ISOFENPHOS
EXPLANATION
Isophenphos was evaluated for acceptable daily intake by the
Joint Meeting in 1981 and reviewed in 1982 (Annex 1, FAO/WHO, 1982a
and 1983a). A toxicological monograph was published after the 1981
Meeting (Annex 1, FAO/WHO, 1982b) and a monograph addendum was
published after the 1982 Meeting (Annex 1, FAO/WHO, 1983b). In 1981, a
temporary ADI of 0 - 0.0005 mg/kg b.w. was established, which was
extended in 1982 because of the absence of appropriate delayed
neurotoxicity studies and because of questions raised by the
multi-generation studies submitted at that time. The requested studies
have been submitted and are summarized in this monograph addendum.
EVALUATION FOR ACCEPTABLE INTAKE
BIOLOGICAL DATA
Toxicological studies
Special studies on cholinesterase inhibition
In vitro studies on purified isofenphos indicate no inhibition
of horse serum acetylcholinesterase or human serum butyrylthiocholine-
sterase up to concentrations of 100 mM. It was concluded that direct
inhibitors are not present as impurities in these isofenphos
preparations (Blass, 1982; Wehling, 1983).
Special studies on delayed neurotoxicity
Experiments using a single dose for delayed neurotoxicity studies
were performed using treatment with antidotes (atropine & 2-PAM) for
cholinergic symptoms. Isofenphos (74 mg/kg b.w. orally) given to hens
after pre-treatment with atropine did not produce clinical or
histopathological signs of delayed polyneuropathy. The unprotected
oral LD50 in this study was 21 mg/kg b.w. (Kimmerle, 1972;
Cherry et al., 1972).
In a similar study, isofenphos was given orally (36 mg/kg b.w.)
to hens after pre-treatment with atropine. Nevertheless, 17 hens out
of 30 died of cholinergic symptoms. The surviving hens did not show
clinical or histopathological signs of delayed neurotoxicity
(Hixson, 1982).
Another study measured the response of NTE in humans to oral
dosing with isofenphos (36 mg/kg b.w.) and pre-treatment with
atropine. Substantial inhibition of NTE was measured within 1 - 2 days
after dosing. No concurrent controls for clinical/histological
evaluations were used in this study, and the reference oral
unprotected LD50 was 16 mg/kg b.w. isofenphos (Thymen, 1978;
Thymen & Eben, 1983).
Another study provided evidence for the potential of isofenphos
to cause delayed neurotoxicity in hens. The oral unprotected LD50 in
tested hens was 3 - 5 mg/kg b.w. isofenphos. After appropriate
antidotal treatment (before and several times after dosing) it was
raised to 100 mg/kg b.w. One hundred mg/kg b.w. isofenphos given
orally caused more than 70% inhibition of brain NTE when measured
3 days after dosing and clinical-morphological signs of delayed
neurotoxicity approximately 2 weeks later (Wilson et al., 1984).
In a subchronic delayed neurotoxicity study, groups of 10 adult
white leghorn laying hens (15 to 20 months old) were treated daily by
gavage with technical isofenphos (as an emulsion in water with
Cremophor EL) at 0.25, 1, or 2 mg/kg b.w. for 90 consecutive days. Ten
hens were used as untreated controls and 10 "dose controls" were given
water and Cremophor EL by gavage. After the seventh administration,
clinical signs and 1 mortality occurred in the highest-dose group
(2.5 mg/kg b.w./day). At 1.25 mg/kg b.w./day 1 bird showed clinical
signs after the seventh administration.
A significant decrease in body weight and an inhibition of
cholinesterase activity in plasma, erythrocytes, and whole blood were
observed at 2 mg/kg b.w./day throughout the 90-day treatment period.
Slightly-reduced body weights and very slight inhibition of the
cholinesterases also occurred at 1 mg/kg b.w./day. Histopathological
examination of the brain, spinal cord, and sciatic nerve of all 10
hens in the 2 mg/kg b.w./day dose group revealed minimal changes
that were similar in incidence and severity to those noted in
concurrent controls. Positive controls (10 hens), treated orally with
tri-o-cresyl phosphate (TOCP) at 5 mg/kg b.w./day (as an emulsion
in water and Cremophor EL), displayed clinical signs (ataxia from
the fifth week onwards) and histopathological lesions, such as
severe degeneration of the pathways in the spinal cord and axonal
degeneration in the medulla oblongata and cerebellum, which are
typical of delayed neurotoxicity. The dose of 0.5 mg/kg b.w./day
isofenphos was tolerated without effects (Thyssen, 1978; Flucke
& Kaliner, 1985).
Special study on embryotoxicity/teratogenicity
Rats
A study to investigate embryotoxic and teratogenic effects in
Long Evans rats of the FB30 strain following dermal application was
conducted. Males were between 3 and 6 months of age and weighed
350 - 500 g at the start of the study; females were between
2.5 - 3.5 months of age and weighed 199 - 260 g. Five groups of 25
animals were treated with 0, 0.3, 1.0, 3.0, or 10.0 mg isofenphos/kg
b.w. The compound was applied to shaved dorsal skin for a 5-hour
contact time, the skin being washed at the end of each exposure. The
applied volume was 0.5 ml/kg b.w. in each test group. Males were not
treated; females were treated from days 6 - 15 of gestation.
On the 20th day of gestation the animals were killed by CO2
asphyxiation and the fetuses examined by the Wilson technique for
visceral malformations and by clearing and skeletal staining for
skeletal abnormalities. There were no treatment-related abnormalities
in behaviour or physical appearance. One dam in the 3 mg/kg b.w. group
was killed during treatment because of evidence of illness, of which
it had shown signs before treatment began. All dose groups gained less
weight than controls during treatment and throughout gestation. There
were no significant differences between control and treated groups
with regard to fertilization and pregnancy rates, which corresponded
to those known for the rats used in this study. The malformations seen
in the various groups are shown in Table 1.
Evidence of maternal toxicity was observed, with a significant
failure of treated animals to gain weight. The apparent no-effect
level for maternal toxicity was 0.3 mg/kg b.w., although the mothers
in this group showed poor weight gain during gestation (Schluter,
1981).
Special study on reproduction
A 3-generation rat reproduction study with isofenphos that was
performed at the Huntingdon Research Center in 1976 and 1977, and was
summarized in the 1981 toxicological monograph (Annex 1, FAO/WHO,
1982b), was re-evaluated. Twenty animals per group were fed doses of
0, 1, 10, or 100 ppm isofenphos in the diet. Body weights, food
consumption, and cholinesterase activity were measured. The F0
generation was mated 3 times and cross-mated (control with 100 ppm
group). F1a and F2a rats were mated once. There were no
significant effects on any variables examined, including litter size,
birth weight, viability, or lactation, in animals fed up to 10 ppm. In
the F2a generation successful mating did not occur in the 100 ppm
group because of what was described as "reduced gestation and
lactation" rates.
Comparable gestation and insemination rates between control and
test animals were found in the 1 and 10 ppm groups in all 3
generations. Following the second mating of the F0 generation, 90%
of the control females were pregnant and 70%, 60%, and 40% of the
females in the 1, 10, and 100 ppm groups, respectively, were pregnant.
Taking into account the fact that the insemination rates for this
mating were reduced in all of the study groups, all inseminated
females in the 1 ppm group became pregnant, 12/14 in the 10 ppm group
became pregnant, and all inseminated females in the 100 ppm group
became pregnant. Reduced pregnancy rates in multi-generation studies
are common as a result of reduced willingness to mate, and it occurred
in this study in control animals as well as in the test animals. One
animal in the control group and 1 in the 1 ppm group repeatedly failed
to become pregnant.
The low insemination rate exhibited by controls was considered to
be comparable with the apparently low gestation rate after the second
mating of the F0 animals. The apparent no-effect level in this study
was 10 ppm (Palmer et al., 1977; Eiben, 1983).
Table 1. Embryotoxic and terotogenic malformations in rats following
dermal application of isofenphos.
Number of Nature of
Group malformed fetuses malformations
Control 1 1x BB fissure, rib fusion,
microthalmia resp. anophthalmia
1 1x kinky tail
2 2x nodulations on ribs
1 1x oedematous thorax
0.3 mg/kg b.w. 1 1x nodulations on ribs
1 mg/kg b.w. 1 1x nodulations on ribs
3 mg/kg b.w. 1 1x multiple malformation; WS,
Ri, Be, extra tail, eyes &
umbilical hernia, and general
oedema
1 1x nodulations on ribs
1 1x cleft sternlum, abdominal
fissure, dysplasia of
extremities, cleft palate
10 mg/kg b.w. 1 1x microphthalmia
3 1x hypoplasia of telencephalon,
eye malformations, dysplasia of
extremities
2x hypoplasia of telencephalon,
eye malformations
BB = thorax and abdomen WS = vertebral column
Ri = ribs Be = pelvic bone
COMMENTS
In vitro studies indicate that, in the preparations of
isofenphos used, direct cholinesterase inhibitors are not present as
impurities.
The present Meeting reviewed a 2-generation study and a
re-evaluation of a 3-generation study performed in 1976 and 1977. The
apparent no-effect level in the multi-generation study was 10 ppm. In
a dermal study there was some evidence of anomalies associated with
embryotoxicity at 3 mg/kg b.w.
In the evaluation of the potential of isofenphos to cause delayed
polyneuropathy, the ratios between the unprotected LD50 and the dose
tested for delayed polyneuropathy were considered and are shown in
Table 2.
Table 2. Delayed neuropathy in rats dosed with isofenphos.
Approximate ratio Delayed
Unprotected LD50/ neuropathy Reference
Neurotoxic dose
< 0.3 no Kimmerle, 1972;
Cherry et al., 1972
< 1 (?) no Hixson, 1982
0.04 yes Wilson et al., 1984
Two studies on the biochemical test for delayed neuropathy
(NTE inhibition) can also be compared, as shown in Table 3.
Table 3. NTE inhibition in rats dosed with isofenphos.
Isophenphos Approximate ratio Brain NTE
(oral) Unprotected LD50/ inhibition
mg/kg b.w. Neurotox dose (range) Reference
36 approx. 0.4 23 - 58% Thyssen, 1978;
Thyssen & Eben,
1983
100 approx. 0.04 70 - 80% Wilson et al.,
1984
From these studies it is clear that the dose which causes
substantial inhibition of NTE is approximately twice the unprotected
LD50. This inhibition is, however, below the critical threshold and
does not cause delayed polyneuropathy. The dose which causes NTE
inhibition above the threshold and thus causes polyneuropathy
corresponds to about 25 times the unprotected LD50.
A 90-day repeated dosing study in hens was negative for delayed
polyneuropathy at doses up to 2 mg/kg b.w./day.
On the basis of these results the Meeting concluded that
isofenphos has the potential to cause delayed polyneuropathy. However,
the dose which produces this toxic effect in hens exceeds the LD50
by more than 2 times. The Joint Meeting believes that isofenphos
residues in food are unlikely to cause delayed polyneuropathy in man.
The Meeting appreciated the submission of data on NTE inhibition
for delayed neuropathy studies, in accordance with the recommendations
of the 1984 Joint Meeting. It should be stressed, however, that the
mechanism of initiation of delayed neuropathy through inhibition/aging
of NTE differs fundamentally from that of acetylcholinesterase
inhibition; it is not due to the accumulation of substrate, as with
acetylcholine when the scavenger enzyme is inhibited. Therefore,
unless particular problems in disposal of the chemical arise,
measurement of NTE should be performed 24 - 48 hours after dosing. At
that time the inhibition of NTE (> 70 - 80%) correlates with the
development of delayed neuropathy.
TOXICOLOGICAL EVALUATIONS
LEVEL CAUSING TOXICOLOGICAL EFFECT
Previous levels of administration free of effect were:
Mouse: 1 ppm in the diet, equivalent to 0.15 mg/kg b.w./day
Rat: 1 ppm in the diet, equivalent to 0.05 mg/kg b.w./day
Dog: 1 ppm in the diet, equivalent to 0.05 mg/kg b.w./day
These values were established on the basis of plasma
cholinesterase levels. Although some studies suggest that higher doses
could be considered no-effect levels, there is not an adequate data
base for using an alternative method in this case.
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN
0 - 0.001 mg/kg b.w.
STUDIES WHICH WILL PROVIDE INFORMATION VALUABLE IN THE CONTINUED
EVALUATION OF THE COMPOUND
Observations in man.
REFERENCES
Blass, W. I50 value of isophenphos. Unpublished report from Bayer AG,
1982 Institut fur Produktinformation und Qualitatsuberwachung.
Submitted to WHO by Bayer AG, Bayerwerk, FRG.
Cherry, C.P., Newman, A.J., & Urwin, C. Pathology report of SRA 12869
1972 - acute neurotoxicity experiment in hens. Unpublished report
from Huntingdon Research Centre, Huntingdon, UK. Submitted
to WHO by Bayer AG, Bayerwerk, FRG.
Eiben, R. Comments on the three-generation study with SRA 12869.
1983 Unpublished report from Bayer AG, Institute of Toxicology.
Submitted to WHO by Bayer AG, Bayerwerk, FRG.
Flucke, W. & Kaliner, G. SPA 12869 - Study for subchronic
1985 neurotoxicity. Unpublished report No. 13474 from Bayer AG,
Institute of Toxicology, submitted to WHO by Bayer AG,
Bayerwerk, FRG.
Hixson, E.J. Acute delayed neurotoxicity of technical isofenphos in
1982 hens. Unpublished report from Mobay Chemical Corporation,
USA. Submitted to WHO by Bayer AG, Bayerwerk, FRG.
Kimmerle, G. SRA 12869 acute neurotoxicity studies on hens.
1972 Unpublished report from Bayer AG, Institute of Toxicology.
Submitted to WHO by Bayer AG, Bayerwerk, FRG.
Palmer, A.K., Killick, M.E., & Allen, T.R. Effect of SRA 12869 on
1977 reproductive function of multiple generations in the rat.
Unpublished report from Huntingdon Research Centre,
Huntingdon, UK. Submitted to WHO by Bayer AG,
Bayerwerk, FRG.
Schluter, G. Evaluation for embryotoxic and teratogenic effects in
1981 rats following dermal application. Unpublished report
No. 9801 from Bayer AG, Institute of Toxicology. Submitted
to WHO by Bayer AG, Bayerwerk, FRG.
Thyssen, J. SRA 12869 acute toxicity studies on hens and quail.
1978 Unpublished report from Bayer AG, Institute of Toxicology.
Submitted to WHO by Bayer AG, Bayerwerk, FRG.
Thyssen, J. & Eben, A. Isophenphos (SRA 12869) neurotoxic esterase
1983 activity in hen. Unpublished report No. 11402 from Bayer AG,
Institute of Toxicology. Submitted to WHO by Bayer AG,
Bayerwerk, FRG.
Wehling, K. In vitro biochemical test with purified isofenphos for
1983 cholinesterase effect. Unpublished report No. 11418 (P) from
Bayer Pharmacology Division. Submitted to WHO by Bayer AG,
Bayerwerk, FRG.
Wilson, B.W., Hooper, M., Chow, E., Higgins, R.J., & Knaak, J.B.
1984 Antidotes and neuropathic potential of isofenphos.
Bulletin of Environmental Contamination and Toxicology,
33, 386 - 394.