FAO/PL:1968/M/9/1
WHO/FOOD ADD./69.35
1968 EVALUATIONS OF SOME PESTICIDE RESIDUES IN FOOD
THE MONOGRAPHS
Issued jointly by FAO and WHO
The content of this document is the result of the deliberations of the
Joint Meeting of the FAO Working Party of Experts and the WHO Expert
Committee on Pesticide Residues, which met in Geneva, 9-16 December,
1968.
FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS
WORLD HEALTH ORGANIZATION
Geneva, 1969
PHOSPHAMIDON
This pesticide was evaluated toxicologically by the 1965 Joint Meeting
of the FAO Committee on Pesticides in Agriculture and the WHO Expert
Committee on Pesticide Residues (FAO/WHO, 1965). Additional
toxicological data was presented and discussed at the 1966 Joint
Meeting (FAO/WHO, 1967). Since the previous evaluations, additional
data have become available and are summarized and discussed in the
following monograph addendum.
IDENTITY
Additional information on identity and properties
Phosphamidon is a mixture of approximately 30 per cent alpha-isomer
(trans-phosphamidon) and 70 per cent beta-isomer (cis-phosphamidon),
the latter form being the more active biologically. Technical
phosphamidon contains: cis- and trans-phosphamidon, 89 per cent;
gamma-chlorophosphamidon, 2 per cent; dechlorophosphamidon, 1 per
cent; and inert by-products, 8 per cent (Chevron Chemical Co., 1968;
Ciba Ltd, 1968).
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Biochemical aspects
In vitro gamma-chlorophosphamidon, forming two to four per cent of
technical phosphamidon, is about 50 times as potent an inhibitor of
human plasma cholinesterase as pure phosphamidon (Voss, 1967).
In vivo, however, there was no difference in inhibitory effect on
plasma between the technical and pure phosphamidon when fed to rabbits
in their diet (Rose, 1968a). These findings may be explained by the
effect of rabbit liver homogonates which metabolized
gamma-chlorophosphamidon extremely rapidly: five minutes incubation
was sufficient to degrade 95 per cent, while only 60 per cent of
phosphamidon was decomposed in one hour (Rose, 1968b).
gamma-chlorophosphamidon is degraded in plants at least as rapidly as
phosphamidon (Ciba Ltd, 1968), Dechlorophosphamidon has a lower
mammalian toxicity and lower anti-cholinesterase activity than
phosphamidon (Ciba Ltd, 1968).
The metabolism of phosphamidon by rats and a goat has been recently
investigated. In addition to the metabolites previously reported, two
additional ones were identified, namely "phosphamidon amide"
(N,N-bisdesethylphosphamidon) and "dechlorophosphamidon amide" (the
dimethyl phosphate ester with 3-hydroxycrotonamide). Although these
two compounds and N-desethylphosphamidon are more toxic than
phosphamidon, they do not persist in urine and milk. After the oral
administration of 3 mg/kg of a mixture of 32P-labelled and
14C-labelled phosphamidon to a goat, its milk was fractionated and
the chloroform-soluble component (containing the toxic residues)
reached a maximum after four hours of 1.2 ppm equivalents of 32P but
this level had dropped to 0.1 ppm after 32 hours and had disappeared
after 64 hours. One major metabolite, although transitory, remains to
be identified (Clemons and Menzer, 1968).
Additional data on the metabolites of phosphamidon are included in the
section entitled "Residues in food and their evaluation, Fate of
residues".
Acute toxicity
The following provides additional data on the acute toxicity of
phosphamidon to mice and rats.
LD50 (mg/kg Reference
Animal Route body-weight)
Mouse oral 10 May and Baker Ltd, 1959
s.c. 8 May and Baker Ltd, 1959
Rat (M) oral 19 May and Baker Ltd, 1959
(F) oral 12 May and Baker Ltd, 1959
(M) s.c. 7 May and Baker Ltd, 1959
(F) s.c. 4 May and Baker Ltd, 1959
The acute oral toxicity to rats of the more common metabolites of
phosphamidon is given below:
LD50 (mg/kg
Metabolite body-weight) Reference
cis-N-desethylphosphamidon 8.5 Ciba Ltd, 1968
trans-N-desethylphosphamidon 250 Ciba Ltd, 1968
alpha-chloro-N,N-diethylacetoacetamide 3 000 May and Baker Ltd, 1959
alpha-chloro-N-ethylacetoacetamide 735 May and Baker Ltd, 1959
Two groups, each containing 10 mice, inhaled an aerosol containing
five per cent or 10 per cent of phosphamidon for 90 minutes. All the
mice inhaling the 10 per cent aerosol died but there were no deaths
from the five per cent aerosol, although serious signs of
intoxication were evident (May and Baker Ltd, 1959).
In acute toxicity studies in mice, there was no potentiation of the
toxicity of phosphamidon when it was administered in equal quantities
with each of the following organo-phosphorus insecticides: dimethoate,
endothion, ethion, malathion mevinphos, oxydemeton-methyl, parathion
and phenkapton (May and Baker Ltd, 1959).
Special studies
Studies on the metabolite, N-desethylphosphamidon
Rat. N-desethylphosphamidon was applied to groups of 10 male and 10
female rats at daily doses of 1.5 and 10 mg/kg (stomach tube) over a
90-day period. Growth was retarded at the 10 mg/kg level only.
Differences in food consumption did not occur. Blood and urine were
found to be normal and no pathological changes were observed
(Industrial Bio-Test Laboratories Inc., 1964a).
Dog. N-desethylphosphamidon was given orally in capsules to groups
of three male and three female dogs over 98 days at daily doses of
0.2, 1 and 5 mg/kg. No deaths occurred at the two lower dose levels.
Pathological changes in succumbed or sacrificed animals were not
detected. In particular no degenerative changes occurred in peripheral
nerves, spinal cord or brain. Urine and blood were normal (Industrial
Bio-Test Laboratories Inc., 1964b).
Comments
Additional data from 90-day experiments on rats and dogs did not
reveal any new results permitting a change in the acceptable daily
intake.
Further studies are desirable on the metabolites of phosphamidon and
their toxicity.
TOXICOLOGICAL EVALUATION
Remains the same as in 1966, i.e. acceptable daily intake, 0-0.001
mg/kg body-weight.
RESIDUES IN FOOD AND THEIR EVALUATION
Use pattern
Pre-harvest treatments
Phosphamidon is a systemic insecticide used to control a wide variety
of plant-feeding arthropods. It is employed as a pre-harvest treatment
for many agricultural and horticultural crops as well as in forestry.
The Joint Meeting had no information on post-harvest treatments or
other uses.
Residues resulting from supervised trials
Pre-harvest Residue
Per cent. No. of interval at harvest
Crop concentration treatments (days) (ppm)
Apples 0.03 4 20 0.3*
Pears 0.04 3 31 0.2*
Grapefruit
and lemon 0.15 4 15 0.1*
Oranges 0.4 4 15 0.3*
Strawberries 0.12 1 20 0.2*
Grapes 0.06 3 21 0.2*
Broad beans 0.04 1 10 0.1**
Cucumbers 1.1 1 14 0.1*
Water-melons 0.06 3 3 0.1*
Spinach 0.25 1 16 0.1*
Carrots 0.03 1 0 0.1*
Wheat 0.06 1 60 0.1*
Rice 0.55 1 7 0.05*
* Chevron Chemical Co., 1968.
** Ciba Ltd, 1968.
Fate of residues
Phosphamidon and its most important toxic intermediate are rapidly
degraded in plants, the rate being influenced by age and environmental
factors, particularly temperature. The major pathways of metabolism in
animals are qualitatively the same with rapid degradation to non-toxic
substances.
The more important metabolites detected in beans were
N-desethylphosphamidon, alpha-chloro-N,N-diethylacetoacetamide and
alpha-chloro-N-ethylacetoacetamide (May and Baker Ltd, 1959).
The main toxic metabolite, N-desethylphosphamidon (Anliker et al.,
1961) is more rapidly degraded in the bean plant than the parent
compound, while its mammalian toxicity and plasma cholinesterase
activity are similar to phosphamidon. This observation has been
confirmed for other fruit and vegetables, except apples where the rate
of degradation is of the same order of magnitude (Chevron Chemical
Co., 1968),
The comparative fate of the two geometrical isomers of phosphamidon in
plants and insects was studied (Bull et al., 1967). Although the
cis- is much less toxic than the trans-isomer the difference could
not be entirely accounted for by the more extensive oxidative
N-dealkylation of the cis-isomer. The main metabolites of both
isomers were O-demethyl and N-desethyl phosphamidon and dimethyl
phosphate, of which only the N-desethyl derivative is toxic.
From petiole injections of cotton plants with cis- and
trans-isomers of phosphamidon the biological half-life for the toxic
compounds was slightly less than one day.
The metabolism of a cis-trans mixture (30:70) by excised cotton
leaves and alfalfa sprigs followed a similar pattern.
No information is available on losses of phosphamidon during storage
and processing, although it is known to be readily degraded on
cooking.
Per cent. of applied dose recovered after
various time intervals
Product cis-isomer trans-isomer
Day Day
1 2 4 8 1 2 4 8
Dimethyl phosphate 17.2 23.0 27.8 24.0 7.4 10.8 14.3 16.3
O-demethylphosphamidon 26.4 11.8 4.9 2.9 9.2 4.5 4.2 2.3
N-desethylphosphamidon 18.4 8.6 5.8 2.5 0 1.3 3.0 3.2
Phosphamidon 15.6 7.2 0.7 3.2 38.6 13.9 3.8 4.0
Per cent. of applied dose recovered after
various time intervals
Product Cotton Alfalfa
Day Day
1 2 4 8 1 2 4 8
Dimethyl phosphate 5.6 13.3 20.9 20.6 12.6 27.3 29.8 32.2
O-demethylphosphamidon 9.7 6.9 6.3 6.8 4.9 3.0 1.6 1.1
N-desethylphosphamidon 16.0 9.1 4.5 1.7 10.6 9.5 4.6 2.1
Phosphamidon-cis 10.4 1.5 0.4 0.4 13.4 4.4 2.6 0.9
Phosphamidon-trans 28.3 6.7 1.5 1.4 39.3 14.5 6.5 2.1
Methods of residue analysis
Residues have been determined by measuring the extent of inhibition of
human plasma cholinesterase (Chevron Chemical Co., 1963). The original
method did not distinguish between active impurities and metabolites
but subsequent refinements allow the separation and estimation of
gamma-chlorophosphamidon and the active metabolite,
desethylphosphamidon. The original method detects 0.1 ppm with a
certain loss of precision at 0.05 ppm.
Another enzymatic method is based on the hydrolysis of
acetylthiocholine followed by coupling with dithiobisnitrobenzoic acid
and measurement of the resulting colour. By introduction of thin-layer
chromatography the individual components can be measured separately
with a sensitivity between 0.05 and 0.1 ppm (Voss and Geissbühler,
1967). A double-paper chromatography method is highly specific but is
too time consuming for serial analyses (Pack et al., 1964).
Phosphamidon may also be analysed by a gas chromatographic method
developed for related enolphosphates (Bowman and Beroza, 1967).
National tolerances
Country Crop Tolerance (ppm)
Germany Apples 0.5
Vegetables 0.1
Italy General 0.5
Switzerland General 0.5
United States of Apples 1.0
America Citrus 0.75
Various
vegetables 0.5
Cantaloupes,
water-melons 0.25
Walnuts 0.1
RECOMMENDATIONS FOR TOLERANCES AND PRACTICAL RESIDUE LIMITS
Appraisal
There is a wide range of use in vegetable, fruit and cereal crops. No
quantitative information was available to the Joint Meeting on the
amounts used annually for stated purposes.
There is good evidence that phosphamidon, which consists of a mixture
of two geometrical isomers, undergoes relatively rapid metabolism in
plant tissues. The most significant anti-cholinesterase metabolite
appears to be the result of oxidative N-de-ethylation. Other
anti-cholinesterase metabolites have been identified but are rapidly
degraded. On the basis of acute toxicity and cholinesterase inhibition
data, this metabolite has a comparable toxicity to that of the parent
compound. Residue assays should, therefore, take both phosphamidon,
the N-desethyl derivative and the minor anti-cholinesterase
metabolites into account. This has been done by the use of the
cholinesterase inhibition technique for the residue measurements made
in the supervised trials.
Many data on the residues resulting from supervised trials were made
available by the manufacturers; mainly from Switzerland and the United
States of America. As a result of the residue and metabolism studies
it is possible to recommend the following temporary tolerances.
Recommendations
The following temporary tolerances, to be in effect until 1972, are to
apply to raw agricultural products moving in commerce unless otherwise
indicated. In the case of fruit and vegetables the tolerances should
be applied as soon as practicable after harvest and in any event prior
to actual retail to the public. In the case of commodities entering
international trade, the tolerances should be applied by the importing
country at the point of entry or as soon as practicable thereafter.
Temporary tolerances
Raw cereals 0.1 ppm
Apples, pears 0.5 ppm
Citrus fruit 0.4 ppm
Other fruit 0.2 ppm
Cucumbers, lettuce, tomatoes,
water-melons 0.1 ppm
Cole crops, other vegetables
except root vegetables 0.2 ppm
Root vegetables none required
Residues to be determined by cholinesterase inhibition technique and
results to be expressed as phosphamidon.
Further work or information
Required before 30 June 1972
1. Data on the required rates and frequencies of application,
pre-harvest intervals, and the resultant residues from different
countries.
2. Confirmatory studies of the nature and persistence of the
residues in fruits and vegetables, fresh and processed.
3. Development of an analytical method that is specific for
phosphamidon and its N-de-ethyl derivative.
Desirable
1. Biochemical and metabolic fate of phosphamidon in man following
different types of exposure.
2. Reproduction studies in at least one species other than the rat.
REFERENCES
Anliker, R., Beriger, E., Geiger, M. and Schmid. K. (1961) Über die
synthese von phosphamidon und seinen abbau in pflanzen. Helv. Chim.
Acta., 44: 1622-1645
Bowman, M. C. and Beroza, M. (1967) Gas chromatographic analysis of
3-hydroxy-N-methyl-cis-crotonamide dimethylphosphate (Azodrin) and
3-hydroxy-N,N-dimethyl-cis-crotonamide methylphosphate. J. Agric. Food
Chem., 15: 465-468
Bull, D. L., Lindquist, D. A. and Grabbe, R. R. (1967) Comparative
fate of the geometric isomers phosphamidon in plants and animals. J.
Econ, Entomol., 60: 332-341
Chevron Chemical Co. (1963) Analysis of phosphamidon residues. Method
RM-4. Internal report
Chevron Chemical Co. (1968) Unpublished data from Company submission
to Codex Committee on Pesticide Residues
Ciba Ltd. (1968) Unpublished data from Company submission to Codex
Committee on Pesticide Residues
Clemons, G. P. and Menzer, R. E. (1968) Oxidative metabolism of
phosphamidon in rats and a goat. J. Agric. Food Chem., 16: 312-318
FAO/WHO. (1965) Evaluation of the toxicity of pesticide residues in
food. FAO Mtg. Rept. PL/1965/10/1; WHO/Food Add./27.65
FAO/WHO. (1967) Evaluation of some pesticide residues in food. (FAO,
PL:CP/15; WHO/Food Add./67.32)
Industrial Bio-Test Laboratories Inc. (1964a) 90 day subacute oral
toxicity of desethylphosphamidon - albino rat. Unpublished report
Industrial Bio-Test Laboratories Inc. (1964b) 14-week subacute oral
toxicity of desethylphosphamidon - beagle dogs. Unpublished report
May and Baker Ltd. (1959) Phosphamidon. Unpublished data submitted to
the Ministry of Health, London, United Kingdom
Pack, D. E., Ospenson, J. N. and Kohn, G. K. (1965) In: Analytical
methods for pesticides, plant growth regulators and food additives,
2: 375-392 Academic Press
Rose, J. A. (1968a) A preliminary study of factors influencing the
toxicity of pure phosphamidon and technical phosphamidon. Ciba Ltd,
unpublished report
Rose, J. A. (1968b) Degradation of phosphamidon and related vinyl
phosphates by rabbit liver homogenates. Ciba Ltd, unpublished report
Voss, G. (1967) Determination of gamma-chlorophosphamidon in samples
of technical phosphamidon by an automated cholinesterase inhibition
procedure. Ciba Ltd, unpublished report
Voss, G. and Geissbühler, H. (1967) Automated residue determination of
insecticidal enolphosphates. (19th Internat. Sympos. Plant
Protection.) Med. Rijksfaculteit Landbouwwetenschappen, Ghent,
23: 877-889
See Also:
Toxicological Abbreviations
Phosphamidon (ICSC)
Phosphamidon (PIM 454)
Phosphamidon (FAO Meeting Report PL/1965/10/1)
Phosphamidon (FAO/PL:CP/15)
Phosphamidon (FAO/PL:1969/M/17/1)
Phosphamidon (WHO Pesticide Residues Series 2)
Phosphamidon (WHO Pesticide Residues Series 4)
Phosphamidon (Pesticide residues in food: 1982 evaluations)
Phosphamidon (Pesticide residues in food: 1986 evaluations Part II Toxicology)