So perhaps our task ... is to make clear that there is no technical solution, no monetary solution. The only solution is not to repeat the pollution anywhere.
Miyamoto, 1975[1]
NEUROTOXICITY
Organophosphorus ester induced delayed neurotoxicity [OPIDN]
Background
The first recognized episode of OPIDN was the " Ginger Jakes", an epidemic of poisoning that occurred in the U.S.A in 1930. It is estimated that over 10,000 people were paralyzed by a solvent, triortho-cresyl phosphate [TOCP], that was used to used to adulterate a ginger extract. The ginger extract was apparently widely used as an alcoholic beverage during Prohibition. The episode takes its name from the poisoned Jamaican Ginger, and from the "jakes" [shakes], or flaccid paralysis, suffered by the victims. It is now known that the active neurotoxicant in TOCP poisoning is not TOCP itself, but a metabolite: saligenin cyclic phosphate.
The syndrome was characterized by an ascending ataxia/paralysis, generally restricted to the legs, that had its onset 830 days after exposure. The symptoms were to a large part irreversible; the characteristic time course of the syndrome includes [at the least] an 8 day latency, followed by the onset of ataxia, followed by slow recovery for up to one year [in humans]. Ataxia remaining at the end of 1 year is permanent; neither further deterioration nor significant improvement occurs thereafter.
Rodents are almost totally resistant to OPIDN, although they are excellent models for the acute toxicity of OPs. Adult hens were rapidly identified as the best experimental species; this is still true, despite numerous efforts to find a mammalian species - other than the human - of equal sensitivity. Even in hens, young birds remain unaffected by the ataxia. Because of the source of the TOCP, few if any children were exposed in the "Ginger Jakes" episode. In a much later outbreak, occurring in Meknes in Morocco in 1959, the source of the TOCP was food oil that had been adulterated with TOCP. In this episode, which again affected over 10,000 people, it became tragically apparent that human children are also sensitive to the paralysis, although they seemed to be less severely affected than adults. It is certain that human beings are at least as sensitive as hens, and we may well be the most sensitive species.
Except for the identification of saligenin cyclic phosphate as the active neurotoxicant, and the recognition of the sensitivity of children, this outline of the syndrome was complete within 5 years of the Ginger Jakes outbreak. Earlier awareness that saligenin cyclic phosphate, and not TOCP, was the proximate neurotoxicant might have altered the history of the investigation, since TOCP does not inhibit AChE, while saligenin cyclic phosphate does. In the event, the connection between AChE inhibitors and the delayed paralysis was not made for over 20 years.
Recurring episodes of OPIDN in the 1930s and 1940s were invariably due to TOCP exposure. The settings ranged from industrial exposure to TOCP fumes when TOCP was used as a lubricant or solvent; to environmental exposure to TOCP in contaminated drums that were being used to hold water; or of TOCPadulterated foods. In one bizarre case, the source of exposure was a parsley-derived abortifacient, Apiol. Actually a purgative that is used in hope of inducing abortion, Apiol itself is still used in Africa. The TOCP may have been used as a solvent for the other ingredients, or to extract the parsley derivative.[2]
In 1959, the last major episode of TOCP occurred in Meknes, Morocco. The source was a cheap cooking oil, adulterated by TOCP; 10,000 people were affected. Characteristic of OPIDN is the incident reported from the Meknes tragedy: in one family, the oil was recognized as poor quality, and was first fed to the dog. When the dog remained healthy the next day, the entire family used the oil. All, including the dog, became paralyzed a week later. It is also from Meknes that we know that children become ataxic, unlike the young of other species, although there is a suggestion in the data that very young children are at least partially protected.
During the 30 years between "Ginger Jakes" and Meknes, precautions on the use of tri-cresyl phosphates were developed: primarily, that industrial mixtures contain little or no tri-ortho-cresyl phosphate. Meanwhile, World War II saw the development of OPs as nerve gases, and the introduction and proliferation of organophosphate pesticides (cf parathion case history). The extreme toxicity of the early OP insecticides led to conscious attempts to introduce safer OP insecticides. Unfortunately, because TOCP does not inhibit AChE, no connection had been drawn between Ginger jakes and cholinesterase-inhibiting organophosphorus esters; attention focused on the tri-ortho-cresyl configuration.
Among the safer insecticidal OP compounds being developed in the early 1950s was mipafox: a selective insecticide so safe to humans that the developers kneaded the formulation with their hands. Three became paralyzed, and mipafox was immediately withdrawn from commercial development. Despite its small scale, this tragedy led to OPIDN testing of all OP insecticides in Britain; and to an unwritten but scrupulously honored ban of OPIDNcausing pesticides by European chemical companies. The World Health Organization (WHO) formally banned OPIDNcausing pesticides from its malaria-eradication program, leading to a further reduction in the incentive to market such compounds, since WHO provided considerable testing of candidate insecticides as well as a sizeable market for insecticides actually used. In the U.S.A., which had not been directly affected by mipafox, OPIDNtesting of OP insecticides was included in the protocols of the FDA and USDA.
A second wave of research on OPIDN followed the mipafox incident and established that:
1. Most or all phosphofluoridates are delayed neurotoxicants, and other OPs can also cause paralysis. Among the OPs identified as causing paralysis were the nerve gas sarin and several candidate insecticides, such as CELA-K43, with an LD50 of > 1g/kg in rats.
2. All paralytic OPs inhibit AChE, but the correlation between the two forms of toxicity is terrible: parathion [or, to be precise, its oxon] is an excellent AChE inhibitor, but does not cause paralysis. The nerve gas sarin does, as does the laboratory chemical diisopropylfluorophosphate, or DFP. The nerve gas soman, on the other hand, does not seem to cause OPIDN.
3. The paralysis is not, as was assumed until then, due to primary demyelination, but to axonal degeneration followed by demyelination.
In 1970, the U.S. EPA was formed, and, as part of its function, took over pesticide safety evaluation from FDA and USDA. The initial rules for pesticide testing were essentially the same as they had been under USDA. Safety studies were required before pesticides could be registered, but the choice of what studies to carry out, and which of the data to submit to EPA, was still left up to companies to a considerable extent. Somewhere in the transfer to EPA, the USDA/FDA requirement of testing OPs for OPIDN was lost. Against this background, the Velsicol Chemical Company developed leptophos.
CASE HISTORY: LEPTOPHOS [3]
In 1965, chemists at Velsicol Chemical Company synthesized a promising OP insecticide, which was field-tested on a small scale in 1966, followed by largescale testing on deciduous fruits, potatoes, tomatoes, rice, soy beans, sugar beets, and cole (cabbage family) crops. The compound proved to act best against larvae of the Lepidoptera (caterpillars), and was therefore given the generic name of leptophos. The brand name was Phosvel. Because it was not then required for a company to submit all testing data to EPA, there is no way of knowing all the tests that were carried out on leptophos. It is known, however, that safety testing included at least one two-generation reproduction study [see Ch 9_]; a 28 day feeding study in steer; a two year chronic dosing study in dogs and in rats; and a demyelination (OPIDN) study in hens. Only OPIDN study data are available. This demyelination study was carried out by Industrial Biotest, then the largest independent testing laboratory in the U.S.[4]
The OPIDN study was carefully designed, and included 2 treatments, spaced 21 days apart, for each hen. There were 6 hens at each dose level, and 3 doses. Despite the small number of animals at each dose, the study was thorough, including daily observation, repeated weighing, and measurement of the food consumption of each hen throughout the study. Tissues from the hens were also examined histologically. Shorn of verbiage, results were:
oafter 2 doses, 21 days apart, of 100 mg/kg, all hens remained normal;
oafter 2 doses of 200 mg/kg, 1 hen became permanently ataxic, one became mildly ataxic, with apparent recovery; and 3 hens exhibited only the acute effects of acetylcholinesterase inhibition.
oafter a single dose of 400 mg/kg, 4 hens of 6 became ataxic, and a 5th became ataxic after the 2nd dose of 400 mg/kg. One hen never showed any but acute effects. Four of the hens died before the end of study.[5] Histology showed no demyelination, but the histological techniques used make that meaningless.
Despite the data in body of report, the abstract that formed the first page of the IBT report to Velsicol stated that there was no evidence of delayed neurotoxicity. Velsicol forwarded the complete report, with the abstract as its first page, to EPA as part of the evidence for leptophos' registration.
Field testing of leptophos proceeded abroad while registration efforts proceeded in the U. S. Cotton, because it is a cash crop, grown intensively, and subject to numerous pests, provides a considerable challenge to insecticides. This is especially true in tropical climates, where several crops a year are grown and there is no cold period to decrease insect numbers. Resistance to insecticides occurs rapidly among pests of cotton, and there is always a market for new chemicals.[6] In 1971, during leptophos trials in Egyptian cotton fields, an estimated 1200 water buffalo were paralyzed and died after exposure to leptophos, although the cause of the outbreak of ataxia was not immediately identified.
In October of 1971, Velsicol petitioned for registration of leptophos in the U.S. At the same time, a Velsicol factory in Bayport Texas was converted to full-scale leptophos production. Actual synthesis of the insecticide from its precursors occurred in a closed system, with minimal exposure of the workers. Once synthesized, however, the leptophos was poured into flat tins or large drums and left to cool and solidify (the melting point is 71-72oC). The cooled "cakes" were chopped into chunks with hatchets, and the chunks were dumped into an open grinder to produce the powder that could be formulated with inert ingredients to produce the commercial insecticide. Permanent workers used - but also reused - gloves, hats and safety glasses. Temporary help used no protective clothing. Workers frequently felt nauseated, which they attributed to the odor of the leptophos: actually, nausea is a typical symptom of OP poisoning.
In 1971, at the time of the registration petition, a memo was sent from the World Health Organization (WHO) to EPA, notifying EPA that leptophos was a delayed neurotoxin that should not be registered. The existence of the memo is known through references to it in other memos. Professor Robert L. Metcalf of the University of Illinois, a member of the WHO pesticide-evaluating team, was alerted to the delayed neurotoxicity of leptophos by Dr. Wendell Kilgore of California, who knew about the water buffalo. EPA took no official action. However, the WHO memo may well have been the reason Dr. Ronald Baron of EPA undertook OPIDN testing of leptophos. In 1973, Baron of EPA notified the Toxic Chemicals Branch of EPA that leptophos was a TOCPlike neurotoxin; that it caused demyelination in hens, and that he was planning further studies. As far as is known, those further studies were never done, or at least never reported.
In the same year, Velsicol requested crop tolerances for leptophos of 10 ppm on lettuce and of 2 ppm on tomatoes. Such tolerances are the legal maximum residues of a pesticide that can be present in a food crop for the crop to be sold. At their best, tolerances represent a balance between the persistence of a chemical and its hazard: more persistent pesticides need higher tolerance of residue levels, but these can be granted only if the residues do not present a hazard. Leptophos is a relatively persistent OP, as Velsicol obviously knew. We now know that the residue levels they proposed might have led to observable nerve damage in consumers; Velsicol probably did not know that. They did know, however, that more water buffalo died in Egypt during 1973. Certainly the information was spreading among pesticide toxicologists, and also between companies.
In 1974 the first published data on the delayed neurotoxicity of leptophos appeared in Experientia.[7] The study also included negative results for two other insecticides that had been suggested as culprits in the water buffalo deaths. Kilgore meanwhile reported his information about the paralyzed water buffalo to Dr. Gunter Zweig of EPA, including a report on controlled feeding studies in which water-buffalos were fed leptophos-treated forage, proving that leptophos was actually responsible for the paralysis. A conference was then held between Zweig (EPA), Kilgore (U. California, Davis), and Clara Williams (Head of the EPA Toxicology Branch). Williams concluded, after the conference, that EPA should approve Velsicol's requested tolerances. She wrote that:
"If sometime in the future we get data which...lead us to feel that these tolerances should be revoked, we will take action at that time."
She considered the feeding studies in water buffalo to be "inadequate...no convincing evidence...".
Several memos were sent in 1974 and remained in the files, including one from Metcalf at the University of Illinois to Dr. Donna Kuroda of EPA, warning that leptophos poses a hazard if exported to Indonesia; and a second from Dr. J. Barnes at WHO to EPA, warning that leptophos had proven to be a delayed neurotoxin. As a result of the pressure from these world-renowned pesticide toxicologists, a memo from the EPA Coordination Branch concluded that Zweig and Kuroda were worried because Metcalf and Kilgore were worried, and that Metcalf and Kilgore were worried because Barnes was worried. The memo's authors concluded, however, that, although there was evidence for ataxia in both hens and water buffalo, there was no evidence of "permanent" nerve damage because there was no histological confirmation of axonal degeneration.[8] A recommendation of possibly restricted use was made, and close review of data suggested.
In another memo, Metcalf reported "rumors" of human nerve damage to EPA.[9] EPA's Williams dismissed the new information with a reiteration of the "anecdotal" nature of the information and referred to the possibility that another insecticide, not leptophos, was responsible for the water buffalo deaths. As a result of the slow but definite progress of leptophos through the regulatory maze, European chemical companies became aware of the plausibility of marketing OPIDN-inducing insecticides. CIBA management asked their toxicologist why CIBA refused to market its OPIDN-inducing chemicals when the Americans obviously did not care. Calo, chief toxicologist for Velsicol, went to Egypt and sent back reports claiming that he could not confirm the water buffalo rumors. Velsicol even suggested that the incident was the result of "industrial espionage". At EPA, Baron sent a four page internal memo to Kuroda, saying [among much else] that "although the test did show toxicity at high level after 2nd dosing, the predominant 'tunnel vision' .... suggests that demyelination was not a problem ...."
In this memo he also claimed leptophos caused no demyelination in hens, despite his own earlier data showing demyelination.
In 1975, new tolerances, lower than those originally requested by Velsicol, were published in the Federal Register. Although not synonymous with registration, the publication of such tolerance levels usually precedes actual registration by a very short period. Metcalf says of that period that whenever he or other concerned toxicologists attempted to delay leptophos registration, they only seemed to speed it up. Nonetheless, time was actually running out for leptophos.
It was in 1975 that an internal Velsicol memo about the Bayport plant that produced leptophos described a "series of unusual CNS illnesses... among employees at this plant..." The symptoms included weakness, ataxia, bizarre behavior, central nervous system symptoms, impotence, nervousness, wheezing and choking. A number of workers had gone to private doctors, and the diagnoses included asthma, multiple sclerosis, encephalitis, psychosis, and alcoholism. A more accurate diagnosis was provided by the other workers in the Bayport factory, who referred to the affected men as "Phosvel zombies". The workers, unlike the private doctors, saw more than one case at a time, and recognized the occupational link.[10]
Despite the internal memo, and in the same month, Velsicol requested a National Academy of Sciences Panel to adjudicate leptophos registration, complaining that EPA was too slow. Almost simultaneously, a prestigious consulting firm, hired by Velsicol to look at the functioning of the Bayport plant, warned of problems in the factory, and recommended changes in production with the goal of decreasing worker exposure to chemicals. In November of 1975, Velsicol informed EPA that there were illnesses in plant.