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  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 see
m 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.