PYRAZOPHOS
First draft prepared by S. Caroldi
Istituto di Medicina del Lavoro
Padova, Italy
EXPLANATION
Pyrazophos is an organophosphorus systemic fungicide used on a
wide range of crops and cereals in the control of powdery mildew.
Pyrazophos was scheduled for evaluation at the 1985 Joint Meeting
but the data base available at that time was insufficient for the
estimation of an ADI (Annex 1, reference 44).
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
Biochemical aspects
Absorption, distribution, and excretion
Rats
Eight Wistar rats/sex were intubated with a single 2 mg dose of
14C-pyrazophos (3a-14C) in 1 ml sesame oil. Radioactivity was
measured at different times in blood, plasma, urine, exhaled air,
faeces and after 168 hours post-dosing, in several tissues and
organs.
Blood and plasma levels of radioactivity peaked between 4 and 6
h post-dosing in both sexes; the calculated half-life was
approximately 5 h. Over a period of 24 h, approximately 71% of the
original dose was eliminated via urine and 24% via faeces.
Radioactivity was completely eliminated within 168 h (73% via urine
and 27% via faeces). A negligible amount of radioactivity (less than
0.01%) was found in exhaled air within 24 h and in tissues and
organs at 168 h. Metabolites were detected in urine and faeces
collected during the first 24 h when approximately 95% of total
radioactivity was eliminated. Pyrazophos was not detected in urine
but it was the main substance found in the faeces (66% of
radioactivity in faeces and 16% of total radioactivity). Four
metabolites were identified in urine; the most concentrated was a
pyrazophos P-O hydrolysis product present in urine as free compound
or sulphate and glucoronide conjugates (Lachmann, 1986).
Eleven Wistar rats/sex were intubated with a single dose of 0.4
mg 14C-pyrazophos (3a-14C) in 1 ml sesame oil. A further 10
Wistar rats/sex received 14 daily doses of 0.4 mg of pyrazophos
followed by a single dose of 0.4 mg 14C-pyrazophos. Radioactivity
was measured at different times in blood, plasma, urine, exhaled
air, faeces and at 168 h post-test substance in several tissues and
organs. Peak blood and plasma radioactivity levels were measured
within 4 h post-dosing. The calculated half-life was about 5 h. Over
the initial 24 h, 75% of the original dose was excreted via urine
and 18% via faeces. No differences between single or repeated doses
were detected. Negligible amounts of radioactivity were present in
tissues and organs at 168 h from the last pyrazophos administration.
Six different metabolites were found in urine. The most important
was the product of a double hydrolysis 2-hydroxy-5-methyl-
pyrazolo(1,5-a)pyrimidine-6-carboxylic acid. Other metabolites were
O-pyrazophos and the products of hydrolysis either at the phosphate
or carbonate bond. Two metabolites remained unknown. Pyrazophos was
the only radioactive component present in faeces (Lachmann,1987).
Toxicological studies
Acute toxicity studies
Results of acute toxicity tests with pyrazophos are given in
Table 1.
Table 1. Acute toxicity of pyrazophos
Species Sex Route LD50 LC50 Reference
(mg/kg bw) (mg/m3)
Rats M oral 242-778a Hollander & Weigand (1978a,b,c)
F 151-468 Otaka et al. (1981a,b)
Hollander & Weigand (1977f)
Scholz & Weigand (1972a)
M i.p. 280 Hollander & Weigand (1977a,b)
F 305
M 172 Otaka et al. (1981e,f)
F 135
M s.c. 202 Otaka et al. (1981c,d)
F 193
M 221 Hollander & Weigand (1977g,h)
F 266
F dermal > 2000 Hollander & Weigand (1981)
(24 h exp)
M&F inhalation > 1220 Hollander & Weigand (1982)
(4 h exp)
Mice M oral 214 Hollander & Weigand (1977d,e)
F 205
M 413 Otaka et al. (1981g,h)
F 321
M i.p. 581 Scholz & Weigand (1972b)
F 438
M 167 Otaka et al. (1981m,n)
F 146
M s.c. 339 Otaka et al. (1981i,l)
F 348
M 181 Hollander & Weigand (1977i,l)
F 199
Dogs M&F oral > 500b Hollander & Weigand (1978d)
M&F > 100c Hollander & Weigand (1979b)
a Symptoms were mostly of the cholinergic type. Pathology showed atrophy of spleen
and haemorrhage of intestine. Histopathology was negative.
Table 1 (continued)
b Clear cholinergic symptoms were observed from 200 mg/kg bw, no animals died up to
the highest tested dose. At 500 mg/kg bw, emesis occured within 2 h from dosing which
likely reduced the absorbed dose.
c Clear cholinergic symptoms were observed from 50 mg/kg bw, no animal died up to
the highest tested dose. At 100 mg/kg bw, emesis occured within 2 h from dosing which
could have reduced the absorbed dose.
Guinea-pigs
Pyrazophos produced no indications of any allergenic properties
in the sensitization test in the guinea-pig according to the Buehler
test (Leist & Weigand, 1982).
Rabbits
Undiluted pyrazophos was non-irritant to the rabbit skin.
Undiluted pyrazophos and a 10% dilution of pyrazophos in sesame oil
resulted in slight irritantion to the eye. A 1% dilution of
pyrazophos in sesame oil resulted in no irritantion to the eye
(Hollander & Weigand, 1977c)
Short-term toxicity studies
Mice
Five groups of ten SPF mice/sex were treated with pyrazophos
for 28 days at dietary concentrations of 0, 1, 5, 25 and 125 ppm
equal to 0, 0.2, 0.9, 4.7, 23.3 mg/kg/day and 0, 0.2, 0.9, 5.0, and
22.2 mg/kg bw/day for males and females, respectively. No signs of
toxicity were observed; body-weight gain and food intake were not
affected by pyrazophos administration. Dose-related inhibition of
both plasma (from 5 ppm) and erythrocyte (from 25 ppm)
cholinesterase was measured in animals of both sexes. Marginal
inhibition (approximately 20%) of brain acetylcholinesterase
activity was detected after 28 days of treatment at 125 ppm in male
mice only. The NOAEL in this study was 25 ppm equal to 4.7 mg/kg
bw/day and 5.0 mg/kg bw/day for male and female mice, respectively
(Hollander & Weigand, 1978e).
Four groups of twenty Charles River CD-1 mice/sex were treated
with pyrazophos technical at dietary concentrations of 0, 1, 5, or
25 ppm for 28 days. No signs of toxicity were observed; body-weight
gain was not affected by pyrazophos administration. Inhibition of
both plasma and erythrocyte cholinesterase was observed in animals
of both sexes from the 1 ppm dose level. Brain cholinesterase
activity was not reduced by pyrazophos administration up to the
highest dose. The NOAEL in this study was 25 ppm equivalent to 4
mg/kg bw/day (Estes, 1979).
Rats
Seven groups of ten albino rats/sex from the CIVO-colony
(Wistar-derived) were treated with pyrazophos (technical) at dietary
concentrations of 0, 1, 2.5, 5, 15, 45 or 150 ppm for 28 days in a
range-finding toxicity study. Ten additional rats per sex at 0 and
150 ppm dietary concentrations were discontinued from the feeding of
pyrazophos for 21 days to examine recovery of cholinesterase
activities. No signs of toxicity were observed; body-weight gain and
food and water intake were not affected by pyrazophos
administration. Haematology and pathology did not show any adverse
effects. Dose-related inhibition of both plasma and erythrocyte
cholinesterase was observed at 15 ppm and higher in both sexes.
Plasma cholinesterase activity returned to normal levels in a week
after discontinuing pyrazophos administration at 150 ppm. Inhibition
(approx. 20%) of brain acetylcholinesterase activity was detected
after 28 days of treatment at 150 ppm in female rats only. The NOAEL
in this study was 45 ppm equivalent to 4.5 mg/kg bw/day in both
sexes (Til et al., 1978)
Five groups of ten weanling Wistar-derived rats/sex were fed
pyrazophos (technical substance, purity 90%) at dietary
concentrations of 0, 5, 8, 10 or 50 ppm for 14 weeks. The diets were
prepared every two weeks and stored at room temperature. Data on
stability and homegeneity of test substance in diets are not
reported. Growth, symptoms, food consumption, haematology, blood
chemistry (whole blood cholinesterases included), urinalyses, organ
weights and pathology were the parameters checked in this study. No
mortality occurred throughout the duration of the study. Food and
water intake and body-weight were not affected by pyrazophos
administration. Scattered differences of no biological relevance
between groups were observed on haematology, biochemical blood
values, urinalysis and pathology. Dose-related inhibition of whole
blood cholinesterase activity was observed at 10 and 50 ppm
pyrazophos in both sexes. The NOAEL in this study was 8 ppm of
pyrazophos equivalent to 0.8 mg/kg bw/day (deKnecht-vanEekelen &
Dreef-vanderMeulen, 1978).
Four groups of thirty-six F344 Charles River rats/sex were
dosed with technical pyrazophos (92.8% purity) for 13 weeks at
dietary concentrations of 0, 2.5, 50 or 1000 ppm equal to 0, 0.21,
4.2 or 90 mg/kg bw/day and to 0, 0.21, 4.0 or 100 mg/kg bw/day for
males and females, respectively. Twelve rats sex/group were killed
at the end of the exposure period, the remaining 24 rats/sex/dose
were killed, 12 after two weeks and 12 after four weeks of recovery.
The diets were prepared every three weeks and stored at 4 œC.
Homogeneity and accuracy of the test diets were checked (analytical
results not reported). Survival was not affected by pyrazophos
administration. Clinical symptoms of the cholinergic type were
observed during the first 4 weeks of treatment at the highest dose
level in both sexes. Body-weight gain was decreased at the highest
dose level during the first 7 weeks of treatment in males and
throughout the duration of the study (recovery period included) in
females. Scattered differences of no biological relevance in food
intake, food efficiency and water intake were observed throughout
the study in all groups. Reduced erythrocyte counts, haematocrit and
haemoglobin were measured at the highest dose level at the interim
examination and at the end of the exposure (also an increased number
of reticulocytes was measured at this time) but these parameters
recovered to normal level within the 4 weeks of recovery period.
Cholesterol and albumin concentrations in blood were also
reduced during pyrazophos administration at 1000 ppm and returned to
normal level after cessation of exposure. Dose-related inhibition of
plasma and erythrocyte cholinesterase was measured at 50 and 1000
ppm dose levels. At the end of the exposure period at 1000 ppm,
brain cholinesterase were inhibited by 64% and 93% in males and
females, respectively. Brain cholinesterase was also marginally
affected (22% inhibition) at 50 ppm in female rats but not in males.
Inhibition of brain cholinesterase was detectable at 1000 ppm 4
weeks after the end of exposure. Several organ weights of the 1000
ppm group differed from control values: the absolute and relative
weights of adrenals, spleen and liver (only the relative weight of
liver was increased in females) were increased in both sexes, testis
and pituitary (females only) weights were decreased. Most of the
differences in organ weights disappeared during the 4 weeks of
recovery. Proliferation of large mononuclear cells of spleen and fat
deposition in fascicular zone of adrenals were considered as dose-
related, but they disappeared after the 2 weeks recovery period. The
NOAEL in this study was 2.5 ppm, equal to 0.21 mg/kg bw/day, in both
sexes, based on marginal brain cholinesterase inhibition measured at
the termination of the study in females at 4.0 mg/kg bw/day (Otaka
et al., 1981o)
Four groups of forty F344 rats/sex were fed technical
pyrazophos (purity 92.9%) for 52 weeks at dietary concentrations of
0, 2, 20 or 200 ppm equal to 0.1, 1.0 or 11 mg/kg bw/day and 0.1,
1.4 or 14 mg/kg bw/day in males and females, respectively. Diets
were prepared every three weeks and were stored at 4 œC until use.
Homogeneity and accuracy of the test diets were checked just before
starting the study and thereafter every three months. Actual
concentrations of pyrazophos (mean values) were within ± 10% of
nominal concentrations. General conditions, body-weight, food and
water intake were recorded throughout the study. Parameters
investigated were: haematology, blood biochemistry, PSP and BSP
test, in plasma in erythrocytes and brain cholinesterase activity
and urinalysis. Pathology (organ weights) and histopathology
(incomplete) were performed at the end of the study. Moreover the
concentration of unmetabolized pyrazophos was measured in plasma,
brain, liver, kidneys and fat at weeks 13, 26 and 52 (detection
limit: 0.05 ppm).
No clinical signs of toxicity or excess mortality related to
administration of the test compound were detected. No differences in
food and water intake and body-weight gain were observed among
different groups. Scattered differences of some parameters were
observed throughout the duration of the study. Among them, increased
erythrocyte counts and haematocrit and decreased cholesterol
concentration in blood were observed at the highest pyrazophos level
in both sexes. Urinalysis and the excretory function for PSP and BSP
did not reveal toxic effects. Plasma and erythrocyte cholinesterase
activities were reduced in both sexes at 20 and 200 ppm pyrazophos.
A slight inhibition of brain cholinesterase activity (30%) was noted
at 200 ppm of pyrazophos in females only. Pathology (organ weights)
and histopathology did not show biologically relevant changes.
Pyrazophos was measured only in fat tissue of male and female rats
fed 200 ppm. Pyrazophos concentration in fat was higher in females
than in males and it was higher in both sexes at the end of the
study than at 13 week and 26 week determinations. The NOAEL in this
study was 20 ppm equal to 1.0 and 1.4 mg/kg bw/day in males and
females, respectively. The NOAEL was based on 30% inhibition of
brain cholinesterase activity detected in the brain of female rats
fed dietary concentrations of 200 ppm. The results of this study
were obtained at the interim sacrifice of a carcinogenicity study
discontinued because of the high mortality rate occuring in all
groups, including controls (Otaka, 1983).
Dogs
Four groups of four pedigree English beagle dogs/sex were fed
pyrazophos at dietary concentrations of 0, 0.5, 2.0, 5.0 or
10/125/320 ppm for 92 days. The highest dose level of 10 ppm was
increased to 125 ppm on day 60 from the beginning of the study and
further increased to 320 ppm on day 75. Two animals/sex/dose (except
0.5 ppm group) were observed for approximately 6 weeks after the end
of pyrazophos administration and cholinesterase activities were
estimated.
The following parameters were investigated: clinical
observations (general conditions, body-weight, behaviour, reflex
excitability, eye examinations, visible mucosae, dentition),
laboratory examinations (haematology, cholinesterase activity in
serum and in erythrocytes, other serum enzymes, urinalyses) and
pathology (organ weights, gross pathology, histopathology). Slight
reduction of food intake, impairment of general conditions, tetanic
cramps of the cervical muscles, disturbances of motility and slight
miosis were observed in the highest dose group when the dietary
concentration was increased to 320 ppm. Toxic effects were not
observed in animals of the other groups. Slight anaemia was found at
the highest dose level after the dose of pyrazophos was raised to
320 ppm; other clinical chemistry tests did not show any adverse
effects. A dose-related inhibition of serum cholinesterase activity
was observed at 5 ppm and higher in both sexes. At 5 ppm
approximately 20% inhibition of erythrocyte cholinesterase activity
was measured (range of 6 determinations 15%-25%) in females only. At
the highest dose, erythrocyte cholinesterase activity was reduced in
both sexes. Increased mean weight of the pancreas was noted in all
treated groups which did not correspond to any microscopic
abnormalities. The NOAEL in this study was 5 ppm, equivalent to 0.4
mg/kg bw/day based on marginal inhibition of erythrocyte
cholinesterase activity measured at the next highest doses (Scholz &
Brunk, 1973).
Four groups of eight English beagle dogs/sex, 13 months old at
the beginning of the study, were fed pyrazophos (92.9% purity grade)
at dietary concentrations of 0, 1.2, 18 or 320 ppm for 6 months.
Diets were prepared daily, immediately before feeding. The following
parameters were investigated: clinical observations (general
conditions, body-weight, behaviour, reflex excitability,
ophthalmoscopic examinations, hearing test, visible mucosae,
dentition), laboratory examinations (haematology, clinical
chemistry, cholinesterase determinations, hepatic and renal function
tests, urinalyses) and pathology (organ weights, gross pathology,
histopathology). Clinical symptoms of the cholinergic type were
observed at 320 ppm as occasional diarrhoea and tetanic spasm. Some
dogs of this group developed atrophy of temporal muscles. Decline of
general health conditions, reduction of both food intake and body-
weight were also detected. One dog in poor nutritional state died on
day 107 of treatment, possibly due to respiratory muscle failure
resulting from extreme cholinesterase reduction. Animals showed
slight anaemia and increase of ASAT, ALAT and alkaline phosphatase
in serum. Apart from occasional diarrhoea, no other toxic symptoms
were observed at 18 ppm and dogs in the 1.2 ppm group were
comparable to controls. Serum and erythrocyte cholinesterase
activities were reduced in animals at 18 and 320 ppm throughout the
duration of the study. At the end of the study, dose-related
inhibition of brain cholinesterase was observed at 18 and 320 ppm
(20% and 76%, respectively). The relative weights of several organs
differed from control values at the 320 ppm dose level only.
Pathology did not reveal compound related organic lesions. The NOAEL
in this study was 1.2 ppm equivalent to 0.09 mg/kg bw/day, based on
occasional diarrhoea and marginal brain cholinesterase inhibition
observed at 18 ppm (Brunk et al., 1982).
Four groups of four pedigree English beagle dogs/sex (13 months
old at the beginning of the study) were fed pyrazophos (purity grade
92%) at dietary concentrations of 0, 2, 5 or 10/125/320 ppm for 2
years. The highest dose level of 10 ppm was increased to 125 ppm on
day 14 from the beginning of the study and further increased to 320
ppm on day 28. The test substance was mixed with food and given
daily during the mid-day meal.
The following parameters were investigated: clinical
observation (general conditions, body-weight, behaviour, reflex
excitability, eye examinations, hearing test, visible mucosae,
dentition), laboratory examinations (haematology, cholinesterase
activities, serum enzymes, urinalysis), pathology (organ weights,
gross pathology, histopathology).
One male dog in the 320 ppm group died of a chronic suppurating
pancreatitis and a chronic ascending pericholangitis after 462
administrations of pyrazophos. Several parameters were affected in
this animal but since the cause of death was not dependent on
pyrazophos administration they were not considered. All remaining
dogs survived till the end of the study. Food intake was not
affected by administration of the test compound. At the end of the
study, body-weight gain of the animals of the 320 ppm group was
approximately 50% less than that in the remaining groups. Behaviour,
reflex responses, ophthalmic findings, hearing test, dentition and
visible mucosae were not affected. Slight anaemia and increased
alkaline phosphatase were observed in animals at 320 ppm dose level.
Dose-dependent decreases of serum cholinesterase activity were
observed at 5 and 320 ppm. Erythrocyte cholinesterase activity was
reduced only at 320 ppm. Pathology and organ weights did not show
significant difference between groups. Histopathology was comparable
to controls in the 2 and 5 ppm groups. At 320 ppm, calcifications in
some regions of the basement membranes of the Bowman's capsules of
numerous renal glomeruli, in tubular basement membranes and in the
interstitium between some renal tubules were observed.
The NOAEL in this study was 5 ppm, equivalent to 0.4 mg/kg
bw/day based on erythrocyte cholinesterase inhibition, reduced body-
weight gain and histopathological abnormalities in kidneys observed
in dogs fed 320 ppm (Brunk et al., 1976)
Long-term toxicity/carcinogenicity studies
Mice
Four groups of 70 Charles River CD-1 mice/sex, 4 weeks of age
were treated with pyrazophos technical (92.8% purity grade) at
dietary concentrations of 0, 1, 5 or 25 ppm, equal to 0, 0.14, 0.7
or 3.5 mg/kg bw/day for males and 0, 0.16, 0.8 or 4 for females. Ten
animals/sex/dose were sacrificed after 12 months of treatment. The
remaining animals were sacrificed on week 92 (females) and on week
96 (males). Pyrazophos was stored under refrigeration until used;
test substance was offered to mice after being mixed with basal
laboratory diet. Due to difficulties in obtaining homogeneous
mixture, dietary preparation procedure was changed during the study.
Actual content of the test substance in diets was 65% (0-101), 100%
(55-241) and 85% (42-100) of nominal (mean of 12 determination,
range in brakets) at 1, 5 and 25 ppm, respectively. Daily
observations for overt toxicity signs, abnormalities and masses and
weekly observations for feed consumption and body-weights were
recorded. Haematology determinations and brain cholinesterase
activity on 10 mice/sex/ dose level were performed at 12 months and
at termination of the study. Serum and erythrocyte cholinesterase
activities were measured on 10 mice/sex/dose at 0, 3, 6, 12, 18
months and at termination of the study. Pathology was performed on
all animals found dead or sacrificed at the end of the study.
In both sexes, food intake and body-weight gain were unaffected
by pyrazophos administration. The incidence of clinical signs did
not differ between groups. The low survival rate noted at 1 ppm
(week 92) for the females and at week 96 for the males, prompted the
early termination of the study. The mortality rate was 67%, 75%,
65%, 70% (males) and 72%, 80%, 63% and 75% (females) at 0, 1, 5 and
25 ppm pyrazophos, respectively. Pyrazophos did not effects
haematological parameters tested. Dose-related inhibition of serum
and erythrocyte cholinesterase activities was observed in mice at 5
and 25 ppm (marginal inhibition of erythrocyte cholinesterase at 5
ppm in both sexes). There were no significant changes of brain
cholinesterase activity up to the highest dose level.
Trivial differences in organ weights were observed between
controls and dosed mice. Both neoplastic and non-neoplastic
histopathological observations were spontaneous or naturally
occurring lesions of aging mice. Renal amyloidosis was considered to
be the most frequent "cause of death" among control and treated
animals, the incidence being comparable in all groups. A higher
incidence of testicular atrophy and degeneration was observed in
treated mice but a dose-effect relationship was lacking. No
differences in the incidence of neoplastic lesions were found when
treated mice were compared to controls.
Pyrazophos did not cause adverse effects up to the highest
nominal dose of 25 ppm, equal to 3.5 and 4.1 mg/kg bw/day in males
and females, respectively. However, the poor correspondence of
actual and nominal concentrations of pyrazophos in the diets and the
lack of toxic effects up to the highest dose level hampered a
thorough evaluation of this study (Griggs et al., 1982).
Rats
Five groups of 30 Wistar albino rats/sex were treated with
technical grade pyrazophos (purity 90%) at dietary concentrations of
0, 5, 8, 10 and 50 ppm for 104 weeks (except the 8 ppm group which
was discarded at week 20). General condition, behaviour, body-
weight, food and water intake were recorded throughout the duration
of the study. Haematology, blood biochemistry, cholinesterase
activity in plasma, erythrocytes and brain and urinalysis were
measured. Pathology (organ weights) and histopathology (incomplete)
were performed at the end of the study.
Pyrazophos administration did not affect general condition,
behaviour, body-weight gain, food and water intake of animals at any
dose level. At the end of the study the mortality rate was 40%, 70%,
47% and 60% in males and 27%, 47%, 30% and 33% in females at 0, 5,
10 and 50 ppm, respectively. In the 50 ppm dose group, white blood
cell counts significantly decreased in males at week 102 only
(differential count normal). Glicemia was increased in the 10 and 50
ppm dose groups in males at week 52. Significant inhibition of
plasma and erythrocyte cholinesterase activity was measured in both
sexes at the 10 and 50 ppm dietary levels. Brain cholinesterase
activity was unaffected at the end of the study. A decrease in the
relative spleen weight was observed in females only at all dietary
levels. It was not dose-related and was not accompanied by
treatment-related histological changes. Both neoplastic and non-
neoplastic histopathological observations were those of spontaneous
or naturally occurring lesions of aging albino rats. No differences
in the incidence of neoplastic or non-neoplastic lesions were found
when treated rats were compared to controls. In conclusion scattered
differences in blood parameters were not suggestive of a clear toxic
effect of pyrazophos. The NOAEL in this study was 50 ppm, equivalent
to 2.5 mg/ kg bw/day. (Til et al., 1979a)
Four groups of fifty Wistar rats/sex, 4 weeks of age, were
treated via pelleted feed with technical pyrazophos (95.7% purity)
at dietary concentrations of 0, 2, 80 or 320 ppm equal to 0, 0.1,
4.0 or 15.9 mg/ kg bw/ day for males and 0, 0.1, 4.8 or 19.3 mg/kg
bw/ day for females for 117 weeks. Ten and 20 additional
rats/sex/dose were treated with pyrazophos for 52 and 105 weeks,
respectively. Pyrazophos was dissolved in acetone, mixed with
microgranulated feed and pelleted. This preparation was performed
every two weeks. Pyrazophos was stable at room temperature for at
least 21 days. Homogeneity (range of variation between -15% and 22%
of the mean concentration) and content of the test article in the
feed (mean concentrations 98.5%, 95.6% and 94.5% of the nominal
concentrations at 2, 80 and 320 ppm, respectively) were determined
before the beginning of the study and every two months throughout
the duration of the study. Parameters monitored were
viability/mortality, clinical signs, food and water consumption,
body-weight, ophthalmoscopic examination, hearing test, haematology,
clinical biochemistry including cholinesterase activity
determination, urinalysis and pathology.
A slight but statistically significant increase of body-weight
was detected in both sexes at 320 ppm during the second year of the
study which corresponded to a slight increase of food intake.
Absolute water intake was not affected in any group resulting in a
slight decrease of relative water intake at the highest dose level.
The incidence of clinical signs and palpable masses during life and
the results of ophthalmoscopic examinations and hearing tests were
comparable between groups. At the end of the 117 weeks treatment,
mortality rate was 60%, 40%, 54% and 52% (male) and 60%, 56%, 38%
and 48% (female) at 0, 2, 80 and 320 ppm, respectively.
Slight decrease of total lipid content in blood which
corresponded to lower cholesterol and triglyceride concentrations
was measured in both sexes at 80 and 320 ppm but was not seen
consistently throughout the duration of the study. Other haemato-
logical and biochemical parameters and urinalysis measured at 26,
52, 78 and 105 weeks were unaffected by pyrazophos administration.
Dose-related inhibition of plasma and erythrocyte cholinesterase was
detected at 80 and 320 ppm throughout the duration of the study.
Absent or marginal (between 13% and 23%) inhibition of brain
acetylcholinesterase was measured at 320 ppm in both sexes. No
biologically significant differences in organ weights were detected
at 52, 105 or 117 weeks. Histopathology showed a slight increase of
gastric lesions (ulcers and erosions) in males at 320 ppm; several
other non-neoplastic lesions were considered unrelated to test
substance administration. An increased incidence of hemangiomas in
mesenteric lymph nodes was observed at 105 and 117 week
necroscopies. Hemangiomas occurred at incidences of 12%, 13%, 30%
and 33% (males) and 6%, 4%, 26% and 16% (females) at 0, 2, 80 and
320 ppm, respectively. This type of neoplasm was pratically absent
at 52 weeks necroscopies. A statistically significant positive trend
was present for males only. Hemangiosarcomas were not reported in
mesenteric lymph nodes. The incidence of this type of neoplasm in
historical controls is 8% (0-24) and 2% (0-8) in males and females,
respectively (mean, range in brackets). Increased incidence of skin
fibroma (still within the range of historical controls) was detected
in males at the highest dose level (10%) in comparison with controls
(0%). The NOAEL was 2 ppm, equal to 0.1 mg/kg bw/day in both sexes
based on a higher incidence of hemangiomas in mesenteric lymph nodes
detected in males at the higher doses (Tennekes et al., 1991).
In a supplementary study, two groups of 20 Wistar rats/sex were
treated via pelleted feed with technical pyrazophos (95.7% purity)
at dietary concentrations of 0 or 1 ppm equal to 0 or 0.05 mg/kg
bw/day for males and 0 or 0.06 mg/kg bw/day for females for 104
weeks. Monitored parameters were observation for
viability/mortality, clinical signs, food and water consumptions,
body-weight, ophthalmoscopic examination, hearing test,
cholinesterase determination in plasma, erythrocytes and brain and
pathology (organ weights and macroscopic examinations). Mortality
rate at 104 weeks was 25% in both groups for males and 25% and 15%
at 0 and 1 ppm, respectively for females. All tested parameters were
unaffected by pyrazophos treatment. The NOAEL was 1 ppm of
pyrazophos, equal to 0.05 mg/kg bw/day and 0.06 mg/kg bw/day for
male and female rats, respectively (Tennekes et al., 1991)
Reproduction studies
Rats
In a 3-generation, 2-litter/generation study, four groups of 10
male and 20 female rats (CIVO-colony) approximately 3-4 weeks old at
the beginning of the study were treated with pyrazophos (90% purity
grade) at dietary concentrations of 0, 5, 10 or 50 ppm. The diets
were freshly prepared every two weeks and stored at room temperature
(homogeneity and stability of test substance in diets not reported).
Rats of the F0 generation were maintained on their respective
diets for 13 weeks prior to mating and then up to weaning of the
F1b generation. Litters were culled to 8 pups on day 1. Rats from
F1b litters (10 males and 20 females selected at weaning) were
mated at weeks 14 and 23 post-weaning. Ten male and 20 females of
F2b litters were selected at weaning and mated at weeks 13 and 22
after weaning. F1a, F2a and F3a litters were killed post-
weaning. Rats from the F3b generation (10 males and 10 females)
were selected and continued on the same diet for a 90-day feeding
study.
The following parameters were evaluated in the present study:
- Reproduction phase: litter size, pup survival and litter
weight at day 1, 10 and 20.
- 90-day study: signs of toxicity, food intake, body-weight,
haematology, blood chemistry (included cholinesterase
activity, brain cholinesterase activity, urinalysis and
pathology.
No relevant changes in reproductive parameters were observed.
An increased percent mortality of pups both at day 10 and 20 was
detected at the highest dose level after the second mating of the
F0 generation only. Signs of toxicity were not detected during the
90-day study. Dose-related inhibition of whole blood cholinesterase
activity was measured at 10 and 50 ppm but brain cholinesterase
activity was normal up to the highest dose level. Blood chemistry
and urinalysis did not show changes related to the administration of
the test substance. Total leucocyte counts were increased at 50 ppm
in both sexes (likely because of lymphocyte increase). The absolute
and relative weights of the thymus were increased at the two highest
dose groups in both sexes. This change did not correspond to any
histological abnormality. Pathology did not show changes
attributable to pyrazophos administration. The overall NOAEL in this
combined study (reproduction study and 90-day study) was 5 ppm equal
to 0.45 and 0.42 mg/kg bw/day for males and females, respectively,
based on increased weight of thymus observed at 10 and 50 ppm and
increased lymphocyte count observed at 50 ppm in both sexes (Til et
al., 1979b)
In a two-generation one-litter study/generation, groups of 25
male and 25 female rats (Wistar/HAN) approximately 6 weeks old at
the beginning of the study were treated with pyrazophos technical
(95.7% purity) at dietary concentrations of 0, 2, 20 or 200 ppm.
Homogeneity and stability of pyrazophos in the diet were checked and
found acceptable. Parental rats (F1-generation) were maintained on
their respective diets for 70 days prior to mating. The F0
generation received the test compound in the diet for 126 days prior
to mating. Both F0 and F1 generations received the test
substance during the pairing, gestation and lactation periods.
Trivial differences in food intake and body-weight gain were
observed throughout the duration of the study in F0 and F1
generations. Fertility index, gestation index and viability index
were unaffected by pyrazophos administration in both generations.
Lactation index was not affected in litters of the F0 generation
but it was significantly reduced in litters of the F1 generation
at 200 ppm. In both the F1 and F2 pups, no treatment-related
abnormal findings were observed at external examination and the sex
ratio of the pups was unaffected by treatment. At 200 ppm, reduced
body-weight gain of the pups during lactation was evident in both
the F1 and F2 pups; additionally, in the F2 pups, the initial
body-weight was significantly reduced. Trivial differences in body-
weight gain were also observed at 20 ppm. Dose-related inhibition of
both plasma and erythrocyte cholinesterase activity was measured in
both sexes at 20 and 200 ppm. The inhibition was evident in F0 and
F1 generations and also in pups (plasma cholinesterase only was
measured in pups). The extent of cholinesterase activity inhibition
was higher in erythrocyte than in plasma. Slight inhibition of brain
cholinesterase activity was measured only in females, at the highest
dose level in both generations. Statistically significant inhibition
of brain cholinesterase of no biological relevance was measured in
F2 pups of both sexes at 200 ppm. Several statistical differences
of organ weights between groups were judged of no biological
relevance. Pathology did not show treatment-related abnormal
findings either in parent animals or in pups. The NOAEL in this
study was 20 ppm, equivalent to 1 mg/kg bw/day, based on retardation
of body-weight gain of pups and brain cholinesterase inhibition
measured in F0 and F1 generations (females) and in pups of the
F2 generation. However, neither thymus weight nor lymphocyte
counts (affected parameters in the previously performed reproduction
study) were checked in this study (Suter et al., 1991).
Special studies on delayed neuropathy
Hens
Six white Leghorn hens were treated orally with 150 mg/kg of
pyrazophos diluted (10%) in sesame oil on two separate occasions 21
days apart. Another six hens received a single dose of
atropine/toxogonin i.v. before being similarly dosed with
pyrazophos. In preliminary studies of acute toxicity in hens, the
LD50 was set between 150 and 400 mg pyrazophos/kg bw . A third
group of 6 hens received a single dose of 500 mg tri-o-
cresylphosphate/kg bw (positive controls) and a fourth group of 6
animals received sesame oil (negative controls). All hens were
sacrificed on day 42 after first dosing and histological examination
of cerebrum, cerebellum, spinal cord and the nerves of the brachial
and lumbosacral plexus was performed.
Pyrazophos-treated hens (both groups) showed symptoms of the
type expected for cholinergic toxicity. Positive controls showed
symptoms suggestive of delayed neuropathy. Negative controls did not
develop toxic symptoms. Histopathology did not reveal significant
changes in pyrazophos treated hens and in negative controls. In
positive controls demyelinization and glial cell proliferation were
observed in the optic tract, in the medulla and in the cervical,
thoracic and lumbar segments of the spinal cord. Peripheral nerves
were unaffected in all animals. According to the results of this
study, pyrazophos did not cause delayed neuropathy (Hollander &
Weigand, 1979a)
Special studies on embryotoxicity and teratogenicity
Rats
Four groups of twenty mated female Wistar rats were dosed with
pyrazophos at concentrations of 0, 0.5, 1.6 or 5.0 mg/kg bw/day
orally by gavage (vehicle, starch mucilage) on days 7, 10 and 16 of
gestation. On day 21 of gestation, animals were killed and fetuses
were examined for developmental disorders. Neither toxic signs nor
mortality were observed in pyrazophos-treated or control pregnant
rats. There were no significant differences in body-weight or feed
consumption between the groups. Pathology did not reveal induced
changes. There were no significant differences in the number of
implantations per litter, incidence of live, dead or resorbed
fetuses per litter or in the average weight of live fetuses between
groups. The fetuses developed normally and showed no pyrazophos
related external abnormalities, anomalies of the internal organs, or
skeleton. The NOAEL in the present study was 5.0 mg pyrazophos/kg
bw/day for both maternal toxicity and teratogenicity. Cholinesterase
activity was not measured (Baeder et al., 1978).
Rabbits
Groups of fifteen pregnant Himalayan rabbits were treated by
gavage with pyrazophos (purity 95.6%) during gestation days 6-18 at
doses of 0, 10, 32 or 100 mg/kg bw/day (vehicle, starch mucilage).
On day 29 of gestation, the dams were killed. Internal, external and
skeletal examination were performed on the fetuses. A slight
reduction of food intake was observed in the pregnant rabbits at the
highest dose level which corresponded to some reduction of body-
weight gain (not statistically significant). Intra-uterine
developement of the embryos and the viability of fetuses were not
affected at any dose level of pyrazophos. The morphological
examination of the fetuses for development, external anomalies,
alterations of internal organs and skeleton yielded no evidence of a
teratogenic effect of pyrazophos. The NOAEL was 100 mg/kg bw/ day
for both maternal toxicity and terato-genicity. Cholinesterase
activity was not measured (Baeder & Kramer, 1985; Baeder et al.,
1986).
Special studies on genotoxicity
Table 2. Results of genotoxicity assays on pyrazophos
Test system Test object Concentration of pyrazophos Purity Results Reference
Ames test (1) S. typhimurium 0.2-5000 µg/plate dissolved in ? Negative (2) Gericke (1977)
TA98, TA100, TA1535, DMSO
TA1537
Reversion assay (1) E. coli WP2 hcr 10-5000 µg/plate dissolved in 92.8% Negative (2) Shirasu et al. (1979)
S. typhimurium DMSO
TA98, TA100, TA1535,
TA1537, TA1538
Rec-assay B. subtilis 20-2000 µg/disc dissolved in 92.8% Negative (3) Shirasu et al. (1979)
H17 Rec + M45 Rec- DMSO
Mitiotic gene- S. cerevisiae D4 (1) 800-1600 µg/ml Afugan Negative (4) Bertoldi et al. (1980)
conversion A. nidulans (5) 200-690 µg/ml Afugan Negative
CHO/HGPRT mutation Chinese hamster lung 6.25-75 µg/ml (-act.) 97.3% Negative (6) Muller (1988)
assay (1) fibroblasts V79 25-150 µg/ml (+ act.)
Chromosome Human lymphocytes 1-50 µg/ml x 47 h (- act.) 95.6% Negative (7) Taalman et al. (1985)
aberrations 10-175 µg/ml x 1 h (+ act.)
dissolved in DMSO
Micronucleus test Mouse (NMRI strain) 50, 75, 100 mg/kg p.o. treated 95.6% Negative (8) Mayer et al. (1985)
twice 24 h apart
Table 2 (continued)
(1) Both with and without metabolic activation.
(2) Positive controls yielded expected positive responses.
(3) Kanamycin (10 µg/disc) and Mitomycin C (0.1 µg/disc) gave expected negative and positive response, respectively.
(4) Positive control (methylmethanesulphonate) gave expected positive response at 500 µg/ml.
(5) Also using germinating conidia.
(6) Positive control (EMS 1 mg/ml (- act.); DMBA 7.7 µg/ml ( + act.) gave expected positive responses.
(7) Positive controls (EMS 0.2 µl/ml (- act.); cyclophosphamide 18.7 µg/ml (+ act.) gave expected positive responses.
(8) Positive control (cyclophosphamide) gave expected positive response at 50 mg/kg bw (single dose).
Special study on antidote effect
Rats
Female SPF-Wistar rats were dosed orally with a single
pyrazophos (94% pure) dose of 800 mg/kg bw (4 x LD50). A single
dose (10 minutes after pyrazophos) of either atropine methyl
nitrate, atropine sulphate or obidoxime or combinations of these
antidotes given i.p. delayed the time of death without modifying
survival rate. The best treatment was a combination of atropine
sulphate (3.75 mg/kg bw) and obidoxime (4 mg/kg bw) repeated several
times starting 10 min after intoxication up to 57 h later, which
increased survival rate from 0% to 90% (Sholz & Weigand, 1971).
Male and female Wistar rats were dosed orally with a single
pyrazophos dose (93.6% pure), approximately 2 x LD50. Where no
therapeutic measures were taken, all animals with the exception of
one female, died within three days. Prolonged treatment with
atropine sulphate only did not increase survival rate. The
combinations of both atropine sulphate + 2-PAM methiodide or
atropine sulphate plus obidoxime proved to be efficient antidotes by
reducing mortality to zero (Leist & Weigand, 1983).
Observations in humans
Five healthy humans received 0.15 mg/kg bw of pyrazophos daily
for 10 days. Other 6 subjects received 0.15 mg/kg bw of pyrazophos
for 3 days, followed by 7 daily doses of 0.07 mg/kg bw. Another 11
subjects received 0.07 mg/kg bw of pyrazophos for 10 days.
Pyrazophos was always administered orally, in orange juice, at
breakfeast. Males and females were similarly represented in the
groups. Controls received orange juice only. The great majority of
controls were the same subjects who had previously taken pyrazophos.
All subjects were checked for appearance of clinical symptoms,
alterations of cardiovascular parameters (blood pressure, pulse
rate, ECG). Moreover, haematology, blood biochemistry, urinalysis
and quantitative assay of cholinesterase in plasma, whole blood and
erythrocytes were performed on several occasions before, during and
up to 7 days after the last administration of pyrazophos.
At the highest dose, only two subjects completed the study as two
withdrew because of upper respiratory infection and dosing was
discontinued in one subject because of low baseline cholinesterase
level after 3 doses (sic). Three out of six subjects in the
0.15/0.07 mg/kg bw group reported adverse reactions of the type:
mild headache, "feeling faint" and slight abdominal disconfort. Two
subjects in the lowest dose group referred: dizziness and
lightheadedness. The other two in the same group referred symptoms
which could be related to upper tract respiratory infection. No
alterations of cardiovascular parameters were found. Blood and urine
analysis were within the normal range except for a sharp increase of
serum CPK measured in one subject of the lowest-dose level. CPK was
increased from the first up to the last day of pyrazophos
administration (a pre-dosing normal level was reported) showing a
maximum on day 4 (CPK was not measured during the 7 day recovery
period). This subject showed similar inhibitions of plasma and whole
blood cholinesterase activities of about 25% during the last 3 days
of dosing (average inhibition calculated by comparison of the mean
of cholinesterase activity on days 9-10-11 of the study with the
mean of the three pre-dosing values). Cholinesterase activities in
plasma and erythrocytes were normal on the day when CPK was at its
maximum. Other biochemical parameters were all in the normal range.
The possible explanation for increased CPK offered by authors was
running activity performed by the subject during the study.
Cholinesterase values within each group showed that, at 0.15
mg/kg bw inhibition of plasma cholinesterase was observed starting
on day 6 (approximately 20-50%) and persisting up to 4 days after
the last dose of pyrazophos. In this group marginal inhibition of
whole blood cholinesterase was noted but cholinesterase activity was
not affected in erythrocytes. Cholinesterase activity was not
affected in the other groups.
Evaluation of this study is hampered by the presence of several
methodological problems and lack of details. Controls were mainly
the same subjects who had previously taken pyrazophos. Upper
respiratory disorders are not substantiated by alterations of
haematological parameters like ESR or increased WBC and cholinergic
toxicity was not ruled out. Cholinesterase activities show a great
intra-individual variability (greater than 10-20% which is the
normal intra-individual variation in day-by-day analysis). The NOAEL
for erythrocyte cholinesterase inhibition in this study is 0.15 mg
of pyrazophos/kg bw/day for 10 days, however 3/5, 3/6 and 5/11
subjects in the three groups reported symptoms of the type expected
for cholinergic toxicity (Dinsdale & Protheroe, 1983).
An unknown quantity of Afugan 30 EC squirted out of a damaged
metal container into the face of a 36-year-old male worker, mainly
in the orbital region. The worker was soon hospitalized. Both eyes
developed conjunctivitis. Mild systemic clinical effects of the
cholinergic type developed within 2 hours, namely miosis, tremors,
muscle twitchings, isolated spasms, psychic agitation, bronchial
spasticity and bradycardia. The patient received a total of 4 mg
atropinum sulfuricum i.v. and as soon as systemic symptoms
disappeared on the same day of poisoning, he was transferred to the
out-patients department. Corrosion of the conjunctivae of both eyes
and corrosion of the cornea of the right eye were diagnosed.
Cholinesterase activity measured both in the serum and in
erythrocytes at approximately 3 and 7 h after intoxication, in the
presence of clinical symptoms, resulted in the normal range (Stasik,
1987).
COMMENTS
In rats after a single or 14 daily (only the last dose
radiolabelled) oral administrations of 14C-pyrazophos, blood
radioactivity peaked within six hours after administration. The
half-life was approximately five hours. Radioactivity was eliminated
mainly via urine (71-78%) and faeces (16-24%). The parent compound
accounted for most of the radioactivity detected in faeces,
indicating incomplete absorption from the gastrointestinal tract.
Intact pyrazophos was not found in urine, where the radioactivity
mostly corresponded to its hydrolysis products.
Pyrazophos was moderately toxic after single oral doses to
mice, rats and dogs. No significant differences between sexes or
routes of administration were detected. WHO has classified
pyrazophos as moderately hazardous (WHO, 1992).
In two 28-day studies in mice at dietary concentrations of 0,
1, 5, 25 or 125 ppm (the highest dose in only one study), the NOAEL
was 25 ppm equal to 4.7 and 5.0 mg/kg bw/day for males and females,
respectively. The NOAEL was based on 20% inhibition of brain
cholinesterase observed at 125 ppm.
In a 13-week study in rats at dietary concentrations of 0, 2.5,
50 or 1000 ppm the NOAEL was 2.5 ppm, equal to 0.21 mg/kg bw/day, in
both sexes, based on brain cholinesterase inhibition at the end of
the study at 50 ppm.
In a 52-week study in rats at dietary concentrations of 0, 2,
20 or 200 ppm the NOAEL was 20 ppm, equal to 1.0 and 1.4 mg/kg
bw/day in males and females, respectively. The NOAEL was based on
30% inhibition of brain cholinesterase activity in females at 200
ppm.
In a 92-day (moist semi-solid diet) study in dogs at dietary
concentrations of 0, 0.5, 2.0, 5.0 or 10/125/320 ppm the NOAEL was 5
ppm, equivalent to 0.4 mg/kg bw/day, based on inhibition of
erythrocyte cholinesterase activity at the next highest dose (brain
acetylcholinesterase activity was not determined). Clinical signs of
the cholinergic type were observed when the dietary concentration
was raised to 320 ppm.
In a 6-month study in dogs at dietary levels of 0, 1.2, 18 or
320 ppm pyrazophos (moist semi-solid diet), the NOAEL was 1.2 ppm,
equivalent to 0.09 mg/kg bw/day, based on marginal brain
acetylcholinesterase inhibition observed at 18 ppm.
In a two-year study in dogs at dietary concentrations of 0, 2,
5 or 320 pm (moist semi-solid diet), the NOAEL was 5 ppm, equivalent
to 0.4 mg/kg bw/day based on erythrocyte cholinesterase inhibition,
reduced body-weight gain and histopathological abnormalities
observed in the kidneys of dogs fed 320 ppm.
In a 92/96 (female/male)-week study in mice at dietary
concentrations of 0, 1, 5 or 25 ppm, pyrazophos did not cause
adverse effects up to the highest nominal concentration of 25 ppm,
equal to 3.5 and 4.1 mg/kg bw/day in males and females,
respectively. Inhibition of serum and erythrocyte cholinesterase
activities, but not of brain acetylcholinesterase activity, was
observed at 5 ppm and above. The poor correspondence between actual
and nominal concentrations of pyrazophos in diets hampered
definitive evaluation of this study.
In a two-year study in rats at dietary levels of 0, 2, 80 or
320 ppm, the NOAEL was 2 ppm equal to 0.1 mg/kg bw/day, based on a
higher incidence of hemangiomas in mesenteric lymph nodes detected
in males at the higher doses. Marginal brain acetyl cholinesterase
inhibition was noted at 320 ppm only.
In a two-year study in rats at dietary concentrations of 0, 5,
8, 10 or 50 ppm, the NOAEL was 50 ppm, equivalent to 2.5 mg/kg
bw/day, based on the absence of adverse effects including brain
acetyl cholinesterase inhibition at this dose level. No compound-
related abnormalities were detected in mesenteric lymph nodes.
In a two-litter, three-generation study in rats (a 90-day
toxicity study was also conducted on the F3b generation) at
dietary levels of 0, 5, 10 or 50 ppm, the NOAEL was 5 ppm, equal to
0.45 and 0.42 mg/kg bw/day for males and females, respectively,
based on increased thymus weight observed at 10 and 50 ppm and
increased lymphocyte counts observed at 50 ppm in both sexes in the
90-day toxicity study.
In a two-generation reproduction study in rats at dietary
concentrations of 0, 2, 20 or 200 ppm of pyrazophos, the NOAEL was
20 ppm, equivalent to 1 mg/kg bw/day, based on retardation of body-
weight gain of pups of both generations, reduced lactation index of
the F1 generation and slight inhibition of brain acetyl
cholinesterase activity in parental females and in pups of the F2
generation.
Pyrazophos did not cause delayed neuropathy in hens.
Pyrazophos was not teratogenic in rats or rabbits. The NOAELs
for both maternal and embryofetal toxicity in rats was 5 mg/kg
bw/day and in rabbits 100 mg/kg bw/day, the highest doses tested.
Maternotoxicity was not observed. However, cholinesterase activity
was not measured.
Male and female human volunteers received pyrazophos orally at
0.07, 0.07/0.15 or 0.15 mg/kg bw/day for 10 days. At the highest
dose level only plasma cholinesterase activity was inhibited (20-
40%), with marginal inhibition of erythrocyte cholinesterase
activity. Symptoms which could be attributed to cholinergic toxicity
were observed in all groups. The study was considered inadequate
because of deficiencies in its design and conduct.
After reviewing the available genotoxicity data, it was
concluded that pyrazophos was not genotoxic.
The Meeting concluded, after consideration of the long-term
studies and the genotoxicity data that pyrazophos was unlikely to
pose a carcinogenic hazard for humans.
An ADI was allocated on the basis of NOAELs in the two-year
study in dogs and the three-generation study in rats, using a 100-
fold safety factor.
TOXICOLOGY EVALUATION
Level causing no toxicological effect
Mouse: 25 ppm, equal to 4.7 mg/kg bw/day (28-day study)
Rat: 5 ppm, equal to 0.4 mg/kg bw/day (three-generation
reproduction study)
Dog: 5 ppm, equivalent to 0.4 mg/kg bw/day (two-year
study)
Estimate of acceptable daily intake for humans
0-0.004 mg/kg bw
Studies which will provide information valuable in the continued
evaluation of the compound
Further observations in humans.
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Leist, K. H. & Weigand, W. (1982) Test for sensitizing properties of
Hoe 02873- Active ingredient (Code: Hoe 02873 OF AS207) in the
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Otaka, T., Nakayoshi, H., & Abe, M. (1981d) Acute toxicity study on
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