Pesticide residues in food - 2002 - Joint FAO/WHO Meeting on Pesticide Residues

First draft prepared by
Dr T.C. Marrs
Chief Toxicologist, Food Standards Agency, London, England

Fenamiphos was first evaluated toxicologically by the 1974 JMPR (Annex 1, reference 22), when an ADI of 0–0.0006 mg/kg bw was allocated on the basis of the results of a 2-year study in dogs in which inhibition of plasma cholinesterase activity was observed. In 1985, following a review of additional data, a temporary ADI of 0-0.0003 mg/kg bw was allocated (Annex 1, reference 44). A concern at that Meeting was fetotoxicity, for which a LOAEL of 0.1 mg/kg bw per day in rabbits was identified. The 1985 JMPR requested submission of an on-going carcinogenicity study in rats, more data on a teratogenicity study in rats, and a new teratogenicity study in rabbits. These data were evaluated by the 1987 JMPR (Annex 1, reference 50), when an ADI of 0-0.0005 mg/kg bw was allocated. Fenamiphos was toxic to the dams, but it was not embryotoxic or teratogenic in these studies. The 1997 JMPR re-evaluated fenamiphos (Annex 1, reference 80) and established an ADI of 0-0.0008 mg/kg bw on the basis of the results of a new 1-year study of toxicity in dogs and a 100-fold safety factor. The NOAEL in this study was based on inhibition of brain cholinesterase activity and anaemia at the next highest dose. The available data did not permit the Meeting to establish an acute reference dose (RfD) different from the ADI. Although short-term studies of toxicity in rats, including a study of neurotoxicity after a single dose, were available, there was evidence that dogs were more sensitive to fenamiphos than rats.

A study was conducted in which beagle dogs were given fenamiphos (purity, 95.4%) in single doses of up to 2 mg/kg bw, as shown in Table 1. The study was divided into three trials. In the first, two males and two females were given single doses of 0.063, 0.12, 0.25 and 0.5 mg/kg bw, with a 3-week wash-out period between each dose and starting with the highest dose. Blood was taken for determination of plasma and erythrocyte cholinesterase activity 0, 20, 60, 90, 120, 180 and 360 min and 1, 7, 14 and 21 days after administration of fenamiphos. Three weeks later, the four dogs were given fenamiphos again at the highest dose used in trial 1, namely 0.5 mg/kg bw, and 1 h later were killed for determination of brain cholinesterase activity. In trial 2, fenamiphos was administered to groups of two males and two females at a dose of 0.5 or 2 mg/kg bw. Blood was taken for determination of plasma and erythrocyte cholinesterase activity before administration of the test material and 1 and 2 h later. The animals were then killed, and their brains were taken for determination of cholinesterase activity. In the third trial, fenamiphos was administered at a dose of 1 mg/kg bw to two males and two females, blood being taken for estimation of plasma and erythrocyte cholinesterase activity before administration of the test material and 1, 2, 4 and 24 h later. The dogs were then killed and their brains taken for estimation of cholinesterase activity (i.e., 24 h after administration of fenamiphos); two samples were taken from each brain, one was frozen immediately and the other after 3 h. Plasma and erythrocyte cholinesterase activity was expressed as a percentage of the activity at time 0, each dog serving as its own control. Plasma cholinesterase activity was measured by the method of Ellman et al. (1961), erythrocyte cholinesterase activity by the method of Okabe et al. (1977) and brain cholinesterase activity by the method of Ho & Ellman (1969), after deep-freezing the brain to – 70 ºC. In all three trials, the dogs were monitored for cholinergic signs and symptoms, food intake was determined daily and body weight was measured weekly. Eight control dogs from two other studies were used to establish ‘control’ brain cholinesterase activity.

Table 1. Groups of dogs receiving single doses of fenamiphos in a study of acute toxicity

No clinical signs of cholinergic toxicity were seen at doses up to 0.25 mg/kg bw, but clinical signs were seen at doses > 0.5 mg/kg bw, as was vomiting. No effect was seen on feed intake or on body weight. In trial 1, plasma cholinesterase activity was consistently and significantly reduced at doses > 0.125 mg/kg bw but had largely recovered by 24 h, whereas significant inhibition (by 21%) was observed at only one time (2 h after dosing) at 0.063 mg/kg bw. Recovery was nearly complete at all doses by 24 h. In trial 1, erythrocyte cholinesterase activity was reduced at 0.5 mg/kg bw, but only by a maximum of 18% (at 1 h). Maximum effects were seen 60–90 min after administration of the material. Biologically significant inhibition of erythrocyte cholinesterase activity was not seen at lower doses. In trial 2, considerable depression of plasma cholinesterase activity was recorded at 0.5 mg/kg bw (almost 80% at 60 min), and virtually complete inhibition was observed at 2 mg/kg bw. In this trial, erythrocyte cholinesterase activity was inhibited by 42% 60 min after dosing at 0.5 mg/kg bw and by 36% at 120 min; > 90% inhibition was seen at the dose of 2 mg/kg bw. In trial 3, erythrocyte cholinesterase activity was inhibited by > 80% 60 and 120 min after dosing and by 66% at 240 min, but biologically significant inhibition of erythrocyte cholinesterase activity was not seen at 24 h at the dose of 1 mg/kg bw.

Brain cholinesterase activity was measured in the four dogs in the first trial 1 h after administration of fenamiphos at 0.5 mg/kg and in the four dogs in trial 2 given fenamiphos at the same dose but 2 h after administration. In both cases, the activity was greater than in the brains of control animals. Brain cholinesterase activity in dogs killed 2 h after administration of fenamiphos at 2 mg/kg bw was 6 U/g tissue (control value, 2.7 U/g). However, in trial 3, in which animals given fenamiphos at a dose of 1 mg/kg bw were killed 24 h after dosing, brain cholinesterase activity was 2 U/g when the brain was taken immediately and frozen and 2.2 U/g when the brain was frozen 3 h after death (see Table 2 for a summary of the data on brain cholinesterase activity). The NOAEL for inhibition of erythrocyte cholinesterase activity was 0.25 mg/kg bw, and this is an acceptable overall NOAEL for the study. A NOAEL for inhibition of brain acetylcholinesterase activity was not identified because of deficiencies in the experimental design (Detzer, 2002).

Table 2. Brain cholinesterase activity (U/g tissue, mean ± SD) in dogs given single doses of fenamiphos

From Detzer (2002)

^b Samples frozen after 3 h

Homogenized brain samples from untreated dogs were incubated with saline (control), fenamiphos or paraoxon for 1–24 h, and brain cholinesterase activity was measured according to the method of Ho & Ellman (1969). Inhibition of brain cholinesterase activity was expressed as a percentage of the activity in homogenate that had been incubated with saline, when a 21% decrease in activity was seen after 24 h. Paraoxon (10^–7 mol/l) produced inhibition rapidly, by 29% at 1 h, 27% at 2 h and 4 h and 28% at 24 h. Fenamiphos (at 10^–4 mol/l) produced 38% inhibition at 1 h, with an increase in inhibition to 96% at 24 h. Thus, fenamiphos appeared to be a considerably weaker inhibitor of brain cholinesterase activity than paraoxon, at least at early sampling times. However, 1 or 2 h may not have been the optimal time for sampling in view of the results of the study in vivo. These studies are difficult to compare because the bolus effect of a single dose in vivo could not be predicted from results obtained in vitro, where the inhibitor remained in contact with the enzyme for 24 h (Loof, 2002).

Fenamiphos is an organophosphorus compound and virtually all its toxicological effects are due to acetylcholinesterase inhibition.

In response to a request from the 1997 JMPR, a single-dose study in dogs was performed to aid in the establishment of an acute reference dose. The study was divided into three trials, during the first of which 2 males and 2 females received doses of 0.063, 0.12, 0.25 or 0.5 mg/kg bw, with wash-out periods in between dosing. In the second trial doses of 0.5 or 2 mg/kg bw were used and in the final trial the dose was 1 mg/kg bw. Plasma, erythrocyte and, at sacrifice, brain cholinesterase activities were measured and, for plasma and erythrocyte activity, dogs’ own pre-dosing activity was used as the control. Brain cholinesterase activity was compared with that of dogs from other studies. Clinical signs of cholinergic toxicity were seen at 0.5 mg/kg bw and above, while plasma cholinesterase activity was significantly inhibited at 0.12 mg/kg bw and above and also transiently at the lowest dose. Erythrocyte cholinesterase activity was inhibited at 0.5 mg/kg bw and above, the greatest inhibition occurring 60-90 min after dosing. The data on brain cholinesterase activity could not be interpreted because the controls (which were from other studies) had considerably lower activity than the test animals, with the exception of those dosed with fenamiphos at 1 mg/kg bw and killed after 24 h. Moreover, there was no evidence of a dose-related inhibition of brain cholinesterase activity in the test animals. Accordingly, the NOAEL was considered to be 0.25 mg/kg bw based on inhibition of erythrocyte cholinesterase activity at 0.5 mg/kg bw. The Meeting concluded that data on inhibition of brain cholinesterase activity were not critical to the evaluation because there was evidence from a study of absorption, distribution, metabolism and excretion, evaluated by the 1997 JMPR, of minimal ability of fenamiphos to cross the blood-brain barrier.

The Meeting established an acute RfD of 0.003 mg/kg bw based on the NOAEL of 0.25 mg/kg bw in the single-dose study in dogs reviewed at the present Meeting and a safety factor of 100. This acute RfD was supported by the NOAEL of 0.37 mg/kg bw (on the basis of clinical signs) in an oral single-dose neurotoxicity study in rats that was evaluated by the 1997 JMPR.

Detzer, K. (2002) Acute toxicity study in beagle dogs. Unpublished report No. T 6069380. Bayer AG, Institute of Toxicology, Wuppertal, Germany. Submitted to WHO by Bayer AG. GLP (OECD, 1997, USA 40 CFR 160; Japan Ministry of Agriculture, Fisheries and Food, German Chemicals Act) (with some deviations that did not affect the assessment).

Ellman, G.L., Courtney, D. & Andres, V. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88–95.

Ho, I.K. & Ellman, G.L. (1969) Triton solubilized acetylcholinesterase of brain. J. Neurochem., 16, 1505–1513.

Loof, I. (2002) Action of fenamiphos and paraoxone on acetylcholinesterase of homogenated samples of brain from untreated dogs. Bayer AG, Department of Clinical Pathology, Institute of Toxicology, Wuppertal, Germany. Submitted to WHO by Bayer AG.

Okabe, H., Sagesaka, K., Nakajima, N. & Noma, A. (1977) New enzymatic assay of cholinesterase activity. Clin. Chim. Acta, 80, 87–94.

    See Also:
       Toxicological Abbreviations
       Fenamiphos (ICSC)
       Fenamiphos (WHO Pesticide Residues Series 4)
       Fenamiphos (Pesticide residues in food: 1977 evaluations)
       Fenamiphos (Pesticide residues in food: 1978 evaluations)
       Fenamiphos (Pesticide residues in food: 1980 evaluations)
       Fenamiphos (Pesticide residues in food: 1985 evaluations Part II Toxicology)
       Fenamiphos (Pesticide residues in food: 1987 evaluations Part II Toxicology)
       Fenamiphos (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental)