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    CHLORMEQUAT

    First draft prepared by
    M. Watson,
    Pesticides Safety Directorate,
    Ministry of Agriculture, Fisheries and Food,
    York, United Kingdom

         Explanation
         Evaluation for acceptable daily intake
              Biochemical aspects
                   Absorption, distribution and excretion
                   Biotransformation
                   Effects on enzymes and other biochemical parameters
              Toxicological studies
                   Acute toxicity
                   Short-term toxicity
                   Long-term toxicity and carcinogenicity
                   Reproductive toxicity
                   Embryotoxicity and teratogenicity
                   Genotoxicity
                   Special studies
                        Skin and eye irritation and skin sensitization
              Observations in humans
              Comments
              Toxicological evaluation
         References

    Explanation

         Chlormequat (2-chloroethyltrimethylammonium chloride) was
    evaluated by the Joint Meeting in 1970 and 1972 (Annex I, references
    14 and 18). In 1972, an ADI of 0.05 mg/kg bw was established on the
    basis of the NOAEL in a study of reproductive toxicity in rats. The
    compound was reviewed at the present Meeting as a result of the CCPR
    periodic review programme. This monograph summarizes new data on
    chlormequat and studies that were not reviewed previously; it also
    includes relevant data from the previous monograph and monograph
    addendum on this pesticide (Annex I, references 15 and 19).

    Evaluation for acceptable daily intake

    1.  Biochemical aspects

    (a)  Absorption, distribution and excretion

         Experiments are described in articles published in the open
    literature in which the absorption, distribution and excretion of
    chlormequat were investigated. The level of detail in these papers
    did not permit a complete evaluation by the present Meeting. In the
    first study, the bulk (60.6%) of an oral dose of 14C-chlormequat
    administered to male rats was excreted in the urine within 4 h, and
    96% was eliminated within 46.5 h; faecal excretion accounted for
    2.3%, and less than 1% was expired as 14C-carbon dioxide. The
    remainder was found in the tissues, with the largest amounts in the
    carcass (0.25%), intestines (0.11%) and liver (0.08%). Analysis of
    urine samples by four different thin-layer and paper chromatographic
    systems showed that all of the radiolabel was in chlormequat (Blinn,
    1967).

         In a second study, rats were administered a single (60 mg) oral
    dose of 15N-chlormequat or received 2 mg of the labelled compound
    daily for 100 days. After the single dose, the amount of compound in
    the brain decreased quickly, but there was considerable accumulation
    in the kidneys over the 20 days of the investigation. After
    continuous administration, chlormequat was found particularly in
    active muscles such as those of the heart and diaphragm (Bier &
    Ackermann, 1970).

         In a third study, a lactating cow received a single oral dose
    of 1000 mg of 15N-chlormequat. The compound was found in the milk
    and urine 3 h after administration; most (489 mg) was found 15-39 h
    after administration. Only 22 mg were excreted in the milk, and the
    concentration never exceeded 1 ppm; the peak concentration was found
    12-60 h after administration (Lampeter & Bier, 1970).

    (b)  Biotransformation

         It was reported in a short published article that only
    chlormequat and two other compounds, which may have been other salts
    of chlorcholine, occurred in the urine of rats that had received 200
    mg/kg bw of chlormequat orally. Choline itself was not identified
    (Bronisz & Romanowski, 1968).

    (c)  Effects on enzymes and other biochemical parameters

         General pharmacological tests were carried out to determine the
    physiological effects of chlormequat injected intravenously.
    Oligopnoea, salivation and a tendency to inhibition of intestinal
    propulsion were observed in mice immediately after they were dosed
    with 7.4 mg/kg bw. In cats, there was mild inhibition of the

    vasopressor effect of norepinephrine 30 min after administration of
    1 mg/kg bw. In rabbits, neuromuscular junctions were blocked by
    doses of 1 mg/kg bw and more; this effect was counteracted by
    administration of 10 mg/kg bw of D-tubocurarine and potentiated by
    administration of 1 mg/kg bw neostigmine. Coagulation of rat blood
    was unaffected by concentrations of up to 3 mg/ml. In dogs, doses of
    3 mg/kg bw and more caused a drop in blood pressure; at higher
    doses, increased respiratory and heart rates were observed. These
    effects were mitigated by prior intravenous administration of 1
    mg/kg bw atropine (Mutoh  et al., 1987).

         The action of chlormequat was tested  in vitro with the patch
    clamp technique for electrophysiological measurements described by
    Hamill  et al. (1981). Muscles were excised from the feet of adult
    NMRI mice and dissociated enzymatically to obtain individual muscle
    cells. Chlormequat (purity, 95.6%) activated the nicotinic
    acetylcholine receptor channel at all concentrations between 10 and
    100 mmol/l (Franke & Mellert, 1991).

         The affinity of chlormequat for subtypes of muscarinic
    acetylcholine receptors was investigated  in vitro on membranes
    from bovine cerebral cortex, from rat heart and from rat
    submaxillary gland. The results were compared with those obtained
    for subtype-specific reference substances, and atropine was included
    as a high-affinity reference compound with no subtype selectivity.
    Chlormequat had low affinity for the muscarinic receptors in
    comparison with the reference substances (Weifenbach, 1991).

    2.  Toxicological studies

    (a)  Acute toxicity

         Clinical signs of toxicity seen after treatment with
    chlormequat (Table 1) generally consisted of salivation, writhing,
    chromodaccryorrhoea, decreased activity, tremors, diuresis and
    piloerection. Mortality generally occurred within 24 h of treatment;
    animals that survived recovered within 48 h. The findings at autopsy
    were not consistent or related to treatment.

         Rabbits and dogs may be more sensitive to the toxic effects of
    chlormequat than rats and mice. The acute toxicity in monkeys was
    similar to that seen in rats and mice.
        Table 1.  Acute toxicity of chlormequat
                                                                                
    Species     Route            LD50 or LC50  Purity  Reference
                                 (mg/kg bw or  (%)
                                 mg/l air)
                                                                                

    Mouse       Oral             215-1020      NR      Oettel, 1965
                                               NR      Levinskas & Shaffer, 1966
                                               NR      Ignatiev, 1967
                                               98.0    Hattori, 1981
    Mouse       Intraperitoneal  60-68         NR      Shaffer, 1970
                                               98.0    Hattori, 1981
    Mouse       Subcutaneous     88-92         98.0    Hattori, 1981
    Rat         Oral             330-750       NR      Oettel, 1965
                                               NR      Levinskas & Shaffer, 1966
                                               NR      Ignatiev, 1967
                                               NR      Stefaniek, 1969
                                               98.1    Hattori, 1981
    Rat         Oral             522           66.1a   Fischer & Lowe, 1990
    Rat         Intraperitoneal  53-75         98.1    Hattori, 1981
    Rat         Subcutaneous     113-118       98.1    Hattori, 1981
    Rat         Dermal           > 4000        NR      Gelbke & Freisberg, 1978
    Rat         Dermal           > 5000        98.1    Hattori, 1981
    Rat         Inhalation       > 5.2         99      Zeller & Klimisch, 1979
    Rat         Inhalation       > 4.6         66.1    Hershman, 1990
    Hamster     Oral             1070          NR      Levinskas & Shaffer, 1966
    Guinea-pig  Oral             215-620       NR      Oettel, 1965
                                               NR      Levinskas & Shaffer, 1966

    Table 1 (contd)
                                                                                
    Species     Route            LD50 or LC50  Purity  Reference
                                 (mg/kg bw or  (%)
                                 mg/l air)
                                                                                

    Rabbit      Oral             60-81         NR      Oettel, 1965
                                               NR      Levinskas & Shaffer, 1966
    Rabbit      Dermal           1250          66.1a   Fischer  et al., 1990a
    Cat         Oral             7-50          NR      Oettel, 1965
                                               NR      Levinskas & Shaffer, 1966
    Dog         Oral             < 50          NR      Levinskas & Shaffer, 1966
    Sheep       Oral             150-200       NR      Schulz  et al., 1970
    Monkey      Oral             > 800         NR      Costa  et al., 1967
                                                                                

    NR, not reported
    a Technical material consisting of 66.1% aqueous solution
    
    (b)  Short-term toxicity

    Rats

         Two studies of short-term toxicity are described in the 1972
    JMPR monograph addendum (Annex I, reference 19), but detailed
    reports were not available for evaluation at the present Meeting.
    The summary of the first study states that groups of 10 male rats
    were fed chlormequat at dietary levels of 0, 500, 1000 or 2000 ppm
    (equivalent to 25, 50 or 100 mg/kg bw per day) for 29 days. There
    was no mortality and no clinical signs of reaction to treatment;
    body-weight gain and food intake remained undisturbed by treatment,
    and no gross pathological changes were observed at termination of
    the study (Levinskas & Shaffer, 1962).

         In the second study, groups of 20 male and 20 female rats were
    fed chlormequat at dietary levels of 0, 200, 600 or 1800 ppm
    (equivalent to 10, 30 or 90 mg/kg bw per day) for 90 days. There was
    no mortality and no clinical signs of reaction to treatment, and no
    treatment-related changes in blood chemistry were seen. Body-weight
    gain of males fed 1800 ppm was slightly depressed in comparison with
    that of controls. Slightly increased kidney weights were recorded in
    treated female rats and slightly increased liver weights in treated
    males, particularly at 1800 ppm; however, histopathological
    examination of major organs revealed no treatment-related changes
    (Levinskas, 1965).

         In a recent experiment of acceptable scientific quality, groups
    of five male and five female Wistar rats were fed chlormequat at
    dietary levels of 0, 500, 1500, 3000 or 4500 ppm (equal to 46, 137,

    274 or 411 mg/kg bw per day) for four weeks. The test material was a
    66.7% technical formulation, but the dietary levels were expressed
    as pure chlormequat. Clinical signs of general deterioration in
    health were seen in males and females receiving 4500 ppm and,
    temporarily, in one male and one female receiving 3000 ppm. Reduced
    body-weight gain and food intake were seen in animals fed 4500 ppm,
    and slightly reduced weight gain was seen among those fed 3000 ppm.
    Serum creatinine levels in males and females receiving 4500 ppm and
    in females receiving 3000 ppm were lower than those of controls.
    Decreased serum concentrations of total protein (in males) and of
    urea (in females) were also seen at the high dietary level.
    Examinations of locomotor activity, swimming tests, gross
    pathological examinations, organ weight analysis and
    histopathological examination revealed no reaction to treatment. The
    NOAEL was 1500 ppm, equal to 137 mg/kg bw per day (Schilling  et
     al., 1990).

    Rabbits

         In a recent experiment of acceptable scientific quality, groups
    of 10 male and 10 female New Zealand white rabbits received
    chlormequat (purity, 99%) by repeated, occluded dermal applications
    on shaven skin, five days per week for three weeks at doses of 0,
    20, 50 or 150 mg/kg bw per day. Possible reactions to treatment at
    the application site were limited to erythema during the first two
    weeks of the study; however, these reactions, were no more severe
    than reactions frequently seen after repeated, dermal, occluded
    applications of control compounds. There were no other clinical
    signs of reaction to treatment, and body-weight gain and food intake
    remained undisturbed by treatment. Investigations of haematological
    parameters and blood chemistry, gross pathology, organ weight
    analysis and histopathology revealed no reaction to treatment (Buch
    & Finn, 1981).

    Dogs

         In an experiment for which no detailed report was available to
    the Meeting for evaluation (Annex I, reference 19), groups of two
    male and two female dogs were fed chlormequat at dietary levels of
    0, 20, 60 or 180 ppm (equivalent to 0.5, 1.5 or 4.5 mg/kg bw per
    day) for 106-108 days. There were no deaths and no clinical signs of
    reaction to treatment, and body-weight gain and food intake were
    unaffected. Organ weight analysis and histopathology at termination
    of the experiment revealed no treatment-related changes (Levinskas,
    1965).

         In a study reported in 1967, which was not conducted to
    currently acceptable scientific standards, groups of three male and
    three female beagle dogs were fed chlormequat (technical grade
    purified twice by recrystallization) at dietary levels of 100, 300
    or 1000 ppm (equivalent to 2.5, 7.5 or 25 mg/kg bw per day) for two

    years; groups of 10 males and 10 females served as controls.
    Excessive salivation and hind limb weakness were seen in some
    animals receiving 1000 ppm; in this group, one male died after 22
    days, and one female died after 38 days. The deaths were considered
    by the authors of the report to be secondary to the clinical signs
    of hind-limb weakness. Analysis of blood chemistry and urinalysis
    revealed no treatment-related changes, other than the presence of
    chlormequat in the urine of treated animals. At termination, gross
    pathology, organ weight analysis and histopathology revealed no
    changes attributable to treatment. The NOAEL was probably 300 ppm
    (equivalent to 7.5 mg/kg bw per day), but the lack of further
    investigation into the signs of hind-limb weakness precluded
    establishment of a definitive NOAEL (Oettel & Sachsse, 1967).

    (c)  Long-term toxicity and carcinogenicity

    Mice

         In a study reported in 1971, the design of which was clearly
    not in accordance with currently acceptable scientific standards,
    groups of 52 male and 52 female CFLP mice were fed chlormequat
    (purity, about 98.5%) at dietary levels of 0 or 1000 ppm (equivalent
    to 150 mg/kg bw per day) for 78 weeks. Survival was unaffected, and
    there were no clinical signs of reaction to treatment, except that
    treated animals gained less weight than controls. Histopathological
    examination was initially restricted to 10 males and 10 females from
    each group but was extended to all animals with respect to tissues
    in which a treatment-related effect was seen. The incidence of
    benign lung tumours was higher (20 out of 52) in treated males than
    in controls (10 out of 51), but the incidence was considered to be
    within the normal range in untreated mice. The incidences of lung
    tumours in females and of tumours in all other organs examined in
    animals of each sex were not significantly higher in the treated
    group than in controls (Weldon  et al., 1971).

         Groups of 50 male and 50 female B6C3F1 mice were administered
    diets containing chlormequat (purity, 97-98%) at 500 or 2000 ppm
    (equal to 70 or 286 mg/kg bw per day) for 102 weeks. The dietary
    levels were set on the basis of the results of an eight-week
    subchronic study with 1200-20 000 ppm designed to provide a
    statistical estimate of the dose that would depress body-weight gain
    by 10%. The control group consisted of 20 males and 20 females.
    Body-weight gain remained largely unaffected by treatment, and there
    were no treatment-related clinical signs. Survival was unaffected by
    treatment and was adequate for assessment of carcinogenicity, as at
    least 80% of animals in each group survived until termination of the
    experiment. The incidence of haemangiomas and haemangiosarcomas was
    slightly increased in treated females (1/20 in controls, 4/50 at 500
    ppm and 5/50 at 2000 ppm), but the authors of the report concluded
    that there was no clear evidence for the carcinogenicity of
    chlormequat in these mice (National Cancer Institute, 1979).

    Rats

         The Meeting reviewed a short summary of a two-year study
    reported in 1967, the design of which was not in accordance with
    contemporary scientific standards. Groups of 50 male and 50 female
    Sprague-Dawley rats were fed chlormequat (technical grade, purified
    twice by recrystallization) at dietary levels of 0, 500 or 1000 ppm
    (equivalant to 25 or 50 mg/kg bw per day) for two years. The authors
    reported that survival was unaffected, there were no clinical signs
    of reaction to treatment and food intake and body-weight gain
    remained undisturbed by treatment. Haematological examinations,
    analysis of blood chemistry and urinalysis carried out after three
    and 12 months and before termination revealed no reaction to
    treatment. Gross pathological examination, analyses of liver and
    kidney weights and histopathological examination revealed no
    abnormalities attributable to treatment. Normal, age-related
    pathological changes were seen; in particular, the tumour profile in
    treated and control animals was indistinguishable. Detailed
    evaluation of this report was not possible (Oettel & Froberg, 1967).

         A similar summary was available of another long-term study in
    which groups of 50 male and 50 female Sprague-Dawley rats were fed
    diets containing chlormequat (technical grade purified twice by
    recrystallization) and choline chloride in a ratio of 10:7 for two
    years. The dietary levels of chlormequat were 0, 500, 1000 or 5000
    ppm (equivalent to 25, 50 or 250 mg/kg bw per day). The control
    group consisted of 100 male and 100 female rats. Survival was
    unaffected by treatment; the survival rates after two years were
    72-82% in male rats and 48-64% in females. There were no clinical
    signs of reaction to treatment, and body-weight gain and food intake
    remained undisturbed. Haematological examination, limited analyses
    of blood chemistry and urinalysis carried out after three and 12
    months and before termination revealed no indication of any reaction
    to treatment. Gross pathological examination, analysis of liver and
    kidney weights and histopathological examination of a range of
    organs and tissues revealed no abnormalities attributable to
    treatment. Normal, age-related pathological changes were seen; in
    particular, the tumour profile in treated and control animals was
    indistinguishable (Oettel & Sachsse, 1974).

         In a study designed to assess the tumorigenicity of chlormequat
    in rats, groups of 50 male and 50 female Fischer 344 rats were
    administered diets containing chlormequat (purity, 97-98%) at 1500
    or 3000 ppm (equivalent to 75 or 150 mg/kg bw per day) for 108
    weeks. The dietary levels were set on the basis of the results of an
    eight-week subchronic study with 3150-14 700 ppm designed to provide
    a statistical estimate of the dose that would depress body-weight
    gain by 10%. The control group consisted of 20 males and 20 females.
    Body-weight gain of treated rats was slightly lower than that of
    controls, but there were no treatment-related clinical signs.
    Survival was unaffected by treatment and was adequate for assessment

    of carcinogenicity, as at least 64% of animals in each group
    survived until termination of the experiment. The incidence of
    leukaemia or malignant lymphoma was slightly increased in treated
    females (3/20 in controls, 11/50 at 1500 ppm and 14/50 at 3000 ppm),
    and there was an apparently dose-related increase in the incidence
    of islet-cell adenomas of the pancreas in treated males (0/18 in
    controls, 1/47 at 1500 ppm and 7/45 at 3000 ppm), but the authors of
    the report concluded that there was no clear evidence for the
    carcinogenicity of chlormequat in these rats (National Cancer
    Institute, 1979).

    (d)  Reproductive toxicity

         In a study conducted between 1965 and 1967 which was not in
    accordance with currently acceptable scientific standards, groups of
    20 male and 20 female rats received diets containing chlormequat at
    0, 100, 300 or 900 ppm (equivalent to 5, 15 or 45 mg/kg bw per day)
    throughout three generations. No abnormalities were seen in the
    appearance, behaviour, food intake, body-weight gain, fertility,
    gestation, lactation or viability of the offspring, and no fetal
    malformations occurred that could be attributed to treatment.
    Histopathological examination of 10 rats of each sex of the F3
    generation in each dose group after nine weeks of treatment revealed
    the presence of giant cells in the testicular tubules of four rats
    treated with 900 ppm and two treated with 300 ppm. The authors
    suggested that the cells were an expression of delayed maturation
    during spermatogenesis; however, the Meeting found it difficult to
    assess the significance of the finding and concluded that the NOAEL
    was 100 ppm, equivalent to 5 mg/kg bw per day (Leuschner  et al., 
    1967).

    (e)  Embryotoxicity and teratogenicity

    Mice

         The results of three experiments in mice were summarized in the
    1972 monograph addendum (Annex I, reference 19), but detailed
    reports were not available for evaluation by the present Meeting. In
    the first experiment, groups of pregnant mice received
    intraperitoneal injections of chlormequat at 30 mg/kg bw on days 14
    and 15 or days 11-15 of gestation. Another group of mice received
    200 mg/kg bw per day by gavage on days 11-15 of gestation. All mice
    were sacrificed on day 19. The number of fetuses per dam, fetal
    size, frequency of resorptions and incidence of malformations were
    similar in the test and control groups. In the second experiment,
    pregnant mice were fed chlormequat at dietary levels of 0, 1000 or
    10 000 ppm (equivalent to 150 or 1500 mg/kg bw per day) on days 1-15
    of gestation or 25 000 ppm (equivalant to 3750 mg/kg bw per day) on
    days 11-15. All animals were sacrificed on day 19. The number of
    fetuses per dam, fetal size and frequency of resorptions were

    similar in the test and control groups. Dams fed 10 000 or 25 000
    ppm had a slightly greater number of malformed fetuses than
    controls. In the third experiment, groups of mature male and female
    mice were fed chlormequat at dietary levels of 0, 1000 or 5000 ppm
    (equivalent to 150 or 750 mg/kg bw per day) and mated after 1, 3, 4
    and 10 weeks. All mice were killed on day 19 of gestation. Feeding
    of chlormequat was found to have no effect on the fertility of the
    mice, and no evidence of teratogenicity was seen in the offspring
    (Shaffer, 1970).

    Rats

         In a study described in the 1972 monograph addendum (Annex I,
    reference 19), groups of pregnant rats were fed chlormequat at
    dietary levels of 0, 1000 or 5000 ppm (equivalent to 50 or 250 mg/kg
    bw per day) on days 1-21 of gestation. When the animals were
    sacrificed the day before parturition, no teratogenic effects were
    observed (Shaffer, 1970).

    Hamsters

         In a report published in the literature, groups of eight
    pregnant Syrian golden hamsters were given chlormequat (purity
    unspecified) by gavage at levels of 0, 25, 50, 100, 200, 300 or 400
    mg/kg bw once on day 8 of gestation. Another group received 100
    mg/kg bw daily on days 7, 8 and 9 of gestation. The control group
    consisted of 15 animals. Clinical signs of toxicity were seen in the
    groups receiving the higher doses. All animals were sacrificed on
    day 14 of gestation. Animals fed 100 mg/kg bw or more had fewer
    fetuses than controls and more fetal resorptions. In animals fed 200
    mg/kg bw or more, fetal size and weight were reduced. No
    abnormalities were observed in animals treated with a single dose of
    0, 25, 50 or 100 mg/kg bw. Malformations (including anophthalmia,
    microphthalmia, cleft palate and polydactylism) and evidence of
    developmental retardation were seen in the offspring of dams treated
    with three doses of 100 mg/kg bw or a single dose of 200, 300 or 400
    mg/kg bw. The limited details presented in the publication make it
    difficult to assess the occurrence of maternal toxicity; historical
    data on the occurrence of malformations in controls were not
    presented (Juszkiewicz  et al., 1970).

    Rabbits

         In a study described in the 1972 monograph addendum (Annex I,
    reference 19), groups of pregnant rabbits were fed diets containing
    chlormequat at 0 or 1000 ppm (equivalent to 30 mg/kg bw per day) on
    days 1-28 of pregnancy and were killed two days before parturition.
    No evidence of teratogenicity was seen (Shaffer, 1970).

         In a study of acceptable design, groups of 15 inseminated
    female Himalayan rabbits were given chlormequat at 0, 1.5, 3, 6 or
    12 mg/kg bw per day by gavage on days 6-18 of gestation. Fetuses
    were delivered by caesarian section after sacrifice of the dams on
    day 28 of gestation and were examined for abnormalities
    macroscopically and by X-ray. In addition, the heads of all fetuses
    were fixed in Bouin's solution, and transverse sections were
    assessed. Treatment did not affect mortality. Rapid breathing,
    salivation and apathy were seen on single occasions in single
    animals receiving 6 or 12 mg/kg bw per day; body-weight gain of
    animals given 12 mg/kg bw per day was reduced temporarily, and food
    intake was temporarily depressed in all treated groups. Treatment
    had no effect on the number of fetuses, conception rate, resorption
    rate, size or weight of the fetuses or placental weights. No
    teratogenic effect was seen. Minor variations and developmental
    retardation were seen to the same extent in all groups, including
    controls. The NOAEL for maternal toxicity was thus 6 mg/kg bw per
    day, while there was no evidence of fetotoxicity or teratogenicity
    at the highest dose tested, 12 mg/kg bw per day (BASF, 1979).

    (f)  Genotoxicity

         The results of tests for the genotoxicity of chlormequat are
    summarized in Table 2.

         All of the assays for gene mutation carried out in bacteria and
    mammalian cells gave negative results. No cytogenetic anomalies were
    found in human lymphocytes  in vitro, and unscheduled DNA synthesis
    was not seen in rat hepatocytes. Neither dominant lethal mutation
    nor micronuclei were seen in mice  in vivo, and no chromosomal
    aberrations occurred in rats.

    (g)  Special studies

    Skin and eye irritation and skin sensitization

         The irritancy of chlormequat chloride to the skin was tested in
    Vienna white rabbits. About 0.5 ml of the test material (purity
    unspecified) was applied to intact and abraded sites on each of six
    rabbits and left in place for 24 h under an occlusive dressing. At
    the intact sites, erythema and oedema were observed at the end of
    the application period, but these signs were almost fully reversed
    within two days. More severe signs were observed at the abraded
    sites; the signs were only partly reversible, and superficial
    necrosis was seen in three animals after three days (Gelbke, 1978).

         About 500 mg of the same material were tested in the same way
    in New Zealand white rabbits. Dermal reaction at the treatment sites
    was limited to very slight or well-defined erythema, which was
    evident only at the end of the application period. All reaction had
    resolved completely within 72 h of treatment (Buch & Gardner, 1980).

         In another experiment in New Zealand white rabbits, about 0.5
    ml of chlormequat chloride (a technical material consisting of a
    66.1% aqueous solution) was applied to intact sites on each of six
    rabbits and left in place for 4 h under an occlusive dressing.
    Dermal reaction at the treatment sites was limited to barely
    perceptible or slight erythema, which was evident in three animals 1
    h after treatment and in one animal at 24 h. All reaction had
    resolved completely within 48 h of treatment (Fischer  et al.,
    1990b).


    
    Table 2.  Results of tests for the genotoxicity of chlormequat
                                                                                                                  
    End-point        Test system            Concentration         Purity     Results    Reference
                                            of chlormequat        (%)
                                                                                                                  

    In vitro
    Reverse          S. typhimurium TA98,   Up to 5000 g/plate   66.1b      Negative   Traul & Mulligan, 1990
     mutationa       100,1535, 1537, 1538
                     E. coli WP2uvr A-

    Reverse          S. typhimurium TA98,   Up to 2500 g/plate   92.4       Negative   Zeller & Engelhardt, 1979
     mutationa       100, 1535, 1537, 1538

    Reverse          Chinese hamster ovary  Up to 5000 g/ml      66.1b      Negative   Traul & Johnson, 1990
     mutationa       cells, hprt locus

    Reverse          Chinese hamster V79    Up to 5000 g/ml      94.5-98.9  Negative   Debets  et al., 1986
     mutationa       cells, hprt locus

    Chromosomal      Human lymphocytes      Up to 5000 g/ml      94.5-98.9  Negative   Enninga  et al., 1987
     aberrationsa

    Unscheduled      Rat hepatocytes        Up to 7.5 l/ml       66.1b      Negative   Pant & Law, 1990
     DNA synthesis

    Unscheduled      Rat hepatocytes        Up to 10 000 nl/ml    72c        Negative   Cifone & Myhr, 1987
     DNA synthesis

    Table 2 (contd)
                                                                                                                  
    End-point        Test system            Concentration         Purity     Results    Reference
                                            of chlormequat        (%)
                                                                                                                  

    In vivo
    Dominant lethal  Male NMRI mice         1 x 261 mg/kg bw      99.6       Negative   Gelbke & Engelhardt,
     mutation                                                                            1979

    Micronucleus     Male and female        Up to 2 x 202.5       94.5-98.9  Negative   Geunard  et al., 1983
     formation       NMRI mice              mg/kg bw

    Chromosomal      Male and female        Up to 1 x 500         66.1       Negative   Sharma & Caterson, 1991
     aberration      Sprague-Dawley         mg/kg bw
                     rats
                                                                                                                  

    a With and without metabolic activation
    b Technical material consisting of 66.1% aqueous solution
    c Technical material consisting of 72% aqueous solution


    
         The irritancy of chlormequat chloride to the eye was tested in
    Vienna white rabbits by applying about 0.1 ml of the test material
    (purity unspecified) to the conjunctival sac of the right eyelids of
    six rabbits. Conjunctival redness was seen in five rabbits 24 h
    after treatment. After 48 h, conjunctival redness was seen in two
    rabbits, one of which had a conjunctival discharge. All reactions
    had resolved 72 h after treatment (Gelbke & Grundler, 1981).

         The irritancy of chlormequat chloride (a technical material
    consisting of a 66.1% aqueous solution) was also tested in New
    Zealand white rabbits. About 0.1 ml was applied to the conjunctival
    sac of the left eyelids of six rabbits and left for 24 h. One hour
    after treatment, slight reactions were seen in all the rabbits.
    These signs had resolved by 48 h in two animals and by four days in
    all rabbits (Lowe & Boczon, 1990).

         The potential of chlormequat to cause delayed contact
    hypersensitivity was tested in albino guinea-pigs by the method of
    Buehler. Induction was performed by applying 0.4 ml of the test
    material to a shaven flank and leaving it for 6 h under an occlusive
    dressing. This process was repeated three times per week for a total
    of nine applications. After a two-week rest period, challenge was
    performed by applying the test material to the opposite flank. No
    erythema or oedema was observed after the challenge, whereas a
    positive control compound (dinitrochlorobenzene) included in the
    study gave the expected results. It was concluded that chlormequat
    is not a skin sensitizer (Ventura & Moore, 1990).

    3.  Observations in humans

    No information was available.

    Comments

         In experiments with 14C-labelled chlormequat in rats, which
    were reported only in summary form, absorption was rapid and
    elimination was essentially complete within 48 h, occurring almost
    entirely via the urine; less than 1% of the administered dose
    remained in the tissues. Accumulation of 15N-labelled material in
    the kidneys was reported, but the experimental details were
    incomplete and detailed evaluation was not possible. The
    biotransformation of chlormequat has been little studied; it was
    suggested that the only metabolites found in rat urine may have been
    other salts of chlorcholine.

         Pharmacological tests in mice, rats, rabbits and cats
    administered chlormequat intravenously revealed a stimulatory effect
    on the parasympathetic nervous system and a myoneural blocking
    action. Further work revealed that chlormequat is a partial agonist
    of the nicotinic acetylcholine receptor; the affinity for muscarinic
    receptors was low and rather unselective.

         Chlormequat was of moderate acute oral toxicity in rats, mice,
    hamsters and guinea-pigs (LD50 = 200-1000 mg/kg bw), but there was
    some indication that rabbits and dogs are more sensitive (LD50 =
    50-80 mg/kg bw). Signs of toxicity may have been associated with
    pharmacological action, and there were no consistent
    treatment-related findings at autopsy. WHO (1992) classified
    chlormequat as slightly hazardous.

         In a recently completed four-week study of dietary toxicity in
    rats, the NOAEL was 1500 ppm, equal to 137 mg/kg bw per day, on the
    basis of reduced body-weight gain and depression of serum creatinine
    concentration. These results are largely in agreement with those of
    older studies in rats of up to 90 days' duration. In dogs, the NOAEL
    in an unsatisfactory two-year study was 300 ppm, equal to 7.5 mg/kg
    bw per day.

         The potential carcinogenicity of chlormequat was investigated
    in dietary studies in rats and mice carried out in the early 1970s.
    Those experiments do not comply with contemporary standards. In
    studies of carcinogenicity in rats and mice reported by the National
    Cancer Institute in the USA in 1979, rats were fed dietary levels of
    0, 1500 or 3000 ppm for 108 weeks and mice were fed dietary levels
    of 0, 500 or 2000 ppm for 102 weeks. There were no signs of reaction
    to treatment and chlormequat was not carcinogenic in either species.

         In a multigeneration study in rats, which was not conducted
    according to currently acceptable scientific standards, chlormequat
    had no effect on reproductive performance at dietary levels up to
    900 ppm; however, histopathological examination revealed giant cells
    in the testicular tubules in 4 of 10 rats of the F3 generation
    treated with 900 ppm and 2 of 10 rats of the F3 generation treated

    with 300 ppm. The report suggests that this finding may be an
    expression of delayed maturation during spermatogenesis; the Meeting
    found the significance of this finding difficult to assess, but the
    NOAEL may thus be 100 ppm, equivalent to 5 mg/kg bw per day.

         The teratogenic potential of chlormequat has been investigated
    in mice (following administration by intraperitoneal injection,
    gavage and via the diet), in rats by dietary administration and in
    hamsters and rabbits by gavage. Many of the study reports were
    available only in summary form, and detailed evaluation was not
    possible. In a dietary study in mice, the number of malformations in
    animals fed 10 000 ppm on days 1-15 of gestation or 25 000 ppm on
    days 11-15 was reported to be slightly higher than that seen in
    controls; however, the exact significance of this observation was
    difficult to assess. In hamsters, malformations and evidence of
    delayed development were seen following three doses of 100 mg/kg bw
    per day (on days 7-9 of gestation) or a single dose of 200, 300 or
    400 mg/kg bw on day 8. Evidence of maternal and fetal toxicity was
    also obtained at these doses. Further evaluation of these data in
    hamsters was not possible, owing to lack of detail in the
    publication. A well-conducted study in which rabbits were dosed
    orally with up to 12 mg/kg bw per day was available for detailed
    review. Signs of maternal toxicity were seen at the highest dose
    level, but there was no evidence of teratogenicity or fetotoxicity.

         Chlormequat has been adequately tested for genotoxicity  in
     vitro and  in vivo in a range of assays. The Meeting concluded
    that it was not genotoxic.

         Although review of the available information raised no
    suspicion of significant toxicological concern, the Meeting
    concluded that in view of the inadequacy of the database in
    comparison with acceptable contemporary standards, it was impossible
    to maintain the ADI for chlormequat.

    Toxicological evaluation

    Levels that cause no toxic effect

         Mouse:    2000 ppm, equal to 286 mg/kg bw per day (102-week
                   study of carcinogenicity)

         Rat:      1500 ppm, equal to 137 mg/kg bw per day (four-week
                   study of toxicity)
                   3000 ppm, equivalent to 150 mg/kg bw per day
                   (108-week study of carcinogenicity)

         Rabbit:   6 mg/kg bw per day (maternal toxicity in a study of
                   teratogenicity)
                   12 mg/kg bw per day (fetotoxicity and teratogenicity
                   in a study of teratogenicity)

    Studies that would provide information useful for continued
    evaluation of the compound

         Updating of the database to acceptable standards

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    See Also:
       Toxicological Abbreviations
       Chlormequat (AGP:1970/M/12/1)
       Chlormequat (WHO Pesticide Residues Series 2)
       Chlormequat (Pesticide residues in food: 1976 evaluations)
       Chlormequat (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental)
       Chlormequat (JMPR Evaluations 1999 Part II Toxicological)