Pesticide residues in food -- 1999
Sponsored jointly by FAO and WHO
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)