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    Pesticide residues in food -- 1999



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    with the support of the International Programme
    on Chemical Safety (IPCS)



    Toxicological evaluations




    Joint meeting of the
    FAO Panel of Experts on Pesticide Residues
    in Food and the Environment
    and the
    WHO Core Assessment Group

    Rome, 20-29 September 1999

    CHLORMEQUAT (addendum)

    First draft prepared by
    T.C. Marrs
    Department of Health, London, United Kingdom


            Explanation 
            Evaluation for acute reference dose 
                Short-term studies of toxicity
                Long-term studies of toxicity
                Reproductive toxicity
            Comments
            Toxicological evaluation
            References


    Explanation

         Chlormequat was evaluated by the JMPR in 1970, 1972, 1994, and
    1997 (Annex 1, references 14, 18, 71, and 80). In 1972, an ADI of
    0-0.05 mg/kg bw was established on the basis of a study of
    reproductive toxicity in rats, but in 1994 the Meeting withdrew this
    ADI on the grounds that the data package was inadequate. In 1997, an
    ADI of 0-0.05 mg/kg bw was allocated. The compound was considered by
    the present Meeting to determine an acute reference dose, as requested
    at the Thirty-first Session of the Codex Committee on Pesticide
    Residues (Codex Alimentarius Commission, 1999), and short-term studies
    of toxicity in dogs, long-term studies of toxicity in rats and mice, a
    study of developmental toxicity in rabbits, and a two-generation study
    in rats were reviewed.

    Evaluation for Acute Reference Dose

    1.  Short-term studies of toxicity

    Dogs

         A 1-year study of toxicity was carried out in groups of five male
    and five female beagles fed diets containing chlormequat chloride
    (purity, 67.4%) at doses of 0, 150, 300, or 1000 ppm, equal to 0, 4.7,
    9.2, and 31 mg/kg bw per day in males and 0, 5.2, 10, and 32 mg/kg bw
    per day in females. The animals were initially observed daily and
    specifically for diarrhoea, salivation, lachrymation, and
    incoordination before and 2, 4 and 6 h after feeding. After study day
    91, the animals were examined weekly. Food consumption was determined
    daily, while the animals were weighed weekly. Clinical chemical and
    haematological examinations were carried out before the start of
    administration of the test material and three times during the study.
    Ophthalmological examinations were performed before the start of the
    study and near its end. At autopsy, the organs were examined grossly
    and histopathologically. Of the animals at the highest dose, one male
    died at 42 days and one female at 20 days; diarrhoea was seen in all

    males and in three females at this dose, starting in the first week.
    During the fourth week, the dog that died showed vomiting, spasm,
    staggering, emaciation, and apathy, and pulmonary oedema and atrophy
    of the thymus were found  post mortem. This animal also showed some
    abnormalities in clinical chemistry and haematology (increased
    prothrombin time, alanine aminotransferase and alkaline phosphatase
    activity, and urea, creatinine, total protein, and cholesterol
    concentrations and decreased polymorph and lymphocyte counts). The
    bitch that died at 20 days also had pulmonary oedema and thymic
    changes (multiple haemorrhages). Diarrhoea was also seen in two males
    at 300 ppm in the first and second weeks of the study, and salivation
    was seen at this dose, starting at week 1 and intermittently
    thereafter. No treatment-related effects were seen at 150 ppm, the
    NOAEL, equal to 4.7 mg/kg bw per day, on the basis of diarrhoea and
    salivation at the next highest dose. Because these effects occurred
    early in the study, they were considered relevant to setting an acute
    reference dose. No abnormality was observed on ophthalmological
    examination and, in the animals that survived to term, no gross or
    histopathological abnormality was seen that was related to the
    treatment ( Mellert et al., 1993).

    2.  Long-term studies of toxicity

    Mice

         Groups of 50 male and 50 female B6C3F1/CrlBr (VAF) mice were
    given diets containing chlormequat chloride (67.4% pure) at doses of
    0, 150, 600, or 2400 ppm for 100 weeks, equal to intakes of
    chlormequat of 0, 21, 84, and 340 mg/kg bw per day in males and 0, 23,
    91, and 390 mg/kg bw per day in females. Satellite groups comprising a
    further 10 mice of each sex received chlormequat at the same dietary
    concentrations for 52 weeks, with measured intakes of chlormequat of
    0, 23, 89, and 355 mg/kg bw per day for males and 0, 28, 109, and 445
    mg/kg bw per day for females. The animals were inspected daily and
    more thoroughly weekly for clinical status. Body weight and food
    consumption were recorded weekly during the first 14 weeks of the
    study and thereafter every 4 weeks. The animals in the satellite group
    were killed at 52 weeks and those in the main group at 110 weeks.
    Blood was taken from all sacrificed animals and differential blood
    counts carried out. The mice were examined  post mortem both grossly
    and histopathologically. No clinical signs were observed that appeared
    to be related to treatment. Survival was not affected by the test
    material at any dose. Animals of each sex killed at 52 weeks showed a
    reduction in weight gain at the highest dose at some times, but the
    significance of this finding is hard to assess because of the small
    group sizes. No such reduction was seen in the main groups, killed at
    110 weeks. No significant differences in feed consumption were seen
    between groups. Minor inter-group differences in leukocyte counts
    lacked consistency and are unlikely to be of biological significance.
    No treatment-related differences in organ weights were seen between
    groups. The only organ-specific findings that appeared to be related
    to treatment were found in the female reproductive tract at autopsy,
    which were increased incidences of ovarian tubular downgrowth and of

    cystic endometrial hyperplasia at the two higher doses. There was no
    treatment-related increase in tumour incidence. The NOAEL was 150 ppm,
    equal to 23 mg/kg bw per day, on the basis of histopathological
    effects in the uterus and ovaries at the next highest dose (Mellert et
    al., 1994).

    Rats

         Groups of 20 male and 20 female Wistar rats received diets
    containing chlormequat (purity, 67.4%) as the chloride for 18 months
    at nominal concentrations of 0, 280, 940, or 2800 ppm (calculated
    erroneously on the basis of 72% purity), equal to 0, 13, 43, and 140
    mg/kg bw per day in males and 0, 17, 56, and 170 mg/kg bw per day in
    females. The rats were examined daily and more thoroughly once a week.
    Ophthamological examinations were carried out at the beginning and end
    of the study. Food consumption and body weight were determined weekly
    for the first 14 weeks and thereafter every 4 weeks. Clinical
    chemistry, haematology, and urinalysis were performed 3, 6, 12, and 18
    months after the start of the study. The animals were killed after
    18 months and examined grossly and histopathologically. No
    treatment-related clinical signs were observed in any group, and
    ophthalmological examination showed no adverse effects. The mortality
    rate was unaffected by treatment. The body-weight gain of males was
    reduced throughout the study and that of females at the highest dose
    from the 58th week, so that by the end of the study the weight of the
    males was 18% less than that of concurrent controls and that of the
    females was 10% less. The feed consumption of males was reduced from
    week 54 and that of females from time to time. The lower doses had no
    effect on weight gain or feed consumption. Although some differences
    in clinical chemical and haematological parameters were seen between
    groups, they were marginal, sporadic, and showed no relation to dose;
    they were therefore considered not to be of toxicological
    significance. In particular, no changes were seen in plasma,
    erythrocyte, or brain cholinesterase activity. At autopsy, the only
    finding attributable to treatment was decreased body-weight gain in
    animals at the highest dose by comparison with controls and consequent
    differences in the relative weights of the kidney, brain, and liver.
    No treatment-related histopathological changes were found. The NOAEL
    was 940 ppm, equal to 43 mg/kg bw per day (Schilling et al., 1992).

         Chlormequat chloride (technical grade; purity, 67.4%) was
    administered to groups of 50 male and 50 female Wistar rats at a
    nominal dietary concentration of 0, 280, 940, or 2800 ppm for 2 years.
    The mean daily intakes were 0, 13, 42, and 120 mg/kg bw per day for
    males and 0, 16, 55, and 170 mg/kg bw per day for females. The animals
    were observed daily and inspected thoroughly weekly. Body weight and
    intake of food were determined weekly for the first 14 weeks and every
    4 weeks thereafter. At termination, the survivors were examined
    grossly, and selected tissues were taken for histopathological
    examination. Clinical chemistry, haematology, and urinalysis were
    undertaken on 20 animals of each sex per group, and the brains of 20
    animals of each sex per group were taken for measurement of
    acetylcholinesterase activity. No treatment-related clinical signs

    were seen, and the mortality rate was not affected. Reduced
    body-weight gain and food consumption were seen in animals of each sex
    at the highest dose. Weight gain was decreased throughout the study,
    by 14% in males and 22% in females by comparison with concurrent
    controls. The food consumption of males was reduced throughout the
    study and that of females during the latter part. Some inter-group
    differences were seen in clinical chemical and haematological
    findings, but they were inconsistent and probably not related to
    treatment. Chlormequat did not affect plasma, erythrocyte, or brain
    cholinesterase activity. The material was not carcinogenic, and no
    treatment-related histopathological changes were seen. The NOAEL was
    940 ppm, equal to 42 mg/kg bw per day, on the basis of reduced weight
    gain and feed consumption at the highest dose (Mellert et al., 1992).

    3.  Reproductive toxicity

    Rats

         In a two-generation study, groups of 24 male and 24 female rats
    were given diets containing chlormequat (purity, 67.4%) at
    concentrations of 0, 300, 900, or 2700 ppm, equal to 0, 29, 86, and
    250 mg/kg bw in males and 0, 23, 69, and 230 in females. At least 70
    days after the beginning of treatment, the F0 parents were mated to
    produce the F1a litters and, subsequently, the F1b litters, only
    the latter being retained until weaning. Groups of 24 males and 24
    females from the F1a litters were used as the F1 parents and were
    given chlormequat as above to produce the F2 litters. The F1
    adults and the F2 weanlings were killed at the end of the study. All
    animals, including pups, were examined daily. The feed consumption of
    the F0 and F1 parents was determined weekly before mating and
    during gestation and lactation. Body weights were determined weekly,
    but during gestation and lactation the body weights of the females was
    determined on days 0, 7, 14, and 20 of gestation, on the day of
    parturition, and on days 4, 7, 14, and 21 after delivery. Clinical
    chemical and haematological variables were measured in 12 F1 animals
    in each group. All parental animals were examined after death both
    grossly and histopathologically. The clinical signs seen at the
    highest dose included tremor. The body-weight gain of F0 females at
    900 ppm was slightly reduced while they were suckling the F1a pups,
    and reduced body-weight gain was seen in animals of each sex and of
    both parental generations at the highest dose. At this dose, feed
    consumption and creatinine concentration were also reduced in F1
    females, which had decreased mean numbers of pups per dam and of total
    delivered pups. The NOAEL for reproductive toxicity was 900 ppm, equal
    to 69 mg/kg bw per day, and that for systemic toxicity was 300 ppm,
    equal to 23 mg/kg bw per day, on the basis of reduced body-weight gain
    (Hellwig & Hildebrand, 1993).

    Rabbits

         In a study of developmental toxicity stated to have been
    conducted in accordance with 1966 US Food and Drug Administration
    guidelines and the 1975 Association of the British Pharmaceutical

    Industry guidelines, inseminated Himalayan ChBB:HM rabbits were given
    chlormequat chloride (purity, 99%) at doses of 0, 1.5, 3, 6, or 12
    mg/kg bw per day by gavage on days 6-18 after insemination. The group
    sizes were not the same at each dose, there being 15 controls, 15
    animals at 1.5 mg/kg bw per day, 21 at 3 and 6 mg/kg bw per day, and
    14 at the highest dose. The animals were killed 28 days after
    insemination; the fetuses were removed, sexed, and weighed, and the
    placentas were weighed. The fetuses were eviscerated and their organs
    examined macroscopically. Additionally, their skeletons were X-rayed,
    and the heads were fixed and sectioned for examination. The two higher
    doses caused rapid respiration, salivation, and apathy in one animal
    in each group. These effects were observed on day 13 after
    insemination in animals at 6 mg/kg bw per day and on day 14 after
    insemination in those at 12 mg/kg bw per day. One animal at 3 mg/kg bw
    per day and two at 6 mg/kg bw per day died. The body-weight gain of
    rabbits at the highest dose was decreased, and feed consumption was
    affected in all treated groups. Two animals at 6 mg/kg bw per day and
    one at 12 mg/kg bw per day aborted. No treatment-related effects were
    seen on the numbers of corpora lutea or implants or the weight or sex
    of the fetuses. No teratogenic effects were seen. It might be
    concluded that there was no NOAEL for maternal toxicity, as feed
    consumption was reduced at all doses; however, this effect may have
    been due to reduced palatability. The NOAEL for maternal toxicity was
    6 mg/kg bw per day on the basis of decreased weight gain at 12 mg/kg
    bw per day, provided that the single instance of rapid respiration,
    salivation, and apathy at 6 mg/kg bw per day can be ignored. This
    study was difficult to interpret: the clinical signs observed at 6 and
    12 mg/kg bw per day were consistent with a cholinergic agonist effect,
    but comparison of the findings in these two groups showed little
    evidence of a dose-response relationship (BASF, 1979).

    Comments

         The oral LD50 of chlormequat was 200-1000 mg/kg bw in rodents
    and > 800 mg/kg bw in monkeys but was much lower, approximately 50
    mg/kg bw, in cats and dogs.

         In a 1-year study in dogs given chlormequat chloride (purity,
    67.4%) in the diet at concentrations of 0, 150, 300, or 1000 ppm,
    diarrhoea was seen at 300 ppm in two males during the first and second
    weeks of the study, and salivation was also seen at this dose,
    starting at week 1 and intermittently thereafter. Consequently, the
    NOAEL was 150 ppm, equal to 4.7 mg/kg bw per day, on the basis of
    diarrhoea and salivation at the next highest dose. Because these
    findings were seen early in the study, they were considered relevant
    to setting an acute reference dose.

         Three long-term studies -- two in rats and one in mice -- showed
    that chlormequat was not carcinogenic. None of the studies showed
    acute effects.

         In a two-generation study of reproductive toxicity in rats given
    chlormequat in the diet, clinical signs such as tremor were seen at
    the highest dose (250 mg/kg bw per day). Reproductive toxicity was
    also seen at this dose and systemic toxicity at the intermediate dose
    (69 mg/kg bw per day). The NOAEL for systemic toxicity was 23 mg/kg bw
    per day, and the NOAEL for reproductive toxicity was 69 mg/kg bw per
    day.

         In a study of developmental toxicity in rabbits given chlormequat
    chloride at doses of 0, 1.5, 3, 6, or 12 mg/kg bw per day by gavage on
    days 6-18 after insemination, the body-weight gain of animals at the
    highest dose was decreased and the feed consumption of all treated
    animals was affected, possibly because of reduced palatability. The
    NOAEL for maternal toxicity was 6 mg/kg bw per day and that for
    developmental toxicity was 12 mg/kg bw per day, the highest dose
    tested. 

         An acute reference dose of 0.05 mg/kg bw was established on the
    basis of the NOAEL of 4.7 mg/kg bw per day in the 1-year study in
    dogs, as the clinical signs that were found were considered to be
    acute. A 100-fold safety factor was used.

    Toxicological evaluation

     Levels that cause no toxic effects 

    Mouse:    150 ppm, equal to 23 mg/kg bw per day (2-year study of
              toxicity and carcinogenicity)

    Rat:      940 ppm, equal to 42 mg/kg bw per day (2-year study of
              toxicity and carcinogenicity)

              900 ppm, equal to 69 mg/kg bw per day (reproductive toxicity
              in a two-generation study of reproductive toxicity)

              300 ppm, equal to 23 mg/kg bw per day (systemic toxicity in
              a two-generation study of reproductive toxicity)

    Rabbit:   6 mg/kg bw per day (maternal toxicity in a study of
              developmental toxicity)

              12 mg/kg bw per day (developmental toxicity in a study of
              developmental toxicity)

    Dog:      150 ppm, equal to 4.7 mg/kg bw per day (1-year study of
              toxicity)

     Estimate of acute reference dose 

         0.05 mg/kg bw

    References

    BASF (1979) Study of the prenatal toxicity of
         2-chloroethyltrimethylammonium chloride (chlormequat chloride) on
         rabbits (Document 79/051), Department of Toxicology, BASF,
         Ludwigshafen, Germany. Unpublished paper dated 16 March 1979,
         submittedto WHO by BASF, Ludwigshafen, Germany. 

    Codex Alimentarius Commission (1999)  Report of the Thirty-first 
          Session of the Codex Committee on Pesticide Residues, The 
          Hague, 12-17 April 1999. Rome, Food and Agricultural
         Organization of the United Nations (unpublished FAO document
         ALINORM 99/24A).

    Hellwig, J. & Hildebrand, B. (1993) Reproduction toxicity study of
         chlormequat chloride in rats; continuous dietary administration
         over two generations (Document 93/10982), Department of
         Toxicology, BASF, Ludwigshafen, Germany. Unpublished report
         submitted to WHO by BASF, Ludwigshafen, Germany.

    Mellert, W., Deckardt, K., Kaufmann, W. & Hildebrand, B. (1992) Study
         of the potential carcinogenic effect of chlormequat-chloride in
         Wistar rats; administration via the diet over 24 months (Document
         92/11094), Department of Toxicology, BASF, Ludwigshafen, Germany.
         Unpublished report submitted to WHO by BASF, Ludwigshafen,
         Germany.

    Mellert, W., Deckardt, K., Kaufmann, W., Pappritz, G. & Hildebrand, B.
         (1993) Study of the toxicity of chlormequat-chloride in beagle
         dogs; administration via the diet over 12 months (Document
         93/11109), Department of Toxicology, BASF, Ludwigshafen, Germany.
         Unpublished report submitted to WHO by BASF, Ludwigshafen,
         Germany.

    Mellert, W., Deckardt, K., Kaufmann, W. & Hildebrand, B. (1994) Study
         of the potential carcinogenic effects of chlormequat-chloride in
         B6C3F1 mice; dietary administration for 100 weeks (Document
         94/10024), Department of Toxicology, BASF, Ludwigshafen, Germany.
         Unpublished report submitted to WHO by BASF, Ludwigshafen,
         Germany.

    Schilling, K., Deckhardt, K., Kaufman, W. & Hildebrand, B. (1992)
         Study of the chronic toxicity of chlormequat-chloride in Wistar
         rats; administration via the diet over 18 months (Document
         92/10627), Department of Toxicology, BASF, Ludwigshafen, Germany.
         Unpublished report submitted to WHO by BASF, Ludwigshafen,
         Germany. 
    


    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: 1994 evaluations Part II Toxicology)
       Chlormequat (Pesticide residues in food: 1997 evaluations Part II Toxicological & Environmental)