissue sarcoma and m alignant lymphoma. Br. J. Cancer. 65:442-448. 62. Sterling, T. D. and Arundel A. V. 1986. H ealth effects o f phenoxy herbicides: areview. Scand. J. W ork Environ. Health. 12:161-173. 63. U nited States Environm ental Protection Agency. 1986. Integrated Risk Inform ation System file for 2,4-D dichlorophenoxyacetic acid (2,4-D). Prepared by theOffice o f H ealth and Environmental Assessment, Environmental Criteria andAssessment Office, Cincinnati, OH for the Office o f Drinking W ater, USEPA, W ashington, DC. 64. U nited States Environm ental Protection Agency, Science Advisory Board. 1994. AnSAB report: Page 24 of 24 assessment o f potential 2,4-D carcinogenicity, review o f theepidem iological and other data on potential carcinogenicity o f 2,4-D by the SAB/SAPJoint Committee. USEPA, W ashington, DC. 65. V ineis, P., B. Terracini, G. Ciccone, A. Cignetti, E. Colombo, A. Donna, L. M affi,R. Pisa, P. Ricci, E. Zanini, and P. Comba. 1986. Phenoxy herbicides and soft-tissue sarcomas in female rice weeders: a population-based case-referent study. Scand. J.W ork Environ. H ealth. 13:9-17. 66. W iklund, K., J. Dich, and L-E. Holm. 1987. R isk o f m alignant lymphoma inSwedish pesticide appliers. Br. J. Cancer. 56:505-508. 67. W iklund, K., J. Dich, and L-E. Holm. 1988. Soft tissue sarcoma risk in Swedishlicensed pesticide applicators. J. Occup. M ed. 30:801-804. 68. W iklund, K., J. D ich, and L-E. Holm. 1989. R isk o f soft tissue sarcoma, H odgkin's disease, and non-Hodgkin's lymphoma among Swedish licensed pesticide applicators. Chemosphere. 18:395-400. 69. W ilkinson, C. F. and W orking Group Chairpersons. 1990. Introduction andoverview. p. 5-33. jn B aker, S. R. and C. F. W ilkinson, eds. The Effects o f Pesticides onHuman Health. Advances in M odem Environm ental Toxicology, Vol. X V m . Princeton Sci. Pub. Co., Inc., Princeton, N J. 70. W oods, J. S., L. Polissar, R. K. Severson, L. S. H euser, and B. G. Kulander. 1987. Soft tissue sarcoma and non-Hodgkin's lymphoma in relation to phenoxyherbicide and chlorinated phenol exposure in W estern W ashington. J. N atl. Cancer Inst. 78:899-910. 71. W oods, J. S. 1989. Non-Hodgkin's lymphoma among phenoxy herbicide-exposed farm workers in w estern W ashington State. Chemosphere. 18:401-406. 72. W orld H ealth Organization. 1984.2,4-Dichlorophenoxyacetic acid (2,4-D). Environmental Health Criteria 29. Published under the joint sponsorship o f theUnited N ations Environment Programme, the International Labour Organisation,and the W orld Health Organization, Geneva, Switzerland. 73. W orld H ealth Organization. 1989.2,4-Dichlorophenoxyacetic acid (2,4-D) --environmental aspects. Environm ental Health Criteria 84. Published under the joint sponsorship o f the United Nations Environm ent Programme, the International Labour Organisation, and the W orld Health Organization, Geneva, Switzerland. 74. Zahm, S. H ., D. D. W eisenburger, P. A. Babbitt, R. C. Saal, J. B. Vaught, K. P.Cantor, and A. Blair. 1990. A case-control study o f non-Hodgkin's lymphomaand the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in eastern Nebraska. Epidemiology. 1:349-356. Page 1 o f3 Table 1. Summaiy of key case-control studies of phenoxy herbicides and soft-tissue sarcomas (STS), non-Hodgkin's lymphoma (NHL), and Hodgkin's disease (HD). Odds ratio First author,reference, Cases, number, and year and type C o n tro ls, num ber, and type Y ears encom passed for cases Type o f interview (95% Conjdence interval) Phenoxy 2,4-D herbicides specifically Harden (30) 1979. 52 STS, hospital-based. 208, 1970-1977 G eneral population. M ail w ith 5.3 (2.4 telephone 11.5) supplem ent. Eriksson (25) 1981. 110 STS, 220, 1974-1978 population-based. General population. M ail with 6.8 (2.6 telephone 17.3) supplem ent. Sm ith (58,59) 1984. 82 STS, 92, Other 1976-1980 population-based. types o f cancer. Telephone. 1.3a (0.7 - 2.5)b H oar (34) 1986. 133 STS, 948, 1976-1982 population-based. General population. Telephone. 1.4c (-)d 1.3c (--)d Smith (60) 1986. 51 STS, 315, population-based Other types o f cancer. 1981-1982 Telephone. 0.7a (0.3 - 1.5)b Vineis (65) 1986. 68 STS, population-based. 158, G eneral population. 1981-1983 In-person and mail. 2.4e (0.6 - 10.3)b and l .l f (0 .2 5.1)b |l.lf W oods (70, 71) 128 STS, 694, 1981-1984 1987. population-based. General population. In-person. 0.9g (0.4 -1 .9 ) Harden (33) 1988. Smith (61) 1992. 54 STS, 311, 1978-1983 population-based. General population and 179, other types o f cancer. 30 STS, hospital-based. 82, 1976-1987 G eneral population M ail with 3.3h (1.4 telephone -8 .1 ) supplement. and 2.2a (0 .9 5.3) In-person. 0.8h (0.2 -3 .7 ) and 2.5a Page 2 o f3 Hardell (32) 1981. 169 HD and NHL, hospital-based. and 82, other types o f cancer. 338, 1974-1978 G eneral population. H oar (34) 1986. 121 HD, 948, 1976-1982 population-based. General population. H oar (34) 1986. 170 NHL, 948, population-based. General 1979-1981 population. Pearce (53) 1986. 83 NHL, 228, population-based. General 1977-1981 population and 168, other types o f cancer. Pearce (54) 1987. 183 NHL, 338, population-based. Other types o f cancer. 1977-1981 Persson (56) 1989. 54 HD, hospital-based. 275, 1964-1986 G eneral population. Persson (56) 1989 106 NHL, hospital-based. 275, 1964-1986 General population. W oods (70, 71) 576 NHL, 694, 1981-1984 1989. population-based. General population. Zahm (74) 1990. 201 NHL, 725, 1983-1986 population-based. General population. Cantor (21) 1992. Smith (61) 1992. 622 NHL, 1245, 1980-1983 population-based. General population. 52 HD and NHL, hospital-based. 82, 1976-1987 G eneral population and 82, other (0 .5 12.9) M ail w ith 4.8 (2.9 - -- telephone 8.1) supplem ent. Telephone. 1.0c 1.0c ( ~ ) d ( )d Telephone. 2.2 (1 .2 - 2.3 (1.3 4.1) 4.3) Telephone. l .l h (0.6 - 2.2)b and 1.3a (0 .7 2.2)b Telephone. 1.0a (0.7 -- - 1.5)b M ail. 3.8 (0 .7 - -- 21.0)b M ail. 4.9 (1 .3 - -- 18.0)b In-person. 0.9 (0.5 - 0.7 (0.4 1.5) 1.3) Telephone. Similar 1.5 (0 .9 to risk 2.5) estim ate for 2,4-di In-person. 1.2 (0.9 - 1.2 (0 .9 1.6) 1.6) In-person. 0.8h (0.3 -2 .2 ) and 1.8a (0 .5 6.0) types of cancer. a Odds ratio calculated using cancer controls. b 90% confidence interval. c Results not reported in original article. As cited by Blair et al. (7, 8). d Confidence interval not provided. e Odds ratio calculated using self-respondent subjects. f Odds ratio calculated using next-of-kin for subjects. 8 High exposure. h Odds ratio calculated using population-based controls. 1Numeric results not reported in original article. Top of Page Next Table Page 3 of 3 Page 1 of 1 T able 2. Summary of key cohort studies of phenoxy herbicides and sofl-tissue sarcomas (STS), non-Hodgkin's lymphoma (NHL), and Hodgkin's disease (HD). First author reference, and Cohort population, number and type year Comparison population Relative risk (95% Confidence interval) Years of follow-up STS HD NHL Coggon(24) 1986 5,754 Phenoxy herbicide England and manufacturers and Wales general applicators. population. 1947-1983 1.1 0.3 (0.0 - 5.9) (0 .0 - 1.6) 0.4 (0.01.3) 2,187 Phenoxy herbicide U.S. general Ott (52) 1987 manufacturers. population. 2.5a 1940-1982 (0.113.9) 0.9a (0.05.1) 1.9 (0.64.5) Bond (15) 1988 U.S. general 878 2,4-D manufacturers. population and 2.7b 1945-1982 None observed. (0.0 - internal referents. 14.7) 3.9b (0.414.1) Wiklund (66, 20,245 Applicators. 67, 68) 1989 Sweden general population. 0.9 1.5 1965-1984 (0.8- (0 .4 - 1.9) 2.4) 1.1 (0.71.6) 2.0b Bloemen (14) 1993 (0.2- 7.1) U.S. General 878 2,4-D manufacturers. population and 1945-1986 None Not observed. reported.0and internal referents. 3.0d (0.811.9) 4,461 Phenoxy herbicide Lynge (43, 44, manufacturers, of which 45) 1993 2,119 were potentially exposed. Denmark general 1947-1987 2.3e population. (0.6 - 5.8) 1.3e (0.4 3.3) a Relative risk not reported in original article. As cited by Blair et al. (7, 8). b Relative risk calculated using general population as comparison group. c One case of HD had been observed in initial study (15), but was not reported separately in update. d Relative risk calculated using internal referents as comparison group. e Among cohort members with potential exposure to phenoxy herbicides.. Table of Contents Previous Table Next Table Table 3 Summary of completed studies on the reliability and validity of pesticide use data obtained from interviews. First Study author, population reference, and year H ardell (30) 1979 STSa cases and controls. Comparison Study type population Employers. Validity. ' Hoar (6, STS, HDb, 12,13, and NHLC 34)1986 cases and controls. _ Suppliers. Validity. M ethods used M ajor findings Notes Contacted Agreem ent good M ethods not employers o f for chlorophenols. clearly defined; subjects who low employer had worked in response rate; forestry, reliance on sawmill, or memories o f pulp employers; industries to employers may corroborate not be "gold reported standard"; no exposures to data provided fc phenoxy overall level o f herbicides and agreement or chlorophenols; specifically for inform ation cases and obtained via controls; mailed reporting questionnaires. differences for cases and controls not assessed. Contacted Overall agreement M ethods not suppliers for for use o f clearly defined; 110 subjects pesticides was unclear as to who had about 50%; which pesticides farmed to agreem ent better were assessed f< corroborate for the last 10 agreem ent; reported years; suppliers reliance on exposures to usually reported memories o f herbicides and less pesticide use suppliers; insecticides; than subjects; suppliers may n< information overall agreement be "gold obtained via for herbicides was standard"; in-person about 60%, but recalculated OR interviews was lower for not provided for w ith suppliers years used; 2,4-D; only and records agreements for lim ited data Page 2 of 4 W oods (70) 1987 B row n (19) 1991 o f suppliers. herbicides was similar for cases and controls; provided in several publications. recalculated ORd for NHL and herbicide use was higher; agree-ment for 2,4-D was 83% for NHL cases and 74% for controls. STS and NHL Supervisors Validity. cases and and controls. cow orkers. Contacted Agreement M ethods not supervisors obtained in clearly defined; and essentially all agreem ent cow orkers o f instances in which assessed mostly subjects who supervisor or for recent jobs; had jobs with coworker was reliance on potential reached; memories o f exposures to agreement similar supervisors and corroborate for cases and 1 reported controls. cow orkers; supervisors and exposures to coworkers may phenoxy not be "gold herbicides, standard"; chlorophenols, supervisor and and other cow orker chemicals; response rate nc inform ation provided; no dal obtained via 1 telephone provided for overall level o f interview s. agreement or specifically for cases and controls. Controls for Proxies. NHL and leukemia cases. Comparability o f data from selfand proxyrespondents. Interviewed Agreem ent about Respondents 95 controls 95% for 2,4-D asked only abou who had use; agreem ent frequently used farmed and better for m ore pesticides their proxies recent tim e reported in earii about periods; study; could not controls' use proxy-respondents assess reporting o f specific reported m ore differences pesticides subjects as between cases reported by exposed than the and controls. the controls in subjects earlier case- themselves; Boyle (17) 1992 STS, HD, Proxies. NHL, liver, nasal, and nasopharyngeal cancer cases. Johnson NHL and Proxies. (39, 40) leukemia cases 1993 and controls. Johnson NHL and Same. (39) leukemia cases 1993 and controls. Page 3 of 4 control study. agreement percentages for days per year o f use for herbicides and 2,4-D specifically were about 50%. Comparability o f Interviewed data from self- 270 proxies, and for those proxy-respondents. cases who died during 4-year study period, about cases' use of pesticides, herbicides, and phenoxy herbicides containing 2 ,4-D . For herbicides used in farming, sensitivity was about 45% and specificity was about 95%; for phenoxy herbicides containing 2,4-D, sensitivity was 35% and specificity was 100%. Could not asses; reporting differences between cases and controls. Comparability o f Interviewed Agreement Proxy-responde: data from self- 328 proxies, percentages for interviewed and for those use o f herbicides several years aft proxy-respondents. cases and ranged from about self-respondents controls who 70 to 80%; for died or use o f 2,4-D, became agreem ent incompetent percentages follow ing ranged from about interview in 60 to 75%; for earlier both NHL and case-control leukemia, OR's for study, about herbicides and use o f 2,4-D specifically pesticides, were higher for herbicides, proxy-respondents and specific then herbicides self-respondents. such as 2,4-D. L o n g -term reliability. Reinterviewed Agreement 758 cases and percentages for controls from 2,4-D use were earlier about 80% for case-control self-respondents study about and between 55 use of and 75% for R einterview s conducted up to decade after initial interviews Johnson Farmers. (39) 1993 Same. Short-term reliability. Johnson Farmers. (39) 1993 Suppliers. Validity. a Soft-tissue sarcoma. b Hodgkin's disease. c Non-Hodgkin's lymphoma. d Odds ratio. Table of Contents Next Table Previous Table________ Page 4 of 4 pesticides, proxy-respondents. herbicides, and specific herbicides such as 2,4-D. Interviewed Agreement for use Could not assess 157 farmers of herbicides was reporting on 2 94%; agreement differences occasions. for 2,4-D use was between cases less than 1 80%; agreement and controls. year apart. for usual days per about use of year o f 2,4-D use pesticides, was 71%. herbicides, and specific herbicides such as 2,4-D. Abstracted Agreement, Suppliers may nc data related sensitivity, and be "gold to 2,039 specificity for standard"; could pesticide herbicides were not assess purchases 81, 89, and 20%, reporting from 14 respectively; for differences suppliers for 2,4-D specifically, between cases 42 farmers to agreement was and controls. corroborate 60%, sensitivity reported was 63%, and exposures to specificity was pesticides, 53%. herbicides, and specific herbicides such as 2.4-D. \ Page ! of 2 Table 4. Summary of NOAEL's3 and LOAEL's*5from key toxicity studies of 2,4-D. Study type, species, and reference Form o f 2,4-D 21-day dermal, >toxicity, rabbit acid (36) 1000 2-ethylhexyl ester dimethylamine > NOAEL(mg/kg/day) LOAEL(mg/kg/day) N otes . 1000 1627.5 16.3 540.5 18 Systemic toxicity. Dermal irritation. ... 162.8 ... 180.1 Systemic toxicity. Dermal irritation. Systemic toxicity. Dermal irritation. 13-week oral, toxicity, rat (26) acid, purified acid, technical grade Subchronic oral, toxicity, rat acid -- -- 15 Subchronic oral, toxicity, mouse0 acid 15 13-week oral, toxicity, dog (22) acid (96.7% pure) 1 1-year oral, toxicity, dog (22) 2-year oral, toxicity, rat0 dimethylamine (66.7% pure) 2-ethylhexyl ester (95.1% pure) 1 1 acid 1 acid 1 15 Kidney effects. 15 Kidney effects. H isto p ath o lo g y , 100 body weight, and clinical pathology effects. D ecreased glucose and T4 100 levels and increased kidney w eights. Testes, body w eight, clinical 3.75 chemistry, and liver effects from all three forms. 3.75 3.75 Liver and kidney 5 effects. 5 Kidney effects. 2-year oral, toxicity and oncogenicity, rat (36) 2-year oral, oncogenicity, female mouse (36) 1-year oral, neurotoxicity, rat (36) acid acid acid Two-generation oral, reproductive acid toxicity, rat (36, 49) Gavagei*1, developmental acid toxicity, rat (36) 5 5 5 75 5 25 Gavage, developmental toxicity, rabbit (36) acid 30 triisopropanolamine 10 isopropylamine 10 butoxyethyl ester 10 Page 2 of 2 Decreased body 75 weight, no indication o f oncogenic effects. Kidney effects, no treatment-related 150 increases in the incidence of neoplasms. 75 Decreased body weight. 150 Retinal degeneration. 20 Decreased pup weight. Maternal toxicity. 75 No teratogenic or embryotoxic effects observed. Maternal toxicity for all four forms. 90 No developmental effects observed for all four forms. 30 30 30 a No-observed-adverse-effect-level, which is the highest experimental dose that does not cause toxicity. b Lowest-observed-adverse-effect-level, which is the lowest experimental dose that causes toxicity. c Personal communication, Jess Rowland, Office o f Pesticide Programs, EPA. 1995. d Stomach tube. Table of Contents Previous Table