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Dermal absorption of a dilute aqueous solution of malathion

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Title:
Dermal absorption of a dilute aqueous solution of malathion
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English
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Scharf, John E
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Malathion -- analysis   ( mesh )
medfly
malaoxon
organophosphates
swimming pool
toxic exposure
Dissertations, Academic -- Public Health -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Malathion is a commonly used organophosphate pesticide on field crops, fruits, nut trees, vegetables, livestock, agricultural premises, and land. The approved uses also include mosquito and medfly control. These uses can result in human skin contact. The purpose of this study is to evaluate the human skin absorption of malathion for the purpose of assessing the risks associated with aqueous solution exposures following applications. Aerial applications can result in solubilized malathion in swimming pools and other waters that may be contacted. Human volunteers were selected and exposed to aqueous solutions of malathion at various concentrations. Participants submerged their arms and hands in twenty liters of dilute malathion solution in either a stagnant or stirred environment. The "disappearance method" was applied by measuring malathion concentrations in the water before and after human subject exposure to the water for various periods of time. Malathion was measured using Gas Chromatography. No measurable skin absorption was detected in 42% of the participants. Measurable skin absorption among the remaining 58% of participants resulted in doses that were more than an order of magnitude less than the minimal dose necessary to cause a measurable change in red blood cell acetylcholinesterase (RBC-AChE). Extrapolation of these results to a mathematical model for recreational swimmers and bathers exposed to contaminated swimming pools and surface waters typically detected after bait application again are an order of magnitude below the doses needed to cause a detectable change in RBC-AChE. These data indicate that exposure to aqueous malathion following usual aerial bait applications is not appreciably absorbed, and therefore, it is not a public health hazard.
Thesis:
Thesis (M.S.P.H.)--University of South Florida, 2003.
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Includes bibliographical references.
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by John E Scharf.
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Title from PDF of title page.

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Dermal Absorption Of A Dilute Aqueous Solution Of Malathion By John E. Scharf, MD A thesis submitted in partial fulfillment Of the requirements for the degree of Master of Science College of Public Health University of South Florida Major Professor: Raymond D. Harbison, Ph.D. Thomas E. Bernard, Ph.D. Stuart M. Brooks, M.D. Date of Approval: July 11, 2003 Keywords: Organophosphates, Malaoxon, Medfly, Toxic exposure, Swimming pool Copyright 2003, John E. Scharf

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i Table of Contents List of Tables iii List of Figures v Abstract vi Chapter One: Organophosphate Pesticides 1 Introduction 1 Physical Properties 2 Organophosphate 2 Malathion 3 Malaoxon 4 Isomalathion 5 O,O,S-trimethyl phosphorothioate 5 Diethyl Fumarate 5 Parathion 6 Chlorpyrifos 7 Methamidophos 8 Exposure Pathways 9 Biologic Fate 10 Physiologic Effects 12 Biologic Indicators 12 Demyelination and Phospholipid Peroxidation 13 Delayed Neuropathy 14 Racemic Isomalathion 19 Chapter Two: Skin Absorption 21 Introduction 21 Stratum Corneum 21 Fick’s Law 23 Rate of Absorption 26 Total Immersion 27 Vertebrate Models 30

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ii Chapter Three: Medfly Eradication Exposure Data 31 Background 31 Technical Malathion 32 Environmental Fate 33 Soil 34 Water 34 Air 35 Hillsborough County, Florida, 1997 35 Dade, Lake, Marion, Manatee a nd Highland County, Florida, 1998 37 Los Angeles, California, 1990 38 Chapter Four: Hazard Index 40 Chapter Five: Significance of Research 43 Chapter Six: Methods 45 Background 45 Design 45 Measurements 45 Subjects 45 Test Solution 46 Method 46 Example Calculation 48 Chapter Seven: Volunteer Safety 49 Screening of Volunteer Subjects 49 Medical Observation 49 Hazard Index Model 50 Biological Exposure Indices 50 Dermal Exposure Indices 51 Theoretical Mathematical Exposure Model 51 Over-the-counter Exposure Model 52 Carcinogen Status 52 Chapter Eight: Results 53 Chapter Nine: Discussion 62 Chapter Ten: Conclusion 66 References 67

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iii List of Tables Table 1 Physical Properties and Partition Coeff. (K) of Malathion vs. Malaoxon. 9 Table 2 Organophosphates, OPIDN, a nd AChE / NTE Inhibition Ratio. 16 Table 3 Reference Permeability Constants (Kp). 24 Table 4 Malathion Anatomical Dermal Ab sorption Rates and Ratios in Humans. 25 Table 5 Body Surface Area by Relative Absorption. 26 Table 6 Pesticide Rates of Dermal Absorption in Mice. 27 Table 7 Skin Absorption Rates of Chloroform in 500 ppb Aqueous Solution. 28 Table 8 Technical Malathion. 33 Table 9 Tampa Swimming pool sampling by FL-EPC (ppb). 36 Table 10 Tampa Pond sampling by FL-EPC (ppb). 37 Table 11 Medfly Spray-related Adverse Ev ents, Florida Department of Health. 38 Table 12 Los Angeles Swimming pool sampling by CA-EPA (ppb). 39 Table 13 Los Angeles Pond sampling by CA-EPA (ppb). 39 Table 14 Los Angeles Storm-wa ter runoff by CA-EPA (ppb). 39 Table 15 Parameters for Kp = Dd*Mb / (Dc*De*Fs*As). 48 Table 16 Parameters for Dd = Kp*Dc*De*Fs*As / Mb. 48 Table 17 General Experimental Results. 53 Table 18 Dermal Absorption by Volunteer 54 Table 19 Dermal Absorption by Dosage 54 Table 20 Dermal Absorption by Volunteer 55

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iv Table 21 Dermal Absorption by Dosage 55 Table 22 Hand / Forearm Exposure in Aquarium; Nominal Contamination: Mala thion=50 ppb + Malaoxon=0 ppb; Disappearance Method: Solution Concen trations. 56 Table 23 Hand / Forearm Exposure in Aquarium; Nominal Contamination: Mala thion=50 ppb + Malaoxon=0 ppb; Disappearance Method: Experime ntal determination of Kpmalathion. 57 Table 24 Swimming Pool M odel using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=0 ppb; Adult Swimmer immersed for thr ee hours. 58 Table 25 Theoretical Swimming P ool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=0 ppb; Child Swimmer immersed for three hours. 59 Table 26 Theoretical Swimming P ool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=30 ppb; Adult Swimmer immersed for thr ee hours. 60 Table 27 Theoretical Swimming P ool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=30 ppb; Child Swimmer immersed for three hours. 61

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v List of Figures Figure 1. Organophosphate. 2 Figure 2. Malathion. 3 Figure 3. Malaoxon. 4 Figure 4. Isomalathion. 5 Figure 5. O,O,S-trimethyl phosphorothioate. 5 Figure 6. Diethyl Fumarate. 5 Figure 7. Parathion. 6 Figure 8. Chlorpyrifos. 7 Figure 9. Methamidophos. 8

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vi Dermal Absorption of a Dilute Aqueous Solution of Malathion John E. Scharf, MD ABSTRACT Malathion is a commonly used organophosphate pe sticide on field crops, fruits, nut trees, vegetables, livestock, agricultural premises and land. The approved uses also include mosquito and medfly control. These uses can result in human skin contact. The purpose of this study is to evaluate the human skin absorption of malathion for the purpose of assessing the risks associated with aqueous solution exposures following applications. Aerial applications can result in solubi lized malathion in sw imming pools and other waters that may be contacted. Human voluntee rs were selected and exposed to aqueous solutions of malathion at vari ous concentrations. Participan ts submerged their arms and hands in twenty liters of dilute malathion solution in either a stagnant or stirred environment. The “disappearance met hod” was applied by measuring malathion concentrations in the water before and af ter human subject expos ure to the water for various periods of time. Malathion was measured using Gas Chromatography. No measurable skin absorption was detected in 42% of the participants. Measurable skin absorption among the remaining 58% of particip ants resulted in doses that were more than an order of magnitude less than the minimal dose necessary to cause a measurable change in red blood cell acetylc holinesterase (RBC-AChE). Extr apolation of these results to a mathematical model for recreational swi mmers and bathers expos ed to contaminated swimming pools and surface waters typically detected after bait application again are an order of magnitude below the doses needed to cause a detectable change in RBC-AChE. These data indicate that exposure to aque ous malathion following usual aerial bait applications is not appreciab ly absorbed, and therefore, it is not a public health hazard.

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1 Chapter One Organophosphate Pesticides Introduction The use of organophosphate compounds as insecticides began in the 1930s and has increased markedly since many organoc hlorine insecticides were banned in the 1970s. In contrast to organochlorine insectic ides, organophosphate insecticides degrade rapidly in the environment and do not accumula te or concentrate in the food chain. Thus, organophosphates have less potential for chr onic health effects or environmental contamination than do organochlorines and pos e less risk to consumers of food products. However, organophosphate compounds have a gr eater potential for acute toxicity in humans than do chlorinated compounds. Ev en among the organophosphate pesticides, however, a wide spectrum of potency exists. As insects de velop greater resistance, the trend is to use more potent, and conseque ntly, more lipid-solubl e and longer-lasting insecticides. Malathion has a generally low mammalian toxic ity in spite of its strong insecticidal properties. Malathion itself has little or no cholinesterase ac tivity. Like many other organophosphates, malathion is activated by mono-oxygenase attack to produce the potent anticholinesterase malaoxon. Malathi on and malaoxon are rapi dly detoxified in mammals by carboxylesterase atta ck (but not insects) to produce their respective monacids. If the carboxyleste rase detoxification pathway is inhibited, mammals may be made almost as sensitive as insects to malathion (Caldwell, 1983). Malathion is metabolized in human liver by one of two pathways: (1) hydrolysis to nontoxic metabolites; or (2) oxidation to the active metabolite, malaoxon. Malaoxon has 68x the inhibition of plasma cho linesterase activity as malathion. Furthermore, malaoxon is

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2 generally acknowledged to be the actual inhibito r of plasma cholinesterase and to be the primary compound responsible for the full spectrum of cholinergic symptoms observed in poisoned subjects. Normally, the balance of me tabolic reactions in the gut is heavily in favor of oxidation to malaoxon in insects, but favors hydrolys is to non-toxic metabolites in humans; thus the selective toxicity of malathion on insects vs. humans. Physical Properties In organophosphate molecules, R1 and R2 are usually alkyl groups (typically ethyl or methyl). X is commonly an alkyl group that is replaced by a hydrogen atom during irreversible inhibition, or “ag ing”, of the organophosphate-enzyme complex. O (or S) || R1O---P---OR2 | O---X Figure 1: Organophosphate.

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3 S || CH3O---P---OCH3 | S---CHCOOC2H5 | CH2COOC2H5 Figure 2: Malathion. € Appearance: Clear, amber liquid with penetrating garlic odor. € Chemical Name: diethyl (dimethoxy thiophosphorylthio) succinate € CAS Number: 121-75-5 € Molecular Weight: 330.36 € Water Solubility: 130 mg/L € Solubility in Other Solvents: very soluble € Melting Point: 2.85 C € Vapor Pressure: 5.5 x 10-6 mmHg at 20 C € Log Kow Partition Coefficient: 2.75 € Specific Gravity: 1.23

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4 O || CH3O---P---OCH3 | S---CHCOOC2H5 | CH2COOC2H5 Figure 3: Malaoxon. € Appearance: Colorless, oily liquid. € Chemical Name: diethyl ((dimet hoxy phosphinyl) thio) butanedioate € CAS Number: 1634-78-2 € Molecular Weight: 314.36 € Water Solubility: 209 mg/L € Soluble in ethanol, methanol and acetone. € Melting Point: < 20 C € Vapor Pressure: 9.8 x 10-6 mmHg € Log Kow Partition Coefficient: 2.02 € Specific Gravity: 1.23

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5 O || CH3S---P---OCH3 | S---CHCOOC2H5 | CH2COOC2H5 Figure 4: Isomalathion. O || CH3O---P---OCH3 | Figure 5: O,O,S-trimethyl phosphorothioate. CHCOOC2H5 | | CH2COOC2H5 Figure 6: Diethyl Fumarate.

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6 S || CH3CH2O---P---OCH2CH3 | O | Benzene – NO2 Figure 7: Parathion. € Appearance: Deep brown to yellow liquid w ith a faint odor of garlic. € Chemical Name: O,O-diethyl O-4-nitrophe nyl phosphorothioate € CAS Number: 56-38-2 € Molecular Weight: 291.3 € Water Solubility: 24 mg/l € Soluble in alcohols, oils, ethers. € Melting Point: 6 C € Vapor Pressure: 1.9 x 10-5 mm Hg € Log Kow Partition Coefficient: 3.83

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7 Figure 8: Chlorpyrifos. € Appearance: Amber to white crystalline solid with a mild sulfur odor. € Chemical Name: O,O-diethyl O-3,5,6-trichloro2-pyridyl phosphorothioate € CAS Number: 2921-88-2 € Molecular Weight: 350.62 € Water Solubility: 2 mg/L € Soluble in benzene, acetone, chloroform, ethers. € Melting Point: 44 C € Vapor Pressure: 2.5 mPa € Log Kow Partition Coefficient: 4.70

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8 O || CH3S---P---OCH3 | Figure 9: Methamidophos. € Appearance: Crystalline solid, with off-white color and pungent odor. € Chemical Name: O,S-dimethyl phosphora-midothiolate € CAS Number: 10265-92-6 € Molecular Weight: 141.12 € Water Solubility: 90 g/L € Solubility in solvents unavailable. € Melting Point: 44 C € Vapor Pressure: 3 x 10-4 mmHg € Log Kow Partition Coefficient: NA (Extoxnet, 2003).

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9 Table 1: Physical Properties and Partition Coefficients (K) of Malathion vs. Malaoxon. System Malathion Malaoxon Lipid Solubility More soluble Less soluble Log Kow 2.75 2.02 Log K Chloroform-water 64 5.8 Log K Hexane-water 19 0.42 Log K Petroleum ether-ethanol 11 1.8 Log K CCl4-water 39 2.9 Cuticle Penetration Better Worse Alkaline Hydrolysis Stability More stable Less stable AntiAChE activity Poor Good I50 Inhibit AChE Conc. (M) 1 x 10-4 4 x 10-7 Oxidation of malathion to malaoxon greatly incr eases partitioning into polar solvents, but less likely to penetrate skin (O’Brien, 1957) (Miller, 1998). Exposure Pathways The site of introduction of a xenobiotic into the organism qualitatively governs the initia l exposure. An orally admi nistered compound has to traverse the intestines, the liver, and the lung before it reaches the sy stemic circulation. Due to this ‘first-pass effect ’, the amount that reaches the systemic circulation is much less than the amount administere d. In contrast, this ‘first-pas s effect’ is b ypassed through dermal absorption. The entire amount systemi cally absorbed through the skin is passed through the lungs by the venous blood before it is retuned to the heart and distributed by arterial blood to the vital organs (Caldwell, 1983). Most exposures to organophosphates occur fr om skin absorption. Skin absorption can occur when dermal contact is made during ha ndling and application, and when contact is made with chemical residues on plants, fruits and foliage; in soil; and in dust particles after spraying. Organophosphate insecticides such as malathion are applied in the sulfurcontaining (-thion) form, but readily undergo desulfuration to form a more toxic oxygen-

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10 containing (-oxon) form. In the field, this c onversion occurs slowly under the influence of oxygen and light, producing residues with altered physical prope rties and potentially more toxic as well. Biologic Fate Skin absorption of malathion is in complete because much of the applied dose is lost from the skin surface by washi ng, evaporation and gradual exfoliation of the outer layers of the stratum corneum. The amount absorbed into the body depends on a relationship between the speed w ith which it penetrates the sk in and the speed with which it is lost from the skin surface. Importantly, systemic absorption afte r skin application can be delayed, and the resultant absorption rate across skin is much slower than across mucous membranes. Typically, experimental s ubjects differ by a factor of five in the amount of malathion percutaneous penetration, with standard deviations up to one half the mean value. Thus, assuming a normal distribution, one person in ten will absorb twice the mean value, while one in twenty will absorb three times that amount. Dary exposed human forearms to 1% malathion aqueous solution, placing an occlusive patch over the exposure site after 4 hours of observation. This occlusive patch was removed at 24 hours, analysis of which reveal ed 65% of the applied dose. This amount in the occlusive patch may represent evaporative and mechanical loss potential. Preliminary mass balance over the initial 24-48 hour post-exposure period was as follows: € 70% of initial applied dose removed by washing skin surface-epidermis; € 40% of the remaining dose (12% of initial dose) slowly lost through evaporation; € 60% of the remaining dose (18% of initial dose) retained in dermis; € 90% of the dermal to systemic dose is eliminated in urine; € 15% of the initial applied dose is systemically absorbed and eliminated in urine. The slow rate of dermal absorption and efficient rate of elimination by liver carboxylesterases and kidneys reduce the risk of acute toxicity; however, repeated exposures could burden these same organs of metabolism and elimination (Dary, 1994). Although the skin is often consid ered as merely acting as a pa ssive barrier to the entrance of chemicals, it is clearly capable of me tabolizing endogenous substances as well as

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11 drugs and other exogenous chemicals. Despite the low activity of metabolic enzymes in skin, it must be appreciated that skin is the largest organ in the body, comprising 5% of total body weight. Thus, the skin can contribute significan tly to total metabolism of chemicals. For example, skin is well known to contain cytochrome P-450, NADPHcytochrome c-reductase and glutathione Stransferase. Cutaneous enzymes with monooxygenase activities such as these can be induced by a variety of xenobiotics. Immunohistochemical investigations have doc umented the epidermis, sebaceous glands, and hair follicles as the sites of xenobiotic activation and detoxificat ion within the skin (Caldwell, 1983). Parathion, a closely related organophosphate to malathion, has demonstrated unusual percutaneous absorption kineti cs and systemic toxicity patterns. For example, in an isolated perfused porcine skin flap model of parathion, almost 70% of the parent compound parathion penetrated the skin into the perfusate as paraoxon. Thus, biotransformation of topically dosed orga nophosphates by skin-based cytochrome p450 enzymes to the more toxic –oxon form may play a crucial role in risk assessment (Carver 1988) (Kao, 1990). In the mammal, vigorous hepatic hydrolytic degradation of organophosphates at the carboxylic ester link typically outstrips the process of accumulation caused by oxidation. Hepatic conversion is brought a bout chiefly by microsomal esterases. This hydrolyzing system of the liver is more active against malathion than malaoxon; ultimately, however, both –thion and –oxon forms are metabolized to non-toxic alkyl phosphate mono-acids and excreted rapidly. In the rat model, afte r oral malathion ingestion, twelve hour urine samples contained 1% of the dose as ma laoxon mono-acid and 67% as malathion acids (Kizer, 1991). On the other hand, in th e cockroach insect, both fat body and midgut metabolic systems oxidize malathion to malaoxon, but only the fat body hydrolyzes malaoxon to the non-toxic alkyl phosphate. In the insect, then, oxida tion is much more rapid than hydrolysis, thus malaoxon may accumulate steadily to a lethal level (O’Brien, 1957).

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12 Physiologic Effects Organophosphates combine with and inhibit cholinesterase enzymes, of which acetylcholinesterase (ACh E) in nerve tissue is the most important. After conversion of malathion to its oxygen analog, malaoxon, the activ e site of AChE is phosphorylated (and inhibited) by dimethyl or methyl phosphate. In activation of AChE results in accumulation of acetylcholine at th e neuroreceptor transmission site, resulting in massive overstimulation of the cholinergic system as follows: € Accumulation of acetylcholine in the mu scarinic autonomic receptors cause miosis, tearing, salivation, perspirati on, nausea, vomiting and diarrhea. € Accumulation of acetylcholine in the ni cotinic neuromuscular receptors cause fasciculations, cramps and weakness. € Rising accumulation of acetylcholine in the central nervous system cause sensory and behavioral disturbances, incoordina tion, depressed cognition, and respiratory failure. Biologic Indicators Detection of intact organophos phate compounds in blood is usually not possible. In general, organophosphates do not remain unhydrolyzed in the blood more than a few minutes. Alkyl phosphate metabolites can often be detected in the urine up to 48 hours after exposure. The appearance of these urinary metabolites can demonstrate pesticide absorption at lower dosages than those required to depr ess cholinesterase activity. Occupational malathi on exposures have resulted in dimethyl-dithiophosphate (DMDTP) serum concentrations ranging to 3.5 mg/L (Shafnik and Enos, 1969). Up to 23% of a single oral dose of malathion is excreted in the 16 hour urine as etherextractable phosphates, where bi ological activity was estimat ed via colorimetry (Mattson and Sedlak, 1960). Red Blood Cell (RBC) cholinesterase, the same enzyme found in the nervous system, is a useful surrogate indicator fo r AChE activity at neurorecep tor sites. RBC cholinesterase activity is generally only restored as new red blood cells are form ed at 1% per day. However, it is notoriously di fficult to interpret ch olinesterase inhibiti on without baseline values because normal values vary widely. The laboratory normal range is not useful because upper and lower limits typically diffe r by a factor of four RBC cholinesterase

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13 inhibition from 25-50% of the i ndividual’s baseline is generally regarded as evidence of toxicity, or the Lowest Observable Adverse Effect Level (LOAEL). In the best available No Observable A dverse Effect Level (NOAEL) study, volunteers ingested 16 mg of malathion daily for 47 days without effect on RBC Cholinesterase. Doses of 24 mg, however, when administered for 56 days caused 25% depression of RBC cholinesterase, with maximal effects occurring three weeks after administration. No clinical signs or symptoms were noted in spite of the RBC cholinesterase depression (Moeller and Rider, 1962). Demyelination and Phospholipid Peroxidation In humans and othe r vertebrates, reaction of organophosphates with neuropathy target es terase (NTE) may initiate events which culminate in axonal degeneration. Or ganophosphates inhibit biosynthesis of phospholipids which in turn may cause peroxi dation of the myelin sheath. Thus, toxic organophosphate exposure may result in de myelination-induced neurotoxicity. For example, toxic malathion exposure in rats has induced spinal cord phospholipid peroxidation (Haque, 1987). Chlorpyrifos is heavily used for agricultural and domestic purposes due to its persistence and relative safety compared to other or ganophosphate insecticid es. Unlike parathion, chlorpyrifos evokes delayed ne uropathies only with very hi gh exposures. Animal studies confirm that chlorpyrifos has an order of ma gnitude higher systemic toxicity in neonates. Perhaps more alarmingly, recent studies in rats have shown that chlorpyrifos, in doses below the threshold for symptoms of systemic toxicity, nevertheless can compromise the basic cellular processes of brain development. In the neonate, programming of brain cell reactivity and synap tic connections are at their peak and are thus most vulnerable. In the ne onate, the adverse effects of cholinesterase inhibition are exacerbated by poor synaptic ad aptability to cholinergic hyperstimulation. In a neonatal rat animal model after chlor pyrifos exposure at do ses that do not cause

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14 systemic toxicity, there was alteration in co ordination skills and lo comotor activity (Dam, 2000). In a similar neonatal animal model, chlorpyr ifos exposure during the period of brain cell acquisition and axonogenesis resulted in neuronal loss and abnormalities of synaptogenesis. The adverse effects on cho linergic neurotransmission are a particular concern since cholinergic pathwa ys are critically important for cognitive function. In fact, neonatal exposure in rats produced lastin g reductions in hippocampal cholinergic presynaptic activity. Short-term behavioral studies in this animal model indicated alternations in several activity measures, a nd additional effects on activity emerged well after the end of cholorpyrifos administration (Levin, 2001). These results (e.g. alterations in behavior and locomotion) stress the importance of looking beyond the acute period of organophos phate exposure for adverse effects. Indeed, developmental deficits may appear well after organophosphate exposure and cholinesterase inhibition have disappeared. Delayed Neuropathy Shortly after organophosphate expo sure, a weak reve rsible bond is formed with AChE. With time, however, a more permanent AChE-phosphate bond forms (“aging”) that inactivates the enzyme and requi res an antidote to break. If an antidote such as pralidoxime [2-PAM] is not given within 24-48 hours after exposure, the AChEphosphate bond becomes so strong that p hysiologic recovery depends on de-novo synthesis of AChE. At the nerve junction, AChE is restored in an average of two weeks; in the body as a whole, it may require up to th ree months to restore enzyme activity to near normal levels (Pope and Rall, 1995). Symmetric distal axonal de generation is a systemic health effect caused by some organophosphates that is not due to AChE inhi bition. The degeneration is a dying back of large diameter axons and their my elin sheaths in distal parts of the peripheral nerves and in long spinal cord tracts. This degeneration is caused by inhibition of a neuronal, nonspecific carboxylesterase known as neuropathic target esterase (NTE), essential for

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15 lipid metabolism in neurons. The resulting clinical syndrome, organophosphate-induced distal neuropathy (OPIDN), typically begins one to two weeks after exposure, and consists of flaccidity, paralysis and paresthesias of the lower extremities. All organophosphates inhibit ace tylcholinesterase (AChE), but only a subset of these inhibit neuropathy target esterase (NTE), the enzyme definitively associated with OPIDN. In essence, a sufficient amount of NTE must be irreversibly inhibited by the organophosphate before the OPIDN syndrome may occur. In vitro (n euroblastoma cells) and in vivo (hen, cat, and sheep) testing ha ve established organophosphate-specific NTE / AChE inhibition ratios, and correlated this ra tio precisely with the ability to produce the OPIDN syndrome. Previous work has shown th at if AChE / NTE Inhibition Ratio >> 1, the dose required for producing OPIDN >> LD 50. Accordingly, malathion, parathion, chlorpyrifos, dichlorvos and trichlor fon have not been linked to OPIDN.

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16 Table 2: Organophosphates, OPIDN, and AChE / NTE Inhibition Ratio. Organophosphates linked to OPIDN: AChE / NTE Inhibition Ratio: € DFP [diisopropyl fluorophosphonate] 5.3 € TSP [cyclic tolyl saligenim phosphate] 1.5 € PSP [cyclic phenyl saligenim phosphate] 0.12 € Mipafox 1.1 € DBVP 0.81 € DOVP 0.56 € EPN [ethyl nitrophenyl phenylphosphonate] € Leptophos € Merphos € Tri-ortho-cresyl phosphate [TOCP] € Tri-ortho-tolyl phosphate [TOTP] € Triaryl phosphate € Methamidophos Organophosphates not linked to OPIDN: AChE / NTE Inhibition Ratio: € Parathion 700,000 € Malathion 76,000 € Isomalathion (1R, 3R) 49000 € Isomalathion (1R, 3S) 150000 € Isomalathion (1RS, 3RS) 9100 € Isomalathion (1S, 3R) 2500 € Isomalathion (1S, 3S) 1500 € Chlorpyrifos 200 € Dichlorvos [DDVP] 42 € Trichlorfon 68 (Rosenstock, 1991) (Jianmongkol, 1996) (Ehrich, 1997).

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17 Although there are no known case reports of OP IDN specifically lin ked to malathion, malathion exposure has been listed at least as a contributory factor in a significant OPIDN case series examination. In this seri es, exposed patients had moderate increases in vibrotactile thresholds in the lower extrem ities. These results suggest that previously reported OPIDN cases may represent only the worst disease in a spectrum of polyneuropathy impairment, a sequelae of or ganophosphate exposure that may be much more common than previously thought (McConnell and Rosenstock, 1994). To evaluate the latent neurological effects of organophosphate poisoning, an epidemiological study matched 100 pairs of individuals with previous acute organophosphate poisoning and non-poisoned controls. Ten different organophosphates were listed as the cause of primary poisoni ng, most being parath ion, but of which six were due to malathion. In the neurologica l examination component of the study, clear abnormalities were demonstrated in the organophosphate exposed group in memory, abstraction, mood, motor reflexes and si mple motor skills. In the psychological examination component, clear abnormalities were demonstrated in intellectual functioning, academic skills, abstraction and flexibility in thinking. Minnesota Multiphasic Personality Inventory analysis re vealed greater distress and complaints of disability for the poisoned subjects. Twice as many cases as controls had Halstead-Reitan Battery summary scores in the range characteris tic of individuals with cerebral damage or dysfunction (Savage EP, 1988). Delayed polyneuropathy (DP) is typically caused by organophosphate induced inhibition of NTE, the enzyme essential for norma l neuronal and axonal processes. DP is characterized as a distal sensori-motor axonopathy, beginning about two weeks after toxic exposure, with recovery beginning six to twelve months later. The best available case report of malathion-induced delayed polyneuropathy (DP) occurred in 1990 as follows: A 65yo female was admitted to the hospital in cholinergic crises with muscarinic, nicotinic and central eff ects after deliberate i ngestion of 100 ml of malathion (235 mg/kg)

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18 dissolved in isoproponal. Artific ial ventilation, atropine an d pralidoxime were initiated immediately. The patient was found to have complete and total depression of serum cholinesterase. The patient regained conscious ness and strength a few days later, and was noted to have complete normalization of serum cholinesterase on day 12. Her hospitalization was further complicated by pneumonia and acute respiratory distress syndrome. At day 10, her muscle strength bega n to deteriorate agai n with loss of deep tendon reflexes. Distal muscles were more involved than proximal ones. On day 35, a gastrocnemius muscle biopsy showed eviden ce of denervation and reinervation. A sural nerve biopsy showed degenerating axons, moderate loss of myelinated fibers and clusters of small regenerating axons. Six weeks after admission, muscle streng th began to recover and was normalized after three months. This patient was found to have exposure to a significant pesticide residue as the key DP causative factor: isopropyl-malathion (Argiles, 1990). Organophosphate intoxication will cause chol inergic stimulation acutely, but delayed neuropsychiatric sequelae may also result in a subacute paralytic syndrome known as the intermediate syndrome. Intermediate syndr ome is characterized by deterioration of muscle strength and mental status about 48 hours after the acute presentation. Typically weakness manifests itself within the neck and proximal muscles, which may include the cranial nerves and the diaphragm. Motor reco very may take 5-20 days and most patients will not develop polyneuropathy. Behavior al manifestations include depression, psychosis, aggression, irritability, and memo ry and concentration problems. The best available case report of malathion-induced intermediate syndrome occurred in 1998 as follows: A 44yo female was found in cholinergic crises with muscarinic, nicotinic and central effects after deliberate ingest ion of 125 ml of malathion (50% solution). Artificial ventilation, atropine and pralidoxime were in itiated immediately. Her hospitalization was further complicated by bronchorrhea requiri ng glycopyrollate and pneumonia. She was eventually transferred out of intensive care to the medical ward on day 13. On day 23, she began displaying agitation, disorientation, ha llucinations and somatic delusions. She

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19 became aggressive and inappropriate, voiding on the floor and throwing feces, and was transferred to a psychiatric ward. Her deliri um resolved over 48 hours with psychiatric treatment, and she was discharged from the hospital on day 35 (Choi, 1998). Racemic Isomalathion Commercial malathion is supplie d as a racemic (RS) mixture of the R and S forms arising from an asymmetr ic carbon in its diethyl thiosuccinate group. The corresponding steroisomers of malaoxon diffe r 8.6-fold in their inhibitory potencies against rat brain AChE. Thermal or photoc hemical isomerization may occur during storage of racemic malathion to yield r acemic isomalathion, which is 1000-fold more potent than the parent compound. Shelf storage of malathion at 38C increases both its isomalathion content and its toxicity. For example, unusually high le vels of isomalathion were linked to 2800 poisonings including 5 fatalities among 7500 Pakistani spraymen using malathion for malaria control (Baker, 1978). Contamination of malathion by isomalathion acts synergistically to increase toxicity by inhibi ting the detoxification potenti al of liver carboxylesterase enzymes. Isomalathion contains two asymmetric cente rs, one at phosphorus, and the other at the carbon in the diethyl thiosuccinate moiety, yielding four stereoisomers. Inhibitory potencies of these isomers against AChE differ by as much as 29-fold. Individual stereoisomers each form Michaelis complexes w ith this enzyme prior to the expulsion of the primary leaving group and organophosphor ylation of the active site serine. Subsequently, the inhibited enzyme could unde rgo either reactivatio n (displacement of the entire organophosphoryl group) or aging (s cission of a ligand fr om the inhibitor, leaving a negatively charge d organophosphoryl group). Aging to yield a negative char ge in the active site leaves the enzyme intractable toward reactivation. Diethyl thiosuccinate is the pr imary leaving group for R-isomalathion, while thiomethyl is the primary leaving group for S-isomalathion. This unexpected stereochemically determined switch in the inhibition mechanism potentially extends the

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20 range of toxicological conse quences arising from exposures to the isomalathions. Aging of both electric eel AChE and equine se urm butyrylcholinesterase (EBChE) by Smalathion via an SN2 reaction with P-S bond cleavage to yield an O-methyl phosphate adduct has recently been reconfirmed (Doorn, 2001). Apart from cholinergic toxicity elicite d by AChE inhibition, some organophosphate compounds inhibit and age >70% NTE, producing the OPIDN syndrome. Certain structural requirements have been elucidated for organophosphate inhi bitors of NTE. For a given primary leaving group, longer-chain dialkyl ligands on phosphorus are preferred over dimethyl or diethyl analogs, and sma ll leaving groups are favored among longerchain dialkyl-substituted organophosphates. Considering these structure activity relationships, malaoxon isomers and the isomal athions would be expected to be poor inhibitors of NTE if diethyl thiosuccinate were the primary leaving group. However, if thiomethyl were the primary leaving group, as found in the S-isomal athion configuration, then inhibition of NTE would appear to be a reasonable possibility. In view of unexplained reports of neuropa thic sequelae associated with malathion intoxication in humans, and the fact that isomalathions can arise as impurities in malathion preparations, and the fact that isom alathions are potent inhibitors of esterases other than NTE, the possibility exists that these compounds could be potentiators, rather than initiators, of neuropath ic processes (Healy, 1959) (H arell, 1978) (Rivett, 1987) (Komori, 1991) (Jianmongkol, 1996).

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21 Chapter Two Skin Absorption Introduction Interest in skin permeability arises mainly from the capacity of the skin to limit the body’s accumulation or elimination of substances by percutaneous transport. Although a complex process, dermal uptake of compounds occurs mainly through passive diffusion, involving a selective mechanism in the lipid and protein structures of the stratum corneum. This epidermal skin barrier is the major factor in this process and can reduce overall rates of accumula tion by several orders of magnitude. For example, watersoluble, low molecular weight, non-electrol ytes can diffuse into the bloodstream 1000 times more rapidly if the epidermis is diseas ed, damaged, or removed. Even with the skin intact, there are still 10,000 fold differences in the rates of penetration of different substances. Stratum Corneum The principal barrier function of the epidermis resides almost entirely in the stratum corneum, the thin coherent me mbrane of keratinized, epithelial cells that comprise the “dead” surface layer of the epid ermis. The phenomenon of percutaneous absorption is essentially one of adsorption onto the stratum corneum (10 um thickness), diffusion through it and through the viable epidermis (100 um thickness), and finally through the papillary dermis (100-200 um th ickness) and into the microcirculation. The composite skin layer is pierced by two types of diffusi on shunts: hair follicles and sweat glands. Sebaceous glands are attached to follicular walls at distances of 500 um below the surface, and for this reason their effectiveness as shunts is probably negligible. Hair follicles (40-70/cm2) and sweat glands (200-250/cm2) do act as diffusion shunts, but their role is more subtle than one mi ght think. Although the fractional area covered by

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22 these appendages is only 0.1%, diffusion cons tants of low-molecular weight molecules moving through the appendages are greater than through stratum corn eum. Before steadystate diffusion is established, and particularly during the first few minutes after the application of a substance, the follicles a nd ducts cannot always be ignored even when the ultimate steady-state flux through these shunts is small. The slow rate of malathion dermal absorption and its efficient rate of elimination reduces the risk of acute toxicity for all exposures except when involving substantial portions of unprotected skin. Water as a common vehicle may play an important role in increasing the permeability of malathion in the stratum co rneum. If penetrants are easily dissolved in their oil vehicles applied to the skin, penetration is more difficult. For example, carbon disulfide and analine are abso rbed through the skin about 100 times faster from aqueous solutions compared with oil solutions. The co mparatively high rate of skin absorption of compounds from their aqueous solu tions seems to be a rule th at could be related to the hydration of stratum corneum (Scheuplein, 1965) (Baranowska-Dutkiewicz, 1982). If the skin is hydrated, or the compound is in solution, diffu sion and penetration will be enhanced. On the other hand, stratum co rneum may be subject to compaction and dehydration when in contact with pure solven ts. In addition, the ne crotizing effects of concentrated solvents in contact with skin act to limit absorp tion. Such data indicate that skin absorption rates from dilute aqueous solutions may be seriously underestimated. The entire skin and not just the stratum co rneum can interact in important ways with solvent mixtures. One might conceptualize percutaneous absorption as a process of diffusion through the stratum corneum, follo wed by systemic uptake at the epidermaldermal junction. Furthermore, evaporation from the skin surface, reservoir formation in the dermis, and percutaneous penetration are in terrelated processes that are important in determining the fate of topically applied co mpounds. Rapid penetration into the dermis may occur with pesticides even after attempted soap and water decontamination (Reifenrath, 1991).

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23 Intercellular spaces in the stratum corneu m are normally filled and mechanically coherent. The conversion of aqueous epidermal cells into dried, compact, keratincontaining stratum corneum cells is the crucial event in the continuously developing epidermis that largely determines the low permeability of skin. The viable skin tissue layers and the capillaries are relatively permeable, and the pe ripheral circulation sufficiently rapid so that fo r the great majority of subs tances diffusion through the stratum corneum itself is rate limiting. The us ual diffusion laws of physics pertaining to passive diffusion processes can therefore be applied to skin permeability phenomena and greatly aid in its description. The rate-limiting barrier within the stratum corneum is typically the hydrated intercellular keratin. This keratin has an affinity for both water-soluble and lipid-soluble compounds. Keratin’s bi-functional solubility arises from its inherently mosaic, filament-matrix ultrastructure which allows aqueous and lipid re gions to exist separately. For this reason, attempts to predict permeability constants from correlations with oil-water partition coefficients (Kow) have proved only marginally succes sful. For larger molecules and molecules with several polar groups, the re lative importance of the stratum corneum increases still further (Scheuplein, 1971). Fick’s Law Many investigators have reported on the toxicity and unexpectedly high penetration rates of volatile organic compound s. Through relatively simple analysis of absorption from specific compounds, it is possi ble to estimate potential absorption using Fick’s Law. Dose may then be calculated using experimentally-derived skin absorption rates and constants (Kp). Fick’s Law essentially describe s the behavior of dilute aqueous solutions, such that absorption of the solute will be directly proportional to concentration. In more detail, Fick’s Law may be used to de termine the permeation rate of solvents in an aqueous solution, and is expressed by the formula:

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24 Js = Kp C Where: Js = permeation rate (flux) of th e solute expressed as mcg/cm2; Kp = permeability constant e xpressed as liters / cm2 / hr; C = concentration difference of the solute across the specified tissue in mcg/L. For example, using Fick’s Law, permeability constants for the following compounds in weak aqueous solutions were experimentally derived: Table 3: Reference Permeability Constants (Kp). Compound Aqueous Solution Permeability Constant Concentration: (mg/L) Kp (L / cm2 / hr) Ethylbenzene 112 .00100 Toluene 180 .00090 Styrene 66 .00060 Carbon Disulfide 1000 .00050 Trichloroethylene 100 .00020 Chloroform 0.5 .00012 Aniline 10000 .00004 (Dutkiewicz and Tyras, 1967, 1968). Skin absorption rates are typica lly derived from experimental situations where the hands and wrists are immersed in test solution. Rates obtained in this manner, however, will underestimate actual absorption in cases of whole-body immersion during swimming. The epidermis of the hand represents a relativ ely greater barrier to penetration than many other parts of the body, especial ly hair-follicle-rich areas. For example, the scrotum has an imperfect stratum corneum because of the enormous demands made on it in terms of flexibility, and is well known to absorb almost all of several pesticides. In addition, the face, forehead, scalp and neck absorb 2-6 times more than the forearm. Hands and feet absorb about as much as the forearm, but ab sorption is slower. Larger areas of the body,

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25 such as the back, allow greater absorpti on than the forearm (Feldmann and Maibach, 1974). Table 4: Malathion Anatomical Dermal Absorption Rates and Ratios in Humans. Ratio Rate: %Dose Recovered in Urine at 0-5 days Palm 0.9 Forearm 1.0 6.8 2.3% Foot 1.0 6.8 3.2% Abdomen 1.4 9.4 7.9% Hand, Dorsum 1.8 12.5 4.0% Hand, Palm 5.8 2.9% Scalp 3.7 Jaw Angle 3.9 Forehead 3.4 23.2 9.1% Ear 4.0 Axilla 4.2 28.7 13.7% Scrotum 11.8 Malathion concentration at 4 mcg/cm2 in a non-occlusive acetone vehicle. Subjects requested not to wash the site of application for 24 hours. (Maibach, 1971). Quick reference to a Lund and Browder Chart shows that forearm and hand surface area compared to total body surface area is 11:100, or Fs = 0.11. Adjusting this figure for Maibach’s relative absorption characteristics in different body surface areas yields an adjusted factor of 11:142, or Fsadj = .0775.

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26 Table 5: Body Surface Area by Relative Absorption. Anatomic Surface Relative Multiplication Location Area Absorption Factor Head 7% 3.7 25.9 Neck 2% 2.0 (est) 4 Ant Trunk 13% 1.4 8.9 Post Trunk 13% 1.4 (est) 8.9 Buttocks 5% 1.0 (est) ` 5 Genitalia 1% 11.8 11.8 Both Upper Arm 8% 1.0 (est) 8 Both Lower Arm 6% 1.0 6 Both Hands 5% 1.0 5 Both Thighs 19% 1.0 (est) 19 Both Lower Legs 14% 1.0 (est) 14 Both Feet 7% 1.0 7 Total 100% 142.1 (Brown, 1984). Rate of Absorption The entire skin, not just the stratum corneum or epidermis, can interact in important ways with topically applied compounds. Evapor ation from the skin surface, reservoir formation in the dermis, a nd percutaneous penetration are interrelated processes that are important in determining the fate of topically applied compounds. Rapid penetration into the dermis may o ccur with organophosphates and may account for percutaneous absorption even after one minut e of decontamination with soap and water. (Reifenrath, 1991). Percutaneous penetration rates are determ ined by the physiochemical properties of a compound such as molecular weight, solubility, charge distribution, partition coefficients, and various other parameters. Correlations are consistent for compounds having closely related chemical structures. However, meani ngful results are ofte n inconsistent where

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27 chemicals of diverse structur e are compared. The rate of dermal uptake of malathion compared to other insecticides was asse ssed in a shaven mouse skin model (upper shoulder). As shown in Table 6, and compared to the 13 other insec ticides investigated, malathion exhibited the longest hal f-life and the leas t penetration. Table 6: Pesticide Rates of Dermal Absorption in Mice. Compound: Half-life: Penetrati on: Excretory GI Carcass 1 hr 8 hr 8 hr 8 hr 8 hr Carbaryl 13 min 72% 88% 73% 8% 6% Chlordecone 41 min 54% 66% 4% 20% 25% Parathion 66 min 32% 85% 50% 10% 17% Dieldrin 72 min 34% 83% 4% 12% 62% DDT 105 min 34% 71% 4% 15% 41% Malathion 130 min 25% 67% 30% (urine) 2% 33% Penetration is disappearance of radioactivity from 4 cm2 shaved skin, upper shoulder. (Shah, 1981). It is notable that malathion dermal penetra tion rates are quite vari able in nature among different mammalian species. In Yorkshir e swine, after non-occlusive malathion application to the ventral abdominal skin, co rrected systemic absorption was only 5% at 6 days. Systemic absorption for malathion as a percentage of the original dermal dosage was less than all other compounds tested (b enzoic acid > progesterone > caffeine > testosterone > parathion > malathion) in Yo rkshire swine (Carver, 1989). In rats, after non-occlusive malathion application to 10 cm2 shaven dorsal back for 8 hours, autoradiograms demonstrated 28% of the dose concentrated at the application site, 29% in the skin at large, and 23% containe d in the urinary bladder (Saleh, 1997). Complex mathematical models for the dermal absorption of a dilute malathion solution in humans are available. A physiologically-based pharmacokinetic (PBPK) model has been created that utilizes tissue volume compar tments, air and blood flows, and chemicalspecific partition coefficients. PBPK in corporates seven body compartments (skin

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28 surface, skin perfused, fat, muscle, kidney-ve ssel rich group, intestine, and liver) and four external compartments (air, urine, feces, and acid metabolites). Skin evaporation and oxidative metabolism of malathion to malaoxon ar e incorporated into this model as well. This adult PBPK model, which notably assumes Kp-malathion = 0.001 L/cm2/hr, predicts during total immersion scenarios that 7-31% of the applied dose will be systemically absorbed. Furthermore, 70% of that system ically absorbed dose is metabolized and excreted within 24 hours (Kizer, 1991). PBPK rate constants were chosen to approxi mate experimental data. The model’s skin permeability constant (Kp = 1 x 10-5 /min to 5x10-4 /min, skin surface to viable epidermis) was less than the skin evaporation constant (Kevap = 8 x 10-5 /min to 5 x 10-4 /min, skin surface to air)). This important ratio, Kp/Kevap 1, was based upon several classic studies. Maibach demonstrated in three st udies that 4%, 8%, and 7% of [C14]-malathion applied to human forearm skin was excreted in 07 day urine samples. Peak excretion was 8 hours post-application. As shown in Table 4, up to 29% urinary recovery came from more absorptive (but smaller surface area) parts of the body (Webster and Maibach, 1983) (Feldmann and Maibach, 1974) (Maib ach, 1971). Increasing air flow over the surface of excised pig skin undoubtedly in creases evaporative loss for malathion. Significant evaporative loss for malathion occu rred from both the skin surface-epidermis layer and the dermis layer. In fact, incr eased air flow decreased malathion dermal residues but not parathion, DDT, nor DEET derm al residues. Penetration of the applied malathion dose at 48 hours in the excised pig skin model decreased from 21% to 9% as air flow was increased from 60 to 600 ml/min (Reifenrath, 1991). Total Immersion Swimming pool and bathing imme rsion models show that skin absorption represents a significant route of exposure. In fact, de rmal absorption can contribute from 29-91% of the total daily dose, for an aver age contribution of 64%. This information suggests that when doses from dilute aqueous solutio ns are calculated through Fick’s Law, and carefully considere d, margins of safety may be significantly more narrow than anticipated. On this basi s, regulatory guidelines and policies may need to be reconsidered (Brown, 1984).

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29 In another swimming pool exposure model, ch lorinated pools were found to contain an average of 156-500 mcg/L (156-500 ppb) chlo roform contamination. A worst case dermal exposure model was built utilizing Fick’s Law kinetics. Swimmers were assumed to be totally immersed for three hours daily. During immers ion, some absorption of the aqueous chloroform was presumed to o ccur through the dermal and aural routes. Accelerated dermal absorption would be exp ected from skin that has been freshly wounded, abraded or sunburned. During some of the time the swimmer was immersed, the internal tissues of the nose, mouth and eyes would also be exposed. For example, about five milliliters of water is taken into th e mouth and squirted out with each breath when a child swims. In summary, dermal absorption from the dilute aqueous solution represented 60% of the dose, as shown in the table below: Table 7: Skin Absorption Rates of Chloroform in 500 ppb Aqueous Solution. Route Relative Absorption Dermal 60% Oral 3% Buccal / Sublingual 3% Orbital / Nasal 3% Aural 3% Inhalation 28% (Beech, 1980). An improved methodology to predict in vivo Kp values for dilute aqueous solutions based upon molecular weight and oil-water partition coefficien ts was recently derived by (Bogen, 1994) as follows: Log10Kp = -0.812 – 0.0104 MW + 0.616 log10Kow cm/hr Kp = (10^(-0.812 – 0.0104 MW + 0.616 log10Kow)) / 1000 L/cm2/hr Kp Toluene = (-0.812 – 0.0104 92.1 + 0.616 2.73 ) / 1000 = .0008200 L/cm2/hr Kp Chloroform = (-0.812 – 0.0104 119.4 + 0.616 1.97 ) / 1000 = .0001400 L/cm2/hr Kp Parathion = (-0.812 – 0.0104 291.3 + 0.616 3.83 ) / 1000 = .0000300 L/cm2/hr

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30 Kp Chlorpyrifos = (-0.812 – 0.0104 350.62 + 0.616 4.70 ) / 1000 = .0000300 L/cm2/hr Kp Malathion = (-0.812 – 0.0104 330.36 + 0.616 2.75 ) / 1000 = .0000030 L/cm2/hr Kp Malaoxon = (-0.812 – 0.0104 314.36 + 0.616 2.02 ) / 1000 = .0000015 L/cm2/hr Vertebrate Models Vertebrate behavioral studies after exposure to dilute malathion solutions are effective indicators of contamination and reflect sub-lethal toxicity. Because behavior is an integrated result of endogenous and exoge nous processes, behavioral studies provide a way to addres s the question of effect in contaminant work. When using fish as a bio-indicator, it has been suggested that a stressful condition is detected when one or more physiological variab les are altered to the point where long-term survival may be impaired. Swimming behavior in fish is fr equently assessed as a response in toxicity investigations because altered locomotor ac tivity can indicate effects to the nervous system. Fish were exposed to 20-50 mcg/L (20-50 ppb) dilute aqueous malathion solutions for 24 hours. Malathion exposure caused dramatic d ecreases in distance swam and swimming speed as well as pronounced changes in comp lexity of swimming paths. A highly positive correlation was found between 26% decreased br ain cholinesterase act ivity and decreased swimming speed. Significant fish mortality wa s found when the dilute aqueous malathion solution concentration was increased to an 50 ppb exposure level and as a result caused 50% brain acetylcholinestera se inhibition (Cook 1976) (Brewer, 2001). The sensitivity of verteb rate brain cholinestera se to malaoxon is as follows: sculpin fish > chicken > sparrow > flounder > sunfish > bass > rat > mouse > monkey > duck > bullhead > guinea pig. In summary, as far as brai n cholinesterase sensi tivity is concerned, there is no clear grouping according to vertebra te class. However, sensitivity to poisoning by malathion is as follows: fish > chickens > mice. Mouse liver hydrolyzes more than ten times as much malathion as sunfish liver and about three times as much as chicken liver. Therefore, the greater susceptibility of fi sh to poisoning by malathion can best be accounted for on the basis of their relativ e inability to detoxify malaoxon (Murphy, 1968).

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31 Chapter Three Medfly Eradication Exposure Data Background Certain malathion formulations are registered by EPA for aerial spraying over urban areas in mosquito-c ontrol programs. The use of ma lathion in these programs provides an important public health benefit by controlling mosquitoes that transmit human disease (e.g. encephalitis, dengue feve r, malaria). However, the spraying of malathion bait over urban populations for Medf ly eradication has generated controversy in part because these applications are dire cted not at preventing human illness but at eradicating an agricultural pest. Federal law does not permit spraying malath ion bait over urban areas without an emergency EPA exemption. If and when the EP A grants said exemption, the responsible government authorities post advance notifica tion of spray schedules schedule malathion spraying episodes at night, and advise the public to stay indoors during spraying events and wash any skin surface that may contact the malathion bait (Shafey 1999). Aerial malathion bait may be used as a regul atory control method to establish freedom of nursery or orchard premises from living fru it fly stages, as a condition for movement of produce. To accomplish this, the establishm ent undergoes a series of treatments at intervals, designed to provide continued fr eedom from fruit flie s during the quarantine period. Bait spray applications normally are limited to locations producing regulated commodities within a quarantined area, but located outside the infested core area. Treatments must start at a sufficient time, at least 30 days, before harvest (to span the interval that normally w ould include the completion of egg, larval, and pupal

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32 development), then continue throughout the harvest period The required pre-harvest treatment makes this option useful for only those commodities remaining in the field for more than 30 days after an area is quarantined (Smith, 2001). Full foliar coverage bait spray of host trees and other plants immediately reduces fruit fly populations by 90 percent or more and reduces subsequent reproduction. This decreases fruit fly numbers in the succeeding generation and reduces the risk that gravid female fruit flies will move to uninfested areas. In this manner, the malathion bait applications reduce wild fruit fly populations to a level of infestation where mating thresholds are not achieved or where continued releases of sterile fruit flies can be effective in reducing the rest of the emerging pest population. Technical Malathion Numerous compounds are found in technical grade formulations of pesticides. These co-products occur from breakdown or conversion of the active ingredient, and from unintentional reactions occurring during pest icide synthesis. Isomalathion is one of the largest potentiators of malathion toxicity, and is equipotent to malaoxon. The in vivo mechanism of pot entiation is inhibition of B-type carboxylesterases (CEBs), the gr oup of liver enzymes responsible for the metabolism of malathion and malaoxon into their non-toxi c alpha and beta monacids. Technical malathion mixtures stored for extended peri ods of time under warm, humid conditions may contain isomalathion at concentrations as high as 7%. Each one percent increase in isomalathion content increases mammalian t oxicity by an order of magnitude (Baker, 1978). The concentration of technical malathion co-p roducts varies from ba tch to batch, but the major co-products are typically as shown in the following table.

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33 Table 8: Technical Malathion. € Isomalathion 0.20% € Malaoxon 0.10% € Diethyl fumarate 0.90% € O,O,S-trimethyl phosphorothioate [OOS(O) ] 0.04% € Diethyl methylthiosuccinate 1.00% Concentrations of isomalathion in technical mixtures are generally quite low, rarely exceeding 0.5% of the nominal malathion cont ent. This conversion to isomalathion is most prone to occur in water dispersible powders, where levels exceeding 3.0% are common. Isomerization is a function of stor age temperature, relative humidity, and storage time. Increasing relativ e humidity from 41 to 65% increased the conversion rate, as did storage for six months at 40C. The World Health Organization recommends that technical malathion mixtures exceeding 1.8% isomalathion be discar ded and not used. Trimethyl phosphorothioates [OOS(O)] are pres ent as impurities, reaction products and breakdown products in technical grade malath ion. OOS(O) is known as a potent inhibitor of cholinesterases, beta-car boxylesterases, and neuropathy ta rget esterases, especially when reaching concentrations of 0.2-1.0% in th e technical grade malathion mixture. Diethyl fumarate is known as the causat ive agent of non-immunologic contact urticaria (NICU) in humans. Skin irritation and rash es have been reported following malathionbait applications, both of which are symptomatic of NICU. (Kizer, 1991). Environmental Fate Most organophosphate insecticid es are used for crop spraying in commercial agriculture. Approximately 75% of all insecticides are used for cotton, corn and soybeans. Malathion is the most wi dely used organophosphate insecticide. Organophosphate compounds degrade in the envi ronment at varying rates: half-lives

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34 range from days to months, although generally half-lives are longest in dry climates and low temperature. Soil Malathion is of low persistence in soil with field half lives of up to 25 days. Degradation in soil is rapid and related to the degree of soil binding. Breakdown occurs by a combination of biological degradation an d non-biological reaction with water. If released to the atmosphere, malathion will br eakdown rapidly in sunlight, with a reported half-life in air of about 1.5 da ys. It is moderately bound to soil s, and is soluble in water, so it may pose a risk of groundw ater or surface water contam ination in situations which may be less conducive to breakdown. Water Malathion when dissolved in bodies of water is readily oxidized to malaoxon by a variety of mild oxidizing reagents. Thus, it is generally recognized that malathion is easily oxidized to malaoxon by swimming pool chlorine concentrations. For example, malaoxon has greatest persistenc e when pool water is acidic, and malathion is stable in oxygen saturated water at acidic pH for up to two weeks. Sun light shortens malathion and malaoxon half-lives in pools to 3 days. These data suggest that li ttle accumulation of malathion or malaoxon in swimming pools occurs but does indicate that they can persist at low levels for a considerable period of time. In river water, the half-life of malathion is generally less than one week. For example, in the Suwanee River with large amounts of tannins, malathion was 50% degraded by sunlight within 16 hours. However, malathion may remain stable in distilled water for three weeks and its photolysis half-life is 41 days. Applied at up to 6 pounds per acre in log ponds for mosquito control, it is genera lly effective for 2.5-6 weeks. In seawater, degradation increases with salinity. Breakdow n products in acidic water are monoand dicarboxylic acids such as dimethyl phopho rothionic acid and 2-mercaptodiethyl succinate (Howard, 1991) (Wolfe, 1975). Hydrolysis of malathion in basic pH aqueous solution yiel ds primary breakdown products diethyl fumarate and dimethyl phophorodith ioic acid (DMPTA). At pH 8.0, diethyl

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35 fumarate remains stable for 1.5-4 days. In the fathead minnow animal model, two-way and three-way interactions of the breakdown products caused large synergistic increases in toxicity. In partic ular, diethyl fumarate could be produced as a breakdown product in significant quantities in basic pH aqueous solutions to prod uce synergistic toxic effects with malathion in vivo (Cook, 1976). Air Malathion bait is oxidi zed rapidly in the atmosphe re by ozone and dinitrogen tetraoxide to malaoxon, but not by mol ecular oxygen. Thus, two days post-spray application the outdoor air concentrati on of malaoxon generally exceeds that of malathion. Air concentrations of malathi on also increase at two days post-spray application by volatilization of the bait droplets. Outdoor pa rticle diameter is ~100 um with particles ranging up to 350 um. Indoor particle diamet er is ~3 um and lower, evenly distributed on walls, ceilings and floors. Malathion bait found indoors is 3% of the amount found in the adjacent outdoor area (Caplan PE, 1956) (Kizer, 1991). Hillsborough County, Florida, 1997 Medfly eradication commenced via aerial malathion protein bait application on June 5, 1997. About 2.4 ounces by weight of malathion with 9.6 ounces of corn syrup protein bait was targeted to each acre in the quarantine area. About 2.6 mg/ft2 of malathion was targeted into nine square mile areas centered around each Medfly find. During th e ten week time period under study, thru August 1997, vintage military aircraft spraye d 31,000+ gallons of malathion bait over 400+ square miles of Hillsborough County landscape. One million people lived and worked in the aerial spray zone areas (Smith, 2001). This malathion bait concentration transferring from the air into a one meter deep body of water typically yields a 22 ppb dilute aqueous solution. Import antly, however, bait spray applications are made at 5-10 day intervals until er adication is achieved. Certain areas in Lakeland, for example, were sprayed at least 8 times. As a result, la rge fluctuations in concentration are known to have occurred in “Medfly hot zones”, DC-3 flight pattern overlapping zones, and bodies of wate r subject to storm-water runoff.

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36 A registry for people reporting increased hea lth problems associated with the malathion spray program was established. The “Malathion Health Survey” is a cooperative effort between the USF College of Public Hea lth, Poison Control Center, and Hillsborough County Health Department. As of 10/6/97, more than 700 calls from people reporting exposure to malathion had been received. Most reported some kind of symptoms, including nausea and headaches. The Tampa Tribune has featured possible vi ctims with adverse r eactions temporally related to the applicat ion of malathion bait: (1) A 7yo diver and swimmer had 3 days of headache, nausea and sore throat when practicing the day after a DC-3 spraye d malathion in and around Brandon Swim and Tennis Club (BSTC) pool. The swimming pool was found the day after spraying to have 12 ppb malaoxon contamination; (2) An elementary school teacher sprayed with malathion on campus had a rash; (3) A restaurant worker caught in the pa th of a DC-3 had nausea and chest pain; (4) Three children had blisters/rashes on s houlders/arms and diarrh ea after playing in a Riverview lime tree the day after malathion spraying; (5) A man’s arm had angry red rash when immersed in family pool 3 hours after malathion spraying. Only limited environmental sampling has been accomplished during the aerial application of malathion spray in Hillsborough County. The Environmental Protection Commission (EPC) of Hillsborough County ha s published a limited number of water sample results as follows: Table 9: Tampa Swimming pool sampling by FL-EPC (ppb). € Malathionrange 01; mean 0. € Malaoxonrange 1-19; mean 5. € Malaoxon at BSTC on Day 0=24 ppb; Day 1=12ppb; Day 2=5ppb; Day 3=2.5ppb. € BSTC Swim and Diving Team (n=15-20): 33-50% became ill when swimming the day after aerial applica tion of malathion bait to the swimming pool area.

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37 Table 10: Tampa Pond sampling by FL-EPC (ppb). € Malathionrange 21-79; mean 49. € Malaoxonrange 00-01; mean 01. € Malathion city water treatment plant = 0.7, and malaoxon = 0.5. € Malathion at Riverview apartment pond = 25. € Malathion at Alafia River = 12. € Malathion at six fish kill sites = 2.3-25.0. Dade, Lake, Marion, Manatee and Highland County, Florida, 1998 During the spring and summer of 1998, aerial malathion and ground diazinon application was used by federal and state agriculture authorities to er adicate Medfly infestations that had been detected in portions of five Florida counties. All insecticide application was complete on September 6 with an estimated 132,000 persons residing in the pestic ide treatment area. Reports of potential adverse he alth effects attributed to th e Medfly Eradication Program were solicited by state health and agricultu re authorities. Duri ng the Spring and Summer months, 230 reports of illness were investigat ed and classified by the Florida Department of Health as outlined in the following table.

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38 Table 11: Medfly Spray-related Adverse Ev ents, Florida Department of Health. € Probable: 34 (15%), based upon abnormal medical signs observed by a licensed health-care professional; € Possible: 89 (39%), based upon abnormal symptoms compatible with organophosphate toxicity repo rted to health-care work ers or a state health authority. € Probable plus Possible: 123 (54%), or cr ude incidence rate of 9 / 10,000 residents; € Eight reports involved children < 5yo; € Twenty reports involv ed elderly > 65yo; € Four reports involved work -related illnesses such as pesticide applicators; Of the signs and symptoms reported in the “Probable plus Possible” group: € 71% were respiratory (dyspnea, wheezing, coughing); € 63% were gastrointestinal (cramping, nausea, vomiting, diarrhea); € 60% were neurologic (headache, verti go, ataxia, paresthesia, confusion); € 23% were dermatologic (erythema, rash, pruritis); € 23% were ophthalmic (lacrimation, conjunctivitis, blurred vision). In the case reports, specific exposures were reported while: € Conducting lawn maintenance business, wh ere grass trimmings stuck to skin; € Working on roof of house; € Removing pool cover, which was folded and carried under sleeveless arm. (Shafey, 1999). Los Angeles, California, 1990 In California, more extensiv e environmental sampling has shown the amount of malathion bait actuall y sprayed on some occasions was 40-350% more than would be predicted from uniform coverage calculations:

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39 Table 12: Los Angeles Swimming pool sampling by CA-EPA (ppb). € Malathionrange 4-90; mean 18. € Malaoxonrange 0-46; mean 16. € Malaoxon was found in two pools one w eek after application at 7 ppb. Table 13: Los Angeles Pond sampling by CA-EPA (ppb). € Malathionrange 21-79; mean 49. € Malaoxonrange 00-01; mean 01. Table 14: Los Angeles Stormwater runoff by CA-EPA (ppb). € Malathionrange 0-703; mean 151. € Malaoxonrange 018; mean 2. In summary, there is solid evidence for poten tial toxic exposure to malathion and its coproducts while swimming in outdoor bodies of water during and after aerial malathion bait application. Furthermore, Los Angele s County exposure data has found swimming malathion pool contamination as high as 90 ppb. Many malathion ba it spray scenarios may converge to account for high environmental concentrations: e.g. physical-chemicalbiological reactions; over-lapping spray areas; repeat spray areas, human error; technical limitations; and weather fluctuations.

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40 Chapter Four Hazard Index The aerial application of mala thion bait results in exposure by three routes: inhalation, ingestion, and dermal absorption. Exposure may be acute, sub-acute, or chronic in nature. Exposure to malathion and/or its three si gnificant co-products, isomalathion, malaoxon, and diethyl fumarate, may occur depending upo n the specific scenario under study. The scope of this analysis will focus only upon an adult and child’s acute dermal exposure to dilute aqueous concentrations of malathi on and one of its co-products, malaoxon, while immersed in a contaminated swimming pool after a typical aerial malathion bait spray episode. In presenting exposure and dosage scenarios, the concept of reference exposure level (REL) is utilized: the level at which no advers e health effects are anticipated. Therefore, health protection is achieved if the estimated dosage is below the relevant REL. In order to make REL data easier to interpret, a Hazar d Index (HI) format is chosen, where HI = (estimated dosage / REL). Note that h azard index > 1 represents possible exposure scenarios greater than the relevant REL. Es timated dosages > REL or hazard indexes > 1 are not necessarily hazardous, and do not absolute ly result in signifi cant health effects; however, further analysis may be warranted. Several independent studies and guidelines are av ailable to define the “incipient toxicity” level for malathion exposure. In the best av ailable study, depression of cholinesterase activity was observed two weeks after the oral administration of malathion 24 mg to five human subjects for 55 consecutive days. Maxi mum depression of plasma cholinesterase activity (25%) and red blood cel l (RBC) cholinesterase ac tivity (30%) occurred three

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41 weeks after cessation of malathion. In spite of the depression in both plasma and RBC cholinesterase activity, no clinical side e ffects were observed. In summary, this report agrees with the State of CA assessment of about 0.2 mg/kg/day as the preliminary REL. The best available approach for estimating de rmal exposure to malathion in a swimming pool is the model utilized for chloroform (CF) and trichloroethylene (TCE) for baths and showers. This type model is specific for d ilute aqueous solutions of lipophilic, organic contaminants. In vitro and in vivo measurements of CF and TCE uptake by human skin from dilute aqueous solution indicate that short-term, non-steady-state, dermal uptake kinetics are consistent with a first-order partition mode l. The first-order partition coefficient estimation assumes that the stratum corneum is the effective skin compartment volume of distribution, as it likely is with most lipoph ilic, organic compounds such as malathion. Blood perfusion to skin is lik ely to cause relatively rapi d equilibrium of chemical concentrations within the epidermis, and pe rhaps within the underlying dermal tissue as well. This scenario is expected particularly in the shower, bath and swimming pool scenarios, involving relatively high water temper atures known to elicit a ten fold increase in local dermal blood perfusion rates (Bogen, 1995). For example, Bogen’s method can be utilized, with some key assumptions, to derive an exposure estimate for a child in a contam inated swimming pool model as follows: Ds = Kp*Dc*De*Fs*As*Sys / Mb Where: € Ds = systemic dose rate in mg/kg/day And:

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42 € Kp-malathion = permeability constant across the skin = 0.001 L/cm2/hr; € Dc = concentration of malathion in water = 30 mcg/L or 30 ppb; € De = duration of contact with contaminated water = 3 hr; € Fs = fraction of skin in contact with water = 1.0; € As = exposed skin surface area = 12 000 cm2; € Mb = body mass = 34.5 kg; € Sys = Dermal absorption to Systemic Absorption = 0.10. The dermal absorption to systemic absorpti on conversion factor (Sys = 0.10) is an important parameter that deserves further explanation. This factor is derived from Maibach’s three human st udies of non-occlusive concentrated dermal malathion absorption versus Dary’s contrasting occlusive study of human forearms utilizing dilute malathion aqueous solution exposures (W ebster and Maibach, 1983) (Feldmann and Maibach, 1974) (Maibach, 1971) (Dary, 1994). Dary determined that 15% of the initial applied dose is systemically absorbed and eliminated in urine, but this figure may be adjusted downward towards Maibach’s figur es (4%, 8%, and 7%) to account for the nonocclusive setting of the swimmer. That is, it is assumed that swimmers first shower with soap and water after three hours of swimming, in effect mechanically removing malathion from the skin surface-epidermis la yer, then secondly move into environmental settings conducive to dermal layer eva poration over the remainder of the day. Assuming Kp-malathion is reasonably estimated from similar Fick’s Law studies of ethylbenzene, toluene, styrene, and xylene in dilute aqueous solutions, the specific dose rate in a swimming pool with contamination of 30 ppb malathion to a 7 yo child during a 3 hour work-out is about: 0.003 mg/kg/day, or 0.1 mg total. Thus, swimming pool exposure to malathion in this model is a bout 70 times less than the REL, yielding a hazard index of 0.015. These preliminary data with key assumptions, indicate that exposure to aqueous malathion at this concentration in th is model is not appreciably absorbed, and therefore, it is not a public health hazard.

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43 Chapter Five Significance of Research It is interesting to note that many factors in a swim ming pool exposure model may actually enhance Kp over published values. For example, increased water retention in the stratum corneum layer, increased skin surf ace temperature and increased skin blood flow from exercise all tend to increase Kp. For lipid soluble substances like malathion, lowered viscosity of tissue lipids at higher temperatures reduces activation energies for diffusion. In addition, sunburned or wounded skin accelerates dermal absorption. Finally, skin sites in children appear to be mo re permeable than skin sites in adults. Most of these factors are operational in the sw imming pool model in a way that tends to increase the dermal ab sorption of malathion. Nevertheless, the main area of uncertainty in the swimming pool exposure model is within the estimate for Kp-malathion. Unfortunately, specific experimental data for Kpmalathion do not exist. Fortunately, Kp has been determined for similar compounds in dilute aqueous solutions such as ethylbenzene, styrene, and toluene, all of which were determined to have similar values to the Kp used for malathion above. Further experimental determination of Kp-malathion as used in Bogen’s derm al exposure equations is necessary before models for toxic exposures in dilute aqueous solutions of malathion while bathing are convincing. The balance of malathion and malaoxon e xposure in humans may be swayed by concomitant exposure to exogenous malaoxon. Swimmers may be exposed to malaoxon in addition to malathion as the aerial bait lands in the swimming pool and is oxidized by its chlorine to this much more potent ac tive metabolite. Let’s recalculate the dermal

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44 absorption for the 7yo swimmer, except this time assuming exposure to 30 ppb malathion + 30 ppb malaoxon. Ds = Kp*Dc*De*Fs*As *Sys/ Mb Note that the chosen concen trations of malathion and ma laoxon are within one standard deviation of the mean actual California swim ming pool data, and are both well within the UCL values. In addition, let’s use potency of malaoxon = 68 malathion. € Kpmalathion = permeability constant across the skin = 0.0010 L/cm2/hr; € Kpmalaoxon = permeability constant across the skin = 0.0005 L/cm2/hr; € Dc = concentration of malathion in water = 30 mcg/L or 30 ppb; € Dc = concentration of malaoxon in water = 30 mcg/L or 30 ppb; € De = duration of contact with contaminated water = 3 hr; € Fs = fraction of skin in c ontact with water = 1.0; € As = exposed skin surface area = 12 000 cm2; € Mb = body mass = 34.5 kg; € Sys = Dermal absorption to Systemic absorption = 0.10. Assuming Kp-malathion and Kp-malaoxon are reasonable, the specific dose rate in a swimming pool with contamination of 30 ppb malathion and 30 ppb malaoxon to a 7 yo child during a 3 hour work-out is about: 0.11 mg/kg/day, or about 3.8 mg total. Important assumptions in this first pass analysis include swimmers first showering with soap and water after three hours of swimming, in effect mechanically removi ng malathion and malaoxon from the skin surface-epidermis layer, then sec ondly moving into environmental settings conducive to dermal layer malathion and mala oxon evaporation over the remainder of the day. This swimming pool model dosage for malathion + malaoxon is about half the adjusted REL, yielding HI = 0.5. Therefore, by definition, this dose may be sufficiently close enough to HI = 1 to cause a specific ailm ent or disease related to malathion toxicity, and further analysis is warranted.

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45 Chapter Six Methods Background : Historical studies have measured hu man dermal uptake of related aromatic chemicals from aqueous solutions, using the “disappearance method” in concert with the analysis of excreted metabolites. In all th e studies that examined uptake from aqueous solutions using the disappearance method, both whole hands of each person was immersed for two hours in a one -liter beaker. For example, disappearance measurements of aqueous ethylbenzene at 125 ppm were compared to th e absorbed amount computed by utilizing the urine metabolite mandelic aci d. Chemical flux into sk in was validated as the difference in chemical mass contained in the exposure solution at the beginning and end of the exposure period, divided by the corresponding dermal surface area (Dutkiewicz and Tyras, 1967). Design : Prospective, controlled, and limited de rmal exposure of hands and forearms of human volunteers to dilute aque ous solutions of 50-ppb (mcg/l) malathion in a laboratory setting. Measurements : EPA-approved, commercial assays to 0.5-ppb (mcg/l) malathion will be utilized in order to measure the concentrati ons in water before and after hand and forearm exposure. Subjects : Twenty volunteers

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46 Test Solution : Twenty liter tanks were prepared w ith dilute aqueous so lutions of 50-ppb (mcg/l) malathion. The solution was prepar ed by placing 1 mg of laboratory grade malathion (Supelco PS86, Malathion, N eat) into the dist illed water bath. A Standard Operating Procedure (SOP) wa s carefully developed, documented and verified to ensure volunteer subject safety. Se veral quality assurance based trial runs were undertaken prior to the first vol unteer subject exposure period During each trial run, each twenty liter tank prepared under the SOP was verified to contain a 50-ppb malathion concentration via an EPA-approved, commer cial assay sensitive to 0.5 ppb. During the quality control evaluation period, the tank malath ion concentrations were verified to be within 30% (15 ppb) of the nominal ta rget concentration (50 ppb or 50 mcg/L). The malathion exposure tank was stagnant, hand -stirred, and then mechanically stirred during three different stirring exposure phases of the protocol. The tanks were at room temperature (21 C) and swimming pool temperature (29 C or 90 F) during two different temperature exposure phases of the protocol as well. Volunteer exposure time was gradually increased during several different phases of the protocol from 30 minutes to 120 minutes. All upward adjustments in exposure level were carefully made in series to ensure maximum volunteer safety. Method : Subjects place both hands and forearms (total body surface area: Fs = 0.11) into a dilute aqueous 50-ppb (mcg/l) malathion so lution for up to two (2) hours. Malathion solution assays are then made via the “disa ppearance method” on the tank solution before and after the exposure period. Malathion assays are completed by passi ng the tank exposure solution through Bakerbond C18 Speedisks (Part Number 8055-06). Five hundred milliliters of the exposure solution is drained through the Sp eedisk via full vacuum suction (about 25 mm Hg) applied to a Speedisk extrac tion station (for about five minutes). The Speedisks with extracted malathion solute were then shi pped in a sealed cont ainer to Ecology and Environment, Inc., for completion of the EPA assay method.

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47 Precise concentrations in the dilute aqueous malathion solution were determined before and after exposure using EPA SW-846 Method 8270C at Ecology and Environment, Inc (Lancaster, NY). This EPA-approved commer cial assay method is sensitive to 0.5 ppb. The semi-volatile compound is introduced from a sample extract into the gas chromatograph by direct injection of 2 mcl of the extract. Sample extracts are prepared for analysis by transferring a measured aliquot of extract to an autosampler vial and adding the appropriate amount of internal standard solution. Chromatographic conditions are such that the compounds are separated by the gas chromatograph. The compounds are detected using a mass spectrometer from which both qualitative and quantitative information is obtained. Quantitation is by the internal standard technique using relative response factors. Calculations of chemical flux into skin from the exposure solution are estimated according to Fick’s Law as the difference in chemical mass contained in the exposure solution at the beginning and end of the exposure period, divided by the corresponding exposed dermal surface areas. In summary, a Mass Balance System was implemented upon the exposure solution, with the assumption that all lost malathion in solution was driven down the concentration gradient into the exposed human subject. Kp-malathion (permeability constant across the skin) wa s then derived from the actual Fs=0.11 exposure model. Kp-malathion was then theoretically applie d to an Fs = 1.00 swimming pool model for both theoretical child and adult swimming pool models. Finally, hazard index (HI) for theoretical three-hour swimming pool exposures were calculated based upon experimentally derived Dd and Kp-malathion values.

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48 Table 15: Parameters for Kp = Dd*Mb / (Dc*De*Fs*As). € Dd = skin dosage of malathion absorbed in mcg € Dc = average concentration gradient of malathion from water to skin € De = duration of contact with contaminated water € Fs = fraction of skin in contac t with exposure solution = 0.11; € Fsadj = adjustment for Maibach’s re lative skin absorption = 0.0775; € Fs = fraction of skin in contact with water in swimming pool = 1.00; Table 16: Parameters for Ds = Kp*Dc*De*Fs*As*Sys / Mb. Adult: € As = total skin surface area = 21 345 cm2; € Mb = body mass = 85 kg; € HI = Ds / (0.2 mg/kg/day)*(85 kg) = Ds / 17 mg; Child: € As = exposed skin surface area = 12 000 cm2; € Mb = body mass = 34.5 kg; € HI = Ds / (0.2 mg/kg/day)* (34.5 kg) = Ds / 7 mg; Example Calculation : Utilize the theoretical exposur e model Dd = Kp*Dc*De*Fs*As / Mb, and assume approximate Kp-malathion = 0.001 L/cm2/hr. An 85 kg subject typically has hand area Fs=.025 (each) and forearm area Fs =.03 (each). Therefore, immersing both hands and forearms in the malathion solution yields a total exposure area Fs=.11, with Fsadj = 0.0775 after adjustment for Maibach’s re lative skin absorpti on data. Therefore, this theoretical volunteer would derm ally, but not systemically, absorb: Dd = (.001 L/cm2/hr)(50 ppb or mcg/l)(3 hr)(.0775)(21 345 cm2) = 248 mcg. The 20 liter immersion tank, the n, would change from the or iginal concen tration of 50 ppb malathion to 37.6 ppb (mcg/l) or (1000 mcg 248 mcg) / 20 liters. This change in malathion concentration over the time period of the volunteer subject exposure period is well within the sensitivity of the NI OSH/EPA laboratory assay procedure.

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49 Chapter Seven Volunteer Safety Screening of Volunteer Subjects: Each female participant had a proven negative pregnancy test within 72 hours of study pa rticipation. Each vol unteer subject was screened for a history of acute or chronic disease or disability, Gu lf War Syndrome, or previous significant exposure to malathion, organophosphate or other pesticide. If the screening history is positive, the subject is eliminated from participation in the study protocol. Medical Observation: Each subject is intermittently obs erved for acute cholinergic signs and symptoms by a licensed physician during the two (2) hour experimental protocol period as follows: € Mild exposure symptoms include headache, gi ddiness, dizziness, anxiety, weakness, tremors of tongue and eyelids, and miosis. € Moderate exposure symptoms include beha vioral disturbances, sweating, nausea, vomiting, salivation, lacrimati on, diarrhea, paresthesias, muscle tremors, and incoordination. € Severe exposure symptoms include fecal and urinary incontinence, pulmonary edema, heart block, respiratory failure, convulsions, coma and death. Although highly unlikely at the malathion dosage selected for use in this experimental protocol, a volunteer exhibiting signs or symptoms of organophosphate exposure would be referred to a local emergency room for tr eatment at once. In addition, a resuscitation cart equipped for atropine and pralidoxime (2-PAM) administra tion was immediately available for use by a licensed physic ian should emerge ncy reversal of

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50 cholinergic events become necessary. Furtherm ore, volunteers exhibi ting any cholinergic signs or symptoms would be reported as an adverse event to the Institutional Review Board at once. Hazard Index Model: Worst-case-scenario analysis assumes the subject volunteer absorbs all the malathion in the exposur e tank, 1 mg, or 12 mcg/kg/day. Since the Reference Exposure Level is 200 mcg/kg/da y, Hazard Index = 12/200 or 0.06. Therefore, health protection is achieved since the estimated dosage is an order of magnitude below the relevant REL and HI << 1. Biological Exposure Indices: The ACGIH recommended BEI for organophosphorus cholinesterase inhibitors (e.g. malathion) is RBC Cholinesterase Activity. Significant exposure is widely recognized to have occu rred if RBC Cholines terase activity is decreased more than 30% from an individual’ s baseline condition. Continued depression may occur in dose-dependent fashion for up to 12 weeks post-exposure. RBC cholinesterase activity is the preferred measure as it is the same enzyme found within the nervous system. In Moeller and Rider’s NOAEL / LOAEL landmark malathion study, the defined objective was to determine the maximum amount of malathion which can be ingested daily for a prolonged period of time without depressing the pretest level of RBC Cholinesterase Activity. The subjects were healthy men in San Quentin State Prison. Malathion was orally administered at 8 mg /day for 32 days, 16 mg/day for 47 days, and 24 mg/day for 56 days. RBC Cholinesterase wa s analyzed at baseline then twice weekly. Neither 8 mg/day nor 16 mg/day affected RBC Cholinesterase Activity in a statistically significant manner. However, 24 mg/day fo r 56 days depressed RBC Cholinesterase Activity to about 75% of baseline. Based upon Moeller and Rider’s study, BEI m onitoring with RBC Cholinesterase Activity during this protocol appears unwarrant ed. That is, in the worst case scenario, the volunteer subject would absorb all the malathion in the expos ure tank: 1 mg. This dosage

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51 is well below the known dosage to cause RBC Cholinesterase depre ssion. Furthermore, volunteer subjects in this protocol have only a single exposure. In the Moeller and Rider study, volunteer subjects required 24 mg oral ingestion expos ures on 56 consecutive days before RBC Cholinesterase was depressed. Dermal Exposure Indices: Dermal absorption of neat malathion (24.6 mg), a 50% emulsifiable concentrate (25.7 mg), and a 1% (0.16 mg) and 10% (5.5 mg) aqueous mixture was examined in human volunteers. Each volunteer subject received a single application of the nominal dose of malathion applied to a 4.6 cm2 area of the ventral forearm. The 1.0% aqueous mixture appeared to be more readily absorbed than the other formulations: 6.42% of the external concentration was absorbed per hour (Dary, 1994). Based upon this study analysis, <20% of the dilute malathion aqueous experimental solution will be absorbed after 2 hours tim e by the volunteers, or about 0.2 mg of Malathion. This dosage of malathion is well under the Reference Exposure Level (REL) and Hazard Index (HI) for Malathion Theoretical Mathematical Exposure Model: Utilizing the exposure model described once again with assumed Kp-malathion =0.001 L/cm2/hr, and the formula Ds = Kp*Dc*De*Fsadj*As*Sys / Mb, the subject volunteers in the protocol will be exposed to about: Ds = (.001 L/cm2/hr)(50 ppb)(3 hr)(.0775)(21 345 cm2)(0.10) / (85 kg); = 25 mcg / 85 kg adult; = 0.3 mcg/kg/day. Reference Exposure Level (REL) = 200 mcg/kg/day; Hazard Index (HI) = 0.3 / 200 = 0.0015. Therefore, health protection is achieved since the estimated dosage is below the relevant REL and the HI is << 1.

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52 Over-the-counter Exposure Model: Household over-the-counter malathion formulations (Spectracide, manufactured for Spectrum Group, United Industries Corporation, St. Louis, EPA Reg. No. 10370-291-8845) are ava ilable in 50% xylene solution, or 500 mg/ml. The worst-case-scenario exposure of a subject volunteer in this protocol is equivalent to exposure to about 1/500 ml or 0.002 ml of the over-the-counter solution. Carcinogen Status: At the time of the study protoc ol approval, malathion was not classified as a carcinogen by any certified organizati on including the ACGIH.

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53 Chapter Eight Results Table 17: General Experimental Results. € Demographics: All volunteer subjects were between ages 25-50 yo, weight 70 – 100 kg, and 2/20 (10%) were female. € Subject eleven was removed from furt her data analysis due to the null concentration of malathi on during the exposure period. € Eight of nineteen subject s (42%) absorbed no malathion from solution, thus receiving no malathion do sage during exposure. € Eleven of nineteen subj ects (58%) absorbed malath ion from solution, thus receiving a malathion dosage during exposure. € Begin versus end malathion concentrations were highly variable from volunteer to volunteer, but not significantly different (p = 0.185). € Volunteer Safety: No volunteer expe rienced signs or symptoms of organophosphate exposure or toxicity at any time. € Experimental Kpmalathion derived from human voluntee r exposures ranged from 0 0.0051 L/cm 2 /hr (ave=0.0005, SD=0.0011, UCL=0.0028). € Hazard Index (HI) for adult swimmers immersed for three hours in Malathion=30 ppb+Malaoxon=0 ppb (utilizing experimentally-derived Kpmalathion) ranged from 0 – 0.06 (ave=0.01, SD=0.01, UCL=0.03). € Hazard Index (HI) for child swimmers immersed for thr ee hours in Malathion=30 ppb+Malaoxon=0 ppb (utilizing experimentally-derived Kpmalathion) ranged from 0 – 0.08 (ave=0.01, SD=0.02, UCL=0.04).

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54 € Hazard Index (HI) for adult swimmers immersed for three hours in Malathion=30 ppb+Malaoxon=30 ppb (utilizing experimentally-derived Kpmalathion) ranged from 0 – 2.0 (ave=0.21, SD=0.45, UCL=1.11). € Hazard Index (HI) for child swimmers immersed for thr ee hours in Malathion=30 ppb+Malaoxon=30 ppb (utilizing experimentally-derived Kpmalathion) ranged from 0 – 2.8 (ave=0.29, SD=0.62, UCL=1.53). Table 18: Dermal Absorption by Volunteer I0 50 100 150 200 250 300 350 400 1234567891011121314151617181920Volunteer NumberMalathion (mcg) Table 19: Dermal Absorption by Volunteer II0 0.001 0.002 0.003 0.004 0.005 0.006 1234567891011121314151617181920Volunteer Number

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55 Table 20: Dermal Absorption by Dosage0 1 2 3 4 5 6 7 8 9 Zero1 to 4041 to 8081 to 120 121 to 160 161 to 200 201 to 240 241 to 280 281 to 320 321 to 360Malathion (mcg)Frequency (# volunteers) Table 21: Dermal Absorption by Kp-malathion0 1 2 3 4 5 6 7 8 9 zero1 to 56 to 10 11 to 15 16 to 20 21 to 25 26 to 30 31 to 35 36 to 40 41 to 45 46 to 50 51 to 55Kp-malathion x 104 (L/cm2/hr)Frequency (# volunteers)

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56 Table 22: Hand / Forearm Exposure in Aquarium; Nominal Contamination: Mala thion=50 ppb + Malaoxon=0 ppb; Disappearance Method: Solution Concentrations. subject prepoststir temp time malathion malathion (mcg/l) (mcg/l) ( C) (min) 1 38.6 31.2 no 21 30 2 51.6 52.6 no 21 60 3 46.8 43.6 no 21 60 4 40.4 37.2 no 21 90 5 42.4 44.0 no 21 90 6 50.0 48.4 no 21 90 7 39.2 36.6 no 21 135 8 32.4 34.8 no 21 135 9 47.2 45.6 no 21 135 10 68.0 60.0 yes 21 120 11 0.0 0.0 yes 21 120 12 126.0 108.0 yes 21 120 13 46.0 46.0 yes 21 120 14 46.0 46.0 yes 21 120 15 44.0 46.0 yes 21 120 16 50.0 48.0 yes 29 120 17 56.0 52.0 yes 29 120 18 34.0 48.0 yes 29 120 19 50.0 50.0 yes 29 120 20 50.0 44.0 yes 29 120

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57 Table 23: Hand / Forearm Exposure in Aquarium; Nominal Contamination: Mala thion=50 ppb + Malaoxon=0 ppb; Disappearance Method: Experime ntal determination of Kpmalathion. subject Skin Kp-malathion Dosage (Dd in mcg) (L/cm2/hr) 1 148 .00513 2 0 0 3 64 .00086 4 64 .00067 5 0 0 6 32 .00026 7 52 .00037 8 0 0 9 32 .00019 10 160 .00076 11 0 0 12 360 .00093 13 0 0 14 0 0 15 0 0 16 40 .00025 17 80 .00045 18 0 0 19 0 0 20 120 .00077

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58 Table 24: Swimming Pool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=0 ppb; Adult Swimmer immersed for three hours. subject Skin Systemic Hazard Dosage Dosage Index (Dd in mg) (Ds in mg) 1 9.85 1.0 0.06 2 0 0 0 3 1.64 0.2 0.01 4 1.28 0.1 0.01 5 0 0 0 6 0.50 0.05 0.003 7 0.71 0.1 0.004 8 0 0 0 9 0.36 0.04 0.002 10 1.45 0.1 0.01 11 0 0 0 12 1.79 0.2 0.01 13 0 0 0 14 0 0 0 15 0 0 0 16 0.47 0.05 0.003 17 0.86 0.1 0.005 18 0 0 0 19 0 0 0 20 1.48 0.1 0.01

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59 Table 25: Theoretical Swimming Pool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=0 ppb; Child Swimmer immersed for three hours. subject Skin Systemic Hazard Dosage Dosage Index (Dd in mg) (Ds in mg) 1 5.54 0.5 0.08 2 0 0 0 3 0.92 0.1 0.01 4 0.72 0.1 0.01 5 0 0 0 6 0.28 0.03 0.004 7 0.40 0.04 0.006 8 0 0 0 9 0.20 0.02 0.003 10 0.82 0.08 0.01 11 0 0 0 12 1.00 0.1 0.01 13 0 0 0 14 0 0 0 15 0 0 0 16 0.27 0.03 0.004 17 0.48 0.05 0.007 18 0 0 0 19 0 0 0 20 0.83 0.08 0.01

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60 Table 26: Theoretical Swimming Pool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=30 ppb; Adult Swimmer immersed for three hours. subject Skin Systemic Hazard Dosage Dosage Index (Dd in mg) (Ds in mg) 1 345 34.5 2.0 2 0 0 0 3 57 5.7 0.3 4 45 4.5 0.3 5 0 0 0 6 18 1.8 0.1 7 25 2.5 0.2 8 0 0 0 9 12 1.2 0.1 10 51 5.1 0.3 11 0 0 0 12 63 6.3 0.4 13 0 0 0 14 0 0 0 15 0 0 0 16 17 1.7 0.1 17 30 3.0 0.2 18 0 0 0 19 0 0 0 20 52 5.2 0.3

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61 Table 27: Theoretical Swimming Pool Model using experimental Kpmalathion; Contamination: Malathion=30 ppb + Malaoxon=30 ppb; Child Swimmer immersed for three hours. subject Skin Systemic Hazard Dosage Dosage Index (Dd in mg) (Ds in mg) 1 194 19 2.8 2 0 0 0 3 32 3.2 0.5 4 25 2.5 0.4 5 0 0 0 6 10 1.0 0.1 7 14 1.4 0.2 8 0 0 0 9 7 0.7 0.1 10 29 2.9 0.4 11 0 0 0 12 35 3.5 0.5 13 0 0 0 14 0 0 0 15 0 0 0 16 9 0.9 0.1 17 17 1.7 0.2 18 0 0 0 19 0 0 0 20 29 2.9 0.4

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62 Chapter Nine Discussion It is not uncommon in dermal absorption studi es for wide Kp variation within individual experimental results. Absorption constants a nd as a result dosage received can vary by several orders of magnitude between human subjects. For example, during this protocol, eight of nineteen subjects absorbed nil mala thion, or at least so little that no absorption could be detected by the disa ppearance technique. In contra st, the absorption constant derived from the first volunteer was an orde r of magnitude greate r than the following nineteen subjects, this despite the most cons ervative set of experimental conditions. This wide variation in results is similar to othe r investigators experien ce in similar published dermal absorption study settings. As compared to malathion and malaoxon, to luene and chloroform are smaller in molecular weight but with similar oil-wate r partition coefficients. Thus, according to Bogen’s equation, toluene and chloroform should have consid erably larger permeability constants (Kp). However, th e experimentally derived K pmalathion= .0005 L/cm2/hr obtained in this study is in fact similar to those previously obtained by other investigators for Kptoluene=.0008 L/cm2/hr and Kpcholoroform= 0.0001 L/cm2/hr. It is interesting to note that many factors in the swim ming pool exposure model may enhance the typical Kpmalathion,. For example, water, ambient, and skin temperatures may well be greater in swim exer cise. These increased temperat ures enhance skin absorption. Also, during swimming or bathing, greater hydra tion of skin surfaces takes place, again promoting Kp. For lipid-soluble malathion, lo wered viscosity of tissue lipids at higher temperatures reduces activation energies ne eded for diffusion. In addition, sunburned or

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63 wounded skin, as well as hair follicles and sweat glands, accelerate dermal absorption. Finally, skin sites in children appear to be mo re permeable than skin sites in adults. Thus, the experimental K pmalathion derived from this study is likely a conservative value. Utilizing the experimentally-derived Kpmalathion, the Hazard Index (HI) for adult and child swimmers immersed for three hours in a ma lathion only contaminated pool ranged from 0 – 0.08. These data indicate that exposure to dilute aqueous malathion solutions following usual aerial bait applications is not appreciably absorbed, and therefore, not a public health hazard. Importantly, a potential problem arises in the analysis from the statement ‘usual aerial bait application’. An atypical problem scenario exists with aerial bait application in that malathion is easily oxidized to the more potent malaoxon by chlorine within swimming pools. This fact is evidenced by the fact th at both California and Florida environmental agencies and public health authorities f ound considerable malaoxon contamination of public and private swimming pools in the days and weeks following aerial bait application. In vivo, parent malathion must be meta bolically activated to malaoxon by oxidative desulfuration to be an effectiv e AChE inhibitor. This activation step is carried out by both mammals and insects, but insects are relativ ely deficient in the carboxylesterases used by mammals for detoxification. Thus, insects have higher toxicity to malathion generally speaking. However, humans may have similar mala thion toxicity profiles to insects if and when liver carboxylesterase is inactivate d. One compound well known to inactivate liver carboxylesterase is malaoxon, which may do so if absorbed ex vivo from contaminated swimming pools. Since malaoxon is 68 times as toxic as ma lathion, human voluntee r exposure to same, with or without concurrent e xposure to malathion, is generally deemed too dangerous for human experimentation. Therefore, in order to analyze the potential toxicity of external malaoxon contamination and exposure in the swimming pool model, it is necessary to

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64 estimate Kpmalaoxon. Fortunately, Bogen’s empirical fo rmula for Kp values based upon molecular weight and oil-water partition coe fficient values can be utilized for same. In this case, owing to the more polar qualities of malaoxon, Kpmalaoxon is estimated to be one-half that of Kpmalathion. The Hazard Index for swimmers immersed for three hours in a malathion plus malaoxon contaminated pool was modeled utiliz ing the experimentally-derived Kpmalathion, and Bogen’s technique to estimate Kpmalaoxon. This methodology yielde d an average HI=0.21 for adult swimmers, with HI for child swimmers similarly averaging 0.29. Although HI>1 (albeit slightly) at the UCL (two standard deviations) for the volunteer group modeled in this malathion plus malaoxon exposur e model, even these data indicate there is no appreciable public health hazard. In order to lessen uncertainty in the hazar d index estimate for malathion plus malaoxon exposure scenarios, further animal or huma n experimental dermal absorption data is clearly indicated in order to determine Kpmalaoxon with more certainty. Since human volunteers should not be utilized in experiments with this toxic compound, the next best available alternative is hairless guinea pig sk in. Bogen and others have determined that the hairless guinea pig is a superb model for human dermal skin absorption, and a similar disappearance method to that used in this pr otocol can be effectively used with this guinea pig animal model. Thus, potentially sy nergistic contaminant toxicity profiles for malathion plus malaoxon may be more precis ely determined in the future (Bogen, 1992). Additional routes of entry for malathion and its co-products to enter into a swimmers body exist but have not yet been integrated with in the theoretical mode l described in this paper. EPA estimates that children who swim fo r 3 hours daily take in and squirt out of their mouths 16 liters of pool water and ingest 1% of that amount. The residence time for each 5 ml aliquot in the mouth will be a bout 1.75 seconds, and during this time period about 1% of the malathion will be absorb ed through the buccal and sublingual mucous membranes. Additional absorption is possible ag ain through the orbital, nasal, aural, and

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65 inhalation routes. These unaccount ed for routes of ingestion, absorption, and inhalation in the swimmer may increase total dosag e exposure scenarios by up to 50%. The most neglected aspect of pesticide toxicity investigation in mammals remains zeroing in on the toxicity of breakdown pr oducts of the parent compound. In fact, the toxicity profile of mixtures of parent and breakdown products may be synergistic in nature. Measurements of skin absorption rely on data obtained from single compound solutions. This technique may underestimate ab sorption in the more common scenario of multiple exposures to solvent mixtures in the contaminated water. Studies show that combinations of compounds have greater effect on the stratum corneum, and are absorbed more readily. In addition, potent impurities such as malaoxon, isomalathion, O,O,S-trimethyl phosphorothioate and diethyl fumarate may c ontribute synergistically to the toxicity of technical malathion formula tions both before and after usual aerial bait application (Aldridge, 1979). For example, a diffe rence of a day or two in bait application on two adjacent areas coul d result in a condition in whic h a considerable quantity of the breakdown product along with a substantial qu antity of the parent compound could be washed by rainfall into the common water s ource. In a single body of water, daily pH fluctuations could conceivably be responsib le for producing synergistic conditions as well. The rapid production of di ethyl fumarate could occur du ring the period when pH is high, its rate of production being reduced af ter the active photosynt hetic period, leaving enough malathion to produce synergistic t oxic effects (Bender, 1969). These data suggest that the toxic exposur e profile of the population swimming in the spray area may be significantly different than the profile measured in the original bait spray tank. As a minimal standard, prior to spray application, analysis should be considered for potent and toxic contamin ants (e.g. isomalathion, malaoxon, diethyl fumarate) within the technical malathion form ula. In addition, after spray application, similar analysis should be considered fo r swimming pools within these same designated spray areas. In fact, partially as a result of this type of analysis, California ultimately cut its target aerial spray malathion concentr ations from 2.4 to 1.2 ounces per acre, then subsequently abandoned malathion completely in favor of biological controls in 1994.

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66 Chapter Ten Conclusion Malathion skin absorption measured among 58% of the human volunteers by the disappearance method allowed for e xperimental determination of Kpmalathion=.0005 L/cm2/hr. The dosage in human volunteers from this controlled exposure to a contaminated aquarium solution was several orders of magnitude less than the minimal dose necessary to cause measurable change in red blood cell acety lcholinesterase (RBCAChE). Following experimental determination of Kpmalathion, a mathematical model was evaluated for swimmers using dilute aqueous malathion contamination concentrations typically detected af ter bait application. Extrapolation of Kpmalathion into swimming pool exposures (both adults and children, with a nd without malaoxon) resulted in dosages an order of magnitude below that needed to ca use a detectable cha nge in RBC-AChE. These data indicate that exposure to aqueous mala thion following usual aeri al bait applications is not appreciably absorbed, and th erefore, not a public health hazard.

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67 References Aldridge WN et al, “The t oxicological properties of impu rities in malathion”, Arch Toxicol 1979, 42: 95-106. Argiles AM, “Acute polyneuropathy after malathion poisoning”, Acta Neurol Belg 1990, 90: 190-99. Baker EL, “Epidemic Malathion Poisoning in Pakistan Malaria Workers”, Lancet 1978, Jan 7: 31-34. Baranowska-Dutkiewicz B, “Skin absorption of aniline from aqueous solutions in man”, Toxicol Letters 1982, 10: 367-72. Baselt RC, Disposition of Toxi c Drugs and Chemicals in Man pp. 604-6, Sixth Edition, Biomedical Publications Foster City, CA, 2002. Beech JA, “Estimated worst case trihalomethane body burden of a child using a swimming pool”, Medical Hypotheses 6: 303-307, 1980. Bender ME, “The toxicity of the hydrolysis and breakdown products of malathion to the fathead minnow”, Trans Am Fish Soc 1969, 98: 571-82. Bogen KT, “Health risk assessment of trichl oroethylene in Califor nia drinking water”, Lawrence Livermore National Labora tory (LLNL) 1988. Report #UCRL-21007. Bogen KT, Colston BW, Machicao LK, “D ermal absorption of dilute aqueous chloroform, trichloroethylene, and tetrachlor oethylene in hairless guinea pigs”, Fundam Appl Toxicol 1992, 18: 1: 30-9. Bogen KT, “Models based on steady-state in vitro dermal permeability data underestimate short-term in vivo exposures to organic chemicals in water”, J Exposure Analysis Environ Epidem 1994, 4: 4: 457-75. Bogen KT, Keating G, Vogel JS, “Chlorofor m and trichloroethylen e uptake from water into human skin in vitro: kinetics and risk implications, LLNL 1995. #UCRL-JC-120107. Brewer SK et al, “Behaviora l dysfunctions correlate to altered physiol ogy in rainbow trout (Oncorynchus mykiss) exposed to chol inesterase-inhibiting chemicals”, Arch Environ Contam Toxicol 2001, 40: 70-76.

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68 Brown HS, “The role of skin absorption as a route of exposure for volatile organic compounds (VOCs) in drinking water”, Am J Public Health 1984, 74: 479-484. Caldwell J et al, Biological Basis of Detoxification Acad Press, 1983, 125-7, 244, 352. Caplan PE et al, “Human exposures in popul ated areas during airp lane application of malathion”, Arch Ind Health 1956, 14: 326-32. Carver MP, Levi PE, Riviere JE, “Significan t first-pass bioactivation of parathion (P) during percutaneous absorption in the isolated perfused porcine skin flap (IPPSF)”, The Toxicologist 1988: 8: 125. Carver MP, Riviere JE, “Percutaneous abso rption and excretion of xenobiotics after topical and intravenous administratio n to pigs”, Fundam Appl Toxicol 1989, 13: 714-22. Choi PTL et al, “The use of glycopyrrolate in a case of intermediate syndrome following acute organophosphate poisoning”, Can J Anaesth 1998, 45: 4: 337-40. Cook GH, Moore JC, Coppage DL, “The relations hip of malathion and its metabolites to fish poisoning”, Bull Environ Contam Toxicol 1976, 16: 3: 283-90. Dam K et al, “Chlorpyrifos exposure during a critical neonatal period elicits genderselective deficits in the development of coordination skills and locomotor activity”, Dev Brain Research 2000, 121: 179-87. Dary CC et al, “Dermal Absorption and Disposition of Formulations of Malathion in Sprague-Dawley Rats and Humans”, Chapter 15, Biomarkers of Human Exposure to Pesticides American Chemical Society, 1994. Doorn JA et al, “Identifica tion of butyrlcholinesterase a dducts after inhibition with isomalathion using mass spectrometry: difference in mechanism between (1R)and (1S)Stereoisomers”, Tox Appl Pharm 2001, 176: 73-80. Drill VA, Lazar P, Cutaneous Toxicity 1977. Academic Press, 63-77. Dutkiewicz T, Tyras H, “A study of skin ab sorption of ethylbenzene in man”, Br J Ind Med 1967: 24: 330-332. Dutkiewicz T, Tyras H, “Skin absorption of to luene, styrene, and xylene”, Br J Ind Med 1968: 25: 243. EXTOXNET primary files maintained and archiv ed at Oregon State University, logged in via http://ace.orst.edu/info/extoxnet/pips/ghindex.html on April 10, 2003. Feldmann RJ, Maibach HI, “Pecutaneous penetrat ion of some pesticides and herbicides in man”, Toxicol Appl Pharm 1974, 28: 126-32.

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69 Gaines TB, “Acute toxicity of pe sticides”, Toxicol Appl Pharmacol 1969. 14: 515-534. Gershon S, “Psychiatric sequel ae of chronic exposure to organophosphorus insecticides”, Lancet 1961, June 24: 1371-4. Gitelson S, “Poisoning by a mala thion-xylene mixture”, JAMA 197: 819-821, 1966. Goldman H, “Malathion poi soning in a 34-month old child following accidental ingestion”, J Pediatr 52: 76-81, 1958. Haque N, Rizvi SJ, Khan MB, “Malathion induced alterations in the lipid profile and the rate of lipid peroxidation in rat br ain and spinal cord”, Pharm Toxicol 1987, 61: 12-15. Harell M et al, “Bilateral sudden deafness fo llowing combined inse cticide poisoning”, Laryngoscop e 1978, 88: 1348-51. Healy JK, “Ascending paralysis followi ng malathion intoxication”, Med J Austr 1959, 1: 765-7. Hollingsworth J, Tampa Tribune: € “Malathion: savior or scourge?”, 6/24/97; € “Malathion overdose”, 6/27/97; € “Pool of spray knowledge leaves swimmers in dark”, 6/27/97; € “More tainted water reported”, 7/1/97; € “Malathion hits water supply”, 7/15/97; € “Crawford agrees to pact with EPC”, 7/18/97; € “Water tests confirm traces of pesticide”, 7/19/97; € “Pesticide effects ge t attention”, 7/23/97; € “Attention turns to spra ying’s effects”, 9/2/97; € “Dangers of Medfly spraying not fully explored”, 10/5/97; € “Pool maladies stir rash of complaints”, 10/5/97. € “Malathion risks uns ettling”, 10/6/97. Howard, P. H., Ed. Handbook of Environmen tal Fate and Exposure Data for Organic Chemicals Vol 3: Pesticides. Lewis Pub lishers, Chelsea, MI, 1991. pp. 5-13. Jianmongkol S, Berkman CE, Thompson CM, Rich ardson RJ, “Relativ e potencies of the four stereoisomers of isomal athion for inhibition of hen brain acetylcholinesterase and neurotoxic esterase in vitro”, Tox Appl Pharm 1996, 139: 342-8. Kamori T et al, “A case of delayed myeloneuropathy due to malathion intoxication”, No To Shinkei 1991, 43: 969-74. Kao J, Carver MP, “Cutaneous metabolis m of xenobiotics”, Drug Metabolism Reviews 1990, 22: 4: 393-4.

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70 Kizer KW, “Health Risk Assessment of Aerial Application of Malathion-Bait”, State of California Department of Health Services, February 1991. Levin ED et al, “Persistent behavioral conse quences of neonatal chlo rpyrifos exposure in rats”, Dev Brain Research 2001, 130: 83-89. Mattsmura F, Toxicology of Insecticides Plenum Press, New York, p. 224-6, 1975. Mattson AM, Sedlak VA, “Ether-extractable urinary phosphates in ma n and rats derived from malathion and similar compounds”, J Agr Food Chem 8: 107-10, 1960. McConnell R, Keifer M, Rosenstock L, “E levated quantitative vi brotactile threshold among workers previously poisoned with methamidophos and other organophosphate pesticides”, Am J Ind Med 1994, 25: 325-334. Maibach HI, “Regional variation in percutane ous penetration in man”, Arch Environ Hlth 1971, 23: 208. Miller T, “Organophosphorous In secticides”, posted for Entomology 128 Section 001 at www.wcb.ucr.edu/wcb/school s/CNAS/entm/tmiller/1/modules/page25.html University of California, Riverside, Fall 1998. Moeller HC, Rider JA, “Plasma and Red Blood Cell Cholinesterase Activity as indications of the threshold of incipient toxicity of EPN and Malathion in Human Beings”, Toxicology and Applied Pharmacology 1962, 4: 123-130. Murphy SD et al, “Comparative antic holinesterase acti on of organophosphorus insecticides in vertebrates”, Toxic Appl Pharm 1968, 12: 22-35. Murphy SA, “Aerial pest eradication in Mass achusetts and California and the pesticide malathion”, Environmental Affairs 1992, 19: 851-884. O’Brien RD, “Properties and metabolism in the cockroach and mouse of malathion and malaoxon”, J Economic Entomology 1957, 50: 2: 159-163. Pelligrini G, “Potentiation of toxicity of organophosphorus compounds containing carboxylic ester functions toward warm-b looded animals by some organophosphorus impurities”, J Agric Food Chem 1972, 20:944-950. Pope AM, Rall DP, Environmental Medicine: Integrating a missing element into medical eduction Chapter 22: “Cholinestera se-Inhibiting Pesticide T oxicity”, Institute of Medicine, National Academy Press, Washington DC, 1995. Reifenrath WG, Hawkins GS, Kurtz MS, “Percu taneous penetration and skin retention of topically applied compounds: an in vitro – in vivo study”, J Pharm Sci 1991:80:6:526-32.

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71 Rivett K, Potgieter PD, “Diaphragmatic pa ralysis after organophosphate poisoning”, S Afr Med J 1987, 72: 881-2. Rosenstock L, “Chronic central nervous system effects of acute organophosphate pesticide intoxification”, Lancet 1991, 338: July 27: 223-237. Saleh MA et al, “Determination of the distri bution of malathion in rats following various routes of administration by whole-body elect ronic autoradiography”, Tox Indust Health 1997, 13: 6: 7518. Savage EP, “Chronic neurol ogical sequelae of acute organophosphate pesticide poisoning”, Arch Env Health 1988, 43: 1: 38-45. Scheuplein RJ, “Mechanism of percutaneous ab sorption I: routes of penetration and the influence of solubility”, J Invest Derm 1965, 45: 334-5. Scheuplein RJ et al, “Permeability of the skin”, Physiological Reviews 1971,51:4:702-47. Shafey O et al, Florida Department of Health, NIOSH, CDC, MMWR Weekly November 12, 1999, 48: 44: 1015-1018, 1027. Shafnik MT, Enos HF, “Determination of metabolic and hydrolytic products of organophosphorus pesticide chemicals in human blood and urine”, J Agr Food Chem 17: 1186-9, 1969. Shah PV et al, “Comparative rates of dermal penetration of insecticides in mice”, Toxicol Appl Pharmacol 1981, 59: 414-23. Smith HT, “Fruit fly cooperative control program”, Final Environmental Impact Statement, U.S. Department of Agricu lture (USDOA), Animal and Plant Health Inspection Service (APHIS), 2001. Tregear RT, Physical Function of Skin London: Academic Press, 1966. Webster RC, Maibach HI et al, “Malathion percutaneous absorption after repeated administration to man”, Toxicol Appl Pharmacol 1983, 68: 116-9. Webster RC, Maibach HI, “In vivo percutan eous absorption and decontamination of pesticides in humans”, J Toxicol Environ Hlth 1985, 16: 25-37. Weeks MH, “Preliminary assessment of acute toxicity of malathion in animals”, Report 99-002-74/76. Aberdeen Proving Ground, US Army, Env Hygiene Agency, 1975, 1-25. Wolfe NL et al, “Kinetic investigation of malathion degradation in water”, Bull Environ Contam Toxicol 1975, 13: 6: 707-13.


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Dermal absorption of a dilute aqueous solution of malathion
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ABSTRACT: Malathion is a commonly used organophosphate pesticide on field crops, fruits, nut trees, vegetables, livestock, agricultural premises, and land. The approved uses also include mosquito and medfly control. These uses can result in human skin contact. The purpose of this study is to evaluate the human skin absorption of malathion for the purpose of assessing the risks associated with aqueous solution exposures following applications. Aerial applications can result in solubilized malathion in swimming pools and other waters that may be contacted. Human volunteers were selected and exposed to aqueous solutions of malathion at various concentrations. Participants submerged their arms and hands in twenty liters of dilute malathion solution in either a stagnant or stirred environment. The "disappearance method" was applied by measuring malathion concentrations in the water before and after human subject exposure to the water for various periods of time. Malathion was measured using Gas Chromatography. No measurable skin absorption was detected in 42% of the participants. Measurable skin absorption among the remaining 58% of participants resulted in doses that were more than an order of magnitude less than the minimal dose necessary to cause a measurable change in red blood cell acetylcholinesterase (RBC-AChE). Extrapolation of these results to a mathematical model for recreational swimmers and bathers exposed to contaminated swimming pools and surface waters typically detected after bait application again are an order of magnitude below the doses needed to cause a detectable change in RBC-AChE. These data indicate that exposure to aqueous malathion following usual aerial bait applications is not appreciably absorbed, and therefore, it is not a public health hazard.
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