Pesticide residues in food 2001
First draft prepared by
Food Standards Agency, London, United Kingdom
Diazinon was evaluated by the Joint Meeting in 1963, 1965, 1966, 1970 and 1993 (Annex 1, references 2, 3, 6, 14 and 68). In 1966, an ADI of 0–0.002 mg/kg bw was allocated on the basis of a NOAEL of 0.02 mg/kg bw per day in a study with volunteers and a 10-fold safety factor. Diazinon was last reviewed by the 1993 JMPR, when the ADI of 0–0.002 mg/kg bw was reaffirmed. The present review was undertaken to consider the need for establishing an acute reference dose (RfD).
Diazinon (purity, 88%) was administered as a single dose by gavage to Harlan Sprague-Dawley rats in order to determine the time course of inhibition of serum, erythrocyte and brain cholinesterase activity. Groups of 15 animals of each sex were given a dose of 0, 2.5, 150, 300 or 600 mg/kg bw. Clinical observations were made immediately before blood sampling from the orbital plexus for determination of serum and erythrocyte cholinesterase activity. Five animals of each sex were killed at 3, 5 and 9 h and the remainder at 24 h to obtain brains and spinal cord for determination of cholinesterase activity, the samples being stored at –70 to –90 °C. Acetylcholinesterase activity was measured in the cerebellum, cerebral cortex, striatum and hippocampus and in the thoracic spinal cord by a modification of the method of Ellman et al. (1961).
Survival was unaffected by treatment. At the highest dose, clinical signs were seen at 3 h, which were maximal at 9 h in males, with some recovery after 24 h, and maximal after 24 h in females. No significant differences were seen in body weight. Plasma cholinesterase activity was decreased by more than 20% at the lowest dose at 3 and 9 h, maximally so at 9 h in animals of each sex. At 24 h, the cholinesterase activity in animals at the lowest dose was decreased in comparison with that of concurrent controls by 17% in males and 42% in females. Erythrocyte cholinesterase activity was decreased in females at 2.5 mg/kg bw, 9 h after dosing (60% of the control value). At other times in females and at all times in males, the erythrocyte cholinesterase activity was never less than 80% of that of controls. At the higher doses, the activity was depressed to less than 80% of the control activity at all times in animals of each sex. In an analysis of cholinesterase activity in regions of the central nervous system in animals at the lowest dose, that of males was never less than 80% that of controls but in one instance (cerebral cortex at 9 h) it was equal to 80% that of controls. In females at the lowest dose, no substantial decrease in central nervous system cholinesterase activity was observed. At the higher doses, significantly decreased activity was observed in all regions and at all times but was usually greater at 9 and 24 h than at 3 h. The NOAEL for inhibition of brain cholinesterase activity was 2.5 mg/kg bw. No NOAEL could be identified for erythrocyte cholinesterase activity (Potrepka, 1994).
Single doses of diazinon (purity, 88%) were administered by gavage to groups of 15 male and 15 female Hsd:Sprague-Dawley rats at a dose of 0, 2.5, 150, 300 or 600 mg/kg bw. Of the 15 animals, 10 were used for neurological testing and five for measurement of cholinesterase activity. Triadimefon (purity, 99%) was administered similarly to groups of 10 male and 10 female rats, as a positive control for neurological effects. On the day of treatment, all the rats were observed before and after dosing and thereafter twice daily, for general appearance, behaviour, signs of toxicity, morbidity and mortality. All animals were examined in detail weekly, including palpation for tissue masses. Body weights were estimated before dosing and weekly thereafter, and food consumption was estimated weekly. A functional observational battery (FOB) test was performed 1 week before administration of the test materials, at the time of peak effect after administration of the test materials (9–11 h for diazinon, 1 h for triadimefon) and 7 and 14 days after administration (of diazinon) on the 10 animals intended for neurological testing. Blood samples were obtained at the estimated time of peak effects and at 14 days from the five animals intended for cholinesterase testing in each group, and plasma and erythrocyte cholinesterase activity was determined, while brain cholinesterase activity was estimated in whole brain samples at termination 14 days after administration. The animals used for neurological examination (both survivors to termination and decedents) were subjected to necropsy. Sections were made at 10 levels of the brain, cervical, thoracic, lumbar and sacral spinal cord with ganglia and right and left sciatic nerves, right and left fibular nerves, right and left tibial nerves and right and left lateral cutaneous sural nerves as well as the Gasserian ganglion. Sections were also taken of skeletal muscle from the right thigh, eyes with the optic nerve and any gross lesions identified. Only the first five animals at the highest dose of diazinon, the controls and those given triadimifon were processed for histopathological examination.
Two males and one female died during the study. These deaths were attributed to diazinon, and all occurred at the highest dose. There was a single accidental death in the group of five animals at 300 mg/kg bw and intended for cholinesterase measurement during blood sampling. One control animal was removed from the study as it was thought to have been wrongly dosed, since it had signs of cholinergic poisoning and low cholinesterase activity. The clinical observations included chromodacryorrhoea, reduced activity and tremors at doses ž 300 mg/kg bw, while the group at 600 mg/kg bw also had chromorhinorrhoea, diarrhoea and pallor. Significant decreases in body-weight gain were observed in males at doses > 300 mg/kg bw, while no effects were observed on body weight or weight gain in the females. Food consumption was decreased in males at doses > 300 mg/kg bw and in females at doses > 150 mg/kg bw. In both males and females, effects on parameters of the FOB test were seen only at the estimated time of peak effect after administration of the test materials (9–11 h for diazinon, 1 h for triadimefon) and not on day 7 or 14 after exposure.
The autonomic parameters affected by diazinon at the time of peak effects in males were faecal consistency and soiled fur at doses > 150 mg/kg bw, and these effects were dose-related. Increased salivation, staining of the nose and repeated opening and closing of the mouth were observed at doses > 300 mg/kg bw. Additionally, impaired respiration, lachrymation and staining of the mouth were seen at 600 mg/kg bw. In females, repeated opening and closure of the mouth were observed at doses > 150 mg/kg bw and altered faecal consistency, soiled fur and staining of the nose at doses > 300 mg/kg bw. At the highest dose, impaired respiration and lachrymation were observed. The neuromuscular parameters that were affected in males were: abnormal gait at > 150 mg/kg bw; ataxic gait, impaired righting reflex, impaired hindlimb extensor reflex and decreased hindlimb footsplay at > 300 mg/kg bw and reduced forelimb grip strength at 600 mg/kg bw. In the females, ataxic and abnormal gait was observed at > 150 mg/kg bw and impaired righting reflex at > 300 mg/kg bw. Impaired hindlimb extensor reflex, abnormal hindlimb positioning when held by the tail and reduced forelimb and hindlimb grip strength were observed at 600 mg/kg bw. Central nervous system excitability parameters were also affected. In males, tremors were observed in the home cage and open field at > 300 mg/kg bw, as was twitching or muscle fasciculation. At 600 mg/kg bw, the arousal level was decreased. Females had tremors in the home cage at the highest dose only and in the open field at > 300 mg/kg bw. Twitching in the open field was seen at the highest dose and a lowered arousal level at > 300 mg/kg bw. Touch response was reduced in females at the highest dose, and the tail pinch response was reduced in animals of each sex at this dose. Reduced body temperature was observed in males at > 300 mg/kg bw and in females at > 150 mg/kg bw. Additionally, females at > 300 mg/kg bw were dehydrated. Locomotor activity in the figure-of-eight maze decreased over time in all groups. At the estimated time of peak effect, the activity of males at doses > 300 mg/kg bw and of females at doses > 150 mg/kg bw was decreased. At 7 and 14 days after dosing, the mean total activity counts were similar for all groups.
At the time of peak effect after intake of diazinon (9–11 h), plasma cholinesterase activity was diminished in all treated groups; however, no differences were observed between groups 14 days after dosing. Erythrocyte cholinesterase activity was inhibited at doses > 150 mg/kg bw in both males and females at the time of peak effect. In males, the activity was 18%, 17% and 15% of that of concurrent controls at 150, 300 and 600 mg/kg bw, respectively, while in the females the activity was 24%, 23% and 24% that of concurrent controls at the three doses. Partial recovery was seen 14 days after dosing: in males, the activity at 150 mg/kg bw was 91% that of concurrent controls, while it was 66 and 53% that of concurrent controls at 300 and 600 mg/kg bw, respectively; in females, the activity was 89%, 57%, 74% and 65% that of concurrent controls at 2.5, 150, 300 and 600 mg/kg bw, respectively. No significant differences in brain cholinesterase activity were seen among the groups of males. Significantly reduced brain cholinesterase activity at termination was seen in females that had received diazinon at 150 mg/kg bw, in which the activity was 92% that of concurrent controls; however, as the activity in the groups given the higher doses was comparable to that in the concurrent controls, this finding is unlikely to be of biological significance. No gross or microscopic treatment-related abnormalities were seen at necropsy. Triadimefon decreased weight gain and food consumption during week 1 of the study. At the time of peak effect after exposure (1 h), changes in central nervous system excitability parameters (increased incidence of rearing; increased arousal level) were the only changes seen in the FOB test. The NOAEL was 2.5 mg/kg bw on the basis of depressed erythrocyte cholinesterase and behavioural changes at 150 mg/kg bw (Chow & Richter, 1994).
Diazinon (purity, 87.9%) was administered by gavage to albino Crl:CD BR/VAF/Plus rats. In phase 1 of the study, five animals of each sex were given 100, 250 or 500 mg/kg bw. Subsequently, in order to identify an NOAEL, groups of five females receiving diazinon at 25 or 50 mg/kg bw were added. In phase 2 of the study, groups of five males received diazinon by gavage at a dose of 0, 0.05, 0.5, 1, 10, 100 or 500 mg/kg bw, and females received a dose of 0, 0.05, 0.12, 0.25, 2.5, 25 or 250 mg/kg bw. In phase 1, clinical observations were carried out 1, 2, 4 and 8 h after administration of the test material and daily thereafter. Body weights were determined before treatment and on days 7 and 14 and also on animals that died after 1 day. Cholinesterase activity was not measured in these animals. In phase 2, clinical observations were made 1, 2 and 4 h after adminstration of diazinon, while body weights were measured before treatment and on day 1. The surviving animals from phase 1 were killed on day 14 and subjected to gross necropsy; abnormal tissues were retained for possible histopathological examination. Plasma and erythrocyte cholinesterase activity was measured 24 h after treatment in the animals in phase 2, and the animals were then killed and subjected to gross necropsy, abnormal tissues being retained for possible histopathological examination; additionally, the right half of the brain was removed for determination of cholinesterase activity.
One female that received diazinon at the highest dose died during phase 1 of the study. There was no treatment-related effect on body weight. Clinical signs were seen in males at 250 and 500 mg/kg bw and in females at doses > 50 mg/kg bw, which included miosis, hypoactivity, absence of the pain reflex, red-stained face, yellow-stained urogenital region and soft stools. No pathological changes were observed that could be attributed to treatment. In phase 2, no deaths occurred. Body-weight loss during 24 h after dosing was greater in males at 500 mg/kg bw and in females at 250 mg/kg bw than in concurrent controls. Clinical signs of toxicity were seen in males at 500 mg/kg bw and in females at 250 mg/kg bw, which included miosis, hypoactivity, absent pain reflex, staggering gait, excessive salivation, red-stained face and yellow-stained and/or wet urogenital region. The only gross pathological findings of note were yellow staining of the perineum and red paranasal discharge in males at 500 mg/kg bw and in females at 250 mg/kg bw.
Plasma cholinesterase activity was reduced in males at doses > 10 mg/kg bw and in females at doses > 2.5 mg/kg bw. A statistically and biologically significant reduction in erythrocyte cholinesterase activity was seen in males at 100 mg/kg bw (51% of concurrent control value) and 500 mg/kg bw (64% of concurrent control value). In females, erythrocyte cholinesterase activity was depressed at 25 mg/kg bw (65% of concurrent control value) and 250 mg/kg bw (55% of concurrent control value). Brain cholinesterase activity was reduced in males at 500 mg/kg bw (31% of concurrent control value) and in females at 250 mg/kg bw (30% of concurrent control). In females at 25 mg/kg bw, the brain cholinesterase activity was 64% that of controls; while this difference was not statistically significant, it may be biologically significant. At necropsy in phase 2, staining of the perineum and red paranasal discharge were seen in animals of each sex at the highest dose. No treatment-related histological lesions were seen. The NOAEL was 2.5 mg/kg bw, on the basis of inhibition of brain and erythrocyte cholinesterase activity in females at the next highest dose (Glaza, 1993).
A preliminary report was available of a double-blind, placebo-controlled study in which healthy, informed male volunteers were given single, ascending doses of diazinon (purity, 97.8%) in corn oil in gelatine capsules. Some volunteers received only corn oil in gelatine capsules. The lowest dose used was 0.03 mg/kg bw, given initially to one volunteer receiving the test material and one the placebo. In the next phase, one volunteer received the placebo, six received diazinon at a dose of 0.03 mg/kg bw and one received a dose of 0.12 mg/kg bw. In the next phase, one volunteer received the placebo, six received diazinon at a dose of 0.12 mg/kg bw and one received a dose of 0.21 mg/kg bw. In the next phase, one volunteer received the placebo, six received diazinon at a dose of 0.21 mg/kg bw and one received a dose of 0.30 mg/kg bw. In the final phase, three volunteers received the placebo and seven received diazinon at a dose of 0.20 mg/kg bw. A complete physical examination was carried out before and 2 and 15 days after dosing, which included an electrocardiogram. Vital signs (respiratory parameters, oral temperature, blood pressure and pulse) were recorded before administration of the test material and 1, 2, 4, 6, 8, 12, 24 and 48 h and 4, 7 and 14 days afterwards. The volunteers were asked to report all adverse events. Blood was taken for clinical chemistry and haematological examination before dosing and 1, 2 and 15 days after dosing. Blood was collected 1 and 2 days and immediately before dosing, and plasma and erythrocyte cholinesterase activity was determined by a modification of the method of Ellman et al. (1961). Further samples for cholinesterase determination were taken 1, 2, 4, 6, 8, 12, 24 and 48 h after dosing and on days 5, 8 and 15 after dosing. Urine samples were collected over 24-h periods before dosing and for 0–6, 6–12, 12–24 and 24–48 h after dosing.
The only self-reported adverse event considered to be related to intake of the test material was back pain in a man at the highest dose. No treatment-related effects on haematological or clinical chemical parameters were observed, except for changes in cholinesterase activity. Plasma cholinesterase activity was inhibited by more than 20% at doses > 0.12 mg/kg bw. No significant inhibition of erythrocyte cholinesterase activity was seen at any dose; at 0.21 mg/kg bw, 7% inhibition was observed at 4 h, 4% inhibition at 8 h and 6–7% inhibition at days 5, 8 and 15. At other times, the erythrocyte cholinesterase activity was greater than or equal to that of the group given the placebo. Furthermore, data for the man given the highest dose did not suggest any significant inhibition of erythrocyte cholinesterase activity. The NOAEL was 0.21 mg/kg bw (the highest dose was ignored, as only one man received it). However, the studies in laboratory animals indicated a sex difference in response to diazinon, while this study was performed with male volunteers only (Meyer, 1999).
Diazinon is an organophosphate, and virtually all its toxicological effects are attributable to anticholinesterase activity.
The oral LD50 of diazinon in mice and rats was > 200 mg/kg bw. Diazinon has been classified by WHO (1999) as ‘moderately hazardous’.
After preliminary studies to establish the time course of clinical signs and cholinesterase inhibition, a study of acute neurotoxicity in rats was undertaken. This study involved a two-phase protocol. In phase 1, clinical observations were made, but plasma, erythrocyte, and brain cholinesterase activities were not measured. The doses used were 100, 250, and 500 mg/kg bw for males and 25, 50, 100, 250 and 500 mg/kg bw for females. In phase 2, wider ranges of doses were used (0.05–500 mg/kg bw for males and 0.05–250 mg/kg bw for females), and plasma, erythrocyte and brain cholinesterase activity were estimated. The NOAEL was 2.5 mg/kg bw in females and 100 mg/kg bw in males on the basis of inhibition of brain cholinesterase activity. The LOAELs were 25 mg/kg bw in females and 500 mg/kg bw in males.
A preliminary report was available of a study in male volunteers given ascending single doses of diazinon in gelatine capsules, both the volunteers and the investigators being unaware of who had received diazinon or placebo. Plasma cholinesterase activity was inhibited by > 20% at doses > 0.12 mg/kg bw. Erythrocyte cholinesterase activity was not inhibited. The NOAEL was 0.21 mg/kg bw, the second highest dose studied; the highest dose was ignored, as the group comprised a single volunteer.
After considering the previous evaluation of diazinon and the new data submitted, the Meeting established an acute RfD of 0.03 mg/kg bw. This was based on the NOAEL of 2.5 mg/kg bw in the studies of acute neurotoxicity in rats and a 100-fold safety factor. The study in male volunteers was considered of limited value in establishing the acute RfD, as the studies in rats provided evidence of a considerable sex difference in sensitivity to the inhibition of acetylcholinesterase activity by diazinon.
Chow, E. & Richter, A.G. (1994) Acute neurotoxity study with DZN® diazinon MG87% in rats. Unpublished report No. F-00175 from Ciba-Geigy Corp., Plant Protection Division, Farmington, Connecticut, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. GLP Compliant EPA-FIFRA 40 CFR, OECD GLP Principles and MAFF Japan. Guideline USEPA-FIFRA, pesticide assessment guidelines, subdivision F, hazard evaluation, section 81-8.
Ellman, G.L., Courtney, K.D., Andres, V. & Featherstone, R.M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacol., 7, 88.
Glaza, S.M. (1993) Acute oral toxity study with D.z.n® diazinon MG87% in rats. Unpublished report No. HWI 6117-221 from Hazleton Wisconsin Inc., Madison, Wisconsin, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. GLP Compliant EPA-FIFRA 40 CFR 160, OECD GLP Principles Annex 2, C(81)30 final proposed and MAFF Japan. Guideline USEPA 81-1.
Meyer, L. (1999) Preliminary summary of a randomized, double-blind ascending, acute, oral dose study of diazinon to determine the no effect level (NOEL) for plasma and RBC cholinesterase activity in normal, healthy volunteers. Unpublished report No. 8373 from Covance Clinical Research Unit Inc., Madison, Wisconsin, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. Consistent with the Declaration of Helsinki, GCP compliant 21 CFR 50, 54, 56, 312, 314. Not GLP compliant as not audited by Quality Assurance Unit.
Potrepka, R.F. (1994) Acute cholinesterase inhibition time course study with D.Z.N® diazinon MG87% in rats. Unpublished report No. F-00185 from Ciba-Geigy Corp., Farmington, Connecticut, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. GLP Compliant EPA-FIFRA 40 CFR 160, OECD GLP Principles Annex 2, C(81)30 and MAFF Japan.
WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998–1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety.
See Also: Toxicological Abbreviations Diazinon (EHC 198, 1998) Diazinon (ICSC) Diazinon (FAO Meeting Report PL/1965/10/1) Diazinon (FAO/PL:CP/15) Diazinon (FAO/PL:1967/M/11/1) Diazinon (FAO/PL:1968/M/9/1) Diazinon (AGP:1970/M/12/1) Diazinon (WHO Pesticide Residues Series 5) Diazinon (Pesticide residues in food: 1979 evaluations) Diazinon (Pesticide residues in food: 1993 evaluations Part II Toxicology)