First draft prepared by Dr S. Caroldi
    University of Padua, Padua, Italy

         The present Meeting reviewed available data evaluated at the 1987
    Meeting, together with data submitted to the present Meeting. 
    Coumaphos has been evaluated toxicologically seven times previously,
    in 1968, 1972, 1975, 1978, 1980, and 1987 (Annex 1, FAO/WHO, 1969ab,
    1973ab, 1976a, 1979a, 1981ab, and 1987b).  The temporary ADI was
    withdrawn in 1980.  This monograph addendum summarizes the data
    submitted in 1987 and 1990.



    Long term/carcinogenicity studies

         Fifty male and 50 female Wistar rats (Bor: WISW strain.
    Winkelmann-Borcher) were treated with coumaphos (technical grade,
    99.2% purity) at concentrations of 0, 1, 5, 25 ppm, equal to 0, 0.05,
    0.25, and 1.22 mg/kg bw/day for males and 0, 0.07, 0.36, and 1.70
    mg/kg bw/day for females (calculated as average daily intake througout
    the duration of the study) for 24 months.  Twenty additional rats per
    sex per dose were treated for 12 months and then sacrificed for
    intermediate investigations.  Rats were four to five weeks old at the
    beginning of the study.  Coumaphos was mixed with the feed up to the
    nominal concentrations.  The actual content of coumaphos in the
    formulations was checked every three months and were found to be
    similar to the nominal levels.

         In male rats, food and water intake and weight gain were not
    influenced by coumaphos administration up to 25 ppm.  In female rats
    marginal reduction in food and water intake was observed at 25 ppm
    coumaphos (about 12% for both food and water, the difference being 
    statistically significant only for water intake).  Body weight gain
    was reduced in females, compared to controls.

         Neither sex showed any changes in the incidence of clinical
    signs, or mortality rate at any dose level.  At the end of the
    treatment the mortality rates were 8, 16, 22, and 18% (males) and 23,
    24, 7, and 18% (females) at 0, 1, 5, and 25 ppm coumaphos,
    respectively.  Differences in haematological, clinical chemistry and
    urinalysis parameters were trivial and not related to coumaphos
    administration.  Cholinesterases were measured in plasma and
    erythrocytes (method by Ellman et al.,  1961) after 5, 14, 27, 53,
    80, and 104 weeks and in brain after 53 and 104 weeks from the
    beginning of the study.  In males the 25 ppm dose reduced
    cholinesterase activity in plasma by 27-43% while in females the
    average inhibitions varied between 45-53%.  The 25 ppm dose reduced
    cholinesterase activity in erythrocytes by 16-28% in males and 18-34%
    in females (range of average inhibitions at different times).  At 5
    ppm trivial and sporadic differences were detected.  Brain
    cholinesterase was significantly higher in all treated females after
    53 weeks of treatment (normal at the end of the study) and in all
    dosed males at the end of treatment (a marginal increase had been
    detected after 53 weeks only in the group dosed with 5 ppm).  The
    increased activity of brain cholinesterase was dose related ranging
    between 15-42% in females after 53 weeks and between 23-43% in males
    at the end of the study. 

         Ophthalmological examination was performed at the beginning of
    the study, after 12 months and at the end of the study.  Pupillary
    reflexes were evaluated together with the regions around the eyes and
    the anterior segments.  Refractive media of the eyes and fundus oculi
    were observed after pupil dilatation.  Scattered alterations were not
    dose related.

         Pathology was performed on all animals.  Gross pathological
    effects were comparable between groups.  Organ weights (brain, heart,
    lung, spleen, kidneys, adrenal glands, testicles and thyroid gland)
    were not different at 12 and 24 months.  In males treated with
    coumaphos at 25 ppm, liver weight was lower than in controls at 12
    months but not at 24 months.

         There were no statistically significant differences in the
    incidence of neoplasms.  No non-neoplastic changes appeared to be
    compound-related.  The dose of 5 ppm coumaphos was tolerated without
    damage under the conditions described.  The  NOAEL for coumaphos in
    the diet in this study was 5 ppm, equal to 0.25 mg/kg bw/day and to
    0.36 mg/kg bw/day for male and female rats, respectively.  The study
    indicates absence of any evidence of carcinogenicity under the test
    conditions (Eiben,  1988).

    Special studies on delayed neurotoxicity

         Groups of 1-8 hens (Leghorn laying hens, 12 months old) received
    single (50 or 100 mg/kg bw) or up to 90 daily (5 or 10 mg/kg bw) doses
    of coumaphos (97.1% purity) by oral route in gelatin capsules. 
    Controls received empty gelatin capsules.  Other hens received single
    (50, 100, 500 mg/kg bw) or daily (100 mg/kg bw for either 30 or 90
    days) dermal applications of coumaphos (dissolved in acetone and
    applied on a 5.3 cm› area on the unprotected back of the neck).

         All birds dosed orally with coumaphos showed acute cholinergic
    symptoms despite atropine treatment.  All 4 hens dosed with 100 mg/kg
    bw died within 1-5 days.  The single hen dosed with 50 mg/kg bw
    survived the 30 day observation period.  At 10 mg coumaphos/kg bw
    daily 3/3 hens died because of acute toxicity while 2/3 hens survived
    the 5 mg/kg bw coumaphos daily doses up to the scheduled 90 doses.
    Animals dosed dermally with coumaphos did not show acute cholinergic

         All hens which survived oral coumaphos and all hens which
    received coumaphos dermally developed clinical signs of delayed
    neuropathy.  The severity of toxic effects (measured on a 1-4 scale)
    was dose related varying between a minimum of gross ataxia (grade 2),
    detectable after a single oral or dermal dose of 50 mg/kg bw of

    coumaphos, and a maximum of complete paralysis (grade 4), reached
    after 48-50 days of dermal applications of 100 mg coumaphos/kg bw.
    Some improvement was detected in birds which received 90 doses (either
    oral or dermal), after the treatment was stopped.

         Animals dosed with coumaphos per os did not show
    histopathological changes either in peripheral nerves or in spinal
    cord (equivocal alterations were observed in the spinal cord of a
    single hen). Histopatological changes were not detected in peripheral
    nerves of dermally treated birds (except a single hen dosed daily with
    100 mg coumaphos/kg bw which died after 49 doses).  Unequivocal
    alterations in spinal cord were detected in about 50% of hens dosed by
    dermal route, the other 50% showing either equivocal alterations or
    being unaffected.

         It was concluded that dermally applied coumaphos is capable of
    causing delayed neurotoxicity in adult hens.  Acute toxicity of orally
    administered coumaphos does not allow the administration of dosages
    large enough to produce delayed neuropathy without killing the hens
    (Abou-Donia et al., 1982).

         Thirty hens (Leghorn, Brinkschulter, Senden, 12-18 months old)
    were dosed orally with 50 mg/kg bw of coumaphos (Asuntol 99.1% purity
    grade dissolved 2% v/v in deionized water and given by gavage)
    followed, three weeks later, by a second oral dose of 100 mg/kg bw
    Coumaphos.  All birds were treated with atropine against cholinergic
    toxicity.  Five other hens were dosed orally with tri-ortho-cresyl
    phosphate (TOCP, 375 mg/kg bw) as positive control and 6 hens received
    vehicle only.

         The animals dosed with coumaphos showed cholinergic symptoms
    starting a few hours after dosing and 4/30 and 3/26 died after the
    first and the second dose, respectively, in spite of atropine therapy.
    Surviving hens were observed for abnormal gait until day 42 after
    first dosing but none of them developed ataxia.  Histopathology
    performed in 6 hens randomly chosen from the 23 survivors showed no
    signs of degeneration in peripheral nerves, lumbar spinal cord,
    medulla oblongata and cerebellum. Scattered alterations observed in
    thoracic and cervical spinal cord were not different from those
    observed in controls.

         Hens dosed with TOCP did not show acute cholinergic symptoms. 
    All of them developed delayed neuropathy starting on day 10, and
    progressed to complete paralysis between day 16 and 20. Histopathology
    (performed in 4 birds) showed marked degeneration in all observed
    sections.  Therefore, a double oral dose of coumaphos (50 + 100 mg/kg
    p.o.),  which resulted in severe cholinergic toxicity,  did not cause
    delayed neurotoxicity in hens (Flucke et al., 1987).  

         The effect of coumaphos administration on neuropathy target
    esterase (NTE) was investigated in nervous tissue of hens. Single
    doses of coumaphos (97.7% purity) were given to chickens (Lohmann
    selected Leghorn, Brinkschulter, Senden, older than 9 months) either
    by gavage (100 mg/kg bw to 20 birds in two different studies) or by
    dermal application (500 mg/kg bw to 6 birds).  Animals were killed 24
    and 48 hours after dosing and NTE activity was measured in the brain
    and spinal cord.  NTE activities in nervous tissue of hens dosed with
    coumaphos were compared with those measured in vehicle treated
    controls.  Other hens were dosed by gavage with TOCP (300 mg/kg bw),
    a known inhibitor of NTE, and NTE was measured.  All animals were
    treated with atropine and pralidoxime against cholinergic toxicity.

         Acute symptoms were recorded but not reported in the study.
    Cholinesterase activities were not measured and no spontaneous deaths
    occurred within 48 hours.  NTE activity in nervous tissue of controls
    was within the expected range in hens.  As expected NTE inhibition was
    between 80 and 90% in animals dosed with TOCP.

         Forty-eight hours after 100 mg/kg bw coumaphos p.o., a single hen
    showed about 60% inhibition of NTE in brain and spinal cord. Two other
    hens showed NTE inhibition between 20 and 30% after 24 hours in both
    tissues.  In remaining hens NTE activity was normal or marginally
    affected.  After dermal application of 500 mg coumaphos/kg bw dermally
    (area of application not reported) inhibition, of NTE was below 10% in
    all birds (Bomann et al., 1989a, 1989b).

         This study is insufficient to draw conclusions regarding
    neurotoxicity for the following reasons:  NTE was measured in brain
    and spinal cord but not in peripheral nerves, the real target for
    delayed neuropathy; NTE was measured only up to 48 hours which may not
    be sufficient in the case of delayed absorption/disposal of coumaphos;
    no hens were observed for symptoms of delayed neuropathy; and no
    histological examination was performed at the time when the onset of
    delayed neuropathy is expected. 

    Special study on haematological effects


         The effects of the oral administration of a single dose of 0, 1,
    3 and 10 mg/kg bw coumaphos (purity not given) in 1% tylose were
    investigated on several haematological parameters in groups of 5 WISW
    (SPF Cpb)/Winkelmann rats (sex not given).  Analysis of blood samples
    collected 90 min after dosing showed no treatment-related effects on
    haematocrit, haemoglobin, thromboelastogram, platelet number and
    aggregation, blood sedimentation rate, fibrinogen, thrombin and
    thromboplastin time (Seuter and Perzborn, 1981).

    Special study on urinary function


         The effect of coumaphos on urine and electrolyte excretion was
    studied in groups of male Bor:  WISW (SPF Cpb)/Winkelmann rats
    administered a single oral dose of 0, 1, 3 and 10 mg/kg bw coumaphos
    (purity not given) suspended in a 1% methylcellulose solution.  The
    volume of urine and the amount of urinary K+ and Na+ excreted in
    the 6 hours after dosing were measured.  There was no significant
    difference in the mean volume of urine excreted or in the amount of
    either of the 2 electrolytes between any of the treated groups and the
    controls (Garthoff, 1981a).

    Observations in humans

         The effects of the human exposure to coumaphos resulting from the
    spraying of a 0.1% Asuntol emulsion for veterinary use in a piggery
    was investigated in 4 human subjects (male, 30-50 years).  These
    subjects stayed in the piggery continuously for the first 2 hours of
    the study and intermittently thereafter.  No acetylcholinesterase
    activity inhibition was observed in the whole blood of these subjects
    or in plasma and erythrocytes of 6 treated pigs after 1, 2, 4, 6, or
    24 hours after spraying.  Measurement of coumaphos concentration in
    the air at 50 and 150 cm above the floor showed that only small amount
    of the compound was present during the first 50 minutes after spraying
    (0.123 and 0.097 mg/m3, respectively).  No detectable amount of
    coumaphos could be found in the air after 135 minutes from the
    spraying (Eben et al., 1981).


         The data base is incomplete in that the teratogenicity studies
    were submitted too late for evaluation.  Further, questions existed
    with respect to the acceptability of a mouse reproduction study
    reviewed by the JMPR in 1968.  Consequently an ADI could not be

    Studies without which the determination of an ADI is impracticable

         -    Rat teratogenicity study
         -    Rabbit teratogenicity study
         -    Adequate multigeneration study


    Abou-Donia, M.B., Makkawy, H., and Graham, D.J., (1982). Coumaphos:
    delayed neurotoxic effect following dermal administration in hens. J.
    Toxicol. Environ. Health, 10: 87-99.

    Bomann, W., Eben, A.. Asuntol (1989a). Investigations into the effect
    on neurotoxic esterase (NTE) after dermal application to chickens.
    Unpublished report No. 18415 from Bayer AG, Institute of Toxicology.
    Submitted to WHO by Bayer.

    Bomann, W., Eben, A.. Asuntol (1989b). Investigations into the effect
    on neurotoxic esterase (NTE) after dermal application to chickens.
    Unpublished report No. 18423 from Bayer AG, Institute of Toxicology.
    Submitted to WHO by Bayer.

    Eben, A., Machemer, L., Dorn, H. and Lorke, D., (1981).  Influence of
    a spray treatment with a 0.1% aqueous Asuntol emulsion on the operator
    and the treated animals (pigs).  Unpublished report No. 9860 from
    Institute for Toxicology, Bayer AG.  submitted to WHO by Bayer AG.

    Eiben, R. (1988). Studies on chronic toxicity and carcinogenicity in
    Wistar rats (administration with feed for 24 months). Unpublished
    report No. 17131 from Bayer AG, Institute of Toxicology. Submitted to
    WHO by Bayer.

    Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, M., 1961.  A
    new and rapid colorimetric determination of acetylcholinesterase
    activity. Biochem. Pharmacol., 7: 88-95.

    Flucke, W., Kaliner, G.. Asuntol-Wirkstoff (1987). An examination of
    acute neurotoxicity after oral dosing in the hen. Unpublished report
    No 15430 from Bayer AG, Institute of Toxicology. Submitted to WHO by

    Garthoff B., (1981a).  Bay B 9134:  Study of diuretic action in rats. 
    Unpublished report No. 10072 from Dept. of Cardiovascular
    Pharmacology, Pharmacological Institute, Bayer AG.  Submitted to WHO
    by Bayer AG.

    Garthoff B., (1981b).  Bay B 9134:  Effect on hemodynamics and
    myocardial contractility in anesthetized dogs after oral
    administration.  Unpublished report No. B/B 9134/002 from Dept. of
    Cardiovascular Pharmacology, Pharmacological Institute, Bayer AG. 
    Submitted to WHO by Bayer AG.

    Goodman D.G., Ward J.M., Squire R.A., Chu K.C., and Linhart M.S.,
    (1979).  Neoplastic and nonneoplastic lesions in aging F344 rats. 
    Toxicol. Appl. Pharmacol. 48: 237-248.

    Green L.L., (1977).  Coumaphos levels in tissues and feces from 13
    week; 180 day; and 103 week studies.  Unpublished report No. 76-36-
    106002 from Gulf South Research Institute, New Iberia, Louisiana, USA. 
    Submitted to WHO by Tracor Jitco, Inc. Rockville, Maryland., USA.

    Herbold B., (1981a).  Asuntol:  Salmonella/microsome test for the
    detection of point mutagen effects.  Unpublished report No. 10188 from
    Institute of Toxicology, Bayer AG.  Submitted to WHO by Bayer AG.

    Herbold B., (1981b).  P-O analogue of Asuntal:  Salmonella/microsome
    test for the detection of point mutagen effects.  Unpublished report
    No. 10094 from Institute for Toxicology, Bayer AG.  Submitted to WHO
    by Bayer AG.

    Herbold B., (1981c).  Chlorferron:  Salmonella/microsome test for
    point-mutagenic activity.  Unpublished report No. 10095 from
    Toxicological Institute, Bayer AG.  Submitted to WHO by Bayer AG.

    Herbold B., (1981d).  Potasan:  Salmonella/microsome test for the
    detection of point mutagen effects.  Unpublished report No. 10093 from
    Institute of Toxicology, Bayer AG.  Submitted to WHO by Bayer AG.

    NCI, (1979a).  Bioassay of coumaphos for posible carcinogenicity. 
    Veterinary Medical Review, 2: 179-181 (Abstract).

    NCI, (1979b).  Bioassay of coumaphos for possible carcinogenicity. 
    Unpublished report No. 96 from Carcinogenesis Testing Program,
    Division of Cancer Cause and Prevention, National Cancer Institute,
    National Institutes of Health, Bethesda, Maryland USA.  Submitted to
    WHO by Bayer AG.

    Neuser V. and Jacobi H., (1981).  Bay B 9134:  Effect on central
    nervous system.  Unpublished report No. 10548 from Troponwerke GmbH
    and Co. KG.  Submitted to WHO by Bayer AG.

    Seuter F. and Perzborn E., (1981).  Bay B 9134:  Haemopharmacological
    investigations.  Unpublished report No. 10070 from Institute for
    Pharmacology, Bayer AG.  Submitted to WHO by Bayer AG.

    See Also:
       Toxicological Abbreviations
       Coumaphos (ICSC)
       Coumaphos (FAO/PL:1968/M/9/1)
       Coumaphos (WHO Pesticide Residues Series 2)
       Coumaphos (WHO Pesticide Residues Series 5)
       Coumaphos (Pesticide residues in food: 1978 evaluations)
       Coumaphos (Pesticide residues in food: 1980 evaluations)
       Coumaphos (Pesticide residues in food: 1983 evaluations)