In the preceding chapters we have followed several episodes in the development of a notion of environmental risk. Along with other early cases, the Elixir Sulfanilamide tragedy refined scientific methodology with an analytical technique for deriving LD50s and prompted passage of the Food, Drug, and Cosmetics Act of 1938. With the advent of World War II there was renewed interest in insecticides that could control the spread of malaria and other insect-borne diseases. DDT was the most promising of these, and its potential effects on target organisms, lab animals, wildlife, and humans underwent extensive analysis. Much of the interest in DDT was concentrated in governmental organizations—the PHS, the FWS, the USDA, and the U.S. armed forces. Such scrutiny demonstrated that DDT had opened a new era in insect control and toxicology. No other insecticide killed such a broad spectrum of insects without damaging the crops it was protecting. No other insecticide inspired such extensive investigation. For DDT, scientists extended the scope of toxicology to include effects on wildlife populations.
The wartime pursuit of an effective insecticide against malaria-carrying mosquitoes was just part of the fight against malaria (one of DDT’s important uses). Scientists in the Toxicity Laboratory at the University of Chicago also sought antimalarial drug therapies and contributed new techniques to measure the joint toxicity of drugs and drug resistance. In addition to antimalarial drug therapies, Tox Lab scientists developed methods to trace minute quantities of drugs like bufagin and digitoxin by rendering them radioactive. Equally important, the Tox Lab laid the foundation for an independent discipline of toxicology by training graduate students and supporting research. Through his paternalistic direction, E. M. K. Geiling inspired these and other developments.
Despite the extensive publicity focused on it, DDT was only one of many insecticides that scientists developed during World War II.1 Governmental organizations exhaustively tested DDT, but the task of evaluating other pesticides fell mainly to a young scientist named Kenneth DuBois (1917–73) who was working in the Tox Lab. Like many other scientists, DuBois joined the war effort shortly after completing his Ph.D. in physiology and biochemistry at the University of Wisconsin. At the Tox Lab, DuBois’s research revealed several strong commitments. First, DuBois pursued the biochemical aspects of toxicology and stressed the importance of in vivo confirmation of the effects of toxic agents observed in in vitro enzyme studies. Second, he endeavored to develop methods to measure these effects quantitatively.2
Like DDT and other chlorinated hydrocarbons, the organic phosphate insecticides (later, “organophosphates” or “OPs”) were first examined by German chemists as potential nerve gases to be used in combat.3 Organic phosphate compounds link many phosphorous atoms to oxygen atoms (termed esters of polyphosphoric acids). Among the compounds investigated was diisopropyl fluorophosphate (DFP), which contained only one phosphorous atom. The Germans eventually discarded DFP as a nerve gas, but their experiments indicated that it inhibited cholinesterase, a critically important enzyme needed for the proper functioning of the nervous systems of humans, other vertebrates, and insects. It was the quality of cholinesterase inhibition that convinced physicians to use DFP to treat glaucoma, by reducing the abnormally high tension of the eyeball, and also myasthenia gravis, an autoimmune neuromuscular disease, for which it was more effective than treatment with eserine.4 The first organic phosphate, HETP (hexaethyl tetra phosphate), emerged from Gerhard Schrader’s laboratory at Farbenfabriken, Germany, in the early 1940s. Schrader discovered the insecticidal properties of the organic phosphates during the war, and the chemicals reached America in 1945, when the British and American Technical Intelligence Committee interrogated German chemists in the immediate aftermath of the war. One of the first groups to gain access to the organic phosphates was the Tox Lab, where DuBois and his associates recognized cholinergic symptoms (i.e., changes in the action of the neurotransmitter acetylcholine) produced by the new chemicals and found that atropine would be an effective antidote.5
Kenneth Dubois, studying the effect of radioactive digitoxin on beating mammalian heart. Courtesy of the University of Chicago Photographic Archive apf 1–05876, Special Collections Research Center, University of Chicago Library.
In the U.S., there was considerable interest in the in the new insecticides because the organic phosphates could allegedly control aphids, against which DDT was proving ineffective. The Tox Lab assumed the responsibility for testing the toxicity of the new chemicals largely because the University of Chicago was near one of the major labs, Chemagro, where organic phosphates were synthesized. HETP (C12H30P4O13) was one of the organophosphorous chemicals that German chemists had developed, and under interrogation they compared this new compound to nicotine for its action in destroying aphids. Tox lab researchers could not locate any references to HETP’s mechanism of action other than these possible nicotinic effects. Several researchers at the lab noted during routine testing, however, that animals showed symptoms similar to those produced by DFP. Such symptoms included muscular twitching, tonic and tonic-clonic convulsions, involuntary defecation, micturition (urination), and salivation. The case for parallel actions of the two chemicals was reinforced when researchers produced miosis by placing a dilute solution of HETP in the eyes of rabbits. In the case of HETP, the dilation effect lasted for five to twelve hours compared to three days in response to DFP. The similarities in the action of the two chemicals prompted DuBois to consider their possible effects on cholinesterase.
Through a series of in vitro and in vivo tests, DuBois and George Mangun (also a researcher at the Tox Lab and its director from 1946 to 1953) investigated the effect of HETP on cholinesterase. In the in vitro experiments, they measured HETP’s effect on the cholinesterase of rat and cockroach tissue by adding solutions of the inhibitor dissolved in the buffer to the test system, which facilitated manometric measurement of the cholinesterase activity. The final concentration of 1 × 10−7 M HETP inhibited cholinesterase by 47 percent in the brain tissue of rats, by 53 percent in the submaxillary, 60 percent in serum, 45 percent in erythrocytes, and 58 percent in cockroach tissue. In comparisons of HETP with two recognized cholinesterase inhibitors (DFP and carbamic acid ester), DuBois and Mangun found HETP to be the most effective. For the in vivo experiments, the researchers administered HETP intraperitoneally (directly into the body peritoneum or body cavity) to rats and then measured the cholinesterase activity of the brain, submaxillary glands, and serum with the manometric test system. The results of the in vivo experiments verified the in vitro experiments, revealing cholinesterase inhibition in all of the tissues. Thus DuBois and Mangun concluded: “Hexaethyl tetraphosphate exerts a strong inhibitory effect on mammalian and insect cholinesterase in vitro and in vivo. This finding, in conjunction with its gross effects on animals, suggests that its physiological effects may be at least in part due to its inhibition of this enzyme.”6 This research connected the new insecticides with cholinesterase inhibition. The comparison with carbamic ester anticipated by nearly a decade the development of carbamate insecticides (see below).
DuBois and other researchers at the Tox Lab examined the toxicity of other organic phosphate insecticides as well. For much of this research, DuBois was joined by John Doull. For his doctoral dissertation, Doull used radioisotopes to evaluate cardiotoxic and other effects of bufagin. He received his Ph.D. in 1950 and his M.D. in 1953. Later Doull recalled that he had initially contributed to the analysis of the toxicity of organic phosphate chemicals.7
One of the most important chemicals investigated at the Tox Lab was a new insecticide called parathion. Parathion appeared to be particularly effective against plant insects, and its potential use stimulated researchers to examine its toxicity and pharmacologic action in mammals. Their approach to the analysis of parathion shared many similarities with the toxicological analyses of DDT. Along with Paul R. Salerno and Julius M. Coon, DuBois and Doull evaluated the acute and subacute toxicity of parathion as well as its inhibitory action on cholinesterase. They determined that LD50s were low (less than 20 mg/kg) in all species (rats, mice, cats, and dogs), whether parathion was administered intraperitoneally or orally. Recall that a low LD50 corresponds to a very high toxicity. When sublethal doses of parathion were administered daily, its toxic action was cumulative. DuBois and his team also noted that the symptoms produced by parathion were similar in all species tested. These symptoms were typical of parasympathomimetic drugs (i.e., cholinergic drugs or those that mimic acetylcholine and inhibit cholinesterase). Like HETP, the Tox Lab researchers showed parathion to be a strong inhibitor of cholinesterase. In vitro a final concentration of 1.2 × 10−6 M inhibited by 50 percent rat brain cholinesterase. Finally, they explored possible antidotes to the lethal effects of the drug.8 A picture of consistency within the class of organic phosphate insecticides gradually emerged from toxicological assessments like this one.
DuBois and his team also conducted the first toxicological evaluation of OMPA or Pestox III (Octamethyl Pyrophosphoramide). Like other organic phosphates, OMPA was first synthesized by Schrader, who demonstrated its insecticidal properties. He also noted that plants absorbed OMPA from the soil, which rendered them insecticidal. In fact, one group of researchers showed that OMPA had limited value as a contact insecticide, though plants grown in soil containing the chemical became highly toxic to insects for several weeks. This same group of researchers claimed that OMPA was toxic to mammals when mixed with food and administered orally.9 On the whole, DuBois and his colleagues found the toxicity of OMPA similar to that for parathion. LD50 values for several species were virtually identical to those for parathion. All of the species exhibited symptoms typical of parasympathomimetic drugs except symptoms linked to the stimulation of the central nervous system. In regards to OMPA’s potential as a systemic insecticide, DuBois’s group noted that plants grown in soil containing OMPA contained an anticholinesterase agent. This finding indicated that plants converted OMPA much like the mammalian liver.10
In the Tox Lab, DuBois mainly studied the toxicity of organophosphates to animals, while other labs assessed the risks posed to humans from information gained through occupational accidents. David Grob and other researchers at the Johns Hopkins University School of Medicine reviewed the toxic effects of parathion in thirty-two men and eight women following accidental exposure. The research at Johns Hopkins, like that at the Tox Lab, was supported by the Medical Division of the Chemical Corps of the U.S. Army. Grob and his colleagues identified a disturbing characteristic of parathion: it could be readily absorbed through the skin, respiratory tract, conjunctivae, gastrointestinal tract, or following injection, most likely due to its high solubility in lipids (fats). Moreover, parathion did not produce inflammation in the skin so absorption could remain undetected.11 That parathion could be absorbed through the skin contrasted with DDT, which had a low rate of dermal absorption, a real advantage in the eyes of economic entomologists. This basic difference between the two chemicals accounted for parathion’s much higher level of toxicity. More troubling than dermal absorption was the absence of an inflammatory reaction, which suggested that an individual could suffer a toxic exposure without being aware of it. Still, the Johns Hopkins researchers were able to determine several general warning symptoms—intermittent nausea, vomiting, giddiness, weakness, drowsiness, and fasciculations (muscle twitches) of the eyelids—which appeared for one to seven days before the more severe manifestations developed.
Grob and his colleagues also addressed symptoms that were particular to organic phosphate chemicals and specifically to parathion. They classified the symptoms with respect to two classes of action on the sympathetic nervous system: muscarine-like symptoms (anorexia and nausea, vomiting, abdominal cramps, excessive sweating, and salivation) and nicotine-like symptoms (nausea and vomiting, muscular fasciculations or twitches in the eyelids and tongue, followed by fasciculations in the muscles of the face and neck, in the extra-ocular muscles, and finally generalized fasciculations and weakness). Although categorizing symptoms may seem esoteric, the symptoms suggested that the organophosphates affected both the muscarinic receptors and the nicotinic receptors in the nervous system. The term “muscarine” tied the organophosphates to the long history of poisons. First isolated from a mushroom, Amanita muscaria, in 1869, muscarine was the first parasympathomimetic substance ever studied; it causes profound activation of the peripheral parasympathetic nervous system that may end in convulsions and death. More troubling still, the subjects developed all these symptoms and some of them died, despite the fact that most of them had worn carbon filter respirators, rubber gauntlets, and protective coveralls during their exposure to parathion. Some of the subjects had even worn hip-length rubber boots and rubber aprons. The Johns Hopkins researchers suspected that breaks in these safety procedures had occurred. Moreover, they discovered that the face piece respirators did not fully protect the workers from inhaling organophosphates as aerosols, dusts, or sprays.
Grob and his associates were able to isolate factors that accounted for the fact that parathion was more efficient than Tetraethyl Pyrophosphate (TEPP) as an insecticide even though its anticholinesterase activity and toxicity were lower. These factors also explained the greater danger of parathion to humans and domestic animals in comparison with other organic phosphates. The Johns Hopkins researchers noted that parathion hydrolyzed (broke down into chemical components) more slowly than TEPP, so that once sprayed on trees or fields, parathion remained active for weeks despite contact with moisture, whereas TEPP would hydrolyze within several hours. Another factor they noted was parathion’s higher solubility in lipoid (fat), which meant that it would accumulate in the waxy outer layer of fruit and leaves, where it had been found up to nine days after spraying. Finally, the oxygen analogue of parathion was more toxic and active against cholinesterase than parathion, but Grob and his team could not determine the extent to which exposure to the air (as in spraying), or in plant tissues, or after absorption into the body converted parathion into its oxygen analogue.
By way of conclusion, the Hopkins researchers suggested a series of precautionary measures to be taken when dealing with parathion. Their recommendations included adequate warning labels, complete protective clothing, respirators, and a change of clothing before eating or smoking. They made specific recommendations regarding the use of parathion on crops for consumption: fruits and vegetables should be sprayed only with very dilute solutions, harvested no less than three weeks after the last spraying, and thoroughly washed prior to use.12 Even within a class of highly toxic chemicals, parathion, as Grob and his team showed, was extremely hazardous to humans in the workplace and probably also as a food residue.
English physicians also examined the effects of poisoning by organophosphate chemicals. One of them, Lesley Bidstrup, reported only four organophosphate poisonings up to 1950, but each case stood out for the rapidity with which the insecticides wrought havoc on human systems. In one case, a plant foreman and fellow worker were splashed with parathion. Although the foreman made his assistant wash with soap and water and change his clothes at once, the foreman neglected to follow his own advice. After eight hours, he developed nausea, vomiting, abdominal cramps, diarrhea, and constriction of his pupils. By the time the foreman was admitted to the hospital nine hours later, he had developed fibrillary twitching of voluntary muscles and signs of pulmonary edema. Despite treatment with atropine, he died twenty-one hours after the accident.13 With accounts such as this one in mind, Bidstrup bemoaned the lack of knowledge regarding long-term exposure to small amounts of parathion, although he suggested that if the need to leave sufficient time between spraying and harvesting were strictly observed, the food supply would remain safe. The potential exposure of workers was more worrisome, and Bidstrup concluded: “Experience in the United States of America in the 1949 spraying season has demonstrated that, unless all the recommendations made for the safe handling of organic phosphorus insecticides are carried out in detail, serious illness and even death will occur.”14
Officials at the FDA also studied the new chemicals. From 1946 on, Arnold J. Lehman served as chief of the Division of Pharmacology at FDA. That year, a reorganization of the FDA established specialized sections. Under the new arrangement, the division of toxicology encompassed the acute, chronic, and dermal toxicity sections. Lehman assumed his post at FDA after a distinguished career in research and academia. Before joining the FDA, Lehman served as professor of pharmacology and director of the teaching and research activities in the Pharmacology Department at the University of North Carolina Medical School. In addition, he served as a consultant to the Federal Security Agency and as a member of the Committee on Atomic Research.15 In June 1948 he presented a paper to the Association of Food and Drug Officials of the United States in Portland, Maine, titled “The Toxicology of the Newer Agricultural Chemicals.” Lehman compared the toxicities of about two dozen insecticides including DDT, TEP (or TEPP), parathion, HETP, nicotine, chlordane, and heptachlor. Using DDT as a reference standard for insecticide toxicology, he listed the newer insecticides according to their acute oral toxicities. In this hierarchy, DDT had a median lethal dose of 250 mg/kg. In comparison, TEPP’s LD50 was 2 mg/kg (or 125 times more toxic than DDT), parathion’s LD50 was 3.5 mg/kg (70 times more toxic than DDT), and HETP’s was 7 mg/kg (35 times more toxic than DDT). Lehman’s hierarchy highlighted the relatively low acute toxicity of DDT.
Lehman also addressed three aspects of dermal toxicity: skin irritation, quantities dangerous upon skin application (single exposure and multiple exposure), and quantities dangerous to man (estimated). In a visually powerful manner, Lehman demonstrated that the organic phosphates were at least one order of magnitude more toxic than DDT. For example, Lehman estimated that it would take a single dermal exposure of 169 grams, and multiple exposures of 9 grams/day of DDT to be harmful to man, whereas for TEPP and parathion, he estimated single exposures of only 0.6 and 3 grams, respectively, of TEPP and parathion, and multiple exposures of 0.3 grams/day, were harmful. Despite their relatively higher dermal toxicity, the two organic phosphates irritated the skin only slightly (compared to no irritation for DDT). Thus an individual could suffer toxic exposure to an organic phosphate without noticing it.
Lehman also described chronic toxicity in rats in his address, but an accompanying table revealed the paucity of data available from long-term studies. By 1948, few insecticides had undergone toxicity experiments lasting more than 52 weeks. DDT was one of the few insecticides that had been subjected to a two-year study of chronic toxicity to rats (see chapter 2). Thus Lehman could state that the lowest level of DDT producing gross effects was 100 parts per million (ppm), as demonstrated in a study lasting 104 weeks. For parathion, by way of contrast, Lehman listed 25 ppm as the lowest level producing gross effects. He based this claim on a study of only 4 weeks duration. Remarkably, even at levels of 1,000 ppm, HETP produced no effect over the course of a study lasting 12 weeks.16
Beyond the considerable value of his comparative tables, Lehman anticipated some of the most significant problems associated with the newer chemical insecticides. First, he undercut one of the fundamental beliefs behind the expanding use of pesticides: “It is a fairly safe assumption that chemicals which are toxic to insects are also toxic to man and animals. The great emphasis which has been placed on the specificity of DDT for insects loses its importance when fatal doses are compared on a body-weight basis with warm-blooded animals. On this basis the quantities required are practically identical.”17 Although he was extrapolating from limited data, Lehman’s statement drew on his vast experience in pharmacology. He also expressed concern that the body stored certain insecticides like DDT in fat. Even more disturbing was the secretion of DDT and other chlorinated hydrocarbons in milk: “This is especially important in cases of infants, where the chief diet is milk.”18 Lehman’s concern was not limited to DDT and the chlorinated hydrocarbons. Parathion was known to have a cumulative action, which pointed to its storage in tissues.19 He reserved his most disturbing comment for the end of his paper: no one knew the dangers of using such chemicals in aerosol form. This information was available only for DDT, which had a safety factor several hundred times greater in such conditions. Lehman effectively outlined a comparison of the toxicology of the new agricultural chemicals and from this review identified some of the significant concerns regarding their widespread utilization. Moreover, he anticipated the litany of problems Rachel Carson attributed to pesticides in Silent Spring more than a decade later.
In 1949 Lehman listed the insecticides in descending order of potential harmfulness to the public health, with emphasis placed on risks other than those related to the spray residue on foods. He arranged the toxicity of insecticides as follows: “TEPP > Parathion > Compound 497 > Nicotine > Compound 118 > Chlordane > Toxaphene > DDT > Rote-none.”20 On the important issue of spray residues (typically small amounts of pesticides that remained on foods), he noted that the values established as safe by the current experimental evidence were subject to change, and that they applied only to a single item of food:
Rotenone |
5 parts per million |
Pyrethrins |
10 parts per million |
TEPP |
rapidly decomposed; known decomposition products not considered as a hazard |
Parathion |
2 parts per million |
Gamma isomer |
3–5 parts per million |
DDT |
less than 1 part per million if all of the food consumed is contaminated; 5 parts per million approaches the upper limit in any single item21 |
According to this table, the most toxic of the organic phosphates also decomposed the most rapidly into harmless, nontoxic products. So quickly would TEPP decay that Lehman and other scientists saw no need to set a residue level. Because of its slower rate of decay, the residue limit for parathion was 2 ppm. DDT, the subject of the most extensive scrutiny, received the lowest residue level. Moreover, Lehman cautioned that, because specific chemical methods for the isolation of many of the chlorinated hydrocarbon insecticides had not been developed, the detection of their presence in foods depended on generic organic chloride determinations. Thus food containing any detectable organic chloride residues should be regarded as contaminated.22
The most notable difference between the organic phosphates and other synthetic insecticides, DuBois and his colleagues at the University of Chicago had found, was that the organic phosphates inhibited cholinesterase in all species, including humans. All of the organic phosphates caused cholinesterase inhibition to some extent, but the new insecticides varied considerably in other aspects, such as persistence.
The Committee on Pesticides of the Council on Pharmacy and Chemistry of the AMA reviewed the available information on the known organic phosphates in 1950. After a general description of three organic phosphates, DuBois and Grob, members of the committee, summarized their pharmacology and toxicity (both were recapitulations of earlier papers).23 Additional committee members contributed to the review. For example, two doctors from American Cyanamid (one of the chief producers of organic phosphate chemicals) and another from the California Department of Health discussed clinical experience, briefly presenting eight fatal cases, which were mostly occupational exposures of various sorts resulting from lack of protective clothing, but included also a German biologist who attempted to determine the human tolerance for parathion through self-experimentation. Another tragic case involved a ten-year-old child who drank from a whiskey bottle containing TEPP and died in about fifteen minutes, before medical assistance became available.24 Such accounts revealed the greater toxicities of most organic phosphates in comparison with the chlorinated hydrocarbons like DDT.
The rapid hydrolization of most of the organic phosphates appeared to reduce their risk in soil, but in their contribution to the committee’s review, Lehman, Albert Hartzell, and J. C. Ward noted that the relatively slower rate of hydrolization of parathion posed a health hazard when it was used on turf. They also introduced evidence from animal studies: “Life-time feeding studies in rats at low dietary levels of parathion indicate no detectable cumulative effects below 25 parts per million. Animals fed levels above 25 parts per million and up to 100 parts per million, although they survived, displayed symptoms of nervous system poisoning and possessed an inhibition of blood cholinesterase in proportion to the increase of parathion over 25 parts per million in the diet.”25 Drawing on this information, Lehman extrapolated the risk to humans and recommended a safe residue level on any one item of the diet of approximately 2 parts per million of parathion.26 Even as they proposed a safe residue level, Lehman, Hartzell, and Ward cautioned that only if parathion was applied strictly in accordance with the recommendations of the BEPQ of the USDA, with “particular reference to the time between the last spraying and the harvesting of the fruit,” would normal weathering reduce parathion residues to this level of safety.
Another question that Lehman and his collaborators raised was whether or not the peel of a fruit would be used in the preparation of a particular foodstuff. Even at the lowest effective spray concentrations, the peel of a fruit taken alone could carry a load of 2 to 3 ppm of parathion; this same concentration constituted 0.16 ppm extended to the entire fruit. This distinction was crucial: peeling the fruit before use, utilizing the whole fruit, or using the peel alone could change the level of exposure to parathion by an order of magnitude. In light of these variables, they concluded by underscoring the importance of adherence to recommended spray schedules: “If spray schedules recommended by qualified entomologists are followed, it is quite unlikely that a parathion spray residue problem will become serious.”27
Given the composition of the AMA Committee on Pesticides—two industry doctors, two university toxicologists, and two government representatives from the USDA and the FDA—the conclusion probably reflects a compromise among committee members. As an FDA employee, Lehman may have realized that the political climate for the agency was less than favorable. For example, Clarence Cannon, who had shut down FDA’s pesticide research in 1937, had become chairman of the House Appropriations Committee. From this vantage point, which he held from 1947 to 1964, Cannon and like-minded southern and midwestern conservatives wielded considerable influence over the federal government and especially the FDA.28
In an address before the Chicago Dietetic Association on March 15, 1950, DuBois took up the issue of food residues and food contamination by new insecticides, such as DDT and organic phosphates, as well as by the new systemic insecticides. He succinctly reviewed the state of knowledge in 1950 regarding the acute and chronic toxicity of each of the insecticides. From the practical standpoint of chronic toxicity, the chlorinated hydrocarbons had been a problem of major concern since their introduction: “The chlorinated hydrocarbons are stable toward hydrolysis, and spray residues may remain on fruits and vegetables for a long time. Continued ingestion of these contaminated products may thus produce a health hazard. Furthermore, these materials are fat-soluble, and the ingestion of contaminated forage by dairy cattle results in the appearance of insecticides in the milk where they are concentrated in the fat.”29 All of these factors associated with the chlorinated hydrocarbons contributed to the significant risk of chronic toxicity. DuBois cited one of the few studies of chronic dietary exposure to DDT, which showed that levels of 100 mg/kg DDT in food produced chronic poisoning (in the form of liver damage) in rats during the two-year study. Like Lehman, DuBois urged caution in the face of scientific uncertainty, noting that chronic poisoning by these chemicals was a distinct possibility.30
In contrast to the chlorinated hydrocarbons, food contamination had not been a problem with organic phosphate chemicals, such as HETP and TEPP, because of their rapid hydrolysis when they came into contact with moisture; spray residues on fruits and vegetables would lose their toxicity before the foods were consumed. Even parathion, DuBois explained, although more stable toward hydrolysis than the other organic phosphates, was rendered nontoxic before foods were harvested. But DuBois drew a sharp distinction between typical organic phosphates and the systemic insecticides, such as OMPA (organic phosphate chemicals applied to the soil and taken up by the plants rendering the plants themselves insecticidal). What did this mean for possible food contamination? DuBois noted that the insecticidal agent formed within plants from OMPA rapidly lost its toxicity rendering plants nontoxic to insects by the time the plants reached maturity. DuBois wondered, however, about the potential risk from plants harvested before they finished growing. Because those plants could be dangerously contaminated, DuBois advised restraint in the application of systemic insecticides, restricting use to non-food crops or food crops that were never harvested before maturity.31
Thus, DuBois underscored the fundamental differences between chlorinated hydrocarbons and organic phosphates. Chlorinated hydrocarbons like DDT did not cause acute poisoning after a single dose. Research had demonstrated, however, that animals ingesting the new insecticides for a long time could be poisoned. In contrast, acute toxicity posed the most significant risk with the organic phosphate insecticides, but their rapid hydrolysis greatly limited the threat of chronic toxicity and food contamination. Finally, DuBois noted that organic phosphates used as systemic insecticides presented greater risk than contact with organic phosphates because of their ability to be absorbed by plants. DuBois’s simple taxonomy of the risks associated with the three new classes of insecticides captured their essential differences.
Most of the research activity on organic phosphates discussed to this point was concentrated in two locations: the University of Chicago Toxicity Laboratory under the supervision of DuBois and the FDA Division of Pharmacology with Lehman as its chief. Additional contributions to the literature of organic phosphates came from Grob at Johns Hopkins. Both DuBois and Lehman presented hierarchies of the risks posed by the various new insecticides within the broad categories of chlorinated hydrocarbons, organic phosphates, and systemic insecticides. Although the various groups seemed to work independently from each other, in fact, DuBois, Doull, and other researchers at the Tox Lab worked closely with Dan MacDougall and Dallas Nelson, the scientific staff at Chemagro (later Bayer Corporation) in Kansas City to plan and execute studies and eventually to defend new insecticides before the FDA. John Doull recalled interactions with the FDA: “These meetings were usually held in the FDA commissioner’s office with Drs. Arnold Lehman, Garth Fitzhugh, Bert Vos and Arthur Nelson representing FDA and DuBois, Doull and MacDougall representing Chemagro. In contrast to the complex and lengthy procedure currently required to obtain pesticide tolerances, these meetings were short, informal and focused on the science (toxicology and pathology) rather than on any of the legal or political considerations that often seem to be of primary importance today.”32 It was not until Rachel Carson published Silent Spring in 1962 that the political nature of pesticide regulation came to the forefront of attention among the wider American public.
In 1952, DuBois and Julius M. Coon, a doctor in the Tox Lab, returned to the toxicology of organic phosphorous-containing insecticides to mammals. DuBois and Coon classified the organic phosphates into three groups based on the chemical formula of each insecticide: alkyl pyrophosphates, alkyl thiophosphates, and phosphoramides. Among the alkyl pyrophosphates, TEPP was the most important, and DuBois and Coon reconfirmed the considerable toxicology of TEPP and particularly cholinesterase inhibition. In an analysis of additional alkyl pyrophosphates, DuBois and Coon demonstrated that they all exhibited cholinergic properties similar to TEPP. Several important organic phosphates, including parathion, malathon, and systox, were classified as alkyl thiophosphates. In light of the extensive use of parathion as an agricultural insecticide, DuBois and Coon reviewed the akyl thiophosphates to find a compound as toxic as parathion to insects but less toxic to mammals. They showed that the LD50 for rats for parathion was 5.5 mg/kg while that for malathon (later, malathion) was much higher, at 750 mg/kg. This was one of the first references to the toxicity of this newly developed insecticide. DuBois and Coon urged that these results be interpreted cautiously, since chemicals with a low toxicity for mammals generally exhibited a lower toxicity for insects and thus required use of higher concentrations in the formulations used in insect control. An ideal compound would have a high toxicity for insects and a low mammalian toxicity.33 Thus, the two scientists pointed to one of the paradoxes of insecticide development. Insecticides of a lower toxicity to mammals often necessitated higher concentrations or quantities to produce the same measure of insect control. Raising the concentration or quantity undermined the advantage in toxicity.
In their consideration of the phosphoramides, DuBois and Coon shed light on one such biochemical interaction. They pointed out pharmacologic properties of OMPA (the only phosphoramide released for use at the time) that were unusual among the organic phosphates: “It exhibits no appreciable anticholinesterase action in vitro but is converted by the mammalian liver and by plants into a strong cholinesterase inhibitor. A further differentiating feature is its inability to gain access to the brain in vivo, its cholinergic action being therefore limited to peripheral tissues.”34 Of the organic phosphates, only OMPA, according to DuBois’s experience, could be converted by the mammalian liver and plants into a cholinesterase inhibitor.
Their reference to malathon indicates that in the Tox Lab DuBois and Coon had access to the newest insecticides, even those that were still in development stages. The first complete review of the toxicity of malathon did not appear until 1953, when Lloyd W. Hazleton and Emily G. Holland of the Hazleton Laboratories in Falls Church, Virginia, summarized mammalian investigations of the new chemical. In collaboration with the American Cyanamid Company, Hazleton Laboratories selected malathon from a coordinated screening program. From entomological data, Hazleton and Holland believed that malathon would find wide use as an insecticide and that this might lead to appreciable human exposure. Because their preliminary data suggested considerable variation between insect and mammalian toxicity, they conducted further experiments on the acute toxicity of the substance to several different kinds of animals: “Regardless of technical grade, solvent, species, sex or route of administration, the acute signs of toxicity are characteristic of the anticholinesterase activity. In rats, mice, guinea pigs, and dogs, salivation, depression, and tremors predominate. The signs are of short duration, and unless death occurs within a few hours recovery appears to be complete. This observation should be emphasized, for later studies indicate that cholinesterase inhibition endures far beyond any gross evidence of toxicity.”35 This statement suggests the possibility of a threshold for the effects of cholinesterase inhibition. Above a certain level of exposure, laboratory animals died. Below that level, Hazleton and Holland claimed, animals recovered completely from the exposure. The ability of animals to recover from the anticholinesterase activity of malathon served as a theme of their research.
Hazleton and Holland injected various concentrations of the new chemical into guinea pigs, dogs, and albino rats and monitored the animals to determine the exposure levels that produced cholinesterase inhibition of 50 percent (Inhibition50 or IN50) in the animals’ red blood cells, plasma, and brain. As one example, the Hazleton Laboratory researchers subjected rats to intraperitoneal dosages of malathon, which varied from 50 to 500 mg/kg. The IN50 for red blood cells was 480 mg/kg and 500 mg/kg for the brain. They compared these figures to the IN50’s for parathion (determined after 1.5 hours): 1.65 mg/kg for red blood cells and 3 mg/kg for brain. According to these experiments, malathon was at least two orders of magnitude less toxic than parathion. In chronic feeding experiments conducted on rats, Hazleton and Holland found no evidence of cholinesterase inhibition at 100 ppm malathon in the diet, but it did inhibit cholinesterase by 73 percent in red blood cells at 1,000 ppm/day and 100 percent at 5,000 ppm/day. In a two-year feeding experiment, rats on a daily diet of 100 ppm malathon showed slight evidence of cholinesterase inhibition. The results of the experiments conducted at the Hazleton Laboratories certainly demonstrated not only that malathon was much less toxic than parathion, but that it seemed to be the least toxic of all the organophosphate insecticides.
Hazleton and Holland used their results with malathon to challenge DuBois and Coon’s opinion regarding the organophosphate insecticides: “These data suggest that it would be timely to reconsider the view expressed by DuBois and Coon that those materials which have a low toxicity for mammals generally exhibit a low toxicity for insects.”36 Hazleton and Holland argued that while parathion was approximately 100 times as potent in vitro and 135 times as toxic to rats as malathon, “Under usage conditions, no more than two to three times as much malathon as parathion is recommended.”37 Hazleton and Holland believed that they had discovered an insecticide that was highly toxic to insects but minimally toxic to mammals. To determine whether or not that was the case required significantly more experimentation on both target and nontarget organisms. Would malathon control insects effectively at nontoxic levels? Hazleton and Holland harbored even greater hopes for the new chemical. Beyond its specific value as an insecticide, they expected it to transform thinking about insecticide toxicity: “It is to be hoped that this compound will serve to point the way toward a better understanding of the difference between mammalian and insect toxicity and to free our thinking from the dogma that anticholinesterase activity in vitro is necessarily an index to mammalian toxicity.”38 It is tempting to conclude that these findings seem tainted by the fact that American Cyanamid funded research at the Hazleton Laboratory or that the Hazleton Laboratories researchers failed to place a convincing distance between their objective findings and their chief source of support. The findings at Hazleton Labs suggested that toxicity of malathion constituted an exception to the rule that placed most organophosphates among the most toxic chemicals known to mankind.
Two years later, Kenneth DuBois returned to the subject of malathon (renamed malathion in 1953). With Robert Bagdon, DuBois examined the pharmacologic effects of chlorthion, malathion, and tetrapropyl dithionopyrophosphate in mammals. Bagdon and DuBois cited DuBois’s earlier work on the low toxicity of these compounds as well as Hazelton and Holland’s determination of the low toxicity of malathion. For this study, they considered the effects of thionophospates on blood pressure, respiration, the isolated heart, and the intestine in vitro and in vivo. Bagdon and DuBois concluded: “On the basis of toxicity and associated pharmacologic effects the newer thionophosphates employed for this investigation possess a distinct advantage over others such as parathion and systox from the standpoint of the dose required to produce acute poisoning. Hence, the possibility of accidental poisoning during handling is considerably less than with agents such as parathion.”39 This statement was cautiously couched in terms of toxicology and pharmacology (DuBois’s expertise), but it does not address the other part of the equation: would these insecticides necessitate greater quantities to affect the same control of target insect populations? DuBois restricted his conclusions to his area of specialization (mammalian toxicity). He and Bagdon did take up the issue of purity, however. They acknowledged that toxicity rose with impurity or contamination and cited Hazelton and Holland’s finding that malathion became less toxic with increasing purity. Although Bagdon and DuBois clarified the toxicity of thionophosphates, including malathion, they left open the question of the quantity required to achieve an effectiveness equivalent to that of a more toxic cholinesterase inhibitor like parathion.
In addition to malathion, chemical companies developed other insecticides. Carbamate insecticides were a promising new class of pesticides. Union Carbide developed and released carbaryl or Sevin in 1956. Like organophosphates, carbamates inhibited cholinesterase. However, researchers at the Mellon Institute in Pittsburgh found that Sevin’s anti-cholinesterase activity was greater against insects than against mammals. Tests with cats, guinea pigs, rats, rabbits, and chickens revealed LD50s for various routes of exposure (oral, intravenous, intraperitoneal, and subcutaneous) in the range of 125 mg/kg in cats to greater than 500 mg/kg in rats and rabbits. Two-year chronic feeding studies showed that rats tolerated daily doses of Sevin at levels up to 200 ppm. Similar studies demonstrated that dogs tolerated up to 400 ppm of Sevin in their diets on daily basis.40 Thus, like malathion, the toxicity of Sevin to mammals was relatively low. Early researchers also noted that stability, anticholinesterase activity, and insect toxicity were different for the organophosphates and the carbamates.41
As additional organophosphates and other chemicals entered the public market during the 1950s, DuBois and his research team at the University of Chicago continued to evaluate their toxicity. Among the chemicals that they evaluated were Systox, Di-Syston, and other organophosphates. At the close of the decade, DuBois sought to extend the implications of more than a dozen years of research on the organic phosphate insecticides. Together with his student Sheldon Murphy, who had become a University of Chicago Fellow, he assessed the influence of various factors on the enzymatic conversion of organic thiophosphates to anticholinesterase agents. Their research transcended the limits of research on organic phosphates and merited further study. They concluded: “The results of the present investigation have provided some information on the mechanisms responsible for age and sex differences and other factors which influence susceptibility to cholinergic thiophosphates. The findings suggest that further research along similar lines may aid in gaining an understanding of the reasons for age, sex, species and individual differences in susceptibility to drugs and other chemical agents which have been observed frequently but have not been adequately explained.”42 They found that the enzyme activity of the livers of adult male rats was two to three times greater than that of adult females of the same age. There were no observable differences between the sexes of animals less than thirty days old. Yet Murphy and DuBois noted a dramatic increase in the liver activity of male rats between thirty and sixty days of age. This time period corresponded with the age of puberty.
The Chicago researchers next increased the low enzyme activity in adult females and young males by administering testosterone for a prolonged period. They also reduced the high enzyme activity in adult males by castration and through the extended administration of progesterone and diethylstilbestrol. These experiments indicated that sex hormones influenced the synthesis of the thiophosphate-oxidizing enzyme. Equally important, Murphy and DuBois determined the role of diet and nutrition in enzymatic activity: “Feeding a protein-free diet to adult male rats reduced the ability of the liver to convert guthion to an anticholinesterase agent by 75 percent. The increase in activity of the thiophosphate-oxidizing enzyme which occurs after the administration of carcinogenic hydrocarbons was inhibited when the animals were fed a protein-free diet.”43 By enabling the liver to convert guthion, dietary protein contributed to cholinesterase inhibition.44
Research on the toxicity of the organophosphate insecticides also continued at the FDA. In many respects, the FDA research complemented DuBois’s numerous studies on the toxicity of the organophosphates. Scientists at the FDA endeavored to develop and test methodologies for the analysis of the toxicology of the new synthetic pesticides. J. William Cook was a biochemist in the Division of Food at the FDA from 1951 until 1972. He also served as the director of the Division of Pesticide Chemistry and Toxicology.45 Cook searched for ways to employ the enzyme systems he had devised in his previous position in the San Francisco regional office. It occurred to him that one of the best applications of his enzyme research would be the analysis of organophosphate compounds because they were toxic by virtue of the fact that they inhibited cholinesterase. Cook explained the nature of cholinesterase inhibition: “The cholinesterase enzymes hydrolyze to a compound called acetylcholine. Acetylcoline is involved in the transmission of nerve impulses. Therefore muscle activity is based on acetylcholine being formed and hydrolyzed quickly. When those enzymes are inhibited, the person becomes rigid or has tremors.”46
As Cook surveyed the literature on the esterase systems and their inhibition by the organophosphates, he discovered that such research required relatively complicated, expensive pieces of equipment, which he knew the FDA, with a total budget of five million dollars for all of its programs, could not afford to acquire. It was clear to Cook that budgetary restrictions put the FDA at a real disadvantage in its efforts to regulate pesticides when chemical companies had much more sophisticated analytical equipment. But fiscal constraints at the FDA inspired methodological creativity. Having developed a test for urea by putting the enzyme urease in paper along with a dye that would change with acid-base, Cook considered how he might develop a similar test for the organic phosphates—but the enzyme spot test was not his first thought. Initially he analyzed the organic phosphates using chromatography.47 Learning from a colleague that the sulfur in most organic phosphates might be sensitive to bromine, Cook developed a spot test for organic phosphates. As in the urea test, he sprayed a paper with a bromine-containing compound and superimposed a dye chemical. Wherever the sulfur in the organophosphate used up the bromine, it was not available to change the color of the dye.48 Moreover, the bromination technique converted the non-cholinesterase in vitro inhibitors to in vitro inhibitors of cholinesterase. With this knowledge, Cook could visualize some of the general chemical characteristics of these compounds. Using this approach, he learned to look for many useful signs when petitions came in for new organic phosphate compounds. He was thus able to accept or reject the data that companies submitted in their petitions based on the bromination technique.49
Cook combined the two tests (the anticholinesterase method of analysis with paper chromatography and his newly devised brominated spot test technique) to analyze numerous organic phosphate chemicals, including parathion. The literature indicated that parathion was highly toxic to dogs (exposures as low as 1 ppm depressed cholinesterase). In contrast, large quantities fed to cows did not inhibit cholinesterase or cause it to appear in the cow’s milk. Cook believed that something was happening to the parathion before it reached the bloodstream of the cow because in most mammals parathion fed at toxic levels moved from the bloodstream into the milk and meat. Cook fed parathion to a cow with an opening in its rumen (where he assumed the cow would break down the parathion). By the time he returned to his laboratory, the parathion had disappeared from the samples. In a review of the literature, Cook discovered that parathion had been reduced to a far less toxic amino group. From the combination of his own experimental data and his review of the literature, Cook felt confident that he could approve the use of parathion on plants fed to dairy cows because he knew it would not be transferred to their milk.50
In addition to the paper chromatography test and the test for anticholinesterase activity, Cook and a colleague, D. F. McCaulley, resurrected Edward Laug’s fly bioassay (see chapter 2) for determining organic phosphate pesticides. Other researchers had developed bioassays using flies, but most of them were based upon mortality induced by graded amounts of pesticides. Such bioassays were very sensitive but unspecific. McCaulley and Cook felt that by linking a measurement of in vivo depression of fly cholinesterase to a fly mortality count, group specificity might be added to the assay’s sensitivity. This procedure demonstrated the presence of any chemicals from the group of phosphate pesticides with legal tolerances for food residues (parathion, systox, methyl parathion, guthion, phosdrin, trithion, diazinon, malathion, and OMPA). The fly bioassay was effective as a screening procedure. Those samples showing significant mortality could be checked later for cholinesterase inhibition. Cholinesterase inhibition roughly equal to mortality indicated a phosphate as the main toxic factor; a mortality figure much higher than inhibition indicated the presence of a combination of toxicant, not all phosphate; and the absence of inhibition in the presence of considerable mortality would reveal a toxic factor other than a phosphate.51 A technique that had been abandoned in favor of chemical methods was revived and effectively redeployed for use in a new context (detection of pesticide residues on foods). Refining such techniques ultimately led to the development and publication of the Pesticide Analytical Manual, which became the standard reference for testing the toxicity of pesticides.
Another concern for toxicologists was the toxicity of insecticides used in combination, or potentiation. A team of FDA pharmacologists led by John P. Frawley analyzed the greater than additive toxicity, or potentiation, resulting from simultaneous administration of two anticholinesterase compounds, which was essentially a study of joint toxicity. After reviewing the rather sparse literature on the toxicity of organophosphate insecticides, Frawley and his colleagues noted: “In all these studies, the observations have been based on the continued administration of a single compound. In practice a worker may be exposed to two or more compounds on the same or alternating days, and the average consumer may ingest at the same meal several different food products each containing a different insecticide.”52 Unlike the joint toxicity of antimalarial drugs as studied at the Toxicity Laboratory, which would have to be prescribed by a physician, individuals could be exposed to two organophosphates inadvertently through occupational exposure and possibly even normal daily consumption.
Frawley and his team chose two organophosphates, EPN and malathion, because they were each less toxic than other organophosphates. First they determined the acute toxicity (LD50) of each chemical for rats and dogs, and then they established the toxicity of the two chemicals in combination. In dogs, EPN and malathion administered simultaneously caused up to fifty times the potentiation (additive toxic effects) of separate exposures. And they noted potentiation in rats as well. From these findings, Frawley and his team concluded: “However, of broader significance is the conclusion that in some cases the hazard associated with the administration of chemical and drug combinations cannot be evaluated from the toxicity of the individual compounds. The results point out the need for caution in the use of drug combinations in this phase of pharmacology and toxicology which is frequently overlooked.”53 The FDA group also investigated the joint toxicity of malathion and EPN combined in several ratios to house flies, again using Laug’s fly bioassay, but found no indication of potentiation. This finding suggested that potentiation involved complex chemical reactions between the two phosphates and the biological system.
At the Tox Lab, DuBois also addressed the potentiation of organophosphates. He reasoned that the simplest method for detecting potentiation by acute toxicity tests would be to administer half of the LD50 of each of two organic phosphates. If mortality due to the combination of the two compounds was additive (50 percent) or less than additive, no potentiation had occurred. DuBois used this approach to test for potentiation in several organic phosphates and found that most showed additive or less than additive acute toxic effects. This meant that the combination of half of the LD50 of the two chemicals produced a toxic effect that was equal to or less than the full LD50 dose for either chemical. DuBois anticipated these results when the compounds had the same mode of action, parallel dosage-mortality responses, and a similar time of onset of toxic effects. From the results of the tests of acute toxicity, it became clear to DuBois that he had to clarify the mechanism of toxicity for each organic phosphate involved in potentiation to fully explore subacute effects. Such research revealed that some agents inhibited hydrolytic detoxification reactions. DuBois thought this discovery was potentially valuable for basic research into normal metabolism, but it left unresolved the implications for food residues and occupational exposures.54 He noted, “Our present knowledge of the problem of potentiation of the toxicity of organophosphates does not provide an answer to the question of whether or not this effect constitutes a health hazard in connection with consumption of contaminated food.”55
The organophosphate insecticides, like DDT and other chlorinated hydrocarbons, demanded novel toxicological techniques and strategies. During World War II, the Toxicity Laboratory at the University of Chicago responded to this considerable need. In particular, Kenneth DuBois and his students and colleagues recognized the major toxicological effects of the organophosphates: cholinesterase inhibition. DuBois and his research group developed toxicological profiles for many of the new insecticides, including OMPA and parathion. In addition to the research conducted at the Tox Lab, David Grob at Johns Hopkins examined the toxicity of parathion to humans, drawing on occupational cases of exposure. Arnold Lehman at the FDA also evaluated the risks of the organophosphates, particularly in comparison to other insecticides like the chlorinated hydrocarbons. Like DuBois, Lehman constructed hierarchies of toxicity for the new chemicals. In general, the organophosphate insecticides had a greater acute toxicity (due to cholinesterase inhibition) but considerably reduced chronic toxicity in comparison with the chlorinated hydrocarbons. One promising exception to this developing rule was malathion, or so American Cyanamid and scientists associated with the company argued. In the mid-1950s, Union Carbide introduced the first of yet another promising class of insecticides: Sevin. The carbamates inhibited cholinesterase like the organophosphates. Like malathion, Sevin had a relatively low mammalian toxicity. Combination of certain organophosphates exacerbated their effects as researchers at the Tox Lab and the FDA independently discovered. As toxicologists at the Tox Lab and the FDA strove to assess the risks associated with exposures to the new organophosphates, legislators held hearings to determine the implications for public health.