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    AGP:1970/M/12/1

    WHO/FOOD ADD/71.42

    1970 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
    Group on Pesticide Residues, which met in Rome, 9-16 November, 1970.

    FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS

    WORLD HEALTH ORGANIZATION

    Rome, 1971

    MANCOZEB

    Explanation

    Mancozeb was considered by the 1967 Joint Meeting. Since that time,
    additional information on metabolism and methods of analysis has
    become available and, along with some pertinent older data, is
    summarized in this monograph addendum. Because much of this new
    information is related to the comparative metabolism of mancozeb in
    animals and plants, mancozeb has been considered separately from the
    other dithiocarbamates. However, there is a chemical similarity of
    mancozeb to many of the other dithiocarbamates, particularly maneb,
    and biochemical and toxicological information on those compounds
    should be borne in mind. Therefore, this monograph should be read in
    conjunction with the monograph on dithiocarbamates other than
    mancozeb.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Six rats received a dose of 20 mg of mancozeb labelled with 14C in
    the ethylene group for seven days (equivalent to approximately 100
    mg/kg body-weight/day or 1000 ppm in the diet). After sacrifice on the
    eighth day, the following metabolites were detected as percentage
    radioactivity recovered: unchanged mancozeb, urine 0, faeces 47;
    ethylene bis-isothiocyanate sulfide, urine 5.6, faeces 7.5; ethylene
    thiourea, urine 28, faeces 6; ethylene urea, urine 12, faeces 2;
    N-acetylethylenediamine, urine 19, faeces 0; N-formylethylene-diamine,
    urine 1, faeces 0; ethylenediamine, urine 3.59 faeces 0. Information
    indicating a similar metabolic fate in cows and in plants is given in
    the section on "Fate of residues" (Lyman, 1970).

    Further information on the metabolism of a number of dithiocarbamates
    in rats is reported to be in progress in the Netherlands (Verschuuren,
    1970).

    High dose levels of mancozeb are reported to inhibit the activity of
    redox enzymes containing sulfhydryl groups (Ivanova-Chemishanka,
    1969).

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

    Rat

    A three generation rat reproduction study at dietary levels of O, 25,
    100 or 1000 ppm previously reported (FAO/WHO, 1968) indicated that
    except for reduced fertility and variations in thyroid weight at the
    1000 ppm level in the first and second filial generation, there was no

    effect on reproduction. In particular, there was no report which would
    indicate an embryotoxic or teratogenic effect (Larson, 1965a).

    In the short-term study described below, where male and female rats
    were fed one tenth or one fifth of the LD50, respectively, twice
    weekly for four months (equivalent to 400 or 700 mg/kg body-weight per
    day), there were five deliveries; some of the young were stillborn
    and/or showed structural malformations (Ivanova-Chemishanka, 1969).

    Rabbit

    A study on rabbits employing the Somers test is reported to have
    revealed no teratogenic effects at 25 and 250 mg/kg body-weight, but
    details were not available (Swisher, 1970).

    Special studies on carcinogenicity

    In the study with groups of 18 mice of two hybrid strains (see the
    monograph on dithiocarbamate fungicides apart from mancozeb),mancozeb
    per se was not included. However, when the metabolite ethylene
    thiourea was administered at 215 mg/kg body-weight per day from day 7
    of birth until weaning and then incorporated into the diet at 646 ppm
    until the mice were aged 18 months, the incidence of tumours was 14
    out of 16 for the males and 18 out of 18 for the females in one strain
    and 18 out of 18 and 12 out of 16, respectively, for the males and
    females in the other strain (Innes et al., 1969).

    Special studies on affect upon thyroid function

    Groups of 15 male and 15 female rats were fed dietary levels of 0,
    100, 300 and 1000 ppm of mancozeb for three months. Positive control
    groups of rats were fed propylthiouracil at 300 ppm for three months
    or 1333 ppm for five weeks. Oxygen consumption studies were conducted
    during the third month, and at the end of the study 131I uptake was
    determined for each animal, following which the thyroid gland was
    examined microscopically. At 1000 ppm, terminal body weights were
    adversely affected, and protein-bound iodine (PBI) was depressed in
    the females; thyroid to body-weight ratios were elevated and the
    metabolic rate was lowered in the males; one animal of each sex showed
    significant thyroid hyperplasia; 131I uptake was not affected. At 100
    and 300 ppm, PBI was significantly increased in both sexes, but
    thyroid weights and histology and metabolic rates were unaffected. In
    the groups fed propylthiouracil, weight-gain depression, increased
    thyroid weight ratios, lowered 131I up-takes, lowered metabolic rates
    and severe thyroid hyperplasia were seen in both sexes.
    Propylthiouracil did not affect PBI (Larson, 1965b).

    Non-dose related variations in thyroid to body-weight ratios in 8 of
    20 rats fed 100 or 1000 ppm of mancozeb in the diet have been reported
    (Larson, 1965a). In rats fed levels up to 1000 ppm of mancozeb for 90
    weeks, there was hyperplasia of the thyroid acinar epithelium in the
    group fed 1000 ppm but not at 100 ppm or lower levels (Larson, 1965c).
    High dose levels of mancozeb administered to rats (equivalent to 400

    or 700 mg/kg body-weight per day) resulted in thyroid hypertrophy and
    reduced absorption of 131I (Ivanova-Chemishanka, 1969). In dogs fed
    levels up to 1000 ppm of mancozeb for up to two years, the only
    deviations from the controls in thyroid function were seen at 24
    months in lower 131I uptake, at 48 and 72 hours in the group fed 100
    ppm and at 72 hours in the group fed 1000 ppm (Larson,1965d).

    Acute toxicity

    The following additional information is available:

                         LD50 mg/kg
    Animal     Route     body-weight    Reference
                                                        

    Rat (M)    oral      14 000         Ivanova-
                                        Chemishanka,1969

    Rat (F)    oral      12 800         Ivanova-
                                        Chemishanka,1969
                                                        

    Short-term studies

    Rat

    Groups of 24 male and 20 female rats were fed one tenth or one fifth
    respectively of the LD50, in twice weekly doses for four months.
    (This level is equivalent to 400 or 700 mg/kg body-weight per day or
    8000 and 14,000 ppm in the diet.) Mortality was up to 50 percent in
    the males and 35 percent in the females. After four months, signs of
    paralysis were evident. There was also tracheitis and interstitial
    pneumonia followed by a general toxic reaction affecting primarily the
    liver and the kidneys less (Ivanova-Chemishanka, 1969).

    Long-term studies

    Rat

    Studies are reported to be underway in the Netherlands with rats.
    Preliminary results indicated that certain dithiocarbamate fungicides
    may affect the reticuloendothelial and haematopoletic systems as
    evidenced by the occurrence of slight anaemia, as well as effects on
    the thymus, mesenteric lymph nodes and thyroid function. Details are
    not yet available (Verschuuren, 1970).

    OBSERVATIONS IN MAN

    In a study on plant workers engaged in the manufacture of mancozeb, 54
    employees exposed to varying levels of the fungicide were examined.
    Tests included physical examinations, haematology (including
    protein-bound iodine) and urinalysis. No abnormalities which could be

    related to mancozeb were encountered, except for incidences of
    dermatitis which were occasionally observed in a few sensitive
    subjects (Megowan, 1965).

    COMMENTS

    Since mancozeb was last considered, some further information has been
    forthcoming on the metabolism in certain plants. The indication from
    those studies is that the metabolites are the same as those found in
    animals. In particular, the content of ethylene thiourea in the
    residues was similar. A considerable amount of work on a number of
    dithiocarbamates is now in progress in the Netherlands. This work
    involves short-and long-term studies and studies on mechanisms of
    action and metabolism. Preliminary information from this work
    indicated the existence of some effects on the reticuloendothelial and
    haematopoietic systems as evidenced by slight anaemia, as well as
    changes in the thymus, mesenteric lymph nodes and thyroid function in
    rats. Work reported from eastern Europe also indicates that certain
    dithiocarbamate fungicides affect reproductive physiology, may have a
    carcinogenic potential and disturb the thyroid function. These
    studies, particularly those involving maneb and zineb (see the
    monograph on dithiocarbamates), may or may not be relevant in
    assessing the toxicity of mancozeb. However, in the three generation
    rat reproduction study with mancozeb previously reported (FAO/WHO,
    1968), there was reduced fertility at 1000 ppm, but no indication of
    embryotoxic or teratogenic effects.

    For the above-mentioned reasons, it was considered that the temporary
    acceptable daily intake should remain at the same figure as previously
    established, but because some information is now available comparing
    the similarity of metabolites in plants and animals, the temporary
    acceptable daily intake for mancozeb need no longer be considered to
    be applicable to the parent compound only.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effects

         Rat: 100 ppm in the diet, equivalent to 5 mg/kg body-weight/day

    ESTIMATE OF TEMPORARY ACCEPTABLE DAILY INTAKE FOR MAN

         0-0.025 mg/kg body-weight

    RESIDUES IN FOOD AND THEIR EVALUATION

    Identity of residues

    As recorded in the report of the 1967 meeting, the following metabolic
    intermediates from both plant and animals have been identified:
    ethylene bis-isothiocynate sulfide (EBIS), ethylene thiourea (ETU),
    ethylene diamine (EDA), ethylene urea (EU), N-formyl ethylenediamine
    and N-acetyl ethylenediamine (from rats). EBIS is the proposed new

    structure for ethylenethiuran monosulfide (EMS) - formerly suggested
    as an alternate (Thorn, 1960) and subsequently substantiated (Rohm and
    Base, 1970).

    From experiments with the application of mancozeb to potato, radish
    and tomato plants, metabolism to ethylenediamine was followed by
    oxidation to glycine and ultimate incorporation of fragments into the
    metabolic pool.

    RESIDUES FOLLOWING USE IN THE FIELD

    Analytical methods

    In view of the modified carbonbisulfide liberation method (Gordonet
    al., 1967) not being specific for dithiocarbamates and neglecting
    metabolites such as ETU and EDA, a method has been developed (Rohm and
    Haas, 1970) for the determination of EDA, which is liberated from
    known components of residues (mancozeb, EDA, EU, ETU, EBIS and
    N-acetyl ethylenediamine). The sensitivity of detection for the method
    is 0.01 ppm (as EDA) or 0.05 ppm (as mancozeb). Because of the
    importance of knowing the magnitude of the ETU component in the
    residue, a separate method was developed (Rohm and Haas, 1970) that is
    sensitive to 0.01 ppm for milk and cow tissues and originally 0.05 ppm
    for potatoes.

    Analysis of field-treated potatoes for mancozeb residues

    All residue results included in this summary were determined by the
    ethylene thiourea (ETU) method and calculated as ETU (sensitivity,
    0.05 ppm). Most of the same samples were analyzed for mancozeb
    residues determined as ethylene diamine (sensitivity, 0.05 ppm as
    mancozeb) (See Table I).

    NDR signified "no detectable residue" at the sensitivity of the
    residue method.

    Over 306 samples were collected in 1969 from eleven different states.
    Treatments ranged from 1 to 3 lb/acre with total quantities ranging
    from 8 to 36 lb/acre, depending on number of treatments. Among the 40
    different sets of samples collected, in two sets no residue was
    detected (<0.05 ppm). In a group having the highest treatment rate
    (36 lb/acre total) with an eight day harvest interval, the results
    ranged from non-detectable to 0.60 ppm, with an average of 0.17 ppm.
    The highest level found was 0.78 ppm with a grand average of 0.17 ppm.
    No ETU was detected, as noted at the sensitivity level of 0.05 ppm.
    This checks with a 14C tracer experiment where a level of 0.0022 ppm
    was found using the reverse isotope dilution technique.

    Although traces of residues were found for some unexplainable reason
    in the control samples, the ingestion of mancozeb residues as might
    occur on leafy crops will not lead to residues in milk until the level
    exceeds 10 ppm.

        TABLE I

    Mancozeb residues in field treated potatoes
                                                                                            

    Sample          Treatment rate     Number         Final
    Source          Lb/Acre            of             Treatment          Residues, ppm
                    Total Lb for       Treatments     Harvest
                    Season                            Interval       ETU            Mancozeb
                                                        Days         Max            Max
                                                                                            

    ALABAMA
    Grove Oak
    70-88           2/4                    2              0          NDR            -

    CALIFORNIA
    Shafter
    69-79           2/12                   6             55          NDR            0.19

    Arvin
    70-76           2/2                    1              0          NDR            -

    FLORIDA
    Sanford
    2-69-32         1.5/10.5               7              0          NDR            0.21

    Sanford
    2-69-266        2/22                  11              0          NDR            0.28

    IDAHO
    Penghoe         2,3,2.5/18.5           6             17          NDR            0.19
    69-50

    Blackfoot
    69-50           2,3/11                 5             12          NDR            0.63

    Blackfoot                                                                       0.78
    69-50           2.5/20                 8              0          NDR         Highest Value

    Nowya
    69-52           2/12                   6             14          NDR            0.48

    INDIANA
    Rensselear
    69-55           1,1.5,2/12             8             31          NDR            0.55

    NEW JERSEY
    C. Bullock
    Allentown
    2-69-194        2/8                    4             46          NDR            NDR
                    2/8                    4             46          NDR            NDR

    TABLE I (cont'd)

    Mancozeb residues in field treated potatoes
                                                                                            

    Sample          Treatment rate     Number         Final
    Source          Lb/Acre            of             Treatment          Residues, ppm
                    Total Lb for       Treatments     Harvest
                    Season                            Interval       ETU            Mancozeb
                                                      Days           Max            Max
                                                                                            

    John Field
    Allentown
    2-69-194        2/10                   5             34          NDR            NDR

    Imlaystown
    2-69-194        2/12                   6             39          NDR            0.44

    NEW YORK
    Prattsburgh
    2-69-192        3/36                  12              8          NDR            0.60

    OHIO
    Columbienia
    2-69-206        2/16                   8             14          NDR            0.12

    Lisbon
    2-69-206        2,2.5,3/22.5           9              8          NDR            0.12

    PENNSYLVANIA
    Orefield
    68-105          2/12                   6              2          NDR            0.35
                    2/18                   9              4          NDR            0.50
                    2/6                    3              4          NDR            0.35

    WISCONSIN
    Antigo
    68-109          1.5,2/21              12              7          NDR            0.40

    Eagle River
    68-109          2/24                  12              7          NDR            0.38

    MAINE
    Washburn
    2-69-202        1,1.5/12.5            12              6          NDR            0.16

    2-69-203        1,1.5/16              12              2          NDR            0.15

    Garibou
    2-69-204        1.5/19.5              13              0          NDR            0.15

    TABLE I (cont'd)

    Mancozeb residues in field treated potatoes
                                                                                            

    Sample          Treatment rate     Number         Final
    Source          Lb/Acre            of             Treatment          Residues, ppm
                    Total Lb for       Treatments     Harvest
                    Season                            Interval       ETU            Mancozeb
                                                        Days         Max            Max
                                                                                            

    MICHIGAN
    Greenville
    69-55           2/8                    4             20          NDR            0.35

    Samara
    69-55           1.5/10.5               7             15          NDR            0.33
                                                                                            


    Analyses of milk for mancozeb residues1
                                                                                            

                                                      Feed Level (ppm Mancozeb)

                                    Control     0.78       9.72       38.9         97.2
                                                                                            

    No. of samples                     35       45        35          35           35
    No. of values >0.05 ppm2/           4        0        15          28           33
    Highest value ppm                0.10        -         0.08        0.29         0.71
    Av. of residues found ppm        0.06        -         0.06        0.11         0.32
                                                                                            

    1/ calculated as mancozeb from EDA method
    2/ sensitivity of method

    Analyses of milk for ETU
                                                Feed Level (ppm Mancozeb)
                                                                                            
                                    Control     9.72      38.9        97.2
                                                                                            
    No. of samples                     24       28        23          28
    No. of values >0.01 ppm1/           2        5        11          26
    Highest value found ppm          0.01        0.01      0.02        0.23
    Av. of residues found ppm        0.01        0.01      0.01        0.05
                                                                                            

    1/ sensitivity of method
    
    Residues in soil

    Radioassay of 14C-labelled mancozeb in soil where two varieties of
    potatoes were grown showed a drop in activity, calculated as ppm of
    mancozeb, from 0.72 to 0.16 and 1.88 to 0.33 in the interval Oct. 7/69
    to March 17/70.

    RECOMMENDATIONS FOR TOLERANCE

    TEMPORARY TOLERANCE

    With the demonstration of the similarity of metabolites in plants and
    animals, their quantitative measurement and the absence of detection
    of ethylenethiourea in extensive field testing of potatoes, it is
    possible to recommend a temporary tolerance of 1 ppm.

    FURTHER WORK OR INFORMATION

    REQUIRED (by June 1973)

    1. Elucidation of the effect on the reticuloendothelial and
       haematopoietic systems.

    2. Further clarification of the possible carcinogenic effect of these
       compounds.

    3. Elucidation of the effect on thyroid function.

    REQUIRED (Before further tolerances can be recommended)

    Further studies on the biotransformation of the compound in plants to
    determine the chemical nature of the residues, followed by appropriate
    toxicological studies. The extensive residue data already submitted
    need to be validated by the diethylamine method and by measuring the
    level of ethylene thiourea. (Note: Some of these data are known to be
    available but were not submitted in time for detailed scrutiny by the
    Meeting.)

    REFERENCES

    FAO/WHO (1968) 1967 Evaluation of some pesticide residues in food.
    FAO/PL: 1967/M/11/1; WHO/Food Add./68.30

    Gordon, C.P., Schukert, R.J. and Bornak, W.E. (1967) Improved method
    for the determination of ethylene bis-dithiocarbamate residues in
    plants, fruits and vegetables.

    Innes, J.R.M., Ulland, B.M., Valerio, M.G., Petrucelli, L.  Hart,
    E.R., Pallotta, A.J., Bates, R.R., Falk, H.C., Gart, J.J., Klein, M.,
    Mitchell, I. and Peter, J. (1969) Bioassay of pesticides and
    industrial chemicals for tumorigenicity in mice: a preliminary note.
    J. nat. Cancer Inst., 42: 1101-1114

    Ivanova-Chemishanka, L. (1969) Toxicologic characteristics of 
    mancozeb (in B¨lgarian).Higiena i Zdraveopazvane, 12: 418-426

    Larson, P.S. (1965a) Three generation reproduction study on rats
    receiving Dithane M-45 in their diet. Unpublished report from the
    Department of Pharmacology, Medical College of Virginia, submitted to
    Rohm and Haas Co.

    Larson, P.S. (1965b) Correlative study of functional and morphologic
    changes in the thyroid glands of rats receiving Dithane M-45 or
    propylthiouracil in their diet. Unpublished report from the Department
    of Pharmacology, Medical College of Virginia, submitted to Rohm and
    Haas Co.

    Larson, P.S. (1965c) Toxicological study on the effect of adding
    Dithane M-45 to the diet of rats for a period of 90 weeks. Unpublished
    report from the Department of Pharmacology, Medical College of
    Virginia, submitted to Rohm and Haas Co.

    Larson, P.S. (1965d) Toxicologic study on the effect of adding Dithane
    M-45 to the diet of beagle dogs for a period of two years. Unpublished
    report from the Department of Pharmacology, Medical College of
    Virginia, submitted to Rohm and Haas Co.

    Lyman, W.R. Dithane M-45. (1970) Chemistry and metabolism of terminal
    residues. Unpublished report, submitted to FAO and WHO by Rohm and
    Haas Co.

    Megowan, R. (1965) Dithane health study. Unpublished report from
    Bridesburg Medical Department, submitted to Rohm and Haas Co.

    Rohm and Haas Unpublished data, submitted to FAO 1967 and 1970 

    Swisher, E. (1970) Unpublished information on work conducted at the
    University of Toronto, provided by Rohm and Haas Co.

    Thorn, G.D. (1960) Some observations on the structure of
    ethylenethiuram monosulphide. Can. J. Chem., 38: 2349-2358

    Verschuuren, H.G. (1970) Information provided verbally to the 1970
    Joint FAO/WHO Meeting on Pesticide Residues on work being conducted on
    dithiocarbamate fungicides in The Netherlands.
    


    See Also:
       Toxicological Abbreviations
       Mancozeb (ICSC)
       Mancozeb (FAO/PL:1967/M/11/1)
       Mancozeb (WHO Pesticide Residues Series 4)
       Mancozeb (Pesticide residues in food: 1993 evaluations Part II Toxicology)