Isophenphos was evaluated for acceptable daily intake by the
    Joint Meeting in 1981 and reviewed in 1982 (Annex 1, FAO/WHO, 1982a
    and 1983a). A toxicological monograph was published after the 1981
    Meeting (Annex 1, FAO/WHO, 1982b) and a monograph addendum was
    published after the 1982 Meeting (Annex 1, FAO/WHO, 1983b). In 1981, a
    temporary ADI of 0 - 0.0005 mg/kg b.w. was established, which was
    extended in 1982 because of the absence of appropriate delayed
    neurotoxicity studies and because of questions raised by the
    multi-generation studies submitted at that time. The requested studies
    have been submitted and are summarized in this monograph addendum.



    Toxicological studies

    Special studies on cholinesterase inhibition

         In vitro studies on purified isofenphos indicate no inhibition
    of horse serum acetylcholinesterase or human serum butyrylthiocholine-
    sterase up to concentrations of 100 mM. It was concluded that direct
    inhibitors are not present as impurities in these isofenphos
    preparations (Blass, 1982; Wehling, 1983).

    Special studies on delayed neurotoxicity

         Experiments using a single dose for delayed neurotoxicity studies
    were performed using treatment with antidotes (atropine & 2-PAM) for
    cholinergic symptoms. Isofenphos (74 mg/kg b.w. orally) given to hens
    after pre-treatment with atropine did not produce clinical or
    histopathological signs of delayed polyneuropathy. The unprotected
    oral LD50 in this study was 21 mg/kg b.w. (Kimmerle, 1972;
    Cherry et al., 1972).

         In a similar study, isofenphos was given orally (36 mg/kg b.w.)
    to hens after pre-treatment with atropine. Nevertheless, 17 hens out
    of 30 died of cholinergic symptoms. The surviving hens did not show
    clinical or histopathological signs of delayed neurotoxicity
    (Hixson, 1982).

         Another study measured the response of NTE in humans to oral
    dosing with isofenphos (36 mg/kg b.w.) and pre-treatment with
    atropine. Substantial inhibition of NTE was measured within 1 - 2 days
    after dosing. No concurrent controls for clinical/histological
    evaluations were used in this study, and the reference oral
    unprotected LD50 was 16 mg/kg b.w. isofenphos (Thymen, 1978;
    Thymen & Eben, 1983).

         Another study provided evidence for the potential of isofenphos
    to cause delayed neurotoxicity in hens. The oral unprotected LD50 in
    tested hens was 3 - 5 mg/kg b.w. isofenphos. After appropriate
    antidotal treatment (before and several times after dosing) it was
    raised to 100 mg/kg b.w. One hundred mg/kg b.w. isofenphos given
    orally caused more than 70% inhibition of brain NTE when measured
    3 days after dosing and clinical-morphological signs of delayed
    neurotoxicity approximately 2 weeks later (Wilson et al., 1984).

         In a subchronic delayed neurotoxicity study, groups of 10 adult
    white leghorn laying hens (15 to 20 months old) were treated daily by
    gavage with technical isofenphos (as an emulsion in water with
    Cremophor EL) at 0.25, 1, or 2 mg/kg b.w. for 90 consecutive days. Ten
    hens were used as untreated controls and 10 "dose controls" were given
    water and Cremophor EL by gavage. After the seventh administration,
    clinical signs and 1 mortality occurred in the highest-dose group
    (2.5 mg/kg b.w./day). At 1.25 mg/kg b.w./day 1 bird showed clinical
    signs after the seventh administration.

         A significant decrease in body weight and an inhibition of
    cholinesterase activity in plasma, erythrocytes, and whole blood were
    observed at 2 mg/kg b.w./day throughout the 90-day treatment period.
    Slightly-reduced body weights and very slight inhibition of the
    cholinesterases also occurred at 1 mg/kg b.w./day. Histopathological
    examination of the brain, spinal cord, and sciatic nerve of all 10
    hens in the 2 mg/kg b.w./day dose group revealed minimal changes
    that were similar in incidence and severity to those noted in
    concurrent controls. Positive controls (10 hens), treated orally with
    tri-o-cresyl phosphate (TOCP) at 5 mg/kg b.w./day (as an emulsion
    in water and Cremophor EL), displayed clinical signs (ataxia from
    the fifth week onwards) and histopathological lesions, such as
    severe degeneration of the pathways in the spinal cord and axonal
    degeneration in the medulla oblongata and cerebellum, which are
    typical of delayed neurotoxicity. The dose of 0.5 mg/kg b.w./day
    isofenphos was tolerated without effects (Thyssen, 1978; Flucke
    & Kaliner, 1985).

    Special study on embryotoxicity/teratogenicity


         A study to investigate embryotoxic and teratogenic effects in
    Long Evans rats of the FB30 strain following dermal application was
    conducted. Males were between 3 and 6 months of age and weighed
    350 - 500 g at the start of the study; females were between
    2.5 - 3.5 months of age and weighed 199 - 260 g. Five groups of 25
    animals were treated with 0, 0.3, 1.0, 3.0, or 10.0 mg isofenphos/kg
    b.w. The compound was applied to shaved dorsal skin for a 5-hour
    contact time, the skin being washed at the end of each exposure. The
    applied volume was 0.5 ml/kg b.w. in each test group. Males were not
    treated; females were treated from days 6 - 15 of gestation.

         On the 20th day of gestation the animals were killed by CO2
    asphyxiation and the fetuses examined by the Wilson technique for
    visceral malformations and by clearing and skeletal staining for
    skeletal abnormalities. There were no treatment-related abnormalities
    in behaviour or physical appearance. One dam in the 3 mg/kg b.w. group

    was killed during treatment because of evidence of illness, of which
    it had shown signs before treatment began. All dose groups gained less
    weight than controls during treatment and throughout gestation. There
    were no significant differences between control and treated groups
    with regard to fertilization and pregnancy rates, which corresponded
    to those known for the rats used in this study. The malformations seen
    in the various groups are shown in Table 1.

         Evidence of maternal toxicity was observed, with a significant
    failure of treated animals to gain weight. The apparent no-effect
    level for maternal toxicity was 0.3 mg/kg b.w., although the mothers
    in this group showed poor weight gain during gestation (Schluter,

    Special study on reproduction

         A 3-generation rat reproduction study with isofenphos that was
    performed at the Huntingdon Research Center in 1976 and 1977, and was
    summarized in the 1981 toxicological monograph (Annex 1, FAO/WHO,
    1982b), was re-evaluated. Twenty animals per group were fed doses of
    0, 1, 10, or 100 ppm isofenphos in the diet. Body weights, food
    consumption, and cholinesterase activity were measured. The F0
    generation was mated 3 times and cross-mated (control with 100 ppm
    group). F1a and F2a rats were mated once. There were no
    significant effects on any variables examined, including litter size,
    birth weight, viability, or lactation, in animals fed up to 10 ppm. In
    the F2a generation successful mating did not occur in the 100 ppm
    group because of what was described as "reduced gestation and
    lactation" rates.

         Comparable gestation and insemination rates between control and
    test animals were found in the 1 and 10 ppm groups in all 3
    generations. Following the second mating of the F0 generation, 90%
    of the control females were pregnant and 70%, 60%, and 40% of the
    females in the 1, 10, and 100 ppm groups, respectively, were pregnant.
    Taking into account the fact that the insemination rates for this
    mating were reduced in all of the study groups, all inseminated
    females in the 1 ppm group became pregnant, 12/14 in the 10 ppm group
    became pregnant, and all inseminated females in the 100 ppm group
    became pregnant. Reduced pregnancy rates in multi-generation studies
    are common as a result of reduced willingness to mate, and it occurred
    in this study in control animals as well as in the test animals. One
    animal in the control group and 1 in the 1 ppm group repeatedly failed
    to become pregnant.

         The low insemination rate exhibited by controls was considered to
    be comparable with the apparently low gestation rate after the second
    mating of the F0 animals. The apparent no-effect level in this study
    was 10 ppm (Palmer et al., 1977; Eiben, 1983).

    Table 1.  Embryotoxic and terotogenic malformations in rats following
              dermal application of isofenphos.


                     Number of                    Nature of
    Group            malformed fetuses            malformations

    Control                1             1x BB fissure, rib fusion,
                                         microthalmia resp. anophthalmia

                           1             1x kinky tail

                           2             2x nodulations on ribs

                           1             1x oedematous thorax

    0.3 mg/kg b.w.         1             1x nodulations on ribs

    1 mg/kg b.w.           1             1x nodulations on ribs

    3 mg/kg b.w.           1             1x multiple malformation; WS,
                                         Ri, Be, extra tail, eyes &
                                         umbilical hernia, and general

                           1             1x nodulations on ribs

                           1             1x cleft sternlum, abdominal
                                         fissure, dysplasia of
                                         extremities, cleft palate

    10 mg/kg b.w.          1             1x microphthalmia

                           3             1x hypoplasia of telencephalon,
                                         eye malformations, dysplasia of

                                         2x hypoplasia of telencephalon,
                                         eye malformations

    BB = thorax and abdomen       WS = vertebral column
    Ri = ribs                     Be = pelvic bone


         In vitro studies indicate that, in the preparations of
    isofenphos used, direct cholinesterase inhibitors are not present as

         The present Meeting reviewed a 2-generation study and a
    re-evaluation of a 3-generation study performed in 1976 and 1977. The
    apparent no-effect level in the multi-generation study was 10 ppm. In
    a dermal study there was some evidence of anomalies associated with
    embryotoxicity at 3 mg/kg b.w.

         In the evaluation of the potential of isofenphos to cause delayed
    polyneuropathy, the ratios between the unprotected LD50 and the dose
    tested for delayed polyneuropathy were considered and are shown in
    Table 2.

    Table 2.  Delayed neuropathy in rats dosed with isofenphos.

    Approximate ratio           Delayed
    Unprotected LD50/           neuropathy         Reference
    Neurotoxic dose

             < 0.3              no                 Kimmerle, 1972;
                                                   Cherry et al., 1972

             < 1 (?)            no                 Hixson, 1982

               0.04             yes                Wilson et al., 1984

         Two studies on the biochemical test for delayed neuropathy
    (NTE inhibition) can also be compared, as shown in Table 3.

    Table 3.  NTE inhibition in rats dosed with isofenphos.

    Isophenphos     Approximate ratio     Brain NTE
    (oral)          Unprotected LD50/     inhibition
    mg/kg b.w.      Neurotox dose         (range)        Reference

        36           approx. 0.4          23 - 58%       Thyssen, 1978;
                                                         Thyssen & Eben,

        100          approx. 0.04         70 - 80%       Wilson et al.,

         From these studies it is clear that the dose which causes
    substantial inhibition of NTE is approximately twice the unprotected
    LD50. This inhibition is, however, below the critical threshold and
    does not cause delayed polyneuropathy. The dose which causes NTE
    inhibition above the threshold and thus causes polyneuropathy
    corresponds to about 25 times the unprotected LD50.

         A 90-day repeated dosing study in hens was negative for delayed
    polyneuropathy at doses up to 2 mg/kg b.w./day.

         On the basis of these results the Meeting concluded that
    isofenphos has the potential to cause delayed polyneuropathy. However,
    the dose which produces this toxic effect in hens exceeds the LD50
    by more than 2 times. The Joint Meeting believes that isofenphos
    residues in food are unlikely to cause delayed polyneuropathy in man.

         The Meeting appreciated the submission of data on NTE inhibition
    for delayed neuropathy studies, in accordance with the recommendations
    of the 1984 Joint Meeting. It should be stressed, however, that the
    mechanism of initiation of delayed neuropathy through inhibition/aging
    of NTE differs fundamentally from that of acetylcholinesterase
    inhibition; it is not due to the accumulation of substrate, as with
    acetylcholine when the scavenger enzyme is inhibited. Therefore,
    unless particular problems in disposal of the chemical arise,
    measurement of NTE should be performed 24 - 48 hours after dosing. At
    that time the inhibition of NTE (> 70 - 80%) correlates with the
    development of delayed neuropathy.



         Previous levels of administration free of effect were:

         Mouse:  1 ppm in the diet, equivalent to 0.15 mg/kg b.w./day
         Rat:    1 ppm in the diet, equivalent to 0.05 mg/kg b.w./day
         Dog:    1 ppm in the diet, equivalent to 0.05 mg/kg b.w./day

         These values were established on the basis of plasma
    cholinesterase levels. Although some studies suggest that higher doses
    could be considered no-effect levels, there is not an adequate data
    base for using an alternative method in this case.


         0 - 0.001 mg/kg b.w.


         Observations in man.


    Blass, W. I50 value of isophenphos. Unpublished report from Bayer AG,
    1982      Institut fur Produktinformation und Qualitatsuberwachung.
              Submitted to WHO by Bayer AG, Bayerwerk, FRG.

    Cherry, C.P., Newman, A.J., & Urwin, C. Pathology report of SRA 12869
    1972      - acute neurotoxicity experiment in hens. Unpublished report
              from Huntingdon Research Centre, Huntingdon, UK. Submitted
              to WHO by Bayer AG, Bayerwerk, FRG.

    Eiben, R. Comments on the three-generation study with SRA 12869.
    1983      Unpublished report from Bayer AG, Institute of Toxicology.
              Submitted to WHO by Bayer AG, Bayerwerk, FRG.

    Flucke, W. & Kaliner, G. SPA 12869 - Study for subchronic
    1985      neurotoxicity. Unpublished report No. 13474 from Bayer AG,
              Institute of Toxicology, submitted to WHO by Bayer AG,
              Bayerwerk, FRG.

    Hixson, E.J. Acute delayed neurotoxicity of technical isofenphos in
    1982      hens. Unpublished report from Mobay Chemical Corporation,
              USA. Submitted to WHO by Bayer AG, Bayerwerk, FRG.

    Kimmerle, G. SRA 12869 acute neurotoxicity studies on hens.
    1972      Unpublished report from Bayer AG, Institute of Toxicology.
              Submitted to WHO by Bayer AG, Bayerwerk, FRG.

    Palmer, A.K., Killick, M.E., & Allen, T.R. Effect of SRA 12869 on
    1977      reproductive function of multiple generations in the rat.
              Unpublished report from Huntingdon Research Centre,
              Huntingdon, UK. Submitted to WHO by Bayer AG,
              Bayerwerk, FRG.

    Schluter, G. Evaluation for embryotoxic and teratogenic effects in
    1981      rats following dermal application. Unpublished report
              No. 9801 from Bayer AG, Institute of Toxicology. Submitted
              to WHO by Bayer AG, Bayerwerk, FRG.

    Thyssen, J. SRA 12869 acute toxicity studies on hens and quail.
    1978      Unpublished report from Bayer AG, Institute of Toxicology.
              Submitted to WHO by Bayer AG, Bayerwerk, FRG.

    Thyssen, J. & Eben, A. Isophenphos (SRA 12869) neurotoxic esterase
    1983      activity in hen. Unpublished report No. 11402 from Bayer AG,
              Institute of Toxicology. Submitted to WHO by Bayer AG,
              Bayerwerk, FRG.

    Wehling, K. In vitro biochemical test with purified isofenphos for
    1983      cholinesterase effect. Unpublished report No. 11418 (P) from
              Bayer Pharmacology Division. Submitted to WHO by Bayer AG,
              Bayerwerk, FRG.

    Wilson, B.W., Hooper, M., Chow, E., Higgins, R.J., & Knaak, J.B.
    1984      Antidotes and neuropathic potential of isofenphos.
              Bulletin of Environmental Contamination and Toxicology,
              33, 386 - 394.

    See Also:
       Toxicological Abbreviations
       Isofenphos (Pesticide residues in food: 1981 evaluations)
       Isofenphos (Pesticide residues in food: 1982 evaluations)
       Isofenphos (Pesticide residues in food: 1984 evaluations)