experimental - j-stage

THE KURUME MEDICAL JOURNAL Vol. 5, No. 3, 1958

EXPERIMENTAL STUDIES ON THE TOXICITY OF BENZENE

HEXACHLORIDE (BHC) AND

DICHLORODIPHENYLTRICHLOROETHANE (DDT)

II. TOXICITY TESTS BY THE DUST INHALATION OF

THE INSECTICIDES TO LABORATORY ANIMALS

MITSURU SHIRAKAWA

Department o f Hygiene and Public Health, Kurume UniversitySchool o f Medicine, Kurume, Japan

PREFACE

The author reported previously in the first reportl on the toxicity of the oil solutions of insecticides , BHC and DDT, by means of various administrations such as: subcutaneous and intraperitoneal injections , and peroral and percutaneous administra-tions.

According to these tests, both BHC and DDT were ascertained to have acute, subacute, and chronic toxicities for the laboratory animals and each specific toxic

symptom was expressed. So the uses and the quantities of these insecticides must be

restricted to a certain extent in their practical uses. Accordingly, it is considered that the powdered BHC and DDT have undeniable toxic effects for living bodies dur-

ing their producing process or at the time of use . Then the author tried the inhalation tests of powdered BHC as well as powdered

DDT using laboratory animals to find its effects on living bodies when a large quan-tity of these powders was inhaled at one time or f or a long period of time.

EXPERIMENTAL MATERIALS

The chemical used was BHC powder (f or agricultural applications) containing 0.5 °o gamma isomer . This chemical, consisted of 0.5% of gamma isomer, 4.5% of the other isomers, 85% of bentnite and 10% of talc, was in white, powder form over 250 mesh and under 0.06 ttn in diameter, its temporary specific gravity 50 to 60 per water 100, its pH 6 to 7. It was difficult to dissolve into water and had the pun-

gent odor peculiar to BHC. The DDT powder used to compare with BHC was a white powder containing 10%

crystal of DDT diluted by 90% of talc, and it had an aromatic odor peculiar to DDT.The experimental animals were 10 rats and 49 mice.

METHODS OF TEST

The experimental apparatus was eoiisf ed of three parts, as illustrated by Figure 1, namely, equipments to raise dust, to inhale dust, and to measure dust quantity.

190

TOXICITY OF BHC AND DDT (II) 191

Fig. 1 Diagram of apparatus for exposing rats and mice to the dusts.

The dust-raising equipment was a Gaede's vacuum pump (A) to be used as an

air-blast. This was equipped by a motor of 1/3 H. P. and its air-blast capacity was 66 litres a minute. This was measured by a wet process experimental precision gas-

meter made by S. Elster. The exhaust pipe was tied to one end of the desiccator

(B), replenished with CaC12 for the purpose of exhausting the mobiloil, which some-times spouted from the exhaust pipe as well as ventilating and drying, and put absor-

bent cotton on the upper part in order to prevent flying of minute powder of CaC12 in all directions in the inhaling box. The other end was opened through two glass

tubes to two dust raising cylinders (C1 and C2). The structure of the dust raising cylinder was an upright hard glass cylinder of

4.5 cm diameter and 56 cm length, tightly plugged by rubber stoppers at the upper

and lower ends penetrated at the middle part by a hard glass tube of 1.0 cm dia-

meter and 30 cm length. The tube in the cylinder was swollen in a spherical shape

of 2 cm diameter, and at the top and neck of the sphere, twelve minute openings

were cut.

In the middle of the rubber stopper on the upper end of the dust raising cy-linder, a 1.5 cm diameter hard glass tube penetrated, the top of it remained cut, and it projected about 1 cm into the cylinder also penetrating the rubber stopper.

When a fixed quantity of the powder was put into the cylinder and the motor was moved, the powder dust was scattered upwards in a cloud. The dust was blown

up by the air spouted through the minute openings at the head sphere of the lower

ventilator and the dust filtered through the upper pipe into the next buffer (D). In the buffer, the surplus dust, which was ejected, heaped up at the bottom of

the buffer, while the scattered powder dust filtered into the next inhalation box. The quantity of dust in the dust raising cylinder, however, was extremely exces-

sive so one more buffer (D') was used and the quantity was controlled.

192 MITSURU SHIRAKAWA

The inside dimensions of the inhalation box was 30 cm high, 50 cm long and 50

cm side long, and was made of cryptomeria wood, of good quality. The front was a sliding glass door used as the doorway for the laboratory animals. A bamboo hurdle

was put on the floor. At the lower middle part of the front side of the box was a

1.0 cm diameter glass pipe, and its outer end was tied to the dust sending pipe of the buffer, the inner end stood erect in the middle of the box floor and opened five

cm from the bottom. On the upper wall on the other side of the box a water mano-

meter (E) was placed. On the lower part a dust exhausting pipe was set to make

it possible to collect the powder dust in the water. Through the middle of the box ceiling a mercurial thermometer was inserted to measure the temperature inside.

The atmospheric pressure inside the box was scarecely different from that in the

outside during the dust raising.

During the test, the body temperature of the animals rose only from 0.2•‹ to 0.5•‹C

and had almost no influence on them.

To measure dust quantity, impinger tubes (F and F') were used. In order to

use it, on the middle of the same wall where the dust exhausting pipe was, a 1.0 cm

TABLE 1

Remarks: * marks showed the ones which did not die during the experiments but died

by sacrifice. The other showed the ones died by the inhalation test.


Fig. 2 The survival periods and transition of the animals exposed to the dusts.

diameter glass pipe was inserted. At the end o:f this pipe inside the box, a rubber

tube of the same diameter was placed at the same height as the nose of the animal.

The other end of the glass pipe tied to the impinger tube was a dried rubber tube of

good quality. In order to catch the dust completely into the impinges tube, one tube is not enough, two tubes attained this purpose. As the dust-catching liquid, twice

distilled water, by 125.0 cc respectively, was poured into the two impinger tubes,


Next, these tubes were tied by rubber tubes with the gasmeter (G), which was con-nected to a suction pump (H). The air containing the powder dust in the box was

let in to the tube during the dust raising and the quantity of the air was measured exactly. At the inhalation tests, from 6 to 11 animals were let in to inhale the

dust for 5 to 120 minutes at one time and their action and the symptoms were ob-

served. After the test they were transferred into the breeding box. The quantity of dust was measured once every five days and the mean value of the concentration

of dust was determined.

The animals were subjected to the inhalation test from 1 to 45 days, the numbers of inhalation varied from 1 to 62 times and the test were extended over a period of 1 to 92 days. The dust concentration, inhalation time, survival days, the transition of the animals were shown in Table 1 and the Figure 2.

MANNERS, SYMPTOMS AND TRANSITIONS OF

THE LABORATORY ANIMALS

As soon as the BHC powder dust began to be scattered in the inhalation box,

the animals showed unrest. They moved for a while, shook their heads, sneezed, and scratched the nasal parts. Each one pushed his head into the abdomen in order

to avoid the inhalation of the dust.

Soon they stood silently in one place, with eyelids halfway or completely shut.

The breath was quickened numbering about 140 counts per minute.

The nasal parts became inflamed and wet; some were observed to hemorrhage

from the nostrils. Tears made the eyelids wet, and in extreme cases eye hemor-rhages were clearly noticed. This eye hemorrhage was not noticed in mice, but was

especially noticed in rats and was excessive when the period of inhalation exceeded more than thirty minutes. After each inhalation test, the animals were transferred

into a breeding box; but they did not take food immediately.

In rats, extraordinary excitement and activity were noticed, but in mice such symp-

toms and excessive secretion of nasal mucous or eye hemorrhages were not observed. Both rats and mice, whose symptoms were advance by the frequent inhalation

tests, suffered all over body convulsions and died, the smaller ones dying earlier.

They died as a result of the toxicity caused by BHC.

As f or the group of inhaling DDT powder, the symptoms of poisoning of the

mice were generally a little weaker than that of the BHC group, but they caused

almost the same tremors and convulsions, and then died.

The growth of the mice during the test did not show any extreme difference

between the two inhalation groups and the control group.

HISTOPATHOLOGICAL FINDINGS OF TISSUES

1. Macropathological findings

Congestions or hemorrhages of the lungs loaded with great quantities of air were

able to be seen in almost all cases. As for the other organs, extreme changes of the trachea, liver, kidney, spleen and stomach were not observed macropathologically.


2. Microscopic findings (all the specimens were dyed by hematoxylin-eosin) After rats inhaled BHC, the changes of the lungs were examined microscopically.

In every case with the differences of degree being permitted, emphysema, atelektasis

and alveoral pneumonia were recognized. Predominating were the pneumonic symp-toms.

As for the mice which inhaled BHC powder, the changes of the lung were slight atelektasis and resembled collapse, and also hemorrhages and infiltration of lympho-cytes, white blood cells, and monocytes around the bronchus were recognized.

It was seen that the lung of the mice which inhaled the DDT powder was the

same: that i, alveoral pneumonia and an increase of lymphocytes and white blood cells around the bronchus and in the alveolus of lung.

Excluding the lungs, in the organs of the rats which inhaled BHC powder, the

blood quantity of spleen increased a little, in the whole lobule of the liver was seen

slight hyperemia with congestion and swelling of the liver cells in the middle of the lobule, and fat infiltration and vacuolation were recognized. Throughout most of the

kidney, cloudy swelling of the epithelial cells of nephritic ducts was recognized. As

for the surviving animals, which did not die by poisoning, the liver cells were found to be unequal in size. However, these changes were quite insignificant or entirely

noticeable. The same was observed in the group of mice inhaling DDT powder. Considering

the organs except the lungs, in the middle parts of lobule of the liver, there were

swelling of liver cells, karyorrhexis, vacuolations and cell infiltrations.

In the kidney the cloudy swelling of epithelial cells of nephritic ducts were sligh-

tly observed. The surviving animals showed none of these changes or a little.

No changes were observed in the spleen, trachea and stomach.

SUMMARY AND DISCUSSION

Summarizing the above mentioned experimental results, in the inhalation group of

BHC powder, the rats showed symptoms of temporary excitability, eye hemorrhage and excess secretion of nasal mucous. Except a youngest and smallest of the 6 rats

which died of convulsions, the remaining 5 rats survived and were not poisoned. The symptoms of mice were the same toxic symptoms accompanying each admini-

stration as was described in the first report. Slight tremors were observed and re-

markable convulsions caused the younger ones to die early.

In the DDT group, the symptoms of the mice showed the same results as the

foregoing administrations. Severe tremors appeared and at last death resulted with

convulsions.

According to these results, it was shown that the inhalation of BHC and DDT

powder was toxic to laboratory animals.

Concerning the dust inhalation tests, the first problem is to be undeniable about the injurious effects of the inhaled powder dust itself.

On the effects of powder or dust f or living bodies are reported too many to enu-merate, but in short they are: (1) intoxications or allergic diseases, (2) mucous dis-eases, (3) pulmonary diseases, (4) dissemination of infectious diseases and (5) der-


matological diseases.

The conditions which control the noxiousness of the dust are: (1) the size of the

dust particles, (2) number, (3) hardness, (4) shape, (5) solubility, (6) electric charge-

abilty, (7) deep-reaching action, and (8) chemical reaction and ingredient. Under

these conditions the influences to living body varies greatly in degree. The atmospheric dusts enter into the body through the nose, mouth and throat.

However, the skin and the mucous membrane are also concerned with that. When the dusts are inhaled, by the filtering function or self protective function

of the body the dusts are discharged out of the body. When the bodies are forced to inhale an excessive quantity of dust, these self-protective functions gradually decline

and the influence of the dust increases.

Thus the results are that when the powder is inhaled into the lungs the soluble

particles are dissolved into the humours of the surface of the alveoles and insoluble ones are stuck on the surface or are caught by the cells and entered into the organs,

stimulated them, and caused inflammations and other changes.

According to the author's experiments, in the inhalion test of BHC and DDT

powder, remarkable specific toxic symptoms of BHC and DDT were caused by the in-halation and the changes were confirmed histopathologically. That is, the changes of

the lung due to the inhalation of both powders were alveolar pneumonia and catarrhal

pneumonia, in addition to emphysema, atelektasis, and hemorrhage. The effects of various dusts upon the lung and its histologic.al findings are agreed

to by many research workers in the past. The changes of the lung in this test and the effects of the dust itself are not denied.

As to the histological findings about the effects upon other organs than the lungs,

both BHC and DDT powder showed toxicological changes in the liver and the spleen. Thus it is understood that these changes are not caused by the dusts alone. Each

constituent of BHC and DDT, the organic compounds contained in the dust, are ab-sorbed and the toxicity appears.

Letard and Sacy (1945) reported that when the powder containing 3% BHC was sprinkled over the cornea of rabbits eye for 5 days, eye mucous membranes and nose mucous membranes did not show any symptom nor were tears recognized.

According to the author's experiment, many lacrimations and eye hemorrhages

were noticed in the rats exposed to the dusts. Consequently these symptoms seem to depend on the stimuli to eye mucous membranes.

Moreover, Uruno et al. (1949) did a minute physical examination of the workers

(male 94, female 41) of the DDT manufacturing industry and found that about 1/3 of the workers suffered from functional disorders of the liver.

CONCLUSION

By means of the apparatus devised by the author, dust inhalation tests with pow-dered BHC and DDT, synthetic organic insecticides, were performed on rats and mice. The BHC powder contained 0.50 of gamma isomer, 4.5% of the other isomers, 85% bentnite, and 10% talc; while the DDT powder contained 10% DDT and 90% talc.

The results obtained were as follows:


1. For the laboratory animals the inhalation of both powders were toxic in similar ways both the subcutaneous and intraperitoneal in jections and in peroral and percut-aneous administrations described already in the first report.

According to the amount of the powder inhaled, the kinds of experimental ani-mals, the growth and body weight of the animals, the intensity of the toxicity was

different. 2. Symptoms of the animals poisoned by inhalation were similar to those poisoned

by peroral or parenteral administrations. Namely, BHC powder caused a stimulant symptom for the mucous membranes of nose and eyes; and especially in rats a hyper-

excitability was observed.

3. Moreover, in the case of extreme toxicity, tremors and convulsions were observ-

ed using both chemicals and these were gradually followed by death.

4. According to the histopathological changes, severe pulmonary disorders were

caused by dust inhalation. These disorders showed evidence of septal interstitial and catarrhal pneumonias in addition to emphysema and atelektasis and hemorrhage. In

the liver and kidneys toxicological changes in various degrees were observed. These

were clearly caused by the toxic effects of the dust inhalation. From these studies, it must be concluded that the commercial insecticides powders

of BHC and DDT are toxic when inhaled in excess.

(This work was directed by Prof. H. Mizushima and Prof. N. Saruta, carried out in the Department of Hygiene and Public Health, Kyushu University Medical School,

and published previously on the FUKUOKA ACTA MEDICA, Vol. 43, No. 3, 1952

(in Japanese). At the end of this treatise, the author expresses deep gratitude to his respected

professors Mizushima and Saruta, who have always kindly guided the author, advised him and aided him with revision.)

REFERENCES

1. SHIRAKAWA, M.: Experimental studies on the toxicity of benzene hexachloride (BHC) and dichlorodiphenyltrichloroethane (DDT). I. Toxicity tests of the insecticides fol-lowing various administrations to laboratory animals. Fukuoka Acta Medica. 43(1) : 15-29, 1952; The Kurume Medical Journal. 5 (2): 65-86, 1958.

2. ISHIKAWA, T.: Theory and Practice of Dust Hygiene. 19-43, 1938, Kanehara Book

Co. Ltd.

3. LETARD, H. et SACY, G. S. de: Etude toxicologique du benzene hexachlore. Comptes

rendus des Seances de la Societe de Biologie. 139: 353-354, 1945.

4. URONO, K. et al.: Hygienic investigation on workers in a DDT manufacturing factory.

Japanese Journal of Hygiene. 3(1): 30, 1949.

experimental - j-stage

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