産 業 医 学
18巻,
Jap.
J.
Ind.
Health,
Vol.
TOXICITY
18,
375
1976
OF PROPOXUR
SUBACUTE Georg
Propoxur
TO
RATS
BY
INHALATION
KIMMERLE†
and
Akihiko
IYATOMI†
†
(2-isopropoxyphenyl-N-methyl-carbamate) の ラ ッ ト で の3か Georg
月 亜 急 性 吸 入 毒 性 試 験
Kimmerle*,彌
富
耿
彦**
Male and female rats were exposed to propoxur (2-isopropoxyphenyl-N-methyl-carbamate) aerosol, concentrations averaging 5.7, 18.7 and 31.7mg/m3 air, for 6 hours daily on five days in a week over a period of 12 weeks. The effects were depressions of plasma by 20 to 30%, and of erythrocyte and brain cholinesterase activities which were caused by the highest air concentration of 31.7mg/m3. On the basis of the physical, biochemical, phamacokinetic and metabolic behaviors of propoxur, a maximum allowable concentration of 2.5mg/m3 air is suggested.
formula:
INTRODUCTION Propoxur (2-isopropoxyphenyl-N-methyl-carbamate; common name in Japan being PHC) is an insecticide which is effective against plant pests, household pests, stored-product insects and hygienic pests." It is marketed under the trademarks Unden, Baygon, Bolfo, Blattanex and Suncide.(1) The compound has widely been used, under the designation OMS-33, not only by the World Health Organization in large-scale field trials in El Salvador, Iran and Nigeria, but and is also recommended by other agencies, for the control of malaria.2,3) Propoxur has the following structural † Bayer
AG,
Institut
†† Agricultural Nihon
fur
Chemicals
Tokushu
Toxikologie, Insttute,
Noyaku
Seizo
K.K.
(Present
ad-
dress) * **
Received for publication, バ イ エ ル 社 毒 性 研 究 所
19,
日 本 特 殊 農 薬 製 造 株 式 会 社 農 薬 研 究 所(現 昭 和51年2月19日
(1)
February
trademark K.K.
在)
受 付
Baygon, Blattanex, ed trademarks of tered Seizo
1976
Results of subacute inhalation studies with propoxur on experimental animals have not been reported. Studies conducted at our institute, in which male rats were exposed to propoxur aerosols for periods of one hour and four hours, showed LC50 values of>1440 and >832mg/m3 air, respectively.4) Without knowledge and without availability of data on the inhalation toxicity of propoxur, the American Conference of Governmental Industrial Hygienists suggested in 1973, on the basis of mere analogical evaluation from chemical structure, a threshold limit value in the workroom environment (TLV) of 0. 5 mg/ m3 air. In order to obtain a better assessment of the effects of propoxur, subacute inhalation studies were conducted on rats.
Bolfa and Bayer AG. of
Nihon
Unden are registerSuncide is a regisTokushu
METHODS AND MATERIALS
Noyaku
The studies were conducted
with
technical
376
産 業 医 学
grade
propoxur
(98.9%).
cream-coloured 87•Ž)
with
slight
0.2%
soluble
in
all
organic The
phenolic
Wister
II
and bred
body 180
The
flow
Propoxur
weight
was
in In
During
187
grams
and
the an the
the
spray and
wash
was using a aerosol
cascade
impactor
droplets
had
the
showed
a
that
diameter less
of than
flow
con-
extracted up
into
2
to-75℃.
gas
detector.* size by
showed
Dynamic
was
by
nitrogen droplet
only inhaled.
determined
cooled
determined
of
could
caught
bottles
aero-
airborne
mist
air
ethanol
the
were
a
1).5)
chromatoMeasuremeans of
95%
of
a
1.0±0.5μm,
the while
5.0μm.
inhalation
apparatus.
Three experimental groups of 10 male and 10 female rats were exposed to inhalation of propoxur concentrations averaging 5.7 (3.77.0), 18.7 (17.3-20.2) and 31.7 (26.2-36.3) mg/m3, respectively, for 6 hours daily on 5 days per week over a period of 12 weeks (360 * Thanks rying
are out
extended
these
to
Miss
determinations.
A .
Eben
for
car.
J.
Ind.
Health,
Vol.
18, 1976
groups of 5 in Makrolon cages, Type III.6) The experimental animal housing was lit daily from 7 a.m. to 7 p.m. The housing was airconditioned by means of artificial air circulation, the room temperature being kept at 21 ±2℃.
cham-
rats with
exposures,
series-connected
1.
the
in
and
inhalation
propoxur The
of
(1:1),
together
inhaled
out (Fig.
a mixture 400
apparatus,
the
the
Fig.
Winof
had
flow
aerosol
of
Propoxur
in
dynamic
the
rest
by
beginning
carried
glycol
analytically.
graphy ment
supplied
apparatus
dissolved
centrations
from
rats,
rats
were
these
air.
these
male of
inhalation
the
in
in
Jap.
hours in total). A control group of 10 male and 10 female rats was exposed only to the solvent mixture (ethanol/polyethylene glycol , 20ml/m3). After the exposure, the rats were housed in
feed
polyethylene
take
in
albino
the
the
experiments
bers.
ca.
grams.
dynamic
solized
used
and At
studies,
female
is
soluble
SPF
female
(Borchen).
average
to 84-
It
readily
animals
male
inhalation
white (m.p.
solvents.
strain,
kelmann
a
odour.
and
experimental were
is
powder
water,
polar
studies
and
It
crystalline
18巻,
The and
rats water
received ad
Altromin
R
standard
libitum.
During the experimental period, the animals were inspected daily and details of their behaviour were recorded, and their body weights were measured weekly. Haematological and clinical-chemicaltests and urinalyses were performed on 5 male and 5 female rats of each group at 24 hours after the finalexposure. Haematology included measurement of haematocrit value and haemoglobin content, counts of erythrocytes, leucocytes, thrombocytes and reticulocytes, and a differential blood count. Clinical chemistry included measurements of the following parameters: urea, blood sugar, GPT, GOT, alkaline phosphatase, creatinine, cholesterol,and bilirubin. Plasma and erythrocyte cholinesterase activities were measured in 5 male and 5 female rats of each group immediately after the termination of the 5th, 10th, 20th, 30th, 45th and 60th exposures by the method of Pilzand Eben7); brain cholinesterase activity was measured at autopsy by the method of Ellmann , modified by Voss and Sachsse.8) In order to collect urine required for the analysis, 5 male and 5 female rats of each group were caged singly in metabolism cages after the final exposure. The urine collected over a 6-hour period during the daytime was tested semiquantitatively for albumin, sugar, blood and pH, using Combi-Uristix reagents (manufactured by Ames). The urine deposit was examined microscopically. Twenty-four hours after the finalexposure, the rats were narcotized with ether and sacrificedby exsanguination. The tissueswere grossly examined ,and the thyroids, thymus, heart, lungs, liver,spleen, kidneys, adrenals, and gonads were weighed and histopathologicallyexamined. For histopathologicalexamina-
産 業 医 学
18巻,
Jap.
tions,* the tissues in 10% buffered of
these
stained
J.
Ind.
Health,
were fixed formalin.
tissues
were
cut
with haematoxylin
sections
of liver
and
Vol.
377
1976
for 4 to 8 weeks Paraffin sections at
ca.
5μm
and eosin.
kidneys
18,
were
cut
and
Cryostat at
12μm
and stained with Oil Red O. Kidney sections were also stained with PAS. For the statistical treatment of the results, the nonparametric ranking test of Wilcoxon9) was used.
RESULTS Male and female rats tolerated sixty 6-hour exposures within 12 weeks to propoxur aerosols at concentrations of 5. 7, 18. 7 and 31. 7 mg/m3 air, without any development of symptoms. Body weight gains of the rats exposed. to the propoxur aerosols did not differ significantly from those of the control rats during the 12 weeks (Fig. 2). Haematological values were all within the normal range and provided no indication of damage to the blood profile from inhalation of propoxur (Table 1).
measurement of pH value of the urine, analyses of the urine for content of glucose, albumin, blood, bilirubin and urobilinogen, and the examination of urine deposit gave results which were within the physiological range for both the exposed and the control rats. Erythrocyte and plasma cholinesterase activities measured at different times during the study and brain cholinesterase activity levels measured at autopsy are presented in Table 3. Clearly significant depression of the cholinesterase activity in the brain, erythrocytes and plasma was noted only in the male and female rats which had been exposed to the concentration of 31.7mg/m3 air. The depressions amounted to 20 and up to 30% of the control activities. The gross examinations of the tissues at autopsy showed no signs of deviations from the physiological norm. The absolute autopsy weights of thyroid, thymus, heart, lungs, liver, spleen, kidneys, adrenals and gonads, and their relative weights were presented in Table 4. The absolute autopsy weights and the relative weights of the organs of the rats exposed to propoxur at concentrations of 5.7, 18.7 and 31.7mg/m3 air did not differ significantly from those of the control rats. The histopathological examinations of the tissues provided no finding of any specific changes considered to be due to exposure to propoxur. DISCUSSION The signs of poisoning caused by propoxur correspond to those induced by other cholinesterase-depressing carbamates. In contrast to the mechanism of reaction involved in cholinesterase activity depression by organo-
Fig. and for
2.
Average female
12
body rats
exposed
weight to
curves propoxur
of
male
aerosols
weeks.
The results of the clinical-chemical tests are presented in Table 2. Values for urea, glucose, GPT, GOT, alkaline phosphatase (AP), creatinine, cholesterol and bilirubin are all within the physiological range. The * P .J. Gallagher and C.P. Cherry, Huntingdon Research Centre, Huntingdon, England.
phosphate pesticides, the carbamate-enzyme complex is stable for a short period. In chronic feeding experiments on rats and dogs10,11) and in experiments to study the effects of oral administrations to human volunteers,12) propoxur was not seen to have a cumulative effect. Propoxur have exhaustively been tested for its acute and chronic toxicity in animal experiments,13) and displayed no neurotoxic effects,14) no teratogenic effects,15) no mutagenic effects,16) no carcinogenic effects,10) and no adverse effects on reproduction.17) Atropine
378
産 業 医 学
8巻,
Jap.
J.
Ind.
Health,
Vol.
18, 1976
産 業 医 学
18巻,
Jap.
J.
Ind.
Health,
Vol.
18,
1976
379
380
産 業 医学
18巻,
Jap.
J.
Ind.
Health,
Vol.
18, 1976
産 業 医 学
18巻,
sulphate effect
was in
The 30,
40
Jap.
found
J.
Ind.
to
Health,
have
a
Vol.
18,
good
1976
381
antidotal
rats.18,19)
air and
concentrations 50℃
of propoxur*
amount
to
0.045,
at
0.21,
20, 0.90
and 3.5mg/m3, respectively, which was given by the volatility of the compound. According to our results, these rather lower concentrations do not seem to have any practical significance. This means that a practical hazard is seemingly possible only from inhalation of aerosols and dusts. In our experiments involving exposure to a propoxur aerosol concentration averaging 31.7mg/m3 air for 12 hours, not any somatic damage was found out among rats. The only abnormal biocnemical sign was from 20 to 30% depression of cholinesterase activity for plasma, red. blood cells and brain. Such a slight depression of this enzyme can be considered only as an indication of an exposure and not as parenchymal damages,20) and should serve as a threshold value. In this case a safety factor of ten was applied and this would mean the value 3.17mg/m3 air. As propoxur displays the same pharmacokinetic, metabolic and biochemical behaviour towards man and mammals, a maximal allowable concentration of 2.5mg propoxur/m3 air ought to constitute a sufficiently large safety margin. Therefore, this value is of practical significance only for aerosols and dusts. Acknowledgement. to
Mrs.
for
their
Marion
We
wish
Schmutter
technical
to and
express Mr.
our Franz
thanks Tepper
assistance.
REFERENCES
1)
Behrens, W. and Bocker, E.: Blattanex, ein neues aussichtsreiches Hygiene-Insektizid aus der Grup-
pe der organischen Carbaminsaureester, Pflanzenschutznachr. Bayer, 18: 53, 1965. 2) Wright, J.W., Fritz, R.F., Hocking, K.S., Babione, R., Gratz, N.G., Pal, R., Stiles, A.R. and Vandekar, M.: Ortho-isopropoxyphenylmethylcarbamate (OMS-33) as a residual spray for control of anopheline mosquitoes, Bull. W.H.O., 40: 67, 1969. 3) World Health Organization: Safe use of pesticides. 20th report of the WHO expert committee
on insecticides, WHO Technical Report Series No. 513, 17, Geneva, 1973. 4) Kimmerle, G.: Inhalationstoxizitat, 1966, (unpublished). 5) Kimmerle, G. and Eben, A.: Metabolism, excretion and toxicology of trichloroethylene after inhalation, Arch. Toxicol., 30: 115, 1973. 6) Spiegel, A. and Gonnert, R.: Neue Kafige fur Mause und Ratten, Z. Versuchstierkd., 1: 38, 1961. 7) Pilz, W. and Eben, A.: Die gleichzeitige Bestimmung der beiden Acetylcholinesterase im Rattenvollblut bei zwei verschiedenen pH-Werten, Arch. Toxicol., 23: 17, 1967. 8) Voss, G. and Sachsse, K.: Red cell and plasma cholinesterase activities in micro samples of human and animal blood determined simultaneously by a modified acetylthiocholine/DTNB procedure, Toxicol. Appl. Pharmacol, 16: 764, 1970. 9) Wilcoxon, F.: Probability tables for individual comparisons by ranking methods, Biometrics, 3: 119, 1947. 10) Loser, E.: Chronischer Futterungsversuch an Ratten, 1968, (unpublished). 11) Loser, E.: Chronischer Futterungsversuch an Hunden, 1968, (unpublished). 12) Vandekar, M., Plestina, R. and Wilhelm, K.: Toxicities of carbamates for mammals, Bull. W.H.O., 44: 241, 1971. 13) World Health Organization: 1973 Evaluations of some pesticide residues in food, WHO Pesticide Residues Series No. 3, 330, Geneva, 1974. 14) Kimmerle, G.: Neurotoxizitatsuntersuchungen, 1966, (unpublished). 15) Lorke, D.: Embryotoxizitatsuntersuchungen, 1970, (unpublished). 16) Arnold, D.: Mutagenic study, 1971, (unpublished). 17) Loser, E.: Generationsversuch an Ratten, 1968, (unpublished). 18) Kimmerle, G.: Comparison of the antidotal actions of tetraethylammonium chloride and atropine in acute poisoning of carbamate insecticides in rats, Arch. Toxicol., 27: 311, 1971. 19) Verschoyle, R.D. and Barnes, J.M.: The therapeutic effect of atropine and tetraethylammonium bromide against anticholinesterase insecticides in mice and rats, Bull. W.H.O., 41: 306, 1969. 20) Barnes, J.M. and Denz, F.A.: Experimental methods in determining chronic toxicity, Pharmacol. Rev., 6: 191, 1954. 和 Propoxur(カ
文
要
旨
ーバ メ イ ト系 殺 虫 剤)の 亜 急 性 吸 入 毒 性
試 験 を,雌 雄 ラ ッ トを 用 い て,12週 * The nicken.
determinations
were
performed
by
D .I.
Men-
間(週5日,毎
時 間 暴 露)に わ た って 行 な っ た. 実 験 期 間 中 の平 均 気 中 濃 度 は,最
高 濃 度 群31.7mg/
日6
382
m3,次 この3群
産 業 医 学
い で18.7mg/m3,低
濃 度 群 の5.7mg/m3で,
と対 照群 の計4群
18巻,
Jap.
J.
Ind.
Health,
Vol.
18,
そ の 結 果,全 調 査 項 目の うち,最 高 濃 度 群 に お いて,
で 実 験 を 行 な っ た.
血 漿,血 球 お よび脳 の コ リンエ ス テ ラ ー ゼ 活 性 値 が20 ∼30%の
実 験 期 間 中,外 観 的 中毒 症 状 の 観 察,体 重 変 化 お よ び 血 漿,血 球 コ リ ンエ ス テ ラー ゼ 活 性 値 の測 定 を,ま た 実
低 下 を きた した こ とを 除 い て ,雌 雄 各 濃度 群
と も対 照 群 と比 較 して 有 意 な差 は認 め られ な か った.
験 終 了 時 に一 般 血 液 学 的 検 査,血 液 化 学 的 検 査,解 剖 時
この 成 績 な らび に,こ
の化 合 物 の物 性,生 化 学 的,薬
の 臓 器 肉眼 観 察,臓 器 重 量 等 の 測 定 な らび に 検索 を 実 施
理 学 的 また 代 謝 等 の諸 性 質 に か ん が みpropoxurの
し た.
最 大 許 容 濃 度 を2.5mg/m3と
Reprint
requests
Toyoda
3-chome,
to Agricultural Hino-shi,
Tokyo,
1976
Chemicals 191
東 京 都 日 野 市 豊 田3-1-1日
Institute,
Japan
(A.
Nihon Iyatomi)著
Tokushu
Noyaku
者 へ の 通 信 先:彌
本特殊 農薬製造株式 会社農薬研究所
提 案 した.
Seizo
K.K.,
1-1,
富 秋 彦 ,〒191
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