The toxicology of phorate was considered by Joint Meetings in
1977, 1982 and 1983 (Annex 1, FAO/WHO, 1978a, 1983a, 1984). A
toxicological monograph was prepared by the Joint Meeting in 1977
(Annex 1, FAO/WHO, 1978b) and a monograph addendum was prepared in
1982 (Annex 1, FAO/WHO, 1983b). Due to the lack of an appropriate
delayed-neurotoxicity study, only a temporary acceptable daily intake
of 0-0.002 mg/kg b.w. was allocated in 1982. In 1983, the required
study was not available. The meeting extended the temporary ADI,
however, to 1985. Since then, the required neurotoxicity study and
additional mutagenicity studies have become available. These studies
are reviewed in this monograph addendum.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
Special study on Neurotoxicity
Phorate of 89.5% purity was dissolved in corn oil and orally
administered by gavage to fasted white leghorn hens (22 to 23 months
old). The single dose oral LD50 was determined to be 14.2 mg/kg b.w.
Fifty fasted hens were then atropinized by intramuscular injection of
10 mg/kg b.w. of atropine sulfate and 1 hour later given single oral
doses of phorate at a dosage level of 14.2 mg/kg b.w. (test group). An
additional 15 fasted and atropinized hens were given corn oil only
(vehicle control group) and 15 more non-atropinized hens were given
tri-o-tolyl phosphate (TOTP) at a dosage level of 500 mg/kg b.w.
(positive control group). Twenty-one days later, the dosing was
repeated for all suviving hens in the test and vehicle control groups
except that atropine sulfate was administered at a dosage level of
30 mg/kg b.w. All hens were observed daily for mortality, clinical
signs and evidence of neurotoxic reactions. Body weights and food
consumption were recorded every 3 days. Gross necropsies were
performed on all hens that died during the study and on all hens that
were sacrificed at termination of the study at 42 days. Hens
sacrificed at termination of the study were perfused with 10%
neutralized formalin. Brains, vertebral columns (with spinal cord
in situ) and the entire lengths of right and left sciatic nerves
were excised and fixed. Microscopic slides of neural tissue were
prepared as follows: a sagittal section of the entire brain (corpus
striatum, cerebellum, pons), a longitudinal and cross section of three
levels of spinal cord (cervical, thoracic, lumbrosacral) and both
sagittal and longitudinal sections of the right and left sciatic
nerves. Sections were stained with hematoxylin and eosin and replicate
sections with luxol fast blue. The tissues from 10 hens in each of the
test, vehicle-control and positive-control groups were
None of the 15 vehicle-control hens died during the 42-day study.
All 15 positive-control hens were sacrificed in extremis on day 16
of the study after clinical signs of neurotoxicity, first observed on
day 11, progressively became more severe. These signs included
generalized weakness, ataxia and paralysis of the legs and wings. Of
the 50 test hens, 27 died within 24 hours of the first dose of phorate
and 13 more within 24 hours after the second dose. Ten test hens
survived to termination of the study at 42 days. Vehicle control and
test hens displayed slight generalized weakness of the limbs, lasting
about 2 hours, shortly after each treatment with atropine sulfate.
Test hens had slightly more severe reactions and, in addition, slight
to moderate ataxia for up to 2 hours after dosing with phorate. No
clinical signs of delayed neurotoxicity, however, were observed in any
vehicle-control or test hens. Mean body-weight gains of test hens were
greater at 0 to 21 days and less at 21 to 42 days than those of
vehicle-control hens. Mean feed consumption of test hens was less at 0
to 21 days and greater at 21 to 42 days than that of vehicle-control
hens. Gross necropsies were negative for any effects attributable to
the test material.
Histopathological examination of neural tissues from positive-
control hens revealed treatment-related lesions involving the brain,
spinal cord, and/or sciatic nerves in all 10 hens. Generally mild to
moderate axonal degeneration was observed in the brains of 4/10 hens,
in the spinal cords of 10/10 hens, and in the sciatic nerves of 10/10
hens. In addition, demyelination was observed in the spinal cords of
7/10 hens and in the sciatic nerves of 7/10 hens. Schwann cell
hyperplasia was also observed in the sciatic nerves of 3/10 hens.
These lesions were compatible with a TOTP-induced delayed neuro-toxic
response. Minimal to mild focal lesions of axonal degeneration of the
sciatic nerves were noted in 3/10 test hens. No axonal degeneration
was noted in any vehicle-control hens. The axonal degeneration
observed in 3/10 test hens was associated with interstitial
infiltrations of lymphoid cells, which was also observed in additional
test and vehicle-control hens. This syndrome, which was distinctly
different than that observed in the positive-control hens, was
ascribed to lesions of naturally occurring disease (i.e. Marek's
disease) and was not considered to be related to the test material.
Treatment of hens with phorate did not induce clinical or
histopathological signs indicative of acute delayed neurotoxicity
Special studies on mutagenicity
Technical grade phorate of unspecified purity was tested in the
mutagenicity studies reported in Table 1.
Table 1. Results of mutagenicity assays with phorate
Study type Dosage level and/or Results Reference
Reverse mutation, dosage levels up to 1000 negative Simmon et al.,
S. typhimurium, mg/plate* 1977
Preferential 1 mg (on filter disc)/ negative Simmon
toxicity, E. coli, plate, w/o metabolic et al., 1977
strains p3478, activation
strains M45, H17
Mitotic recombination, 5% w/v for 4 hours negative Simmon
S. cerevisiae D3 incubation followed by et al., 1977
Cultured mammalian cells
Unscheduled DNA dosage levels up to negative Simmon
synthesis, human 10-3 M* et al., 1977
In vivo study
Dominant lethal, 0, 5, 10, & 20 mg/kg/ negative Simmon
male mice day in the diet for et al., 1977
7 weeks, followed by
weekly matings for 8
* without and with metabolic activation
Phorate did not induce clinical or histophathological signs of
neurotoxicity in a neurotoxicity study in hens. No evidence of
mutagenic potential was observed in a series of mutagenicity studies.
Since all required toxicity studies on phorate have been
submitted and evaluated, the Meeting estimated an ADI for phorate.
LEVEL CAUSING NO TOXICOLOGICAL EFFECT
Rat: 1 ppm in the diet, equivalent to 0.05 mg/kg b.w.
Dog: 0.01 mg/kg b.w./day
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN
0-0.0002 mg/kg b.w.
FURTHER WORK OR INFORMATION DESIRABLE
Observations in man.
Fletcher, D.W. 42-Day neurotoxicity study with phorate in mature white
1984 leg horn chickens. Unpublished report from Bio-Life
Associates, Ltd., BLAL No. 83 DN 103. AC Protocol No.
981-84-114. Submitted to WHO by American Cyanimid Company.
Simmon, V.F., Mitchell, A.D. & Jorgenson, T.A. Evaluation of selected
(1977) pesticides as chemical mutagens in in vitro and in vivo
studies. Unpublished report from Stanford Research
Institute, SRI report No. LSU-3493, for U.S. Environmental
Protection Agency, EPA report No. EPA-600/1-77-028.
Submitted to WHO by American Cyanamid Company).