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    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

    CRUFOMATE

    IDENTITY

    Chemical name

         4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate

         O-(4-tertbutyl-2-chlorophenyl) methyl methylphosphoramidate
         (IUPAC)

    Synonyms

         Ruelene(R),  Dowco(R)132, Montrel(R), Rulex(R)

    Structural formula

    CHEMICAL STRUCTURE 

    Other information on identity and properties

    The recrystallized technical product melts at 52.5-59.2C. When pure,
    crufomate is a white solid melting at 62-62.5C. The compound is
    stable from room temperature to 60C. It is unstable in strongly acid
    and in alkaline media. At room temperature, it is freely soluble in
    most of the common organic solvents except for the paraffin solvents
    in which its solubility is about one to three per cent by weight. Its
    solubility in water is 0.5 per cent by weight.

    Crufomate is formulated in several concentrations either for direct
    use or for application after suitable dilution (emulsifiable
    concentrates, pour-ons, systemic insecticide, anthelmintic, sheep and
    goat wormer).

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    Biochemical aspects

    Following an 11 mg/kg intramuscular dosage of 32P labelled crufomated
    to a yearling steer, radioactivity at the site of the treatment
    dropped from 16 milliroentgens per hour immediately following the
    treatment to 1.5 within two days and 0 after 13 days. Residues of
    crufomate were practically non-existent after 14 days. Peak levels of
    radioactivity occurred in the blood, urine and faeces in two to eight
    hours, 12-18 hours, and 18-30 hours respectively. Fat tissues

    contained less than 0.2 ppm of radioactive compounds after 14 days.
    Red blood cholinesterase activity was depressed no more than 25 per
    cent. The end product of the metabolism of crufomate is phosphoric
    acid (Plapp. 1960).

    A 21-day study of the metabolism of radioactive crufomate using acute
    oral dosages of 100 and 200 mg/kg of body-weight in sheep shows
    residues in the urine consisting of crufomate and several hydrolysis
    products. Over 85 per cent of the administered 32P was recovered in
    the excreta. The 32P in the urine, amounting to 75 per cent of the
    dose, was primarily in the form of the hydrolysis products of
    crufomate. Some of the crufomate was hydrolyzed completely to
    inorganic phosphate and retained in the animal tissues and bones.
    Crufomate itself was not found in the tissues after seven days,
    indicating fairly rapid metabolism. Residues of crufomate and several
    hydrolysis products were found in the blood and plasma shortly after
    treatment, but within two days the crufomate decreased to a low level
    (Bauriedel and Swank, 1962).

    Other metabolic studies in sheep (Chamberlain and Gatterdam, 1960),
    and in poultry (Buttram and Arthur, 1961) showed rapid breakdown of
    crufomate. The likely mammalian metabolites of crufomate do not cause
    inhibition of cholinesterase (Smith, 1968).

    Acute toxicity (oral)

                                                                 
    Animal        Sex      LD50 (mg/kg          References
                           body-weight)
                                                                 

    Rat           male           950      McCollister et al., 1968

    Rat           female         770      McCollister et al., 1968

    Rabbit        mixed          490      McCollister et al., 1968

    Guinea-pig    male         1 000      McCollister et al., 1968

    Dog           male        >1 000      McCollister et al., 1968

    Dog           female        >750      McCollister et al., 1968
                                                                 

    Sheep. Lambs received a single dose of 100, 200 or 400 mg/kg. Signs
    of intoxication appeared at 200 mg/kg or more. Whole blood
    cholinesterase activity was depressed to 50 per cent at 100 mg/kg,
    and was markedly depressed at higher doses (Galvin et al., 1960).

    Symptoms are highly variable and mainly caused by central nervous
    system disturbances. At necropsy severe congestion of lungs and
    kidneys, enlargement of liver and scattered haemorrhages were found
    (Radeleff, 1964).

    Short-term studies

    Rat. Rats (10 males and 10 females per group) were started at the
    age of 45 days on diets containing 10, 30, 100, 300 or 1000 ppm
    crufomate. During a 90-day experimental period no evidence of
    ill-effect was found when judged by general appearance and behaviour,
    growth, mortality or food consumption records, haematological
    determinations, final body and organ weight ratios, and gross and
    microscopic examination of tissues. Results of measurements of
    cholinesterase activity in blood and brain show that at 41 days there
    was a peculiarly uniform depression of cholinesterase activity in the
    plasma of both male and female rats over the entire range of 10-1000
    ppm. The same phenomenon, although not as marked, was seen at 69 days,
    indicating that a higher intake of chemical per unit of body-weight
    during the early period way have been partially responsible.
    Measurement of the erythrocyte-and brain-cholinesterase activities
    showed little or no effect at the 30 ppm level of crufomate. In the
    groups of rats from each dietary level which were placed on the
    control ration for 48 days following the 90-day test period, recovery
    of blood and brain cholinesterase was complete (McCollister et al.,
    1968).

    Dog. Groups comprising one male and one female dog were given diets
    containing 0, 15, 40, 125 or 250 ppm crufomate for 75 days. Dogs that
    received 250 ppm crufomate in their diets for 75 days showed no
    evidence of adverse effect by any of the criteria examined except
    cholinesterase activity and liver histopathology. Plasma-,
    erythrocyte- and brain-cholinesterase levels were decreased to about
    65 per cent of the control values. Slight centrilobular granular
    degeneration was observed on microscopic examination of the liver.
    Cholinesterase measurements were within the normal range in dogs at
    the 125 ppm level. There was no evidence of adverse effect in dogs
    that received 15 or 40 ppm dietary crufomate for 75 days, as judged by
    general appearance and behaviour, growth, mortality, food consumption,
    haematological indices, cholinesterase activities, final body and
    organ weights and gross and microscopic appearance of the tissues
    (McCollister et al., 1968).

    Groups of dogs, each containing four males and four females per group,
    were given diets containing 0, 10, 20, 200 or 2000 ppm of crufomate
    for up to two years. The dogs on 2000 ppm crufomate were transferred
    to the control diet after 30 days and sacrificed on days 94 and 95 of
    the study. After one year, one male and one female from each of the
    other groups were sacrificed for tissue examination. Cholinesterase
    determinations were made at 30, 60 and 90 days after the start of the
    study and at six-week intervals up to one year and every three months
    thereafter. The animals on the highest level of crufomate manifested
    gross signs of intoxication, values for serum alkaline phosphatase and
    transaminase were elevated, plasma cholinesterase was reduced to 55
    per cent of the pre-test average, and red cell cholinesterase was
    depressed to 19 per cent of the pre-test values. In those dogs that
    received 200 ppm crufomate in their diet plasma and erythrocyte

    cholinesterase activity was depressed slightly. Other biochemical,
    haematological findings and gross and microscopic examinations of the
    tissues were comparable to the controls. Groups of male and female
    dogs that received diets containing 10 or 20 ppm for two years showed
    no evidence of adverse effect according to any of the criteria applied
    (McCollister et al,, 1968).

    Long-term studies

    Rat. Groups of rats (25 of each sex) were started on diets
    containing 1, 10, 100 or 1000 ppm of crufomate. Fifteen rats of each
    sex from each group were designated for a two-year study. The
    remaining 10 of each sex were killed at 12 or 18 months. Additional
    groups of rats (five of each sex) were given diets containing 20, 40,
    60 or 80 ppm of crufomate for 20 months.

    Both male and female rats that received 1000 ppm crufomate in their
    diets showed retardation of growth during the second year of the
    study. Necropsy and microscopic examination of these rats after 24
    months revealed atrophy of the muscles of the hindlegs and slight
    degeneration of the sciatic nerve. A reduction in testis weight to
    about one half, reflecting degeneration and atrophy of seminiferous
    tubules, was seen at the 1000 ppm level after 12 and 18 months and
    again at 24 months. The only evidence of my effect at dosage levels
    below 1000 ppm was depression of cholinesterase. Brain cholinesterase
    activity was depressed to 38 to 50 per cent of control for the rats
    fed 1000 ppm but was within normal range at the lower dosage levels.
    Male rats showed no significant effect on plasma cholinesterase at 100
    ppm and below, or in red cells at 40 ppm and below. Corresponding
    values for female rats were 40 ppm for plasma and 60 ppm for red cell
    cholinesterase (McCollister et al., 1968).

    Special studies

    (a) Fertility and reproduction

    Groups of four male and 12 female rats were maintained on diets
    containing 0, 10 and 100 ppm crufomate through a three-generation
    study involving two litters per generation and assessing fertility,
    reproduction and lactation. In addition, a fourth group received 1 ppm
    crufomate for the first generation and 500 ppm for the remaining two
    generations of the experiment. In all these groups no evidence was
    found of adverse effect on fertility, gestation, viability or
    lactation, as well as body-weights at weaning and mating.
    Cholinesterase inhibition was not investigated (McCollister at al.,
    1968).

    Forty bulls, three to seven years old, were given a single oral dose
    of crufomate at 50 mg/kg or sprayed to wetness with 0.75 per cent
    dispersion of a crufomate wettable powder without affecting the
    motility of the spermatozoa after one and four weeks (McGregor 1960).

    An acute oral dosage of 100 mg/kg of crufomate had no effect on the
    fertility of male rats, rabbits or guinea-pigs (McCollister, 1959).

    (b)  Neurotoxicity

    Intraperitoneal injections of crufomate into 25 mature hens at dosage
    rates of 500, 750 and 1000 mg/kg caused mortalities in one out of
    seven, one out of four and 10 out of 14, respectively, of the birds
    treated. Of the surviving 31 treated hens 26 developed ataxia and
    paralysis. Those able to take food and water made uneventful and
    complete recovery within 90 days of treatment. Histopathological
    examination of nerve tissue revealed no departure from normal (Hymas
    and Stevenson, 1961).

    (c)  Potentiation

    Single oral doses of crufomate were administered jointly to rats with
    each of 14 commercial cholinesterase inhibiting insecticides (13
    organo-phosphorus compounds and one carbamate). There was no effect
    other than additive (ratios 0.7 to 2.2) for the LD50 values except
    for crufomate-malathion (fivefold increase). Dietary feeding studies
    in dogs for 12 weeks with diets containing 100 ppm malathion plus 20
    ppm crufomate, or 10 ppm malathion plus 20 ppm crufomate, showed no
    significant evidence of potentiation (McCollister et al., 1968).

    (d) Studies on the metabolite

    Rat. Groups of rats (25 of each sex) were fed 100, 300, 1000, and
    3000 ppm 4-tert-butyl-2-chlorophenol (the hydrolytic metabolite of
    crufomate) in the diet for two years. Rats fed the dosage levels of
    100, 300 or 1000 ppm showed no significant evidence of adverse effects
    as judged by general appearance, behaviour, growth, mortality,
    incidence of tumorous growths, haematological studies, serum urea
    nitrogen and alkaline phosphatase determination. Final average body
    and organ weights and gross and microscopic effects after examination
    of the tissues were normal. Rats fed dietary levels of 3000 ppm showed
    moderate microscopic liver changes and increased senility in the
    heart, kidney and liver (McCollister, 1964).

    Dog. Groups of eight dogs (four of each sex) were fed diets
    containing 20, 200 or 2000 ppm 4-tert-butyl-2-chlorophenol for two
    years. The male dogs on 2000 ppm showed atrophy and degenerative
    changes in the seminiferous tubules of the testes. No other gross or
    microscopic effect was observed at any dose level (Dieterich et al.,
    1965).

    Observations in man

    Groups of three adult males per test were given one, two or four
    tablets containing 200 mg of crufomate daily for seven days.
    Laboratory tests consisted of complete blood counts, urinalysis, red

    blood cell cholinesterase response and serum glutamic pyruvic
    transaminase determinations. The subjects were interrogated concerning
    side effects. The second and third groups showed a delayed 50-80 per
    cent red blood cell cholinesterase depression after completion of the
    treatments. No other abnormalities were noted in the laboratory tests
    (Campbell, 1962).

    Comments

    The most sensitive criterion upon which to judge the safety of
    crufomate upon ingestion by animals or human subjects is its
    cholinesterase-inhibiting properties. The short-term and long-term
    studies are adequate. Because the only study available in man was too
    limited, it is desirable that further metabolic studies in man should
    be carried out to show that 4-tert-butyl-2-chlorophenol is the main
    metabolite also in this species.

    TOXICOLOGICAL EVALUATION

    Level causing no significant toxicological effect

         Rat: 40 ppm in the diet, equivalent to 2 mg/kg per day

         Dog: 40 ppm in the diet, equivalent to 1 mg/kg per day

         Man: 200 mg/day, equivalent to about 3 mg/kg per day

    Estimate of acceptable daily intake for man

         0-0.1 mg/kg per day

    RESIDUES IN FOOD AND THEIR EVALUATION

    Use pattern

    Crufomate is used on or in cattle, sheep, and goats to control
    internal cattle grubs and external horn flies and cattle lice (Anon.,
    1966). Applications of crufomate for cattle grub control have been by
    injection, oral administration, and spraying (McGregor et al., 1959),
    but the simple pour-on application of a low volume of insecticide on
    the backs of cattle has been found best (Rogoff and Kohler, 196O).
    Formulations of crufomate may be sprayed or poured on to cattle during
    the warmer months of the year. However, in the United States of
    America during October through February, treatment is restricted
    depending on the locality. The dosage rate ranges from about 15 to 50
    mg/kg of body-weight.

    As a pour-on applied to the backline of cattle, an emulsifiable
    concentrate of crufomate is diluted with water to apply about 39 mg/kg
    for grubs, horn flies and lice, and about 52 mg/kg for internal
    helminth parasite control.

    Another treatment of internal helminth parasites of livestock involves
    the oral drench (introduction into rumen directly) of 50 to 75 mg/kg.
    The following internal parasites of livestock are examples of genera
    for which this treatment has been recommended: Haemonchus and
    Osteragia (stomach worms), Tricho-strongylus (hairworms),
    Strongyloides (threadworm), Bunostomum (hookworm),
    Oesophagostomum (nodular worms), Nematodirus (thin-necked
    strongyle), Trichuris (whipworms), Cooperia (Cooper's worms),
    Oestrus ovis (head grub) and others.

    Retreatment for horn flies and cattle lice should not be done more
    often than every 28 days and not within 28 days of slaughter. Animals
    under stress from sickness, crowding, excitement or surgery should not
    be treated. Lactating dairy cows or dry dairy cows within 28 days of
    freshening should not be treated. General recommendations for the
    current use of cruformate are given in Anon. (1967, 1968).

    Residues resulting from supervised trials

    When crufomate is used as directed, its residues decrease to a
    negligible level 28 days after external treatment and 14 days after
    internal treatment. Residues of crufomate found in supervised trials
    are summarized in the following table:

                                                                                            
                   Rate of        Post-treatment     Residues*             Reference
    Animal         application    interval (days)      (ppm)
                   (mg/kg)
                                                                                        

    Cattle            75              14             No residues         Anon. (1962b)
                                                     (<0.04) fat,
                                                     lean meat

    Sheep             89.8            14             No residues         Claborn et al.
                                                     (<0.02) fat,        (1960)
                                                     internal organs

    Lambs            200               7             No residues         Bauriedel and
                                                     (<0.05) fat,        Swank (1962)
                                                     lean meat

    Cattle            50               7             0.3-1.3**           Mussel and
                                                     fat, less in        Ludwig (1961)
                                                     other tissues

    Cattle           100               7             0.6-1.6**           Mussel and
                                                     fat, less in        Ludwig (1961)
                                                     other tissues

    (continued)
                                                                                        
                   Rate of        Post-treatment     Residues*             Reference
    Animal         application    interval (days)      (ppm)
                   (mg/kg)
                                                                                        

    Cattle            50               7             0.07-0.2** fat      Mussel and
                                                                         Ludwig (1961)

    Cattle           100               7             0.1-2.4** fat       Mussel and
                                                                         Ludwig (1961)

    Cattle            52               7             0.05-0.14 fat       Dishburger and
                                      14             <0.02-0.05 fat      Rice (1968)
                                      21             no residues
                                                     (<0.02) fat

    Calves         52 and 104         28             0.1-0.22 fat        Rice (1964)

    Lactating         49-66            1             0.195 milk          Leahy and
    cow                                2             no detectable       Brown (1963)
                                                     residues
                                                     (<0.05) milk

    Lactating         37.5             1             0.14-0.3 milk       Anon. 1962a
    cow                                2             0.0 milk

    Steers            50               7             0.1 phenol in       Kutchinski
                                                     fat (limit of       (1960)
                                                     sensitivity, 0.1)
                                                                                        

    *  When one figure is given, residue is maximum value.
    ** No residues (<0.1 ppm) after 28 days.
    
    Fate of residues

    In animals

    Six possible routes for the hydrolysis of crufomate to inorganic
    phosphate are shown below (Bauriedel and Swank, 1962).

    CHEMICAL STRUCTURE 

    B, C, D, F and G have been identified, definitely or tentatively, as
    metabolites in addition to the 4-tert-butyl-2-chlorophenol
    hydrolysis product.

    Several studies with P32 labelled crufomate administered orally and
    by injection to cattle, sheep, and hens have shown that crufomate is
    eliminated rapidly as hydrolysis products or hydrolyzed to inorganic
    phosphate which may be retained. (Bauriedel and Swank, 1962; Brady and
    Arthur, 1962; Buttram and Arthur, 1961; Plapp, 1960; Chamberlain and
    Gatterdam, 1960).

    The two products that may be expected as residues are cruformate and
    its phenolic hydrolysis product. The other hydrolysis products
    (metabolites A, B, C, D, E, F) appear to be too polar to be stored
    appreciably; the studies with P32 labelled crufomate verify the rapid
    elimination of these products.

    Evidence of residues in food in commerce or at consumption

    No residues of crufomate were found in 149 tissue samples taken in
    1968 from slaughter-houses in five states (Indiana, Louisiana,
    Nebraska, Oklahoma, and Texas) in the United States of America
    (Stewart, 1968).

    Methods of residue analysis

    Because crufomate is used almost exclusively for the control of
    arthropods and worms on or in animals, residue analyses for this
    chemical and its metabolites have been conducted primarily on meat,
    milk, blood, and animal tissues.

    For the residue analyses at the present time, the method of choice
    based on sensitivity, specificity, and speed of analysis would appear
    to be gas chromatography with a thermionic or flame photometric
    detector for sensing phosphorus compounds, after removal of fat (if
    necessary) by a hexane-acetonitrile or similar solvent partition.
    Burke (1965) and Bowman and Beroza (1965) determined the compound
    itself by gas chromatography. Bowman and Beroza (1967) later
    determined it in milk and silage by temperature-programmed gas
    chromatography with the flame photometric detector as part of a
    multicomponent analysis. No clean-up other than a hexane-acetonitrile
    partition was needed to remove the fat from the milk extract. The
    fraction partitioning into the upper fat-containing phase is readily
    calculated from the p-value, which is 0.031 in this system (Bowman
    and Beroza, 1965). Recoveries at levels of fortification of 0.05 and
    0.2 ppm were generally better than 90 per cent with a sensitivity of
    about 0.01 ppm. In an unpublished communication Dishburger and Rice
    (1968) determined crufomate in animal fat tissue. They used liquid
    chromatography on silicic acid for a clean-up prior to analysis of the
    eluate by gas chromatography with thermionic detection. Sensitivity
    was 0.02 ppm and recoveries averaged 92 per cent at levels of 0.02 to
    0.06 ppm. The use of electron capture, microcoulometric, and
    electrolytic-conductivity detectors for the gas chromatographic
    analysis of crufomate appears feasible but has not been evaluated.

    Early methods used in the analysis of crufomate residues were
    manometric housefly-head cholinesterase inhibition procedures
    developed by The Dow Chemical Co. They required modifications
    depending on the substrate being analysed. A procedure for extracting
    crufomate from milk developed by Timmerman et al. (1961) was about 90
    per cent efficient. Leahy and Taylor (1963) determined crufomate in
    milk by paper chromatography of the milk extract, acid digestion of
    the portion of the paper chromatogram containing the pesticide, and
    determination of the phosphorus content colorimetrically.

    For confirmation of identity of residues, Elgar (1967) suggested
    independent parameters that might be used. When enough material can be
    isolated, spectroscopy (infra-red, ultra-violet, mass) is excellent
    for such confirmations. Bowman and Beroza (1967), reported gas
    chromatographic retention times on three liquid phases for crufomate.

    They also gave partitioning data (p-values) in six binary solvent
    systems, which may be used for identification or for establishing the
    quantitative validity of gas chromatographic peaks. Rf values in
    paper (Leahy and Taylor, 1963; McKinley and Read, 1962) and thin-layer
    chromatography (El-Refai and Hopkins, 1965; Ragab, 1967) can be
    useful. The combined use of these and other methods (chemical
    derivatives, bio-assay) will make identifications more certain.

    The 4-tert-butyl-2-chlorophenol metabolite of crufomate was
    determined spectrophotometrically in beef fat and meat with a
    sensitivity of 0.1 ppm and an average recovery of 80 per cent after a
    clean-up by liquid chromatography and steam distillation, coupling
    with diazotized p-nitroaniline, and chromatography of the resulting
    dye (Anon., 1960). The presence of a chlorine atom may allow
    quantification by electron-capture gas chromatography.

    National tolerances

    Registration in the United States on a non-residue basis (Anon., 1965)
    is to be superseded by the establishment of negligible or finite
    residue tolerances. In other countries such as Great Britain, Ireland,
    European Economic Community countries, all Latin American countries,
    Australia, New Zealand, and Canada, products containing crufomate are
    registered on a non-residue basis when used for external parasite
    control. Crufomate is registered for internal parasite control of
    livestock in Great Britain, Ireland, Spain, Australia, New Zealand,
    Brazil, Uruguay, Argentina, Colombia, Ecuador, Venezuela, Paraguay,
    Central American countries, Mexico, Canada and the United States of
    America.

    The Dow Chemical Company is presently reviewing their residue and
    toxicological data on crufomate and may soon recommend shortening or
    elimination of withdrawal time specifications.

    RECOMMENDATIONS FOR TOLERANCES AND PRACTICAL RESIDUE LIMITS

    Appraisal

    Crufomate is used internally and is applied externally to livestock as
    an insecticide; it is also used as an anthelmintic. In its principal
    uses, it is administered long before slaughter so there will be no
    residues from such applications in meat. Another use, of secondary
    importance, is accompanied by a recommended minimum of 14 days between
    treatment and slaughter.

    Lactating cattle experimentally treated by pour-on oral drench yielded
    milk that did not contain residues in excess of 0.05 ppm after two
    days.

    A monitoring survey, conducted in 1968 on cattle tissue from five
    states of the United States of America, detected no residues in any of
    the 149 samples examined.

    Several gas chromatographic methods for the compound, which appear to
    be suitable for regulatory purposes or for evaluation as a referee
    procedure, have been reported.

    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 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

         Meat (on fat basis) (at slaughter)           1 ppm
         Whole milk                                   0.05 ppm

    Further work or information

    Required before 30 June 1972

    1. Data from countries other than the United States of America and the
    United Kingdom on the use pattern and resultant residues.

    2. Further data on the use pattern and resultant residues in milk from
    treated animals, using gas-liquid chromatographic methods.

    3. Data on residues in the non-fatty portion of meat and meat
    products.

    4. Comparative evaluation of gas-liquid chromatographic methods for
    regulatory purposes.

    Desirable

    1. Collaborative studies to establish a referee method.

    2. More extensive studies on cholinesterase effects in man.

    3. Studies on the metabolism in man to show that
    4-tert-butyl-2-chlorophenol is the main metabolite in that species.

    REFERENCES

    Anon. (1960) Determination of 4-tert-butyl-2-chlorophenol in the
    meat and the fat of cattle. Residue determination method ACR 60.8. The
    Dow Chemical Co. Midland, Michigan

    Anon. (1962a) Residues of Ruelene (R) in milk from a single oral
    drench application of Ruelene wormer sheep and goat drench. The Dow
    Chemical Co., Midland, Michigan. Unpublished report

    Anon. (1962b) Residues of Ruelene in fat and lean meat from cattle
    treated with Ruelene in the feed and by oral drench. The Dow Chemical
    Co., Midland, Michigan. Unpublished report

    Anon. (1965) U.S.D.A. Summary of registered agricultural pesticide
    chemical uses (4-tert-butyl-2-chlorophenyl methyl
    methylphosphoramidate). p. 767, Issued October 1, 1965, Pesticides
    Regulation Division, Agricultural Research Service, U.S. Department of
    Agriculture, Washington, D.C.

    Anon. (1966) Ruelene (R) systemic insecticide, The Dow Chemical Co., 
    Midland, Michigan

    Anon. (1967) Suggested guide for the use of insecticides to control
    insects affecting crops, livestock, households, stored products,
    forests, and forest products, 1967. Agriculture Handbook No. 331,
    U.S.D.A. Supt. of Documents, U.S. Government Printing Office,
    Washington, D.C.

    Anon. (1968) Technical information bulletin. 1968 registered livestock
    and household uses for Ronnel and registered livestock uses for
    "Ruelene". The Dow Chemical Co., Midland, Michigan

    Bauriedel, W. R. and Swank, M. G. (1960) Residue and metabolism of
    radioactive Ruelene (R), 4-tertiary-butyl-2-chlorophenyl methyl
    methylphosphoramidate, administered as a single oral dose to cattle.
    Dow Chemical Co. Unpublished report

    Bauriedel, W. R. and Simmons, B. L. (1961) Residue and metabolism of
    radioactive Ruelene (R), 4-tertiary-butyl-2-chlorophenyl methyl
    methylphosphoramidate, administered to a steer by intrarumenal
    injection. Dow Chemical Co. Unpublished report

    Bauriedel, W. R. and Swank, M. G. (1962) Residue and metabolism of 
    radioactive 4-tert-butyl-2-chlorophenyl methyl
    methylphosphoramidate, administered as a single oral dose to sheep. J.
    Agr. Food. Chem., 10: 150-154

    Bowman, M. C. and Beroza, M. (1965) Extraction p-values of
    pesticides  and related compounds in six solvent systems. J. Ass.
    Offic. Agr. Chem., 48: 943-952

    Bowman, M. C. and Beroza, M. (1967) Temperature programmed gas
    chromatography of twenty phosphorus-containing insecticides on four
    different columns and its application to the analysis of milk and corn
    silage. J. Ass. Offic. Anal. Chem., 50: 1228-1236

    Brady, U. E., jr and Arthur, B. W. (1962) Absorption and metabolism of 
    Ruelene(R) by Arthropods. J. Econ. Entom., 55: 833-836

    Burch, G. R. (1963) Report on Ruelene(R) study in dogs. Pitman-Moore 
    Division, The Dow Chemical Co., Indianapolis, Indiana. Unpublished
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    Burke, J. A. (1965) Gas chromatography of pesticide residue analysis: 
    Some practical aspects. J. Ass. Offic. Agr. Chem., 48: 1037-1058

    Buttram, J. R. and Arthur, B. W. (1961) Magnitude and nature of
    residues in tissues and eggs of poultry receiving Ruelene (R) in the
    feed. J. Econ. Entom., 54: 456-460

    Campbell, P. J. (1960) Study of Ruelene. Stough-Wisdom Research Inc. 
    Unpublished report

    Chamberlain, W. F. and Gatterdam, P. E. (1960) Third report on studies
    with P32 labeled Ruelene. Livestock Insects Investigations,
    Entomology Research Division, U.S.D.A., Kerrville, Texas. Unpublished
    report

    Claborn, H. V., Mann, H. D. and Ivey, M. C. (1960) Second report on
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    Dieterich, W. H., Paynter, O. E. and Weir, R. J. (1965) The chronic
    toxicity of Ruelene and a Ruelene derivative in beagle dogs. Hazleton
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    Dishburger, H. J. and Rice, J. R. (1968) Residues in omental fat of
    cattle following pour-on application of Ruelene (R) 8R and 25E
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    Unpublished report

    Elgar, K. E. (1967) Confirmation of identity, in report by Egan, H.,
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    El-Refai, A. and Hopkins, T. L. (1965) Thin-layer chromatography and 
    cholinesterase detection of several phosphorothiono insecticides and
    their oxygen analogs. J. Agr. Food Chem., 13: 477-481

    Galvin, T. J., Bell, R. R. and Turk, R. D. (1960) Anthelmintics for 
    ruminants. II. Anthelmintic activity and toxicity of Ruelene in sheep.
    Amer. J. vet. Res., 21: 1058-1061

    Hymas, T. A. and Stevenson, G. T. (1961) Attempt to produce
    neurotoxicity by the injection of Ruelene or Ronnel into mature
    Leghorn hens. Dow Chemical Co. Unpublished report

    Jackson, J. B., Drummond, R. O., Buck, W. B. and Hunt, L. M. (1960) 
    Toxicity of organic phosphorus insecticides to horses. J. Econ.
    Entomol., 53: 602-604

    Kutschinski, A. H. (1960) Determination of
    4-tert-butyl-2-chlorophenol  residues in the meat and fat of cattle
    sprayed with 4-tert-butyl-2-chlorophenyl methyl

    methylphosphoramidate (Ruelene(R)). The Dow Chemical Co., Midland,
    Michigan. Unpublished report

    Leahy, J. S. and Brown, F. G. (1963) Residues of
    4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate in the milk
    of dairy cows following dermal application. The Veterinary Record,
    75: (39) 1000-1001

    Leahy, J. S. and Taylor, T. (1963) The determination of residues of 
    "Ruelene" in milk.  Analyst, 88: 882-885

    McCollister, D. D. (1959) Fertility studies with male laboratory 
    animals given single doses of Ruelene. Dow Chemical Co. Unpublished
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    McCollister, D. D. (1964) Results of two-year dietary feeding studies 
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    report

    McCollister, D. D., Olson, K. J., Rowe, V. K., Paynter, O. E., Weir,
    R. J. and Dieterich, W. H. (1968) Toxicology of
    4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate (Ruelene)
    in laboratory animals. Food and Cosmetic Toxicology, 6: 185-198

    McGregor, W. S., Ludwig, P. D. and Wade, L. L. (1959) Progress report
    on Ruelene for cattle grub control. Down to Earth, Fall, 1959, pp. 1-2

    McGregor, W. S. (1960) The effects of Ruelene (R) treatments on the 
    fertility of range bulls. Dow Chemical Co. Unpublished report

    McKinley, W. P. and Read, S. I. (1962) Esterase inhibition technique
    for the detection of organophosphorus pesticides. J. Ass. Offic. Agr.
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    Mussell, D. R. and Ludwig, P. D. (1961) Residues of Ruelene(R) in
    animal tissues when applied as a spray to cattle. The Dow Chemical
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    Plapp, F. W. (1960) Studies on the metabolism of P32 Ruelene (R) in a 
    Hereford steer. Agricultural Research Service, U.S.D.A., Corvallis,
    Oregon. Unpublished report

    Radeleff, R. D. (1964) Organophosphorus compounds. Ruelene (R),
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    Veterinary Toxicology, Lea and Febger, Philadelphia, Pa., pp. 209-210

    Ragab, M. T. H. (1967) Direct fluorescent detection of
    organothiophosphorus pesticides and some of the sulfur-containing
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    Anal. Chem., 50: 1088-1098

    Rice, J. R. (1964) Residue determination of Ruelene in bovine tissue
    and Ruelene TF-88 pour-on application. The Dow Chemical Co., Lake
    Jackson, Texas. Unpublished report

    Rogoff, W. M. and Kohler, P. H. (1960) Effectiveness of Ruelene (R) 
    applied as localized "pour-on" and as spray for cattle grub control.
    J. Econ. Entomol.,  53: 814-817

    Sherman, M. and Ross, E. (1961) Acute and subacute toxicity of 
    insecticides for chicks. Toxicol. appl. Pharmacol., 3: 521-533

    Skerman, K. D. (1962) Mintrel(R). A new anthelmintic for sheep. 
    Aust. vet. J., pp. 324-334

    Smith, G. N. (1968) Cholinesterase inhibition of crufomate (Ruelene
    (R)  insecticide) and metabolites. Dow Chemical Co. Unpublished report

    Stewart, J. H. (1968) U.S. Department of Agriculture, Washington, D.C. 
    Private communication

    Timmerman, J. A., jr, Dorough, H. W., Buttram, J. R. and Arthur, B. W.
    (1961)  In vitro stability and recovery of insecticides from milk.
    J. Econ. Entomol. 54: 441-444

    Weidenbach, C. P., Radeleff, R. D. and Buck, W. B. (1962)
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    See Also:
       Toxicological Abbreviations
       Crufomate (ICSC)
       Crufomate (WHO Pesticide Residues Series 2)