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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

    OXYTHIOQUINOX

    IDENTITY

    Chemical names

    6-methyl-2-oxo-1,3-dithiolo-[4,5-b]quinoxaline (IUPAC)

    Synonyms

    Chinomethionat
    Oxythiochinox
    Morestan(R) (trade name)

    Formula

    CHEMICAL STRUCTURE 

    Other information on identity and properties

    The technical compound contains at least 80 per cent pure active
    ingredient and the following by-products in varying concentrations:

    CHEMICAL STRUCTURE 


    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    Biochemical aspects

    No data available.

    Acute toxicity

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

    Mouse (M)          i.p.          650       DuBois and Raymund, 1962

    Mouse (F)          i.p.          700       DuBois and Raymund, 1962

    Rat (M)            oral       > 2500       Bayer, 1959

    Rat (F)            oral         3000       DuBois, 1961

    Rat (M)            i.p.          700       DuBois, 1961

    Rat (F)            i.p.          600       DuBois, 1961

    Guinea-pig (M)     oral         1500       DuBois and Raymund, 1962

    Guinea-pig (F)     i.p.          350       DuBois and Raymund, 1962

    Chicken            oral         >500       DuBois, 1962

    Cat                oral        >1000       Bayer, 1959
                                                                         


    Short-term studies

    Rat. A group of 10 male rats was treated daily with 100 mg/kg(in one
    per cent aqueous tragacanth suspension) by stomach tube, for 28 days.
    One death occurred after 16 days; autopsy of this animal revealed
    kidney damage. Loss of appetite and body-weight were observed in the
    other animals after 20 doses. The haematogram was normal (Bayer,
    1959).

    Four groups of 12 male and 12 female rats were fed 0, 10, 25 or 50 ppm
    in the diet for 16 weeks without any deaths. Food intake was
    unaffected in both sexes, as was body-weight gain in the females. A
    slight transient depression of body-weight gain was observed between
    four and 12 weeks, in the males fed 50 ppm. Based on data from five
    male and five female animals per group, gross and histopathology were
    comparable to the controls. Depression of absolute liver and kidney
    weights, apparent in all male test groups was not dose related (Doull
    et al., 1963).

    Six groups, each comprising five male weanling rats were fed for 90
    days, dietary levels of 0, 10, 25, 60, 150 and 500 ppm active
    ingredient of oxythioquinox as the 25 per cent wettable powder.
    Body-weight gain was depressed at the 500 ppm level, probably due, at
    least in part, to diet rejection. Liver to body-weight ratio was
    significantly increased at 500 ppm. Investigations of liver microsomal
    enzyme activity (EPN detoxification, O-demethylase, and acetoacetic
    acid synthesis) showed depressed activity in all systems at 500 ppm,
    and acetoacetic acid synthesis was also inhibited at 150 ppm. Analysis
    of liver tissue failed to reveal the presence of oxythioquinox using a
    method sensitive to the detection of 0.5 ppm (Carlson and DuBois,
    1968).

    Oral administration of an aqueous emulsion five times weekly for four
    months to groups of 10 male rats at dose levels of 0, 10, 25, 50, 100
    or 250 mg/kg did not cause any mortality. Haematograms and urinalysis
    were normal at all dose levels. However, clinical symptoms (hair loss)
    were apparent at 250 mg/kg after seven weeks. Body-weight gain was
    reduced in the 100, and 250 mg/kg groups and liver-weight ratios were
    increased at these levels. Histological damage to liver cells was
    apparent at 250 mg/kg (Kimmerle, 1963).

    In the three generation rat study, described under "Special studies.
    Reproduction", liver damage was observed in the FO generation in the
    animals fed 500 ppm. In this group, gross pathology was normal but
    histopathology showed 60 per cent of the animals displaying toxic
    injury (periportally arranged swollen liver epithelial cells, with
    several pyknotic nuclei) with a further eight per cent showing
    questionable injury (Hecht and Grundmann, 1964).

    Dog. Groups of two male and two female dogs were fed 0, 10, 25 or 50
    ppm in dry diet for 28 months. Body-weight, food intake, general
    behaviour and appearance, haematograms, serum glutamic oxaloacetic
    transaminase, and serum glutamic-pyruvic transaminase determinations,
    organ weights (absolute and organ to body-weight ratios) and gross
    pathology of treated animals were all comparable to the controls.
    Histological examination revealed inflammatory cell foci in the livers
    of all the dogs which received 25 and 50 ppm; but the same effect was
    noted in two-thirds of the 10 ppm group and in three of the four
    controls (Doull et al., 1966).

    Long-term studies

    Rat. Technical oxythioquinox (91 per cent pure) was administered to
    groups of 50 male and 50 female rats at dietary levels of 10, 25, 60,
    125 or 500 ppm, for two years. One hundred male and 100 female rats
    served as untreated controls. Clinical symptoms in the form of yellow
    staining of the hair of the paws were apparent at 60 ppm, the
    frequency increasing with increasing dose level. At 500 ppm, abdominal
    and facial fur were also stained yellow. Body-weight gain was
    depressed in both sexes, and food intake was depressed in the female
    at 500 ppm. Incidence of mortality, haematograms, urinalysis, and

    gross pathology were all comparable to the controls. Marked liver
    hypertrophy was shown, as evidenced by absolute organ weights, the
    increase being significant in all test groups except the females fed
    25 ppm. Thyroid weights were also significantly increased in males at
    150 and 500 ppm, and in females at 25 and 60 ppm. There was a tendency
    towards decreased adrenal weights in males at 150 and 500 ppm, and in
    females at 500 ppm. Pituitary enlargement was, however, comparable in
    all groups (Lorke and Loser, 1966a).

    Histopathologically, vacuolar cytoplasmic swelling was noted in the
    liver at all levels, the incidence tending to increase with increasing
    dose level. Necrotic lesions were observed only at 500 ppm. Bile duct
    hyperplasia occurred in six out of 38 of the rats fed 500 ppm. In the
    testes reduced spermatogenesis was significantly more frequent at 150
    and 500 ppm. Tumour incidence was unrelated to the dose level
    (Grundmann and Hobik, 1966a),

    Special studies

    Reproduction

    Rat. In a three-generation rat study, 91 per cent pure technical
    oxythioquinox was incorporated in the diets of groups of eight male
    and 16 female rats (except for the F2b breeding animals where 10
    males and 20 females were utilized). At levels of 0, 10, 25, 60 and
    150 ppm, no effects on adult body-weight, incidence of pregnancy,
    litter size, birth and weaning weights, or survival to four weeks were
    noted up to and including 60 ppm. At 150 ppm, incidence of pregnancy
    was reduced in both first generation litters and litter size was
    reduced in one litter in each generation. At 500 ppm, pregnancy was
    inhibited. Cross breeding studies showed the inhibition to be due to
    male infertility. Treated females mated with untreated males produced
    litters. but the litter size was markedly reduced. The 500 ppm group
    was discontinued after the first attempted mating. Body-weight gain
    was reduced in female rats and doubtfully in male rats at 500 ppm.

    Abnormalities observed during the study included uni- and bilateral
    anophthalmia, and unspecified abnormalities of the incisor teeth.
    These abnormalities were randomly distributed between groups. In the
    F1a litters, one hairless offspring occurred in the 60 and 150 ppm
    groups.

    Organ weights and histopathology on two male and two female animals in
    the F3b litter were comparable to the controls (Lorke and Loser,
    1966; Grundmann and Hobik, 1966).

    Observations in man

    Compressions containing dry or moist oxythioquinox were applied to the
    forearms of nine human volunteers for varying periods of two, four,
    eight or 24 hours. Exposures for 24 hours resulted in reddening of the
    skin and occasional swelling and blistering, the incidence and

    severity of these injuries being more marked with the moist product
    (Bayer, 1959).

    Comments

    Adequate data are available on acute and short-term studies. However,
    a no-effect level has not been demonstrated in long-term studies in
    rat. Liver hypertrophy is extensive at 10 ppm, the lowest dose tested.
    At present it is not possible to estimate a daily acceptable intake
    for man. Until further data are available, foods should not be
    permitted to contain any residues of oxythioquinox and its possible
    metabolites from treated plants.

    Further information is needed concerning the metabolic fate of this
    pesticide in various animals including man; particularly further
    research is needed to ascertain its effect on spermatogenesis and
    whether this phenomenon occurs in primates. Since it has been reported
    that there is an injurious effect to skin upon prolonged application,
    it will be necessary to do further studies on the cutaneous toxicity
    of this compound including studies related to the question of
    photo-sensitization.

    RESIDUES IN FOOD AND THEIR EVALUATION

    Use pattern

    Pre-harvest treatment

    Oxythioquinox is used as fungicide and acaricide on a large number of
    crops. As a fungicide, it is used against mildew diseases on pome,
    stone and soft fruit, strawberries, grapes and cucurbits and has a
    protective and curative action (Grewe et al., 1965; Wäckers et al.,
    1965). As an acaricide, it is effective not only against susceptible
    mite strains, but also against strains which are resistant to other
    acaricides (Unterstenhöfer et al., 1965). A good insecticidal side
    effect has been observed against Psylla piri (Unterstenhöfer et al.,
    1965).

    Established pre-harvest intervals are as follows:

                                                                      
                                                          Pre-harvest
    Country             Crop                            interval (days)
                                                                      

    Denmark             Tree fruit                             8
                        Cucumbers                              4

    Germany             Tree fruit and vegetables             14
                        Cucumbers                              4

    United Kingdom      Tree fruit                            21
                        Gooseberries, black currants          14

                                                                      
                                                          Pre-harvest
    Country             Crop                            interval (days)
                                                                      

                        Marrow                                 7
                        Cucumbers grown under glass            3

    Finland                   -                               14

    Holland             Tree fruit                            28
                        Cucumbers                              3

    Israel              Apples, citrus fruit, grapes          14
                        tomatoes, strawberries,
                        egg-plants, peppers                    7

    Italy                     -                                5

    Yugoslavia          Tree fruit                            14
                        Cucumbers                              7

    Austria             Tree fruit                            14
                        Vegetables                             4

    Poland              Tree fruit and vegetables             21
                        Cucumbers                              3

    Sweden              General                                7
                        Cucumbers                              4

    Switzerland         Tree fruit                            21
                        Vegetables                             5

    Spain               Cucumbers                             10
                        Other crops                           15
                                                                      


    Post-harvest treatments

    No use.

    Other uses

    Oxythioquinox is used against mildew and mites on ornamentals (Grewe
    et al., 1965; Wäckers et al., 1965; Besemer et al., 1963).

    Residues resulting from supervised trials

    In the following table, residue values are given after application at
    the recommended concentrations:

                                                                         
                                                 Residue at harvest
                                  Pre-harvest           (ppm)
    Crop             Number of    interval
                     treatments   (days)         Range         Average
                                                                         

    Apples           1-4          7-10           n.d.-1.5       0.2
    (United
    States of        1            7              0.8            0.8
    America)

    Pears            3            7-8            0.1-0.8        0.4
    (United
    States of        1            7-8            0.2-2.1        1.2
    America

    Strawberries     2            7-8            n.d.-2.2       1.0
    (United
    States of                     7              n.d.           n.d.
    America

    Grapes           2            7              n.d.-9.4       6.0
    (United
    States of                     7              12.5           12.5
    America)

    Cucumbers        2            4              n.d.           n.d.

    Summer           4            1-7            n.d.-0.9       0.6
    squash           4            1-7            n.d.-1.2       0.9

    Winter           4            1-8            n.d.-0.1       n.d.
    squash           4            1-8            0.5-0.6        0.5

    Alfalfa          1            7              0.3-1.1        0.9
    (United                       7              3.6            3.6
    States of                     14             0.1-0.3        0.1
    America                       14             3.0            3.0
                                                                         


    Fate of residues

    General comments

    The half-life of oxythioquinox on apples, pears, and goose berries is
    between five and nine days (Grewe et al., 1965). Loss probably is
    caused not only by enzymatic processes, but also by physical and
    chemical influences in the environment. Wash-off may occur only a
    short time after application. After a few days even heavy rainfall
    causes no further noticeable loss. Probably oxythioquinox is dissolved
    in the wax layers of the plant surfaces (Grewe et al., 1965).

    In diluted ammoniacal solution, oxythioquinox is instantly saponified
    to form 2,3-dithiol-6-methylquinoxaline with liberation of carbonate
    (Grewe et al., 1965). This substance, which is also formed in plants
    (Chemagro Internal Report), is probably unstable to light, as shown by
    experiments with its analogue 2,3-dithiolquinoxaline, which is formed
    under similar conditions from Eradex, a compound related to
    oxythioquinox (Tietz et al., 1962).

    In soils

    The half-life of oxythioquinox in soils is about 60 days
    (Farbenfabriken Bayer A.G., private communication).

    In plants

    When oxythioquinox C14 was applied to growing oranges and apples,
    there was a steady decrease in the amount of total radioactivity.
    While activity on the surface was due only to oxythioquinox itself
    free and bound 2,3-dithio-6-methyl quinoxaline and other, unknown
    metabolites were found in the peel, reaching a maximum at 7-14 days
    and then decreasing slowly. The unknown metabolites were not
    extractable with organic solvents. Reduction followed by treatment
    with boron trifluoride-methanol and diazomethane solubilized
    approximately 80 per cent of the insoluble material. Perhaps this
    consists of conjugated compounds with carboxylic groups or phenolic or
    glycosidic hydroxyls. In the pulp only traces of radioactivity were
    found (Chemagro Internal Report).

    In animals

    When rats were fed carbonyl-14C-labelled oxythioquinox. it was found
    that most of the radioactivity was exhaled as 14CO2. After
    2,3-14C- and 35S-labelled active ingredient was fed to rats, no
    14CO2 was found in the air expired. In this particular case, most
    of the activity was present in the urine and in the faeces. A small
    proportion of the activity was found in the blood plasma in
    protein-bound form. The activity is not present as oxythioquinox
    itself, but partially as 2,3-dithiol-6-methyl quinoxaline, in traces
    as the corresponding dihydroxy compound, and as other still
    unidentified metabolites. The quinoxaline ring is apparently not
    metabolized in the animal body (Chemagro Internal Report).

    In storage and processing

    Washing of oranges containing 0.4 ppm oxythioquinox reduced the
    residue to the non-detectable level (Chemagro Internal Report).
    Peeling reduces residues to values below 1 ppm.

                                                                        
                        ppm in peel    ppm in pulp (seven
                        (seven days    days after
                        after          application)
           Crop         application)

                                                                        
           Oranges      0.2-4.4        n.d.-0.2
           Lemons       0.2-1.2        n.d.
           Grapefruit   n.d.-1.8       n.d.-0.2
           Apples       1.0-4.1        0.1-0.7
                                                                        
    (Farbenfabriken Bayer A.G., private communication)

    After processing oranges containing 0.4 ppm oxythioquinox, residues of
    less than 0.1 ppm were detected in frozen and heated juice, chopped
    peel and cattle feed, press liquor and molasses. A residue level of
    1.7 ppm was found only in cold pressed citrus oil (Chemagro Internal
    Report).

    Evidence of residues in food in commerce or at consumption

    No data available.

    Methods of residue analysis

    Analytical methods appear to be sufficient for present purposes. A
    colorimetric method has been developed by Havens et al., 1964. It is
    an adaption of a method to determine residues of "Eradex New" in plant
    material (Tietz et al., 1962). The procedure involves a hydrolysis
    with concentrated ammonium hydroxide to give
    2,3-dithiol-6-methylquinoxaline. Subsequent treatment with ammonical
    nickel reagent gives a red-coloured chelate which is measured at
    540 mµ. The limit of sensitivity is 0.14 ppm. This method gives poor
    results, when large amounts of oil are present in the sample.

    For the determination of residues in orange peel. a method has been
    described based on a combination of thin-layer chromatography and
    cathode ray polarography (Hearth et al., 1966). Sensitivity: 0.5 ppm.

    Gas chromatographic determination with an electron capture detector is
    used for moist crops, dry hops, and for crops with high oil content.
    Sensitivity: 0.1 ppm for most crops (Vogeler et al., 1967; Chemagro
    Internal Reports).

    National tolerances

    No registrations have been agreed in the United States of America. A
    petition will be filed for the following tolerances:

    Walnuts: 0.1 ppm
    Cucumbers, water melons, winter squash: 0.75 ppm
    Apples, pears, melons (except water melons), summer squash: 1.5 ppm
    Citrus, grapes: 2.5 ppm
    Papayas: 5.0 ppm
    Strawberries: 6 ppm

    In Germany the tolerance for cucumbers is 0.1 ppm. For pome fruit, a
    tolerance of 0.3 ppm has been proposed.

    In the Netherlands and Switzerland, a tolerance of 0.1 ppm has been
    agreed for fruit, vegetables, and cucumbers.

    RECOMMENDATIONS FOR TOLERANCES AND PRACTICAL RESIDUE LIMITS

    Appraisal

    This compound is widely used as a fungicide and an acaricide in many
    countries. The technical product contains at least 80 per cent pure
    active ingredient and at least six by-products in varying
    concentrations. Further information on the composition of the
    technical product is required, together with information on the
    quantities being used in each country. The residues consist of the
    parent compound, one identified metabolite and other metabolites not
    yet identified. Accordingly further information on the chemical nature
    of the terminal residues is also required together with the nature of
    degradation products in plants and animals.

    A variety of analytical methods are available which would be adequate
    to detect residues down to 0.1 ppm, including gas-liquid
    chromatography, thin-layer chromatography, polarography. However, if
    tolerances of 0.1 ppm or below are to be considered, improved methods
    of analysis would be required.

    Recommendations

    Since an acceptable daily intake has not been established, tolerances
    cannot be recommended.

    Further work or information

    Required (before an acceptable daily intake or tolerances can be
    established)

    1.     Information on the nature of terminal residues in plants and
           animal products.

    2.     Data from countries other than the United States of America on
           the required rates and frequencies of application, pre-harvest
           intervals, and the resultant residues.

    3.     Further data on residue levels in raw agricultural products
           moving in commerce.

    4.     Data on residue levels in total diet studies.

    5.     Comparative evaluation of methods of analysis for regulatory
           purposes.

    6.     Experimental studies on the metabolism responsible for liver
           hyperplasia in rats.

    7.     Biochemical studies on excretion and metabolism.

    8.     Two-year studies on rats at lower dosage.

    9.     Further information on anti-spermatogenic effects.

    Desirable

    1.     Collaborative studies to establish a referee method.

    2.     Studies of metabolism in various animals including man.

    3.     Further studies on the cutaneous toxicity, including studies
           related to the question of photosensitization.

    REFERENCES

    Bayer. Active ingredient Dr Sasse Ss2074. Farbenfabriken Bayer

    Basemer, A. F. H. and Immikhuizen, E. (1963) Biologisch Veld-en
    Kasonderzoek van Insekticiden, Acariciden en Fungiciden in
    Tuinbouwgewassen. P.D.-Jaarboek 1962, Wageningen, No. 138: 131-146

    Carlson, B. P. and DuBois, K. P. (1968) Effects of feeding various
    dietary levels of Morestan to male rats for 90 days. University of
    Chicago. Unpublished report

    Doull, J., DiGiacomo, R., Root, M., Vesselinovitch, D. and Meskauskas,
    J. (1966) Chronic oral toxicity of Morestan (Bayer 36205) to male and
    female dogs. University of Chicago. Unpublished report

    Doull, J., Root, M. and Gowan, J. (1963) Subacute oral toxicity of
    Morestan (Bayer 36205) to male and female rats. University of Chicago.
    Unpublished report

    DuBois, K. P. (1961) Intraperitoneal and oral toxicity of Bayer 36205
    to rats. University of Chicago. Unpublished report

    DuBois, K. P. (1962) The acute oral toxicity of Bayer 36205 to
    chickens. University of Chicago. Unpublished report

    DuBois, K. P. and Raymund, A. B. (1962) The acute toxicity of Bayer
    36205 to mice, guinea pigs and rats. University of Chicago.
    Unpublished report

    Grewe, F. and Kaspers, H. (1965) Morestan, a new fungicide of the
    2,3-disubstituted quinoxaline group for controlling powdery mildews.
    Pflanzenschutz-Nachrichten "Bayer", 18: 1-23

    Grundmann, E. and Hobik, H. P. (1966a) Bay 36205, two-year feeding
    test with rats. Histology. Farbenfabriken Bayer. Unpublished report

    Grundmann, E. and Hobik, H. P. (1966b) Bay 36205 Generation test.
    Histology. Farbenfabriken Bayer. Unpublished report

    Havens, R., Adams, J. M. and Anderson, C. A. (1964) Colorimetric
    determination of 6-methyl-2,3-quinoxalinedithiol cyclic carbonate
    (Morestan) residues in apples and pears. J. Agr. Food Chem.,
    12: 247-248

    Hearth, F. E., Ott, D. E. and Gunther, F. A. (1966)
    Oscillopolarographic analysis of Morestan residues in Valencia orange
    rind following thin layer chromatography. J. Assoc. off. agric.
    Chemists, 49: 774-778

    Hecht, G. and Grundmann, E. (1964) Report on testing of Morestan for
    toxic effects on the liver. Farbenfabriken Bayer. Unpublished report

    Kimmerle, G. (1963) Four months' feeding test on rats with the active
    ingredient Ss2074. Farbenfabriken Bayer. Unpublished report

    Lorke, D. and Loser, E. (1966a) Bay 36205 studies of chronic toxicity
    to rats. Farbenfabriken Bayer. Unpublished report

    Lorke, D. and Loser, E. (1966b) Bay 36205 studies of chronic toxicity
    to rats. Farbenfabriken Bayer. Unpublished report

    Tietz, M. et al. (1962) Method of determining residues of the
    acaricide Eradex New in plant material. Pflanzenschutz-Nachrichten
    "Bayer", 15: 166-171

    Vogeler, K. and Niessen, H. (1967) Gas chromatographic determination
    of Morestan residues in plants. Pflanzenschutz-Nachrichten "Bayer",
    20: 550-556

    Wäckers, R. and van den Berge, C. (1965) Experiences with Morestan in
    Dutch fruit farming and market gardening. Pflanzenschutz-Nachrichten
    "Bayer", 18: 34-44
    


    See Also:
       Toxicological Abbreviations