PESTICIDE RESIDUES IN FOOD - 1997 Sponsored jointly by FAO and WHO with the support of the International Programme on Chemical Safety (IPCS) TOXICOLOGICAL AND ENVIRONMENTAL EVALUATIONS 1994 Joint meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group Lyon 22 September - 1 October 1997 The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data submitted for the purpose of the JMPR assessment. A registration authority should not grant a registration on the basis of an evaluation unless it has first received authorization for such use from the owner who submitted the data for JMPR review or has received the data on which the summaries are based, either from the owner of the data or from a second party that has obtained permission from the owner of the data for this purpose. MALATHION First draft prepared by T.C. Marrs Medical Toxicology and Environmental Health Department of Health, London, United Kingdom Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution, and excretion Biotransformation Effects on enzymes and other biochemical parameters Cholinesterases Other enzyme systems Interactions with other organophosphates Toxicological studies Acute toxicity Short-term toxicity Long-term toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration reproductive toxicity Developmental toxicity Special studies Dermal and ocular irritation and dermal sensitization Macrophage and mast cell function Ocular function Neurotoxicity Antidotes Observations in humans Comments Toxicological evaluation References Explanation Malathion was evaluated by the JMPR in 1963, 1965, and 1966 (Annex 1, references 2, 4, and 6). An ADI of 0-0.02 mg/kg bw was established in 1963, which was confirmed in 1965 and 1966. It was evaluated at the present Meeting within the CCPR periodic review programme. Malathion is S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethyl phosphorodithioate. It is likely that the results of earlier toxicological studies on malathion have been substantially affected by impurities. Of particular interest are isomalathion [ S-1,2-bis(ethoxycarbonyl)ethyl O,S-dimethyl phosphorodithioate] and various trialkyl phosphorothioates [for reviews, see Aldridge et al. (1985) and Dinsdale (1992)]. These compounds are notable for their pulmonary toxicity. Furthermore, isomalathion has a greater than additive effect when administered with malathion, probably due to carboxylesterase inhibition (Ryan & Fukuto, 1984). In fact, isomalathion appears to be the major impurity of malathion and affects the LD50 of the commercial formulation. O,O,S-Trimethyl phosphorothioate and O,S,S-trimethyl phosphorothioate produce disorders of blood clotting (Keadtisuke et al., 1990), and O,O,S-trimethyl phosphorothioate produces an unusual neurotoxic syndrome with hypophagia, weight loss, and hypothermia (Ohtako et al., 1995). After an epidemic of malathion poisoning among spraymen in Pakistan (Baker et al., 1978), WHO issued specifications for malathion water-dispersible powders, which required that a 50% powder contain no more than 0.9% isomalathion after storage at 54 ¡C for six days (Miles et al., 1979; WHO, 1985). Subsequently, major manufacturers, under the auspices of FAO, adopted a code of conduct which requires that, inter alia, the active ingredient and co-formulant of commericial formulation be the same as those tested toxicologically (FAO, 1986). It is likely that esterase-inhibitory activity attributed to technical-grade malathion is due largely to the action of malaoxon (WHO, 1986). Evaluation for acceptable daily intake 1. Biochemical aspects (a) Absorption, distribution, and excretion The disposition of 14C-malathion (purity, > 98%; specific activity; 90 µCi/mg (3.3 MBq/mg)), labelled at the carbonyl carbons of the ethoxycarbonyl groups, was studied by Reddy et al. (1989). After a preliminary study, 14C-malathion in corn oil was administered by gavage as single doses of 40 or 800 mg/kg bw to groups of five male and five female Sprague-Dawley (Crl:CD BR) rats. Disposition was also assessed after administration of oral doses of unlabelled malathion (purity, 94.6%) at 40 mg/kg bw per day for 15 days, followed by a 16th dose of 14C-malathion. In the preliminary study, very little of the radiolabel appeared in expired air, and most was eliminated within 72 h; consequently, in the main study, the animals were killed at 72 h. Malathion was rapidly absorbed, biotransformed, and excreted, predominantly in the urine but also in the faeces. After the low dose, 84% appeared in the urine of males and 88% in that of females within 72 h, mostly within 12 h: faecal elimination was 11 and 5.9% in males and females, respectively. Less than 1% of the administered dose was recovered in the tissues. At the high dose, urinary excretion was 76% for the males and 85% for the females; faecal elimination was 14 and 6.6%, respectively. Low concentrations were present in tissues at 72 h. After the repeated doses, 85 and 88% of the label was excreted in the urine within 72 h, mostly within the first 12 h, and faecal elimination was 6.8% in males and 5.8% in females. Less than 1% of the dose was present in the tissues. Malathion, either of 94.6% purity or a 50% emulsifiable concentrate, was labelled with 14C in one methoxy group and given orally to male Sprague-Dawley albino rats at a dose of 280 mg/kg active ingrdient (approximately one-tenth of the LD50). More than 90% of the dose was excreted in the urine within 24 h; the rest of the label was detected in the faeces, intestines, liver, and kidney, in descending order of concentration. The disposition of the pure malathion and the 50% emulsifiable concentrate was not significantly different (Abou Zeid et al., 1993). The toxicokinetics of malathion was studied by Garcia-Repetto et al. (1995) after oral administration of a dose of 467 mg/kg bw (stated to be one-third of the LD50) to male albino Wistar rats. A two- compartment model was discerned, the central compartment being blood, adipose tissue, and muscle and the peripheral compartment, brain and liver. The half-life in blood was 1.4 ± 0.25 days. In a fatal case of malathion poisoning, malathion and the mono- and dicarboxylic acids were found in cardiac blood and tissues, malaoxon being found additionally in most tissues (Morgade & Barquet, 1982). (b) Biotransformation In the study of Reddy et al. (1989) cited above, the metabolites of malathion were studied by high-perfomance liquid chromatography and gas chromatography-mass spectrometry. 14C-Malathion was excreted in urine and faeces as the a and b monocarboxylic acids and the dicarboxylic acid of malathion. Minor metabolites were the oxon of malathion (malaoxon), O,O-dimethylphosphorodithioic acid, 2-mercaptosuccinic acid, fumaric acid, monoethyl fumarate, O,O-dimethylphosphorothioic acid, and desmethylmalathion. Figure 1 shows the proposed pathway for the metabolism of malathion in rats. (c) Effects on enzymes and other biochemical parameters (i) Cholinesterases Groups of 27 male and 27 female Sprague-Dawley Crl:CD:BR rats were given malathion (purity, 96.4%) by gavage in corn oil at doses of 0, 500, 1000, or 2000 mg/kg bw; 20 animals of each sex were used to measure cholinesterase activity, and seven of each sex for determination of neuropathological effects. Viability, clinical signs, body weights, and the results of a functional observational battery of tests and locomotor activity were recorded before treatment, 15 min after treatment, and on days 7 and 14 in seven animals of each sex per dose of those reserved for neuropathology and five of each sex at each dose of those destined for cholinesterase measurements. Cholinesterase activity was determined in plasma, erythrocytes, and brain regions in five animals of each sex per group before the start of the study, 15 min after treatment, and on day 15. Similar measurements were made on day 7, but as one male was killed in extremis, only four remained at this time. Treatment-related clinical signs consisting of salivation and/or anogenital staining occurred after one or two days of treatment in all groups. Additionally reduced hindlimb extensor strength wasseen in one male at the highest dose and decreased ambulatory and motor activity counts in males at this dose on day 0. These males also showed a > 20% reduction in plasma cholinesterase activity in comparison with controls at day 7; although no reduction was seen at 1000 mg/kg bw, a marginal reduction was seen at 500 mg/kg bw. No reductions were seen at other times. In females, reductions > 20% were seen at both 500 and 2000 mg/kg bw at day 7 and at the latter dose at day 15. Erythrocyte acetylcholinesterase activity was reduced by > 20% in the males at the highest dose at day 7, while in females it was reduced by > 20% at day 0 (500 and 2000 mg/kg bw), day 7 (1000 and 2000 mg/kg bw) and day 15 (2000 mg/kg bw only). No consistent biologically significant depressions in brain acetylcholinesterase activity were seen, although there were 10-20% decreases in activity in comparison with concurrent controls, mainly in the group at the high dose. There were no treatment-related neuropathological lesions. There was no NOAEL, as clinical signs occurred in all groups (Lamb, 1994a). Malathion (purity, 96.4%) was given to groups of 25 male and 25 female Sprague-Dawley Crl:CD:BR rats at dietary concentrations of 0, 50, 5000, or 20 000 ppm (equal to 0, 4, 350, or 1500 mg/kg bw per day in males and 0, 4, 400, or 1600 mg/kg bw per day in females) for 13 weeks. Clinical signs, body weight, and food consumption were recorded, a functional observational battery of tests was carried out, and locomotor activity was evaluated. Plasma, erythrocyte, and regional brain cholinesterase activities were measured in five animals of each sex per dose before treatment, at weeks 3 and 7, and at the end of the study. Tissues from the remaining five animals in each group were perfused in situ, and neuropathological examinations were carried out on the brains of the controls and animals at the high dose. All animals survived to the end of the study. Anogenital staining was observed in rats at the high dose, and body-weight gain and food consumption were reduced in comparison with controls. No treatment-related effects were seen in functional and locomotor evaluations. Plasma cholinesterase activity was > 20% lower than in concurrent controls in males at 20 000 ppm at all times after the start of treatment, while the activity in rats at 5000 ppm was reduced by 10-20%; erythrocyte acetylcholinesterase activity was reduced by > 20% at all times in rats at doses > 5000 ppm. In the females, reductions of > 20% were seen in plasma cholinesterase at 5000 ppm at week 7 only and at 20 000 ppm at all three times (all by comparison with concurrent controls). Reductions in erythrocyte acetylcholinesterase activity of > 20 % were seen at all times in females at 5000 and 20 000 ppm. Regional brain acetylcholinesterase activity was very variable; significant depressions in activity were seen only in rats at the high dose. Thus, significant depressions were seen in the olfactory lobe (by 34%) and midbrain (by 24%) and a marginally significant depression (18%) in the brain stem of males at week 13; in the cerebral cortex, a 26% depression in activity was seen at week 7 only. No clinically or biologically significant depression in activity was seen in the hippocampus or cerebellum of males. In females, depressed brain acetylcholinesterase activity was observed more often and frequently to a greater extent than in the males. Depressed activity in comparison with concurrent controls was seen in the olfactory lobe at 3 (31%), 7 (27%), and 13 weeks (50%) and in the brainstem at 13 weeks (36%), less depression in activity being seen at the other times. In the midbrain, depressed activity was seen at 7 (34%) and 13 weeks (40%). In the cerebral cortex, depressions were seen at 3 (32%), 7 (40%), and 13 weeks (53%). In the hippocampus, depressed activity occurred at all times, by 44% at 3 weeks, 38% at 7 weeks, and 47% at 13 weeks. In the cerebellum, depressions of 20% at 3 weeks and 32% at 13 weeks were seen. No effects were observed on the absolute or relative weights of the brain or brain regions, and no neuropathological abnormalities were observed. The NOAEL was 5000 ppm, equal to 350 mg/kg bw per day, on the basis of the occurrence of statistically and biologically significant inhibition of brain acetylcholinesterase activity at the highest dose (Lamb, 1994b). Malaoxon is a much more powerful anticholinesterase than malathion, and very pure samples of the latter have little activity (WHO, 1986). Thus, the IC50 values for cholinesterase inhibition in 17-day-old aggregate cultures of rat neural cells were > 12 × 10-4 mol/L for malathion and 2.8 × 10-4 mol/L for malaoxon (Segal & Federoff, 1989). Malaoxon produces a dimethylphosphorylated cholinesterase, however, which rapidly undergoes spontaneous reactivation, as shown ex vivo in the blood of malaoxon-poisoned rats, rabbits, dogs, and monkeys (Abraham & Edery, 1976). Abou Zeid et al. (1993) showed that there was faster recovery of serum cholinesterase activity in Sprague-Dawley rats after dermal application of pure malathion than of the 50% emulsifiable concentrate. Ward et al. (1993) reported a correlation between the anticholinesterase activity of a series of organophosphates, including malathion and malaoxon, and their ability to compete with agonist binding to muscarinic receptors. (ii) Other enzyme systems When rat microsomal suspensions were incubated with 4 mmol/L malathion in vitro, the release of ß-glucuronidase was inhibited (Lechener & Abdel-Rahman, 1985). (iii) Interactions with other organophosphates Feeding Holtzman rats with fenchlorphos potentiated the effect on erythrocyte or brain acetylcholinesterase activity of a single intraperitoneal challenge with malathion at 200 mg/kg bw (Murphy & Cheever, 1968). The combined effect on dioxathion and malathion was more or less than additive, depending on the doses used. Malathion acts synergistically with many other organophosphates, such as ethyl- para-nitrophenyl thionobenzenephosphonate (Frawley et al., 1957), at substantial doses. The LD50 of malathion is markedly reduced by co-administration of tri- ortho-tolylphosphate (Murphy et al., 1959). 2. Toxicological studies (a) Acute toxicity It is likely that the results of earlier studies on malathion were substantially affected by impurities. The LD50 values for these impurities in rats after oral administration are: isomalathion, 89-120 mg/kg bw, O,O,S-trimethylphosphorodithioate, 450-660 mg/kg bw, O,S,S-trimethyl-phosphorodithioate, 26-110 mg/kg bw, and O,O,S-trimethylphophorothioate, 47-260 mg/kg bw (Aldridge et al., 1979). The results of studies on the acute toxicity of malathion are given in Table 1; those on malaoxon are summarized in Table 2. (b) Short-term toxicity Rats Malathion (purity, 96.4%) was administered in the diet to groups of five male and five female albino Fischer (CDF:F-344/CrlBR) rats for 29 or 30 days at concentrations of 0, 50, 100, 500, 10 000, or 20 000 ppm, equal to 0, 5.1, 10, 52, 1000, or 2000 mg/kg bw per day for males and 0, 5.7, 12, 58, 1100, or 2200 mg/kg bw per day for females. The animals were observed weekly for body weight and food consumption. Ophthalmological, haematological, and clinical chemical examinations, including plasma and erythrocyte cholinesterase activity, were undertaken before treatment and at termination of the study; brain acetylcholinesterase activity was measured at termination. Animals were autopsied at the end of treatment, and selected organs were examined and weighed; microscopic examination of organs was carried out only in the controls and rats at 20 000 ppm. No deaths occurred during the study, adverse clinical signs were not seen, and no abnormalities were present on ophthalmological or haematological examination. A number of abnormal biochemical variables were noted, including cholinesterase activity. That in plasma was decreased by > 20% in animals at the two highest doses in comparison with concurrent controls, while erythrocyte acetylcholinesterase activity was decreased in males at 10 000 ppm (by 17%) and 20 000 ppm (by 16%) and only slightly in females. At 10 000 ppm, brain acetylcholinesterase activity was depressed at termination by 11% in males and 17% in females, while at 20 000 ppm there was a 26% decrease in males and 28% in females. Differences were seen between treated groups in total protein and albumin concentrations, and a significant decrease in alkaline phosphatase activity was seen in animals at the two highest doses. Animals at 20 000 ppm had a significant decrease in weight gain in comparison with the control group, while food consumption was decreased only during week 1. The relative and Table 1. Acute toxicity of malathion Species Strain Sex Route LD50 and 95% CI, ±SEM, Purity Reference or range (mg/kg bw, unless (%) otherwise stated) or LC50 Mouse Swiss white F Oral 6100 > 95 Toia et al. (1980) Mouse Swiss-Webster M,F Intraperitoneal 985 NR Menzer & Best (1968) 954-1018 Rate Wistar M Oral 2800 NR Dauterman & Main (1966) 2660-3110 Rat Osborne-Mendel NR Oral 1400 ± 100 98 Frawley (1957) Rat Sherman M Oral 1375 NR Gaines (1969) 1206-1568 F 1000 885-1130 Rat Wistar M,F Oral 1580 92.2 Pellegrini & Santi (1972) Rat Wistar M,F Oral 8000 98.2 Pellegrini & Santi (1972) Rat Wistar M Oral (laboratory chow) 1090 ± 83 95 Boyd & Tanikella (1969) Oral (26% casein) 1401 ± 99 Oral (3.5% casein) 5993 ± 138 Rat Sprague-Dawley M,F Oral 5000 ± 385 NR Terrell et al. (1978) Rat Sprague-Dawley M Oral 3800 (3040-4750) NR Cooper & Terrell (1979a) Rat Lac:P Oral 10 700 (9300-12 300) 99.7 Aldridge et al. (1979) Rat Sprague-Dawley F Oral 4400 (2533-8228) NR Cooper & Terrell (1979a) Table 1. (continued) Species Strain Sex Route LD50 and 95% CI, ±SEM, Purity Reference or range (mg/kg bw, unless (%) otherwise stated) or LC50 Rat Sprague-Dawley M Oral 3200 (2651-3862) NR Cooper & Terrell (1979b) F 3700 (2221-6164) Rat CD Sprague- M Oral 5400 (4100-6900) 96-98 Kynoch (1985a) Dawley-derived F 5700 (4300-7800) Rat Albino Crl:CD M Oral 6156 (4665-8123) 96.8 Fischer (1991) (SD)BR F 4061 (3078-5359) Rat Wistar M Oral 734 NR Jokanovic & Maksimovic F (1995) Rat HSD Sprague- M Oral 8210 (6518-10 342) 99.1 Kuhn (1996) Dawley F 8239 (6239-10 881) Rat Sprague-Dawley M Intraperitoneal 1100 NR Murphy et al. (1959) Rat Sherman M,F Derman (57% > 44 444 NR Gaines (1969) emulsifiable concentrate) Rat CD Sprague- M,F Derman > 2000 96-98 Kynoch (1985b) Dawley-derived Rat Albino Wistar M,F Inhalation (4 h) > 5.2 mg/L 96-98 Jackosn et al. (1986) Rabbit New Zealand M,F Derman 8.79 ± 0.48 NR Imlay et al. (1978) albino Hamster Syrian F Intraperitoneal (30% 24 00 NR Dzwonkowska & commercial Hubner (1986) preparation) Table 1. (continued) Species Strain Sex Route LD50 and 95% CI, ±SEM, Purity Reference or range (mg/kg bw, unless (%) otherwise stated) or LC50 Dog Mongrel NR Oral > 4000 98 Frawley (1957) Dog Mongrel M Intraperitoneal 1.517 ml/kg bw 95 Guiti & Sadoghi (1969) 0.77-2.25 Buffalo Indian (Bubalus M Oral 100-125 NR Gupta (1984) bubalis) Chicken White Leghorn F Oral 775 93.6 Fletcher (1989) 610-984 NR, not reported Table 2. Acute toxicity of malaoxon Species Strain Sex Route LD50 and 95% CI, ±SEM, Purity Reference or range (mg/kg bw, unless (%) otherwise stated) Mouse Swiss white F Oral 215 NR Toia et al. (1980) Rat Sprague-Dawley M,F Intraperitoneal About 25 NR Brodeur & DuBois (1967) Rat Wistar M Oral 158 NR Dauterman & Main (1966) 142-175 NR, not reported absolute weights of the liver were increased in males at the highest dose and in females at the two highest doses, and periportal hepatocytic hypertrophy was seen at the two highest doses in animals of each sex. These changes were considered to be related to treatment. Increased relative kidney weights were observed in males at the two highest doses and in females at the highest dose. The NOAEL was 500 ppm, equal to 52 mg/kg bw per day, on the basis of the increased weight of the livers with histopathological changes and inhibition of brain acetylcholinesterase activity (Daly, 1993a). Malathion (purity, 96.4%) was administered in the diet to groups of 10 male and 10 female albino Fischer (CDF:F-344/CrlBR) rats for 90 days at concentrations of 0, 100, 500, 5000, 10 000, or 20 000 ppm, equal to 0, 6.6, 34, 340, 680, or 1400 mg/kg bw per day for males and 0, 7.9, 39, 380, 780, or 1600 mg/kg bw per day for females. Body weight and food consumption were estimated before treatment and periodically during the study. Ophthalmological, haematological, and clinical chemical examinations, including plasma, erythrocyte, and brain cholinesterase activities, were undertaken before treatment and at termination of the study. The animals were killed at least 90 days after the start of the study and autopsied. Selected organs were examined and weighed, and all animals were examined microscopically. One male at the high dose died during the study from unknown cause. Anogenital staining was seen in four males and six females at the high dose during treatment, and in animals at this dose, body weights and weight gain were consistently lower than in the control group; there was a decrease in food consumption only in week 1, in contrast to greater food consumption by animals at the high dose than by controls later in the study. Haemoglobin count and haematocrit were decreased in males at the high dose, while the mean corpuscular volume and mean cell haemoglobin were decreased in males at doses > 5000 ppm. In females, the erythrocyte count was marginally increased at doses > 500 ppm, while the mean corpuscular volume was decreased at 10 000 and 20 000 ppm, and the mean cell haemoglobin was decreased at doses > 5000 ppm. A number of abnormal biochemical variables were noted, including cholinesterase activity. Plasma cholinesterase activity was decreased marginally (17%) in males at 5000 ppm, while there was clearly significant depression at the higher doses in comparison with the values in concurrent controls. In female rats, plasma cholinesterase activity was depressed by > 20% at doses > 5000 ppm. In the males, erythrocyte acetylcholinesterase activity was marginally but significantly depressed at 500 ppm (by 18% in comparison with concurrent controls) and markedly depressed at higher doses. In the females, depression of erythrocyte acetylcholinesterase activity by > 20% was observed at all doses. At 10 000 ppm, there was marginally significant depression of brain acetylcholinesterase activity at termination (by 13% in males and 17% in females), while at 20 000 ppm there was biologically significant depression, by 20%, in males and 44% in females At 5000 ppm, there was less inhibition of brain acetylcholinesterase activity in animals of each sex (8.8% in males and 10% in females), which was, however, statistically significant. Significant decreases in alkaline phosphatase activity were seen in males at the three highest doses and in females at the highest dose. The activity of gamma-glutamyl transpeptidase was elevated in males at the highest dose and in females at the two highest doses. A reduction in aspartate aminotransferase activity was observed in females at the highest dose. Differences in relative and absolute liver and kidney weights were seen between groups, with associated histopathological changes. The absolute and relative weights of the liver were increased in males at doses > 5000 ppm and in females at the highest dose. Periportal hepatocyte hypertrophy was seen in males at doses > 10 000 ppm and in females at doses > 5000; these changes were considered to be related to treatment. The absolute and relative weights of the kidney were increased in animals of each sex at the highest dose. At 10 000 ppm, the absolute and relative kidney weights were increased in males and the relative kidney weights in females; at 5000 ppm, the relative kidney weights were increased in animals of each sex. Chronic nephropathy was more severe in males at doses > 5000 ppm than in those at lower doses or in controls, but there were no differences between groups in the prevalence of this pathological change. The NOAEL was 500 ppm on the basis of decreased mean corpuscular volume and mean corpuscular haemoglobin, increased liver weights and relative kidney weights, and chronic nephropathy in males at the next highest dose and decreased mean corpuscular haemoglobin, hepatocytic hypertrophy, and increased relative kidney weight in the females at the next highest dose. There were also marginally significant decreases in brain acetylcholinesterase activity at 5000 ppm. The finding of a marginal increase in erythrocyte count at 500 ppm in females is ignored. The NOAEL is equal to 34 mg/kg bw per day (Daly, 1993b). Rabbits Malathion (purity, 94%) was applied to the skin of groups of six male and six female New Zealand white rabbits for 6 h per day on five days per week for three weeks at doses of 50, 300, or 1000 mg/kg bw per day; six males and five females were sham-treated. Effects on the skin, organ and body weights, food consumption, clinical chemical parameters including cholinesterase activity, and haematological variables were evaluated; selected organs were examined pathologically. Two males, one at 50 mg/kg bw per day and one at the high dose, died before termination of the study,. There were no physical alterations or changes in body or organ weights or food consumption attributable to treatment, except for erythema and oedema in treated animals. Decreases in plasma, erythrocyte, and brain cholinesterase activity > 20% were seen at the highest dose in animals of each sex; females also showed depression of erythrocyte activity at 300 mg/kg bw per day. Brain acetylcholinesterase activity was substantially reduced in the cerebrum and cerebellum of animals of each sex at the highest dose. Females at 300 mg/kg bw per day had a 19% reduction in brain acetylcholinesterase activity in comparison with concurrent controls, but this reduction was not statistically significant. The NOAEL was 300 mg/kg bw per day on the basis of inhibition of brain acetylcholinesterase activity at the highest dose (Moreno, 1989). Dogs Malathion (purity, 92.4%) was administered to groups of three male and three female beagles in gelatin capsules at doses of 0, 125, 250, or 500 mg/kg bw per day for 28 days. The animals were observed twice daily, and haematological and clinical chemical measurements were made before treatment and 15 and 29 days after the start of treatment. Selected organs were weighed and examined post mortem. Diarrhoea was observed at all doses, and anorexia at the highest dose. One male at the high dose died. Weight gain was reduced at the highest dose and marginally so at 250 mg/kg bw per day; food consumption was reduced at the highest dose. At 15 days, serum albumin and sodium levels were decreased in dogs at the highest dose, as were blood urea nitrogen, aspartate aminotransferase activity, and creatinine. Decreased plasma and erythrocyte cholinesterase activities were seen at 15 days and at termination: plasma cholinesterase activity was decreased by > 20% in dogs at the intermediate and high doses at 15 days and at all doses at termination; erythrocyte acetylcholinesterase activity was decreased by 20% at the high dose by 15 days and by 17% at all doses at termination. The LOAEL was 125 mg/kg bw per day, on the basis of reduced erythrocyte acetylcholinesterase activity at all doses at termination of the study, with clinical signs (diarrhoea) at all doses. No NOAEL was identified (Fischer, 1988). Malathion (purity, 95%) was administered in capsules to groups of six male and six female beagles at doses of 0, 62.5, 125, or 250 mg/kg bw per day on seven days a week for one year. The animals were observed twice daily, with more detailed examinations weekly; they were weighed before the start of the study, at the beginning of treatment, weekly thereafter, and at sacrifice. Food consumption was measured weekly, and haematological and clinical chemical variables, including plasma and erythrocyte cholinesterase activity, were determined before treatment, at six weeks, three months, six months, and just before the animals were killed. Cerebellar and cerebral acetylcholinesterase activity was determined at termination of the study. Ophthalmological examination was carried before the start of treatment and just before sacrifice. No clinical signs of toxicity were observed, nor was any abnormality seen on ophthalmological examination. No significant difference was seen in body weight or food consumption, although animals of each sex at the high dose showed a small decrease in mean weight. Clinical chemistry revealed perturbations in a number of variables. Plasma and erythrocyte cholinesterase activities were decreased by more than 20% in animals of each sex at all doses and times in comparison with concurrent controls. Brain acetylcholinesterase activity was unaffected, except for some diminution in cerebellar acetylcholinesterase activity at the highest dose (by 16% in males and 11% in females); cerebral cholinesterase activity was unaffected. Serum albumin, total protein, the albumin:globulin ratio, and calcium levels were decreased and lactate dehydrogenase increased in animals of each sex, generally only at the high dose but occasionally in those at the intermediate dose. In females, albumin was reduced at all doses at six weeks. Alkaline phosphatase activity was increased in males at the high dose but not in females. Other changes observed occasionally included low blood urea nitrogen in animals at the high dose and decreased alanine aminotransferase activity in those at the intermediate and high doses, but these did not appear to be of toxicological significance. Significantly lower calcium levels were found in animals at the high dose at six weeks and later. Haematological examination revealed dose-related decreases in erythrocyte and haemoglobin counts and haematocrit. The erythrocyte and haemoglobin counts were decreased at all times in males at the high dose, and the haematocrit was decreased in the males at the high dose at three and six months. In the females, erythrocyte and haemoglobin counts and haematocrit were marginally affected at six weeks in the group at the high dose, but at three months the haematocrits were decreased at the intermediate and high doses and haemoglobin count at the high dose. Additionally there was an increase in mean corpuscular volume at the high dose and in mean corpuscular haemoglobin at the intermediate and high doses. At six months, the erythrocyte and haemoglobin counts and haematocrit were all decreased in females at the high dose and the erythrocyte count in those at the intermediate dose. At termination, only a decrease in erythrocyte count was seen in females at the highest dose. Platelet counts were increased in males and females at all times and all doses in comparison with concurrent controls; many of these changes were statistically significant. Urinalysis revealed no marked changes. The absolute liver weights of females at the low and high doses were increased, and the relative liver weights were raised in males at the intermediate and high doses and in females at all doses. The absolute and relative kidney weights were raised in animals at the intermediate and high doses. No treatment-related pathological alteration was seen macroscopically or microscopically. The NOAEL was 125 mg/kg bw per day on the basis of body-weight depression and haematological and clinical chemical changes. The changes in liver weights and the reduced albumin in females at six weeks are discounted on the grounds that there was no morphological correlate and that there was no clear dose-response relationship (Schellenberger & Billups, 1987). (c) Long-term toxicity and carcinogenicity A number of long-term studies have been carried out in mice and rats by the US National Cancer Institute and others. Many of the studies were reviewed by IARC (1983) and by Rueber (1985). The working group convened by IARC concluded that the available data did not provide evidence that malathion or malaoxon is carcinogenic in humans. This view is in line with those of the study authors but not with those of Rueber (1983). More modern studies are now available and are summarized below, with a brief resumé of the earlier studies for the sake of completeness. Mice Groups of 50 B6C3F1 mice of each sex were given doses of 0, 8000, or 16 000 ppm malathion admixed in the diet for 80 weeks, equivalent to 1200 or 2400 mg/kg bw per day. The animals were killed 14-15 weeks after discountinuation of the malathion-containing diets. Throughout the study, the mean body weights of animals of each sex were lower than those of controls; poor food consumption, hyperexcitability, and abdominal distention were also noted in the second year; tremors were seen in a few female mice. Malathion was reported not to be carcinogenic (US National Cancer Institute, 1978); however, the slides were re-examined by Rueber (1985), who concluded that malathion had increased the incidence of neoplasms of the liver in male mice. Malathion (purity, 96.4%) was administered in the diet to groups of 65 B6C3F1 BR mice of each sex for 18 months at concentrations of 0, 100, 800, 8000, or 16 000 ppm (equal to 0, 17, 140, 1500, or 3000 mg/kg bw per day for males and 0, 21, 170, 1700, and 3500 mg/kg bw per day for females). Animals were observed twice daily and examined in detail weekly. Body weights were determined weekly until week 14, fortnightly to week 26, and monthly thereafter. Plasma, erythrocyte, and brain cholinesterase activity was determined 10 animals of each sex from each group killed at 12 months and at termination; only erythrocyte enzyme activity was determined in 10 mice of each sex per group at week 36, and these mice were retained until terminal sacrifice of the survivors at 18 months. The mice were examined post mortem, and selected organs were processed and examined histologically. There was no treatment-related effect on mortality, but body weights and food consumption were reduced in animals of each sex at 8000 and 16 000 ppm. Plasma cholinesterase activity was reduced by > 20% in comparison with concurrent controls at 12 and 18 months in males at 800 ppm; similar results were seen in females, except that the reduction was 18% in those at 800 ppm by 12 months. Erythrocyte acetylcholinesterase activity was decreased by > 20% in animals of each sex at doses > 800 ppm at 9, 12, and 18 months, while brain acetylcholinesterase activity was decreased in males at the highest dose at 12 and 18 months and in those at 8000 ppm at 18 months. In females, brain acetylcholinesterase activity was not decreased at 8000 ppm at 12 months but was decreased at 18 months; females at 16 000 ppm showed a 20% decrease in brain acetylcholinesterase activity at 12 months and a 43% decrease at 18 months. The absolute and relative liver weights were increased in males at the two highest doses, and the relative liver weight was increased in females at the highest dose. Other changes in organ weights included increased relative kidney weights in certain groups. Macroscopically, an increased incidence of liver nodules was seen at the two highest doses; microscopically, effects were seen on the liver, kidney, adrenal cortex, and bone. Liver hepatocellular hypertrophy was observed in all animals at the two highest doses at termination, and milder hypertrophy was seen in the animals killed at 12 months. The incidences of liver tumours in animals that survived to termination are given in Table 3. There was a significant trend in the incidence of adenomas in animals of each sex, and the incidence was significantly raised by comparison with controls at the two highest doses; the incidences in historical controls at the same laboratory were 14-22% in males and 0-11% in females. There was no significant trend for hepatocellular carcinoma, but the incidence was significantly raised in the males at 100 and 8000 ppm. Proximal tubular vacuolation seen at lower doses in the kidneys was absent in all males at the highest dose and most of those at 8000 ppm. Female mice at 8000 and 16 000 ppm had an increased incidencse of renal cortical mineralization. A treatment-related decrease in fibrous osteodystrophy of the sternum observed in females at the highest dose is of unknown significance. A treatment-related, early disappearance of the X zone of the adrenal cortex was observed in females at 8000 and 16 000 ppm at 12 months. The overall NOAEL was 800 ppm, equal to 140 mg/kg bw per day, on the basis of inhibition of brain acetylcholinesterase activity and an increased incidence of liver adenomas in animals of each sex at the next highest dose (Slauter, 1994). Rats Malathion Three studies were carried out by Hazleton Laboratories (Hazleton & Holland, 1953). In the first, groups of 20 male Colworth Farm rats were given technical-grade malathion (purity, 65%) in the diet at doses of 0, 100, 1000, or 5000 ppm, equivalent to 5, 50, and 250 mg/kg bw per day, for 109 weeks. Body weight and food consumption were decreased in those at the highest dose. Depressed cholinesterase activity was seen in rats at 5000 ppm and to a lesser extent in those at 1000 ppm. The study was not adequate for conclusions to be drawn about carcinogenicity. In the second study, with the same doses, similar criticisms can be made, except that the purity of the malathion was > 90%. A third study was carried out with male and female Colworth Farm rats which received doses of 0, 500, 1000, 5000, or 20 000 ppm of malathion (purity, 99%). The highest dose was lethal to the male rats; the size of the study precluded conclusions about carcinogenicity. Groups of 50 male and 50 female Osborne-Mendel rats were given technical-grade malathion (purity, 95%) in the diet at concentrations of 4700 or 8150 ppm (equivalent to 240 and 410 mg/kg bw per day) for 80 weeks. A pooled control group consisted of 15 matched controls of each sex and 40 untreated male and female rats from bioassays of other chemicals. The rats were killed after 109 weeks. The body weights of female rats receiving malathion were lower than those of controls, and the survival times of those at the higher dose were decreased. Malathion was reported not to be carcinogenic (US National Cancer Institute, 1978). The slides were re-evaluated by Rueber (1985), who Table 3. Incidences of hepatocellular tumours (%) in mice at terminal sacrifice after treatment with malathion Tumour Dose (ppm) Males Females 0 100 800 8000 16 000 0 100 800 8000 16 000 Adenoma 2 11.8 4.2 24.1 98.0 0 1.9 0 17.9 82.4 Carcinoma 0 11.8 4.2 11.1 2.0 1.8 0 3.8 1.9 3.9 found that the incidence of benign and malignant neoplasms at all sites analysed together was increased in treated rats; in particular, the incidence of carcinomas of the endocrine organs was increased, and malignant neoplasms of the brain were observed in seven treated rats. Rueber concluded that malathion was carcinogenic in male and female Osborne-Mendel rats. In a re-evaluation of the slides commissioned by the US National Toxicology Program (Huff et al., 1985), the original interpretation that malathion is not carcinogenic was confirmed. In a second study by the US National Cancer Institute (1979a), malathion (purity, 95%) was fed in the diet to groups of 50 male and 50 female Fischer 344 rats at doses of 0, 2000, or 4000 ppm (equivalent to 100 or 200 mg/kg bw per day; only 49 males at the higher dose) for 103 weeks. The authors concluded that malathion was not carcinogenic. When Rueber (1985) re-evaluated the slides, he found that the incidence of benign and malignant neoplasms analysed together was significantly increased, particularly in males. He concluded that malathion was carcinogenic in Fischer 344 rats. A re-evaluation of the slides commissioned by the US National Toxicology Program (Huff et al., 1985) confirmed that malathion was not carcinogenic. Malathion (purity, 92.1%) was given in the diet to groups of 50 male and 50 female Sprague-Dawley rats at concentrations of 0, 100, 1000, or 5000 ppm, equivalent to 5, 50, or 250 mg/kg bw per day. The animals were observed daily throughout the study, and body weights and food consumption were recorded at the end of weeks 1, 13, 24, 53, 79, and 103. Blood samples for haematological examination and determination of cholinesterase activity and urine samples for urinalysis were collected from five rats of each sex per group in weeks 12, 26, and 53; blood and urine were also collected at week 104, and alanine and aspartate aminotransferase activities, urea nitrogen, and glucose were determined additionally in blood. Brain acetylcholinesterase activity was not determined. Animals that died, were killed in extremis, or killed at termination were examined post mortem. No significant difference in food consumption or survival was seen, and no significant intergroup differences were seen on haematological or biochemical examination, except in cholinesterase activity. During the first year of the study, the body weights of animals of each sex at the highest dose were reduced, while in the second year the body weights of those at 1000 and 5000 ppm were depressed. Moreover, erythrocyte acetylcholinesterase activity was reduced by > 20% in comparison with the controls in rats at the intermediate and high doses at 3, 6, 12, and 24 months; plasma cholinesterase activity was less affected, although there was a depression of > 20% in the females at the high dose at 12 and 24 months. Absolute and relative liver weights were increased in male rats at the high dose and relative kidney weights in males at the two highest doses. Absolute brain weights were decreased in females at the two highest doses and relative kidney weights in those at the high dose. Although foci of cellular alteration were recorded twice in the livers of males at the highest doses and once in females at the intermediate and high doses, this difference was not statistically significant. A significant difference in sinusoidal dilatation was found between the controls (2%) and males at the high dose (16%). Extramedullary splenic haematopoiesis was seen more often in males at the high dose than in controls. There was no evidence of carcinogenic potential. The NOAEL was 100 ppm, equivalent to 5 mg/kg bw per day, on the basis of reduced erythrocyte acetylcholinesterase activity and body weight at the next highest dose (Rucci et al., 1980). After an audit by the US Environmental Protection Agency (1987; Cyanamid, 1990), the Agency requested a re-evaluation of the slides. Seely (1991) found only two treatment-related lesions: periportal hepatocellular hypertrophy and cystic hepatocellular degeneration, both only in male rats at the highest dose. There was no evidence of differences in tumour incidence. The NOAEL for effects on the liver was thus 1000 ppm (equivalent to 50 mg/kg bw per day). This re-evaluation does not alter the overall NOAEL of 5 mg/kg bw per day (see above). Malathion (purity, 96.4%) was administered in the diet of groups of 90 male and female Fischer 344 (CDF:F-344/CrlBR) rats at concentrations of 0, 100/50, 500, 6000, or 12 000 ppm for two years; the lowest dose was reduced from 100 to 50 ppm at week 18 because of inhibition of erythrocyte acetylcholinesterase activity, resulting in mean intakes over the entire study of 0, 4, 29, 360, and 740 mg/kg bw per day for males and 0, 5, 35, 420, and 870 mg/kg bw per day for females. Groups of 10 animals of each sex per group were killed at three and six months, 15 of each sex per group at 12 months, and the remainder at two years. Physical condition, ophthalmoscopic parameters, body weight, and food consumption were determined before treatment and at selected intervals, while electroretinography, haematology, and clinical chemistry (including determination of cholinesterase activity) were performed at selected intervals and on selected animals. Selected organs from animals killed at 12 and 24 months were weighed, and the animals were examined macroscopically. Tissues from those at the high dose and from controls, and certain organs from animals at the low dose were examined histopathologically. Malathion reduced the survival of males at 6000 and 12 000 ppm, early deaths being observed from the 14th month in males at the highest dose and from about the 20th month in those at the next lowest dose. Survival of females at the high dose was impaired towards the end of the study. Nephropathy and mononuclear-cell leukaemia were the main causes of death, although the frequency of neither was treatment-related. Anogenital staining was seen in females at the highest dose. Decrements in body weight and weight gain were seen in animals of each sex throughout the study at the two highest doses, although mean food consumption was greater in these animals than in the controls, throughout the study in the case of the males and in the second year of the study in the case of the females. Decreases in mean haemoglobin concentration, haematocrit, mean corpuscular volume, and mean cell haemoglobin were seen in animals of each sex at the two highest doses at 6, 12, and 18 months, although all parameters were not affected at all the time intervals and there was a tendency for improvement during the study. The mean cell haemoglobin concentration was decreased in males at the two highest doses only at 12 months, accompanied by an increase in platelet count. At 3, 6, 12, and 24 months, animals of each sex showed reductions in plasma, erythrocyte, and brain cholinesterase activity, predominantly at the two highest doses. Thus, animals of each sex at the highest dose had decreased plasma cholinesterase activity at all times. In males at 6000 ppm, decreases in activity > 20% were seen at three and six months and at termination, while there was a marginal decrease at 12 months (83% of control value). Males at 500 ppm had a significant decrease in activity only at termination, when the activity was 71% of that of concurrent controls. In the females, plasma cholinesterase activity was consistently reduced by at the two highest doses; the activity was little affected at 500 ppm, except at termination when there was a marginally significant decrease of 18%. There were consistent reductions in erythrocyte acetylcholinesterase activity at the two highest doses in males and a marginally significant reduction at 500 ppm at termination only, when the activity was 83% that of concurrent controls. Erythrocyte acetylcholinesterase activity was similarly reduced in females at 6000 and 12 000 ppm. Although reductions > 20% were seen in females at 500 ppm at three months and at termination and a marginal reduction at 12 months (86% of control value), no reduction was seen at six months. Erythrocyte acetylcholinesterase activity was also reduced in females at the lowest dose at three months (75% of control value), so that on day 113 the lowest dose was reduced from 100 ppm to 50 ppm. Six weeks later, erythrocyte acetylcholinesterase activity was evaluated in 10 controls and 10 at 50 ppm and found to be comparable. Thereafter, the activity in animals at 50 ppm was unremarkable. Brain acetylcholinesterase activity was reduced by > 20% in males at 6000 ppm at termination. Decreases seen at the highest dose were 84% of the control value at three months, 81% at six months, and 85% at 12 months; no determination was carried out at termination because there were no survivors. At 6000 ppm, the activity was decreased to 88% of the control value at three months, 88% at six months, and 89% at 12 months. In females at 12 000 ppm, the activity of brain acetylcholinesterase was substantially reduced in comparison with that of concurrent controls. Smaller decreases were seen at 6000 ppm at three months (15%), six months (17%), 12 months (12%), and termination (18%). No significant inhibition was seen at lower doses. Alkaline phosphatase activity was reduced in comparison with concurrent controls in animals of each sex at the two highest doses at 6 and 12 months and at the highest dose at 18 months. Aspartate aminotransferase activity was reduced in females at doses > 500 ppm at 12 months and at the highest dose at 18 months. Alanine aminotransferase activity was also decreased in females at the three highest doses at 12 months. gamma-Glutamyl transpeptidase activity was increased consistently in males at the two highest doses from 12 months and at most intervals in females. Cholesterol content was increased in animals of each sex at the two highest doses at 6, 12, 18, and 24 months. Increases in mean and relative liver and kidney weights were observed in animals of each sex at the two highest doses at the interim sacrifice, in females at 6000 and 12 000 ppm at terminal sacrifice, and in males at 6000 ppm at terminal sacrifice. Males also had decreased relative kidney weights at 500 ppm. Relative spleen weight was increased in males at the two highest doses at interim sacrifice, and absolute spleen weight was reduced in males at 6000 ppm and in females at 12 000 ppm at terminal sacrifice. Relative and absolute thyroid and parathyroid weights were elevated in males at the two highest doses at interim sacrifice, in males at 6000 ppm at termination, and in females at 6000 and 12 000 ppm at termination. Microscopic findings of significance were largely confined to nasoturbinal tissues, kidney, and liver. Degeneration and hyperplasia of the olfactory epithelium were seen in animals of each sex at the two highest doses. The hyperplasia was focal, with thickening of the epithelium and proliferation of basal cells, forming clusters in the lamina propria. While focal degeneration was also observed in a few controls and rats at lower doses, the hyperplasia was confined to those at the two highest doses. In several rats, the epithelium was replaced by ciliated and non-ciliated columnar epithelium. Subacute and chronic inflammation and dilated and hyperplastic mucosal glands were seen in some animals; subacute and chronic inflammation and hyperplasia of the respiratory epithelium of the nasopharynx and dilated mucosal glands were also seen. Like the other changes in nasal tissues, inflammatory cells and cell debris were seen most frequently at the two highest doses. Thus, the NOAEL for this effect was 500 ppm. The incidence and severity of nephropathy was greater in rats at 6000 and 12 000 ppm than in controls. A nasal turbinate adenoma was seen in one male at 6000 ppm and a carcinoma in one male at 12 000 ppm; although the numbers observed were small, this is a rare tumour in Fischer rats. Hepatocellular adenomas and carcinomas were seen in some animals. In the female rats, the prevalence of adenomas and cacinomas combined was increased at the highest dose and that of adenomas alone at 6000 ppm (see Table 4). Testicular interstitial-cell tumours were seen in virtually all male rats that survived to termination. The overall NOAEL was 500 ppm, equal to 29 mg/kg bw per day, on the basis of decreased survival and body-weight gain, increased food consumption, changes in haematological parameters, decreased brain acetylcholinesterase activity, increased g-glutamyl transpeptidase activity, increased liver, kidney, thyroid, and parathyroid weights, and degeneration and hyperplasia of the olfactory epithelium at the next highest dose. Although an increased incidence of liver tumours was seen in females, malathion was not considered to be carcinogenic in view of the small numbers of such tumours observed (Daly, 1996a). Table 4. Prevalences of hepatocelular adenomas and carcinomas in rats at termination after treatment with malathion Dose (ppm) Males Females 0 100/50 500 6000 12 000 0 100/50 500 6000 12 000 No. of animals 37 41 29 14 0 38 41 41 34 20 Tumour Adenoma 2 2 3 1 0 0 0 1 3 3 Carcinoma 1 1 0 1 0 0 1 1 0 1 Malaoxon Malaoxon (purity, > 95%) was fed to groups of 50 Fischer 344 rats of each sex in the diet at concentrations of 0, 500, or 1000 ppm for 103 weeks. The authors concluded that malaoxon was not carcinogenic in rats (US National Cancer Institute, 1979b). The slides were re-examined by Rueber (1985), who concluded that the incidence of benign and malignant neoplasms at all sites was increased in the treated animals. The neoplasms in question were in endocrine organs, including the pituitary, adrenal, and thyroid glands; the incidences of hyperplasia, adenomas, and carcinomas of C cells of the thyroid were increased. In a re-evaluation of the slides commissioned by the US National Toxicology Program (Huff et al., 1985), the original conclusion that malaoxon is not carcinogenic was largely confirmed, but there was stated to be equivocal evidence that malaoxon is carcinogenic in that there was an increased incidence of C-cell neoplasms of the thyroid. Groups of 85 Fischer 344 (CDF:F-344/CrlBR) rats of each sex were exposed to malaoxon (purity, 96.4%) admixed in the diet at concentrations of 0, 20, 1000, or 2000 ppm (equal to 1, 57, or 110 mg/kg bw per day in males and 1, 68, or 140 mg/kg bw per day in females); 55 animals were retained for 24 months, while 10 of each sex per group were killed at 3, 6, and 12 months. Cholinesterase activity was estimated in all animals. Clinical chemical and haematological parameters were determined in all animals killed at 6 and 12 months and in 10 animals of each sex per group of animals that were retained at 18 months and termination. Physical observations, ophthalmoscopy, and measurements of body weight and food consumption were carried out before treatment and at selected intervals during the study. The surviving rats were sacrificed at 24 months. The rats were examined post mortem, and selected organs were weighed. Histopathological examinations were perfomed on controls and those at the high dose at 12 and 24 months and on rats that died or were killed in extremis during the study. Selected tissues from animals at the intermediate and low doses were also examined. Survival was curtailed in female rats at 1000 and 2000 ppm and in males at 2000 ppm. The most common causes of death were pneumonitis and mononuclear leukaemia; the occurrence of the former appeared to be dose-related. Anogenital staining was seen in females at the highest dose throughout the study and in males in the latter part of the study. Treatment-related decreases in body weight and weight gain were seen at the highest dose. Food consumption was decreased in males at 1000 and 2000 ppm. Plasma, erythrocyte, and brain cholinesterase activity was affected by malathion. Plasma cholinesterase activity was reduced by > 20% at all times in animals of each sex at the two highest doses. Erythrocyte acetylcholinesterase activity was reduced by > 20% in the same groups and in males at the lowest dose at six months; the reductions at other times in animals of each sex at this dose were 10-20%. Brain acetylcholinesterase activity was reduced by 18-11% in comparison with concurrent controls in males at the highest dose and more clearly reduced in females at earlier times. There were substantial reductions in brain acetylcholinesterase activity in animals of each sex at the highest dose at termination of the study. At the intermediate dose, there was a 30% decrease in brain acetylcholinesterase activity in males at termination. No abnormality was seen on ophthalmoscopic examination. Although there were sporadic differences in clinical chemical measurements between groups, none appeared to be treatment-related. The absolute and relative liver and kidney weights were increased in males at 2000 ppm at 12 months, and the relative and absolute adrenal weights were increased in males at this dose at two years. The absolute and relative spleen weights of females at 2000 ppm were decreased. The incidence of emaciation: was increased in males at 2000 ppm and in females at 1000 and 2000 ppm. Inflammatory changes in the nasal turbinates, lungs, and tympanic spaces, which may have been secondary to increased disposition of food particles, were present in males at the highest dose and females at the two highest doses. Thus, foreign material such as food was found in the nasal lumen with inflammatory cells and cell debris. The nasal mucosa also showed chronic inflammatory changes and hyperplasia and hypertrophy of goblet cells. In a small number of rats, squamous metaplasia was observed. Degeneration of the olfactory epithelium was accompanied by focal replacement by ciliated and non-ciliated columnar epithelium. Mineralization of the stomach was seen in males at the two highest doses and females at the highest dose. No treatment-related neoplasia was observed. Interstitial tumours of the testis were present in > 75% of the animals at all doses and were not considered to be related to treatment. The NOAEL was 20 ppm, equal to 1 mg/kg bw per day, on the basis of decreased food consumption and brain acetylcholinesterase activity at termination in males and emaciation at termination and inflammatory changes in the nasal turbinates in females at the next highest dose (Daly, 1996b). (d) Genotoxicity The results of tests for the genotoxicity of malathion and malaoxon are shown in Table 5. Four impurities in malathion, isomalathion, O,O,S-trimethyl phosphorothioate, O,S,S-trimethyl phosphorodithioate, and O,O,O-trimethyl phosphorothioate of > 99% purity were tested for their potential to induce reverse mutation in S. typhimurium TA97, TA98, and TA100 at doses of 10-1000 µg/plate. Negative results were obtained, with and without metabolic activation and with and without preincubation (Imamura & Talcott, 1985). (e) Reproductive toxicity (i) Multigeneration reproductive toxicity In a small study in Wistar rats given malathion in the diet at about 240 mg/kg bw, a higher incidence of ring-tail disease was seen in treated than in control rats (Kalow & Marton, 1961). Table 5. Results of tests for the genotoxicity of malathion and malaoxon End-point Test system Concentration Purity Results Reference (%) Malathion In vitro Reverse mutation S. typhimurium NR NR Negativea McCann et al. (1975) TA98, TA100, TA1535, TA1537 Reverse mutation S. typhimurium 100-5000 µg/plate 95.2 Negativea Traul (1987) TA98, TA100, TA1535, TA1537, TA1538 Reverse mutation S. typhimurium 5-300 µg/plate NR Positive Shiau et al. (1980) TA98, TA100, (TA 1535 TA1535, TA1536, without S9) TA1537, TA1538 Reverse mutation S. typhimurium 33-165 µg/plate NR Negativeb Pednekar et al. (1987) TA97a, TA98, TA100 Reverse mutation S. typhimurium NR NR Negative Byeon et al. (1976)c TA98, TA100, TA1535, TA1538 Reverse mutation S. typhimurium 80 and 400 ppm/plate 90-95 Negativea Wong et al. (1989) TA98, TA102, TA1535, TA1537 Reverse mutation S. typhimurium < 5000 µg/plate NR Negativea Moriya et al. (1983) TA98, TA100, TA1535, TA1537, TA1538 Table 5. (continued) End-point Test system Concentration Purity Results Reference (%) Reverse mutation S. typhimurium < 10 mg/plate NR Negativea Waters et al. (1982) TA98, TA100, TA1535, TA1537, TA1538 Reverse mutation S. typhimurium NR (preincubation) NR Positive Ishidate et al. (1981) TA98, TA100, (TA 100 with TA1537 S9) Reverse mutation Escherischia coli NR NR Negativea Nagy et al. (1975) Reverse mutation Escherischia coli 100-5000 µg/plate 95.2 Negativea Traul ( t 987) Reverse mutation Escherischia coli < 5000 µg/plate NR Negativea Moriya et al. (1983) Reverse mutation Escherischia coli < 10 mg/plate NR Negativea Waters et al. (1982) Forward mutation Escherischia coli 2 × 20 mol/L NR Negative Mohn (1973) Forward mutation Schizosaccharomyces NR 99 Negative Degraeve et al. (1980) pombe Forward mutation Schizosaccharomyces 30-182 mmol/L NR Negativea Gilot-Delhalle et al. pombe (1983) DNA damage Bacillus subtilis 5-300 µg/plate NR Positive Shiau et al. (1980) rec and exc DNA damage Bacillus subtilis rec 200 µg/plate NR Negative Shirasu et al. (1976) DNA damage Bacillus subtilis rep NR NR Negative Waters et al. (1982) Table 5. (continued) End-point Test system Concentration Purity Results Reference (%) DNA damage Bacillus subtilis rew NR NR Negative Waters et al. (1982) Primary DNA Saccharomyces NR NR Negative Waters et al. (1982) damage cerevisiae Unscheduled Primary rat 0.01-0.16 µl/ml 94 Negative Pant (1989) DNA synthesis hepatocytes Unscheduled Human lung NR NR Negativea Waters et al. (1982) DNA synthesis fibroblasts Chromosomal Chinese hamster NR NR Positivea Ishidate et al. (1981) aberration lung fibroblasts Chromosomal Cultured human 0.02-20 µg/ml NR Positive Balaji & Sasikali (1993) aberrations peripheral leukocytes Sister chromatid Cultured human 0.02-20 µg/ml NR Positive Balaji & Sasikali (1993) exchange peripheral leukocytes Chromosomal Cultured human 33-660 µg/ml NR Positivea Garry et al. (1990) aberrations peripheral leukocytes Sister chromatid Cultured human 33-660 µg/ml NR Positivea Garry et al. (1990) exchange peripheral leukocytes Sister chromatid Human fetal fibroblasts 2.5-40 µg/ml 99 Positive Nicholas et al. (1979) exchange Table 5. (continued) End-point Test system Concentration Purity Results Reference (%) Sister chromatid Chinese hamster V79 10-80 µg/ml 94 Positive Chen et al. ( 1981) exchange/cell cells (high dose) cycle delay Sister chromatid Chinese hamster ovary 0.03-1 mmol/L 99 Positive Nishio & Yueki (1981) exchange cells In vivo Sex-linked recessive Drosophila melanogaster Feeding: adult, 50 ppm; 50% Negative Velazquez et al. (1987) lethal mutation larva, 100 ppm ECd Injection: adult, 10 and 25 ppm Sex-linked recessive Drosophila melanogaster NR NR Negative Waters et at. (1982) lethal mutation Sex chromosome Drosophila melanogaster Feeding: 0-10 ppm 50% Negative Velazquez et al. (1987) loss adult Injection: 0 and 5 ppm ECd Non-disjunction Drosophila melanogaster Feeding: 0-20 ppm Ecd 50% Negative Velazquez et al. (1987) Dominant lethal Mouse NR NR Negative Degraeve et al. (1980) mutation Dominant lethal Mouse 'Maximum lethal dose' NR Negative Waters et al. (1982) mutation Chromosomal Rat bone marrow 0.5-2.0 g/kg 94 Negative Gudi (1990) aberration (Sprague-Dawley) Chromosomal Syrian hamster bone 0.24-1.2 g/kg 30e,f Positive Dzwonkowska & aberration marrow Hubner (1986) Table 5. (continued) End-point Test system Concentration Purity Results Reference (%) Chromosomal CFW mouse spermatocytes 0.45 mg/day for 30f Positive Bulsiewicz et al. (1976) aberration 50 or 100 days Chromosomal Mouse bone marrow and 500-2000 mg/kg bw NR Positive Salvadori et al. (1988) aberration primary spermatocytes single dose or 5 daily doses dermally Malaoxon In vitro Reverse mutation S. typhimurium 100-10 000 µg/plate 94.4 Negativea Zeiger et al. (1988) TA97, TA98, TA100, TA1535, TA1537 Cell mutation Mouse lymphoma 12.5-300 nl/ml NR Positive Myhr & Caspary (1991) tk locus L5178Y cells without S9; equivocal with S9 Sister chromatid Chinese hamster ovary 0.03-1 mmol/L 96 Positive Nishio & Yueki (1981) exchange cells Chromosomal Chinese hamster ovary > 5 mg/ml 94.4 Negative Ivett et al. (1989) aberration cells Sister chromatid Chinese hamster ovary > 5 mg/ml 94.4 Positivea Ivett et al. (1989) exchange cells In vivo Sex-linked recessive Drosophila melanogaster Feeding: 5 ppm 94.4 Positive Foureman et al. (1994) lethal mutation Injection: 2 ppm feeding; negative, injection Table 5. (continued) NR, not reported; S9, 9000 × g supernatant of rodent liver a With and without metabolic activation b With and without metabolic activation with S9 and caecal microbial extract c In Korean; not fully evaluated d 50% emulsifiable concentrate e 30% commercial preparation from Organika-Azot, Poland f Sadofos-30 (approximately 30% solution) In a two-generation study of reproductive toxicity, groups of 25 male and 25 female CD:Sprague-Dawley-derived rats were fed diets containing malathion (purity, 94%) at concentrations of 0, 550, 1700, 5000, or 7500 ppm, equal to 0, 43, 130, 390, or 600 mg/kg bw per day in the F0 males and 0, 50, 150, 440, and 660 mg/kg bw per day in the F0 females. The equivalent intakes for the F1 generation were: 43, 130, 390, or 630 mg/kg bw per day for males and 51, 150, 460, and 750 mg/kg bw per day for females. Each parent generation was mated to produce two litters, and offspring were selected randomly from the second (F1b) litter to be the parents of the next generation. Offspring that were not selected, the offspring of the first litters (F1a and F2a), and the F2b offspring were examined grossly and discarded. One pup of each sex per F1b and F2b litter was selected randomly, killed, and examined post mortem; abnormal tissues were saved. The F0 and F1 adults were killed and examined post mortem, and the reproductive organs and abnormal tissues were saved. Tissues from the controls and animals at the high dose were examined histologically. Treatment had no effect on clinical signs, growth before mating, food consumption, maternal weight gain during gestation, reproductive performance, fertility indices, gestation length, or parturition in the F0 and F1 parental generations. Pup sex ratio and survival were also unaffected. Pup weight was reduced at day 21 in the F1a litters at 5000 and 7500 ppm and in the F1b litters at 7500 ppm. Mean pup weights in the F2a litters were comparable in all groups, except in those at the high dose on day 21, which were decreased. Pup weights were reduced at days 4, 7, 14, and 21 in the F2b litters at 5000 ppm but not at 7500, except on day 21. At the highest dose, mean pup weight at day 21 was lower than that of concurrent controls. Similar effects were not seen at lower doses. Examination post mortem showed no treatment-related effects. The NOAEL for reproductive toxicity was 7500 ppm, equal to 600 mg/kg bw per day, while that for developmental toxicity was 1700 ppm, equal to 130 mg/kg bw per day (Schroeder, 1990). In a study reported briefly, malathion (purity unspecified) was administered to 16 male JIPMER albino rats for 12 weeks at a dose of 45 mg/kg bw per day by gavage. There were 12 appropriate controls. The histological changes observed included interstitial oedema, congestion, desquamation of cells lining the seminiferous tubules, reduced numbers of spermatogonia, and absence of Leydig cells (Balasubramanian et al., 1987). (ii) Developmental toxicity Female Sherman-strain rats were pair-mated with healthy adult male rats of about the same age. On day 11 after insemination, they were given a single intraperitoneal injection of malathion at a dose of 700 or 900 mg/kg bw. On day 20 of gestation, the fetuses were removed. The offspring and placentae were weighed, the numbers of resorptions and dead animals were recorded, and half of the offspring were examined in detail. The higher dose of malathion affected the body weight of the dams but had no effect on the weight of the fetuses and did not induce malformations (Kimbrough & Gaines, 1968). Technical-grade malathion (purity unspecified) was administered by gavage to groups of 20 female Wistar rats at doses of 0, 50, 100, 200, or 300 mg/kg bw on days 6-15 of gestation. Neither maternal nor fetal toxicity was observed at the highest dose used (Khera et al., 1978). After a dose-ranging study, groups of female Crl:CD:(SD)BR rats were given malathion (purity, 94%) by gavage on days 6-15 of gestation at doses of 0, 200, 400, or 800 mg/kg bw per day in corn oil; the groups consisted of 24 rats at the lowest dose and 25 at the other doses. The animals were observed daily for clinical signs, body-weight gain, and food consumption. After 20 days, the rats were killed and examined for pregnancy, implantations, resorptions, live and dead fetuses, and number of corpora lutea; the uterus was weighed, and fetuses were examined for malformations. Cholinesterase activity was not measured in this study. Malathion had no effect on survival, the only early death occurring in the control group. Five rats at the highest dose had urine staining of abdominal fur and decreased mean weight gain and food consumption during treatment; after treatment, the weight gain of animals at the high dose was increased in comparison with the controls. No effect was seen on pregnancy rate or numbers of corpora lutea, implantations, resorptions, or fetuses per litter, fetal body weight, or sex ratio of fetuses. No fetal abnormality attributable to treatment was observed. The NOAEL was 400 mg/kg bw per day on the basis of maternal toxicity at the highest dose. The NOAEL for fetal toxicity was 800 mg/kg bw per day (Lochry, 1989). Malathion (70% with 30% calcium carbonate) was administered at a dose of 100 mg/kg bw per day on days 7-12 of gestation to seven mated New Zealand white rabbits. Five mated rabbits received the vehicle alone. No difference between the treated and control groups was seen in respect of resorptions, fetal size, or external or visceral abnormalities. The NOAEL for effects on the fetus was thus 100 mg/kg bw per day. It is unclear from the paper whether any significant maternal toxicity was observed (Machin & McBride, 1989). After a range-finding study, malathion (purity, 92.4%) was administered at doses of 25, 50, or 100 mg/kg bw per day by gavage in corn oil to groups of 20 mated female New Zealand white rabbits on days 6-18 of gestation; controls received the vehicle alone. The rabbits were examined daily for mortality and for physical and behavioural abnormalities. Body-weight gain was calculated for days 0-6, 6-12, 6-18, 18-29, and 0-29 days after the start of the study and on days 6, 12, 18, and 29. The survivors were killed on gestation day 29 and examined post mortem. The uterus and ovaries were excised and examined, and the number of corpora lutea recorded. The number and position of live and dead fetuses, resorption sites, and the total number of implantation sites was also recorded. Live fetuses were removed, weighed, measured crown to rump, and examined for gross external and visceral abnormalities. Fetuses were then processed and examined for skeletal abnormalities. Cholinesterase activity was not measured. Although there was no statistically significant difference in survival between the treated and controls groups, no deaths occurred among the controls, four in the group at the low dose, three in the group at the intermediate dose, and two in the group at the high dose. In the last group, the deaths resulted from intrapulmonary intubation. Maternal weight gain was reduced at the doses of 50 and 100 mg/kg bw per day during treatment on days 6-18 of gestation. At days 12, 18, and 29, the mean body weight of the animals at the high dose was decreased in comparison with the controls. The mean number and percent of resorptions was slightly increased at doses > 50 mg/kg bw per day. There was no difference in fertility, number of corpora lutea, implantation sites, litter size, or fetal weight or length. No other signs of toxicity were seen in does or fetuses, nor was there any evidence of teratogenicity. The NOEAL was 25 mg/kg bw per day for maternal toxicity and 100 mg/kg bw per day for fetal toxicity, the former being based on decreased weight gain at the next highest dose and the latter on the absence of fetal toxicity at any dose (Siglin, 1985). (f) Special studies (i) Dermal and ocular irritation and dermal sensitization A single semi-occluded application of malathion (purity, 96-98%) to the skin of New Zealand white rabbits elicited slight to well-defined, transient dermal reactions, with very slight oedema in six animals and very slight erythema in five animals; the sixth had well-defined erythema. The skins were all normal by day 2 (Liggett & Parcell, 1985a). Malathion (purity, 96-98%) produced mild conjunctival reactions in the eyes of New Zealand white rabbits. No damage to the cornea or iris was seen at any stage, and the eyes were normal after two days (Liggett & Parcell, 1985b). Malathion (purity, 96-98%) was tested in nine albino guinea-pigs; there were 10 controls. One treated animal with respiratory distress was killed in extremis. There was no evidence of delayed contact hypersensitivity (Kynoch & Smith, 1985). (ii) Macrophage and mast cell function Repeated administration of malathion to female C57Bl/6 mice at a dose of 1 mg/kg bw per day increased macrophage function, while 0.1 mg/kg bw per day caused mast cell degranulation (Rodgers & Xiong, 1997). (iii) Ocular function Malathion (purity, 98%) instilled into the eyes of Long-Evans rats had no effect on responses evoked by a visual pattern and produced no ophthalmological abnormality (Boyes, 1997). (iv) Neurotoxicity The potential of malathion (purity, 93.6%) to induce delayed neuropathy was tested in white Leghorn hens. After determination of the oral LD50, the ability of atropine to antagonize the effects of malathion at doses greater than the LD50 was investigated. In the main study, 60 birds received malathion at a dose of 1000 mg/kg bw (1.3 times the unprotected LD50). They were given atropine sulfate subcutaneously at 10 mg/kg bw 1 h before administration of malathion and then at 30 mg/kg bw 15 min and 1, 3, and 5 h afterwards. A total of 39 birds died with clinical signs consistent with cholinesterase activity poisoning within 15 days. Three weeks after the first dose of malathion, the survivors were dosed again, this time at 850 mg/kg bw (1.1 times the LD50), with atropinization as above. A further seven birds died, but the survivors recovered completely. Positive controls were treated with tri- ortho-tolylphosphate at 500 mg/kg bw. The hens were observed daily; body weights and food consumption were recorded at the start of the study, and body weight was recorded thereafter at three-day intervals. All dead birds were examined with perfusion fixation, and the brain, spinal cord, and sciatic nerve were examined histologically No treatment-related histopathological changes were seen in the birds treated with malathion, whereas those treated with tri- ortho-tolylphosphate showed changes typical of organophosphate-induced delayed polyneuropathy in the spinal cord and sciatic nerve. Clinical signs of delayed polyneuropathy were seen only in the positive control birds (Fletcher, 1989). Groups of 12 retired laying Leghorn hens were given malathion orally at doses of 75, 150, or 300 mg/kg bw, and groups of 12 Long-Evans rats (28 rats at the high dose) received 600, 1000, or 2000 mg/kg bw. All received atropine pretreatment, and some received subsequent treatment with atropine. Clinical assessments were carried out. Cholinesterase and neuropathy target esterase activities were estimated, and sections of the medulla, cervical and lumber spinal cord, and branches of the tibial nerve were examined. Flaccid paralysis was seen in the hens at 300 mg/kg bw for about 24 h, but none died. There was no clinical indication of delayed polyneuropathy in the hens treated with malathion, whereas those given tri- ortho-tolylphosphate, mipafox, or diisopropylphosphorofluoridate developed typical behavioural signs of neuropathy. Malathion at a dose of 2000 mg/kg bw induced clinical signs consistent with cholinesterase poisoning in the rats, and gait changes were observed 14-21 days after administration at the highest dose. No treatment-related histopathological changes were seen in the birds or rats treated with malathion, whereas those treated with tri- ortho-tolylphosphate, mipafox, or diisopropyl phosphorofluoridate showed changes typical of organophosphate-induced delayed polyneuropathy. The activities of both acetylcholinesterase and neuropathy target esterase were inhibited by malathion. In the hens, brain acetylcholinesterase activity was inhibited by 17 ± 3% at the lowest dose of malathion and by 76 ± 1% at the highest; the corresponding inhibition of neuropathy target esterase activity was 0 ± 3% and 50 ± 22%. In the rats, acetylcholinesterase activity inhibition was 26 ± 6% at the lowest dose and 56 ± 2% at the highest; the corresponding figures for neuropathy target esterase inhibition were 19 ± 7% and 75 ± 5%, all compared with concurrent controls (Ehrich et al., 1995). Malathion and malaoxon produced nugatory inhibition of neuropathy target esterase activity in human neuroblastoma cells (3 and 1%, respectively) (Ehrich et al., 1994). The ratio of the IC50 value for neuropathy target esterase to that for acetylcholinesterase was 30 000 in murine neuroblastoma cells and 76 000 in human cells (Ehrich et al., 1997). (v) Antidotes Malaoxon, the oxon analogue of malathion, inhibits cholinesterase activity by producing a dimethylphosphoryl derivative, which is susceptible to oxime-induced reactivation. Experimental evidence indicates that clinically significant reactivation occurs (Hobbiger, 1973). Most authors have found significant reactivation with oximes, but there are notable exceptions. For example, Ganendran and Balabaskaran (1976) found little reactivation of malathion- and malaoxon-inhibited human whole-blood cholinesterase activity. Similar results were obtained for acetylcholinesterase activity in goat brain (Cheema et al., 1989). The efficacy of four pyridinium oximes, trimedoxime, obidoxime, pralidoxime, and HI-6, in the treatment of poisoning by malathion (purity, 96%) was tested in male Wistar rats, which were given malathion at twice the LD50, as determined experimentally during the study, and treated with 30 mol/kg bw of trimedoxime, obidoxime, or HI-6 or 60 mol/kg bw of pralidoxime; atropine and diazepam therapy were also used. Of the oximes, obidoxime was the most effective, followed by trimoxime, and then pralidoxime and HI-6, which were equally effective; however, better survival was achieved with HI-6 at a dose of 150 mol/kg bw than with the other regimes. Thus, malathion posoning can be treated with mono- and bis-pyridinium oximes (Jokanovic & Maksimovic, 1995). Malathion (50% emulsifiable concentrate) was administered at a dose of 100 mg/kg bw or at a minimally lethal dose of 125 mg/kg bw to buffalo calves (Bubalus bubalis). Pralidoxime methiodide combined with atropine was reported to reverse the clinical evidence of toxicity (Gupta, 1984). Pralidoxime chloride or diacetyl monoxime at a dose of 100 mg/kg bw intraperitoneally reversed the rise in blood glucose observed after injection of malathion at 500 mg/kg bw to female albino rats, provided the oximes were given immediately after the malathion. When given 15 min later, the antidotes were ineffective (Agarwal & Matin, 1981). Obidoxime reversed malaoxon-induced inhibition of cholinesterase activity in isolated rat diaphragm and restored the ability to sustain tetany; moreover, obidoxime at 20 mg/kg bw together with atropine raised the LD50 in OF mice by 5.1-fold, the comparable figure for atropine alone being 1.7-fold (Abraham & Edery, 1976). Obixodime has been used successfully in treating malathion poisoning in humans (Dive et al., 1994; see below). 3. Observations in humans Malathion and ethyl- para-nitrophenyl thionobenzenephosphonate (purity of each unspecified) were tested in volunteer male prisoners aged 23-36 years. In phase I of the study, four samples of blood were taken during two weeks before the start of the study for measurements of plasma and erythrocyte cholinesterase activity, and then malathion was administered at a dose of 8 mg/day to five subjects for 32 days. Phase II was begun three weeks after completion of phase I. On the two days before its start, samples of plasma and washed erythrocytes were taken for measurements of cholinesterase activity, and then malathion was administered to the same five subjects at a dose of 16 mg/day for 47 days. In phase III, five new subjects were selected; plasma and erythrocyte cholinesterase activity was determined in blood samples drawn twice weekly for 36 days, before administration of malathion at a dose of 24 mg/day for 56 days. During phase I, no clinical effects were observed, and there were no changes in blood counts or the results of urinalysis. Furthermore, no significant depression of plasma or erythrocyte cholinesterase activity was observed in any subject. Similarly, no clinical effects were observed in phase II. In phase III, depression of plasma cholinesterase activity was observed two weeks after the first administration of malathion, the maximum depression being 25%, seen three weeks after cessation of treatment; erythrocyte acetylcholinesterase activity was depressed to the same extent. The NOAEL was 6 mg/day, approximately equal to 0.27 mg/kg bw per day. No potentiation of malathion by ethyl- para-nitrophenyl thionobenzenephosphonate was observed (Moeller & Rider, 1962). An epidemic of poisoning of spraymen in Pakistan was attributed largely to contaminants, particularly iso-malathion, in the formulation of malathion used (Baker et al., 1978). There are numerous case reports of individual poisoning. Thus, Dive et al. (1994), reported an instance in which an elderly woman consumed about 100 ml of a garden preparation containing 15% malathion in isopropyl alcohol. The preparation also contained isopropylmalathion and O,O,S-trimethylphosphorothioate. A typical cholinergic crisis was followed by cardiac, pulmonary, neurological, and renal manifestations, and the patient was treated with atropine and obidoxime. The cardiac manifestations included arrhythmia and conduction disturbances. Mild interstitial pulmonary fibrosis was observed in a lung biopsy sample. Matsushita et al. (1985) reported allergic contact dermatitis in people exposed to organophosphorus insecticides including malathion. Thomas et al. (1990) reported a study of women exposed to malathion during aerial spraying of large areas of the San Francisco Bay area, USA, in 1981-82. A number of associations were found, of which one, congenital gastrointestinal anomalies, remained statistically significant after control for confounders. An episode of epidemic hysteria was reported at an elementary school in Arizona, USA, in response to the smell of malathion (Baker & Selvey, 1992). During a campaign to eradicate Mediterranean fruit flies with a pesticide based on malathion, a number of cases of urticaria, angioneurotic oedema, and non-specific rashes were reported. Of 10 subjects that received a patch test, none responded. One case of possible immediate reaction to malathion bait was reported (Schanker et al., 1992). No case of organophosphate-induced delayed polyneuropathy due to malathion has been reported in humans. Comments Malathion is rapidly absorbed, biotransformed, and excreted, predominantly in the urine but also in the faeces, largely as its two monocarboxylic acids and the dicarboxylic acid. The oral LD50 values for malathion in laboratory rodents were 1000-10 000 mg/kg bw, the observed differences probably being due to impurities. The most recent LD50 values tend to be higher. The cholinesterase-inhibiting metabolite of malathion, malaoxon, has much lower oral LD50 values of 100-220 mg/kg bw. WHO has classified malathion as slightly hazardous (WHO, 1996). In a study of neurotoxicity in rats receiving single doses of 0, 500, 1000, or 2000 mg/kg bw, there was no NOAEL, as clinical signs were present at all doses. In a 13-week study of neurotoxicity, also in rats, at dietary concentrations of 0, 50, 5000, or 20 000 ppm, the NOAEL was 5000 ppm, equal to 350 mg/kg bw per day, on the basis of inhibition of brain acetylcholinesterase at the highest dose. In a 30-day study of toxicity in rats receiving malathion in the diet at concentrations of 0, 50, 100, 500, 10 000, or 20 000 ppm, the NOAEL was 500 ppm, equal to 52 mg/kg bw per day, on the basis of increased liver weight and histopathological changes in the liver (periportal hepatocyte hypertrophy) at the next highest dose. In a 90-day study of toxicity in rats, malathion was given at dietary concentrations of 0, 100, 500, 5000, 10 000, or 20 000 ppm. The NOAEL was 500 ppm, equal to 34 mg/kg bw per day, on the basis of decreased mean corpuscular volume and mean corpuscular haemoglobin, increased liver weights and relative kidney weights, and chronic nephropathy in males and decreased mean cell volume, hepatocyte hypertrophy, and increased relative kidney weight in females at the next highest dose. A 21-day study of dermal toxicity was carried out in which rabbits were treated with malathion at doses of 0, 50, 300, or 1000 mg/kg bw per day for 6 h per day, five days per week. The NOAEL was 300 mg/kg bw per day on the basis of inhibition of brain acetylcholinesterase activity at the highest dose. In a 28-day study of toxicity in dogs, malathion was fed in gelatin capsules at doses of 0, 125, 250, or 500 mg/kg bw per day for 28 days. There was no NOAEL because of clinical signs at all doses. In a one-year study of toxicity in dogs, malathion was administered orally in capsules at doses of 0, 62.5, 125, or 250 mg/kg bw per day on seven days per week. The NOAEL was 125 mg/kg bw per day on the basis of body-weight depression and changes in haematological and clinical chemical parameters at the highest dose. A number of long-term studies of toxicity and carcinogenicity have been carried out on malathion in both rats and mice. The earlier ones were reviewed by a working group convened by the IARC, which concluded that the available data did not provide evidence that malathion was carcinogenic. In an 18-month study in mice, malathion was administered at dietary concentrations of 0, 100, 800, 8000, or 16 000 ppm. The NOAEL was 800 ppm, equal to 140 mg/kg bw per day, on the basis of inhibition of brain acetylcholinesterase activity at termination and an increased incidence of liver adenomas in animals of each sex at the next highest dose. In a two-year study in rats, dietary concentrations of 0, 100, 1000, or 5000 ppm were used. The NOAEL was 100 ppm, equivalent to 5 mg/kg bw per day, on the basis of reduced erythrocyte acetylcholinesterase activity and body weight. In another long-term study in rats, malathion was given at doses of 0, 100/50, 500, 6000, or 12 000 ppm for two years. The NOAEL was 500 ppm, equal to 29 mg/kg bw per day, on the basis of decreased survival and body-weight gain, changes in haematological parameters, decreased brain acetylcholinesterase activity, increased g-glutamyl transpeptidase activity, increased liver, kidney, and thyroid/parathyroid weights, and changes in the olfactory epithelium at the next highest dose. Numerous tests have been carried out for genotoxicity both in vitro and in vivo. Most of the evidence indicates that malathion is not genotoxic, although some studies indicate that it can produce chromosomal aberrations and sister chromatid exchange in vitro. There was no evidence that malathion induces chromosomal aberrations in vivo. Malaoxon did not induce reverse mutation in bacteria, but it caused sister chromatid exchange in two tests in mammalian cells and induced sex-linked recessive lethal mutation in Drosophila in vivo. The four common impurities of malathion, isomalathion, O,O,S-trimethyl phosphorothioate, O,S,S-trimethyl phosphorodithioate, and O,O,O-trimethyl phosphorothioate, did not induce reverse mutation in bacteria. The Meeting concluded that malathion is not genotoxic. A number of studies of reproductive toxicity have been carried out, only some of which showed NOAELs. In a study in rats, malathion was administered by gavage to groups of pregnant animals on days 6-15 of gestation at doses of 0, 200, 400, or 800 mg/kg bw per day. The NOAEL was 400 mg/kg bw per day on the basis of maternal toxicity at the highest dose; no fetal toxicity was observed. Malathion was administered orally at doses of 0, 25, 50, or 100 mg/kg bw per day to groups of pregnant rabbits on days 6-18 of gestation. The NOAELs were 25 mg/kg bw per day for maternal toxicity and 100 mg/kg bw per day for fetal toxicity; teratogenicity was not seen at any dose. A two-generation study was undertaken in rats in which malathion was given at dietary concentrations of 0, 550, 1700, 5000, or 7500 ppm. The NOAEL was 7500 ppm, equal to 600 mg/kg bw per day, for reproductive toxicity and 1700 ppm, equal to 130 mg/kg bw per day, for developmental toxicity, the latter being based on reduced pup weights. Two studies on the neurotoxicity of malathion in hens were reviewed. In neither was there evidence that malathion can cause delayed neuropathy, although some inhibition of neuropathy target esterase activity was found in the brains of birds at 2000 mg/kg bw. In a study in volunteers with doses of 8, 16, or 24 mg of malathion per day, the NOAEL was 16 mg per day (equivalent to 0.27 mg/kg bw per day) on the basis of inhibition of plasma and erythrocyte cholinesterase activity. Several cases of exposure to impure malathion have been reported, none of which resulted in delayed neuropathy. An ADI of 0-0.3 mg/kg bw was established on the basis of the NOAEL of 29 mg/kg bw per day in the two-year study of toxicity and carcinogenicity in rats, with a safety factor of 100. This ADI is supported by the NOAEL of 25 mg/kg bw per day in the study of developmental toxicity in rabbits. The alternative approach of basing the ADI on the study in humans was not taken, as the study was old and the material was therefore likely to contain toxic impurities. Toxicological evaluation Levels that cause no toxic effect Mouse: 800 ppm, equal to 140 mg/kg bw per day (18-month study of toxicity and carcinogenicity) Rat: 500 ppm, equal to 29 mg/kg bw per day (two-year study of toxicity and carcinogenicity) 1700 ppm, equal to 130 mg/kg bw per day (study of reproductive toxicity) 400 mg/kg bw per day (maternal toxicity in a study of developmental toxicity) Rabbit: 25 mg/kg bw per day (maternal toxicity in a study of developmental toxicity) Dog: 125 mg/kg bw per day (one-year study of toxicity) Human: 0.3 mg/kg bw per day (47-day study of toxicity) Estimate of acceptable daily intake for humans 0-0.3 mg/kg bw Studies that would provide information useful for continued evaluation of the compound Further observations in humans References Abou Zeid, M.M., El-Barouty, G., Abdel-Reheim, E., Blancato, J., Dary, C., El-Sebae, A.H. & Saleh, M.A. (1993) Malathion disposition in dermally and orally treated rats and its impact on the blood serum acetylcholine esterase and protein profile. J. Environ. Sci. Health, B28, 413-430. Toxicological criteria for setting guidance values for dietary and non-dietary exposure to malathion Human exposure Relevant route , study type, species Results, remarks Short-term Oral, toxicity, rat LD50 = 1000-11 000 mg/kg bw (1-7 days) Inhalation, toxicity, rat LC50 > 5.2 mg/L Dermal irritation, rabbit Mildly irritating Ocular irritation, rabbit Mildly irritating Dermal sensitization, guinea-pig Not sensitizing Medium-term Repeated oral, 90 days, rat NOAEL = 34 mg/kg bw per day: systemic toxicity (1-26 weeks) Repeated dermal, 21 days, rabbit NOAEL = 300 mg/kg bw per day: decreased brain acetylcholinesterase activity Repeated oral, developmental toxicity, rabbit NOAEL = 25 mg/kg bw per day: maternal toxicity; NOAEL = 100 mg/kg bw per day: fetal toxicity Repeated oral, reproductive toxicity, rat NOAEL = 600 mg/kg bw per day: no parental toxicity; NOAEL = 130 mg/kg bw per day: developmental toxicity Long-term Repeated oral, 2 years, rat NOAEL = 29 mg/kg bw per day: decreased survival, (> 1 year) reduced body weight, decreased brain acetylcholinesterase activity Abraham, S. & Edery, H. (1976) Rapid spontaneous reactivation of cholinesterase inhibited by malaoxon. Israel J. Med. Sci., 12, 1524. Agarwal, R. & Matin, M.A. (1981) Effect of oximes and atropine on the concentration of cerebral glycogen and blood glucose in malathion-treated rats. J. Pharm. Pharmacol., 33, 795-796. Aldridge, W.N., Miles, J.W., Mount, D.L. & Verschoyle, R.D. (1979) The toxicological properties of impurities in malathion. Arch. Toxicol., 42, 95-106. Aldridge, W.N., Dinsdale, D. & Nemery, B. (1985) Some aspect of the toxicology of trimethyl and triethyl phosphorothioates. Fundam. Appl. Toxicol., 5, S47-S60. Baker P & Selvey D (1992) Malathion-induced epidemic hysteria in an elementary school. Vet. Hum. Toxicol., 34, 156-160. Baker, E.L., Zack, M. & Miles, J.W. (1978) Epidemic malathion poisoning in Pakistani malaria workers. Lancet, i, 31-34. Balaji, M. & Sasikali, K. (1993) Cytogenetic effect of malathion in in vitro culture of human peripheral blood. Mutat. Res., 301, 13-17. Balasubramanian, K., Ratnakar, C., Ananthanarayanan, P.H. & Balasubramanian, A. (1987) Histopathological changes in the testis of malathion treated albino rats. Med. Sci. Res., 15, 509-510. Boyd, E.M. & Tanikella, T.K. (1969) The acute oral toxicity of malathion in relation to dietary protein. Arch. Toxicol., 24, 292-303. Boyes, W.K., Hunter, E., Gary, C. & Peiffer, R.L. (1997) Topical exposure of the eyes to the organophosphate insecticide malathion: Lack of visual effects. Unpublished report. Submitted to WHO by the US Environmental Protection Agency. Brodeur, J. & DuBois, K.P. (1967) Studies on factors influencing the acute toxicity of malathion and malaoxon in rats. Can. J. Physiol. Pharmacol., 45, 621-631. Bulsiewicz, H., Rozewicka, L., Januszewska, H. & Bajko, J. (1976) Aberrations of meiotic chromosomes induced in mice with insecticides. Folio Morphol. (Warsaw), 35, 361-363. Byeon, W.-H., Hyun, Z.H.H. & Lee, S.Y. (1976) Salmonella/microsomal enzyme activation system. Korean J. 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See Also: Toxicological Abbreviations Malathion (ICSC) Malathion (FAO Meeting Report PL/1965/10/1) Malathion (FAO/PL:CP/15) Malathion (FAO/PL:1967/M/11/1) Malathion (JMPR Evaluations 2003 Part II Toxicological) Malathion (FAO/PL:1968/M/9/1) Malathion (FAO/PL:1969/M/17/1) Malathion (AGP:1970/M/12/1) Malathion (WHO Pesticide Residues Series 3) Malathion (WHO Pesticide Residues Series 5) Malathion (Pesticide residues in food: 1977 evaluations) Malathion (Pesticide residues in food: 1984 evaluations) Malathion (IARC Summary & Evaluation, Volume 30, 1983)