PHOSPHAMIDON
EXPLANATION
Phosphamidon was evaluated for acceptable daily intake by the
Joint Meetings in 1963, 1965, 1966, 1968, 1982, and 1985 (Annex 1,
FAO/WHO, 1964, 1965a, 1967a, 1969a, 1983a, and 1986a). Toxicological
monographs were published after the 1963 and 1965 Meetings (Annex 1,
FAO/WHO, 1964, and 1965b), and monograph addenda were published after
the 1966, 1968, and 1982 Joint Meetings (Annex 1, FAO/WHO, 1967b,
1969b, and 1983b). An ADI was established at the 1966 Meeting on the
basis of no-effect levels taken exclusively from data reported by
Industrial Bio-Test Laboratories (IBT), which could not be validated.
For this reason the 1982 Meeting made the ADI temporary and requested
replacement studies. The required data were not available to the
Meeting in 1985 and, because of the lack of support for maintaining
the temporary ADI, the safety factor was doubled. The data have now
been provided and are summarized and discussed in this monograph
addendum.
EVALUATION FOR ACCEPTABLE INTAKE
BIOLOGICAL DATA
Toxicological studies
Special study on reproduction
Rats
Groups of 15 male and 30 female albino rats (CD-CRL, 7 weeks old)
were fed diets containing 0, 5, 30, or 50 (80 for the first 2 weeks)
ppm phosphamidon (technical, 92.1% pure) in a 2-generation (2
litters/generation) study. Weanlings from the second litter were
selected to become parents of the next generation.
In the parental generations unthriftiness, hyperactivity, ocular
and/or nasal discharge, and tremours were observed in animals of the
50 and/or 30 ppm dose groups. There were no compound-related effects
on mortality or food intake. Body weights were significantly reduced
in F0 parental males at 50 ppm and in F1 parental males at 50 and
30 ppm. F1 maternal body weights were slightly reduced at 50 ppm. In
some cases F1 parental body weights were also decreased at 5 ppm,
but this occurred in a non-dose-related manner. F0 and F1 parental
absolute organ weights were significantly affected at 50 ppm. Relative
brain and heart weights were significantly increased in F1 males at
50 and 30 ppm. No treatment-related histopathological findings were
observed in F0 or F1 parental animals. Reproductive performance,
as expressed in indices for mating, fecundity, and male and female
fertility, was decreased at 50 ppm.
Mean litter size and pup survival were decreased in all
generations (significantly for F2b litters and pups) at 50 ppm. Pup
weights (at day 21) were decreased in F1a, F1b, F2a, and
F2b pups at 50 ppm, and a tendency to lower pup weights was observed
in F1a, F1b, and F2b pups at 30 ppm. Significantly-reduced
absolute organ weights were observed in F1b and F2b offspring (in
females only liver and kidney weights were reduced) at 50 ppm; F1b
males receiving 30 ppm phosphamidon also had decreased liver, kidney,
and testes weights. Relative brain weights of F1b males and females
were increased in the 30 and 50 ppm dose groups and of F2b offspring
at 50 ppm. The increase in relative brain weight reflects the
decreased body weights of these animals. This was also true for heart
and kidney weights in F2b weanlings. After macroscopic and
microscopic examinations of F1b and F2b pups, no treatment-related
findings were observed. Examination of craniofacial sections from
F2a pups was performed because of the finding of hydrocephalus in
the first teratogenicity experiment. On day 4 or day 21 postpartum no
malformations or variations were noted.
The NOEL in this study was 5 ppm (Holson, 1985a).
Special studies on teratogenicity
Rats
Groups of 30 pregnant albino Sprague-Dawley rats were dosed by
oral gavage with 0, 1, 2, or 4 mg phosphamidon (92.1% pure) in CMC/kg
b.w./day from days 6 through 15 of gestation. All dams were sacrificed
on day 20 of gestation and the fetuses were removed, weighed, and
examined for sex and for external, visceral, skeletal, and head
abnormalities.
In the 2 and 4 mg/kg b.w./day groups increased incidences of oral
and ocular discharge, hypoactivity, and tremours were observed. There
was no mortality and none of the test animals delivered prematurely or
aborted during the study. At 2 and 4 mg/kg b.w./day, mean body weight
(actual and corrected for uterus weight) was decreased (significantly
at 4 mg/kg b.w./day). The percentages of pregnant dams, number of
corpora lutea, number of implantations, and number of pre-implantation
losses were comparable among control and treated groups.
Prenatal viability and death, mean fetal body weight, and sex
ratios were not affected at any dose level. At 4 mg/kg b.w./day an
increased incidence of fetuses with a body weight 30% less than the
controls was noted, which was probably due to maternal toxicity. The
incidence of external malformations was increased at 4 mg/kg b.w./day.
Visceral and skeletal malformations were not observed. Hydrocephalus
was observed in 2, 2, and 3 fetuses of the 1, 2, and 4 mg/kg b.w./day
groups, respectively. Although the incidences were not statistically-
significantly increased and no external observations of dome-shaped
head were noted in any of the fetuses with suspected hydrocephalus,
the finding of hydrocephalus was considered to be equivocal (Holson,
1985b).
In order to clarify the equivocal findings of hydrocephalus, a
repeat study was initiated in which groups of 30 pregnant female
albino Sprague-Dawley rats were dosed by oral gavage with 0, 0.5, 2,
4, or 6 mg phosphamidon (92.1% pure) in CMC/kg b.w./day from days 6
through 15 of gestation. All animals were killed on gestation day 20.
Maternal gross lesions were observed and recorded as well as external,
visceral, skeletal, and Bouin's head examinations on the removed
fetuses.
In the 2, 4, and 6 mg/kg b.w./day groups, increased incidences of
salivation, ocular discharge (4 and 6 mg/kg b.w./day only), and
tremours were observed. Twenty-four animals in the 6 mg/kg b.w./day
group and 5 animals in the 4 mg/kg b.w./day group died during the
dosing period. Pink pancreas and haemorrhages of the stomach were
observed after necropsy and laparohysterectomy of animals in the 2
highest dose groups.
Statistical analysis was not carried out on maternal or prenatal
data generated from the 6 mg/kg b.w./day group, due to the limited
number of dams (less than 50%) surviving until scheduled death.
The percentages of pregnant dams, number of corpora lutea, number
of implantation sites, and number of pre-implantation losses were
comparable among controls and all teated groups. Mean body weights
(actual and corrected for uterine weight) of the dams were
significantly reduced in the 2 and 4 mg/kg b.w./day dose groups. Mean
fetal weight was significantly reduced at 4 mg phosphamidon/kg
b.w./day. Significant differences were not observed between control
and treated groups in prenatal viability, prenatal death, mean litter
size of viable fetuses, or sex ratios. A range of variations was
observed during skeletal, external, and Bouin's head examinations. In
the 4 mg/kg b.w./day group, a significantly-increased combined
incidence of either incompletely ossified sternebra 2, 5, and 6 or
absent sternebra 2, 5, and 6 were noted. At the same dose level there
was a significant increase in the incidence of fetuses having a body
weight 30% less than the mean control. Both effects were probably
secondary to the maternal toxicity observed in this dose group. No
visceral or skeletal malformations were found, and there was no
finding of hydrocephaly after evaluation of the head specimens of all
fetuses. Fetotoxicity was not observed at 2 mg/kg b.w./day. Based on
these data, the NOEL in this study was 0.5 mg phosphamidon/kg b.w./day
for maternal toxicity. There was no evidence of any teratogenic effect
in this study (Holson, 1985c).
Rabbits
Groups of 18 pregnant New Zealand white rabbits were dosed by
oral gavage with 0, 1, 3, or 10 mg phosphamidon (91.8% pure) in CMC/kg
b.w./day from day 6 to 18 of gestation. The dams were sacrificed on
day 30 of gestation and removed fetuses were examined for external,
skeletal, and visceral malformations. One dam in each dose group died,
1 animal in each of the mid- and high-dose groups gave premature
birth, and 1 animal in the 10 mg/kg b.w./day group aborted. At
10 mg/kg b.w./day, mean body weights of the dams were significantly
decreased. The number of pregnant dams, corpora lutea, implantations,
and pre-implantation losses, as well as litter sizes, number of
resorptions, prenatal viability, and fetal body weights were not
affected by phosphamidon treatment. No treatment-related malformations
were observed during external, visceral, or skeletal examinations. The
NOEL in this study was 3 mg phosphamidon/kg b.w./day, based on
maternal toxicity. Phosphamidon did not show teratogenic activity at
the doses tested (Holson, 1985d).
Special studies on mutagenicity
For the results of mutagenicity studies with phosphamidon, see
Table 1. Additionally, it was found that phosphamidon was mutagenic in
several plant systems (Wuu & Grant, 1966; Reddy & Rao, 1969; Panda &
Sharma, 1979; Srivastava & Sarma, 1979; Singh et al., 1979;
Sharma et al., 1983).
Special study on delayed neurotoxicity
Twelve domestic hens, 12 months old and protected by atropine
sulphate (10 mg/kg), received orally 30 mg phosphamidon (92.1%
pure)/kg b.w. After an interval of 21 days, the same procedure was
repeated. The dose of 30 mg/kg b.w. was chosen based upon the LD50
of phosphamidon in laying hens ranging from 25 to 36 mg/kg b.w. A
negative control group of 10 hens was treated with distilled water
alone. A positive control group of 4 hens received a single oral dose
of 1000 mg TOCP/kg b.w.
No spontaneous mortality occurred, but 2, 2, and all 4 hens of
the vehicle, dose, and positive control groups, respectively, were
sacrificed before the end of the study, due to poor general condition.
The animals of the TOCP group showed signs of severe ataxia. In the
phosphamidon-treated animals, ataxia, hyper-irritability, trismus,
ventral body position, and diarrhoea were observed, but after both
applications (days 1 and 22), they recovered within 6 days. There were
no indications of delayed neurotoxicity. In all hens of the positive
control group, weight loss was observed, as well as progressive ataxia
in the second week after administration. After neuro-pathological
examination, no treatment-related changes were observed in the test
group, whereas the positive control group developed neuropathy (Schoch
& Krinke, 1985).
Short-term study
Dogs
Groups of beagle dogs (8/sex/group) were orally administered in
gelatine capsules 0, 0.05, 0.1, or 2.5 mg phosphamidon (91.8% pure)/kg
b.w./day for 1 year. Interim sacrifice of 2 dogs/sex/group was carried
out after 3 months of treatment. Two male and 2 female control dogs,
and 1 male and 2 female dogs of the high-dose group, were sacrificed
after a 4 week recovery period.
Table 1. Special studies on the mutagenicity of phosphamidon
Test system Test object Concentration Purity Results Reference
used non-activated activated
In vitro
Ames test S. typhimurium 25 - 2025 µg/plate 91.8% negative negative Arni & Müller,
strain: TA98 1982
TA100
TA1535
TA1537
Mitotic gene conversion S. cerevisiae 80 - 10,000 91.8% negative questionable Arni & Müller,
test strain D7 g/plate 1983
Back mutation test S. cerevisiae 80 - 10,000 91.8% negative questionable Arni & Müller,
strain D7 g/plate 1983
Spot test reverse E. coli not given not negative negative Nagy et al., 1975
mutation WP2 given
Mouse lymphoma forward mouse L5178Y 20.6 -33.0 nl/ml 91.8% negative Beilstein &
mutation assay Tk+/- cells 65.0 - 1040 nl/ml 91.8% negative Müller, 1983
DNA repair test human fibroblasts 2, 10, 50, & 250 91.8% negative Puri & Müller,
nl/ml 1983a
rat hepatocytes 1, 5, 25 & 125 nl/ml 91.8% negative Puri & Müller,
1983b
Table 1. (cont'd).
Test system Test object Concentration Purity Results Reference
used
Chromosome aberration human lymphocytes 1.9 - 61 µg/ml not positive Georgian, 1975
test given
In vivo
Sister chromatid Chinese hamster oral, 1x 2.5, 5, & 91.8% negative Hool & Arni, 1983
exchange test bone marrow cells 10 mg/kg b.w.
Nucleus anomaly test Chinese hamster oral, 2x (days 0) 2.5, 91.8% questionable Strasser et al.,
bone marrow cells 5, & 10 mg/kg b.w. 1983
oral, 2x (days 0, 1) 91.8% questionable Strasser et al.,
3.5, 5.0, 7.1, & 1983
10 mg/kg b.w.
Chinese hamster bone oral, 2x (days 0, 1) 92.1% negative Strasser et al.,
marrow cells 1.67, 2.35, & 1985
6.7 mg/kg
Chromosome aberration mouse spermatogonia oral, 5x (days 0 - 4) 92.1% negative Strasser & Arni,
test 1.12 & 3.35 mg/kg 1985
mouse spermatocytes oral, 5x (days 0, 2, 92.1% negative Strasser et al.,
3, 5, & 9) 2.2 & 1986
6.7 mg/kg
Table 1. (cont'd).
Test system Test object Concentration Purity Results Reference
used
Micronucleus test mouse bone marrow oral, 2x (days 0, 1) not positive Rani, 1980
5 mg/kg b.w. given
Host-mediated assay S. typhimurium i.p. 3x (days 0 - 2) not positive Rani, 1980
G-46, mouse 7.5 mg/kg b.w. given
Chromosome aberration rat bone marrow i.p. 0.07 - 9.56 not positive Georgian, 1975
test mg/kg b.w. given
mouse bone marrow i.p. 0.07 - 9.56 not positive Georgian, 1975
mg/kg b.w. given
One male dog in the 2.5 mg/kg b.w./day group had to be killed
during week 24 due to poor general condition. In high-dosed dogs,
vomiting was slightly increased. Mean body weight, food consumption,
and food conversion were not adversely affected. Ophthalmoscopy and
hearing tests showed no abnormal findings. Clinical analysis showed
treatment-related effects on haematology and biochemical parameters.
In high-dose males erythrocyte counts, haemoglobin concentrations, and
haematocrits decreased significantly; alanine aminotransferase
activity was increased, but at week 13 only. In males and females of
the high-dose group, cholinesterase activity (measured by the Ellman
method) was inhibited in plasma (20 - 30%), and erythrocytes
(70 - 81%) at weeks 13 and 52; in the same group, brain cholinesterase
was depressed (50%) at interim sacrifice and at the end of the
administration period. During the 4 week recovery period, plasma and
brain cholinesterase activities recovered completely, but inhibition
in erythrocytes was still found (50%). Organ-weight ratios were not
affected by phosphamidon treatment and no treatment-related effects
were observed at macroscopic or microscopic examination. The NOEL was
0.1 mg/kg b.w./day (Kobel et al., 1985).
Long-term study
Rats
Groups of CD Sprague-Dawley (60 - 70/sex/group) rats were fed
diets containing 0, 0.2, 0.8, 1, 30, or 80 ppm phosphamidon for 24
months. The groups receiving 0.2 and 0.8 ppm phosphamidon were
discontinued after 15 weeks.
In the high-dose group, and to a lesser extent in the 30 ppm
group, the incidences of crusty muzzle, irritability, foot-pad
abnormalities, muscle tremours, yellow/brown-stained fur, and poor
coat quality were significantly increased. Survival rates of rats in
the treated groups were not significantly different from those of
controls. Food consumption at 80 ppm was decreased initially, and
increased in the majority of subsequent intervals, up to 1 year for
males and up to 2 years for females. In males, growth was decreased at
80 ppm throughout the study, and at 30 ppm during the first 3 months.
Only a slight decrease in growth was observed in females at 80 ppm.
Haemoglobin, haematocrit, and erythrocyte counts were decreased in
males at 80 ppm and in females at 80 ppm after 18 and 24 months. A
significant increase in Howell-Jolly bodies in the red blood cells was
observed at final sacrifice in the 30 and 80 ppm male groups, and in
the 80 ppm female group. Serum cholinesterase and brain cholinesterase
activities were markedly inhibited in rats in the 30 and 80 ppm male
and female groups. Erythrocyte cholinesterase activity was inhibited
to a lesser extent at 80 ppm. Organ weights were not affected in a
dose-related manner. In the 30 and 80 ppm dose groups, a variety of
non-neoplastic alterations was observed. These alterations included
ulcerative pododermatitis of the limbs, hyperkeratotic dermatitis of
the tail, and inflammatory changes in the lung and liver. There were
no differences in the incidence of neoplastic lesions between treated
and control groups. The NOEL was 1 ppm phosphamidon in the diet
(Mayhew & Wingard, 1986).
COMMENTS
Phosphamidon induced effects on litter size and pup viability at
maternally-toxic dose levels in a rat-multigeneration study. In
teratogenicity studies, fetotoxicity and maternal toxicity, but no
teratogenic effects, were observed.
A study with hens showed that phosphamidon did not induce delayed
neurotoxicity.
In a 1-year study in dogs, the main effect was inhibition of
serum, erythrocyte, and brain cholinesterase activities at 2.5 mg/kg
b.w./day. At 0.1 mg/kg b.w./day no effects were found. A long-term
toxicity/carcinogenicity study in rats also revealed cholinesterase
inhibition as the predominant effect. There was no increase in tumour
incidence. In this study no effects were observed at 1 ppm.
In mutagenicity studies, phosphamidon was negative in a number of
in vitro test systems, except for 1 test on chromosome aberrations.
Several in vivo tests on chromosome anomalies have been carried out
in rodents. Reports submitted to WHO showed negative or questionable
results, whereas literature data showed a positive effect.
On the basis of the toxicological data and in view of the
negative carcinogenicity study in rats, a full ADI was allocated.
TOXICOLOGICAL EVALUATION
LEVEL CAUSING NO TOXICOLOGICAL EFFECT
Rat: 1 ppm, equivalent to 0.05 mg/kg b.w./day.
Dog: 0.1 mg/kg b.w./day.
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN
0 - 0.0005 mg/kg b.w.
STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE FOR THE CONTINUED
EVALUATION OF THE COMPOUND
Observations in man.
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