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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF CERTAIN 
    VETERINARY DRUG RESIDUES IN FOOD



    WHO FOOD ADDITIVES SERIES 45





    Prepared by the
    Fifty-fourth meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)



    World Health Organization, Geneva, 2000

    TRICHLORFON

    First draft prepared by
    Dr G. Roberts
    Chemical Products Assessment Section, Therapeutic Goods
    Administration, Department of Health and Aged Care, Canberra,
    Australia

            Explanation 
            Biological data 
                Biochemical aspects 
                    Absorption, distribution, and excretion 
                    Biotransformation 
                Toxicological studies 
                    Acute toxicity 
                    Short-term studies of toxicity 
                    Long-term studies of toxicity and carcinogenicity 
                    Genotoxicity 
                    Reproductive toxicity 
                        Multigeneration studies 
                        Developmental toxicity 
                    Special studies 
                        Neurotoxicity 
                        Immunotoxicity 
                Observations in humans 
            Comments 
            Evaluation 
            References 


    1.  EXPLANATION

         Trichlorfon, dimethyl(2,2,2-trichloro-1-hydroxyethyl)phosphonate
    or metrifonate, is an organophosphonate insecticide with insecticidal,
    acaricidal, and anthelmintic properties. It is used as an insecticide
    on food crops and forests. It is given orally, topically, or
    parentally for the control of endo- and ectoparasites in and on
    animals of various species. The recommended dose for treatment of
    cattle orally or with aqueous pour-on, wash, or spray solutions is
    50-75 mg/kg bw. Repeated dosing may be necessary. The preparations for
    use on horses are similar, but the recommended oral dose is 35 mg/kg
    bw. One topical application for use on horses contains febantel.
    Humans may be given trichlorfon orally for infestation with
     Schistosoma haematobium, and the drug has been studied for use in
    the treatment of Alzheimer disease.

         Trichlorfon has not previously been evaluated by the Committee.
    It was evaluated on three occasions by the Joint FAO/WHO Meeting on
    Pesticide Residues (FAO/WHO, 1972a, 1976a, 1979a), which established
    an ADI of 0-0.01 mg/kg bw in 1978. An Environmental Health Criteria
    publication on trichlorfon has been issued (WHO, 1992).

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         Few studies were available of the kinetics of trichlorfon in
    laboratory animals; however, the JMPR reported that trichlorfon is
    absorbed, distributed, degraded, and excreted very rapidly in mammals.
    After oral administration, radiolabel was detected in the blood of a
    cow within 0.5 h. While residues were observed in cattle and sheep
    meat within 1 h, the concentrations decreased rapidly thereafter.
    Secretion into the milk of cows was seen 6-8 h after treatment, with
    peak concentrations at 14-18 h. After intraperitoneal administration
    of trichlorfon to rats, 71% of the total dose was eliminated in urine
    within 16 h. After oral administration to cows, 66% of the dose was
    eliminated within 12 h (FAO/WHO, 1972b).

         After [14CH3]trichlorfon was administered by stomach tube to
    pregnant guinea-pigs on days 35 and 52 of gestation, distribution to
    the main organs was rapid, the highest concentrations being present in
    the liver, kidney, and lung. Thirty minutes after dosing, substantial
    uptake of radiolabel into the fetus was found, which was more
    pronounced after administration at 52 days of gestation, the
    concentration in fetal liver equalling that in the placenta at that
    time (Berge & Nafstad, 1986; WHO, 1992).

         Groups of four healthy volunteers were given trichlorfon at a
    single oral dose of 2.5, 5, 7.5, or 15 mg/kg bw in a randomized
    double-blind study. Peak plasma concentrations were achieved within 2
    h, and the rate of absorption was linear over the dose range. The
    plasma half-time was 2-2.5 h in all cases (Aden-Abdi et al., 1990).
    Similar results were obtained by Nordgren et al. (1980, 1981) in
    patients withschistosomiasis .

    2.1.2  Biotransformation

          In vitro 

         In the pH range 5.4-8.0, 0-60% of a dose of trichlorfon was
    decomposed  in vitro within 2 h (Metcalf et al., 1959). Trichlorfon
    underwent dehydrochlorina-tion and rearrangement to form dichlorvos
    under alkaline, neutral, or slightly acidic conditions  in vitro. 
    Conversion ceased at pH 5. Studies on inhibition of house fly
    acetylcholinesterase and chymotrypsin  in vitro suggested that
    trichlorfon had little or no inhibitory action at any pH. It was
    postulated that the insecticidal action of trichlorfon is due to its
    conversion to the active metabolite, dichlorvos (Miyamoto, 1959). The
    rate of decomposition of trichlorfon correlated with inhibition of
    acetylcholinesterase from bovine erythrocytes (Reiner et al., 1975).

         Incubation of trichlorfon in buffer at pH 7.4  in vitro resulted
    in decomposition to dichlorvos. Inclusion of the soluble fraction
    (105 000 ×  g) from chicken or cow liver led to faster degradation
    and the formation of desmethyl trichlorfon and desmethyl dichlorvos,
    but predominantly the latter. Enzymatic pathways were the major routes
    of degradation (Akhtar, 1982). 

         When 32P-trichlorfon at a final concentration of 0.014 mol/L
    was incubated with 20% rat brain homogenate for 5 h at 37°C, 18% of
    the compound was metabolized to three acidic and one non-acidic
    metabolites. Two of the acidic metabolites, monodemethylated
    trichlorfon and monomethyl phosphate, represented 37% and 7% of the
    total metabolites, respectively. The third acidic metabolite was
    thought to be 2,2,2-trichloro-1-hydroxyethyl phosphonic acid and
    represented 16% of the metabolites. The non-acidic metabolite was not
    identified. The main metabolic pathway in nervous tissue appears to be
    hydrolysis of the methyl ester linkages. Trichlorfon irreversibly
    inhibits rat brain acetylcholinesterase activity  in vitro, and the
    presence of acetylcholine offered competitive protection (Hassan et
    al., 1965a).

         [14CH3]Trichlorfon was incubated with human serum for 3 h at
    37.5°C at a concentration of 1 mg/ml. Precipitated protein was
    hydrolysed, and the resulting amino acids were separated on an ion
    exchanger. The metabolites included dichlorvos, dimethyl phosphate,
    and desmethyl trichlorfon. The 14C content of the amino acid
    hydrolysate represented approximately 15% of the total radiolabel.
    These results suggest cleavage of the methoxy moiety and methylation
    of protein (Dedek & Lohs, 1970a).

          In vivo 

         After an intraperitoneal injection of 125 mg/kg bw trichlorfon to
    mice, dichlorvos was detected in the brain about 15 min later at a
    peak concentration that was less than 1% of the peak brain
    concentration of trichlorfon (Nordgren et al., 1978).

         Mice were given an oral dose of 6.2 mg 32P-trichlorfon, and
    water-extractable metabolites were determined in the whole body. The
    percentages of metabolites after 0.5 and 4 h were, respectively,
    desmethyl trichlorfon (4.3, 4.0), desmethyl dichlorvos (21, 9.9),
    dimethyl hydrogen phosphate (35, 48), methyl hydrogen phosphate (14,
    18), phosphoric acid (22, 21), and unknown compounds (3.6, 0) (WHO,
    1992).

         Sixteen hours after an intraperitoneal dose of 5 mg
    32P-trichlorfon to rats, the urinary metabolites found were
    trichlorfon (0.7%), dimethyl phosphate (38%), phosphoric acid and
    methyl phosphate (0.8%),  O-desmethyl trichlorfon (1.4%), and
     O-desmethyl dichlorvos (0.8%). The remainder was unidentified. (Bull
    & Ridgway, 1969).

         When trichlorfon with 14C in the two methyl groups was
    administered intraperitoneally to rats at 200 mg/kg bw per day, 28% of
    the dose was recovered as 14CO2 in expired air within 24 h. In
    urine, 32% was recovered as formate (2-4%), dimethyl phosphate
    (20-24%), and unidentified substances within 24 h (Hassan & Zayed,
    1965).

         After intravenous or intraperitoneal administration of
    [14CH3]trichlorfon to male rats, unextractable label was found
    principally in the liver but also in other organs. The data suggest
    that methylation of proteins occurs  in vivo (Dedek & Lohs,1970b).

         [32P]Trichlorfon was administered intraperitoneally to rats at
    100 mg/kg bw per day in saline, and 75-85% of the administered dose
    was recovered in urine collected for 24 h. The three metabolites found
    were acidic: monomethyl phosphate accounted for 20-30%, dimethyl
    phosphate for 60-70%, and an unidentified metabolite for 10%.
    Monodemethylated trichlorfon is likely to be formed as an
    intermediate, before loss of trichloroethanol as the glucuronide and
    subsequent formation of monomethyl phosphate (Hassan et al., 1965b).

         [32P]Trichlorfon was injected intravenously to dogs at a dose
    of 150 mg/kg bw, and urine was collected for 48 h. Less than 1% of the
    dose was recovered as unchanged compound, with approximately 65% as
    the glucuronic acid conjugate of trichloroethanol (Arthur & Casida,
    1957).

         Patients with schistosomiasis received oral doses of 7.5-10 mg/kg
    bw trichlorfon on two occasions 14 days apart. Their plasma
    concentrations of trichlorfon peaked between 1 to 2 h, and the
    concentrations of dichlorvos represented about 1% of parent drug
    (Nordgren et al., 1980, 1981).

         The main metabolites of trichlorfon in mammals were desmethyl
    trichlorfon, desmethyl dichlorvos, dimethyl hydrogen phosphate, methyl
    dihydrogen phosphate, and phosphoric acid. Thus, the main degradation
    routes of trichlorfon are demethylation, P-C bond cleavage, and ester
    hydrolysis via dichlorvos. The proposed metabolic pathways for
    trichlorfon, as identified by WHO (1992), are reproduced in Figure 1.

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         The results of studies of the acute toxicity of trichlorofon are
    shown in Table 1. The acute toxicity of trichlorfon in rats and mice
    was similar when it was administered orally, intraperitoneally, or
    subcutaneously. It was less toxic to rats and rabbits after dermal
    application than when given by the other routes. The acute toxicity of
    trichlorfon is due to inhibition of acetylcholinesterase activity at
    nerve endings, leading to accumulation of endogenous acetylcholine.
    The signs of toxicity are sweating, salivation, diarrhoea,
    bronchorrhoea, bradycardia, bronchoconstriction, muscle

    FIGURE 1

        Table 1. Results of studies of the acute toxicity of trichlorfon

                                                                                        
    Species      Sex      Route              LD50             References
                                             (mg/kg bw)
                                                                                        

    Mouse        M&F      Oral               616-950          Klimmer (1955); Mihail 
                                                              (1978); FAO/WHO (1972b); 
                                                              Hirakawa (1983a); Renhof 
                                                              (1997)
    Mouse        M&F      Intraperitoneal    344-600          Dubois & Cotter (1955); 
                                                              Hirakawa (1983a); WHO 
                                                              (1992)
    Mouse        M&F      Subcutaneous       267360           FAO/WHO (1972b); Hirakawa 
                                                              (1983a)
    Mouse        M&F      Intravenous        354-370          Renhof (1997)
    Mouse        M&F      Dermal             > 5000           Hirakawa (1983a)
    Rat          M&F      Oral               136-660          Dubois & Cotter (1955); 
                                                              Klimmer (1955); Kimmerle 
                                                              (1970); FAO/WHO (1972b); 
                                                              Crawford & Anderson (1973); 
                                                              Mihail (1978); Hirakawa 
                                                              (1983b); Heimann (1985); 
                                                              WHO (1992); Renhof (1997)
    Rat          M&F      Intraperitoneal    156-485          Dubois & Cotter (1955); 
                                                              Arthur & Casida (1957); 
                                                              FAO/WHO (1972b); Crawford &
                                                              Anderson (1973); Hirakawa 
                                                              (1983b)
    Rat          M&F      Subcutaneous       290-400          FAO/WHO (1972b); 
                                                              Hirakawa (1983b)
    Rat          M        Intravenous        184              Kimmerle (1970)
    Rat          M&F      Inhalation         LC50, > 533      Kimmerle (1975a)
                                             mg/m3
    Rat          M&F      Dermal             2800-> 5000      Hirakawa (1983b); Mihail 
                                                              (1978); WHO (1992)
    Guinea-pig   M&F      Intraperitoneal    300              Dubois & Cotter (1955); 
                                                              WHO (1992)
    Rabbit                Oral               160              FAO/WHO (1972b)
    Rabbit       M        Dermal             5000             WHO (1992)
    Dog          M        oral               > 300-420        Mihail (1978); WHO (1992); 
                                                              Kawauchi (1997)
                                                                                        
    
    fasciculations, and coma. The primary cause of death is respiratory
    failure. After sublethal doses, animals recover very rapidly,
    suggesting that trichlorfon is detoxified faster than many other
    organophosphorus compounds (WHO, 1992).

         In two separate studies, six New Zealand white rabbits were
    treated dermally with trichlorfon for 24 h on intact and abraded skin.
    No irritation was seen after 24, 48, or 72 h. Further animals received
    trichlorfon in the eye for 5 min ( n = 5) or 24 h ( n = 3). Severe
    redness was seen up to 72 h after dosing for either length of time.
    Slight to moderate chemosis was also observed up to 48-72 h after
    dosing in both groups. The effects did not persist to day 7, and no
    effect was seen in the cornea or iris. The compound is a slight ocular
    irritant (Thyssen, 1981; Bond, 1986).

         Trichlorfon was assessed for skin sensitizing potential in eight
    male BOR: DHPW guinea-pigs by the open epicutaneous test. Dermal
    induction was carried out 5 days a week for 4 weeks with 0, 1, 3, or
    10% trichlorfon, and dermal challenge was conducted with 0.3, 1, 3, or
    10 % trichlorfon 4, 6, or 8 weeks after induction. No skin reactions
    were seen in animals induced with 1% trichlorfon, but approximately
    half the animals induced with 3 or 10% trichlorfon showed skin
    reactions when challenged with each concentration of trichlorfon. The
    compound was therefore considered to have skin sensitizing potential
    (Mihail, 1986).

    2.2.2 Short-term studies of toxicity and carcinogenicity

          Mice 

         Trichlorfon (purity, 98.2%) was administered to groups of 15
    Charles River CD-1 mice of each sex at a dietary concentration of 0,
    100, 300, 900, or 2700 mg/kg for 8 weeks. There were no deaths or
    drug-related clinical signs, and body weights were not affected. Food
    consumption was not affected in males, but appeared to have been
    increased by 18-32% in females. The report suggests that the latter
    finding was due to increased wastage by the treated female mice.
    Cholinesterase activity in plasma and erythrocytes, measured during
    weeks 4, 6, and 8, was not significantly affected at the lowest dose
    but was dose-dependently inhibited at higher doses; the maximum
    inhibition was by about 70% for plasma and 50% for erythrocyte enzyme.
    Brain acetylcholines-terase activity at the end of the study was
    dose-dependently inhibited at doses > 300 mg/kg of diet, by up to
    68%. Gross necropsy revealed no remarkable changes. Haematology, serum
    biochemistry, urinary anlysis, organ weighing, and histopathology were
    not performed (Hayes, 1985).

         Groups of 10 male and 10 female B6C3F1 mice were given
    trichlorfon (purity unknown) in the diet at a concentration of 0, 62,
    180, 560, 1700, or 5000 mg/kg for 13 weeks. The results were reported
    in abstract form only. All but one male at 5000 mg/kg of diet and one
    female at 1666 mg/kg of diet survived. The body weights of mice at
    5000 mg/kg of diet were reduced. Plasma and erythrocyte cholinesterase

    activities were decreased in a dose-related manner, while motor
    activity and grip strength were reduced at 1700 and 5000 mg/kg of
    diet. The absolute and relative weights of the liver, kidney, and
    spleen were increased in mice at 1700 and 5000 mg/kg of diet. The
    neurotoxic effects and changes in organ weight were not accompanied by
    pathological effects (Chan & Peters, 1989; WHO, 1992).

          Rats 

         Groups of 10 male and 10 female rats were exposed for 6 h/day for
    3 weeks (total, 15 exposures) to an atmosphere containing trichlorfon
    (purity unknown) at a concentration of 0, 12.7, 35.4, or 103.5
    mg/m3. The health of the animals at 103.5 mg/m3 was affected, but
    details were not provided. Body-weight gain was unaffected, and
    haematological, clinical chemical, and urinary parameters measured at
    the end of the study were similar to those of controls. Plasma,
    erythrocyte, and brain cholinesterase activities were inhibited in
    males given 103.5 mg/m3 and in females given 35.4 and 103.5 mg/m3.
    Gross and histopathological examinations showed no remarkable changes,
    the only finding at autopsy being increased spleen weights in males at
    35.4 and 103.5 mg/m3 (Kimmerle, 1975b; WHO,1992).

         Groups of 12 male and 12 female Crj: CD SD rats were given
    trichlorfon (purity, 100%) at a dose of 0, 25, 50, or 100 mg/kg bw per
    day by gavage for 30 days. Further groups of 10 rats of each sex were
    treated and then allowed a 2-week recovery period. Additional treated
    groups of 20 rats of each sex were used to determine the profiles of
    the drug in blood after dosing on days 1, 15, and 30. The peak
    concentrations of the individual enantiomers of trichlorfon and
    dichlorvos were dose-related and were reached by about 1 h, declining
    rapidly to become undetectable by 24 h. The concentrations of each
    enantiomer were roughly similar in males, while that of the
    (-)-enantiomer was generally higher than that of the (+)enantiomer in
    females; this was reflected in higher total drug concentrations in
    females. The concentrations of dichlorvos in blood represented only a
    small fraction of those of trichlorfon and were again higher in female
    rats. There was no evidence of accumulation in blood.

         The signs of toxicity included tremor, decreased locomotor
    activity, salivation, bradypnoea, and prone position in many females
    given 100 mg/kg bw per day, in one or two females given 50 mg/kg bw
    per day, and in males given 100 mg/kg bw per day. There were no deaths
    and no effects on body weight, food consumption, or ophthalmological
    or urinary parameters. Slight decreases in erythrocyte counts and
    haematocrit were seen in females at 100 mg/kg bw per day when compared
    with controls at the end of treatment, but these values were not
    significantly different from those of controls at the end of the
    recovery period. Animals at 100 mg/kg bw per day had increased blood
    concentrations of cholesterol, triglycerides, and phospholipids
    (females), and increased activity of lactic dehydrogenase (males). At
    autopsy, the livers of animals at 50 and 100 mg/kg bw per day were
    heavier and/or enlarged and showed hypertrophy of centrilobular

    hepatocytes. The weight of the kidney was increased in males at 100
    mg/kg bw per day. The NOEL was 25 mg/kg bw per day (Toyoshi, 1996).

         Groups of seven Wistar rats of each sex were given trichlorfon
    (purity unknown) in the diet to provide a dose of 0, 1, 5, 10, 30, 50,
    or 100 mg/kg bw per day for 12 weeks. The highest dose inhibited
    cholinesterase activity in all tissues examined (erythrocytes, serum,
    brain, heart, liver, and skeletal muscle), but the extent of the
    inhibition differed. Nevertheless, no changes were detected in
    nocturnal behaviour, reaction to external stimuli, conjunctival or
    pinna reflexes, or the avoidance reflex to painful stimuli. No
    significant histological alterations were observed (reviewed in WHO,
    1992).

         Groups of 10 male and 10 female Fischer rats were given
    trichlorfon (purity unknown) in the diet at a concentration of 0, 62,
    180, 560, 1700, or 5000 mg/kg for 13 weeks. The results were reported
    in abstract form only. All rats survived. The body weights of those at
    5000 mg/kg of diet were reduced. Plasma and erythrocyte cholinesterase
    activities were lowered in a dose-related manner, while motor activity
    and grip strength were reduced at 1700 and 5000 mg/kg of diet. The
    absolute and relative weights of the liver, kidney, and spleen were
    increased in animals at 1700 and 5000 mg/kg of diet. The neurotoxic
    effects and changes in organ weights were not accompanied by
    pathological effects (Chan & Peters, 1989; WHO, 1992).

         Groups of 13 rats of each sex were fed diets containing
    trichlorfon (purity unknown) at a concentration of 0, 20, 100, or 300
    mg/kg for 16 weeks. No effects were observed on growth, behaviour,
    food consumption, organ weights, or gross or microscopic appearance of
    the tissues. At 300 mg/kg of diet, plasma and erythrocyte
    cholinesterase activity was depressed by 20-30% in males and females,
    while that of brain cholinesterase was depressed by 20% in females
    only. Salivary gland cholinesterase activity was not inhibited. At 100
    mg/kg of diet, a slight depression of plasma cholinesterase activity
    was observed (Doull & Rehfuss, 1956; WHO, 1992).

         Groups of 10 male Wistar albino rats were fed diets containing
    trichlorfon (purity unknown) at 0, 1, 5, 25, or 125 mg/kg for 16
    weeks. Erythrocyte cholinesterase activity was not depressed at any
    dose, and no effects were seen on food consumption, growth, or gross
    appearance of the tissues (reviewed in WHO, 1992).

          Guinea-pigs 

         After oral administration of trichlorfon (purity unknown) at 100
    mg/kg bw per day for 60 days to guinea-pigs, the haemoglobin
    concentration but not the haematocrit was decreased. The activity of
    serum cholinesterase was decreased by 50%, and that of alkaline
    phosphatase was increased (reviewed in WHO, 1992).

          Rabbits 

         Groups of five New Zealand white rabbits of each sex received
    dermal applications of 0, 100, 300, or 1000 mg/kg bw per day
    trichlorfon (purity, 99%) onto shaved skin. The drug was left on the
    skin of restrained animals for 6 h/day, 5 days/week for 3 weeks. The
    appearance and behaviour of the rabbits were unchanged, and there were
    no deaths. Body weight, food consumption, and the skin at the
    application sites were unaffected. There were no meaningful changes in
    haematological, clinical chemical, or urinary parameters at the end of
    the study. Plasma, erythrocyte, and brain cholinesterase activities
    were inhibited by 20-30% in animals at 300 and 1000 mg/kg bw per day.
    At autopsy, no treatment-related alterations in liver microsomal
    enzyme activities, organ weights, or histological appearance were seen
    (Heimann & Wood, 1987).

          Dogs 

         Four dogs were fed diets containing trichlorfon (purity unknown)
    for 12 weeks at a concentration of 20, 100, 300, or 500 mg/kg and were
    then left for a further 4 weeks on unmedicated diet. All of the dogs
    remained healthy and maintained their body weight throughout the
    study. After 12 weeks of treatment, the erythrocyte and plasma
    cholinesterase activities of animals at 300 and 500 mg/kg of diet were
    decreased by 40-60% when compared with the concentrations before
    treatment, and the activity in plasma was inhibited by 10% in animals
    at 100 mg/kg of diet. On cessation of treatment, the enzyme levels
    recovered within 2-4 weeks (Doull & Vaughn, 1958; FAO/WHO, 1972b).

         Four dogs were fed diets containing trichlorfon (purity unknown)
    at a concentration of 0, 50, 200, or 500 mg/kg for 12 weeks. Plasma
    and erythrocyte cholinesterase activities were depressed within 2
    weeks in animals at 500 mg/kg of diet, and the effect peaked at 6-8
    weeks, at which time the inhibition was about 60% in plasma and 45% in
    erythrocytes. A gradual increase in plasma cholinesterase activity was
    then seen, to 45% inhibition at termination, even though the compound
    was still present in the feed. At 200 mg/kg of diet, the
    cholinesterase activity was slightly decreased but was within the
    control range. The enzyme activities were restored 6 weeks after
    treatment ceased (Williams et al., 1959; FAO/WHO, 1972b).

         Groups of three male and three female beagles were given
    trichlorfon (purity, 99.2%) in capsules at a dose of 0, 5, 15, or 45
    mg/kg bw per day for 3 months. A further three animals of each sex
    were given the vehicle or the high dose for 3 months and then allowed
    to recover for 2 weeks. The blood concentrations of the enantiomers of
    trichlorfon and dichlorvos were measured after dosing on days 1, 30,
    and 90. Peak concentrations were achieved within 1 h, the
    (-)-enantiomer being detected at 10-100-fold higher concentrations
    than the (+)-enantiomer and dichlorvos. Disappearance from blood was
    rapid, and the compounds were virtually undetectable by 7 h. The drug
    did not accumulate in blood.

         No deaths were observed in any group, and no meaningful effects
    were seen on mean body weight, food intake, or in ophthalmological,
    electrocardiographic, or auditory parameters. Dogs receiving 15 mg/kg
    bw per day or more showed tremor, decreased locomotor activity, and
    crouching, while at 45 mg/kg bw per day, ataxic gait, lateral
    position, vomiting, salivation, licking, liquid, loose, or bloody
    stools, and wasting were observed. The results of blood chemistry and
    urinary analysis were unremarkable. Haemoglobin, haematocrit, and
    erythrocyte count were slightly but progressively decreased during
    treatment in males given 45 mg/kg bw per day. It was reported, with no
    supporting data, that erythrocyte cholinesterase activity was
    inhibited at 15 and 45 mg/kg bw per day. One dog at 45 mg/kg bw per
    day lost weight, and at autopsy was found to have decreased thymus
    weight with lymphocyte depletion in the cortex, small and immature
    testes and prostate, and oligospermia in small epididymides. Similar
    thymic changes were observed in one female at 45 mg/kg bw per day. All
    of the effects were reversible on cessation of treatment. The NOEL was
    5 mg/kg bw per day (Suzuki, 1996).

          Monkeys 

         Groups of five rhesus monkeys of each sex were given trichlorfon
    (purity, 98.7%) at a dose of 0, 0.1, or 0.2 mg/kg bw per day in
    sterile water by oral intubation for 26 weeks in order to establish an
    NOEL for inhibition of erythrocyte cholinesterase activity. There were
    no deaths and no effects on appearance, behaviour, nutritional state,
    feed or water consumption, or body-weight gain. Haematological tests
    showed no changes. No compound-related effects on the liver or kidney
    were seen in clinical chemical or urinary analyses, on gross
    pathology, or in organ weights. At 0.2 mg/kg bw per day, small
    increases and decreases in erythrocyte cholinesterase activity
    (< 20%) were seen, which were within the range of biological
    variation. The NOEL was 0.2 mg/kg bw per day, the highest dose tested
    (Hoffman et al., 1988; WHO, 1992).

    2.2.3  Long-term studies of toxicity and carcinogenicity

          Mice 

         Three studies of carcinogenicity were performed with groups of 30
    AB/Jena mice that received an oral dose of 0 or 30 mg/kg bw twice
    weekly, an intraperitoneal dose of 0 or 28 mg/kg bw per day, or a
    dermal application of 0 or 0.25 ml of a 1% solution, providing 2.5 mg
    trichlorfon per animal. Dosing was continued for 75 weeks, and the
    studies were terminated after 80 weeks. The treated animals showed
    reduced body-weight gain and survival, but the tumour incidences were
    not increased (Teichmann & Hauschild, 1978; FAO/WHO, 1979b).

         Groups of 60 male and 60 female Charles River CD-1 mice were
    given  trichlorfon (purity unknown) in the diet at a concentration of
    0, 100, 300, or 1000 mg/kg for 90 weeks, except for males at the
    highest dose which were treated for 82 weeks. These doses were
    equivalent to 15, 45, and 150 mg/kg bw per day. Inhibition of

    body-weight gain was observed in females given 300 or 1000 mg/kg of
    diet, and cholinesterase activity was depressed in animals of each sex
    at 1000 mg/kg of diet. No significant microscopic alterations were
    reported, and the tumour incidences were not treatment-related
    (reviewed in WHO, 1992).

         Groups of 50 Crl:CD-1(ICR)BR mice of each sex were given
    trichlorfon (purity, 98.2%) in the diet at a concentration of 0, 280,
    890, or 2700 mg/kg for 104 weeks, equal to 49, 160, and 510 mg/kg bw
    per day for males and 66, 240, and 750 mg/kg bw per day for females.
    Increased incidences of urine-stained fur, 'ear lesions' (males), and
    vaginal discharge (females) were observed. The body weights of treated
    females were increased at various times during the study, but a
    consistent effect was observed only at the high dose. There were no
    effects on survival, food intake, or haematological end-points. Plasma
    and erythrocyte cholinesterase activities were depressed by 20-74% in
    the groups given 890 and 2700 mg/kg of diet and in females given
    280 mg/kg of diet. Brain cholinesterase activity was inhibited by
    20-70% at all doses in a dose-related manner. The weights of the liver
    were increased in females at the intermediate and high doses, but this
    effect was not accompanied by microscopic changes. Pathological
    examination revealed no treatment-related alterations (Hayes, 1988;
    WHO, 1992).

          Rats 

         von Gibel and co-workers undertook a series of studies in Wistar
    rats (von Gibel et al.,1971, 1973; Stieglitz et al., 1974), which were
    given trichlorfon three times per week orally at 30 mg/kg bw, twice
    per week by subcutaneous injection at 30 mg/kg bw, twice per week
    orally at 15 mg/kg bw, or twice per week by intraperitoneal injection
    of 15 mg/kg bw. Treatment was continued for the lifetime of the
    animals. The groups given 30 mg/kg bw were reported to have higher
    incidences of liver necrosis, liver cirrhosis, and forestomach
    papillomas than controls. The lower doses caused hyperplasia of the
    blood-forming elements of bone marrow, extraosseal metaplasia in the
    liver and spleen, and hepatotoxicity and carcinogenicity. JMPR
    (FAO/WHO, 1972b, 1976b) and the International Agency for Research on
    Cancer (IARC, 1983) concluded that these studies were difficult to
    interpret because of lack of information on the methods and results
    and the amalgamation of tumour types. Also, the findings contradicted
    those of other studies. Therefore, little use could be made of these
    studies .

         Groups of 25 male and 25 female Sprague-Dawley rats were fed
    diets containing trichlorfon (purity unknown) at a concentration of 0,
    50, 250, 500, or 1000 mg/kg for 17 months for males and for 24 months
    for females. These doses were equivalent to 2.5, 12, 25, and 50 mg/kg
    bw per day. Survival was reduced at 1000 mg/kg of diet, and the
    body-weight gain of males at this dose was retarded. Cholinesterase
    activity was depressed in serum, erythrocytes, and submaxillary gland
    but not in the brain of rats at 1000 mg/kg of diet; at 500 mg/kg of
    diet, only serum enzyme activity was inhibited. Female rats fed 500 or

    1000 mg/kg of diet had no primary follicules and had primitive ova,
    while male rats fed 1000 mg/kg of diet showed depression of
    spermatogenesis. Necrotizing arteritis was observed at the two highest
    concentrations. The frequencies of mammary tumours in females, in
    ascending order of dose, were 14, 8, 20, 21, and 25%. The time to
    appearance of these tumours was reduced in a dose-related manner
    (reviewed in FAO/WHO, 1972b).

         Groups of 25 male and 50 female Sprague-Dawley rats were fed
    diets containing trichlorfon (purity unknown) at a concentration of 0,
    100, 200, or 400 mg/kg for 18 months, equivalent to 5, 10, and 20
    mg/kg bw per day. A significant increase in the mortality rate in all
    groups led to premature termination of the study at 70 weeks. Serum
    and erythrocyte cholinesterase activities were inhibited at 400 mg/kg
    of diet. Cystic granular alterations of the ovaries were seen at 200
    and 400 mg/kg of diet, and ovarian atrophy, absence of primary
    follicules, and primitive ova were noted at 400 mg/kg of diet. Benign
    mammary tumours were found in 8% of controls, 11% of females at 200
    mg/kg of diet, and 15% of females at 400 mg/kg of diet, with no change
    in the time of their appearance. These results and those of the study
    described above were considered to indicate that trichlorfon enhances
    certain normal ageing processes, particularly in reproductive tissues
    (reviewed in FAO/WHO, 1972b).

         Groups of 50 FB30 rats of each sex were fed diets containing
    trichlorfon (purity, 95.3%) at a concentration of 50, 250, 500, or
    1000 mg/kg for 2 years, equivalent to 2.5, 12.5, 25, and 50 mg/kg bw
    per day. The control group comprised 100 males and 100 females. No
    changes in survival, general behaviour, appearance, body-weight gain,
    or food consumption were reported. No significant differences in blood
    count or urinary parameters were reported in samples obtained during
    the final 3 weeks of the study. Tests of liver function showed an
    increase in 'serum dehydrogenase' activity in males at 50 mg/kg of
    diet and in females at 50 and 500 mg/kg of diet. Serum protein
    concentration and alanine and aspartate aminotransferase activities
    were unchanged. Acetylcholinesterase activity in 'total blood' was
    decreased by approximately 20% at 1000 mg/kg of diet in week 90. No
    macroscopic evidence of treatment-related changes was reported at
    necropsy. Liver weights were significantly increased in males at 250
    and 1000 mg/kg of diet, and spleen weights were significantly
    increased in females at 50 and 1000 mg/kg of diet. Histological
    examination was performed on five male and five female rats at 1000
    mg/kg of diet and on three male and three female control animals. The
    ovaries of an additional 15 treated and 30 control animals were
    examined, as previous studies had indicated a possible effect. The
    findings, including tumour incidences, were unrelated to treatment
    (Lorke & Loser, 1966; Grundmann & Hobik, 1966; FAO/WHO, 1972b).

         Groups of 30 male and 35 female albino rats received an oral dose
    of 0 or 30 mg/kg bw or an intraperitoneal dose of 0 or 12 mg/kg bw of
    trichlorfon (purity unknown) twice per week. Dosing was continued for
    90 weeks, and the studies were terminated after 118 weeks. The treated

    animals showed reduced survival, but the tumour incidences were not
    increased (Teichmann et al., 1978; FAO/WHO, 1979b).

         Trichlorfon (purity, 98.6-99.1%) was fed in the diet to groups of
    75 Long-Evans rats of each sex at a concentration of 0, 100, 300, or
    1000 mg/kg, equivalent to 5, 15, and 50 mg/kg bw per day. The animals
    were maintained on their respective diets for 90 days and then mated
    within groups. The resulting F1 litters were reared to 21 days
     post partum, at which time 75 offspring of each sex per group were
    selected to continue on the experiment. The F0 and selected F1
    rats were treated for 2 years. There was no effect on fertility,
    reproduction, food intake, body-weight gain, or mortality rate in
    either generation. Plasma cholinesterase activity was depressed by
    30-60% in both generations at 1000 mg/kg of diet at most times during
    the study. Erythrocyte cholinesterase activity was reduced by 30-60%
    in both generations given 1000 mg/kg of diet, at most sampling times
    in females and on some occasions in males. Brain cholinesterase
    activity was significantly lower (40%) in females given 1000 mg/kg of
    diet and was 25% lower in F1 males. No histological changes
    attributable to treatment were found (Rosenblum, 1981; Griffin et al.,
    1982).

         Groups of 50 Fischer 344 rats of each sex were given diets
    containing trichlorfon (purity, 98.8%) for 2 years at a mean measured
    time-weighted average concentration of 0, 92, 270, or 1500 mg/kg. The
    nominal concentrations were 0, 100, 300, and, at the highest
    concentration, 1000 mg/kg of diet for 27 weeks, 1250 mg/kg of diet for
    5 weeks, 1500 mg/kg of diet for 8 weeks, and 1750 mg/kg of diet for 65
    weeks. These doses are equal to 4.5, 13, and 76 mg/kg bw per day for
    males and 5.8, 17, and 94 mg/kg bw per day for females. Satellite
    groups of 20 animals of each sex in the control and high-dose groups
    were killed after 1 year. The only noteworthy clinical signs were
    paleness and hunched backs in males and rough coat in females at 1750
    mg/kg of diet. Ophthalmic parameters were normal, and the mortality
    rate was not affected. Females at 1750 mg/kg of diet showed slightly
    decreased feed consumption and body-weight gain during the first 66
    weeks, while the body weight of males at this dose was lower than that
    of controls during the final 25 weeks of the study.
    Hypercholesterolaemia occurred in males at 300 and 1750 mg/kg of diet
    and in females at 1750 mg/kg of diet. Plasma, erythrocyte, and brain
    cholinesterase activities were depressed by 20-40% at the highest
    dose. Erythrocyte counts, haemoglobin concentration, and haematocrit
    were slightly decreased at 1750 mg/kg of diet, but this anaemic
    response had recovered in females by the end of the study. Urinary
    parameters were not affected. At 1750 mg/kg of diet, the liver weights
    were increased in males and females and the spleen weights were
    increased in males, but with no associated pathological changes. The
    kidney weights and the incidence of granular kidneys were increased at
    1750 mg/kg of diet, corresponding to a slight enhancement in chronic
    (age-related) nephropathy. The pathological changes included
    hyperplasia of the upper small intestine and gastritis of the
    non-glandular stomach in animals at 300 and 1750 mg/kg of diet and an
    increased incidence of chronic lung inflammation in females at 1750

    mg/kg of diet. The incidence of adrenal phaeochromocytomas was
    increased in males at 1750 mg/kg of diet but was within the historical
    control range. The NOEL was 92 mg/kg of diet, equal to 4.5 mg/kg bw
    per day (Hayes, 1989; WHO, 1992).

         Groups of 50 Fischer 344 rats of each sex were given diets
    containing trichlorfon (purity, 98.5%) for 2 years at a nominal
    concentration of 0 or 2500 mg/kg, equal to 130 mg/kg bw per day for
    males and 160 mg/kg bw per day for females. Satellite groups of 20
    animals of each sex per dose were killed after 1 year. This study was
    initiated to ensure that trichlorfon had been tested at the maximum
    tolerated dose. The body weights of treated animals were 10-15% lower
    than those of controls throughout the study. Treated animals had
    reduced food consumption during about the first half of the study, but
    thereafter it was comparable to that of controls. The mortality rate
    and ophthalmoscopic parameters were unaffected. The clinical signs in
    treated animals included urine staining and enlarged abdomen in
    animals of each sex and pale eyes and rough coat in males. Decreased
    erythrocyte count, haemoglobin concentration, and haematocrit were
    noted in treated rats but had recovered in males by the end of 2
    years. Hypercholesterolaemia was seen in treated groups throughout the
    study. The activities of gamma-glutamine transferase, alanine and
    aspartate aminotransferases, and alkaline phosphatase in blood were
    increased in treated males at various times but not at termination.
    Urinary parameters were not affected. Statistically significant,
    40-70% depressions in plasma, erythrocyte, and brain cholinesterase
    activities were observed in animals of each sex at all times studied.

         The liver and kidney weights were increased in treated groups,
    and the lung weights were increased in females after 2 years; after 1
    year, only changes in liver and kidney were reported. Histological
    examination revealed duodenal hyperplasia, gastritis of the
    non-glandular stomach, myodegeneration of the stomach tunica
    muscularis, pulmonary inflammation and hyperplasia, hepatocellular
    cytoplasmic vacuolation, focal hepatocellular hyperplasia associated
    with sinusoidal dilatation and cystic degeneration (males),
    nasolachrymal duct inflammation (females), chronic nephropathy
    (females), and renal tubular hyperplasia. Non-statistically
    significant increases in the incidences of renal tubular adenomas in
    males and mononuclear-cell leukaemia in females were within the
    historical control range. The increased incidences of
    alveolar/bronchiolar adenomas in males and of carcinomas in females
    were greater than the recorded historical control means, but the
    numbers were small and did not attain statistical significance
    (Christenson, 1990).

         An increased incidence of tumours was thus detected in two
    studies in rats conducted in the same laboratory. The increased
    incidence of mammary tumours in females was slight, and the tumours
    were benign. The strain used was Sprague-Dawley, which has a
    relatively high, variable background incidence of mammary tumours
    (Haseman et al., 1986). The lack of confirmation of this finding in

    several studies in other species indicates that the evidence for the
    carcinogenicity of trichlorfon in animals is limited.

          Hamsters 

         A group of 23 male and 25 female Syrian golden hamsters received
    trichlorfon (purity unknown) at a weekly intraperitoneal dose of 0 or
    20 mg/kg bw for 90 weeks, and the study was terminated after 100
    weeks. The treated animals showed reduced survival and body-weight
    gain, but the tumour incidences were not increased (Teichmann &
    Schmidt, 1978; FAO/WHO, 1979).

          Dogs 

         Trichlorfon (purity unknown) was added to the diet of groups of
    one male and one female beagle at a concentration of 50, 250, 500, or
    1000 mg/kg, equivalent to 1.2, 6.2, 12, and 25 mg/kg bw per day, for 1
    year. Few or no data on individual animals were provided. No effect on
    food consumption or weight gain was reported. Serum and erythrocyte
    cholinesterase activities were significantly depressed (by 40-50% of
    control values) at 500 and 1000 mg/kg of diet. Brain
    acetylcholinesterase activity was depressed by 40% in animals at
    1000 mg/kg of diet. The relative spleen weights were increased and
    showed marked congestion and apparent lymphoid tissue atrophy at
    1000 mg/kg of diet and to a lesser extent at other doses. A slight
    increase in the severity of foci of chronic inflammatory cells in the
    liver and a decrease in spermatogenesis were seen at 1000 mg/kg of
    diet. Hyperplastic adrenal cortical nodules recorded as 'mild' were
    seen in all treated groups but not in controls. Owing to the small
    number of animals in each group, however, the significance of this
    finding is questionable (Doull et al., 1962; WHO, 1992).

         Groups of four beagles of each sex were given diets containing
    trichlorfon (purity, 95.3-98.3%) at a concentration of 0, 50, 200,
    800, or 3200 mg/kg, equivalent to 1.2, 5, 20, and 80 mg/kg bw per day
    in a study designed to last 4 years. All males at 3200 mg/kg of diet
    died within the first year, and only one female at this dose survived
    4 years. At this dose, the animals showed muscular twitching, severe
    abdominal cramps, and salivation. The males in particular quickly
    became emaciated. Autopsy of the animals that died during the
    experiment showed brittle livers of a 'yellowish or loam' colour. Two
    females and three males at 800 mg/kg of diet died during the first
    year. The mortality rates at the two lower doses were similar to those
    of controls, and the appearance and behaviour of the animals were
    normal. Animals at higher doses were weakened and sick, and lower body
    weights were recorded for surviving animals at 800 mg/kg of diet
    during the latter stages and for the surviving female at 3200 mg/kg of
    diet throughout the study. The effects on food consumption were
    similar to those on body weight. Haematological parameters for treated
    dogs were within the normal range. Elevated serum activities of
    alanine and aspartate aminotransferases were noted in females at 3200
    mg/kg of diet at 2 years but not at 4 years. No treatment-related
    effects were found on urinary analysis. Cholinesterase activity in

    whole blood was not inhibited at 50 mg/kg of diet; at 200 mg/kg of
    diet, a 30% depression was seen at 1 month but the level rose again at
    4 months. The group at 200 mg/kg of diet also showed inhibited
    activity (15-20%) in plasma and erythrocytes throughout the study. At
    800 and 3200 mg/kg of diet, dose-related depressions of 30-80% in
    cholinesterase activity in whole blood, plasma, and erythrocytes were
    found. The female at 3200 mg/kg of diet which survived to 4 years had
    an enlarged, swollen liver, an enlarged spleen and adrenals, and small
    ovaries. Dogs fed 800 mg/kg of diet had increased spleen and adrenal
    weights and decreased testis weight. There were no histopathological
    changes that were considered to be related to treatment. The NOEL was
    50 mg/kg of diet, equivalent to 1.2 mg/kg bw per day (Loser, 1970;
    Spicer & Payne, 1971; FAO/WHO, 1972b).

          Monkeys 

         Groups of five male and five female rhesus monkeys  (Macaca 
     mulatta) were given aqueous solutions of trichlorfon (purity,
    98.6-99.1%) by gavage at a dose of 0, 0.2, 1, or 5 mg/kg bw per day, 6
    days/week for 10 years. Two animals at 5 mg/kg bw per day showed
    transient pupillary constriction, and one had muscular fasciculations
    during the first month only; diarrhoea and soft stools were noted
    frequently. At this dose, the body weights were lower towards the end
    of the study, and there was a tendency to lower erythrocyte count,
    haemoglobin concentration, and haematocrit at various sampling times.
    No treatment-related effects on the mortality rate or clinical
    chemical or urinary parameters were recorded. Plasma, erythrocyte, and
    brain cholinesterase activities were inhibited by 30-80% in animals at
    1 and 5 mg/kg bw per day, while males at 0.2 mg/kg bw per day showed
    slight cholinesterase inhibition (16%) only in erythrocytes, which was
    considered not to be biologically significant. Liver morphology in
    biopsy samples taken during the first 3 years was unchanged. Gross and
    microscopic examination of tissues showed no changes related to
    treatment (Griffin, 1988; WHO, 1992).

    2.2.4  Genotoxicity

         The results of genotoxicity assays with trichlorfon are
    summarized in Table 2. Other studies, evaluated by IARC (1983) and WHO
    (1992), showed similar findings. The inconsistencies in a number of
    tests has been suggested to be due to the variable purity of the
    materials tested (Dreist & Mihail, 1990).

    2.2.5  Reproductive toxicity

         (a)  Multigeneration studies

         Trichlorfon (purity, 98.3%) was administered to groups of 10 male
    and 20 female FB 30 rats in the diet at a concentration of 0, 100,
    300, 1000, or 3000 mg/kg for three generations with two litters per
    generation. Females in the F0 generation at 1000 and 3000 mg/kg of
    diet had significantly depressed weight gain. The size of F1a
    litters was reduced at doses > 1000 mg/kg of diet, while pup growth

    and survival during lactation were impaired at 3000 mg/kg of diet. The
    F1b mating resulted in lower pregnancy rates at 1000 and 3000 mg/kg
    of diet. Litter size and pup growth and survival were also reduced at
    3000 mg/kg of diet, and all animals in this group died before mating
    to produce the F2 generation. During the remainder of the study, no
    effects were found on fertility or on litter size or weight. The
    F2a, F2b, F3a, and F3b groups given 1000 mg/kg of diet all had
    reduced body-weight gain during the lactation period. No fetal
    malformations were found in any group. Examination of the F0, F1b,
    and F2b adults revealed no gross or microscopic alterations. The
    NOEL was 300 mg/kg of diet, equivalent to 30 mg/kg bw per day (Loser,
    1969; Spicer & Urwin, 1971; WHO, 1992). 

         (b)  Developmental toxicity

          Mice 

         A single dose of 360 mg/kg bw trichlorfon (purity unspecified),
    injected intraperitoneally into 33 AS/Jena mice on day 1 of gestation,
    was embryotoxic. The frequency of post-implantation loss was increased
    with single doses of 60, 120, or 240 mg/kg bw on day 9 (13-15 mice per
    group) and with 120 or 240 mg/kg bw on days 1-7 (25-30 mice per
    group), and was most pronounced with 240 mg/kg bw on days 7-14 (23
    mice). There were no severe malformations (Scheufler, 1975; WHO,
    1992).

         CD-1 mice were given trichlorfon (purity unspecified) by gavage
    at a dose of 0, 300, 400, 500, or 600 mg/kg bw per day on days 6-10
    (3-25 females per group) or 10-14 (4-21 females per group) of
    gestation and were killed on day 18. The food intake and weight gain
    of dams were depressed at all doses but with no dose-response
    relationship. Fetal body weight was lower at doses > 400 mg/kg bw
    per day, while the frequency of fetal malformations (cleft palate) was
    slightly increased only in the groups treated with 500 or 600 mg/kg bw
    per day during days 10-14 of gestation. The highest dose, 600 mg/kg bw
    per day, was also administered by gavage on day 8 or on days 8-10,
    10-12, or 12-14 of gestation (4-28 females per group). An increase in
    the frequency of cleft palate was seen only in the last group. The
    NOEL was 300 mg/kg bw per day (Staples & Goulding, 1979; WHO, 1992).

         Trichlorfon (purity, > 98%) was given by gavage to CD-1 mice at
    a dose of 0, 200, 300, or 400 mg/kg bw per day on days 7-16 of
    gestation (15-24 females per group), and the animals were killed 1 day
    after the last dose. The two higher doses increased the mortality
    rate, and all doses decreased the weight gain of dams. The number of
    live fetuses and fetal weight were decreased at 400 mg/kg bw per day.
    Delayed development of fetuses was found, as indicated by reduced
    calcification in a number of skeletal areas, rib variations, and
    increased incidences of enlarged renal pelves in treated groups
    (Courtney et al., 1986).


        Table 2. Summary of results of tests for genotoxicity with trichlorfon

                                                                                                  
    End-point          Test object          Concentration           Result     Reference
                                            or dose (maximum)
                                                                                                  

     In vitro 
    Interaction        Determination of     160 mg/kg bw i.p.       Positive   Dedek et al. 
    with DNA           7-methylguanine                                         (1976)
                       in mouse urine

                       Determination of     120 mg/kg bw i.p.       Positive   Dedek (1981)
                       7-methylguanine 
                       in mouse liver and 
                       kidney

    Gene mutation      B. subtilis NIG17,   0.3 mg per disc -S9     Negative   Inukai & Iyatomi 
    (rec)              NIG45                                                   (1977)

                       P. mirabilis         10 mg per spot -S9      Positive   Adler et al. (1976)
                       PG273, PG713

                       B. subtilis H17,     NR -S9                  Negative   Shirasu et al. 
                       M45                                                     (1976)

                       B. subtilis H17,     2 mg per disc -S9       Positive   Shirasu et al. 
                       M45                                                     (1979)

                       S. typhimurium       10 mg/disc ?S9          Positive   Jones et al. (1984)

    Reverse            S. typhimurium       5 mg per plate ±S9      Positive   Byeon et al. (1976)
    mutation           TA100
                       TA98, TA1535,        5 mg per plate ±S9      Negative
                       TA1538

                       TA98, TA100,         0.5 mg per plate ±S9    Negative   Inukai & Iyatomi 
                       TA1535, TA1537                                          (1977)
                                                                                                  

    Table 2. (continued)

                                                                                                  
    End-point          Test object          Concentration           Result     Reference
                                            or dose (maximum)
                                                                                                  

                       TA98, TA100          ~ 8.5 mg per plate      Positive   Batzinger & 
                                            ±S9                                Bueding (1977)

                       TA100, TA1535        10 mg per plate ±S9     Negative   Zeiger et al. (1987)

                       TA98, TA100          2 mg per plate ±S9      Negative   Diril et al. (1990)

                       TA1535, TA1536,      NR -S9                  Negative   Shirasu et al. 
                       TA1537, TA1538,                                         (1976)
                       E. coli WP2, WP2hcr

                       TA1535, TA1536,      2 mg per disc -S9       Negative   Carere et al. 
                       TA1537, TA1538                                          (1978a,b)

                       TA100, E. coli       20 mg per plate ±S9     Positive   Shirasu et al. 
                       WP2hcr                                                  (1979); Moriya et 
                       TA98, TA1535,        20 mg per plate ±S9     Negative   al. (1983)
                       TA1537, TA1538

                       TA100, TA104         25 mg per plate ±S9     Positive   Barrueco et al. 
                       TA97, TA98,          25 mg per plate ±S9     Negative   (1991)
                       TA1535

                       TA98, TA1537         5 mg per plate±S9       Negative   Watabe (1997)
                       TA100, TA1535,       5 mg per plate±S9       Positive
                       E, coli  WP2uvrA

                       S. cerevisiae        NR -S9                  Negative   Guerzoni et al. 
                       632/4, 632/1b,                                          (1976)
                       814/18b

                                                                                                  

    Table 2. (continued)

                                                                                                  
    End-point          Test object          Concentration           Result     Reference
                                            or dose (maximum)
                                                                                                  

    Reverse            S. cerevisiae        10 mg per ml ±S9        Negative   Hoorn (1983)
    mutation           S138, S211a

                       S. cerevisiae D7     40 mg per ml ±S9        Positive   Jones et al. 
                                                                               (1984)

    Mitotic crossing   S. cerevisiae D7     40 mg per ml ±S9        Positive   Jones et al. 
    over, gene                                                                 (1984)
    conversion

    Forward            S. coelicolor        2 mg per disc -S9       Positive   Carere et al. 
    mutation                                                                   (1978a,b)

                       S pombe SP-198       30 mg per ml ±S9        Positive   Gilot-Delhalle et 
                                                                               al. (1983)

                       V79 cells            200 mg per ml -S9       Negative   Aquilina et al. (1984)

                       L5178Y cells         200 µg per ml -S9       Positive   Witterland 
                                            600 µg per ml +S9       Positive   (1984); Jones et 
                                                                               al. (1984)

    DNA damage         E coli pol+/-        10 mg per plate ±S9     Negative   Herbold (1984)

                       E. coli (SOS         NR ±S9                  Negative   Xu & Schurr 
                                            chromotest)                        (1990)

    Unscheduled        EUE cells            1000 mg per ml -S9      Positive   Aquilina et al. 
    DNA synthesis                                                              (1984)
                       Primary rat          50 µg per ml -S9        Negative   Myhr (1983)
                       hepatocytes

                                                                                                  

    Table 2. (continued)

                                                                                                  
    End-point          Test object          Concentration           Result     Reference
                                            or dose (maximum)
                                                                                                  

    Sister chromatid   V79 cells            80 µg per ml -S9        Positive   Chen et al. 
    exchange                                60 µg per ml +S9        Positive   (1981, 1982)

                       CHO cells            100 µg per ml -S9       Positive   Jones et al. 
                                            2 mg per ml +S9         Positive   (1984); Putman 
                                                                               (1987)

    Chromosomal        Don-6 cells          250 mg per ml -S9       Positive   Sasaki et al. 
    damage                                                                     (1980)
                       Human lymphocytes    30 mg per ml -S9        Positive   Herbold (1986)
                                            3000 mg per ml +S9      Positive

     In vivo 
    Reverse            Host-mediated                                           Batzinger & 
    mutation           assay in mice                                           Bueding (1977)
                       S. typhimurium       200 mg/kg bw orally     Negative
                       TA98
                       S. typhimurium       200 mg/kg bw orally     Positive
                       TA100

    Recessive lethal   Drosophila           4.5 mg/kg bw            Negative   Benes & Sram 
    mutation           melanogaster                                            (1969); Brzheskiy 
                                                                               (1973); Lamb 
                                                                               (1977)

    Sister chromatid   Chinese hamster      300 mg/kg bw orally     Negative   Volkner (1987)
    exchange           bone marrow

                                                                                                  

    Table 2. (continued)

                                                                                                  
    End-point          Test object          Concentration           Result     Reference
                                            or dose (maximum)
                                                                                                  

    Chromosomal        Micronucleus         2 x 312 mg/kg bw i.p.   Negative   Paik & Lee 
    damage             formation in mouse   2 x 250 mg/kg bw        Negative   (1977); Herbold 
                       bone marrow          orally                             (1979a); Jones 
                                            2 x 400 mg/kg bw        Negative   et al. (1984); 
                                            orally                             Herbold (1997)
                                            400 mg/kg bw orally     Negative
                                            400 mg/kg bw orally     Negative
                                            of (+)-enantiomer
                                            600 mg/kg bw orally     Positive
                                            of (-)-enantiomer

                       Metaphase analysis   400 mg/kg bw orally     Positive   Kurinnyi (1975); 
                       in mouse             10 mg/kg bw i.p.        Positive   Moutschen-Dahmen
                       bone marrow          100 mg/kg bw i.p.       Negative   et al. 
                                            0.5 mg/ml in drinking,  Negative   (1981); 
                                            water 5 days/week,                 Degraeve et al. 
                                            7 weeks                            (1982, 1984); 
                                            405 mg/kg bw i.p.       Negative   Nehes et al. 
                                                                               (1982)

                       Metaphase analysis   250 mg/kg bw i.p.       Negative   Dzwonkowska & 
                       in hamster                                              Hubner (1986)
                       bone marrow

                       Metaphase analysis   250 mg/kg bw orally     Negative   Bootman & 
                       in  rat bone                                            Hodson-Walker 
                       marrow                                                  (1987)

                                                                                                  

    Table 2. (continued)

                                                                                                  
    End-point          Test object          Concentration           Result     Reference
                                            or dose (maximum)
                                                                                                  

                       Metaphase analysis   1.5 mg/ml in drinking,  Positive   Bulsiewicz et al. 
                       in mouse             water 50-100 days                  (1976); 
                       spermatogonia        100 mg/kg bw i.p.       Negative   Moutschen-Dahmen
                       and spermatocytes    0.5 mg/ml in drinking,  Negative   et al. 
                                            water 5 days/week,                 (1981);  
                                            7 weeks                            Degraeve et al. 
                                            100 mg/kg bw i.p.       Negative   (1982, 1984); 
                                                                               Herbold (1992)

                       Dominant lethal      100 mg/kg bw i.p.       Negative   Epstein et al. 
                       mutation in mice     0.5 mg/ml in                       (1972); Dedek 
                                            drinking-water,         Negative   et al. (1975) ;
                                            5 days/week,                       Fischer et al.
                                            7 weeks                            (1977); Herbold
                                            280 mg/kg bw i.p.       Negative   (1979b,c); 
                                            405 mg/kg bw i.p.       Negative   Becker &
                                            405 mg/kg bw i.p.       Positive   Schoneich 
                                            250 mg/kg bw orally     Negative   (1980); 
                                            NR                      Negative   Moutschen-Dahmen
                                            405 mg/kg bw i.p.       Positive   et al. (1981); 
                                            54 mg/kg bw per day,    Positive   Degraeve et al.
                                            3 weeks i.p.                       (1982, 1984);
                                                                               WHO (1992)
                                                                                                  

    NR, not reported;  S9, metabolic activation; i.p., intraperitoneally
    

          Rats 

         Trichlorfon (purity unspecified) was administered by gavage to
    groups of 11 female Wistar rats at a single dose of 80 mg/kg bw on day
    9 or 13 of gestation or to 10 rats at a dose of 8 mg/kg bw per day
    daily throughout gestation. The animals were killed on day 19 of
    gestation. The frequencies of post-implantation deaths and fetal
    malformations such as exencephaly and non-closing eyelids were
    increased at the single dose on day 13. The other treatments had no
    effect on development (Martson & Voronina, 1976; WHO, 1992).

         Trichlorfon (purity, 98.5%) was administered in the diet to
    groups of 9-26 female CD rats on days 6-15 of gestation, to give an
    intake of 0, 76, 140, 380, 430, or 520 mg/kg bw per day, with necropsy
    on day 21. At the two highest doses, maternal food intake and body
    weight and fetal weights were reduced, while the number of fetal
    deaths was increased at 430 mg/kg bw per day. Major external and
    skeletal malformations were found in fetuses at 430 and 520 mg/kg bw
    per day and relatively minor skeletal changes at 380 mg/kg bw per day.
    The predominant malformations were alterations of the skull and
    central nervous system (exencephaly, meningocoele, hydrocephaly), the
    limbs (syndactyly, markedly shortened radii and ulnae, missing
    digits), micrognathia, cleft palate, facial haematomas, generalized
    oedema, and great vessel defects. The minor skeletal alterations were
    mainly doubled vertebral centra, wavy ribs, fenestrated supraoccipital
    bones. and umbilical hernia. The NOEL was 140 mg/kg bw per day
    (Staples et al., 1976; WHO,1992).

         Trichlorfon (purity, 98.5%) was given by gavage at a dose of 0,
    50, 75, 150, 200, or 250 mg/kg bw per day to groups of 9-30 female CD
    rats on days 6-15 of gestation. Deaths occurred among dams at 150,
    200, and 250 mg/kg bw per day, with overt signs of toxicity at the
    highest dose. The survivors were killed on day 21 of gestation. Fetal
    body weights were reduced significantly at doses > 75 mg/kg bw per
    day, but the incidence of malformations was unaffected. The NOEL was
    50 mg/kg bw per day (Staples et al., 1976).

         Trichlorfon (purity unspecified) was given at a dose of 480 mg/kg
    bw per day to 34 CD rats by gavage on days 6-15 of gestation. Lower
    fetal body weight, a higher fetal mortality rate, and malformations
    such as generalized oedema, herniation of the brain, hydrocephaly,
    micrognathia, cleft palate, and skeletal alterations were seen. A
    single gavage dose of 480 mg/kg bw on day 8 or 10 of gestation to 14
    females per group decreased maternal food consumption but had no
    adverse effect on fetal development (Staples & Goulding, 1979; WHO,
    1992).

         Groups of 25 female Long-Evans rats were given trichlorfon
    (purity, 98.4%) by gavage at a dose of 0, 10, 30, or 100 mg/kg bw per
    day on days 6-16 of gestation and were killed on day 20. There were no
    deaths, but diarrhoea was observed in some animals at 100 mg/kg bw per
    day. Fetal development was not affected. The NOEL for maternal
    toxicity was 30 mg/kg bw per day (Machemer, 1979a; WHO, 1992).

         Trichlorfon (purity, > 98%) was given by gavage to CD rats at a
    dose of 0, 50, 100, or 200 mg/kg bw per day on days 7-19 (10-20
    females per group) or 8-20  of gestation (15-18 females per group).
    The animals were killed 1 day after the last dose. The highest dose
    increased the mortality rate and decreased the weight gain of the
    dams. Delayed development of fetuses was found, as indicated by
    reduced calcification in a number of skeletal areas, but no
    abnormalities were noted (Courtney et al., 1986).

         Groups of 33 female Charles River Crl:CD BR rats were given diets
    containing trichlorfon (purity, 99%) at a concentration of 0, 500,
    1100, or 2500 mg/kg on days 6-15 of gestation. Body-weight gain was
    slightly reduced among animals at the highest dose. Analysis of
    cholinesterase activity in five animals per group killed on day 16 of
    gestation showed inhibition of plasma, erythrocyte, and brain enzymes.
    When the remaining dams were killed on day 20 of gestation, only brain
    acetylcholinesterase activity was significantly inhibited. No effect
    was seen on the numbers of resorptions, live fetuses, fetal body
    weight, or malformation frequency. Increased incidences of delayed
    ossification and curved, wavy, or bulbous ribs were seen at 2500 mg/kg
    of diet. While fetal brain acetylcholinesterase activity was lower in
    all treated groups, there was no dose-response relationship (Kowalski
    et al., 1987; WHO, 1992).

          Hamsters 

         Groups of 5-30 female golden hamsters received trichlorfon
    (purity unspecified) at a dose of 0, 100, 200, 300, or 400 mg/kg bw
    per day by gavage on days 7-11 of gestation, and the survivors were
    killed on day 15. Three of 30 hamsters at the highest dose died, and
    the signs of cholinesterase inhibition were dose-related at doses >
    200 mg/kg bw per day. Maternal food consumption and body-weight gain
    and fetal body weight were decreased at 300 and 400 mg/kg bw per day.
    Fetuses at the highest dose showed an increased frequency of stunting
    malformations, mostly consisting of oedema, cleft palate, patagium,
    and fused ribs. A single dose of 400 mg/kg bw per day by gavage given
    on day 8 of gestation to 16 females reduced maternal food consumption
    and increased the fetal death rate but had no meaningful effect on
    fetal development. The NOEL was 100 mg/kg bw per day (Staples &
    Goulding, 1979; WHO, 1992).

          Guinea-pigs 

         Groups of 10 white female guinea-pigs were given six doses of 100
    mg/kg bw per day trichlorfon (purity, 97%) by gavage on days 36-38 and
    51-53 of gestation. Seven females served as controls, and all animals
    were allowed to deliver naturally. The numbers of abortions and
    stillborn fetuses were increased and fetal body weight was decreased
    in the treated group. The pups of treated dams developed trembling and
    locomotor disturbances. At autopsy on postnatal day 2-3, the total
    weights of the brain, cerebellum, medulla, cerebral cortex,
    hippocampus, thalamus, and colliculi were decreased. The cerebellum
    showed reduction of the external granular and molecular layers, with

    regional absence of Purkinje cells. The activities of the
    neurotransmitter enzymes choline acetyltransferase and glutamate
    decarboxylase in the cerebellum were depressed when compared with
    controls (Berge et al., 1986; WHO, 1992).

          Rabbits 

         Groups of 15 female Himalayan rabbits were given trichlorfon
    (purity, 98.4%) by gavage at a dose of 0, 5, 15, or 45 mg/kg bw per
    day on days 6-18 of gestation and were killed on day 29. Body-weight
    gain was reduced in treated groups, and two abortions occurred at 45
    mg/kg bw per day. Fetal development was not affected (Machemer, 1979b;
    WHO, 1992).

         Groups of 20 female American Dutch rabbits were given trichlorfon
    (purity, 99%) by gavage at a dose of 0, 10, 35, or 110 mg/kg bw per
    day on days 6-18 of gestation and were killed on day 28. The highest
    dose was not well tolerated and resulted in deaths, overt signs of
    toxicity, and reduced body-weight gain. At 35 mg/kg bw per day, there
    were also signs of toxicity, one death, and one abortion. Erythrocyte
    but not plasma cholinesterase activity was inhibited on day 19 of
    gestation in animals at 35 and 110 mg/kg bw per day, while on day 28
    only brain cholinesterase activity was inhibited at these doses. At
    110 mg/kg bw per day, there was a slight increase in the number of
    resorptions, slight decreases in fetal and placental weights, and
    delayed ossification. The frequency of fetal abnormalities was not
    increased. The NOELs were 10 mg/kg bw per day for maternal toxicity
    and 35 mg/kg bw per day for embryo- and fetal toxicity (Clemens et
    al., 1990; WHO, 1992).

          Pigs 

         Several outbreaks of congenital tremor in piglets have been
    described in herds in which the sows had been treated with trichlorfon
    preparations between day 45 and day 63 of gestation. Clinically, the
    syndome was characterized by ataxia, tremor, pronounced hypoplasia of
    the cerebellum, and a reduction in the size of the spinal cord (WHO,
    1992). This syndrome was reproduced experimentaly in sows given diets
    providing one to four doses of 50-100 mg/kg bw per day 55-98 days
    after conception. The offspring had a high incidence of overt
    neurological signs and cerebellar hypoplasia. Histological examination
    revealed patchy loss of Purkinje cells in the cerebellum. Postnatally,
    the rate of increase of brain weight was similar to that of controls,
    but normal weights had not been achieved up to 35 days after birth
    (Knox et al., 1978; Pope et al., 1986; Berge et al., 1987a,b; WHO,
    1992).

    2.2.6  Special studies

         (a)  Neurotoxicity

         The results of these studies are summarized in Table 3. Like
    other organophosphonates, trichlorfon has neurotoxic potential. Rats
    given a dose of about 200 mg/kg bw per day for 13 weeks showed
    decreased locomotor activity and loss of coordination, whereas rats
    given a dose of 30 mg/kg bw per day for 3 weeks showed increased
    locomotion and decreased learning ability and nerve conduction
    velocity. Numerous studies in hens consistently demonstrated the
    neurotoxicity of single doses of trichlorfon. Leg weakness, impaired
    walking ability, and decreased activity were seen immediately after
    treatment. These effects are considered to be secondary to inhibition
    of cholinesterase activity and to be generally unrelated to damage to
    the nervous tissues. Delayed neurotoxicity is usually detected some
    weeks after dosing and is associated with degeneration of nervous
    tissue and marked inhibition of neuropathy target esterase activity.
    Neuropathy target esterase is a protein which is located specifically
    in neurons, but its biological function, other than as a target for
    neuropathic organophosphates, is unknown. The toxicity of an agent can
    be predicted by its relative ability to interact with either
    acetylcholinesterase to produce cholinergic effects or neuropathy
    target esterase to produce delayed polyneuropathy (Ray, 1998). The
    studies gave no evidence of delayed neuropathy in chickens. In a
    monkey given a single oral dose of trichlorfon at 250 mg/kg bw per
    day, nerve conduction was impaired 4 weeks after treatment, and there
    was histological evidence of demyelination of nerves and axonal
    degeneration.

         (b)  Immunotoxicity

         Groups of four female BALB/cByJ and C57BL/6J mice were given
    drinking-water containing trichlorfon at 1.75 mg/ml for 14 days.
    Measurement of a variety of responses to immunization with influenza
    virus indicated that immune function was not impaired (Reiss et al.,
    1987; WHO, 1992).

    2.3  Observations in humans

         Wegner (1970) reviewed data on over 6000 people, obtained mostly
    in South Africa and South America, who had been treated with
    trichlorfon for various intestinal and body parasites  (Ankylostoma, 
     Ascaris, Strongyloides, Trichocephalus, and  Schistosoma), at doses
    varying from 5 to 70 mg/kg bw per day for up to 12 days. A dose of 7.5
    mg/kg bw given two to four times at 2-week intervals was considered to
    be optimal, as the side-effects observed were less severe than at
    other dosages. The symptoms included depression of cholinesterase
    activity, weakness, nausea, diarrhoea, and abdominal pain. A dose of
    24 mg/kg bw caused more severe symptoms, including tachycardia,
    salivation, colic pain, vomiting, nausea, fatigue, tremors, and
    sweating. The effects were not cumulative, and spontaneous recovery
    was rapid in all cases. Spermatogenesis (size and shape of sperm)

    appeared to be affected in a few cases. Inhibition of cholinesterase
    activity was seen at all doses (FAO/WHO, 1972b; WHO, 1992).

         Reduced erythrocyte and plasma cholinesterase activities have
    been reported in groups of patients with various parasitic
    infestations given trichlorfon orally at doses of 5-12.5 mg/kg bw. The
    drug was reported to be well tolerated with few clinical symptoms
    related to treatment. The symptoms that were observed were cholinergic
    in nature and were usually found at doses > 10 mg/kg bw.
    Cholinesterase activity recovered to pretreatment levels within 4-15
    weeks after cessation of treatment (Abdel Aal et al., 1970; Plestina
    et al., 1972; Jewsbury, 1981).

         In a clinical trial, 10 male and 10 female patients with
    Alzheimer disease were given oral doses of trichlorfon for up to 30
    weeks according to the following dose regimen: 2.5 mg/kg bw at week 0,
    5 mg/kg bw in week 1, 7.5 mg/kg bw in weeks 6 and 7, 15 mg/kg bw in
    weeks 8 and 9, and the 'best weekly dose' in weeks 18-30. The 'best
    dose' was defined as that associated with the greatest improvement for
    each patient. This small trial indicated general improvement in a
    variety of functional deficits associated with Alzheimer disease.
    Side-effects were reported at doses > 5 mg/kg bw, which included
    nausea, vomiting, and diarrhoea. Plasma cholinesterase activity was
    inhibited by 45-60% at all doses, and erythrocyte acetylcholinesterase
    activity was inhibited by 30-70% at doses > 5 mg/kg bw with no
    concomitant clinical findings. Measurement of acetylcholinesterase
    activity in the cerebrospinal fluid from two patients 24 h after a
    dose of 5 mg/kg bw revealed inhibition of 37 and 47% below
    pretreatment levels (Becker et al., 1990).

         In a prospective, double-blind, randomized study, four parallel
    groups of 119-121 patients with Alzheimer disease received oral doses
    of placebo or trichlorfon once daily. An initial loading dose of 0,
    0.5, 0.9, or 2.0 mg/kg bw per day was given during weeks 0-2 in order
    to achieve steady-state inhibition of cholinesterase activity more
    quickly, and this was followed during weeks 3-10 by a dose of 0, 0.2,
    0.3, or 0.65 mg/kg bw per day. The intermediate and high doses were
    found to improve cognitive function, but the low dose had equivocal
    effects. Abdominal pain, diarrhoea, flatulence, nausea, and leg cramps
    were the most commonly reported side-effects and were most severe in
    the patients given the high dose. Erythrocyte acetylcholinesterase
    activity was inhibited in a dose-related manner (Table 4). The initial
    dose of 0.5 mg/kg bw per day for 2 weeks inhibited erythrocyte
    acetylcholinesterase activity by 29%, and subsequent administration of
    0.2 mg/kg bw per day for 10 weeks maintained the inhibition at 30-37%,
    with no increase with duration of dosing. Since this dose enhanced
    inhibition over that caused by the initial dose by only 8%, an
    insignificant change, the Committee considered that the NOEL for
    inhibition of acetylcholinesterase activity was 0.2 mg/kg bw per day
    (Cummings et al., 1998).

        Table 3. Summary of studies of neurotoxicity with trichlorfon

                                                                                                
    Species     Treatment               Results                               Reference
                                                                                                

    Rats        200 mg/kg bw per day    Electrophysiological changes          Averbrook &
                i.p. for 5-15 days      indicating enhanced excitability      Anderson (1983)
                                        in sciatic nerve. No pathological
                                        changes in nerves

    Rats        30 mg/kg bw per day     Increased locomotion, decrease        Lehotzky (1982)
                orally for 3 weeks      in rotorod performance, learning 
                                        ability, and nerve conduction
                                        velocity

    Rats        0, 85, 438, or 2275     Rats given 2275 ppm had stained       Sheets &
                ppm in the diet for 13  fur, reduced motor and locomotor      Hamilton (1995)
                weeks                   activity, slightly uncoordinated 
                                        righting response (males only), 
                                        30-80% inhibition of plasma, 
                                        erythrocyte, and brain 
                                        cholinesterase activity, and 
                                        demyelination in spinal nerve roots. 
                                        At 438 ppm, only plasma and 
                                        erythrocyte cholinesterase activity 
                                        was inhibited (20-30%), and no 
                                        behavioural or biochemical 
                                        evidence of neurotoxicity was 
                                        seen at 85 ppm (6 mg/kg bw 
                                        per day)

    Chicken     Single subcutaneous     Leg weakness for 1-2 days, no         Witter & Gaines 
                dose of 90 mg/kg bw     delayed paralysis                     (1963)

    Chicken     Single dose of 25-500   Birds that survived the acute         Lorke & 
                mg/kg bw orally or      toxicity of trichlorfon showed no     Kimmerle (1966)
                75-500 mg/kg bw i.p.    signs of neurotoxicity over 
                                        6-10 weeks

    Chicken     100-5000 ppm in         Body weight loss at > 500 ppm.        Lorke & 
                feed for 30 days        Blood cholinesterase activity         Kimmerle (1966) 
                                        decreased by 40-60% at all            Hobik (1967); 
                                        doses but no signs of                 FAO/WHO 
                                        neurotoxicity and no                  (1972)
                                        degeneration in nervous tissue.
                                                                                                

    Table 3. (continued)

                                                                                                
    Species     Treatment               Results                               Reference
                                                                                                

    Chicken     Single subcutaneous     One hen died 1.5 h after dosing;      Hierons & 
                dose of 200 mg/kg bw    the lone survivor was killed 24 h     Johnson (1978)
                                        after dosing, when NTE was 
                                        inhibited by 46% in brain and 
                                        17% in spinal cord.

    Chicken     Single doses of 50,     Dose-related deaths after oral        Olajos et al. 
                100 mg/kg bw orally,    doses only. All treatments had        (1979)
                50, 100, 200 mg/kg      acute effects; ataxia from 11-17 
                bw subcutaneously,      days after dosing. Brain NTE 
                or 200 +100 mg/kg bw    decreased by 40-60% at high 
                subcutaneously 3        subcutaneous doses but by only 
                days apart              about 20% at <100 mg/kg bw. 
                                        Mild degenerative changes seen 
                                        in cerebellum, brainstem, and 
                                        striatum at higher doses.

    Chicken     185 mg/kg bw orally,    Acute signs of intoxication but       Thyssen et al. 
                survivors given 167     no pathological changes in            (1982)
                mg/kg bw 21 days        nervous tissue
                later

    Chicken     Single subcutaneous     Marked cholinesterase inhibition      Slott & 
                dose of 100 or 300      in plasma, brain, and spinal cord     Ecobichon 
                mg/kg bw                (50-60%) but no inhibition of NTE     (1984)
                                        in brain or spinal chord

    Chicken     100 mg/kg bw            Plasma, brain, and spinal cord        Slott & 
                subcutaneously every    cholinesterase inhibited by           Ecobichon 
                72 h for 6 doses        10-60%, and walking ability           (1984)
                                        slightly impaired. No inhibition 
                                        of NTE in brain or spinal cord 
                                        and only slight oedematous 
                                        effects on nervous tissue

    Chicken     3, 9, or 18 mg/kg bw    Signs of ataxia and decreased         Hayes & Ramm 
                per day for 3 months    activity at 18 mg/kg bw per day       (1987)
                by gavage               but no effect on locomotor activity. 
                                        Blood cholinesterase activity 
                                        decreased in all groups but 
                                        nervous tissue unremarkable.

                                                                                                

    Table 3. (continued)

                                                                                                
    Species     Treatment               Results                               Reference
                                                                                                

    Chicken     Single gavage dose      Acute signs of intoxication,          Bomann &
                of 340 mg/kg bw, 220    deaths, and marked inhibition         Kaliner 
                mg/kg bw                (90%) of brain cholinesterase         (1996)
                (-)-enantiomer,         activity with each compound.  
                or 400 mg/kg bw         < 50% NTE inhibition in brain, 
                (+)-enantiomer          spinal cord, and sciatic nerve; no 
                                        changes in gait or nervous tissue 

    Monkey      Single dose of          Weakness of lower extremities         Shiraishi et al. 
    (Macaca     250 mg/kg bw by         on day 24 and nerve conduction        (1983)
    fuscata)    gavage                  defects by day 28. On day 37,
                                        demyelination of sural and tibial 
                                        nerves with increased axonal 
                                        degeneration
                                                                                                

    NTE, neuropathy target esterase
    

    Table 4. Mean inhibition of erythrocyte acetylcholinesterase 
             activity (%) in patients with Alzheimer disease 
             given trichlorfon orally

                                                               
    Time (week)    Dose of trichlorfon (mg/kg bw per day)
                                                               
    0-2            0           0.5        0.9        2.0
    3-10           0           0.2        0.3        0.65

    2              1.1 ± 14    29 ± 12    49 ± 12    71 ± 82
    8              0.1 ± 21    37 ± 13    52 ±12     68 ± 95
    12             1.0 ± 24    34 ± 14    52 ± 10    72 ± 24
                                                               


         A position paper (Bayer AG, 1999) reported that the results of
    clinical trials involving over 4000 treated patients show that
    trichlorfon at the recommended dose of 0.5-0.9 mg/kg bw is 'a safe and
    effective treatment' for Alzheimer disease. A dose-response
    relationship was found between the dose of trichlorfon and the
    incidence of muscle weakness in placebo-controlled trials, and the
    increased risk for muscular weakness was statistically significant at
    doses > 1.25 mg/kg bw. The recommended dose of 0.5-0.9 mg/kg bw
    resulted in incidences of 0.6% for generalized weakness or myasthenia
    (0.2% with placebo) and 4.3% for fatigue or asthenia (3.3% with

    placebo). The muscular weakness was reported to be reversible on
    discontinuation of therapy.

         Acute poisoning of humans with trichlorfon has been reported to
    manifest as delayed neurotoxicity, severe polyneuritis, and
    polyneuropathy 2-3 weeks after ingestion. These diagnoses were based
    on observations of weakness, loss of feeling in the extremities, and
    difficulty in walking, accompanied by muscular atrophy, myalgia, and
    were related to motor nerve damage (Pollingher et al., 1973; Akimov et
    al., 1975; Eljasz & Kryzyszton-Przekop, 1975; Fukuhara et al., 1977;
    Shiraishi et al., 1977; Hierons & Johnson, 1978; Shiraishi et al.,
    1982; Niedziella et al., 1985; De Freitas et al., 1990). After
    reviewing the literature, Johnson (1981) concluded that only doses of
    trichlorfon that exceed the lethal dose but are survived by the victim
    because of treatment are likely to result in a level of inhibition of
    neuropathy target esterase activity at which delayed neurotoxicity
    would be expected. Development of neuropathy after repeated exposure
    to lower doses was considered to be unlikely (WHO, 1992).

         Five persons exposed to trichlorfon, two while attempting suicide
    and three occupationally, showed increased incidences of chromosomal
    breaks and stable chromosomal rearrangements in cultured lymphocytes.
    The effect was observed immediately after exposure and 1 month later,
    but not 6 months later (Bao et al., 1974). Seventeen workers involved
    in the production of trichlorfon-containing products for at least 6
    months had increased incidences of chromatid breaks in their
    lymphocytes when compared with an unexposed control group, but the
    incidences were lower than those in a control group of factory workers
    who had had no direct exposure to pesticides (Kiraly et al., 1979).

    3.  COMMENTS

         The Committee considered the results of studies on the
    toxicokinetics, metabolism, and acute toxicity of trichlorfon,
    short-term and long-term studies of toxicity, and studies of
    genotoxicity, reproductive and developmental toxicity, neurotoxicity,
    and immune function, and studies in humans. Most of the available
    studies were relatively old, and many were published reports which
    lacked raw data or data on individual animals. Such studies are
    difficult to evaluate, and most could not be assessed independently.
    Unpublished reports of studies conducted during the 1980s and 1990s
    contained sufficient detail and were carried out according to
    appropriate standards for study protocol and conduct.

         The absorption of trichlorfon is rapid in all species tested,
    including humans, irrespective of the route of administration. Peak
    blood concentrations were achieved within 1-2 h but decreased quickly
    thereafter; the half-time of trichlorfon in human plasma is
    approximately 2 h. It is widely distributed. Trichlorfon was detected
    in the milk of lactating cows, and the compound and its metabolites
    were found in fetal tissue in treated guinea-pigs. Trichlorfon
    undergoes conversion to dichlorvos via a dehydrochlorination reaction
    that occurs spontaneously at pH values above 5.5. Although little

    dichlorvos was recovered, it is generally believed to be responsible
    for the anticholinesterase effects of trichlorfon. The metabolism of
    trichlorfon in mammals also occurs through  O-demethylation and
    cleavage of phosphorus-carbon bonds. Therefore, the major metabolites
    are desmethyl trichlorfon, desmethyl dichlorvos, dimethyl hydrogen
    phosphate, methyl dihydrogen phosphate, and phosphoric acid.
    Trichlorfon and its metabolites are excreted primarily in the urine.

         Trichlorfon is moderately toxic when given as a single oral dose,
    the LD50 values being 620-950 mg/kg bw in mice, 140-660 mg/kg bw in
    rats, 160 mg/kg bw in rabbits, and 300-420 mg/kg bw in dogs. The signs
    of toxicity were indicative of inhibition of acetylcholinesterase
    activity, and the cause of death was usually respiratory failure. The
    rapid recovery observed after sublethal doses suggests that
    trichlorfon is readily detoxified.

         Trichlorfon was given orally to mice at doses of 10-750 mg/kg bw
    per day, to rats at doses of 0.05-250 mg/kg bw per day, and to dogs at
    doses of 0.5-45 mg/kg bw per day for periods up to 16 weeks. The
    weights of the liver, kidney, and spleen were increased at doses of
    250 mg/kg bw per day and above in mice and at doses of 50 mg/kg bw per
    day and above in rats, but these changes were usually not associated
    with other evidence of toxicity, except in one study in rats in which
    hypertrophy of centrilobular hepatocytes and biochemical changes
    suggestive of altered lipid metabolism were seen. These effects
    occurred at doses which also induced significant signs of cholinergic
    intoxication. Slight anaemia was observed in one study in rats at 100
    mg/kg bw per day and in one study in dogs at 45 mg/kg bw per day. One
    dog out of three given trichlorfon at a dose of 45 mg/kg bw per day
    had small, immature testes and prostate and oligospermia, but the
    animal had also lost weight and appeared to be severely stressed by
    the treatment. The NOELs were 15 mg/kg bw per day for inhibition of
    cholinesterase activity in brain and erythrocytes in mice, 5 mg/kg bw
    per day for inhibition in brain and erythrocytes in rats, and 5 mg/kg
    bw per day for inhibition in in erythrocytes in dogs.

         In two studies of up to 2 years' duration, mice were given oral
    doses of 15-750 mg/kg bw per day. Body-weight gain was impaired at
    doses of 30 mg/kg bw per day and above; the mean weight of the liver
    was increased at a dose of 240 mg/kg bw per day in one study, but no
    pathological alterations were found. The NOEL for inhibition of brain
    and erythrocyte cholinesterase activity was 49 mg/kg bw per day. The
    incidences of tumours were unaffected by treatment. Neither study was
    suitable for identifying a NOEL.

         Six studies of up to 2 years' duration were conducted in which
    rats were given trichlorfon in the diet at concentrations providing
    doses of 2.5-160 mg/kg bw per day. The weights of the liver and spleen
    were increased in three studies at doses of 50 mg/kg bw per day and
    above. Hypercholesterolaemia, duodenal hyperplasia, and gastritis of
    the non-glandular stomach were seen at 13 mg/kg bw per day and above.
    Anaemia, enhancement of age-related nephropathy, increased renal
    tubular hyperplasia, myodegeneration of the stomach tunica muscularis,

    pulmonary inflammation, and hyperplasia were observed at doses of 75
    mg/kg bw per day and above. Other observations included ovarian
    atrophy at 20 mg/kg bw per day and above, depression of
    spermatogenesis at 50 mg/kg bw per day, and a slight increase in the
    incidence of benign mammary tumours in females with a reduction in the
    time to appearance at doses of 20 mg/kg bw per day and above. The NOEL
    for inhibition of brain and erythrocyte acetylcholinesterase activity
    was 13 mg/kg bw per day. The incidence of mammary tumours was
    increased in female Sprague-Dawley rats in two studies. The increases
    were slight, the tumours were benign, this strain of rats has a
    relatively high and variable background incidence of mammary tumours,
    and the finding could not be confirmed in several other studies in
    various species. The Committee concluded that trichlorfon does not
    have carcinogenic potential in rats. The overall NOEL in rats was 4.5
    mg/kg bw per day on the basis of an increased cholesterol
    concentration and pathological changes to the gastrointestinal tract.

         In two studies of 1 and 4 years' duration, dogs were given
    trichlorfon orally at doses of 1.3-80 mg/kg bw per day. Deaths
    occurred at doses of 20 mg/kg bw per day and above, and signs of
    hepatocellular damage and increased severity of inflammation were seen
    in the liver at doses of 25 mg/kg bw per day and above. In animals
    given 20-25 mg/kg bw per day, the weight of the spleen was increased,
    with atrophy of lymphoid tissue; furthermore, small ovaries, reduced
    testis weight, and decreased spermatogenesis were seen. 'Blood
    cholinesterase' activity was inhibited at doses of 5 mg/kg bw per day
    and above. The overall NOEL in dogs was 1.3 mg/kg bw per day on the
    basis of inhibition of cholinesterase activity.

         Oral administration of trichlorfon to rhesus monkeys at a dose of
    0, 0.2, 1, or 5 mg/kg bw per day for 10 years resulted in clinical
    signs of toxicity indicative of cholinesterase inhibition, reduced
    body-weight gain, and anaemia at the highest dose. Inhibition of
    plasma, erythrocyte, and brain cholinesterase activities was seen at
    the two higher doses. In a separate 6-month study in rhesus monkeys,
    erythrocyte acetylcholinesterase activity was not depressed at 0.2
    mg/kg bw per day, the highest dose tested. The NOEL in monkeys was 0.2
    mg/kg bw per day on the basis of inhibition of brain and erythrocyte
    acetylcholinesterase activity.

         Trichlorfon has been tested in a wide range of assays for
    genotoxicity and DNA damaging activity, with considerable variation in
    the results. Trichlorfon induced point mutations in microorganisms and
    cultured mammalian cells, DNA damage in microorganisms, and
    chromosomal aberrations and sister chromosome exchange in cultured
    mammalian cells. The results of tests for point mutation in
     Drosophila melanogaster and for sister chromatid exchange in bone
    marrow were clearly negative. Although positive results were obtained
    in a few assays for chromosomal damage in bone marrow and in the germ
    cells of male animals exposed at near-lethal doses, the results of
    most studies of micronucleus formation, metaphase alterations, and
    dominant lethal mutations were negative. Since the tests conducted
     in vivo produced mostly negative results when trichlorfon was

    administered orally, the Committee considered that the weight of
    evidence indicates that trichlorfon is unlikely to represent a
    genotoxic risk.

         Trichlorfon was given to rats at a dose of 0, 10, 30 100, or 300
    mg/kg bw per day in a three-generation study of reproductive toxicity,
    with two litters per generation. The body-weight gain of F0 dams at
    100 and 300 mg/kg bw per day was depressed. The slight effects on the
    gonads reported in the studies of general toxicity were not
    consistently reflected in this study. The pregnancy rate of rats of
    the F1b generation at doses of 100 mg/kg bw per day and above was
    reduced, but the fertility of other generations was unaffected. The
    litter size of dams at 300 mg/kg bw per day was reduced and all of the
    offspring died, but reproductive parameters were unaffected at lower
    doses. The NOEL was 30 mg/kg bw per day in adults, on the basis of
    reduced body-weight gain, and 100 mg/kg bw per day for reproductive
    effects, on the basis of reduced litter size and the death of
    offspring.

         Two studies of developmental toxicity were conducted in mice
    given oral doses of 200-600 mg/kg bw per day. Dams at 200 mg/kg bw per
    day showed reduced body-weight gain, and higher doses led to an
    increased mortality rate. The effects of these maternally toxic doses
    included increased embryotoxicity, decreased fetal weight, and delayed
    fetal development. The incidence of cleft palate was slightly
    increased at doses of 500 and 600 mg/kg bw per day. NOELs were not
    identified for maternal toxicity or fetotoxicity.

         Studies of developmental toxicity were conducted in rats given
    oral doses of 8-520 mg/kg bw per day. In three studies, maternal
    toxicity in the form of reduced body-weight gain and/or an increased
    mortality rate was observed at doses of 150 mg/kg bw per day and
    above. The NOEL for maternal toxicity was 75 mg/kg bw per day. In the
    same studies, the incidences of fetal malformations such as
    morphological alterations of the skull, central nervous system, and
    limbs, micrognathia, cleft palate, facial haematomas, generalized
    oedema, and defects in major blood vessels were increased at doses of
    430 mg/kg bw per day and above. At 380 mg/kg bw per day, minor
    skeletal changes were observed. Reduced fetal body weight was seen at
    75 mg/kg bw per day. In three other studies, the incidence of fetal
    malformations was unaffected. The NOEL for developmental effects was
    50 mg/kg bw per day on the basis of fetotoxicity.

         Hamsters given trichlorfon orally at a dose of 0, 200, 300, or
    400 mg/kg bw per day during gestation showed signs of cholinesterase
    inhibition, and depressed body-weight gain was seen at the two higher
    doses. The NOEL for maternal toxicity was 100 mg/kg bw per day. Fetal
    body weight was decreased at the two higher doses, and an increased
    incidence of fetal stunting was seen at the highest dose. The NOEL for
    developmental effects was 200 mg/kg bw per day.

         Guinea-pigs given trichlorfon at an oral dose of 100 mg/kg bw per
    day for 6 days during gestation had abortions and stillborn fetuses,
    and  the offspring showed reduced body weight and brain weight,
    locomotor disturbances, and morphological and biochemical alterations
    in the brain. 

         In two studies of developmental toxicity in rabbits, oral doses
    of 5-110 mg/kg bw per day were given. Overt toxicity and inhibition of
    erythrocyte acetylcholinesterase activity were observed in dams given
    doses of 35 mg/kg bw per day and above, and body-weight gain was
    affected at all doses. The highest dose, 110 mg/kg bw per day,
    slightly increased the rate of resorptions and retarded fetal
    development, but no fetal abnormalities were found. A NOEL for
    maternal toxicity was not identified. The NOEL for developmental
    effects was 45 mg/kg bw per day on the basis of toxicity to embryos
    and fetuses. 

         Several outbreaks of congenital tremor with cerebellar hypoplasia
    were reported in piglets of sows that had been treated with
    trichlorfon, and the syndrome was subsequently reproduced
    experimentally. The piglets of sows treated with trichlorfon at doses
    of 50-100 mg/kg bw per day during appropriate periods of gestation
    showed neurological signs and hypoplasia and loss of Purkinje cells in
    the cerebellum.

         Rats given a dose equivalent to 200 mg/kg bw per day for 13 weeks
    showed decreased locomotor activity and loss of coordination. In
    another study in rats, a dose of 30 mg/kg bw per day for 3 weeks
    increased locomotor activity and decreased learning ability and nerve
    conduction velocity.

         The results of numerous studies in hens consistently demonstrated
    the acute neurotoxicity of trichlorfon. Delayed neurotoxicity,
    associated with degeneration of nervous tissue and marked inhibition
    of neuropathy target esterase, was not seen in hens. A monkey given a
    single oral dose of 250 mg/kg bw showed impaired nerve conduction 4
    weeks after treatment and histological evidence of demyelination of
    nerves and axonal degeneration.

         Trichlorfon has been used as an anthelmintic agent in humans.
    Oral doses of up to 10 mg/kg bw given on two to four occasions were
    well tolerated, with few clinical symptoms. A dose of 24 mg/kg bw
    caused significant cholinergic symptoms, but the effects were not
    cumulative. In a few cases, spermatogenesis appeared to have been
    impaired. The results of clinical trials with trichlorfon in the
    treatment of Alzheimer disease showed dose-related but reversible
    muscle weakness. The recommended dose of 0.5-0.9 mg/kg bw resulted in
    a small increase in the frequency of generalized weakness and fatigue,
    while doses of 1.25 mg/kg bw and higher produced significant weakness.
    Dose-related inhibition of erythrocyte acetylcholinesterase activity
    was also observed in these studies. 

         In 121 subjects, an initial dose of 0.5 mg/kg bw per day for 2
    weeks inhibited erythrocyte acetylcholinesterase activity by 29%.
    Subsequent administration of a dose of 0.2 mg/kg bw per day for
    10 weeks maintained the inhibition of erythrocyte acetylcholinesterase
    activity at levels between 30 and 37%, with no increase with duration
    of dosing. Since this dose enhanced the inhibition caused by the
    initial dose by only 8%, an insignificant change, the Committee
    concluded that the NOEL was 0.2 mg/kg bw per day.

         In some cases of severe human poisoning with trichlorfon,
    weakness and loss of feeling in the extremities, difficulty in
    walking, muscular atrophy, and motor nerve damage have been observed.
    In many of these cases, the doses might have been lethal in the
    absence of medical intervention. The Committee concluded that
    extremely high doses of trichlorfon would be required to achieve the
    level of inhibition of neuropathy target esterase associated with
    delayed neurotoxicity.

    4.  EVALUATION

         The Committee concluded that inhibition of acetylcholinesterase
    activity was the most relevant end-point for establishing an ADI. The
    most appropriate NOEL was 0.2 mg/kg bw per day for inhibition of
    erythrocyte acetylcholinesterase activity in humans treated orally. A
    safety factor of 10 was applied to this figure, giving an ADI of 0-20
    µg/kg bw.

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    See Also:
       Toxicological Abbreviations
       Trichlorfon (EHC 132, 1992)
       Trichlorfon (HSG 66, 1991)
       Trichlorfon (JECFA Food Additives Series 51)
       TRICHLORFON (JECFA Evaluation)
       Trichlorfon (WHO Pesticide Residues Series 1)
       Trichlorfon (WHO Pesticide Residues Series 5)
       Trichlorfon (Pesticide residues in food: 1978 evaluations)
       Trichlorfon (IARC Summary & Evaluation, Volume 30, 1983)