PYRETHRINS JMPR 1972
Explanation
This insecticide was considered at the Joint Meetings held in 1965,
1966, 1967, 1968, 1969 and 1970 (see FAO/WHO 1965; 1967; 1968; 1969;
1970; 1971). Since the last evaluation (FAO/WHO, 1971) results of some
additional experimental studies have been made available, as well as
further information relating to methods of analysis and to residues in
dried codfish.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOCHEMICAL ASPECTS
Absorption, distribution and excretion
Pyrethrins are absorbed from the gastrointestinal tract following oral
administration. Studies in male rats receiving 3 mg/kg orally resulted
in almost complete absorption and metabolism within 100 hours. No
pyrethrin was observed in urine, although substantial quantities of
metabolites were present. In faeces, small quantities of the parent
pyrethrin were observed, again accompanied by metabolites.
Biotransformation
Utilizing an in vitro enzyme system from insects in the presence of
NADPH2, Casida and his coworkers (FAO/WHO, 1971), showed that
pyrethrin I was converted to at least ten metabolites. A major
metabolite was characterized as a product which had undergone
oxidation of a methyl group in the isobutenyl moiety to the carboxylic
acid. In a more comprehensive study, these authors concluded that
oxidation rather than hydrolysis in insects might be the major mode of
metabolism of pyrethroid chemicals. Pyrethrins I and II have also been
shown to be oxidatively metabolized in rats. Oxidation was found to
occur at the trans-methyl group of pyrethrin I as well as on the
pentadienyl side chain to produce two diols. These metabolites were
also found in conjugate form.
The oral administration of radio-labelled pyrethrin I, or pyrethrin
II, to rats produced several urinary metabolites. Each contained a
trans-2-carboxyprop-1-enyl side chain resulting from oxidation of
the chrysanthemate isobutenyl group or hydrolysis of the pyrethrate
methoxy-carbonyl group. Also, the cis-2',4'-pentadienyl side chain
of pyrethrin I and pyrethrin II was modified to give a
cis-4',5'-dihydroxypent-2'-enyl group, a 4' conjugate of this
diol, or a trans-2',5'-dihydroxypent-3'-enyl group
(Elliot et al., 1972).
Pyrethrins, in addition to metabolism by oxidation, were hydrolysed,
as evidenced by 14CO2 in expired air following treatment with
pyrethroid labelled in the carboxyl group attached to the C1 of the
cyclopropane ring. The metabolic route proposed by Elliott et al.
(1972) is shown in Figure 1.
TOXICOLOGICAL STUDIES
Special studies on reproduction
Two groups of nine rabbits each were administered pyrethrins at 0 and
90 mg/kg body-weight/day, orally, from day 8 - 16 of gestation. No
apparent effects were noted on the number and weight of foetuses,
implantation sites or on gross external and internal examination. Two
control pups and one pup in the group given pyrethrins had a club-like
deformed front paw, and one pyrethrin pup had a missing caudal
vertebrae. There appears to be no apparent teratogenic effects
elicited by pyrethrins in rabbits (FAO/WHO, 1971).
Special studies on skin sensitization
Two groups of nine male guinea pigs were used to examine the
sensitizing effect of pyrethrins. Sensitization was assessed by
comparison of the response following a challenge dose with that
elicited by previous sensitizing doses. A positive control
(1-chloro-2,4-dinitrobenzene) produced sensitization in all animals of
one group. No sensitization was obtained with a 1% formulation of
pyrethrins (FAO/WHO, 1971).
Acute toxicity
Acute toxicity of pyrethrins has been studied in animals, and a
summary of the results in four species is given in Table 1.
TABLE 1 Acute toxicity of pyrethrins in animals
Species Route LD50 Reference
(mg/kg
body-weight)
Rat (M) oral 710 FAO/WHO, 1971.
Rat oral 584 - 900 Ibid.
1 440 Bond et al., 1972
i.p. 167 - 798 FAO/WHO, 1971
Mouse oral 273 - 796 Ibid.
i.p. 172 - 452 Ibid.
Chick Perivisceral 240 - 1 262 Ibid.
Dog i.v. 6 - 81 Ibid.
1 Lethal dose.
The five ranges (FAO/WHO, 1971) comprise the various grades of
pyrethrum, including crude oleoresins and refined concentrates.
The relatively low inherent toxicity of pyrethrum should be noted. The
marked difference in the oral and intravenous toxicities may indicate
a rapid degradation or a species susceptibility, with the dog being
extremely susceptible to the toxic effect of pyrethrins. The acute
signs of poisoning in rats include: depression, rapid and/or laboured
respiration, ataxia, incoordination, convulsions and muscular tremors.
Necropsy findings include: congestion of the lungs, liver, kidneys,
adrenals and pancreas and slight gastric inflammation (FAO/WHO, 1965;
1971).
The acute effects resemble veratrine intoxication, proceeding from
excitation to tetanic convulsions, except that pyrethrins also cause
muscular fibrillation. Death is caused by respiratory failure.
Persistent tremor is occasionally seen in animals that recover from a
single large dose (FAO/WHO, 1965).
An acute dermal toxicity test was performed with rabbits using
pyrethrins in combination with synergist. Typical sprays made with the
synergists, tropital or piperonyl butoxide (1%) in combination with
0.1% pyrethrins, exhibited a low order of toxicity when tested
dermally on 6 - 12 male rabbits. The acute dermal LD50 of both
formulations was >10 gm/kg. At 10 gm/kg with tropital, three of 12
rabbits died. At 5 and 10 gm/kg, body-weight gain was reduced, and
transient signs of toxicity were evident with both synergist
combinations. No effects were noted (see Table 2) at a concentration
equivalent to 2 mg/kg pyrethrins and 20 mg/kg synergist (FAO/WHO,
1971).
Short-term studies
Rat
Two groups of rats (10 males and 10 females) were exposed to aerosols
of 1% pyrethrins for one hour. The flow was 50 litre/min. containing 2
mg/litre of air. Gross examination of the lung tissue demonstrated the
presence of haemorrhagic pin point lesions in 9 of 10 male and 10 of
10 female rats exposed. Microscopic examination of the lungs indicated
that the alterations were typical of those found in murine
pneumonitis. No distinguishing pathological observations were reported
which might be attributed to pyrethrins (FAO/WHO, 1971).
Groups of rats (10 males and 10 females/group) were administered
pyrethrin alone and in combination with various synergizers daily for
90 days at high dosages (pyrethrum alone - 360 mg/kg; pyrethrum - 180
mg/kg + piperonyl butoxide - 600 mg/kg; pyrethrum - 180 mg/kg +
sulfoxide - 132 mg/kg; pyrethrum - 90 mg/kg + tropital - 567 mg/kg).
With piperonyl butoxide alone, a higher mortality was observed. The
pyrethrin + sulfoxide combination caused deaths. Growth was reduced by
almost all treatments in both males and females (Bond et al., 1972).
TABLE 2 Acute toxicity of pyrethrin and synergists1
LD50 (mg/kg)
Pyrethrin 1 440
Piperonyl butoxide 6 040
Sulfoxide 1 760
Tropital 3 600
P + piperonyl butoxide 720 + 2 400
TABLE 2 (cont'd)
LD50 (mg/kg)
P + sulfoxide 720 + 528
P + tropital 360 + 2 268
1 Bond et al., 1972.
Rabbit
A group of 10 male and 10 female rabbits were tested by repeated
dermal application to either abraded or intact skin with a 1.0%
formulation of pyrethrins at a dose of 10 mg/kg body-weight/day. Five
male and 5 female rabbits acted as controls. Treatments of 1 ml/kg
body-weight of the formulation were applied daily (6 - 8 h exposure
per day) five days per week for three weeks. No abnormalities were
attributed to the test material. Repeated dermal applications of a 1%
formulation of pyrethrins was not detrimental to rabbits (FAO/WHO,
1971).
Long-term studies
Rat
Groups of 12 male and 12 female rats were fed pyrethrin in soybean oil
at dietary levels of 0, 200, 1 000 and 5 000 ppm for two years. The
daily dosage was, therefore, approximately 0, 10, 50 and 250 mg/kg,
respectively. Even the highest level had no significant effect on
growth or survival. Slight, though definite, liver damage
characterized by bile duct proliferation and focal necrosis was found
at the two highest dosage levels (FAO/WHO, 1966).
OBSERVATIONS IN MAN
Injury to man from pyrethrum has most frequently resulted from the
allergenic properties of pyrethrin flowers and certain extracts rather
to other forms of toxicity. The allergy has been associated primarily
with occupational and therapeutic contact.
Two hundred human subjects (177 females, 23 males) were patch tested
for skin sensitivity and irritation using pyrethrins at 1% in water
simulating formulation. This level did not produce primary irritation
and was not a sensitizer to human skin (FAO/WHO, 1971).
COMMENT
Data requested by the 1970 Joint Meeting have been received in part.
Pathways of metabolism consist of oxidation as well as hydrolysis.
Limited tests on metabolites indicate a lower order of toxicity than
pyrethrins.
Short-term studies of combinations of pyrethrin and synergizers
confirmed the relative safety of high levels of these materials.
Dermal and inhalation toxicity studies of pyrethrins in combination
with synergists, using a commercial formulation, exhibited a low order
of toxicity to rabbits.
The rapid metabolism and apparent lack of storage are reassuring. On
the basis of the new information, an ADI was established. However,
because results of requested short-term studies in the dog and other
species, to elucidate the effect on the liver found in a long-term
study in rats, ware not forthcoming, the ADI has not been increased.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Rats: 200 ppm in the diet, equivalent to 10 mg/kg
body-weight/day.
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN
0 - 0.04 mg/kg body-weight
METHODS OF ANALYSIS
Furmanec et al. (1969) used the colorimetric method of Williams
et al. (1956) with a thin-layer chromatographic separation for
assaying commercial preparations containing pyrethrins. The
insecticidal constituents pyrethrin I and II were separated from inert
material by TLC on silica gel G using hexane: ethyl acetate (75:25).
The quantitative procedure was based on the colour produced with
phosphoric acid and ethyl acetate, which was measured at 550 nm. Rapid
field assay methods for the determination of pyrethrins in pyrethrum
flowers were developed by Donegan et al. (1971). The modified
methods were based on the sulphur colour method of Cueto and Dale
(1953) and the phosphoric acid method of Williams et al. (1956).
Pyrethrins can be determined by gas chromatography using a flame
ionization or electron capture detector. The former detector was used
by Abe and Fujita (1971) with a column of 2% XE-60 on 60/80 acid
washed Chromosorb W. Tetenyi et al. (1971) employed the electron
capture detector with temperature programming and a column of 3%
N.P.G.S. on 60/80 silanized Chromosorb W. Benvenue et al. (1970)
quantitatively measured levels of pyrethrins in commercial
formulations by gas chromatography, the concentrations ranging from
0.05 to 50% of the mixtures.
Moore (1971) cleaned up sample extracts of biological materials
including eggs, tissue, body fat, milk and milk products, with
acetonitrile partitioning, silica gel column chromatography and TLC
before the determination with the electron capture detector cell of
Bruce (1967).
The use of liquid-gel chromatography for the analytical
characterization of pyrethrum extracts was investigated by Stevenson
(1972). The column eluate was examined by three general procedures:
(a) collection of fractions followed by GLC, (b) direct application to
a moving wire flame ionization detector and (c) direct application to
a moving TLC plate and subsequent development to give a two
dimensional chromatographic display of the sample. The thermal micro
separation technique of Stahl (1968) and Stahl and Fuchs (1968) for
the separation of volatile material on to a thin-layer plate was
applied by Stahl (1969) to the rapid separation of pyrethrins,
nicotine and synthetic pesticides.
Shah (1970) compared the standard mercury reduction method of the AOAC
(1955) with the official method of the Pyrethrum Board of Kenya
(P.B.K., 1954, 1957). Variations in reagents and techniques were
discussed. Gomila et al. (1969) tested the suitability of the
A.O.A.C. method (1960) for the determination of pyrethrins in mosquito
coils. The method could be applied with fairly reproducible results.
The application of high speed liquid chromatography combined with
gradient elution shows considerable potential for the analysis of
pyrethrins and related compounds for residue analysis as well as
quality control. Schmit et al. (1971) applied high speed reverse
phase liquid chromatography with an ultraviolet detector to the
determination of the components of the World Standard pyrethrum
extract which did not contain the complex mixture present in a crude
oleo-resin extract. The non-polar stationary phase employed was
"permaphase" ODS with a water/methanol mobile phase. Optimum
separation was achieved under gradient elution conditions (30%
methanol + 70% water increasing to 100% methanol at 3 min.). It is
considered that the requirement for analytical methods capable of
measuring residues of pyrethrins at the 0.1 ppm level has been met.
The gas and liquid chromatographic procedures can be adapted for
regulatory purposes.
RESIDUES IN DRIED CODFISH
Further information relating to residues in dried codfish has been
received from the Norwegian Codex Alimentarius Council (Race, 1971).
This is quoted below:-
"Dried Cod Fish when hung out to dry is at times severely exposed
to attack by blowflies (Calliphora spp). Under certain weather
conditions (dry and warm) 100% spoilage by larvae may occur. The use
of pyrethrum can reduce this spoilage considerably - one aims to
reduce spoilage by up to 90%.
"The use of pyrethrum is under strict control. Special permission
for its use must be applied for to the General Director of Fisheries.
As an illustration it can be mentioned that in 1965, 57 such
applications were granted. Reports from 31 producers show that these
treated altogether 1 480 metric tons of raw fish. Inspection of
production sites has shown that instructions for the use of pyrethrum
are followed. The use of pyrethrum has decreased considerably during
the last few years. In 1966 ca. 1 340 kg of pyrethrum concentrate
(5.3%) was sold, in 1970 ca. 120 kg for treatment of dried cod fish.
"Laboratory and field trials have been carried out by the
Chemical/Technical Research Institute of the Directorate of Fisheries
to find out the beet possible method for the use of pyrethrum.
"Laboratory tests showed that dipping for ´ min. in 0.02%
pyrethrum emulsion gave complete protection against fly larvae.
Practical trials showed however that a concentration of 0.12% was
necessary to attain satisfactory protection. It was also shown that
spraying the hung fish with 0.12% emulsion gave good protection. Lower
concentrations in dip or spray fluid did not give satisfactory
results.
"Residual concentration of pyrethrum in fish treated with 0.12%
pyrethrum emulsion varied somewhat and decreased on storage (ca. 50%
in sunlight and ca. 20% in darkness after 23 weeks).
"Immediately after drying (ca. 10 weeks after treatment) the
concentration was 7 - 8 ppm total pyrethrum determined by chemical
means. Biological tests gave a concentration of around 3 ppm or lower.
It is concluded that treatment with 0.12% is appropriate to give a
product with a residual concentration under 3 ppm. Under most of the
tests residual concentration after treatment proved to be over 1 ppm.
Only in special cases (considerable rainfall immediately after hanging
out) did one find residual concentrations of 0.1 - 0.4 ppm."
This information leads to the inescapable conclusion that, as employed
in Norway, the proposed tolerance for residues of pyrethrins on dried
codfish is more than an order of magnitude too low. Further
information was available regarding similar treatments of dried fish
in various parts of Africa, although no firm residue data was
presented. Further data is required on residues in dried fish from
supervised trials and commercial usage. It seems therefore
appropriate, and reasonable, to raise the tolerance to that already
given for raw cereals, i.e. 3 ppm.
APPRAISAL
Methods of analysis for residues of pyrethrins at the 0.1 ppm level
are available and can be adapted for regulatory purposes. Available
data indicated a need to recommend a tolerance for residues of
pyrethrins on dried fish of 3 ppm in place of the existing temporary
tolerance of 1 ppm in dried codfish.
RECOMMENDATIONS
TOLERANCE
Fish (dried) 3 ppm
FURTHER WORK OR INFORMATION
REQUIRED (Before 30 June 1975)
Further data on residues in dried fish from supervised trials and from
commercial usage.
DESIRABLE
Toxicity studies in a non-rodent mammalian species with special
emphasis on the effects on the liver.
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