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cases, based on unpublished proprietary data submitted for the purpose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.
ABAMECTIN (addendum)
First draft prepared by
D.J. Clegg
Carp, Ontario, Canada
Explanation
Evaluation for acceptable daily intake
Biochemical aspects
Tissue distribution of the 8,9-Z isomer of avermectin B1a
Exploratory study of uptake of the 8,9-Z isomer by fetal
CF-1 mice
Placental P-glycoprotein levels in CF-1 mice of the
+/+ genotype
P-Glycoprotein in immature rats
Development of P-glycoprotein in rat fetuses
Immunohistochemistry of P-glycoprotein in rhesus monkeys
P-Glycoprotein in monkey fetuses
P-Glycoprotein in human fetuses
Toxicological studies
Acute toxicity
Short-term toxicity
Reproductive toxicity
Multigeneration reproductive toxicity
Developmental toxicity
Special study: Hypersensitivity of dogs to ivermectin
Observations in humans
Comments
Toxicological evaluation
References
Explanation
Abamectin (a mixture containing > 80% avermectin B1a and
< 20% avermectin B1b) was evaluated toxicologically by the Joint
Meeting in 1992 and 1994 (Annex 1, references 65 and 71). An ADI of
0-0.0002 mg/kg bw was allocated in 1994 on the basis of an NOAEL of
0.12 mg/kg bw per day in a multigeneration study of reproductive
toxicity in rats, using a safety factor of 500. The safety factor was
increased because of concern about the teratogenicity of the 8,9- Z
isomer (earlier identified as the Delta-8,9 isomer), which is a
photolytic degradation product that forms a variable part of the
residue on crops. In 1995 (Annex 1, reference 74), the Joint Meeting
established a separate ADI of 0-0.001 mg/kg bw for abamectin itself as
the basis for risk assessment when abamectin is used as a veterinary
drug and the residue does not contain the 8,9- Z isomer. The present
Meeting reviewed information that was requested by the 1994 JMPR and
reconsidered the decision of the 1995 JMPR.
Data submitted to the 1994 JMPR indicated that the high
sensitivity of CF-1 mice to the neurotoxicity of avermectins is
associated with P-glycoprotein deficiency in the small intestine and
in the capillary endothelial cells of the blood-brain barrier. It was
speculated that the heterogeneity of the response in CF-1 mice may
explain the absence of a dose-response relationship for maternal
toxicity in the studies of teratogenicity. Data submitted to the
present Meeting resolved the issue of the variability seen in earlier
studies in CF-1 mice.
The 1994 JMPR considered additional data, comprising information
on the photo-oxidative stability of avermectin B1a (the major
component of abamectin) and a study on the relative sensitivities of
CF-1 and CD-1 mice. In addition, because of the close structural
relationship between ivermectin and abamectin and the similarity of
the toxic effects of these compounds in various animal species, data
on ivermectin, comprising two studies in primates and one in humans
were also considered. The JMPR addendum on abamectin does not include
evaluations of these studies. The Meeting commented that in a study of
photo-oxidative stability, the half-life of the 8,9- Z isomer was 4.5
h and that of avermectin B1a, 6.5 h. In comments on the first study
in rhesus monkeys, it was indicated that ivermectin had no effects on
body weight, clinical signs, or ophthalmological, haematological,
clinical chemical or pathological manifestations at the high dose of
1.2 mg/kg bw per day administered orally for two weeks to immature
monkeys. The second study on ivermectin, in neonatal monkeys given a
maximum of 0.1 mg/kg bw per day for two weeks by nasogastric
intubation, also showed no effects.
P-Glycoprotein, a component of the plasma membrane, has been
associated with multi-drug resistance: cells with high P-glycoprotein
levels in the membrane show decreased uptake, decreased drug
steady-state levels, and decreased drug retention. Further, ivermectin
has been shown in mice genetically engineered for disruption of the
gene encoding P-glycoprotein to be a substrate for this protein. These
genetically P-glycoprotein-deficient mice had a dramatic increase in
sensitivity to a variety of P-glycoprotein substrates, including
ivermectin. A five-day study by administration in the diet, reviewed
below and summarized by the 1994 JMPR, indicated the absence of
adverse neurotoxic effects of abermectin at 0.8 mg/kg bw per day in
CD-1 mice and in most CF-1 mice; however, 17% of the CF-1 mice were
highly sensitive, showing severe neurotoxic signs 3-4 h after the
initial dose. Immunohistochemical investigation showed that the
sensitive mice had much lower P-glycoprotein levels in the cerebellum,
cerebral cortex, and jejunum than insensitive CF-1 mice and CD-1 mice.
Using restriction fragment length polymorphism (RFLP), three
P-glycoprotein genotypes can be distinguished, +/+, +/- and -/-, the
incidence being in the 1:2:1 ratio expected on the basis of Mendelian
inheritance.
The 1994 JMPR concluded that CF-1 mice are an inappropriate model
for studying the toxicity (including teratogenicity) of avermectins.
Consequently, the ADI was revised and based on a re-evaluation of the
multigeneration study of reproductive toxicity in rats. The NOAEL in
this study was assessed in 1992 to be 0.05 mg/kg bw per day, on the
basis of a reduction in maternal body weight during early lactation.
The re-evaluation in 1994 was based on the consideration that this
effect was unlikely to be adverse and the NOAEL was therefore
increased to 0.12 mg/kg bw per day on the basis of toxicity seen in
the pups in the same study. Since questions about the potential
teratogenicity of the 8,9- Z isomer remained unanswered, a safety
factor of 500 was again used, giving an ADI for abamectin and its
8,9- Z isomer of 0-0.0002 mg/kg bw.
Abamectin was considered again by the JMPR in 1995. The Meeting
concluded that the existing ADI for abamectin and its 8,9- Z isomer
is appropriate when abamectin is used as a plant protection product
but is inappropriate for residues after veterinary use, since the
8,9- Z isomer is not detectable in such residues. The Meeting
therefore allocated an ADI for abamectin alone, again based on the
NOAEL in the two-generation study of reproductive toxicity in rats of
0.12 mg/kg bw per day, but using a 100-fold safety factor, to give an
ADI of 0-0.001 mg/kg bw.
The 1994 JMPR indicated a number of studies that would provide
information useful for continued evaluation of abamectin:
-- data on P-glycoprotein in other species, including humans;
-- establishment and validation of a more sensitive method to
assess the neurotoxic effects of avermectins in rodents;
-- study of the acute toxicity of the 8,9- Z isomer in CF-1 and
CD-1 mice, with measurements of P-glycoprotein in blood and
brain;
-- study of the teratogenicity of abamectin and the 8,9- Z isomer
in CD-1 and CF-1 mice with concurrent measurements of
P-glycoprotein, in order to correlate its presence with
maternal toxicity and teratogenicity.
Additional data have been received on the toxicity of single doses
of abamectin in CF-1 mice and of the 8,9- Z isomer in CF-1 and CD-1
mice, on the short-term toxicity of abamectin in CF-1 and CD-1 mice,
on the teratogenicity of the 8,9- Z isomer in CF-1 and CD-1 mice, on
the effects of ivermectin on reproduction in rats, on the
immunohistochemistry of P-glycoprotein in rhesus monkeys, on the
toxicity of a single oral dose of radiolabelled 8,9- Z isomer, on the
effect of a single dose on the onset of P-glycoprotein expression in
the developing placenta of CF-1 mice, and on fetal uptake of the
8,9- Z isomer in CF-1 mice. Studies on P-glycoprotein expression in
brain and jejunum of human fetuses, in human placentas, in adult and
fetal rhesus monkeys, and in rat fetuses and pups are also summarized.
Evaluation for Acceptable Daily Intake
1. Biochemical aspects
(a) Tissue distribution of the 8,9-Z isomer of avermectin B1a
Groups of eight 20-28-week-old CF-1 mice of each sex, genotyped
for P-glycoprotein expression by RFLP, were given a single oral dose
of 0.2 mg/kg bw of the radiolabelled isomer (dose volume, 10 ml/kg),
to deliver about 1.2 µCi/mouse. The radiolabelled material was a
sesame-oil solution of 0.02 mg/ml unlabelled isomer mixed with an
equal amount of 8,9- Z[53H] avermectin B1a. Four mice of each sex
of the +/+, +/-, and -/- genotypes were necropsied 8 and 24 h after
treatment. The brains and blood from the vena cava were removed from
all mice, and the testes were removed from males. Radiolabel was
determined in heparinized blood in microfuge tubes and in organs
(rinsed in saline and blotted dry) in preweighed scintillation tubes.
The levels of radiolabel in the brains of male and female -/- mice
were about 60 times higher than those in +/+ mice. By 24 h, the
difference was even greater, since clearance occurred in +/+ and +/-
mice but not in -/- mice. At both 8 and 24 h, +/- mice had higher
levels in the brain than +/+ mice. In the testes, the levels of
radiolabel were highest in mice of the -/- genotype, accumulation
resulting in higher levels at 24 h. In mice of the +/- and +/+
genotypes, radiolabel levels in testes were lower than that in plasma,
but the levels were in approximate equilibrium at 24 h. Plasma
radiolabel levels were also highest in mice of each sex of the -/-
genotype at 8 h, but the difference between the -/- and +/+ genotypes
was smaller than seen in the organ systems. By 24 h, the plasma
radiolabel level was higher in males than in females (Lankas et al.,
1997a).
(b) Exploratory study of uptake of the 8,9-Z isomer by fetal CF-1
mice
Thirty-five female CF-1 mice, about 20 weeks of age, weighing
25-52 g, and of the +/- genotype for P-glycoprotein expression, were
given 1.5 mg/kg bw (about 2.5 µCi/mouse) of radiolabelled 8,9- Z
isomer of avermectin B1a on gestation day 17. Ten mice were killed 4,
8, and 24 h after treatment, and the maternal plasma levels of the
test material were determined; fetuses were removed, sexed, weighed,
genotyped for P-glycoprotein expression (three litters at 4 h and two
at both 8 and 24 h), and analysed for radiolabel. Placentas were
analysed for P-glycoprotein by western blotting.
The maternal plasma levels varied significantly among animals,
even though all of the mice were of the +/- genotype. Maximum plasma
levels were observed 8 h after treatment, the levels at 4 and 24 h
being approximately equivalent. The level of radiolabel (dpm/g fetus)
was clearly highest in -/- fetuses, intermediate in +/- fetuses, and
lowest in +/+ fetuses at all intervals and in all litters, and these
differences increased with time of sacrifice after treatment. These
differences and increases were still clearly apparent when the levels
were expressed as dpm per g fetus/dpm per ml plasma. P-Glycoprotein
was found in all placentas from fetuses with a positive allele but not
in those from from fetuses with only negative alleles, in the one
litter for which data were reported (Lankas et al., 1997b).
(c) Placental P-glycoprotein levels in CF-1 mice of the +/+ genotype
Ten female CF-1 mice homozygous (+/+) for P-glycoprotein were
cohabited with males of the same species, strain, and genotype. Day 0
of gestation was designated as the day a vaginal plug was found. One
or two females were killed on day 9, 11, 13, or 15 of gestation, and
their placentas were removed and placed in dry ice. The placentas from
a single litter on gestation days 9, 11, and 13 were pooled, and those
from days 15 and 17 were assayed individually (the placentas from
gestation day 17 were obtained from a study of fetal uptake reported
elsewhere in this addendum.) Placentas were assayed for P-glycoprotein
by western blotting. P-Glycoprotein was present as early as day 9.
Relative to total placental protein, P-glycoprotein expression
increased with duration of gestation, the highest levels being found
on day 17 (Lankas et al., 1997c).
(d) P-Glycoprotein in immature rats
The levels of P-glycoprotein were measured in the brain and small
intestine of six young (six weeks), sexually immature rats (strain
unspecified) of each sex by immunohistochemistry. P-Glycoprotein
staining was found in cerebral and cerebellar capillary endothelial
cells and in brush-border epithelial cells of the jejunum. It was
reported that rats of this age are insensitive to the toxicity of
avermectin (Lankas & Cartwright, 1995a).
(e) Development of P-glycoprotein in rat fetuses
The development of P-glycoprotein was investigated in
Sprague-Dawley Crl:CD(SD)BR rats by immunohistochemical and western
blot analyses of brain and intestine of female rats, of their fetuses
on day 20 of gestation, and of pups on days 2, 5, 8, 11, 14, 17, and
20 post partum. The 40 F0 females, 10 weeks old (weighing 200-300
g) at the start of the study, were paired 1:1, and mating was
confirmed by the presence of a vaginal plug. (Whether this was
considered day 0 or day 1 of gestation is not stated, although in
previous studies from this laboratory the day on which a vaginal plug
was found was considered to be day 0 of gestation.) Four unmated
females were also studied. Deaths among the pregnant females were
checked daily. One fetus of each sex per litter of four F0 females
was killed on day 20 of gestation by hypothermia, and the brain and
jejunum were snap frozen and analysed for P-glycoprotein. The brain,
jejunum, and uterus of each of the four dams were also analysed for
P-glycoprotein. On postnatal day 0, the litters were culled to five
pups of each sex, with fostering as required. On days 2, 5, 8, 11, 14,
17, and 20, one pup of each sex from each of four litters was killed,
and the brain and jejunum were processed for immunohistochemical and,
in some cases, western blot analyses. The uteri of the four
non-pregnant females were examined by the same procedures as used for
the pregnant animals.
No deaths were observed, and fetal weights were within the normal
range. The litter size of the four rats sacrificed before parturition
ranged from 13-17. No external malformations were seen in any fetuses
or pups. P-Glycoprotein was found in F0 females in a diffuse pattern
on the endothelial-cell surface of the capillaries of the cerebrum and
cerebellum and on the brush border of jejunal epithelial cells;
moderate amounts were present on the luminal surface of the uterine
epithelium. No P-glycoprotein was observed in the uteri of
non-pregnant rats. P-Glycoprotein staining was seen in the brains of
all fetuses and pups; however, the intensity of staining was much
weaker in fetuses on day 20 of gestation and in younger than in older
pups. Virtually no immunohistochemical staining for P-glycoprotein was
observed in the jejunum on gestation day 20 or on days 2 and 5 post
partum; minimal staining was seen on day 8, increasing in intensity
with time. Western blotting of the brain and scanning dosimetry of
P-glycoprotein showed that the levels of P-glycoprotein, expressed as
the percentage of adult levels, were 11, 6.5, 5.7, 4.4, 6.9, 19, 37,
and 89% on gestation day 20 and on days 2, 5, 8, 11, 14, 17, and 20
post partum, respectively. Thus, P-glycoprotein levels are much
lower in the brain and jejunum in the early postnatal phase, and,
since the presence of P-glycoprotein in the tissue barrier reduces
penetration of abamectin, the lower levels in the brain and jejunum
are likely to permit increased penetration of abamectin. Once a
critical level of abamectin is achieved in the brain, the toxicity to
pups seen in the multi-generation studies is observed (Cukierski et
al., 1996).
(f) Immunohistochemistry of P-glycoprotein in rhesus monkeys
The immunohistochemistry of P-glycoprotein was investigated in the
endothelial-cell surface of capillaries in the cerebellum and
cerebrum, in the brush border of jejunal epithelial cells, and in the
canaliculi of the liver of four untreated, one- to two-year-old rhesus
monkeys of each sex. The animals were killed by pentobarbital
injection. P-Glycoprotein staining was observed in a diffuse pattern
in the tissues examined, the staining being least intense in the
jejunal cells and most intense in the liver; staining was
approximately the same in all brain sections (Schinkel et al., 1994).
Lankas and Cartwright (1995a) state that 'It is known that juvenile
monkeys are relatively insensitive to avermectin-related toxicity',
probably due to a well-developed P-glycoprotein export system in the
brain.
(g) P-Glycoprotein in monkey fetuses
In another exploratory study on the immunohistochemistry of
P-glycoprotein, five male and four female fetal monkeys were killed by
phenobarbital injection, and sections of brain and jejunum from the
fetuses and biopsy samples of placenta and endometrium from the
maternal animal were frozen and analysed. A diffuse pattern of
P-glycoprotein staining was seen on the endothelial-cell surface of
capillaries in the cerebellum, cerebrum, cerebellar peduncle, and pons
of the fetal monkeys, with comparable intensity of staining in all
areas of the brain. Diffuse staining was also seen in the syncytial
trophoblasts lining the chorionic villi, and trophoblasts in the
marginal zone of the placenta were also weakly stained. Patchy
P-glycoprotein staining was also seen on the endothelial surface of
maternal uterine blood vessels, the staining being most intense in the
trophoblasts of the chorionic villi. No staining was found in the
jejunum of the fetal monkeys. The intensity of staining in the brains
of fetal monkeys was comparable to that in juvenile monkeys; however,
jejunal P-glycoprotein was observed in juvenile monkeys. Infant
monkeys were stated to be insensitive to avermectin-induced toxicity
(Lankas & Cartwright, 1995b).
(h) P-Glycoprotein in human fetuses
Bohr and Mollgärd (1974) showed that tight junctions composed of
strands are present in human fetuses of a crown-rump length of 60-150
mm. Transepithelial permeability is believed to depend on the presence
of such strands, which develop early in the human fetus. Betz and
Goldstein (1981) investigated changes in the metabolism and transport
properties of capillaries isolated from rat brain 1-45 days post
partum and concluded that various aspects of brain capillary
function show distinct developmental patterns which may be related to
changes in blood-brain barrier permeability during development. The
blood-brain barrier in rats is thus incompletely developed at birth,
in contrast to the early development of tight junctions in the human
fetus.
Van Kalken et al. (1992) examined the expression of P-glycoprotein
in human fetuses at 7-28 weeks of gestation by immunohistochemical
staining. P-Glycoprotein mRNA was detected in seven-week-old fetuses,
but P-glycoprotein expression in the brain was not detected until week
28 of gestation, when the level was comparable to that in adults.
MacFarland et al. (1994) examined the distribution of P-glycoprotein
in the human placenta. In the first trimester, P-glycoprotein was
present in the syncytiotrophoblast microvillus border, and some
placental macrophages showed weak staining. At term, no staining was
seen in the trophoblasts but strong staining was seen in placental
macrophages. These findings indicate that P-glycoprotein is present in
the human fetal brain and in the placenta, but is minimal or absent in
the brains of fetal rats.
2. Toxicological studies
(a) Acute toxicity
(i) Abamectin
Female CF-1 mice weighing 23-29 g, were genotyped by RFLP with
radiolabelled DNA probes to identify mice homozygous (+/+) and
heterozygous (+/-) for P-glycoprotein expression. No homozygous (-/-)
mice were used. Abamectin (purity not specified) in sesame oil was
administered orally to groups of five female mice at doses of 0, 10,
20, 40, or 80 mg/kg bw. All mice homozygous for P-glycoprotein that
were given 40 mg/kg bw died within 3-6 days, and all those at 80 mg/kg
bw within 1-3 days; all mice heterozygous for P-glycoprotein that were
given 20 mg/kg bw died within 2-4 days and all those at 40 and 80
mg/kg bw died within 1-2 days. The physical signs in all treated
animals were tremors, decreased activity, and bradypnoea within hours
of treatment and persisting for 1-4 days. Lateral recumbency was
generally seen before death. The oral LD50 values for abamectin were
28 mg/kg bw for homozygous (+/+) mice and 14 mg/kg bw for heterozygous
(+/-) mice. In previous studies, the oral LD50 values for mice of the
-/- genotype were 0.3-0.4 mg/kg bw (Hall, 1997; Lankas et al., 1997d).
(ii) 8,9-Z Isomer of abamectin
Groups of three randomly selected CD-1 female mice (7-8 weeks old,
weighing 22-28 g) were given doses of 50, 90, 162, 292, or 525 mg/kg
bw, and groups of five randomly selected CF-1 male mice (14 weeks old,
weighing 33-39 g) were given doses of 10, 20, or 30 mg/kg bw of the
8,9- Z isomer orally in sesame oil, and observed for seven days. CD-1
mice at 292 mg/kg bw and above showed decreased activity and
bradypnoea, and some animals had tremors before death; all of these
animals were moribund within 5-6 h of treatment. No deaths occurred at
lower doses, but mice at 90 mg/kg bw and above appeared unkempt, and
those at 162 mg/kg bw had a hunched posture; however, all of these
mice were normal on day 3 (at 90 mg/kg bw) or day 6 (at 162 mg/kg bw).
Of the CF-1 mice, 1/5 at 10 mg/kg bw , 3/5 at 20 mg/kg bw, and 2/5 at
30 mg/kg bw died, all on day 1 after treatment. Surviving animals were
normal by day 2. The signs of toxicity included ptosis (at 10 mg/kg bw
only), decreased activity, and bradypnoea. The acute oral LD50 of the
8,9- Z isomer of abamectin was 217 mg/kg bw in female CD-1 mice and
about 20 mg/kg bw in male CF-1 mice. No sex differences in toxicity
have been reported with any of the avermectins (Lynch, 1996).
(b) Short-term toxicity
Abamectin (purity 96% by weight based on combined analysis for
avermectin B1a and B1b) was administered to groups of 49 male and 50
female CF-1 mice and to groups of five male and five female CD-1 mice
at a dose of 0.8 mg/kg bw per day for four days. Severe tremors and/or
ataxia were seen in 12 female and five male moribund CF-1 mice after
the first dose. These animals were killed 3-4 h after treatment,
together with two female and one male CF-1 controls; 20 of the
remaining (insensitive) CF-1 mice and all of the remaining CF-1
controls were killed on study day 4, when the brain and small
intestine were removed. The cerebral cortex, cerebellum, and jejunum
were then cut into 3-mm sections, flash-frozen, and processed
immunohistochemically for P-glycoprotein (Schinkel et al., 1994) with
a primary antibody specific for the intermembrane binding site for
P-glycoprotein (Kartner et al., 1985). Samples of cerebellum,
cerebrum, and small intestine were also processed for assessment of
P-glycoprotein by western immunoblotting after crude extraction of
lysed cells to obtain cell membranes and other cytoplasmic components.
The protein content of the samples was measured by solubilizing them,
electrophoretic transfer to nitrocellulose and incubation overnight at
4 ¡C with C219, a primary antibody for P-glycoprotein, then reaction
for 1 h at room temperature with horseradish peroxidase-linked
anti-mouse immunoglobulin A. Immunoreactive P-glycoprotein was then
detected with a chemiluminescent substrate. After completion of the
biochemical studies, five male and five female insensitive CF-1 mice
and a random sample of five male and five female or 10 female CD-1
mice were given single doses of 1-10 mg/kg bw abamectin per day.
P-Glycoprotein was not detected in the brain or small intestine of
11/12 female or 5/5 male CF-1 mice killed after the first dose
because of moribundity; the one remaining female had very small
amounts of P-glycoprotein in the brain. The surviving CF-1 mice and
all CD-1 mice had very slight to intense P-glycoprotein staining.
Staining for P-glycoprotein of variable intensity was seen in vehicle
control CF-1 mice. Western immunoblots indicate intense banding of
P-glycoprotein in all three tissues from CD-1 mice and
abamectin-insensitive CF-1 mice but virtually no P-glycoprotein in
sensitive CF-1 mice.
Challenge with higher doses (1, 2.5, 5, or 10 mg/kg bw) of
abamectin resulted in mild signs of toxicity in 6/10 insensitive CF-1
mice at 5 mg/kg bw and in 8/10 at 10 mg/kg bw. The signs included
slight tremors and ataxia 5-6 h after treatment, with recovery within
24 h except for one mouse at 10 mg/kg bw which still showed slight
signs of toxicity but recovered by 48 h. CD-1 mice at 10 mg/kg bw
showed no signs of toxicity (Lankas et al., 1994, 1997d).
(c) Reproductive toxicity
(i) Multigeneration reproductive toxicity
In a multigeneration study of reproductive toxicity, groups of
20 female and 10 male Crl:CD(SD)BR rats were given ivermectin (purity,
97.7%) in sesame oil orally at doses of 0, 0.4, 1.2, or 3.6 mg/kg bw
per day. No effects were seen on parental body weights, either before
treatment (70 days) or during mating and pregnancy. The duration of
gestation was increased in animals at 3.6 mg/kg bw per day. The
incidence of live offspring per litter on day 1 post partum was
slightly increased at 1.2 mg/kg bw per day in F1a litters but not at
other doses or in F1b litters. Deaths of pups by day 7 were increased
at 1.2 and 3.6 mg/kg bw per day in F1a litters and at all doses in
F1b and F2a litters, and the dose of 3.6 mg/kg bw per day was
terminated because of the high mortality rate: 53% by day 14 of
lactation. The weights of pups of the F1a, F1b, and F2a generations
were generally comparable at all doses on day 1 but were decreased by
day 7 and thereafter in F1a litters at 3.6 mg/kg bw per day and in
F1b and F2a litters at 0.4 and 1.2 mg/kg bw per day. Delayed incisor
eruption was also seen at 3.6 mg/kg bw per day. Since treatment-
related increases in pup mortality early in lactation and reduced pup
weight gain were seen at all doses, the study was terminated early,
and a second study with doses of 0, 0.05, 0.1, 0.2, and 0.4 mg/kg bw
per day was initiated. No effects were reported, except for an
increase in mortality in F3a litters at 0.4 mg/kg bw per day between
days 1 and 7.
In order to assess the postnatal toxicity of ivermectin,
cross-fostering was studied. Forty female rats were given 2.5 mg/kg bw
per day for 61 days and then mated with untreated males but continued
to receive ivermectin throughout mating and gestation. On day 1
post partum, the litters of 40 control female rats treated with
sesame oil and those of the treated rats were culled to four pups of
each sex, which were cross-fostered in four groups: control parents ×
control pups, control parents × treated pups, treated parents ×
treated pups, and treated parents × control pups. Postnatal survival,
growth, and development were monitored until weaning on day 25 post
partum. Increased mortality and decreased pup weights were seen in
treated parents × treated pups and treated parents × control pups, but
not in the other crosses. These results indicate that neonatal
toxicity is a function of postnatal exposure, since the progeny
affected were those of dams continuously exposed to ivermectin.
Metabolic studies were also performed in eight-week-old female
rats given tritiated ivermectin orally at 2.5 mg/kg bw per day
(specific activity, 0.2 mCi/mg). Six rats in group 1 were given the
compound for 61 days and then throughout mating and gestation to day 9
post partum, and six in group 2 received the compound on days 1-9
post partum. Blood was collected from the orbital sinus of two rats
in group 1 on days 5, 10, 15, 20, 40, and 60 of exposure and from two
dams in each group on days 1, 4, and 6 post partum. Two pups were
taken from each group 1-5 h and 1, 4, and 6 days post partum, and
radiolabel was measured in blood and in pup brain and carcass. All
animals were killed 24 h after the last dose on day 10 post partum,
and blood, liver, brain, and carcass were collected. Milk samples were
obtained from two dams in each group on days 4, 6, and 10 post
partum.
The levels of radiolabel in plasma in animals in group 1 increased
and plateaued after 10-15 days. The plasma levels during lactation
increased by three to four times between day 60 (before mating) and
day 1 post partum and then gradually diminished to levels comparable
to those before mating by day 10 post partum. In group 2, the plasma
levels were low on day 1 post partum but increased to levels about
equal to those in group 1 by day 10. The levels of radiolabel in milk
were consistently two to three times higher than those in plasma in
both groups. The levels in brain, liver, and carcass on day 10 were
comparable in the two groups. Pups from group 1 had very low plasma
levels on day 1 post partum, which increased rapidly, however, so
that and on days 6 and 10 the level was two to three times greater
than that in the dam. In pups from group 2, no radiolabel was detected
in plasma on day 1; by days 4 and 6, the level was about half that
seen in pups, and by day 10 the levels were comparable to those in
pups from group 1. The residues in pup liver, brain, and carcass on
day 10 post partum exceeded those in adults by two to three times.
The plasma:brain ratios of radiolabel in offspring of the two groups
were about 1 on days 1 and 4 post partum and 2-3 on days 6 and 10.
These results can be interpreted in terms of the development of the
blood-brain barrier in rats and of maternal fat mobilization and
consequent release of lipophilic ivermectin. They are consistent with
development of the blood-brain barrier on days 6-10 post partum, as
also indicated by the mortality rate among pups. In humans, the
blood-brain barrier develops prenatally (Lankas et al., 1989).
(ii) Developmental toxicity
Groups of 25 mated Crl:CF-1 BR mice, 10 weeks old and weighing
19-29 g, were given the 8,9- Z isomer of avermectin B1 (purity, 99%)
in sesame oil at doses of 0, 0.015, 0.03, or 0.06 mg/kg bw per day on
days 6-15 of gestation by gavage. The presence of a vaginal plug was
considered to be day 0 of gestation. Physical signs were reported
daily; body weights were recorded on days 0, 6, 8, 10, 12, 14, 16, and
17 of gestation; and mice were killed on day 17. The uteri were
examined, and implants were counted and reported as resorptions or
dead or live fetuses. Maternal animals were subjected to gross
necropsy, and the fetuses were weighed, sexed, and examined
externally. Every third fetus per litter was dissected, as were all
externally malformed fetuses. The head of every third fetus was fixed
in Bouin's fixative. All fetuses were processed for alizarin staining
and skeletal examination.
No maternal deaths were reported. A single incidence of abortion,
probably occurring on gestation days 10-12 and unlikely to be related
to treatment, was observed in an animal at 0.06 mg/kg bw per day. No
treatment-related physical signs were observed, and no effects were
seen on body weight or body-weight changes between days 6-16 and 16-17
of gestation. Food intake was not significantly affected at any dose.
No gross organ changes were observed in dams, and no compound-related
effects were seen on the incidence of implantations, fetal deaths, or
resorptions, the sex ratio of fetuses, or fetal body weight. The
external malformations reported were exencephaly in three fetuses in
three litters at 0.03 and in three fetuses in two litters at 0.06
mg/kg bw per day. One of the fetuses at 0.03 mg/kg bw per day also had
a ventricle septal defect in the heart. A single fetus at 0.015 mg/kg
bw per day group had a cleft palate. Undated data on historical
control CF-1 mice gave the incidence of exencephaly as 68/25 037
fetuses (i.e. 0.27%), with a maximum of 7/428 (1.6%). In the study
reported, the incidence was 1.3% at 0.03 mg/kg bw per day and 1.4% at
0.06 mg/kg bw per day, i.e. within the upper limits of those of
historical controls. Similarly, the incidence of cleft palate in this
study is well within the range in historical controls. Lack of a
dose-response relationship also mitigates against compound-induced
effects. The NOAEL for both maternal and developmental toxicity was
0.06 mg/kg bw per day, the highest dose tested (Gordon et al., 1985a).
Five groups of 25 mated female Crl:CF-1 BR mice, 9.5 weeks of age
and weighing 21-28 g were given the 8,9- Z isomer of avermectin B1
(purity, 99%) in sesame oil at doses of 0, 0.015, 0.03, 0.1, or 0.5
mg/kg bw per day by gavage on days 6-15 of gestation. The presence of
a vaginal plug was considered to be day 0 of gestation. Physical signs
were observed daily, and body weights recorded on days 0, 6, 8, 10,
12, 14, 16, and 17 of gestation. At sacrifice on day 17 of gestation,
uterine implants were counted and classified as resorptions or live or
dead fetuses. Complete gross necropsies were performed. All fetuses
were weighed, sexed, and examined externally. Every third fetus per
litter was dissected, as were all externally abnormal fetuses. The
head of every third fetus was fixed in Bouin's fixative for
sectioning, and all fetuses were processed for alizarin staining and
skeletal examination.
Physical signs of toxicity (chromodacryorrhoea and lethargy) were
seen in one female at 0.5 mg/kg bw per day on day 9 of gestation,
which continued until sacrifice when moribund on day 12. The effects
were considered to be due to treatment. No effects on body weight,
weight gain, or food intake were seen in other animals. No gross
macroscopic changes were reported. The number of implantations per
pregnant female was slightly (not statistically significantly) reduced
at 0.5 mg/kg bw per day, and the incidence of resorptions was
increased at 0.03 and 0.5 mg/kg bw per day; however, the latter was
not dose related and was statistically significant only at 0.03 mg/kg
bw per day. Fetal weights were not affected, but were slightly
increased (not statistically significantly) at 0.5 mg/kg bw per day,
probably due to the reduced number of live pups arising from the
reduced implantation rate and slightly increased resorption rate.
The external malformations included exencephaly in one fetus in
one control litter, two fetuses in two litters at 0.015 mg/kg bw, five
fetuses in two litters at 0.03 mg/kg bw, no fetuses at 0.1 mg/kg bw,
and two fetuses in two litters at 0.5 mg/kg bw. Cleft palate was found
in none of the controls, in one fetus in one litter at 0.015 mg/kg bw,
in one fetus in one litter at 0.03 mg/kg bw, in six fetuses in one
litter at 0.1 mg/kg bw, and in 23 fetuses in six litters at 0.5 mg/kg
bw. The percentage incidences of fetuses with exencephaly were 0.3,
0.7, 2.1, 0, and 0.8% and of affected litters, 0.4, 0.8, 0.87, 0, and
0.87%, and the percentage incidences of fetuses with cleft palate were
0, 0.35, 0.42, 2.1, and 9.9%, and of litters, 0, 4, 4.3, 4.2, and 26%.
The clumping of incidence of cleft palate within litters at 0.1 and
0.5 mg/kg bw per day is very clear. Visceral and skeletal examinations
revealed a number of minor malformations (cervical ribs, lumbar ribs,
and sternebral ossification defects), which were not considered to be
related to treatment. The NOAEL for maternal toxicity was 0.1 mg/kg bw
per day, and that for teratogenicity was 0.03 mg/kg bw per day (Gordon
et al., 1985b).
A group of 276 female Crl:CF-1 BR mice, 9-10 weeks old and
weighing 22-32 g, was given abamectin (purity, 97.1%) at 0.4 mg/kg bw
in sesame oil in order to distinguish those that were sensitive and
insensitive to this drug. Mice that had tremors or recumbency were
considered to be sensitive. Insensitive mice were further tested by
giving them an additional oral dose of 0.8 mg/kg bw. Sensitive mice
comprised 27% of the tested population, but 69% of the sensitive mice
died during the selection process and only 23 survived. Sensitive and
insensitive mice were mated 1:1 with CF-1 males of unknown sensitivity
two to three weeks after treatment. Four groups of 25 mated,
insensitive animals were given the 8,9- Z isomer (purity, 79.8%) in
sesame oil at 0, 0.5, 1, or 1.5 mg/kg bw per day on days 6-15 of
gestation, day 0 of gestation being considered the day of mating. A
control group of four mated, sensitive female CF-1 mice was treated
similarly with sesame oil. Other mated, sensitive CF-1 female mice
were given one to three doses of 0.2 mg/kg bw per day, followed by two
doses (to all but one female which had not reached gestation day 6) of
0.3 mg/kg bw per day. The dose was increased further to 0.5 mg/kg bw
per day for one day and then to 1 mg/kg bw per day for a further day.
After this dose, 17/18 females showed decreased activity or were
recumbent, and the eighteenth female (last mated) was recumbent after
two doses of 1 mg/kg bw per day. Treatment was therefore withheld for
two days, after which 6/18 females appeared normal and were given one
to three doses of 0.75 mg/kg bw per day. One mouse on day 9, two mice
on day 12, two mice on day 13, three mice on day 14, four mice on day
15, and two mice on day 17 of gestation died or were killed when
moribund. Of the four survivors, three were recumbent for two days
before and on the day of terminal sacrifice (day 18 of gestation).
Animals that died before term were checked for pregnancy and
discarded.
All mice were observed for physical signs of toxicity daily, with
an additional examination 1-5 h after treatment. Body weights were
recorded on gestation days 0, 6, 8, 10, 12, 14, 16, and 18, and food
intake was measured between gestation days 3-5, 6-8, 9-11, 12-14, and
15-17. At term all female mice were killed, internal organs were
examined for gross changes, pregnancy status was determined, and
corpora lutea were counted. The cerebrum and cerebellum of the four
surviving sensitive mice, seven insensitive controls, and 11
insensitive animals receiving 1.5 mg 8,9- Z isomer were processed for
P-glycoprotein immunochemistry. The uterine contents were examined for
the numbers of implants, live fetuses, dead fetuses, and resorption
sites. Fetuses were examined externally after weighing, and then half
of the fetuses per litter (including externally malformed and/or dead
fetuses) were dissected for visceral examination. The heads of live
fetuses were sectioned. All fetuses were processed for skeletal
staining and examination.
Histopathological examination of the brains of CF-1 females at
termination of the study showed diffuse P-glycoprotein staining on the
endothelial-cell surface of capillaries of the cerebrum and cerebellum
of insensitive mice and the absence of such staining in sensitive
mice.
No adverse physical signs were observed, except in the sensitive
females given increasing doses. No effects were seen in this group at
doses of 0.2 or 0.3 mg/kg bw per day, but at 0.5 mg/kg bw per day
closed eyes and decreased activity were reported, and decreased
activity, recumbency, and ptosis were also observed at 1.0 mg/kg bw
per day. These signs persisted when the dose was reduced to 0.75 mg/kg
bw per day. The body weight, weight gain, and food consumption of
insensitive mice were unaffected. In sensitive mice, body weight and
weight gain were reduced from about day 10 of gestation, and food
intake declined throughout the study, the decrease increasing with
time. The incidence of pregnancy, number of corpora lutea, incidence
of resorptions, sex ratio, pup weight, and placental structures were
comparable in all insensitive groups. The four sensitive controls, one
of which had only three implantations, of which one was resorbed
early, had comparable numbers of corpora lutea; the increased
preimplantation loss was due to one mouse that had only three implants
(13 corpora lutea). The placental structure was normal. In the treated
sensitive mice, the numbers of corpora lutea per pregnant female were
comparable to those in controls. All females became pregnant, but
three of the four surviving litters comprised only dead pups, probably
due to immobility of the females during the last two or more days of
gestation. The body weights of the 11 live pups in one litter were
comparable to those of controls.
An increasing incidence of cleft palates was seen in the
insensitive animals, with 2.4% in controls, 4.4% in those at 0.5 mg/kg
bw per day, 6.9% at 1.0 mg/kg bw per day, and 20% at 1.5 mg/kg bw per
day, and also in sensitive mice, with 0% in controls and 45% in the
single surviving live litter of treated animals. Other malformations,
including exencephaly, open eyelids, brachydactyly, and hind-limb
rotation, were observed in insensitive mice, but the highest incidence
of these malformations was in the control group. Malformations found
only in treated animals were limited to insensitive mice and comprised
anencephaly (1/295 fetuses at 0.5 mg/kg bw per day) and macrophthalmia
(1/307 fetuses), micrognathia (1/307 fetuses), and tail malformation
(2/307 fetuses) at 1.5 mg/kg bw per day. Vertebral malformations were
seen in all groups except the sensitive controls, the incidence being
similar and low. Variants included a dose-related increase in the
incidence of pseudopolydactyly in insensitive mice. The occurrence of
extra cervical ribs, supernumary ribs, and sternal ossification
defects did not appear to be associated with treatment. The apparently
dose-related increase in the incidence of cleft palate in the treated
insensitive mice is not totally unexpected, since the expression of
cleft palate is dependent on fetal genotype. Insensitive parental mice
would include both the +/+ and +/- genotypes, and hence pups could
exhibit any of the three genotypes; thus, the sensitivity of the pups
within each test group will vary. In the absence of information on
fetal genotype, NOAELs cannot be determined. There was no NOAEL for
induction of malformations in sensitive or insensitive mice. The NOAEL
for maternal toxicity in insensitive CF-1 mice was 1.5 mg/kg bw per
day, the highest dose tested; that in sensitive mice was much lower
but could not be determined because of the changing doses (Wise et
al., 1996a).
Four groups of 22 mated female Crl:CD-1 BR mice, aged 12 weeks and
weighing 24-32 g, were given the 8,9- Z photoisomer of abamectin
(purity, 98.1%) in sesame oil at doses of 0, 0.75, 1.5, or 3 mg/kg bw
per day by gavage on days 6-15 of gestation. They were cohabited 1:1
with males; the day a copulatory plug was found was considered to be
gestation day 0. The test solution was shown to be of adequate
stability, with 95-100% of the nominal concentrations. Physical signs
were observed daily; body weight was recorded on days 0, 6, 8, 10, 12,
14, 16, and 18, and food consumption on days 3-5, 6-8, 9-11, 12-14,
and 15-17. All mice were killed on day 18 and subjected to gross
necropsy. Implants were counted and classified as resorptions or live
or dead fetuses. Placentas were examined for gross abnormalities.
Fetuses were weighed and examined for gross abnormalities. One-half of
the fetuses and all dead or abnormal fetuses were dissected. The heads
of live fetuses were fixed in Bouin's fluid for subsequent sectioning,
and all fetuses were processed by alizarin staining for skeletal
examination. Distal tail segments were collected from all fetuses and
mating partners for analysis of the P-glycoprotein genotype.
No adverse physical signs or deaths were reported. The mean
body-weight gain of treated animals was slightly (not statistically
significantly) increased, but the effect was not dose related and was
not considered to be toxicologically significant. Food intake was
generally comparable in all groups. No treatment-related findings were
observed on gross necropsy. All of the adult animals examined (12 of
each sex at 3 mg/kg bw per day, 10 males and nine females at 1.5 mg/kg
bw per day, 11 of each sex at 0.75 mg/kg bw per day, and 11 male and
12 female controls) showed the +/+ genotype for P-glycoprotein. The
numbers of preimplantation losses were higher in controls than in
treated groups, mainly due to high losses in 4/20 females, which
resulted in a slightly lower control implantation rate (12) than in
treated animals (> 13). The incidence of resorptions was comparable
in all groups. The birth of 12 stillborn pups to dams at 0.75 mg/kg bw
per day before sacrifice resulted in an increased incidence of dead
offspring. The sex ratios were normal.
The malformations observed included cleft palate, in none of the
controls, two pups (0.73%) at 0.75 mg/kg bw, one (0.31%) at 1.5 mg./kg
bw, and four (1.4%) at 3 mg/kg bw per day; one tail malformation at
0.75 mg/kg bw per day; and hind-limb extension in two fetuses in each
group. The incidence of cleft palates at 3 mg/kg bw per day was four
in three litters, which is within the incidence of historical controls
of this strain drawn from contemporary studies (1995-96) performed
within the same laboratory. The incidences in 14 control groups were
used, but 10 of these were from range-finding studies in which the
mice were injected intravenously with sodium bicarbonate in saline
(the carrier in these studies). The percentage of affected fetuses
ranged from 0.32 to 3.7% (1/312 to 11/296). In the present study, the
percentage incidence of cleft palate at the high dose was 1.5%
(4/272). Two of the control groups exceeded this level (2% and 3.7%).
Further, it should be noted that the incidences at the three doses
were 2/254, 1/251, and 4/272, with no strict dose-effect relationship.
The other variants observed and the limb extension and tail
malformations were probably not related to treatment. The NOAEL for
maternal toxicity and for induction of malformations was 3 mg/kg bw
per day, the highest dose tested (Wise et al., 1996b).
The genotypes of Crl:CF-1 BR female mice, approximately 11 weeks
of age and weighing 23-31 g, were determined by RFLP and Southern
blotting, and groups of 12 female mice were paired according to
genotype, as follows: sesame oil controls, +/- females × +/- males and
-/- females × -/- males; treated, +/+ females × +/+ males, +/- females
× +/+ males, and+/- females × -/- males. After mating, day 0 of
gestation being considered the finding of a copulatory plug, treated
females were given the 8,9- Z photoisomer of abamectin (purity,
94.3%) at a dose of 1.5 mg/kg bw per day in sesame oil on gestation
days 6-15. Clinical signs were recorded daily before treatment and
twice daily during treatment. Body weight was measured on gestation
days 0, 6, 8, 10, 12, 14 16, and 18. Mice were killed on day 18, and a
tail clip was collected for further genotyping if necessary. After
confirmation of pregnancy, implantations were counted and classified
as resorptions or live or dead fetuses, and fetuses were weighed and
examined externally for malformations. They were then killed and a
hind limb was collected for possible P-glycoprotein genotyping.
Samples were also collected of five placentas from a control dam of
the negative genotype (-/- × -/-) and a control dam of the positive
genotype (+/+ × +/+) and of all placentas from two control animals
with the control genotype (+/- × +/-). Heads and placentas were also
collected from all fetuses of four control dams of the +/- × +/-
genotype and four dams at 1.5 mg/kg bw per day of the +/- × -/-
genotype; the heads and placentas were preserved for P-glycoprotein
immunohistochemistry. Finally, the brain of the control dam with the
+/+ × +/+ genotype from which the five placentas were collected was
preserved for immunohistochemistry.
No deaths or adverse clinical signs were observed during the
study. The body weights were not statistically significantly affected,
but the +/- × +/- control group had moderately increased body-weight
gain, which was considered to be incidental. The incidence of
pregnancy was slightly lower in both +/- × +/- and -/- × -/- control
groups (8 and 9/12, respectively) than in the treated groups (12/12 in
all groups). This finding is probably incidental, since treatment did
not commence until day 6 of gestation. The number of implants was
increased slightly in the +/- × +/- cross, resulting in a slightly
larger litter size (13 ± 0.9) than in the second control (-/- × -/-,
12 ± 4.4) or treated groups (10 ± 4.0 to 12 ± 2.8). The incidences of
resorptions were comparable in all groups. No fetal deaths were
recorded, and fetal weights were comparable in all groups.
Genotypic analysis showed that all of the offspring of +/+ × +/+
crosses that were analysed were +/+ and all of those of the -/- × -/-
crosses were -/-. Matings of +/- control males and females resulted in
15 +/+, 32 +/-, and 18 -/- offspring (the expected 1:2:1 Mendelian
ratio). Western blotting of the placentas showed that the amount of
P-glycoprotein in the placenta depended on the genotype of the fetus,
the +/+ genotype having the greatest P-glycoprotein content, the +/-
genotype having less, and the -/- genotype having no P-glycoprotein.
The incidence of cleft palate was 0 in the treated +/+ × +/+ group,
12% in the +/- × +/+ treated group, and 58% in the +/- × -/- treated
group. The incidence was 0.83% in the +/- × +/- control group and 0 in
the -/- × -/- control group. No +/+ offspring of treated dams showed
cleft palate, whereas 30/31 fetuses with the -/- genotype and 29/70
(41%) of fetuses with the +/- genotype had this malformation. One
control fetus of the -/- genotype had exencephaly. The incidence of
postaxial pseudopolydactyly was 8.3% of all fetuses for +/- × +/-
controls and 2.8% for -/- × -/- controls. The incidences were 11% in
treated +/- × -/- offspring, 0.8% in +/- × +/- offspring, and 1.4% in
+/+ × +/+ offspring. The results clearly indicate that the fetal
genotype is closely related to both the incidence of cleft palate and
the presence of P-glycoprotein (Lankas et al., 1996).
(d) Special study: Hypersensitivity of dogs to ivermectin
Beagles and most others strains of dogs tolerate single doses of
3-10 mg/kg bw ivermectin; however, some collie dogs have been reported
to be hypersensitive to doses of 0.1-0.2 mg/kg bw. In order to
investigate this phenomenon, a colony of collies made up of
individuals derived from individual breeding pairs was screened for
sensitivity to ataxia, depression, tremors, and mydriasis at low
doses. Three sensitive and three insensitive dogs were killed, and
their livers and brains were analysed immunohistochemically for
P-glycoprotein. The insensitive dogs showed slight, consistent
staining of brain capillaries for P-glycoprotein, but the sensitive
dogs showed no staining. In the liver, P-glycoprotein was strongly
stained in bile caniculi and hepatocytes of both sensitive and
insensitive dogs. These results suggest that collies have a rare
mutation of the mdr1a gene, which is responsible for brain
P-glycoprotein expression, but have no such mutation in the mdr2
gene, responsible for liver P-glycoprotein expression.
3. Observations in humans
Ivermectin has been administered to humans for the treatment of
parasitic diseases. Fifty million doses of 0.2 mg/kg bw have been
given worldwide. No evidence of toxicity has been reported, even when
ivermectin was used at much higher doses (up to 1.6 mg/kg bw), and no
adverse neurological effects have been reported (personal
communication from Merck Research Laboratories).
Comments
Abamectin
In a study to establish the LD50 of abamectin in CF-1 mice
genotyped for P-glycoprotein expression, similar signs of toxicity
were seen in +/+ (homozygous) and +/- (heterozygous) mice. The oral
LD50 for +/+ mice was 28 mg/kg bw, and that for +/- mice was 14 mg/kg
bw. Separate studies indicated an LD50 for -/- mice of 0.3-0.4 mg/kg
bw. Thus, in CF-1 mice, the oral LD50 appears to be related to the
genotype for P-glycoprotein.
A four- to five-day study with CF-1 and CD-1 mice given abamectin
at 0.8 mg/kg bw per day resulted in severe toxicity in 17% of the CF-1
mice after the first dose. P-Glycoprotein was not detectable in the
brain or jejunum of these mice, except in one mouse which had minimal
expression in the brain. Insensitive CF-1 mice showed
slight-to-intense P-glycoprotein staining, and CD-1 mice showed
intense P-glycoprotein staining. Oral administration of challenge
doses (10 mg/kg bw) to groups of insensitive CF-1 mice after five days
of treatment at 0.8 mg/kg bw per day caused slight toxicity, with
complete recovery within one to two days. The results indicate severe
toxicity in mice of the -/- genotype and variable toxicity in those of
the +/- and +/+ genotypes. No toxicity was seen in CD-1 mice, which
are probably of the +/+ genotype. These results indicate that the
genotype of CF-1 mice with respect to P-glycoprotein expression
governs the toxicity of abamectin. There is no evidence that mutations
of this genotype occur in CD-1 mice.
8,9-Z Isomer
Studies of tissue distribution in genotyped CF-1 mice after
administration of radiolabelled 8,9- Z isomer indicated marked
differences according to genotype. In the brain, the levels in -/-
mice of each sex were about 60 times greater than those in +/+ mice.
By 24 h, the difference was even greater, since clearance occurred in
+/+ mice but not in -/- mice. A similar pattern was seen in the
testes, with the highest levels in -/- mice. At 24 h, the testicular
levels in +/- and +/+ genotype mice were in equilibrium with those in
plasma. In plasma, the level of radioalabel was highest in -/- mice,
but the differences between the +/+ and -/- genotypes were much less
than in the organ systems.
A single oral dose to CD-1 and CF-1 mice resulted in LD50 values
of 220 mg/kg bw for female CD-1 mice and about 20 mg/kg bw for male
CF-1 mice. Data on other avermectins indicate no sex difference for
acute toxicity. Since the signs fit the pattern for neurotoxicity, it
is probable that the low LD50 value (i.e. increased susceptibility)
in CF-1 mice is related to the accessibility of the target organ to
the test material and hence to the presence or absence of
P-glycoprotein expression.
A number of studies of developmental toxicity were performed in
CF-1 mice. In the first study, the NOAEL for both maternal and
developmental toxicity was 0.06 mg/kg bw per day, the highest dose
tested. Cleft palate and exencephaly were observed, but the incidence
was within historical control limits. A second study showed an NOAEL
for maternal toxicity of 0.1 mg/kg bw per day and an LOAEL, based on
signs of toxicity, of 0.5 mg/kg bw per day. The NOAEL for
teratogenicity was 0.03 mg/kg bw per day, the incidences of cleft
palate at doses of 0.1 mg/kg bw per day and above being greater than
those in historical controls; however, at 0.1 mg/kg bw per day, cleft
palates were seen in only one litter; at 0.5 mg/kg bw per day, the
incidence of cleft palate showed clumping within litters. A third
study with the 8,9- Z isomer was performed in female CF-1 mice which
had been screened for sensitivity to abamectin before the start of the
study. Sensitive and insensitive female mice were paired with males of
unknown sensitivity, and the doses given to sensitive female mice were
varied during exposure. Marked effects on sensitive mice occurred at
doses above 0.5 mg/kg bw, only 4/18 animals surviving to term. Of
these, only one mouse had a live litter. No effects were seen on
insensitive mice at doses up to 1.5 mg/kg bw per day; however, cleft
palates were observed at all doses between 0.05 and 1.5 mg/kg bw per
day. This study demonstrates that the incidence of malformations is
not related to maternal toxicity.
Yet another study of developmental toxicity was performed using
parental mice of known genotype for the mdr-1 gene, which encodes
P-glycoprotein expression. This study indicated a relationship between
the parental genotype and the incidence of cleft palate: at 1.5 mg/kg
bw per day, cleft palate was observed in none of the offspring of +/+
× +/+ crosses, in 12% of those of +/+ male × +/- female crosses, and
58% of those of -/- male × +/- female crosses; one cleft palate
occurred in the control +/- × +/- cross and none in the -/- × -/-
cross. Genotypic analysis of the fetuses from treated females showed
no cleft palates in +/+ mice, 41% in +/- mice, and 97% in -/- mice.
Analyses of the placentae for P-glycoprotein showed a correlation with
the genotype of the fetus, the levels being highest in +/+ fetuses and
absent in -/- fetuses; the +/- control matings yielded a Mendelian
distribution of 15 +/+, 32 +/-, and 18 -/- pups. A close relationship
between fetal genotype and the presence of P-glycoprotein in the
placenta would be expected, since much of the placenta is formed from
fetal tissue. The relationship between the incidence of cleft palate
and genotype appears to be a reflection of prevention by
P-glycoprotein expression of penetration of the test material through
placental membranes.
The presence of placental P-glycoprotein was investigated in +/+
male and +/+ female CF-1 mice. Western blotting of the placentae
indicated the presence of P-glycoprotein on day 9 of gestation, the
levels increasing with the duration of gestation. Levels present at
the time of palatal closure (about day 15) would be sufficient to
hinder placental transfer of the 8,9- Z isomer of abamectin. Passage
of the radiolabelled isomer across the placenta was investigated after
administration to +/- female mice on day 17 of gestation. The maternal
plasma levels of radiolabel were variable but maximal 8 h after
treatment. The levels of radiolabel in the fetus depended on the fetal
genotype, being lowest in +/+ fetuses and highest in -/- fetuses,
indicating that blockage of placental transfer depends on genetically
controlled expression of placental P-glycoprotein.
A study of developmental toxicity in CD-1 mice treated by gavage
with doses of 0, 0.75, 1.5, or 3 mg/kg bw per day of the 8,9- Z
isomer showed no adverse effects on the maternal mice. The incidences
of cleft palate were 0, 2, 1, and 4 (or 0, 0.73, 0.31, and 1.4%) at 0,
0.75, 1.5, and 3 mg/kg bw per day, respectively. These incidences were
not dose-related and fell within control incidences seen after oral or
intravenous administration of vehicles in the same laboratory. The
NOAEL for maternal, embryo-and fetal toxicity was 3 mg/kg bw per day.
Ivermectin
A multigeneration study in rats given ivermectin at doses of 0.4
mg/kg bw per day and greater resulted in early mortality of pups
post partum and reduced pup body-weight gain. The study was
terminated early. A further study at doses of 0.05-0.4 mg/kg bw per
day showed no effects, except increased pup mortality in the F3a
litters at 0.4 mg/kg bw per day between days 1 and 7 post partum.
Postnatal toxicity was assessed in a series of cross-fosterings of
newborn pups and was shown to be due to postnatal, not in-utero,
exposure. The postnatal toxicity was further investigated with
radiolabelled ivermectin given either before or throughout mating and
gestation. In the rats dosed post partum, the levels of radiolabel
in plasma were initially low but were comparable to those observed
after long-term exposure by day 9 post partum. The levels of
radiolabel in milk were consistently three to four times the plasma
levels in both groups, which may reflect mobilization of ivermectin
from fatty tissues. In the offspring of parents treated post
partum, radioalabel was not detected in plasma on day 1 post
partum, but was half that in the offspring treated by long-term
exposure on days 4 and 6, and equivalent on day 9. The tissue levels
in the pups on day 9 were two to three times those in the parents. The
plasma:brain ratios of radiolabel in the offspring of both groups were
1 on days 1 and 4 post partum and 2-3 on days 6-9. These findings
can be interpreted to indicate that the development of the blood-brain
barrier in rat offspring is delayed, occurring some time after
parturition. The postnatal toxicity observed in rats may be a function
of the accessibility of the target organ to the toxin, owing to the
late formation of the blood-brain barrier and to possible mobilization
of ivermectin from adult fatty tissues.
P-Glycoprotein distribution in adult and young animals
In the immature rat (about six weeks old), P-glycoprotein is
present in the brain and in the brush-border epithelial cells of the
jejunum. In fetal animals (day 20 of gestation), however, minimal
P-glyco-protein was detected in the brain, the levels being less than
10% of that in adult animals up to day 14 and then increasing rapidly.
No P-glycoprotein was detected in the jejunum of fetal rats or in rats
on days 2 or 5 post partum; P-glycoprotein was detectable by day 8
post partum, and the levels increased with time thereafter. These
data indicate late expression of P-glycoprotein, occurring some 10-15
days post partum. In non-pregnant adult rats, P-glycoprotein was not
observed in the uterus; it was present, however, on the luminal
surface of the uterine epithelium in pregnant rats.
P-Glycoprotein was present on the endothelial surface of
capillaries in the cerebrum, cerebellum, cerebellar peduncle, and pons
of rhesus monkey fetuses. The intensity of staining was comparable in
all areas of the brain. P-Glycoprotein was also present in the
placenta, but none was detected in fetal jejunum. The brain levels of
P-glycoprotein in monkey fetuses were comparable to those in the
brains of one- to two-year-old rhesus monkeys examined in another
study.
In humans, P-glycoprotein was detected in the brain capillaries of
fetuses aborted at 28 weeks, but not at earlier gestational ages. The
levels found were comparable to that in the adult brain. In human
placenta, P-glycoprotein was found in the syncytiotrophoblast
microvillus border and in some placental macrophages in the first
trimester, but mainly in the placental macrophages at term.
Species sensitivity to avermectins
Increased sensitivity has been seen in CF-1 mice and in Collie
dogs. In no other species or strain of animal has increased
sensitivity to avermectins been observed. In humans, 50 000 000 doses
of 0.2 mg/kg bw ivermectin have been administered for treatment of
parasitic diseases, with no report of toxicity directly attributable
to the drug. Higher doses (1.6 mg/kg bw) have also not resulted in
toxicity. Treatment of humans is not usually required more often than
yearly.
The data reviewed on the effects of the 8,9- Z isomer on
developmental toxicity in the CF-1 mouse clearly indicate a strong
relationship between the increased incidence of cleft palate and
reduced expression of P-glycoprotein in this strain of mouse. Since
the phenomenon is seen only in these animals, the Meeting considered
that use of the results of studies with this strain is not appropriate
in establishing an ADI. The NOAEL for teratogenic activity in the CD-1
mouse was 3 mg/kg bw per day.
Data on the avermectins have been used in the overall review of
abamectin. In the multigeneration studies of reproductive toxicity of
ivermectin and abamectin in rats, the critical adverse effects were
those on pups during early lactation. The studies with ivermectin
indicate that the pup mortality and reduced body-weight gains seen
early in lactation may be associated with delayed development of
P-glycoprotein expression. Reduced glycoprotein expression in early
lactation correlates with the mortality of pups. Young pups are not
only more susceptible to abamectin because they lack P-glyco-protein
expression, but they are also exposed to levels of abamectin in milk
that are two to three times those in maternal plasma. P-Glycoprotein
expression in humans is fully developed by week 28 of gestation. The
appropriateness of the multigeneration study of reproductive toxicity
of abamectin as the basis for the ADI is, therefore, questionable.
Because of the hypersusceptibility of rats postnatally, the
Meeting determined that a reduced interspecies safety factor would be
appropriate for establishing an ADI. A safety factor of 50 was
therefore applied to the NOAEL from the multigeneration study in rats
(0.12 mg/kg bw per day) to give an ADI of 0-0.002 mg/kg bw, which is
supported by the NOAEL of 0.24 mg/kg bw per day in the one-year study
in dogs, applying a safety factor of 100. A single ADI for both
abamectin and its 8,9- Z isomer was deemed to be appropriate, since
the potential teratogenicity of the isomer has been satisfactorily
explained.
Toxicological evaluation
Levels that cause no toxicological effect
Abamectin
Mouse: 4 mg/kg bw per day (two-year study of toxicity and
carcinogenicity)
Rat: 1.5 mg/kg bw per day (two-year study of toxicity and
carcinogenicity)
0.12 mg/kg bw per day (two-generation study of
reproductive toxicity)
Dog: 0.25 mg/kg bw per day (one-year study of toxicity)
8,9-Z isomer
Mouse: 3 mg/kg bw per day (study of developmental toxicity in
CD-1 mice)
Estimate of acceptable daily intake for humans (sum of abamectin
and 8,9-Z isomer)
0-0.002 mg/kg bw
Studies that would provide information useful for continued
evaluation of the compound
Further observations in humans, possibly involving repeated
exposure
References
Betz, A.L. & Goldstein, G.W. (1981) Developmental changes in
metabolism and transport properties of capillaries isolated from rat
brain. J. Physiol., 312, 365-376.
Bohr, V. & Mollgärd, K. (1974) Tight junctions in human choroid plexus
visualized by freeze-etching. Brain Res., 81, 314-318.
Cukierski, M.A., Lankas, G.R., Umbenhauer, D.R. & Cartwright, M.E.
(1996) Exploratory study of P-glycoprotein development in rat fetuses.
Unpublished report TT#94-739-0, submitted to WHO by Merck Research
Laboratories, Three Bridges, NJ, USA.
Gordon, L.R., Clark, R.L., Nickell, B.E., Collevechio, K. & Landes,
D.K. (1985a) 8,9 Isomer of avermectin B1: Oral teratology study in
mice. Unpublished report TT#85-710-0, submitted to WHO by Merck
Research Laboratories, Three Bridges, NJ, USA.
Gordon, L.R., Clark, R.L., Nickell, B.E., Collevechio, K. & Geiger,
J.E. (1985b) 8,9 Isomer of avermectin B1: Oral teratology study in
mice. Unpublished report TT#85-710-1, submitted to WHO by Merck
Research Laboratories, Three Bridges, NJ, USA.
Hall, S. (1997) Abamectin: Exploratory acute oral toxicity study in
mice. Unpublished report TT#96-2727, submitted to WHO by Merck
Research Laboratories, Three Bridges, NJ, USA.
Kartner, N., Evemden-Porelle, D., Bradley, G. & Ling, V. (1985)
Detection of P-glycoprotein in multidrug resistant cell lines by
monoclonal antibodies. Nature, 316, 820-823.
Lankas, G.R. & Cartwright, M.E. (1995a) Exploratory P-glycoprotein
immunohistochemistry in rhesus monkeys. Unpublished report (project ID
# 618-936-PGRM), dated 14 March 1995, submitted to WHO by Merck
Research Laboratories, West Point, PA, USA.
Lankas, G.R. & Cartwright, M.E. (1995b) Exploratory P-glycoprotein
immunohistochemistry in fetal rhesus monkeys. Unpublished report,
submitted to WHO by Merck Research Laboratories, Three Bridges, NJ,
USA.
Lankas, G.R. & Cartwright, M.E., (1995c) Exploratory study of
P-glycoprotein in non-pregnant rats. Unpublished report, submitted to
WHO by Merck Research Laboratories, Three Bridges, NJ, USA.
Lankas, G.R., Minsker, D.H. & Robertson, R.T. (1989). Effects of
ivermectin on reproduction and neonatal toxicity in rats. Food
Chem. Toxicol., 27, 523-529.
Lankas, G.R., Cartwright, M.E. & Umbenhauer, D. (1994) Exploratory
5-day oral toxicity study comparing abamectin sensitivity and
P-glycoprotein levels in CF-1 and CD-1 mice. Unpublished report
TT#94-2775, dated 12 September 1994, submitted to WHO by Merck
Research Laboratories, Three Bridges, NJ, USA.
Lankas, G.R., Wise, L.D., Cartwright, M.E., Umbenhauer, D.R.,
Cukierski, M.A., Nickell, B.E., Collevechio, K., Powzaniuk, W.C.,
Kulp, S.H., Jefferson, T., Overs, M.G. & Aversa, D. (1996) L-652,280:
Exploratory oral developmental toxicity study in CF-1 mice of known
P-glycoprotein genotype. Unpublished report TT#96-721-0, submitted to
WHO by Merck Research Laboratories, Three Bridges, NJ, USA.
Lankas, G.R., Hall, S.J. & Umbenhauer, D.R. (1997a) L-652,820:
Exploratory acute radiolabelled oral toxicity study in CF-1 mice.
Unpublished report TT#97-2578, submitted to WHO by Merck Research
Laboratories, Three Bridges, NJ, USA.
Lankas, G., Umbenhauer, D. & Wise, L.D. (1997b) L-652,280: Exploratory
fetal uptake study in CF-1 mice. Unpublished report TT#97-2538,
submitted to WHO by Merck Research Laboratories, Three Bridges, NJ,
USA.
Lankas, G.R., Wise, L.D. & Umbenhauer, D. (1997c) Exploratory acute
breeding study in CF-1 mice. Unpublished report TT#97-2568, submitted
to WHO by Merck Research Laboratories, Three Bridges, NJ, USA.
Lankas, G.R., Cartwright, M.E. & Umbenhauer, D. (1997d) P-Glycoprotein
deficiency in a sub-population of CF-1 mice enhances
avermectin-induced neurotoxicity. Toxicol. Appl. Pharmacol., 142,
357-365.
Lynch, D. (1996) L652,280: Exploratory acute oral toxicity study in
mice. Unpublished report TT#95-2754, submitted to WHO by Merck
Research Laboratories, Three Bridges, NJ, USA.
MacFarland, A., Abramovich, D.R., Ewen, S.W.B. & Pearson, C.K. (1994)
Stage-specific distribution of P-glycoprotein in first trimester and
full-term human placenta. Histochem. J., 26, 417-423.
Schinkel, A.M., Smit, J.J.K., van Tellingen, O., Beijnen, J.M.,
Wagenaar, E., van Deemter, L., Mol, C.A.A.M., van der Valk, M.A.,
Robanus-Maandag, E.C., te Riele, H.P.J., Berns, A.J.M. & Borst, P.
(1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a
deficiency in the blood brain barrier and to increased sensitivity to
drugs. Cell, 77, 492-502.
Van Kalken, C.K., Giaccone, G., van der Valk, P., Kuiper, C.M.,
Hadisaputero, M.M.N., Bosma, S.A.A., Scheper, R.J., Meijer, C.J.L.M. &
Pinedo, H.M. (1992) Multidrug resistance gene (P-glycoprotein)
expression in the human fetus. Am. J. Pathol., 141, 1063-1072.
Wise, L.D., Lankas, C.R., Cartwright, M.E., Cukierski, M.A., Nickell,
B.E., Collevechio, K., Powzaniuk, W.C., Kulp, J.H., Jefferson, T.,
Hipple, A.L. & Duchai, D.M. (1996a) L-652,280: Oral developmental
toxicity study in CF-1 mice. Unpublished report TT#95-741-0, submitted
to WHO by Merck Research Laboratories, Three Bridges, NJ, USA.
Wise, L.D., Lankas, G.R., Umbenhauer, D.R., Stabinski, L.G.,
Cukierski, M.A., Nickell, B.E., Collevechio, K. & McMahon, M.G.
(1996b) Oral developmental toxicity study in CD-1 mice. Unpublished
report TT#96-732-0, submitted to WHO by Merck Research Laboratories,
Three Bridges, NJ, USA.
See Also:
Toxicological Abbreviations
Abamectin (Pesticide residues in food: 1992 evaluations Part II Toxicology)