Pesticide residues in food -- 1999
Sponsored jointly by FAO and WHO
with the support of the International Programme
on Chemical Safety (IPCS)
Toxicological evaluations
Joint meeting of the
FAO Panel of Experts on Pesticide Residues
in Food and the Environment
and the
WHO Core Assessment Group
Rome, 20-29 September 1999
CHLORPYRIFOS
First draft prepared by
D. Wagner
Therapeutic Goods Administration,Department of Health and Aged Care,
Canberra, ACT, Australia
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 studies of toxicity
Long-term studies of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Multigeneration reproductive toxicity
Developmental toxicity
Special studies: Neurotoxicity
Studies on metabolites
Acute toxicity
Short-term studies of toxicity
Genotoxicity
Developmental toxicity
Observations in humans
Experimental studies
Case reports
Monitored field trials
Studies of morbidity
Comments
Toxicological evaluation
References
Explanation
Chlorpyrifos [ O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)
phosphorothioate] is a broad-spectrum organophosphorus pesticide. The
toxicology of chlorpyrifos was first evaluated by the 1972 Joint
Meeting (Annex 1, reference 18), when an ADI of 0-0.0015 mg/kg bw
was established on the basis of a NOAEL of 0.014 mg/kg bw per day in a
1-month study in humans. Further biochemical and toxicological
information was considered by the 1977 JMPR (Annex 1, reference
28), when the ADI was changed to 0-0.001 mg/kg bw. Additional
reports on the toxicology of chlorpyrifos were reviewed by the 1982
Joint Meeting (Annex 1, reference 38), which increased the ADI to
0-0.01 mg/kg bw on the basis of a NOAEL of 0.1 mg/kg bw per day in
humans exposed to chlorpyrifos for 9 days, with a 10-fold safety
factor. This ADI was supported by findings in rats and dogs.
Chlorpyrifos was reviewed at the present meeting within the periodic
review pro-gramme of the Codex Committee on Pesticide Residues.
Evaluation for Acceptable Daily Intake
1. Biochemical aspects
(a) Absorption, distribution, and excretion
Rats
A dose of 50 mg/kg bw [36Cl]chlorpyrifos given orally to male
Wistar rats by intubation was eliminated rapidly in the urine (about
90% of the dose) and faeces (about 10% of the dose). The compounds
excreted in the urine were identified as 3,5,6-trichloro-2-pyridyl
phosphated (5-80%), 3,5,6-trichloro-2-pyridinol (TCP; 15-20%), and
traces of chlorpyrifos. The concentrations of residue were highest in
liver and kidney 4 h after dosing, but the half-time in these tissues
was < 20 h. The longest half-time, 62 h, was recorded in fat (Smith
et al., 1967).
By 72 h after a single oral dose of 19 mg/kg bw
[14C]ring-labelled chlorpyrifos was given by intubation to male
Sprague-Dawley rats, 83-87% had been eliminated, mainly in the urine
(68-70%), faeces (14-15%), and expired air (0.15-0.39%). The residues
found at this time represented about 1.7% of the total dose, and the
concentration, while highest in fat, was < 1 ppm in any tissue
(Branson & Litchfield, 1971a). In a study to investigate the breakdown
of chlorpyrifos by microsomal enzymes in vitro (Branson & Wass,
1970), TCP was again identified as the major metabolite of
chlorpyrifos. In cows fed chlorpyrifos at a dose of 5 ppm in the diet
for four days, hydrolysis to the pyridinol was the major metabolic
pathway (Gutenmann et al., 1968).
Female Fischer 344 rats were given chlorpyrifos by oral gavage or
by inhalation in nose-only or whole-body chambers, while another group
was given [14C]chlorpyrifos by oral gavage to estimate the
efficiency of recovery. Oral dosing led to 30-80% recovery of the
administered dose in urine, while nose-only or whole-body inhalational
exposure led to an average urinary excretion of 0.28-0.58 µg of TCP
per ppb of chlorpyrifos in air, after adjustment for the route of
exposure. Rats with whole-body exposure absorbed more chlorpyrifos
than would have been expected to occur by inhalation alone, probably
due to grooming activity. The total combined recovery of radiolabel
from urine, faeces, and cage wash was 99.8% of the administered dose
(Nolan et al., 1986).
Single doses of [14C]ring-labelled chlorpyrifos (19 mg/kg bw)
or TCP (7 mg/kg bw) were given by oral gavage to Sprague-Dawley rats,
and the radiolabel was analysed in blood samples, expired air, and
excreta. The main compound found in urine was TCP. The biological
half-time of each chemical was estimated to be 8-17 h, and by 72 h
> 98% of each compound had been eliminated, mainly in the urine
(68-70% of chlorpyrifos, 73-76% of TCP) and faeces (14-15% of
chlorpyrifos, 6-7% of TCP). Elimination in the form of radiolabelled
carbon dioxide was a minor (< 1%) pathway. The residue concentrations
were consistently low in the brain, and the highest were found in bone
and intestine after administration of TCP and in fat and intestine
after administration of chlorpyrifos. Administration of single oral
doses of [14C]TCP or [14C]chlorpyrifos did not lead to
accumulation of these compounds in rat tissues (Branson & Litchfield,
1970, 1971b).
Groups of male and female Fischer 344 rats were given
[14C]chlorpyrifos as a single oral dose of 0.5 or 25 mg/kg bw or
unlabelled chlorpyrifos as 15 consecutive doses of 0.5 mg/kg bw per
day followed on day 16 by 0.5 mg/kg bw of [14C]chlorpyrifos. By 3
days after exposure, essentially all of the radiolabel had been
recovered (> 97%), mainly in the urine (84-92% of the administered
dose), with 6-12% in the faeces. Repeated dosing induced a slight
increase (6-7%) in urinary excretion as compared with the single dose
of 0.5 mg/kg bw per day. At sacrifice, the residues in the carcass
accounted for 0.2% of the administered dose of 25 mg/kg bw. The
residues were found mainly in perirenal fat and livers of males and in
the ovaries and fat of females. The half-time for excretion was 12 h
for males and 23 h for females at 25 mg/kg bw. No unchanged
chlorpyrifos was found in urine. The major metabolites were TCP (12%),
its glucuronide conjugate (80%), and, tentatively, a sulfate conjugate
(Nolan et al., 1987).
Hens
Groups of 36 laying hens were fed diets containing chlorpyrifos
at 0 (two groups), 0.3, 1, 3, or 10 (three groups) ppm for 30-45 days
before sacrifice. All control birds and 24 birds at each dose were
killed after 30 days, and the remaining 12 hens at each dose were fed
treated diet for a further 15 days; eggs were collected from these
birds throughout treatment. The additional two groups of birds at 10
ppm were allowed to recover from treatment for 7 and 21 days,
respectively, after the 30-day treatment period and were then killed.
Chlorpyrifos residues were detected only in fat, whereas TCP was found
in liver (0.15 ppm), kidney (0.33 ppm), and eggs (< 0.05 ppm) of hens
given 10 ppm chlorpyrifos. No residues were detected after the 7-day
withdrawal period (Dishburger et al., 1972a).
Goats
Two goats were fed [14C]ring labelled (positions 2 and 6)
chlorpyrifos twice daily in capsules for 10 days at concentrations
equivalent to 15-19 ppm in the feed. The majority (80%) of the
radiolabel was recovered in urine, with smaller amounts in faeces
(3.6%), gut (0.9%), tissues (0.8%), and milk (0.1%). The major urinary
metabolite (> 75% of the residual radiolabel) was the ß-glucuronide
conjugate of TCP, with smaller amounts of unconjugated TCP. The major
residue in fat was chlorpyrifos (0.12 ppm), while TCP was the major
residue in liver and kidney (Glas, 1981a). A similar pattern of
elimination was seen in a study in which lactating goats were fed
[14C]ring labelled chlorpyrifos twice daily by capsule; little
radiolabel (0.05-0.14%), mainly associated with chlorpyrifos, was
recovered in milk (Glas, 1981b).
Pigs
Groups of two boars and one sow were fed basal rations containing
0, 1, 3, or 10 ppm chlorpyrifos for 30 days, and tissue samples were
collected after withdrawal periods of 0, 7, and 21 days. Residues were
initially found predominantly in fat tissues, with lower
concentrations in muscle, liver, and kidney, but no residues were
detectable after 7 days of withdrawal (McKellar et al., 1972).
Weanling piglets of each sex were given daily oral doses of[14C]TCP
equivalent to 75 ppm in the diet for 7 days, and urine and tissue
samples were collected after withdrawal periods of up to 7 days. The
principal urinary excretion product was unchanged TCP. The liver and
kidney initially contained the highest residues; while the
concentrations decreased rapidly after withdrawal, the liver had the
slowest rate of clearance (Bauriedel & Miller, 1981).
Cattle
Calves were fed TCP in the feed at concentrations < 100 ppm
for 28 days, and tissue samples were taken on day 28 or after a 3-,
7-, or 21-day withdrawal period. The concentrations of residues were
highest in liver and kidney, with lower concentrations in fat and
muscle. These concentrations declined rapidly after the return to
untreated feed (Glas, 1977a). One calf fed
2-methoxy-3,5,6-trichloropyridine for 28 days showed significant
concentrations (> 2 ppm) of TCP but no
2-methoxy-3,5,6-trichloropyridine in liver, kidney, and fat
(Glas,1977b).
Female calves were fed one capsule of chlorpyrifos per day for 30
days at doses equivalent to daily concentrations of dry matter in the
diet of < 100 ppm. Tissue samples were taken on day 30 or after a
1-5-week withdrawal period. A dose-related increase in the
concentration of residues was seen in all tissues but especially in
fat, whereas TCP was found predominantly in the liver and kidney.
After the dose of 100 ppm, the residues of chlorpyrifos in fat were
0.93 ppm at 7 days and 0.02 ppm at 35 days after withdrawal of the
test material (Dishburger et al., 1972b, 1977).
Humans
In persons poisoned with chlorpyrifos formulations, chlorpyrifos
was detected in serum samples only and at lower concentration than the
diethylphosphorus metabolites, which were excreted mainly in urine.
The urinary diethylphosphorus metabolites were excreted by first-order
kinetics, with an average elimination half-time of 6.1 ± 2.2 h in the
fastest phase and 80 ± 26 h in the slowest (Drevenkar et al., 1993).
Male volunteers received chlorpyrifos as an oral dose of 0.5
mg/kg bw and 1 month later a dermal dose of 5 mg/kg bw. The time to
the maximal concentration of TCP in blood was 0.5 h after oral dosing
and 22 h after dermal treatment. The elimination half-time,
irrespective of the route of administration, was 27 h. The percentage
of the administered dose recovered from the urine was 70% after oral
dosing and 1.3% after dermal administration (Nolan et al., 1982,
1984).
(b) Biotransformation
A generalized metabolic pathway for chlorpyrifos is shown in
Figure 1. Chlorpyrifos, like many of the most commonly used
organophosphorus insecticides, is a phosphorothionate.
Phosphorothionate insecticides are bioactivated by the microsomal
cytochrome P450 system in the bodies of vertebrates and insects to
their active oxon (phosphate ester) metabolites, which are about three
orders of magnitude more potent as anticholinesterases than the parent
compounds. Most bioactivation takes place in the liver, while
detoxification takes place in the liver and plasma. Chlorpyrifos is
rapidly metabolized by mixed-function oxidases to the highly reactive
chlorpyrifos oxon by oxidative desulfuration (step 1). Oxidative
metabolism involving concurrent activation and degradation via a
common intermediate appears to be the general pattern for P=S esters
(Yang et al., 1971; Wolcott et al., 1972; Nakatsugawa, 1992). The
oxidative desulfuration is believed to proceed via an electrophilic
phosphooxathiiran intermediate (Kamataki & Neil, 1976). The
degradation step occurs by conversion directly to TCP and diethyl
thiophosphate (step 2). The oxon can be deactivated by hydrolysis to
diethylphosphate and TCP (step 4) (Ma & Chambers, 1994; Sultatos &
Murphy, 1983). A minor reaction pathway is hydrolysis to monethyl
3,5,6-trichloro-2-pyridinyl phosphorothioate (step 3).
The moderate toxicity of chlorpyrifos, when compared with some
other phosphorothionates, may be due to hydrolytic detoxification of
the oxon by A-esterases, such as paraoxonase, and high serum
concentrations of paraoxonase may protect against poisoning by
organophosphorus insecticides with paraoxonase substrates as active
metabolites (Omenn, 1987; Geldmacher-von Mallinckrodt & Diepgen,
1988). This effect has been demonstrated in rats directly, in which
injection of purified paraoxonase before dosing with chlorpyrifos oxon
reduced the inhibition of brain acetylcholinesterase in these animals
by 2.5 times when compared with controls (Costa et al., 1990).
Twelve rats given chlorpyrifos at 5 mg/kg bw (3 mCi per rat)
orally by gavage excreted 88% of the administered radiolabel in urine
within 48 h. At least six metabolites were present, three of which
accounted for 97% of the excreted radiolabel. These were identified as
the glucuronide of TCP (80%), a glucoside of TCP (4%), and TCP itself
(12%) (Bakke et al., 1976).
(c) Effects on enzymes and other biochemical parameters
Hydrolysis is the most important route of detoxification of
organophosphorus esters. Hydrolytic esterases are distributed
ubiquitously in the blood and tissues of virtually all animal
organisms and catalyse the hydrolysis of a variety of esters,
including organophosphorus esters, but have little activity on
organophosphorus itself. Esterases that interact with
organophosphoruses have been characterized as A- and B-esterases
according to their sensitivity to inhibition by organophosphorus
compounds. Plasma and tissues of mammals have significant
concentrations of the calcium-activated A-esterases, arylesterase (EC
3.1.1.2), and the high-density lipoprotein-associated paroxonase (EC
3.1.8.1). The B-esterases, including aliesterases (EC 3.1.1.1; also
called carboxylesterases) and cholinesterases (e.g. butyryl
cholinesterase, EC 3.1.1.8), are inhibited by organophosphorus
compounds such as chlorpyrifos oxon. While they bind them, they do not
hydrolyse them (Derelanko & Hollinger, 1995).
Butyrylcholinesterase is the predominant cholinesterase in human
serum (> 99%), while in rats there is an approximately equal
distribution of acetylcholinesterase and butyryl cholinesterase
(Nolan, 1997). These enzymes detoxify organophosphorus compounds by
sequestering them through binding to or phosphorylation by blood
proteins, such as albumin, which stoichiometrically degrades them
(Aldridge, 1953). These protein-bound organophosphorus esters are
secreted into the bile. Erythrocyte acetylcholinesterase (EC 3.1.1.7)
is similar to the acetylcholinesterase in nervous systems and can bind
to and sequester or hydrolyse organophosphorus compounds. The enzyme
activities attributable to A-esterases, B-esterases, and
cholinesterases vary widely within populations and are influenced by
genetic and environmental factors and disease states (Derelanko &
Hollinger, 1995). The serum activity of paraoxonase in rabbits is some
40 times greater than that in rats. Most avians, including hens, have
relatively low activity of A-esterases, and the mean value of serum
chlorpyrifos oxonase activity in humans is 10 times that of rat serum
(Furlong et al., 1989).
In humans, a substrate-dependent polymorphism of serum
paraoxonase is observed, in which one isoform of paraoxonase has a
high turnover for paraoxon and the other a low turnover (Furlong et
al., 1989; Smolen et al., 1991). The polymorphism is also observed
with the oxons of methyl parathion, chlothion, and
phenylphosphonothioic acid O-ethyl O-para-nitrophenyl ester. The
two isoforms appear, however, to hydrolyse chlorpyrifos oxon and
phenylacetate at the same rate. Cloning and sequencing of the human
paraoxonase cDNA has revealed the molecular basis of the polymorphism:
arginine at position 192 determines high paraoxonase activity, and
glutamine at this position encodes low paraoxonase activity (Humbert
et al., 1993). In addition to this polymorphism, a 13-fold variation
in serum enzyme activity of a given genetic class is seen (Furlong et
al., 1989). While chlorpyrifos oxon was hydrolysed by the same plasma
fraction that hydrolysed paraoxon in a study of plasma samples from
320 white blood donors (Furlong et al., 1988), the population
distribution of chlorpyrifos oxon hydrolysis was unimodal. This
contrasted with a bimodal population distribution of the activity for
paraoxon hydrolysis, and the variation in paraoxonase activity was
about 11-fold, while that in chlorpyrifos oxonase activity showed a
four- to fivefold variation.
Correlation of the LD50 values for orally administered
chlorpyrifos in rats with the activation rates measured in brain
rather than liver (Chambers, 1992) suggested that local
biotransformation in target tissues is an important determinant of its
toxicity. In a more recent review, however, Chambers & Carr (1995)
compared published LD50 or LC50 values for a variety of
insecticides in several vertebrate species. Studies in rats indicated
that the sensitivity of brain acetylcholinesterase activity to
inhibition by various phosphorothionate oxons did not correlate with
their acute toxicity. Chlorpyrifos oxon has a greater affinity for rat
brain acetylcholinesterase than does paraoxon, with median inhibitory
concentrations (IC50 values) of 4.0 and 22 nmol/L, respectively, but
a lower LD50. This finding is consistent with the greater affinity
(IC50, 0.75 nmol/L) of chlorpyrifos oxon for plasma aliesterases
than for rat brain acetylcholinesterase, indicating that aliesterases
provide protection against chlorpyrifos oxon.
Further work by Chambers & Carr (1995) indicated longer
inhibition of esterases after exposure chlorpyrifos than to parathion.
This was attributed to the greater lipophilicity of chlorpyrifos than
parathion (hexane:acetonitrile partition coefficients, 0.28 and 0.062,
respectively) and the assumption that a substantial fraction of the
dose of chlorpyrifos would have been sequestered by fat and released
gradually for later bioactivation. Studies of hepatic microsomal
metabolism of parathion and chlorpyrifos indicated that desulfuration
of parathion was favoured over dearylation (activation vs
deactivation), whereas the reverse was seen for chlorpyrifos (Ma &
Chambers, 1994). In channel catfish, however, the sensitivity of
acetylcholinesterase to inhibition by oxons reflects the acute
toxicity of these insecticides, and may be largely responsible for
their toxicity in this species. Thus, the metabolism of insecticides
appears to be more influential in some species than in others in
determining their toxicity.
Chlorpyrifos oxonase is a calcium-dependent A-esterase that
hydrolyses chlorpyrifos oxon, the active metabolite of chlorpyrifos.
As this activity may be related to that of paraoxonase, it was
determined spectrophotometrically by measuring the generation of TCP
(Furlong et al., 1989). Mortensen et al. (1996) hypothesized that
young rats have less chlorpyrifos oxonase activity than adults, and
they measured the activity of this enzyme in the brain, plasma, and
liver of male Long-Evans rats (CRL:(LE)BR) at postnatal day 4 and in
adults. No activity was detected in brain at either age, but the
activities in plasma and liver were markedly lower in younger than in
adult animals, the activities in plasma and liver being 1/11 and 1/2
of those in adults, respectively.
As the Michaelis-Menten constant for chlorpyrifos oxonase
activity was high (210-380 µmol/L), experiments were performed to
determine whether this enzyme could hydrolyse physiologically relevant
concentrations (nanomolar to low micromolar) of chlorpyrifos oxon. On
the assumption that tissue acetylcholinesterase inhibition provides a
sensitive bioassay for the concentration of chlorpyrifos oxon, changes
in the tissue acetylcholinesterase IC50 for chlorpyrifos oxon were
measured in the presence and absence of chlorpyrifos oxonase. Brain
acetylcholinesterase activity was determined spectrophotometrically,
while plasma and liver cholinesterase activities were determined by a
radiometric method with a [3H]acetylcholine iodide substrate. An
increase in the 'apparent' IC50 would indicate that chlorpyrifos
oxonase had hydrolysed substantial amounts of chlorpyrifos oxon during
the 30-min preincubation with the chlorpyrifos oxon. In the adult
rats, the apparent IC50 values for both plasma and liver
cholinesterase were higher in the presence of chlorpyrifos oxonase,
suggesting that the enzyme in those tissues could hydrolyse
physiologically relevant concentrations of chlorpyrifos oxon within
30 min. Young animals, however, showed less of a shift in the IC50
curves than adults, confirming that they have a lower capacity to
detoxify physiologically relevant concentrations of chlorpyrifos oxon
with chlorpyrifos oxonase.
2. Toxicological studies
(a) Acute toxicity
(i) Lethal doses
Numerous studies have been carried out with technical-grade
chlorpyrifos to establish LD50 and LC50 values (Table 1). The
lowest value after oral administration was 96 mg/kg bw (range,
96-480 mg/kg bw) in rats and 100 mg/kg bw (range, 100-150 mg/kg bw) in
mice. The signs of acute intoxication were consistent with
cholinesterase inhibition and included inactivity, salivation,
dyspnoea, flaccid paralysis, vomiting, piloerection, exophthalmia, and
diarrhoea; female animals were generally more sensitive to the acute
effects of chlorpyrifos than males. The LD50 for dermally applied
chlorpyrifos was consistently lower than that for oral or inhalational
exposure and indicated the lower dermal absorption of chlorpyrifos
(about 2% in humans) when compared with the 70-90% absorption after
inhalational or oral exposure.
(ii) Dermal and ocular irritation and dermal sensitization
Ocular irritation: In a paper in which few procedural details
were provided (Taylor & Olson, 1963), slight conjunctival redness was
seen in rabbits treated with chlorpyrifos, regardless of whether the
eyes were washed after administration of the test material. The
redness persisted for more than 7 days in some animals. Transient
conjunctival and iridial irritation, possibly due to a direct physical
irritating effect of cholorpyrifos powder, was seen in three young
adult female New Zealand white rabbits after each had received 100 mg
of a finely ground commercial preparation in the left conjunctival
sac. The eyes of all animals were normal after 24 h (Jones, 1985a).
The ocular irritation potential of technical-grade chlorpyrifos
(purity, 99%) was tested (in accordance with GLP requirements) in six
young male New Zealand white rabbits, 100 mg of test material being
placed into the right eye and the untreated left eye serving as a
control. There were no signs of corneal or iridial irritation. Slight
to moderate conjunctival redness (mean Draize score, 1 at 1 h, 0.83 at
24 h), slight chemosis (mean Draize score, 0.83 at 1 h, 0.16 at 24 h),
and discharge (mean Draize score, 1.6 at 1 h, 0.83 at 24 h) were seen
up to 24 h after instillation, but no signs of irritation were
reported at 48 h (Jackson & Ogilvie, 1994b).
The ocular irritation potential of 0.1 g of chlorpyrifos (purity,
99.3%) was assessed in three male and three female New Zealand white
rabbits by instilling the test substance into the conjunctival sac of
the right eye, the left eye remaining untreated and serving as
control. This study was conducted according to GLP requirements.
Diffuse corneal opacities (score 1 for degree and area of opacity)
were observed in two animals at 24 h, but these effects had
disappeared by 48 h and no other corneal effects were seen. Iridial
inflammation (score 1) was noted in four animals at 1 h, and this
effect persisted for 24 h in one of the animals and for 48 h in
another. No iridial effects were observed after 72 h. Conjunctival
irritation was observed in all animals, with redness (score 2:
diffuse, deeper crimson red at 1-24 h; score 1: vessels definitely
injected above normal at 48 h), chemosis (score 2: obvious swelling
with partial eversion of lids up to 48 h), and discharge (scores 2-3:
moistening of the lids and hairs just adjacent to lids or a
considerable area around the eye only at 1 h). No signs of
conjunctival irritation was observed at 72 h. The maximum individual
irritation scores were seen at 1 h and ranged from 12 to 19, with a
mean score of 16. The mean scores at 24, 48, and 72 h were 9.3, 2.5,
and 0, respectively. The overall scores according to EEC Council
Directive 67/548/EEC were 2 (mean, 0.11) for corneal opacity, 3 (0.17)
for iridial inflammation, 11 (0.61) for conjunctival redness, and 8
(0.44) for conjunctival chemosis (Dreher, 1994d)
Technical-grade chlorpyrifos (100 mg; purity, 96.2%) was
instilled into the conjunctival sac of the left eye of Himalayan
albino rabbits, and the right eyes of the animals served as controls.
A group of control animals was similarly treated with distilled water
only. All animals were observed for signs of ocular irritation at 1,
4, 24, 48, and 72 h, and then daily up to day 7. At the 4-h
observation, very slight iridial irritation (score 1) and conjunctival
irritation (score 1) were reported. It was not clear from the report
whether the conjunctival irritation was redness, discharge, or
chemosis. No signs of irritation were reported at 24 or 72 h. A
'quality assurance statement' was issued for this study (Frederick
Institute of Plant Protection and Toxicology, 1995d).
Table 1. Studies of the acute toxicity of technical-grade chlorpyrifos
Species Strain Sex Vehicle LD50 (mg/kg bw; GLP Reference
95% CI or range) or QA
Oral administration
Mouse SmithWebster M 1% aqueous gum 102 (94-110) Coulston et al. (1971)
tragacanth
Mouse NAMRU F Soya bean oil 152 (143-162) Berteau & Deen (1978)
Mouse Swiss albino M&F Vegetable oil 109 (93-127) QA Fredrick Institute (1995a)
Rat NR M 5% corn oil 163 (97-276) Taylor & Olson (1963)
F 135 (97-188)
Rat Sprague-Dawley F Soya bean oil 169 (146-196) Berteau & Deen (1978)
Rat Sprague-Dawley M Arachis oil 276 (167-455) GLP Dreher (1994a)
F 350 (285-429)
M&F 320 (260-393)
Rat Wistar M&F Vegetable oil 134 (102-163) QA Frederick Institute (1995b)
Rat Sprague-Dawley M Maize oil 264 GLP Wilson & MacBeth (1994)
F 141
M&F 192
Rat Sprague-Dawley M Maize oil 475 (311-727) Buch & Gardner (1981)
F 337 (220-515)
Rat Sprague-Dawley M Corn oil 221 (181-69) Nissimov & Nyska (1984a)
F 144 (105-200)
Table 1. (continued)
Species Strain Sex Vehicle LD50 (mg/kg bw; GLP Reference
95% CI or range) or QA
Rat Sprague-Dawley M Not reported 205 (134-299) Henck & Kociba (1980)
F 96 (72-140)
M 248 (170-379)
F 97 (80-112)
M 270 (188-426)
F 174 (130-244)
Guinea-pig NR M Corn oil 504 (300-850) GLP Lackenby (1985a)
Rabbit NR M Corn oil 1000-2000 GLP Lackenby (1985a)
Chicken NR M Capsule 32 (14-72) GLP Lackenby (1985a)
Chicken Leghorn M Diet 25 (21-31) Sherman et al. (1967)
Chicken Leghorn M Capsule 32 Stevenson (1963)
Chicken White Rock M&F Capsule 50-63 Stevenson (1966a)
Chicken NR M&F Capsule 20-50 Ross & Roberts (1974)
Chicken NR M&F Corn oil 102 (64-169) Ross & Roberts (1974)
Chicken NR M Capsule, 32 (14-72) Taylor & Olson (1963)
undiluted
Turkey Beltsville NR NR 32-63 Stevenson (1967)
small white
Table 1. (continued)
Species Strain Sex Vehicle LD50 (mg/kg bw; GLP Reference
95% CI or range) or QA
Inhalation
Mouse NAMRU F 65% xylene 94 (83-106) Berteau & Deen (1978)
(27-50 min,
whole body,
aerosol)
Rat Albino M&F Vapour (4 h, > 200a GLP Hardy & Jackson (1984)
(HC/CFHB) whole body)
Rat Sprague-Dawley F 65% xylene 78 (57-108) Berteau & Deen (1978)
(60-180 min,
whole body,
aerosol)
Rat Sprague-Dawley M&F Undiluted (4 h, > 230b (max. GLP Anderson et al. (1995)
nose only, attainable
powder) concentration)
Rat Sprague-Dawley M 40% xylene > 4070a Buch (1980)
F (4 h, nose only, 2890b (2010-4160)
aerosol)
Rat Wistar M&F Undiluted (4 h, > 1020a GLP Kenny et al. (1987)
whole body,
aerosol)
Rat Fischer 344 M&F 1% aqueous > 3200a GLP Phillips & Lomax (1989)
solution (4 h,
whole body,
aerosol)
Table 1. (continued)
Species Strain Sex Vehicle LD50 (mg/kg bw; GLP Reference
95% CI or range) or QA
Rat Sprague-Dawley M&F Vapour (4 h, > 36a (max. GLP Blagden (1994)
nose only) attainable
concentration)
Rat Wistar M&F Acetone (4 h, 560b (360-950) Fredrick Institute (1996a)
whole body,
nebulized particles
< 5 µm)
Dermal application
Rat Sprague-Dawley M&F PEG > 2000 Lackenby (1985b)
Rat Sprague-Dawley M&F Undiluted > 2000 GLP Jackson & Ogilvie (1994a)
Rat NR M&F Undiluted > 2000 QA Nissimov & Nyska (1984b)
Rat Fischer M&F Undiluted > 2000 GLP Jeffrey et al. (1986)
Rat Sprague-Dawley M&F Arachis oil > 2000 GLP Dreher (1994b)
Rat Sprague-Dawley M&F Saline > 5000 (intact Buch et al. (1980)
and abraded)
Rabbit Himalayan M&F Water 1233 (993-1531) QA Fredrick Institute (1995c)
Rabbit New Zealand M&F Undiluted 1580 (828-2606) Henck & Kociba (1980)
white M&F 1598 (1243-1919)
M&F 1801 (1023-3152)
Table 1. (continued)
Species Strain Sex Vehicle LD50 (mg/kg bw; GLP Reference
95% CI or range) or QA
Rat CFY M&F Tween 20/ 147 (120-179) Davies & Kynoch (1970)
DMSO/water
2:3:5 (subcutaneous)
M, male; F, female; NR, not reported; GLP, good laboratory practice; QA, quality assurance; PEG, polyethylene glycol;
DMSO, dimethyl sulfoxide
Technical-grade chlorpyrifos (100 mg; fine powder) was placed
into the right eye of nine young adult albino rabbits. The eyes of
three animals were irrigated with saline solution 30 s after
treatment, while the eyes of the remaining animals were not irrigated.
Ocular examinations were conducted after 24, 48, and 72 h and at 4 and
7 days. Most of the animals displayed slight initial pain after
instillation of the test material. No signs of irritation were
observed in the cornea or the iris. Slight conjunctival redness was
observed in all animals with unwashed eyes, and in two animals whose
eyes were irrigated after 30 s. The irritation disappeared in most
animals by days 3-4 but persisted for 7 days in a single animal with
unirrigated eyes. The mean irritation scores in rabbits with unwashed
eyes were 2.3 at 24 h and 0.3 at 7 days, while in the rabbits with
irrigated eyes the scores were 1.3 and 0, respectively (Buch &
Gardner, 1980a).
Dermal irritation: A finely ground commercial preparation of
chlorpyrifos (500 mg) moistened with distilled water was applied under
an occlusive dressing to about 6 cm2 of clipped skin on the back of
three young adult female New Zealand white rabbits as a single 4-h
application. No primary irritation was observed (Jones, 1985b).
The dermal irritation potential of technical-grade chlorpyrifos
(purity, 99%) was tested in a study conducted according to GLP in six
young male New Zealand white rabbits, to which 500 mg of the material
(moistened with water) were applied under a gauze patch on clipped
intact skin on the trunk and then covered with an occlusive dressing.
The dressings were removed after 4 h and the skin wiped to remove
residual material. The skin was examined for signs of irritation 1,
24, 48, and 72 h after removal of the patch and scored for irritation
on the basis of a system recommended by the US Environmental
Protection Agency. Further assessments were conducted after 4, 5, and
6 days to determine the reversibility of the effects. Slight erythema
(score 1) was noted on three of six test sites at 1 h, and this effect
persisted until 72 h at one site and until day 5 in another animal. No
skin irritation was seen on day 6 (Jackson & Ogilvie, 1994dc).
A technical-grade, fine-powder commercial preparation (purity not
stated) was impregnated at a dose of 0.5 g onto unmedicated lint
patches and tested in six young adult male New Zealand white rabbits
on shaved abraded and intact sites on the back. The patches were held
in place under impermeable dressings, which were removed after 23 h.
The reactions at the test sites were assessed by the method of Draize
at 24 and 72 h and 7 days after administration of the test material.
Well-defined or very slight erythema was seen in all animals at 24 h,
on both intact and abraded sites. Very slight oedema was also seen at
the intact and abraded test sites of one animal at 24 h, another at 72
h, and a third at 7 days. No skin reactions were seen in other animals
at these intervals. The primary irritation index was calculated to be
0.67 (Buch & Gardner, 1980b).
Technical-grade chlorpyrifos (purity, 96.2%) was applied at a
dose of 500 g to clipped intact and abraded skin on the trunks of
three male and three female Himalayan albino rabbits, and the trunks
were wrapped with plastic sheets held in place with tapes for 24 h. A
control group was treated with distilled water. After exposure, the
coverings were removed, but the report did not indicate whether the
test sites were washed or cleaned. The skin was observed 24 and 72 h
after application for signs of irritation. No sign of erythema or
oedema was reported. A 'quality assurance statement' was issued for
this study (Frederick Institute of Plant Protection and Toxicology,
1995e).
Technical-grade chlorpyrifos (purity, 99.3%), moistened with
0.5 ml distilled water, was applied at a dose of 0.5 g to clipped
areas on the backs of New Zealand white rabbits in a study carried out
in accordance with GLP. Surgical gauze was applied over the sites and
held in place with a strip of surgical adhesive tape, and the trunk of
each rabbit was wrapped in an elasticized corset. After the 4-h
contact period, the corset and patches were removed, and the treated
skin was wiped with cotton-wool soaked in distilled water. One hour
and approximately 24, 48, and 72 h after removal of the patches, the
test sites were examined for evidence of dermal irritation and scored
according to the method of Draize. Very slight (score 1) to
well-defined (score 2) erythema and very slight (score 1) to slight
(score 2) oedema were observed in all animals 1-24 h after removal of
the patches. At 48 h, very slight erythema was observed in three
animals and very slight oedema in one. No sign of skin irritation was
seen at 72 h. The sum of the readings at 24 and 72 h was 10, and the
primary irritation index (the sum of the readings/12) was 0.8. This
score indicates mild irritation according to Draize (Dreher, 1994d).
Dermal sensitization: The skin sensitizing potential of
technical-grade chlorpyrifos (purity, 99.3%) was assessed in female
albino Dunkin-Hartley guinea-pigs in a study conducted according to
GLP. Very slight (score 1; 10 animals) to well-defined (score 2; 2
animals) erythema and very slight (score 1; 1 animal) oedema were
observed 1 h after topical induction, but no signs of irritation were
observed at 24 h. No skin reaction was observed in control animals,
and no skin reactions were observed at the test or vehicle control
sites after topical challenge 21 days after induction. One animal was
found dead at this stage of the study, but the cause of death was not
determined. The test material was not a skin sensitizer in this study
(Dreher, 1994e).
Technical-grade chlorpyrifos (purity, 96.2%) was prepared as a 1%
solution in 0.5% carboxymethyl cellulose and tested at a dose of
100 mg in male Hartley guinea-pigs. Test and positive control groups
received induction by exposure of the shaven skin of the left flank,
and challenge 14 days after the third induction dose. Control animals
received only a challenge with 0.5 ml of a 0.1% solution. The test
animals had barely perceptible (score 1; 4/10 animals) erythema or
slight oedema (score 1; 2/10 animals) 24 h after the challenge, but no
signs of irritation were observed at 48 h. A single control animal had
barely perceptible erythema at 24 h, but no other signs of skin
irritation were seen in this group. Positive control animals had
barely perceptible to deep-red erythema (score 1-3) and slight to
marked oedema (score 1-3) 24 h after the challenge and barely
susceptible erythema and slight to marked oedema at 48 h. This study
was issued a 'quality assurance statement' (Frederick Institute of
Plant Protection and Toxicology, 1996b).
After a study to determine a suitable dosing regimen, six male
and six female young albino Dunkin-Hartley guinea-pigs were treated
with a commercial preparation of chlorpyrifos in a standard Beuhler
skin sensitization assay. The test substance (0.3 ml, 100% w/v in
polyethylene glycol on a lint pad) was applied for 6 h under an
occlusive bandage to the freshly clipped left flank of the animals on
days 1, 8, and 15. Challenge doses of chlorpyrifos and solvent were
applied for 6 h on day 29 to the right and left flanks, respectively.
The negative controls were treated only on day 29, when they received
the same treatment as the test animals. The challenge sites of both
groups were evaluated 24 and 48 h after removal of the patch. No
adverse skin reactions were seen after exposure to chlorpyrifos or
vehicle alone (Jones, 1985c).
Technical-grade chlorpyrifos (purity, 99%) was tested in young
female Dunkin-Hartley guinea-pigs by the Magnusson-Kligman
maximization test in a study conducted according to GLP. The test
consists of an induction with intradermal injections of the test
material followed after 1 week by topical application, and a challenge
3 weeks after induction. The test sites were assessed for irritation
24, 48, and 72 h after injection and 24 and 48 h after removal of the
patch (48-h exposure). A concentration of 25% of the test material in
maize oil was selected for injection in the induction phase and 75% in
maize oil for the topical induction phase; a concentration of 75% in
maize oil was also selected for the challenge. During the induction
phase, slight or discrete erythema was observed in all test and
control animals 1 and 24 h after injection and 1 h after topical
application. A number of test and control animals also showed slight
or discrete erythema 24 h after topical application. During
application, two test animals became ataxic, thin, and subdued and
were removed from the study, and one test animal was removed from the
study before the challenge application because it had lesions at the
test site. A single test animal showed a positive response to
challenge, with slight to discrete erythema. None of the animals given
the vehicle alone reacted to the challenge application (Jackson &
Ogilvie, 1994a).
Technical-grade chlorpyrifos (purity, 96.8%) dissolved in 100%
dimethylsulfoxide and diluted in the same solvent to achieve the
desired concentrations was tested in a study that conformed to GLP in
albino Dunkin-Hartley guinea-pigs. In animals treated with a single
dose, irritation ranging from scattered mild redness to moderate and
diffuse redness was seen at 24 h in 5/10 animals; only mild redness
was seen at 48 h in 4/10 animals. In 20 animals induced and then
challenged with chlorpyrifos, only one had scattered mild redness
after the challenge dose at 24 h, and no skin reaction was seen at 48
h. In a positive control group, 9/10 animals had mild redness at 24 h
and 4/10 at 48 h (Berman, 1987).
(b) Short-term studies of toxicity
Mice
In a 2-week study conducted according to GLP requirements, young
adult CD-1 mice, were given a commercial preparation of
technical-grade chrlopyrifos (purity, 96.3%) at dietary concentrations
of 0 (control), 75, 150, 300, 600, or 1200 ppm, equaivalent to 0,
14-18, 30-32, 56-67, 89-100, and 130-180 mg/kg bw per day,
respectively. Deaths and treatment-related clinical signs, including
tremor, ocular opacity, ocular staining, hunching, and lachrymation,
were seen mainly in animals at the highest dose. Marked reductions in
body weight and feed consumption were observed in animals at doses
> 600 ppm. In males, cholinesterase activity was reduced at all
doses. At 75 ppm, plasma cholinesterase activity was reduced by
> 95%, erythrocyte acetylcholinesterase activity by almost 40%, and
brain acetylcholinesterase activity by about 50%. Inhibition of
erythrocyte acetylcholinesterase activity was not dependent on dose,
and approximately 35% inhibition being seen at all doses, but plasma
and brain cholinesterase activities were reduced in a dose-related
manner, with an inhibition of 99% in plasma and about 89% in brain at
the high dose. Similar patterns of cholinesterase inhibition were seen
in females. On the basis of the results of this study, doses of 5, 50,
200, 400, and 800 ppm were selected for a 13-week dietary study in
mice (Crown et al., 1984a).
Groups of 40 male and 40 female CD-1 mice were given chlorpyrifos
(purity, 95.7%) in the diet at 0 or 15 ppm, equal to 2.7 mg/kg bw per
day for males and 3.4 mg/kg bw per day for females; 20 animals of each
sex per group were killed after 1 week, and 20 were treated for a
total of 4 weeks. There were no significant clinical signs or
unscheduled deaths. Food consumption was unaffected by treatment, but
the body-weight gain of male mice was decreased by 25% at the end of
the study. Organ weights were not affected by treatment, and there
were no significant differences between groups. The findings at
necropsy were within normal limits for all groups. After 1 week,
plasma cholinesterase activity was depressed by 88-91% and erythrocyte
acetylcholinesterase activity was depressed by 40-53%; after 4 weeks,
plasma cholinesterase activity was depressed by 91% and erythrocyte
acetylcholinesterase activity by 53%. Brain acetylcholinesterase
activity was unaffected by treatment at either time. There was no
difference between the sexes in the alterations in plasma and
erythrocyte cholinesterase activity (Davies et al., 1985).
Groups of 12 Crl CD-1 mice of each sex were given chlorpyrifos
(purity, 93.5%) in the diet at concentrations of 0, 5, 50, 200, 400,
or 800 ppm (equivalent to 0, 0.7-1.3, 7.1-14, 32-53, 41-140, and
110-300 mg/kg bw per day, respectively) for 13 weeks. This study was
conducted to the requirements of GLP. Dose-related increases in the
mortality rate and the frequency of ocular opacities were seen at 400
and 800 ppm. Body weights were reduced by 10-15% in mice at 800 ppm
throughout the study. Plasma cholinesterase activity was markedly
decreased at all doses, and decreased erythrocyte acetylcholinesterase
activity was seen, but with no clear dose-dependency; brain
acetylcholinesterase activity was inhibited in a dose-dependent manner
in males at doses > 200 ppm and in females at doses > 50 ppm
(Table 2).
Table 2. Group mean values for inhibition of cholinesterase activity in
Crl CD-1 mice given chlorpyrifos in the diet (expressed as a
percentage of control values)
Cholinesterase Sex Dose (ppm)
5 50 200 400 800
Plasma Male 45*** 95*** 98*** 99*** 99***
Female 36*** 97*** 98*** 99 *** 99 ***
Erythrocyte Male 34a 21 16 12 26
Female 11 57*** 44** 48** 44**
Brain Male 43a 14* 80*** 85*** 87**
Female 3a 28*** 58*** 84*** 88***
Plasma, butyryl cholinesterase; erythrocyte and brain,
acetylcholinesterase
* p < 0.05; ** p < 0.01; *** p < 0.001
a Increased in comparison with controls
A single malignant lymphoma was reported in a female at the high
dose at terminal sacrifice, but no other neoplastic lesions were
reported. Given the isolated nature of this finding, it was considered
not to be related to treatment. The absolute and relative organ
weights were generally unaffected by treatment, although the relative
liver weights were increased in females at 200, 400, and 800 ppm. The
clinical signs included urogenital staining in males at doses
> 200 ppm and in females at 800 ppm. Ocular opacities occurred in
some animals at 400 and 800 ppm. Dose-related histopathological
findings were seen in the adrenal glands (including lipogenic
pigmentation at doses > 200 ppm in females and at > 400 ppm in
males) and in the eyes (acute or subchronic keratitis in two males and
four females at 800 ppm and in a single female at 400 ppm).
Erythrocyte acetylcholinesterase activity was variable, and the effect
of treatment on this parameter was equivocal. The NOAEL was 5 ppm,
equal to 0.7 mg/kg bw per day, on the basis of inhibition of brain
acetylcholinesterase activity at higher doses (Crown et al., 1987).
Rats
In a 2-week study to establish the doses to be used in a 13-week
study, technical-grade chlorpyrifos (purity, 95.5%) was administered
to groups of five rats of each sex at dietary concentrations of 0
(controls), 10, 30, 84, 240, or 694 ppm for 14 days, equivalent to 0,
1.4-1.9, 4.1-5.2, 12-14, 34-39, and 66-95 mg/kg bw per day,
respectively. At the high dose, clinical signs of intoxication were
reported, consisting of irritability, hunching, tremor, ataxia,
urogenital staining, pigmented orbital secretion, failure to groom,
and proneness. No treatment-related clinical signs were observed at
other doses, and treatment-related deaths were confined to animals at
the high dose. Body weights were reduced in males and females at
694 ppm and in females at 240 ppm, and food consumption was also
reduced in animals at 694 ppm. Dose-related decreases in blood
cholinesterase activity were seen at all doses, the inhibition ranging
from 42% at 10 ppm to 93% at 694 ppm. On the basis of these findings,
the doses selected for the 13-week study were 0.5, 10, and 100 ppm
(Crown et al., 1984b).
Plasma, erythrocyte, and brain cholinesterase activities were
markedly reduced in rats after dietary administration of chlorpyrifos
for 14 days at doses of 5 or 10 mg/kg bw per day in a study carried
out according to GLP. Inhibition of cholinesterase activity occurred
in the absence of clinical signs of intoxication. Feed consumption,
body weights, and gross pathological appearance were similar in
control and treated groups. Statistically significant increases in
absolute and relative adrenal weights (about 20% compared with
controls) were observed in females only at 10 mg/kg bw per day
(Liberacki et al., 1990).
Fischer 344 rats received technical-grade chlorpyrifos (purity,
95%) by nose-only inhalation for 6 h/day for 5 days at a target
concentration of 20 ppb in a study that conformed to GLP. No deaths or
treatment-related clinical signs were reported. Exposure of female
rats to a concentration of 0.34 mg/m3 (23 ppb) resulted in a
significant decrease in plasma cholinesterase activity. No effect on
erythrocyte or brain acetylcholinesterase activity was seen in females
or on plasma, erythrocyte, or brain cholinesterase activity in males
(Newton, 1988a).
Chlorpyrifos was administered by nose-only inhalation to female
Fischer 344 rats at a time-weighted average concentration of 12 ppb
(equivalent to 172 µg/m3) for 6 h/day on 5 days per week for 2 weeks
in a study that met GLP requirements. No clinical signs of
intoxication or treatment-related changes in body weights were
reported, and all rats survived until the scheduled termination.
Clinical chemistry showed no treatment-related changes in
haematological, clinical chemical, or urinary parameters, including
plasma, brain, and erythrocyte cholinesterase activity (Landry et al.,
1986).
In a study to determine a dose range, conducted to GLP
requirements, technical-grade chlorpyrifos was administered by
whole-body exposure of groups of Wistar rats for 6 h/day, 5 days per
week for 2 weeks at target concentrations of 0 (air control), 10, 100,
and 400 mg/m3 (actual concentrations, 0, 10, 94, and 388 mg/m3).
Exposure of animals to the high dose was terminated after five
exposures because they became moribund, with rapid weight loss and
deterioration of their general condition, and all surviving animals
were killed on day 9. No deaths was observed at other doses. There was
no NOAEL in this study, as significant inhibition of plasma,
erythrocyte, and brain cholinesterase activity was seen at all doses
in males and females. Plasma cholinesterase activity was inhibited by
78, 86, and 88% at the three doses, respectively, in males and by 91,
93, and 95%, respectively, in females when compared with controls.
Erythrocyte cholinesterase activity was inhibited by 78, 58, and 75%
in males and by 60, 80, and 73% in females, respectively. Brain
cholinesterase activity was inhibited by 50% at 10 mg/m3, about 70%
at 94 mg/m3, and about 72% at 388 mg/m3 in males and females.
Clinical signs of toxicity (tremors, salivation, and lachrymation),
decreased food consumption, and decreased body weights were observed
at the two higher doses. Increased adrenal weights were seen at 94 and
388 mg/m3, and effects in the forestomach including thickening and
congestion were observed at 388 mg/m3 (Kenny et al., 1988).
Fischer 344 rats were exposed to time-weighted average
concentrations (nominal) of 0, 1, or 5 ppb (0, 0.014, and
0.072 mg/m3) of chlorpyrifos vapour for 6 h/day, 5 days per week for
2 weeks in a study conducted according to GLP. The mean daily
time-weighted average concentrations found by analysis were 0.7 and
5 ppb, respectively. Although two control groups were used, their
cholinesterase activity did not differ statistically significantly,
and the activity in test groups was compared with that of the combined
control group. Statistically significant decreases in plasma,
erythrocyte, and brain cholinesterase activity were observed at 5 ppb,
but as the reductions in brain and erythrocyte acetylcholinesterase
activity did not reach 20% when compared with the control group, the
effect was considered not to be of toxicological significance. Plasma
cholinesterase activity was inhibited by > 20% in females at 5 ppb.
A statistically significant decrease in plasma cholinesterase activity
in females at 0.7 ppb was considered to be incidental to treatment.
There were no treatment-related deaths, clinical signs of toxicity, or
changes in body weight during the study. No treatment-related effects
were seen on body or organ weights, and no lesions were found at gross
examination. No significant adverse treatment-related effects were
observed at 5 ppb (approximately 0.07 mg/m3) (Landry et al., 1985;
Streeter et al., 1987).
Groups of 10 CDF Fischer 344 rats of each sex were fed diets
containing chlorpyrifos (purity, 95.7%) at concentrations that
provided doses of 0, 0.1, 1, 5, or 15 mg/kg bw per day for 13 weeks in
a study that conformed to GLP. The treatment-related effects seen at
the highest dose consisted of decreased body weight and body-weight
gain in males, increased fatty vacuolation of the adrenal zone
fasciculata in males, and changes in haematological and clinical
chemical parameters consisting of decreased erythrocyte counts in both
sexes; increased platelet counts, reduced serum total protein,
albumin, and globulin concentrations, and decreased alanine
aminotransferase and alkaline phosphatase activity in males; and
decreased serum glucose concentration and increased urinary specific
gravity in females. Plasma and erythrocyte cholinesterase activities
were depressed at doses > 1 mg/kg bw per day, and the activity of
brain acetylcholinesterase was depressed at 5 and 15 mg/kg bw per day.
The NOAEL was 0.1 mg/kg bw per day on the basis of depression of
erythrocyte and brain acetylcholinesterase activity (Szabo et al.,
1988).
Groups of 10 rats of each sex were given diets containing
chlorpyrifos at 0, 10, 30, 300, or 1000 ppm, but treatment at the
highest concentration was discontinued after 4 weeks because of severe
clinical signs. Plasma cholinesterase activity was reduced in a
dose-related manner and was significantly lower than that of controls
at all doses. Erythrocyte cholinesterase activity was similarly
significantly reduced at all doses, but no dose-dependency was
demonstrated. Brain cholinesterase activity showed a clear
dose-related depression in animals of each sex at doses > 30 ppm.
At 30 ppm, the activity was inhibited by 22% in males and 24% in
females 41 days after administration. No other significant effects
were found on haematological or clinical chemical parameters, but few
were examined. No histopathological lesions were found that could be
linked to treatment. The NOAEL for inhibition of brain cholinesterase
activity was 0.5 mg/kg bw per day, and the LOAEL was 1.5 mg/kg bw per
day. There was no NOAEL for inhibition of erythrocyte or plasma
cholinesterase activity, as effects were seen at concentrations
> 10 ppm (0.5 mg/kg bw per day) (JMPR 1972; modified by reference
to the original reports of Beatty & McCollister, 1964; Beatty &
McCollister, 1971).
Five groups of 10 rats (strain not specified) of each sex
received chlorpyrifos (purity not specified) in the diet at
concentrations that provided doses of 0.03-10 mg/kg bw per day. The
doses of 0 and 0.3 mg/kg bw per day were fed for 90 days; the doses of
1, 3, and 10 mg/kg bw per day were fed for 28 days, when these animals
were fed control diets for 3 weeks and then diets containing 0, 0.03,
or 0.1 mg/kg bw per day. Behaviour and growth were affected at 3 and
10 mg/kg bw per day. Cholinesterase activity was depressed at 0.3
mg/kg bw per day. During the period of removal from treated diet, the
cholinesterase activity returned to pre-test levels; on resumption of
feeding at 0.1 mg/kg bw per day, only a slight depression was observed
in plasma and erythrocyte cholinesterase activity. In view of of the
unusual design of this study, the toxicological relevance of this
effect is considerede equivocal. No depression of brain or blood
enzymes was seen at 0.03 mg/kg bw per day (Blackmore, 1968).
Groups of 10 Crl rats of each sex were fed diets containing
chlorpyrifos (purity, 97.5%) at concentrations that provided a dose of
0.3 mg/kg bw per day for 13 weeks or 1, 3, or 10 mg/kg bw per day for
4 weeks. No deaths occurred during the study. After 4 weeks, there was
a clear dose-response relationship for inhibition of erythrocyte and
plasma cholinesterase activity at all doses, and animals at higher
doses showed clinical signs of toxicity. These animals were therefore
removed from exposure and allowed to recover for 3 weeks, during which
time the plasma and erythrocyte cholinesterase activities returned to
control values. These groups were then fed chlorpyrifos at doses of 0,
0.03, or 0.1 mg/kg bw per day for 13 weeks. The dose of 0.03 mg/kg bw
per day had no unequivocal effect on cholinesterase activity in
erythrocytes, plasma, or brain in either sex, but the dose of
0.1 mg/kg bw per day significantly reduced the erythrocyte
cholinesterase activity. The NOAEL was 0.03 mg/kg bw per day on the
basis of inhibition of erythrocyte cholinesterase activity after 13
weeks at 0.1 mg/kg bw per day (Hazleton Laboratories, 1968).
Groups of 20 Sprague-Dawley rats of each sex were given
chlorpyrifos (purity not stated) in the diet at 0, 0.03, 0.15, or
0.75 mg/kg bw per day for 6 months; an interim sacrifice of five
animals of each sex per dose was conducted after 3 months. A total of
16 animals across dose groups died from chronic murine pneumonia.
Cholinesterase activity was inhibited at the high dose in both
erythrocytes (50%) and plasma (65%), but the activity in brain was not
inhibited at any dose. Treatment did not change body-weight gain, food
consumption, or haematological or other clinical chemical parameters.
The NOAEL was 0.15 mg/kg bw per day, on the basis of inhibition of
erythrocyte acetylcholinesterase activity at 0.75 mg/kg bw per day
(JMPR, 1972; modified by reference to the original report of Coulston
et al., 1971).
Groups of 20 Crl CD Sprague-Dawley-derived rats of each sex were
given chlorpyrifos (purity, 95.5%) at dietary concentrations of 0
(control), 0.5, 10, or 200 ppm for 13 weeks in a study conducted
according to GLP. The minimum and maximum achieved doses of the test
material were calculated to be 0, 0.03 and 0.078, 0.6 and 1.5, and 13
and 31 mg/kg bw per day, respectively. All animals were inspected once
or twice daily for signs of ill-health and other treatment-related
signs and all were examined closely each week. No deaths or
significant clinical signs were observed at any dose. Clinical
chemistry, urinary analysis, and ophthalmoscopy revealed no findings
that were considered to be related to treatment. At 200 ppm
(approximately 13 mg/kg bw per day), reduced body weights, erythrocyte
count, erythrocyte volume fraction, and haemoglobin and increased food
consumption were observed. Statistically significant reductions
(> 20% inhibition compared with controls) in plasma cholinesterase
activity were observed in males at doses > 0.5 ppm (approximately
0.03 mg/kg bw per day) at 12 weeks and in females at doses > 10 ppm
(approximately 0.6 mg/kg bw per day). The inhibition was dose-related
in males but not in females. Erythrocyte and brain cholinesterase
activities were not measured in this study. The NOAEL was 10 ppm,
equal to 0.6 mg/kg bw per day, on the basis of reduced body weights
and statistically significant reductions in packed cell volume,
haemoglobin, and erythrocyte volume fraction at 200 ppm (Crown et al.,
1985).
Groups of 10 Fischer 344 rats of each sex were exposed to
chlorpyrifos (purity, 100%) by nose only at concentrations of 0, 5.2,
10.3, or 20.6 ppb (0, 75, 150, or 300 µg/m3) for 6 h/day, 5 days per
week for 13 weeks in a study that conformed to GLP. No
treatment-related deaths and only minimal clinical signs were observed
during the study. Body weights and urinary, haematological, and
clinical chemical parameters were unaffected by treatment. No effects
on plasma, erythrocyte, or brain cholinesterase activity were seen at
any dose. Gross and histopathological examination revealed no effects
associated with administration of chlorpyrifos (Corley et al., 1986).
Groups of 15 Fischer 344 rats of each sex were exposed to
chlorpyrifos (purity, 95%) by nose only for 6 h/day, 5 days per week,
for 13 weeks at target concentrations of 0 (control), 5, 10, and
20 ppb (0, 0.07, 0.14, and 0.28 mg/m3, respectively). The study was
conducted according to GLP requirements. There were no
treatment-related deaths or ophthalmic, haematological, or clinical
chemical changes. Plasma cholinesterase activity was inhibited by 23%
in males at the high dose ( p < 0.01), but other changes in plasma
cholinesterase activity were considered not to be related to
treatment. Erythrocyte and brain cholinesterase activities were
unaffected. Pathological and histopathological examination showed no
effect of treatment. The NOAEL was 20 ppb (0.28 mg/m3), on the basis
of inhibition of plasma cholinesterase activity. The absence of frank
toxicity at any dose in this study makes it difficult to draw
conclusions about the toxic potential of inhaled chlorpyrifos at doses
> 0.28 mg/m3 (Newton, 1988b).
In a study in Fischer 344 rats treated dermally, conducted
according to GLP requirements, the animals received chlorpyrifos
(purity, 100%) at 0, 0.1, 0.5, or 5 mg/kg bw per day in corn oil for a
total of 15 days over a 21-day period. No treatment-related effects
were observed at any dose, and plasma, erythrocyte, and brain
cholinesterase activities were not inhibited. Gross and microscopic
examination did not reveal any treatment-related changes, and body
weights and organ weights were unaffected by treatment. In the 4-day
range-finding component of this study, decreased plasma cholinesterase
activity (45%) and erythrocyte cholinesterase activity (16%) were
observed at 10 mg/kg bw per day, in the absence of clinical signs of
intoxication (Calhoun & Johnson, 1988, 1989).
Rabbits
No skin irritation was found when a formulation containing 61.5%
chlorpyrifos and 35% xylene was applied to the skin of the back and
abdomen of groups of two rabbits at doses of 5, 10, 25, and 50 mg/kg
bw 20, 4, 3, and 1 times, respectively. Both plasma and erythrocyte
cholinesterase activities were significantly inhibited in each dose
regimen. The values for plasma recovered more quickly than those for
erythrocytes, which were still significantly inhibited at day 40 after
the 3-, 4-, and 20-day exposure patterns (Pennington & Edwards, 1971).
Three rabbits were exposed for 5 min to a formulation containing
61.5% chlorpyrifos and 34.5% xylene from an ultra-light vapour cold
aerosol fog generator delivering 3.8 L/h. Two rabbits were exposed at
a distance of 8 m at a height of 1.3 m and one at a height of 0.8 m.
Exposure was terminated after 5 min because of ocular and pulmonary
irritation. The recorded concentration of chlorpyrifos in
breathing-space air was about 108 mg/L (range, 83-133 mg/L). By 24 h
after treatment, the rabbits had decreased activities of plasma
cholinesterase (< 33%) and erythrocyte cholinesterase (< 12%),
but these values had recovered almost to the control level by 72 h
after treatment (Pennington & Edwards, 1971).
Chickens
White Leghorn chickens were given chlorpyrifos in their
drinking-water at 1 ppb, 1 ppm, or 100 ppm for up to 84 days. Only
plasma cholinesterase activity was reported because of technical
difficulties with blood sampling. Significant inhibition (54%) of
plasma cholinesterase activity was recorded on day 84 in birds at 100
ppm (Stevenson, 1965).
Dogs
Technical-grade chlorpyrifos (purity, 95.8%) was administered to
groups of two pure-bred beagles of each sex orally in gelatine capsule
at doses of 0 (control; lactose only), 0.01, 0.03, 0.5, or 5 mg/kg bw
per day for 4 weeks, although only one animal of each sex was exposed
at 0 and 0.5 mg/kg bw per day. This study met GLP requirements. No
deaths occurred during the study, and no clinical signs associated
with treatment were observed. All animals gained weight steadily
during treatment, and food consumption was similar in all groups.
Rapid inhibition of plasma cholinesterase activity was observed at 0.5
and 5 mg/kg bw per day at all intervals, and the inhibition was both
dose- and time-dependent. Plasma cholinesterase activity was reduced
at 0.03 mg/kg bw per day, but the inhibition was considered not to be
significant owing to variation between groups and the small group
sizes. In animals at 5 mg/kg bw per day, erythrocyte cholinesterase
activity was 47% that of controls on day 7 and was still inhibited by
> 70% at the end of the study; brain cholinesterase activity was
inhibited by 32% in comparison with concurrent controls at the end of
the study. In animals at 0.5 mg/kg bw per day, erythrocyte
cholinesterase activity was inhibited by 30% on days 27-28 only, and
the effect was considered not to be toxicologically significant owing
to its transient nature. Macroscopic post-mortem examination showed no
changes that were considered to be of toxicological significance. The
NOAEL was 0.5 mg/kg bw per day, on the basis of inhibition of
erythrocyte and brain cholinesterase activity at 5 mg/kg bw per day
(Harling et al., 1989a).
Five dogs received 10 applications of a 0.087% commercial aerosol
formulation of chlorpyrifos in dichlorophene on their backs (with the
fur brushed against the grain) for 2 weeks, each application lasting
30 s. No deaths occurred, and no abnormalities were seen in clinical
signs, body-weight gain, or opthalmoscopic, haematological or clinical
chemical parameters. Erythrocyte cholinesterase activity was not
decreased, but plasma cholinesterase activity was inhibited from the
beginning of treatment, returning to normal 72 days after treatment.
The inhibition of plasma cholinesterase activity was not accompanied
by clinical signs (Sharp & Warner, 1968).
Groups of two young adult beagles of each sex were given daily
doses of chlorpyrifos orally by capsule at doses of 0, 0.03, 0.1, 0.3,
or 1 mg/kg bw per day for 90 days. Plasma cholinesterase activity was
inhibited at all doses. The NOAEL for inhibition of erythrocyte
cholinesterase activity was 0.03 mg/kg bw per day, and that for
inhibition of brain cholinesterase activity was 1 mg/kg bw per day
(Blackmore, 1968).
Technical-grade chlorpyrifos (purity, 95.8%) was administered
orally by capsule to groups of four pure-bred beagles of each sex at
doses of 0 (control), 0.01, 0.22, or 5 mg/kg bw per day for 13 weeks.
There were no unscheduled deaths or unequivocal clinical signs during
the study. The group mean body weights of animals at the high dose
were reduced. Haematology, clinical chemistry, urinary analysis, and
ophthalmoscopy revealed no findings that were considered to be related
to treatment. No treatment-related pathological macroscopic or
microscopic change was found. Statistically significant, dose-related
reductions in plasma cholinesterase activity were seen at 0.01 mg/kg
bw per day (bitches only; week 6 only; < 25%) and above (dogs and
bitches; all sample intervals; < 87%). Erythrocyte cholinesterase
activity was significantly inhibited in a dose-dependent manner at
0.22 mg/kg bw per day (< 46%) and at 5 mg/kg bw per day (< 85%).
At 0.01 mg/kg bw per day, erythrocyte cholinesterase was inhibited by
17-18% at week 12, but the reduction was not statistically significant
at any sample interval. Brain cholinesterase activity was inhibited in
animals of each sex (by 46%) at 5 mg/kg bw per day, and the NOAEL for
this effect was 0.22 mg/kg bw per day. The NOAEL for the study, on the
basis of inhibition of erythrocyte acetylcholinesterase activity, was
0.01 mg/kg bw per day (Harling et al., 1989b).
Beagles aged 10 and 11 months were given diets containing
chlorpyrifos (purity, 97.2%) at concentrations that provided doses of
0, 0.01, 0.03, 0.1, 1, or 3 mg/kg bw per day for 1 year (one animal of
each sex per dose), 1 year with a 3-month recovery period (two animals
of each sex per dose), or 2 years (four animals of each sex per dose).
Clinical signs were recorded daily, and body weights were recorded
weekly for the first 6 months and every 2 weeks thereafter. Food
intake was recorded weekly during the first 3 months and monthly
thereafter. The clinical investigations in the groups receiving 0, 1,
or 3 mg/kg bw per day included haematology (packed cell volume,
haemoglobin, erythrocyte and leukocyte counts, and prothrombin time)
and urinary analyses (specific gravity, occult blood, ketones, solids,
pH, albumin, and sugar). The serum concentration of blood urea
nitrogen and the activities of alkaline phosphatase and alanine and
aspartate aminotransferases were measured in all dogs before treatment
and at various intervals thereafter. Bromsulphalein retention was
measured before treatment and at the 1-year sacrifice of animals at 0,
1, or 3 mg/kg bw per day. Cholinesterase activity was determined in
plasma and erythrocytes in most groups before treatment and at various
intervals during the study, and the activity in brain was measured at
necropsy. Fasted dogs were necropsied and the brain, heart, liver,
kidney, spleen, and testes were weighed. Portions of these organs and
a standard set of tissues were preserved from the controls and animals
at 1 (after 1 year only) and 3 mg/kg bw per day for histopathological
examination. The animals fed chlorpyrifos for 2 years were given
complete physical examinations before the end of the study, including
routine neurological and opthalmoscopic evaluations. The data were
evaluated only with Student's t test.
The body weights were not significantly affected by treatment,
and food consumption was comparable in groups of the same sex.
Consumption of the test compound was monitored regularly and reported
to approximate the nominal concentrations closely. The organ weights
were not affected by treatment, except for an increase in the mean
relative liver weight in males at 3 mg/kg bw per day (3.5 g/100 g vs
2.5 g/100 g in controls) in the 2-year study. Details of the recorded
clinical signs were not presented, and the authors simply stated that
"No clinical signs of toxicity were observed in any of the dogs in
either phase of the experiment." The results of clinical pathology
were presented as individual and summary data. There were no
unequivocal treatment-related differences between control and treated
groups in the limited haematological parameters measured nor in
urinary analyses. No effect of treatment was found on blood urea
nitrogen, alkaline phosphatase or alanine and aspartate
aminotransferase activities or Bromsulphalein retention. The results
of gross pathological examination were reported in the form of a
general summary. None of the findings indicated an effect of
treatment. The results of the limited histopathological examinations
(control and high dose only) were reported in a summary table with no
indication of the severity of lesions or abnormalities; individual
data were not presented. The available data did not indicate any
treatment-related effects.
Cholinesterase activity was presented for individual animals and
for groups (Table 3). Inhibition was evident within nine days (the
first assay time) of the start of the study, and the values recorded
at termination were similar to those recorded at interim assays.
Plasma cholinesterase activity was decreased in a dose-related manner
at both times, and erythrocyte cholinesterase activity was similarly
depressed, females being slightly more severely affected than males.
Brain cholinesterase activity was the least affected by treatment. The
cholinesterase activity of animals that were allowed to recover,
assayed after 2, 6, and 13 weeks on normal diet, showed a return to
control levels after 2 weeks for plasma activity and after 13 weeks
for erythrocyte activity.
Table 3. Percentage inhibition of cholinesterase activity in beagles treated with
chlorpyrifos in the diet in comparison with concurrent controls
Dose Inhibition of cholinesterase activity (%)
(mg/kg bw per day)
Plasma Erythrocytes Brain
Male Female Male Female
One year (males and females)
3 72 70 79 82 8
1 62 62 63 61 3
0.1 39 36 18 30 0
0.03 18 17 2 33 0
0.01 1 1 16 13 0
Two years (males)
3 77 67 83 70 20
1 69 50 68 66 7
0.1 52 38 27 41 8
0.03 23 22 0 6 7
0.01 2 0 14 6 1
Average values at terminal sacrifice
Inadequacies in data collection and recording were noted.
Although the long-term effects of dietary intake of chlorpyrifos could
not be evaluated in the absence of complete pathological and
histopathological examination, the data were adequate for analysis of
inhibition of cholinesterase. Plasma cholinesterase activity was
inhibited in animals of each sex at 0.03 mg/kg bw per day. Erythrocyte
cholinesterase activity was inhibited by > 20% at 0.1 mg/kg bw per
day at both the 1- and 2-year intervals, but the inhibition did not
consistently reach statistical significance when compared with the
values in concurrent controls or with pre-treatment values. This was
due in part to the small group sizes and the variation in this
parameter between groups, including the control group, the activity
differing by as much as twofold between individual animals. Under
these conditions, the erythrocyte cholinesterase activity would have
had to be inhibited by as much as 50% in comparison with controls
before statistical significance was achieved. The NOAEL was 0.1 mg/kg
bw per day on the basis of inhibition of erythrocyte cholinesterase
activity at higher doses. Inhibition of brain cholinesterase activity
was seen only at 3 mg/kg bw per day after 2 years (JMPR, 1972;
modified by reference to the original report of McCollister et al.,
1971a).
A supplementary report provided some of the data that were
lacking or inadequate in the original report, but the observations
made in individual animal during life were limited and did not
consistently include basic observations. Individual body weights,
ophthalmological data, the results of pre-treatment and terminal
physical examinations, and an inventory of histopathological findings
were included. The conclusions of the original study were unchanged,
with the additional observation that no effect of treatment was
detected by ophthalmology (Kociba et al., 1985).
Rhesus monkeys
Groups of one or two rhesus monkeys of each sex were given
technical-grade chlorpyrifos in 1% aqueous gum tragacanth by stomach
tube at doses of 0, 0.08, 0.4, or 2 mg/kg bw per day for 6 months.
There were no treatment-related changes in body-weight gain, food
consumption, clinical findings, or haematological, clinical chemical,
or histopathological parameters. Plasma cholinesterase activity was
depressed at all doses at 16 weeks and at > 0.4 mg/kg bw per day at
24 weeks, and erythrocyte cholinesterase activity was depressed at 0.4
and 2 mg/kg bw per day (Table 4). The NOAEL for erythrocyte
cholinesterase inhibition was 0.08 mg/kg bw per day and that for brain
cholinesterase inhibition was 2 mg/kg bw per day. The usefulness of
this study was limited by the small number of animals tested (JMPR
1972; modified by reference to the original paper of Coulston et al.,
1971).
Table 4. Mean cholinesterase activity (and percent inhibition in
comparison with controls) in rhesus monkeys given chlorpyrifos
by stomach tube
Cholinesterase Time Dose (mg/kg bw per day)
activity (weeks)
0 0.08 0.4 2
Plasma 16 6.0 4.4 (26%) 1.6 (72%) 1.8 (70%)
24 4.4 3.8 (14%) 1.7 (61%) 2.0 (55%)
Erythrocytes 16 9.5 8.2 (13%) 6.5 (32%) 2.6 (72%)
24 8.6 7.6 (13%) 6.3 (27%) 5.4 (38%)
(c) Long-term studies of toxicity and carcinogenicity
Mice
In a study of carcinogenicity conducted in accordance with test
guidelines 83-2 of the US Environmental Protection Agency and OECD 451
and in compliance with GLP standards, technical-grade chlorpyrifos
(stated purity, 95.9%) was mixed in maize oil and incorporated into a
commercially available powdered animal diet to produce dietary
concentrations of 0 (control), 5, 50, and 250 ppm, calculated to be
equal to 0.7-1.1, 6.1-12, and 32-55 mg/kg bw per day in males and
0.7-1.2, 6.6-12, and 34-62 mg/kg bw per day in females. The test diets
were administered to groups of 64 CD-1 of each sex, 3 weeks old, for
at least 79 weeks, terminal sacrifice being undertaken during weeks
80-82. The animals continued to receive the treated diets until
scheduled necropsy. An additional 12 males per group were treated
identically to the mice in the main groups and were used to replace
animals for reasons unrelated to treatment (such as excessive
aggression). All of the unused spares were discarded after 10 weeks of
treatment. Each animal was weighed weekly for the first 13 weeks of
the study and every 2 weeks thereafter, while food consumption was
measured weekly for 13 weeks and monthly thereafter. The animals were
examined daily for clinical signs of intoxication, and a careful
examination, including palpation, was conducted weekly. Blood smears
were prepared from all mice after 12 and 18 months for differential
leukocyte counts, but only those from controls and animals at the high
dose were examined. Plasma, erythrocyte, and brain cholinesterase
activity was determined in five animals of each sex at each dose at 9
and 18 months. All animals were examined grossly, the examination
including all external surfaces, the cranial, thoracic, abdominal, and
pelvic cavities, and the carcass. A large number of organs and tissues
from all animals were fixed for histopathological examination; the
eyes, kidneys, livers, lungs, and thyroids/parathyroids from animals
at all doses were examined, as were other tissues from control and
animals at the high dose and those from animals that died during the
study.
There was no adverse effect on survival, and no treatment-related
increase in the incidence of neoplastic lesions. Treatment-related
clinical signs (ocular opacities, excess lachrymation, and cranial
hair loss) and reductions in body weights and food consumption were
observed at 250 ppm (approximately 32 mg/kg bw per day). The effects
on cholinestrase activity are summarized in Table 5. Plasma
cholinesterase activity was inhibited at all doses. Determinations of
erythrocyte cholinesterase activity revealed > 20% inhibition at 42
weeks and about 30% inhibition at week 78 in animals at 250 ppm but
considerable intra-group variation at other doses. In addition, the
erythrocytes were washed with saline before the cholinesterase
determinations at 78 weeks but not at 42 weeks. The NOAEL for
inhibition of erythrocyte cholinesterase activity was 5 ppm, with a
statistically significant inhibition of 29% in males at 78 weeks and
41% in females at 42 weeks in animals at 50 ppm. Significant
inhibition of brain cholinesterase activity ( p < 0.001 or
p < 0.01) was observed in males and females at 250 ppm at 42 and 78
weeks, the activity being reduced by 80-86% when compared with
controls. At 50 ppm, decreases in activity of 43-47% were seen in
males at 42 and 78 weeks and in females at 42 weeks, but this finding
reached statistical significance ( p < 0.05) in females only at 42
weeks and in males only at 78 weeks. At 5 ppm, brain cholinesterase
activity was reduced in males at 78 weeks (27% reduction), but this
Table 5. Percent inhibition of cholinesterase activity in groups of five mice
fed diets containing chlorpyrifos
Cholinesterase Interval Dose (ppm)
(weeks)
5 50 250
Male Female Male Female Male Female
Plasma 42 4*** 45*** 95*** 97*** 98*** 99***
78 49* 50*** 95*** 96*** 98*** 98***
Erythrocyte 42 1 15 11 41** 22 23
78 - +10a 29* 4 31* 29**
Brain 42 8 1 43 46* 80** 85***
78 27 +13a 47 9 86** 84***
* p < 0.05; ** p < 0.01; *** p < 0.001
a Increased activity in comparison with controls
finding was not statistically significant and was considered to be
incidental to treatment. The NOAEL for inhibition of brain
cholinesterase activity was 5 ppm.
Non-neoplastic effects were observed at the high dose, in the
livers (slight subchronic pericholangitis, histiocytic proliferation,
and centrilobular hepatocytic fatty vacuolation) of males and in the
eyes of males and females. No treatment-related lesions were seen at
lower doses. In males at the high dose, an increased incidence of lung
nodules was seen macroscopically, with rates of 5/31 in controls, 4/26
at 5 ppm, 2/37 at 50 ppm, and 14/49 at 250 ppm. The nodules were
diagnosed histologically as bronchio-alveolar adenomas or carcinomas,
but the incidences were stated to be within the range in historical
controls. Nevertheless, two sets of data were reported for the
incidence of neoplasms in historical controls: values obtained from
24-month studies and a published report on spontaneous neoplasms in
CD-1 mice in a number of testing laboratories. Those used in the
report were less than ideal. The use of background incidence from
24-month studies would be expected to increase the range of
spontaneous tumours over that in the 78-week protocol used in this
study. In addition, the tumour incidence was reported in a published
paper (Maita et al., 1988) as a percentage of the animals examined,
while the historical data from the 24-month study and the tumour
incidence in this study were given as percentages of the number of
organs examined. Histopathological examination did not reveal any
statistically significant increase in the incidence of neoplasms in
treated animals when compared with controls. The NOAEL was 5 ppm
(0.7 mg/kg bw per day) on the basis of inhibition of erythrocyte and
brain acetylcholinesterase activity (Gur et al., 1991).
Groups of 56 CD-1 mice of each sex were maintained on diets
containing 0, 0.5, 5, or 15 ppm of chlorpyrifos (purity, 99.6%) for
105 weeks. They were observed daily for clinical signs, body weights
were recorded monthly, and daily food consumption was calculated from
a 3- or 4-day measurement made once a month. Gross pathological
examination was performed on all animals that died, when the weights
of the brain, testes, heart, kidneys, and liver were recorded and a
range of other tissues were collected. Sections were prepared of all
the these tissues for histopathology. Haematology was limited to
examination of smears of blood collected from the tail vein at
terminal sacrifice.
The body weights and absolute and relative organ weights were not
significantly affected by treatment over the course of the study, and
the mean body weight of all treated female mice was greater (< 13%)
than that of controls from day 84 onwards, although food consumption
was comparable in all groups. The mortality rate was also generally
unaffected by treatment, and treated males had slightly higher
survival rates than controls: 39% in controls, 46% at 0.5 ppm, 50% at
5 ppm, and 48% at 15 ppm; the survival rates of females were lower
than those of controls only at the highest dose: 46% in controls, 59%
at 0.5 ppm, 46% at 5 ppm, and 38% at 15 ppm. There were no clinical
signs or pathological findings that indicated an effect related to
treatment. A significant increase was observed in the incidence of
spindle-cell hyperplasia of the adrenal gland in animals of each sex
given 0.5 ppm chlorpyrifos and in males given 5 ppm. This finding was
not considered to be related to treatment as there was a high
background incidence in ageing mice and no dose-response relationship.
Vacuolation was found in sciatic nerve preparations from 2/56 male and
3/54 female controls, 5/54 males and 8/55 females at 0.5 ppm, 2/56
males and 2/52 females at 5 ppm, and 6/52 males and 8/53 females at 15
ppm. The incidences of alveologenic adenomas and hyperplastic nodules
in the liver were significantly increased only in males at 5 ppm.
These findings were considered to be incidental.
This study was barely adequate for assessing the carcinogenicity
and long-term toxicity of chlorpyrifos, as it had serious shortcomings
in design, method, and data collection. No clinical pathology was
performed, and in particular cholinesterase activity was not measured;
no clinical signs were reported; individual data were not presented
for several parameters, including histopathology; and intestinal
parasites (nematodes) were present in all groups (JMPR, 1982; modified
by reference to the original report of Warner et al., 1980).
Rats
In a 2-year study, groups of 25 male and 25 female Sherman rats
received chlorpyrifos (purity, 97.2%) in the diet at doses of 0, 0.01,
0.03, 0.1, 1, or 3 mg/kg bw per day (dietary concentrations not
provided). Supplementary animals were included at each dose to allow
interim sacrifices. The design of the study is shown in Table 6.
Clinical signs were recorded 'frequently', body weights were recorded
twice weekly for the first 4 weeks, weekly for 2-6 months, and every 2
weeks for the remainder of the study. The food intake of the main
group was recorded continuously during the first 3 months and once a
month thereafter. The clinical investigations included haematology
(packed cell volume, haemoglobin, and erythrocyte and leukocyte
counts) and urinary analysis (solids, pH, albumin, sugar, occult
blood, ketones) for five rats of each sex at 0, 1, and 3 mg/kg bw per
day; the serum concentration of urea nitrogen and the activities of
alkaline phosphatase and alanine aminotransferase were measured in all
rats at sacrifice at 12 and 18 months, in all male survivors and in
four to five females at 24 months. Cholinesterase activity was
determined in plasma, erythrocytes, and brain samples, as shown in
Table 6. Rats in groups A, H, I, and K were necropsied in a standard
manner, and the weights of the brain, heart, liver, kidney, spleen,
and testes were recorded. Portions of these organs and a standard set
of tissues were preserved for histopathology, but only the slides for
the controls, animals at the high dose, and animals at 1 mg/kg bw per
day at the 12-month necropsy were examined. Some tissues from animals
that died during the study and from those animals killed when moribund
were also examined histologically. Statistical evaluation of the data
was limited to Student's t test.
Body weights (data presented only as graphs) were generally not
significantly affected by treatment. No details of the recorded
clinical signs were presented; rather, the authors stated that "No
changes in appearance or demeanour or signs of toxicity were observed
grossly in any of the rats. No evidence of a cholinergic response was
noted at any time." The results of clinical pathology were presented
as individual and summary data, and no unequivocal treatment-related
differences between control and treated groups were found for the
limited haematological parameters measured or for the results of
urinary analyses. Treatment had no effect on serum concentrations of
urea nitrogen or on alkaline phosphatase and alanine aminotransferase
activity.
Plasma cholinesterase activity was significantly decreased at 3
mg/kg bw per day at all assay times, the inhibition ranging from 20 to
40% in males and 55 to 74% in females; the activity was less severely
inhibited at 1 mg/kg bw per day, but from 6 months onwards the
inhibition was 18-38% in males and 50-69% in females when compared
with controls, and these differences were statistically significant at
most assay times. Erythrocyte cholinesterase activity was severely
inhibited in animals of each sex at the higher doses at all assay
times, with rates of 60-100% at 3 mg/kg bw per day, 13-90% at 1 mg/kg
bw per day, and little effect at the other doses. Animals that had
Table 6. Design of the long-term study of toxicity and carcinogenicity in groups of
56 mice fed diets containing chlorpyrifos
Group No. of rats of Sacrifice interval End-point examined
each sex per dose
A 25 2 years Necropsy, blood and brain
cholinesterase
B 5 1 week Blood cholinesterase
C 5 1 month Blood cholinesterase
D 5 3 months Blood cholinesterase
E 5 6 months Blood and brain cholinesterase
F 5 9 months Blood cholinesterase
G 6 12 months Blood and brain cholinesterase
H 5 12 months Necropsy
I 7 12 months, Necropsy, blood and brain
7-8-week recovery cholinesterase
J 7 18 months Blood and brain cholinesterase
K 7 18 months Necropsy
been allowed to recover showed full restoration of cholinesterase
activity. Brain cholinesterase activity was significantly inhibited by
treatment at 3 mg/kg bw per day (30-53% inhibition) at all assay
times, to a lesser degree at 1 mg/kg bw per day (3-16%), and was
comparable to that of controls at other doses. Animals that were
allowed to recover showed restoration of brain cholinesterase
activity.
This study was considered to be inadequate for assessing the
carcinogenicity and long-term effects of chlorpyrifos, other than
clinical chemical findings including cholinesterase inhibition, as
there were serious shortcomings in data collection, and the reports of
gross and histopathology were inadequate and no clinical findings were
reported. The NOAEL for inhibition of plasma and erythrocyte
cholinesterase activity was 0.1 mg/kg bw per day on the basis of
significant inhibition at 1 mg/kg bw per day. The NOAEL for inhibition
of brain acetylcholinesterase activity was 1 mg/kg bw per day on the
basis of significant inhibition at 3 mg/kg bw per day (JMPR, 1972;
modified by reference to the original report of McCollister et al.,
1971b).
A supplementary report provided some of the data that were
lacking or inadequate in the original report, but the observations
made in individual animal during life were limited and did not include
basic observations such as abnormal behaviour or gait. Individual body
weights and gross and histopathological findings were included in the
supplement, but the histopathology was limited and variable, only a
small number of tissues from each rat being examined consistently. The
results were not presented as summary tables of incidence and
severity. The conclusions of the original study remain unchanged by
this supplement (McCollister et al., 1985).
In a 2-year study conducted according to accepted test guidelines
(OECD 451, USEPA 83-5) and GLP, groups of 60 Fischer 344 rats of each
sex were fed diets containing technical-grade chlorpyrifos (purity,
96.1%) at concentrations of 0, 0 (vehicle control), 0.2, 5, or
100 ppm. The vehicle used was 0.04% w/w maize oil. The diets were
prepared weekly and assayed regularly for stability and accuracy of
dosing. Body weights were generally recorded weekly; food consumption
was recorded weekly for 13 weeks and monthly thereafter. Clinical
signs were recorded daily and palpation performed weekly.
Cholinesterase activity was measured before treatment and at weeks 14,
32, 45, 50 (interim sacrifice of five animals of each sex per group
for measurement of brain acetylcholinesterase activity), 78, and 104
weeks (terminal sacrifice with measurement of brain
acetylcholinesterase activity in 10 animals of each sex per group),
and generally on 10 rats of each sex per dose. Haematological
parameters were measured in blood smears from 10 rats of each sex in
the vehicle control group and at 100 ppm at months 12 and 18. Gross
pathological findings were recorded for all animals at necropsy,
including those that died during the study or were killed at early
sacrifices. Necropsy included removal of the eyes with the optic nerve
and adnexa. The weights of the brain, testes, thyroids, adrenals,
kidneys, and liver were recorded, and a range of tissues was
collected. All gross lesions were sectioned, and sections from all
control animal and those at 100 ppm were examined. Appropriate
statistical tests were used to analyse the data.
The mean consumption of chlorpyrifos was estimated to be 0.012,
0.3, and 6 mg/kg bw per day for animals of each sex. Mortality rates
and the incidences of clinical signs or palpable masses were not
affected by treatment. A 5% reduction in the mean body weights of
males at 100 ppm was significant in comparison with controls at most
times during weeks 3-94, and the weights of females at this dose were
significantly lower (about 4%) than those of controls at most times
during weeks 2-64. A variety of non-neoplastic and neoplastic lesions
were recorded, and the incidence of these lesions occasionally showed
a positive trend with dose; however, the incidence was generally
within the range of historical controls in the laboratory or within
the range of published values from the National Toxicology Program in
the USA. The incidences of neoplastic lesions did not show unequivocal
or statistically significant dose-response relationships. The only
observation that was considered to be related to treatment was an
increased incidence of cataracts and diffuse retinal atrophy in
females (Table 7).
Table 7. Incidences of ocular abnormalities found microscopically in rats given
diets containing chlorpyrifos
Abnormality Sex Dose (ppm)
0 0 (vehicle) 0.2 5 100
Diffuse retinal atrophy Male 4/60 0/60 3/23 1/28 3/60
Female 5/60 15/59* 9/60 5/58 24/60*
Cataract Male 31/60 31/60 7/23 6/28 30/60
Female 38/60 38/59 33/60 32/58 51/60*
* Significantly different from controls at p < 0.01 or p < 0.001
Significant (> 20%) inhibition of plasma cholinesterase activity
relative to the control values was observed at doses > 0.3 mg/kg bw
per day (Table 8). While erythrocyte cholinesterase activity was
inhibited in animals at 100 ppm, the variation in the data and the
lack of statistical significance and/or dose-response relationships
mitigated against an unequivocal finding. The NOAEL for inhibition of
brain cholinesterase activity was 0.3 mg/kg bw per day (Crown et al.,
1988).
In a study conducted in compliance with GLP standards, groups of
60 Fischer 344 rats of each sex received diets containing chlorpyrifos
(purity, 98.5%) at concentrations that provided doses of 0 (control),
0.05, 0.1, 1, or 10 mg/kg bw per day for 24 months. Ten rats of each
sex at each dose were randomly designated at the start of the study
for interim sacrifice at 12 months. Blood was collected for
haematology, clinical chemistry, and measurement of plasma and
erythrocyte cholinesterase activity at 6, 12, and 18 months as well as
at the 24-month terminal sacrifice. The groups were observed daily for
deaths and signs of toxicity. All rats were examined clinically at
least once a week from after the sixth month. All animals were
palpated for externally detectable masses before treatment, before the
12-month kill, and monthly thereafter. Body weights and feed
consumption were determined weekly for the first 3 months and monthly
thereafter. All rats were weighed, but feed consumption was determined
for only 20 rats of each sex per group.
All clinical laboratory procedures scheduled for 6 and 12 months
were performed on rats designated for the 12-month interim kill,
whereas tests scheduled for 18 and 24 months were performed on rats
designated for terminal sacrifice. Blood samples were obtained for
haematology and clinical chemistry by orbital sinus puncture under
Table 8. Group mean percent inhibition of cholinesterase activity in comparison
with vehicle controls in rats given diets containing chlorpyrifos
Assay time Dose Cholinesterase inhibition (%)
(weeks) (ppm)
Plasma Erythrocytes Brain
Male Female Male Female Male Female
50 0 0 7 0* 31 0 35
0.2 1 4 0 42 0 14
5 15 51 0* 39 9 10
100 93 98 13 45 57* 80*
78 0 0 3 27 0
0.2 4 3 11 0
5 28* 47* 0 0
100 93* 97* 10 0
104 0 8 9 0 0 18 4
0.2 13 0 0 0 0 0
5 36 37* 17 11 0 0
100 95* 96* 34 18 58* 61*
Values the same as or higher than those of the vehicle control are recorded as
0 inhibition.
* Significantly different from control at p < 0.01 or p < 0.001
light anaesthesia. The haematological determinations consisted of
packed cell volume, haemoglobin concentration, erythrocyte count,
total and differential leukocyte counts, and platelet count. The
clinical biochemical determinations included blood urea nitrogen,
alkaline phosphatase and alanine and aspartate aminotransferase
activities, glucose, total protein, albumin, globulin (calculated),
creatine phosphokinase, total bilirubin, cholesterol, calcium,
phosphorus, sodium, potassium, and chloride. Urine samples were
obtained 1-2 weeks before the scheduled sacrifices at 12 and 24 months
and at 6 and 18 months from 10 rats of each sex per group. The urinary
parameters measured included specific gravity and semi-quantitative
estimates of bilirubin, glucose, ketones, occult blood, pH, protein,
and urobilinogen. Microscopy of a pooled sample was also conducted.
Plasma and erythrocyte cholinesterase activities were assayed in 10
rats of each sex per group at 6, 12, 18, and 24 months, and
acetylcholinesterase activity was measured in half-brain samples
obtained at the 12-month and 24-month scheduled necropsies. All
animals killed at the interim and terminal sacrifices or which died or
were killed when moribund were necropsied and subjected to a complete
gross examination. The brain, liver, kidneys, testes, ovaries, and
adrenal glands were weighed, and many organs and tissues were removed
and preserved in neutral, phosphate-buffered 10% formalin for
subsequent histopathological evaluation. Histological sections of the
formalin-fixed tissues from all controls and those at the highest dose
were prepared, stained with haematoxylin and eosin and examined
microscopically. Histopathological examination of tissues from animals
at the three lower doses was limited to the liver, kidneys, adrenals,
and tissues with gross lesions at both sacrifices. At terminal
sacrifice, the lungs, spleen, testes, pituitary, and
thyroid/parathyroid were also examined microscopically. Appropriate
statistical tests were applied to the data.
Males at the high dose showed a consistent decrease in
body-weight gain relative to controls in the absence of reduced food
consumption, depression of plasma (56-87%), erythrocyte (20-40%) and
brain (56-58%) cholinesterase activities, and an increase in the
weight of their adrenal glands which was characterized microscopically
by exacerbated fatty vacuolation of the zona fasciculata. Similar
effects were observed in females at this dose, but were generally less
pronounced than in males: for example, a transient decrease in
body-weight gain relative to controls with no reduction in food
consumption, depression of plasma (82-95%), erythrocyte (generally
> 20%), and brain (57-61%) cholinesterase activities, and an
increase in adrenal weight at the terminal sacrifice with no
associated histopathological lesions. At 1 mg/kg bw per day, the only
effects attributable to treatment were inhibition of plasma
cholinesterase activity (39-71% in males and 60-86% in females) and
erythrocyte cholinesterase activity (20-40% in males and < 22% in
females); brain cholinesterase activity was not affected. These
results are summarized in Table 9. No treatment-related effects were
observed at the two lower doses. There was no increase in the
incidence of any type of tumour. The NOAEL for inhibition of
erythrocyte cholinesterase activity was 0.1 mg/kg bw per day, on the
basis of toxicologically (> 20%) or statistically significant
inhibition at 1 mg/kg bw per day, and the NOAEL for inhibition of
brain cholinesterase activity was 1 mg/kg bw per day on the basis of
toxicologically and statistically significant inhibition at 10 mg/kg
bw per day (Young & Grandjean, 1988).
Chickens
Hens were fed diets containing chlorpyrifos at concentrations of
0, 25, 50, or 200 ppm (approximately 0, 2.5, 5, and 20 mg/kg bw per
day) for 52 weeks. Mortality was unaffected by treatment, with rates
of 13% in controls, 3% at 25 ppm, 7% at 50 ppm, and 10% at 200 ppm.
Plasma cholinesterase activity was assayed in three hens at each dose
1, 3, 7, 14, 22, and 29 days after the start of treatment, monthly
thereafter, and 1, 2, and 3 weeks after the end of treatment. The
onset of inhibition of cholinesterase activity was rapid and
dose-related, with 22% inhibition in week 1 at 25 ppm, 45% inhibition
at 50 ppm, and 76% inhibition at 200 ppm. It persisted at similar
levels throughout the study but returned to control levels during the
Table 9. Group mean cholinesterase activities in rats given diets containing chlorpyrifos,
expressed as percent of the respective control values for each sampling period
Cholinesterase Sex Sampling period Dose (mg/kg bw per day)
(months)
0 0.05 0.1 1 10
Plasma Male 6 100 96.5 95.1 60.9* 44.2*
12 100 94.5 97.8 28.6* 13.3*
18 100 93.4 80.1 36.6* 22.5*
24 100 92.4 85.5 40.0* 19.7*
Female 6 100 97.9 91.0 34.6* 16.9*
12 100 104 87.0* 13.8* 4.8*
18 100 99.1 85.8* 30.4* 12.4*
24 100 103 94.4 39.7* 17.9*
Erythrocytes Male 6 100 107 88.8 76.1* 75.6*
12 100 107 93.2 67.4 63.1
18 100 109 95.3 66.2* 71.0*
24 100 105 108 86.5 73.5*
Female 6 100 93.3 106 104 87.5
12 100 88.2 109 82.2 59.5*
18 100 114 99.7 78.0 81.9
24 100 107 87.2 83.6 79.9
Brain Male 12 100 93.8* 93.1* 91.0* 42.1*
24 100 102 100 103 44.3*
Female 12 100 97.9 102 95.3* 38.9*
24 100 101 100 96.2 42.9*
* Statistically significantly different from control by Dunnett's or Wilcoxon's test,
alpha = 0.05, one-sided
recovery period. Overall feed consumption, body weight, egg
production, feed efficiency, egg weight, and shell thickness were not
affected by treatment. There was no NOEL for inhibition of plasma
cholinesterase activity in this study, as significant inhibition was
seen at the lowest dose tested (Sherman & Herrick, 1973).
(d) Genotoxicity
Chlorpyrifos was not genotoxic in vitro or in vivo in a range
of studies (Table 10).
Table 10. Results of studies for the genotoxicity of chlorpyrifos
End-point Test object Concentration Purity Results GLP Reference
(%) or QA
In vitro
Gene mutation S. cerevisiae D3 NR NR Negative Poole et al. (1977;
rec locus abstract only)
Gene mutation B. subtilus H17, 20 (100, 200, 500, 1000 95.83 Negative Shirasu et al. (1980)
M45 2000 µg/plate in DMSO
Reverse mutation S. typhimurium TA98, 10, 50, 100, 500, 1000, 95.83 Negativea Shirasu et al. (1980)
TA100, TA1535, 5000 µg/plate in DMSO
TA1537, TA1538
Reverse mutation S. typhimurium TA98, 1, 3.162, 10, 31.62, 95.7 Negativea,b QA Bruce & Zempel (1986)
TA100, TA1535, 100 µg/plate in DMSO
TA1537, TA1538
Reverse mutation S. typhimurium TA98, 0.01, 0.1, 1, 5, 10, 95 Negativea,c Jai Research Foundation
TA100, TA1535, 100 µg/plate in DMSO (1996a)
TA1537
Reverse mutation S. typhimurium TA98, 30, 100, 300, 3000, 96.8 Negativea GLP Loveday et al. (1987)
TA100, TA1535, 10 000 µg/plate in DMSO
TA1537, TA1538
Reverse mutation S. typhimurium TA98, NR NR Negative Poole et al. (1977;
TA100, TA1535, abstract only)
TA1537
Reverse mutation E. coli WP2 NR NR Negative Poole et al. (1977;
abstract only)
Table 10. (continued)
End-point Test object Concentration Purity Results GLP Reference
(%) or QA
Relative toxicity E. coli and NR NR Negative Poole et al. (1977;
B. subtilis abstract only)
Reverse mutation S. typhimurium TA98, 5, 500, 5000 µg/plate 96.2 Negativea,d QA Fredrick Institute
TA100, TA1535, in DMSO (1996c)
TA1537, TA1538
Forward mutation Chinese hamster ovary 10, 20, 25, 30, 40, 95.7 Negativea Mendrala (1985)
cells, hprt locus 50 µmol/L
Forward mutation Chinese hamster ovary 5, 10, 25, 50, 75 µg/ml, 96.8 Negativea,e GLP Tu (1987)
cells, hprt locus 16 h, - S9
5, 10, 20, 30, 40, 50 µg/ml,
16 h, - S9
30, 50, 100, 300, 1000 µg/ml,
+S9, in DMSO
Sister chromatid Human lymphocytes 0.02, 0.2, 2 or 20 µg/ml NR Negativea,f Sobti et al. (1982)
exchange (Laz-007) in ethanol
Chromosomal Chinese hamster ovary 0.975, 1.47, 2.93, 4.89, 96.8 Negativea,g GLP Loveday (1987)
aberration cells 9.75, 14.7, 29.3, 48.9, 97.5,
147 µg/ml, 19 h, - S9
1.56, 3.12, 5.2, 10.4, 15.6,
31.2, 52, 104, 156 µg/ml,
10 h, - S9
9.75, 14.7, 29.3, 48.9, 97.5,
147, 293 µg/ml, 19 h, +S9
1, 1.5, 3, 5, 10, 15, 30, 50,
100 µg/ml, 10 h, + S9
2.95, 4.95, 9.85, 14.8, 29.6,
49.4, 98.5, 296 µg/ml, 10 h,
+S9
Table 10. (continued)
End-point Test object Concentration Purity Results GLP Reference
(%) or QA
Chromosomal Rat hepatocytes 16.7, 50, 167, 500, 1667, 98.6 Negativea,h GLP Linscombe et al. (1992)
aberration 5000 µg/ml harvested after
24 h and 5, 16.7, 50, 167
µg/ml harvested after
24 and 48 h, in DMSO
Sister chromatid Chinese hamster ovary 1, 10, 100 µg/ml in acetone NR Negative Muscarella et al. (1984)
exchange cells
Unscheduled DNA Rat hepatocytes 1, 3.16, 10, 31.6, 95.7 Negative Mendrala & Dryzaga
synthesis 100 µ