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    PYRETHRINS                                 JMPR 1972

    Explanation

    This insecticide was considered at the Joint Meetings held in 1965,
    1966, 1967, 1968, 1969 and 1970 (see FAO/WHO 1965; 1967; 1968; 1969;
    1970; 1971). Since the last evaluation (FAO/WHO, 1971) results of some
    additional experimental studies have been made available, as well as
    further information relating to methods of analysis and to residues in
    dried codfish.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

    Pyrethrins are absorbed from the gastrointestinal tract following oral
    administration. Studies in male rats receiving 3 mg/kg orally resulted
    in almost complete absorption and metabolism within 100 hours. No
    pyrethrin was observed in urine, although substantial quantities of
    metabolites were present. In faeces, small quantities of the parent
    pyrethrin were observed, again accompanied by metabolites.

    Biotransformation

    Utilizing an in vitro enzyme system from insects in the presence of
    NADPH2, Casida and his coworkers (FAO/WHO, 1971), showed that
    pyrethrin I was converted to at least ten metabolites. A major
    metabolite was characterized as a product which had undergone
    oxidation of a methyl group in the isobutenyl moiety to the carboxylic
    acid. In a more comprehensive study, these authors concluded that
    oxidation rather than hydrolysis in insects might be the major mode of
    metabolism of pyrethroid chemicals. Pyrethrins I and II have also been
    shown to be oxidatively metabolized in rats. Oxidation was found to
    occur at the trans-methyl group of pyrethrin I as well as on the
    pentadienyl side chain to produce two diols. These metabolites were
    also found in conjugate form.

    The oral administration of radio-labelled pyrethrin I, or pyrethrin
    II, to rats produced several urinary metabolites. Each contained a
    trans-2-carboxyprop-1-enyl side chain resulting from oxidation of
    the chrysanthemate isobutenyl group or hydrolysis of the pyrethrate
    methoxy-carbonyl group. Also, the cis-2',4'-pentadienyl side chain  
    of pyrethrin I and pyrethrin II was modified to give a
    cis-4',5'-dihydroxypent-2'-enyl group, a 4' conjugate of this
    diol, or a trans-2',5'-dihydroxypent-3'-enyl group
    (Elliot et al., 1972).

    Pyrethrins, in addition to metabolism by oxidation, were hydrolysed,
    as evidenced by 14CO2 in expired air following treatment with
    pyrethroid labelled in the carboxyl group attached to the C1 of the
    cyclopropane ring. The metabolic route proposed by Elliott et al.
    (1972) is shown in Figure 1.

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

    Two groups of nine rabbits each were administered pyrethrins at 0 and
    90 mg/kg body-weight/day, orally, from day 8 - 16 of gestation. No
    apparent effects were noted on the number and weight of foetuses,
    implantation sites or on gross external and internal examination. Two
    control pups and one pup in the group given pyrethrins had a club-like
    deformed front paw, and one pyrethrin pup had a missing caudal
    vertebrae. There appears to be no apparent teratogenic effects
    elicited by pyrethrins in rabbits (FAO/WHO, 1971).

    Special studies on skin sensitization

    Two groups of nine male guinea pigs were used to examine the
    sensitizing effect of pyrethrins. Sensitization was assessed by
    comparison of the response following a challenge dose with that
    elicited by previous sensitizing doses. A positive control
    (1-chloro-2,4-dinitrobenzene) produced sensitization in all animals of
    one group. No sensitization was obtained with a 1% formulation of
    pyrethrins (FAO/WHO, 1971).

    Acute toxicity

    Acute toxicity of pyrethrins has been studied in animals, and a
    summary of the results in four species is given in Table 1.


    FIGURE 1
    TABLE 1  Acute toxicity of pyrethrins in animals

                                                                             

    Species        Route            LD50              Reference
                                    (mg/kg
                                    body-weight)
                                                                             

    Rat (M)        oral             710               FAO/WHO, 1971.

    Rat            oral             584 - 900         Ibid.

                                    1 440             Bond et al., 1972

                   i.p.             167 - 798         FAO/WHO, 1971

    Mouse          oral             273 - 796         Ibid.

                   i.p.             172 - 452         Ibid.

    Chick          Perivisceral     240 - 1 262       Ibid.

    Dog            i.v.             6 - 81            Ibid.
                                                                             

    1  Lethal dose.


    The five ranges (FAO/WHO, 1971) comprise the various grades of
    pyrethrum, including crude oleoresins and refined concentrates.

    The relatively low inherent toxicity of pyrethrum should be noted. The
    marked difference in the oral and intravenous toxicities may indicate
    a rapid degradation or a species susceptibility, with the dog being
    extremely susceptible to the toxic effect of pyrethrins. The acute
    signs of poisoning in rats include: depression, rapid and/or laboured
    respiration, ataxia, incoordination, convulsions and muscular tremors.
    Necropsy findings include: congestion of the lungs, liver, kidneys,
    adrenals and pancreas and slight gastric inflammation (FAO/WHO, 1965;
    1971).

    The acute effects resemble veratrine intoxication, proceeding from
    excitation to tetanic convulsions, except that pyrethrins also cause
    muscular fibrillation. Death is caused by respiratory failure.
    Persistent tremor is occasionally seen in animals that recover from a
    single large dose (FAO/WHO, 1965).

    An acute dermal toxicity test was performed with rabbits using
    pyrethrins in combination with synergist. Typical sprays made with the
    synergists, tropital or piperonyl butoxide (1%) in combination with
    0.1% pyrethrins, exhibited a low order of toxicity when tested
    dermally on 6 - 12 male rabbits. The acute dermal LD50 of both
    formulations was >10 gm/kg. At 10 gm/kg with tropital, three of 12
    rabbits died. At 5 and 10 gm/kg, body-weight gain was reduced, and
    transient signs of toxicity were evident with both synergist
    combinations. No effects were noted (see Table 2) at a concentration
    equivalent to 2 mg/kg pyrethrins and 20 mg/kg synergist (FAO/WHO,
    1971).

    Short-term studies

    Rat

    Two groups of rats (10 males and 10 females) were exposed to aerosols
    of 1% pyrethrins for one hour. The flow was 50 litre/min. containing 2
    mg/litre of air. Gross examination of the lung tissue demonstrated the
    presence of haemorrhagic pin point lesions in 9 of 10 male and 10 of
    10 female rats exposed. Microscopic examination of the lungs indicated
    that the alterations were typical of those found in murine
    pneumonitis. No distinguishing pathological observations were reported
    which might be attributed to pyrethrins (FAO/WHO, 1971).

    Groups of rats (10 males and 10 females/group) were administered
    pyrethrin alone and in combination with various synergizers daily for
    90 days at high dosages (pyrethrum alone - 360 mg/kg; pyrethrum - 180
    mg/kg + piperonyl butoxide - 600 mg/kg; pyrethrum - 180 mg/kg +
    sulfoxide - 132 mg/kg; pyrethrum - 90 mg/kg + tropital - 567 mg/kg).
    With piperonyl butoxide alone, a higher mortality was observed. The
    pyrethrin + sulfoxide combination caused deaths. Growth was reduced by
    almost all treatments in both males and females (Bond et al., 1972).

    TABLE 2  Acute toxicity of pyrethrin and synergists1

                                                                

                                            LD50 (mg/kg)
                                                                

    Pyrethrin                               1 440

    Piperonyl butoxide                      6 040

    Sulfoxide                               1 760

    Tropital                                3 600

    P + piperonyl butoxide                  720 + 2 400

    TABLE 2  (cont'd)

                                                                

                                            LD50 (mg/kg)
                                                                
    P + sulfoxide                           720 + 528

    P + tropital                            360 + 2 268
                                                                

    1  Bond et al., 1972.

    Rabbit

    A group of 10 male and 10 female rabbits were tested by repeated
    dermal application to either abraded or intact skin with a 1.0%
    formulation of pyrethrins at a dose of 10 mg/kg body-weight/day. Five
    male and 5 female rabbits acted as controls. Treatments of 1 ml/kg
    body-weight of the formulation were applied daily (6 - 8 h exposure
    per day) five days per week for three weeks. No abnormalities were
    attributed to the test material. Repeated dermal applications of a 1%
    formulation of pyrethrins was not detrimental to rabbits (FAO/WHO,
    1971).

    Long-term studies

    Rat

    Groups of 12 male and 12 female rats were fed pyrethrin in soybean oil
    at dietary levels of 0, 200, 1 000 and 5 000 ppm for two years. The
    daily dosage was, therefore, approximately 0, 10, 50 and 250 mg/kg,
    respectively. Even the highest level had no significant effect on
    growth or survival. Slight, though definite, liver damage
    characterized by bile duct proliferation and focal necrosis was found
    at the two highest dosage levels (FAO/WHO, 1966).

    OBSERVATIONS IN MAN

    Injury to man from pyrethrum has most frequently resulted from the
    allergenic properties of pyrethrin flowers and certain extracts rather
    to other forms of toxicity. The allergy has been associated primarily
    with occupational and therapeutic contact.
    Two hundred human subjects (177 females, 23 males) were patch tested
    for skin sensitivity and irritation using pyrethrins at 1% in water
    simulating formulation. This level did not produce primary irritation
    and was not a sensitizer to human skin (FAO/WHO, 1971).

    COMMENT

    Data requested by the 1970 Joint Meeting have been received in part.
    Pathways of metabolism consist of oxidation as well as hydrolysis.
    Limited tests on metabolites indicate a lower order of toxicity than
    pyrethrins.

    Short-term studies of combinations of pyrethrin and synergizers
    confirmed the relative safety of high levels of these materials.
    Dermal and inhalation toxicity studies of pyrethrins in combination
    with synergists, using a commercial formulation, exhibited a low order
    of toxicity to rabbits.

    The rapid metabolism and apparent lack of storage are reassuring. On
    the basis of the new information, an ADI was established. However,
    because results of requested short-term studies in the dog and other
    species, to elucidate the effect on the liver found in a long-term
    study in rats, ware not forthcoming, the ADI has not been increased.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Rats:     200 ppm in the diet, equivalent to 10 mg/kg
                   body-weight/day.

    ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN

         0 - 0.04 mg/kg body-weight

    METHODS OF ANALYSIS

    Furmanec et al. (1969) used the colorimetric method of Williams
    et al. (1956) with a thin-layer chromatographic separation for
    assaying commercial preparations containing pyrethrins. The
    insecticidal constituents pyrethrin I and II were separated from inert
    material by TLC on silica gel G using hexane: ethyl acetate (75:25).
    The quantitative procedure was based on the colour produced with
    phosphoric acid and ethyl acetate, which was measured at 550 nm. Rapid
    field assay methods for the determination of pyrethrins in pyrethrum
    flowers were developed by Donegan et al. (1971). The modified
    methods were based on the sulphur colour method of Cueto and Dale
    (1953) and the phosphoric acid method of Williams et al. (1956).

    Pyrethrins can be determined by gas chromatography using a flame
    ionization or electron capture detector. The former detector was used
    by Abe and Fujita (1971) with a column of 2% XE-60 on 60/80 acid
    washed Chromosorb W. Tetenyi et al. (1971) employed the electron
    capture detector with temperature programming and a column of 3%
    N.P.G.S. on 60/80 silanized Chromosorb W. Benvenue et al. (1970)
    quantitatively measured levels of pyrethrins in commercial
    formulations by gas chromatography, the concentrations ranging from
    0.05 to 50% of the mixtures.

    Moore (1971) cleaned up sample extracts of biological materials
    including eggs, tissue, body fat, milk and milk products, with
    acetonitrile partitioning, silica gel column chromatography and TLC
    before the determination with the electron capture detector cell of
    Bruce (1967).

    The use of liquid-gel chromatography for the analytical
    characterization of pyrethrum extracts was investigated by Stevenson
    (1972). The column eluate was examined by three general procedures:
    (a) collection of fractions followed by GLC, (b) direct application to
    a moving wire flame ionization detector and (c) direct application to
    a moving TLC plate and subsequent development to give a two
    dimensional chromatographic display of the sample. The thermal micro
    separation technique of Stahl (1968) and Stahl and Fuchs (1968) for
    the separation of volatile material on to a thin-layer plate was
    applied by Stahl (1969) to the rapid separation of pyrethrins,
    nicotine and synthetic pesticides.

    Shah (1970) compared the standard mercury reduction method of the AOAC
    (1955) with the official method of the Pyrethrum Board of Kenya
    (P.B.K., 1954, 1957). Variations in reagents and techniques were
    discussed. Gomila et al. (1969) tested the suitability of the
    A.O.A.C. method (1960) for the determination of pyrethrins in mosquito
    coils. The method could be applied with fairly reproducible results.

    The application of high speed liquid chromatography combined with
    gradient elution shows considerable potential for the analysis of
    pyrethrins and related compounds for residue analysis as well as
    quality control. Schmit et al. (1971) applied high speed reverse
    phase liquid chromatography with an ultraviolet detector to the
    determination of the components of the World Standard pyrethrum
    extract which did not contain the complex mixture present in a crude
    oleo-resin extract. The non-polar stationary phase employed was
    "permaphase" ODS with a water/methanol mobile phase. Optimum
    separation was achieved under gradient elution conditions (30%
    methanol + 70% water increasing to 100% methanol at 3 min.). It is
    considered that the requirement for analytical methods capable of
    measuring residues of pyrethrins at the 0.1 ppm level has been met.
    The gas and liquid chromatographic procedures can be adapted for
    regulatory purposes.

    RESIDUES IN DRIED CODFISH

    Further information relating to residues in dried codfish has been
    received from the Norwegian Codex Alimentarius Council (Race, 1971).
    This is quoted below:-

         "Dried Cod Fish when hung out to dry is at times severely exposed
    to attack by blowflies (Calliphora spp). Under certain weather
    conditions (dry and warm) 100% spoilage by larvae may occur. The use
    of pyrethrum can reduce this spoilage considerably - one aims to
    reduce spoilage by up to 90%.

         "The use of pyrethrum is under strict control. Special permission
    for its use must be applied for to the General Director of Fisheries.
    As an illustration it can be mentioned that in 1965, 57 such
    applications were granted. Reports from 31 producers show that these
    treated altogether 1 480 metric tons of raw fish. Inspection of
    production sites has shown that instructions for the use of pyrethrum
    are followed. The use of pyrethrum has decreased considerably during
    the last few years. In 1966 ca. 1 340 kg of pyrethrum concentrate
    (5.3%) was sold, in 1970 ca. 120 kg for treatment of dried cod fish.

         "Laboratory and field trials have been carried out by the
    Chemical/Technical Research Institute of the Directorate of Fisheries
    to find out the beet possible method for the use of pyrethrum.

         "Laboratory tests showed that dipping for  min. in 0.02%
    pyrethrum emulsion gave complete protection against fly larvae.
    Practical trials showed however that a concentration of 0.12% was
    necessary to attain satisfactory protection. It was also shown that
    spraying the hung fish with 0.12% emulsion gave good protection. Lower
    concentrations in dip or spray fluid did not give satisfactory
    results.

         "Residual concentration of pyrethrum in fish treated with 0.12%
    pyrethrum emulsion varied somewhat and decreased on storage (ca. 50%
    in sunlight and ca. 20% in darkness after 23 weeks).

         "Immediately after drying (ca. 10 weeks after treatment) the
    concentration was 7 - 8 ppm total pyrethrum determined by chemical
    means. Biological tests gave a concentration of around 3 ppm or lower.
    It is concluded that treatment with 0.12% is appropriate to give a
    product with a residual concentration under 3 ppm. Under most of the
    tests residual concentration after treatment proved to be over 1 ppm.
    Only in special cases (considerable rainfall immediately after hanging
    out) did one find residual concentrations of 0.1 - 0.4 ppm."

    This information leads to the inescapable conclusion that, as employed
    in Norway, the proposed tolerance for residues of pyrethrins on dried
    codfish is more than an order of magnitude too low. Further
    information was available regarding similar treatments of dried fish
    in various parts of Africa, although no firm residue data was
    presented. Further data is required on residues in dried fish from
    supervised trials and commercial usage. It seems therefore
    appropriate, and reasonable, to raise the tolerance to that already
    given for raw cereals, i.e. 3 ppm.

    APPRAISAL

    Methods of analysis for residues of pyrethrins at the 0.1 ppm level
    are available and can be adapted for regulatory purposes. Available
    data indicated a need to recommend a tolerance for residues of
    pyrethrins on dried fish of 3 ppm in place of the existing temporary
    tolerance of 1 ppm in dried codfish.

    RECOMMENDATIONS

    TOLERANCE

         Fish (dried)        3 ppm

    FURTHER WORK OR INFORMATION

    REQUIRED (Before 30 June 1975)

    Further data on residues in dried fish from supervised trials and from
    commercial usage.

    DESIRABLE

    Toxicity studies in a non-rodent mammalian species with special
    emphasis on the effects on the liver.

    REFERENCES

    Abe, Y. and Fujita, Y. (1971) Pyrethroidal compounds I; Quantitative
    determination of natural pyrethrins by GLC. Nippon Nogei Kagaku Kaisi,
    45: 22-28 (in Japanese).

    AOAC (1955) Official methods of analysis of the AOAC, 8th ed., p.
    68-70.

    AOAC (1960) Official methods of analysis of the AOAC, 9th ed., p.
    41-43.

    Bevenue, A., Kawano, Y. and de Lano, F. (1970) Analytical studies of
    pyrethrin formulations by gas chromatography. J. Chromat., 50: 49-58.

    Bond, H., Mauger, K. and DeFeo, J.J. (1972) The oral toxicity of
    pyrethrum, alone and combined with synergisers and common drugs.
    Report, Dept. of Pharmacology and Toxicology, Univ. of Rhode Island.
    (unpublished)

    Bruce, W.N. (1967) Detector cell for measuring picogram quantities of
    organophosphorus insecticides, pyrethrin synergists and other
    compounds by gas chromatography. J. Agr. Fd. Chem., 15: 178-181.

    Cueto, C. and Dale, W.E. (1953) Colorimetric determination of
    pyrethrins, allethrin and furethrin. Anal. Chem., 25: 1367-1369.

    Donegan, L., Morrison, J.N. and Webley, D.J. (1971) Rapid field assay
    for pyrethrum flowers. Pyrethrum Post, 11(1): 36-40.

    Elliott, M., Janes, N.F., Kemmel, E.C. and Casida, J.E. (1972)
    Metabolic fate of pyrethrin I, pyrethrin II and allethrin administered
    orally to rats. J. Agr. Fd. Chem., 20: 300-313.

    FAO/WHO (1965) Evaluation of the toxicity of pesticide residues in
    food. FAO/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.

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

    FAO/WHO (1969) 1968 evaluations of some pesticide residues in food.
    FAO/PL/1968/M/9/1; WHO/Food Add./69.35.

    FAO/WHO (1970) 1969 evaluations of some pesticide residues in food.
    FAO/PL/1969/M/17/1; WHO/Food Add./70.38.

    FAO/WHO (1971) 1970 evaluations of some pesticide residues in food.
    FAO/AGP:1970/M?12/1; WHO/Food Add./71.42.

    Furmanec, D., Schilling, F.A.E. and Brown, B.B. (1969) A rapid
    colorimetric assay for pyrethrins. Pyrethrum Post, 10(1): 21-23.

    Gomila, G., Lakos, E. and Priori, J. (1969) Application of the AOAC
    method, 9th edition, to determine pyrethrins in mosquito coils.
    Pyrethrum Post, 10(2): 22-23.

    Moore, J.B. (1971) Paper submitted to the International Symposium on
    recent advances in research with pyrethrum, the natural insecticide.
    Minneapolis, U.S.A., 30-31 August. (unpublished)

    P.B.K. (1954) Determination of pyrethrins. Official methods of
    analysis of Pyrethrum Board of Kenya. Method for extracts.

    P.B.K. (1957) Determination of pyrethrins. Official methods of
    analysis of Pyrethrum Board of Kenya. Methods for powder and flowers.

    Race, J. (1971) Comments from Norway about the tolerance proposed for
    pyrethrins, in dried codfish. Communication to Codex Committee on
    Pesticide Residues. (unpublished)

    Schmit, J.A., Henry, R.A., Williams, R.C. and Dieckman, J.F. (1971)
    Applications of high-speed reversed-phase liquid chromatography. J.
    Chromat. Sci., 9: 645-651.

    Shah, V.M. (1970) A comparison of the mercury reduction methods of
    pyrethrum analysis. Pyrethrum Post, 10(4): 27-32.

    Stahl, E. (1968) T.A.S. technique. A thermal method for the separation
    of volatile material for application to thin-layer chromatography. J.
    Chromat., 37: 99-102 (in German).

    Stahl, E. (1969) The T.A.S. method for rapid separation of pesticides
    and preservatives. Z. Lebensm.u. Forsch., 140: 321-329 (in German).

    Stahl, E. and Fuchs, J. (1968) Characterisation of pharmacopoeial
    drugs by thin-layer chromatography. III. Direct application to the
    plate of active constituents of drugs containing essential oils.
    Dt.ApothZtg., 108: 1227-1231 (in German).

    Stevenson, D.S. (1972) Application of liquid-gel chromatography to the
    analytical characterisation of pyrethrum extracts. Pyrethrum Post,
    11(3): 90-93.

    Tetenyi, P., Bethelyi, E., Okuda, T. and Szilagyi, I. (1971) Use of
    programmed temperature in analytical determination of pyrethrins by
    electron capture detector. Pyrethrum Post, 11(1): 29-31, 47.

    Williams, H.L., Dale, W.E. and Sweeney, J.P. (1956) A new colorimetric
    method for pyrethrins. J. Ass. Off. Agr. Chem., 39: 872-879.
    


    See Also:
       Toxicological Abbreviations
       Pyrethrins (FAO Meeting Report PL/1965/10/1)
       Pyrethrins (FAO/PL:CP/15)
       Pyrethrins (JMPR Evaluations 2003 Part II Toxicological)
       Pyrethrins (FAO/PL:1967/M/11/1)
       Pyrethrins (FAO/PL:1968/M/9/1)
       Pyrethrins (FAO/PL:1969/M/17/1)
       Pyrethrins (AGP:1970/M/12/1)
       Pyrethrins (WHO Pesticide Residues Series 4)