PESTICIDE RESIDUES IN FOOD - 1997
Sponsored jointly by FAO and WHO
with the support of the International Programme
on Chemical Safety (IPCS)
TOXICOLOGICAL AND ENVIRONMENTAL
EVALUATIONS 1994
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Core Assessment Group
Lyon 22 September - 1 October 1997
The summaries and evaluations contained in this book are, in most
cases, based on unpublished proprietary data submitted for the purpose
of the JMPR assessment. A registration authority should not grant a
registration on the basis of an evaluation unless it has first
received authorization for such use from the owner who submitted the
data for JMPR review or has received the data on which the summaries
are based, either from the owner of the data or from a second party
that has obtained permission from the owner of the data for this
purpose.
CHLORMEQUAT (addendum)
First draft prepared by
J.-J. Larsen
Institute of Toxicology, Danish Veterinary and Food Administration,
Ministry of Food, Agriculture and Fisheries, Soborg, Denmark
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
Genotoxicity
Reproductive toxicity
Special studies: Dermal and ocular irritation and
dermal sensitization
Comments
Toxicological evaluation
References
Explanation
Chlormequat (2-chloroethyltrimethylammonium chloride) was
evaluated by the Joint Meeting in 1970, 1972, and 1994 (Annex 1,
references 14, 18, and 71). In 1972, an ADI of 0-0.05 mg/kg bw was
established on the basis of the NOAEL in a study of reproductive
toxicity in rats. In 1994, this ADI was withdrawn owing to the
inadequacy of the database in comparison with acceptable contemporary
standards. The compound was reviewed at the present Meeting in
response to a request from the manufacturer. New data on the
absorption, distribution, excretion, and biotransformation of
chlormequat and on its long-term toxicity in rats and dogs,
carcinogenicity in mice and rats, reproductive toxicity in rats, and
skin sensitization potential in guinea-pigs were reviewed.
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 (61%) of an oral dose of 14C-chlormequat administered
to male rats was excreted in the urine within 4 h, and 96% was
eliminated within 47 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 on chlormequat (Blinn, 1967).
In a second study, rats were given a single oral dose of 60 mg
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 15N-chlormequat. The compound was found in the milk and urine
3 h after administration; most (490 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).
In a more recent study, 136 male and female rats were treated
intravenously with a single dose of 0.1 mg/kg bw or orally with a
single dose of 0.5 or 30 mg/kg bw 14C-chlormequat (radiochemical
purity, 96.8%). Urine, faeces, organs, bile, and expired air were
collected from all animals at various intervals up to 168 h after
treatment; 82-103% of the radiolabel was recovered. Most was excreted
during the first 24 h. More than 85% of the radiolabel was excreted
with urine and < 6% with faeces; < 1% was eliminated as volatile
compounds. The maximum blood level was reached about 2 h after oral
administration of either the low or the high dose. Less than 1% of the
radiolabel was eliminated in bile during the first 24 h. The maximum
radiolabel appeared 2-5 h after oral administration. Little radiolabel
was found in organs or tissues after 168 h, the highest concentrations
being found in liver and kidney (Giese & Hoffmann, 1989).
(b) Biotransformation
Only chlormequat and two other compounds, which may have been
other salts of chlorcholine, were reported in the urine of rats that
had received 200 mg/kg bw of chlormequat orally. Choline itself was
not identified (Bronisz & Romanowski, 1968).
A group of 74 male and females rats were given a single
intravenous dose of 0.1 mg/kg bw or a single oral dose of 0.5 mg/kg bw
(with or without preteatment with unlabelled chlormequat for 14 days)
or 30 mg/kg bw 14C-chlormequat (radiochemical purity, 96.8%). Urine,
faeces, bile, liver, kidney, gastrointestinal tract, brain, muscle,
spleen, bone, lung, heart, fat, testes, and uterus were examined for
radiolabel for up to 168 h after treatment. Expired air from two males
was examined within 0-24 h. A total of 81% of the dose of radiolabel
was excreted during the test period. In all cases, > 85% of the
radiolabel was found in urine and < 5% in faeces. Chlormequat was
excreted mainly unmetabolized. A very polar but unidentified
metabolite was found in faeces (Giese & Kohl, 1989).
(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 given 7.4
mg/kg bw. In cats, there was mild inhibition of the vasopressor effect
of noradrenaline 30 min after administration of 1 mg/kg bw
chlormequat. In rabbits, neuromuscular junctions were blocked by doses
of > 1 mg/kg bw; this effect was counteracted by administration of
10 mg/kg bw D-tubocurarine and potentiated by administration of 1
mg/kg bw neostigmine. Coagulation of rat blood was unaffected by
concentrations up to 3 mg/ml. In dogs, doses of > 3 mg/kg bw 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, rat heart, and 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,
chromodacryorrhoea, decreased activity, tremors, diuresis, and
piloerection. Death 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.
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)
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
Rabbits, cats 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.
(b) Short-term toxicity
Rats
Two short-term studies of toxicity are described in the 1972 JMPR
monograph addendum (Annex 1, 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 0, 25, 50, or 100 mg/kg bw per day) for 29 days. There were no
deaths 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 0, 10, 30, or 90 mg/kg bw per day) for 90 days. There
were no deaths, no clinical signs of reaction to treatment, and no
treatment-related changes in blood chemistry. The 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 more 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 0, 46,
140, 270, or 410 mg/kg bw per day) for four weeks. The test material
was a technical-grade formulation of 66.7% purity, 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. Tests of
locomotor activity and swimming, gross pathological examinations,
organ weighing, and histopathological examination revealed no reaction
to treatment. The NOAEL was 1500 ppm, equal to 140 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 occluded dermal 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 weights, and
histopathology revealed no reaction to treatment (Buch & Finn, 1981).
Dogs
In an experiment for which no detailed report was available to
the Meeting (Annex 1, 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, 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 histopathological examination 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 0, 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 hindlimb weakness were seen in some animals receiving
1000 ppm, and one male died after 22 days and one female after 38
days. The deaths were considered by the authors to be secondary to the
clinical signs of hindlimb 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 absence of further investigation into the signs of hindlimb
weakness precluded establishment of a definitive NOAEL (Oettel &
Sachsse, 1967).
Groups of five male and five female beagle dogs were given
technical-grade chlormequat (purity, 67.4%) mixed in the diet at doses
of 0, 150, 300, or 1000 ppm, equal to 0, 4.7, 9.2, or 31 mg/kg bw per
day in males and 0, 5.2, 10, or 32 mg/kg bw per day in females, for 12
months. Food consumption was determined daily and body weight once a
week. In addition to clinical, hematological, and ophthalmological
examinations, urinalysis and neurofunctional examinations were carried
out. At the end of study, all animals were subjected to gross
pathological and histopathological examination. Animals at 1000 ppm
had diarrhoea, vomiting, salivation, apathy, and other severe clinical
symptoms as well as many changes in clinicochemical and haematological
parameters. Two dogs at this dose died. Diarrhoea, vomiting, and
salivation were also seen at 300 ppm. The NOAEL was 150 ppm, equal to
4.7 mg/kg bw per day, on the basis of diarrhoea, vomiting, and
salivation (Mellert et al., 1993).
(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 0 or 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 tissues from animals in which a
treatment-related effect was seen. The incidence of benign lung
tumours was higher (20/52) in treated males than in controls (10/51)
but 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 fed diets
containing chlormequat (purity, 97-98%) at 0, 500, or 2000 ppm (equal
to 0, 70, or 290 mg/kg bw per day) for 102 weeks. The dietary levels
were set on the basis of the results of an eight-week study with doses
of 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 concluded that there was no clear evidence for the
carcinogenicity of chlormequat in these mice (National Cancer
Institute, 1979).
Groups of 50 male and 50 female B6C3F1 mice were fed diets
containing technical-grade chlormequat (purity, 67.4%) at 0, 150, 600,
or 2400 ppm, equal to 0, 21, 84, or 340 mg/kg bw per day in males and
0, 23, 91, or 390 mg/kg bw per day in females, for 110 weeks.
Satellite groups of 10 male and 10 female mice at each dose were
killed after 52 weeks. Food consumption and body weight were
determined weekly during the first 14 weeks and every four weeks
thereafter. The health of the animals was checked daily, and clinical
signs were followed up thoroughly once a week. All animals that died
or were killed in a moribund condition during the study or were killed
terminally were subjected to gross pathological and histopathological
examinations. The body weight of animals in the satellite goup at 2400
ppm was reduced. The incidence and severity of tubular down-growth in
the ovaries and the incidence of endometrial hyperplasia were
increased in mice at 600 and 2400 ppm. There was no significant
increase in the incidence of benign or malignant tumours. The NOAEL
was 150 ppm, equal to 21 mg/kg bw per day, on the basis of tubular
down-growth in the ovaries and endometrial hyperplasia (Mellert et
al., 1994).
Rats
In 1994, 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 0, 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
were unchanged. 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 profiles in treated and control animals were
indistinguishable. Detailed evaluation of this report was not possible
(Oettel & Froberg, 1967).
A summary of another long-term study was available 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 0, 25, 50, and 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 unaffected. Haematological examination, limited analyses of
blood chemistry, and urinalysis carried out after three and 12 months
and before termination revealed no indication of 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 profiles in treated and control animals were 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 fed
diets containing chlormequat (purity, 97-98%) at 0, 1500, or 3000 ppm
(equivalent to 0, 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
study with doses of 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. The 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); however, the authors concluded that
there was no clear evidence for the carcinogenicity of chlormequat in
these rats (National Cancer Institute, 1979).
Groups of 20 male and 20 female Wistar rats were fed diets
containing technical-grade chlormequat (purity, 67.4%) at 0, 280, 940,
or 2800 ppm, equal to 0, 13, 43, or 140 mg/kg bw per day in males and
0, 17, 56, or 170 mg/kg bw per day in females, for 78 weeks. Food
consumption and body weight were determined once a week during the
first 14 weeks and then at four-week intervals. The animals were
observed daily and inspected thoroughly once a week for clinical
signs. At the start and end of the study, controls and those at the
highest dose were examined for ophthalmological signs. All animals
were subjected to haematological examination, blood chemical analyses,
and urinalysis at 3, 6, 12, and 18 months. All rats still alive at the
end of study and rats that died intercurrently were subjected to gross
pathological and histopathological examination. Statistically
significant reductions in body weight (18% in males and 10% in
females) and a minor reduction in food intake were observed in animals
at 2800 ppm. Pathological examination revealed no substance-induced
changes in any organ. Higher incidences of tubular mineralization and
tubular atrophy in the kidneys of treated males were considered to be
toxicologically insignificant. Tumour incidences were not enhanced.
The NOAEL was 940 ppm, equal to 43 mg/kg bw per day, on the basis of
reduced body weight in males (Schilling et al., 1992).
Groups of 50 male and 50 female Wistar rats were fed diets
containing technical-grade chlormequat (purity, 67.4%) at 0, 280, 940,
or 2800 ppm (equal to 0, 13, 42, or130 mg/kg bw per day in males and
0, 16, 55, and 170 mg/kg bw per day in females) for two years. Food
consumption and body weight were determined once a week during the
first 14 weeks and then at four-week intervals. The animals were
observed daily and inspected thoroughly once a week for clinical
signs. At the end of the study, all surviving rats (40 controls, 34 at
280 ppm, 36 at 940 ppm, and 32 at 2800 ppm) were subjected to
haematological examination, blood chemical analysis, and urinalysis
and then to gross pathological and histopathological examination.
Reduced body weights (14% in males and 22% in females) and food
consumption (10% in males and 6% in females) were observed in animals
at 2800 ppm at the end of the study. No signs of carcinogenicity were
observed. The NOAEL was 940 ppm, equal to 42 mg/kg bw per day, on the
basis of reduced body weight (Mellert et al., 1992).
(d) 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.
(e) Reproductive toxicity
Mice
The results of three experiments in mice were summarized in the
1972 monograph addendum (Annex 1, 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 killed 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
0, 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 killed 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 slightly more 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 0, 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 fertility, and no
evidence of teratogenicity was seen in the offspring (Shaffer, 1970).
Rats
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 0, 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
Table 2. Results of tests for the genotoxicity of chlormequat
End-point Test system Concentration Purity Result Reference
of chlormequat (%)
In vitro
Reverse S. typhimurium TA98, < 5000 µg/plate 66.1a Negativeb Traul & Mulligan
mutation 100, 1535, 1537, 1538 (1990)
E. coli WP2uvrA
Reverse S. typhimurium TA98, < 2500 µg/plate 92.4 Negativeb Zeller & Engelhardt
mutation 100, 1535, 1537, 1538 (1979)
Reverse Chinese hamster < 5000 µg/ml 66.1a Negativeb Traul & Johnson
mutation ovary cells, hprt locus (1990)
Reverse Chinese hamster V79 < 5000 µg/ml 94.5-98.9 Negativeb Debets et al. (1986)
mutation cells, hprt locus
Chromosomal Human lymphocytes < 5000 µg/ml 94.5-98.9 Negativeb Enninga et al. (1987)
aberration
Unscheduled Rat hepatocytes < 7.5 µg/ml 66.1a Negative Pant & Law (1990)
DNA synthesis
Unscheduled Rat hepatocytes < 10 000 nl/ml 72c Negative Cifone & Myhr (1987)
DNA synthesis
In vivo
Dominant Male NMRI mice 1 × 261 mg/kg bw 99.6 Negative Gelbke & Engelhardt
lethal mutation (1979)
Micronucleus Male and female < 2 × 202.5 94.5-98.9 Negative Geunard et al. (1983)
formation NMRI mice mg/kg bw
Chromosomal Male and female < 1 × 500 66.1 Negative Sharma & Caterson
aberration Sprague-Dawley rats mg/kg bw (1991)
a Technical material consisting of 66.1% aqueous solution
b With and without metabolic activation
c Technical material consisting of 72% aqueous solution
malformations occurred that could be attributed to treatment.
Histopathological examination of 10 rats of each sex of the F3
generation at each dose 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).
In a study described in the 1972 monograph addendum (Annex 1,
reference 19), groups of pregnant rats were fed chlormequat at dietary
levels of 0, 1000, or 5000 ppm (equivalent to 0, 50, or 250 mg/kg bw
per day) on days 1-21 of gestation. When the animals were killed the
day before parturition, no teratogenic effects were observed (Shaffer,
1970).
In a two-generation study of reproductive toxicity,
technical-grade chlormequat (purity, 67.4%) was administered to groups
of 24 male and 24 female Wistar rats at dietary levels of 0, 300, 900,
or 2700 ppm (equal to 0, 29, 86, or 250 mg/kg bw per day in males and
0, 23, 69, or 230 mg/kg bw per day in females). At least 70 days after
the beginning of treatment, F0 animals were mated in order to produce
an F1a litter and subsequently remated to produce an F1b litter.
Groups of 24 males and 24 females from the F1 litter were selected as
the F1 parents and kept on the diet for at least 98 days before they
were mated to produce an F2 litter. The study was terminated after
weaning of the F2 litter. All animals were inspected daily, and the
food consumption of the F0 and F1 parents was determined weekly.
Pups were observed for the usual parameters and also for physiological
development and behavioural effects (gripping reflex, acoustic
startle, and pupillary reflex). A statistically significant reduction
in body weight was observed in F0 and F1 females at 2700 ppm, and
food consumption was moderately reduced in males and females of both
generations; transient tremor and hypersensitivity were also observed
in F0 and F1 females at this dose, mainly during or after the
lactation period. Also at this dose, male fertility was reduced, fewer
pups were delivered by each dam, and the growth and development of the
F1a, F1b, and F2 pups were retarded. The NOAEL for reproductive
toxicity was 900 ppm, equal to 69 mg/kg bw per day, on the basis of
reductions in male fertility and number of delivered pups (Hellwig et
al., 1993).
Hamsters
Groups of eight pregnant Syrian golden hamsters were given
chlormequat (purity unspecified) by gavage at concentrations of 0, 25,
50, 100, 200, 300, or 400 mg/kg bw once on day 8 of gestation or
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 killed 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 single doses 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
maternal toxicity; the occurrence of malformations in historical
controls was not presented (Juszkiewicz et al., 1970).
Rabbits
In a study described in the 1972 monograph addendum (Annex 1,
reference 19), groups of pregnant rabbits were fed diets containing
chlormequat at 0 or 1000 ppm (equivalent to 0 or 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 removed
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; the 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 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) Special studies: Dermal and ocular irritation and dermal
sensitization
In two studies conducted between 1978 and 1980, which were not in
accordance with current test guidelines, the irritancy of chlormequat
chloride to the skin was tested in rabbits. In the first study, about
0.5 ml of the test material (purity unspecified) was applied to intact
and abraded sites on each of six Vienna white 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).
In the second study, 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 a study based on current test guidelines, about 0.5 ml of
chlormequat (a technical material consisting of a 66.1% aqueous
solution) was applied to intact sites on each of six New Zealand white
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 reactions had resolved
completely within 48 h of treatment (Fischer et al., 1990b).
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 eyelid 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
by 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 eyelid 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, the animals were
challenged 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).
The sensitizing effect on the skin of chlormequat was tested in
guinea-pigs in the maximization test based on the method of Magnusson
and Kligman. After intradermal induction with a volume of 0.1 ml
Freund's adjuvant:0.9% saline (1:1), well-defined erythema and slight
oedema were observed at the injection sites of both control and test
animals. Injection of the test substance in 0.9% saline caused
well-defined erythema, and injection of the test substance in Freund's
adjuvant:0.9% saline resulted in well-defined erythema and slight
oedema. The control animals, treated with 0.9% saline, showed no skin
reaction. After percutaneous induction, perfomed only in the test
animals, partially open incrustation was observed in additon to
erythema and slight oedema. After percutaneous challenge with 50% test
substance, no skin reaction was observed in controls or test animals.
The authors concluded that chlormequat has no sensitizing effect on
the skin of the guinea-pig (Rossbacher & Kirsch, 1992).
Comments
In experiments with 14C-labelled chlormequat in rats, absorption
was rapid, and elimination was essentially complete within 24 h,
occurring almost entirely via the urine and mainly as unmetabolized
chlormequat. 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. Studies of the biotransformation of
chlormequat suggested that the only metabolites found in rat urine may
have been salts of chlorcholine. An unidentified polar metabolite was
found in faeces.
Pharmacological tests in mice, rats, rabbits, and cats given
chlormequat intravenously revealed a stimulatory effect on the
parasympathetic nervous system and a myoneural blocking action.
Further work showed that chlormequat is a partial agonist of the
nicotinic acetylcholine receptor; the affinity for muscarinic
receptors was low and relatively unselective.
Chlormequat was of moderate acute oral toxicity in rats, mice,
hamsters, guinea-pigs, and monkeys (LD50 = 200-1000 mg/kg bw), but
rabbits and dogs appeared to be more sensitive (LD50 = 50-80 mg/kg
bw) than the other species. The signs of toxicity may have been due to
pharmacological activity, and there were no consistent
treatment-related findings at autopsy. WHO has classified chlormequat
as slightly hazardous (WHO, 1996).
In a four-week study of toxicity in rats at dietary
concentrations of 0, 500, 1500, 3000, or 4500 ppm, the NOAEL was 1500
ppm, equal to 140 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 a 12-month study of toxicity in
dogs at dietary concentrations of 0, 150, 300, or 1000 ppm, the NOAEL
was 150 ppm, equal to 4.7 mg/kg bw per day, on the basis of diarrhoea,
vomiting, and salivation.
In a 110-week study of toxicity and carcinogenicity in mice at
dietary concentrations of 0, 150, 600, or 2400 ppm, the NOAEL was 150
ppm, equal to 21 mg/kg per day, on the basis of tubular down-growth in
the ovaries and endometrial hyperplasia. In a 78-week study of
toxicity and carcinogenicity in rats at dietary concentrations of 0,
280, 940, or 2800 ppm, the NOAEL was 940 ppm, equal to 43 mg/kg bw per
day, on the basis of reduced body weight. Tumour incidences were not
enhanced. The potential carcinogenicity of chlormequat was
investigated in a 104-week study in rats at dietary concentrations of
0, 280, 940, or 2800 ppm. No carcinogenicity was observed. The NOAEL
was 940 ppm, equal to 42 mg/kg bw per day, on the basis of reduced
body weight.
In a multigeneration study of reproductive toxicity in rats at
dietary concentrations of 0, 300, 900, or 2700 ppm, the NOAEL for
reproductive toxicity was 900 ppm, equal to 69 mg/kg bw per day, on
the basis of reduced numbers of pregnancies and of delivered pups and
retarded growth and development of the pups.
The developmental toxicity of chlormequat has been investigated
in mice after administration by intraperitoneal injection, gavage, or
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. In a study in mice at dietary concentrations of 0, 1000,
or 10 000 ppm on days 1-15 of gestation or 25 000 ppm on days 11-15 of
gestation, the number of malformations in animals fed 10 000 ppm or 25
000 ppm was reported to be slightly higher than that in controls;
however, the significance of this observation was difficult to assess.
In hamsters receiving chlormequat at levels of 0, 25, 50, 100, 200,
300, or 400 mg/kg bw once on day 8 of gestation or 100 mg/kg bw per
day on days 7-9 of gestation, malformations and evidence of delayed
development were seen with doses > 200 mg/kg bw on day 8 and after
the three doses of 100 mg/kg bw per day. Evidence of maternal and
fetal toxicity was also seen at these doses. The study in hamsters
could not be fully evaluated, owing to lack of detail in the
publication. A full report of a well-conducted study in which rabbits
were dosed orally with 0, 1.5, 3, 6 or 12 mg/kg bw per day on days 6-
18 of gestation was available. Signs of maternal toxicity were seen at
the highest dose, but there was no evidence of developmental toxicity.
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.
Chlormequat was not irritating to the skin or eye in rabbits. It
did not cause delayed contact hypersensitivity when tested in albino
guinea-pigs by the method of Buehler or by the method of Magnusson and
Kligman.
An ADI of 0-0.05 mg/kg bw was allocated on the basis of the NOAEL
of 4.7 mg/kg bw per day for diarrhoea, vomiting, and salivation in the
one-year study of toxicity in dogs, and using a safety factor of 100.
Toxicological evaluation
Levels that cause no toxicological effect
Mouse: 150 ppm, equal to 21 mg/kg bw per day (110-week study
of toxicity and carcinogenicity)
Toxicological criteria for setting guidance values for dietary and non-dietary exposure to chlormequat
Human exposure Relevant route, study type, species Results, remarks
Short-term Dermal irritation, rabbit Not irritating
(1-7 days) Eye irritation, rabbit Not irritating
Skin sensitization, guinea-pig Non-sensitizing
Inhalation toxicity, rat LC50 > 5 mg/L air
Dermal toxicity, rabbit LD50 = 1300 mg/kg bw
Oral toxicity, rabbit LD50 = 70 mg/kg bw
Oral toxicity, cat LD50 = 7-50 mg/kg bw
Medium-term Repeated oral, reproductive toxicity, rabbit NOAEL = 6 mg/kg bw per day: maternal
(1-26 weeks) toxicity, no reproductive toxicity
Long-term Repeated oral, one year, dog NOAEL = 4.7 mg/kg bw per day: diarrhoea,
(> 1 year) vomiting, and salivation
Rat: 940 ppm, equal to 42 mg/kg bw per day (104-week study
of toxicity and carcinogenicity)
900 ppm, equal to 69 mg/kg bw per day (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 (fetotoxicity and teratogenicity in
a study of developmental toxicity)
Dog: 150 ppm, equal to 4.7 mg/kg bw per day (one-year study
of toxicity)
Estimate of acceptable daily intake for humans
0-0.05 mg/kg/bw
Studies that would provide information useful for continued evaluation
of the compound
Study of developmental toxicity in rodents that meets current
scientific standards.
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