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Acute and subacute toxicity study of inhaled methyl isocyanate in Charles Foster rats
ECOTOXICOLCGY
AND
ENVIRONMENTAL
SAFETY
(1989)
l&68-74
Acute and Subacute Toxicity Study of Inhaled Methyl lsocyanate in Charles Foster Rats N. SETHI, R. DAYAL, Division
of Toxicology,
Central
Drug
Received
AND R. K. SINGH
Research September
Institute,
Lucknow
226001,
India
24, I988
Charles Foster male and female rats were exposed only once to 3.52 and 35.32 ppm doses of methyl isocyanate in separate experiments for 10 min each in an acute toxicity study, while in subacute toxicity experiments, they were exposed to 0.2 12,0.265, and 0.349 ppm doses for 30 min daily, for 6 days, by inhalation. Clinical signs, mortality, body and organ weights, and changes in hematology and clinical pathology were routinely monitored to determine the principal organ sites damaged on exposure to the gas. During exposure, animals were observed to have congestion in eyes, lachrymation, nasal secretion and dyspnea, progressively increasing ataxia, and immobility. Uncoordinated movements were also observed, indicating effects on the nervous system. Upon microscopic examination of the viscera, pathological findings confined to bronchial tree, hmg parenchyma, liver, and kidneys were observed. o 1989 Academic press, IIIC. INTRODUCTION
Methyl isocyanate (CH,N=C=C=O) is the smallest of all industrially used members of the group and perhaps the most toxic. MIC is toxic following inhalation, ingestion, or topical application although, as with other isocyanates, its toxicity is much greater following inhalation than after exposure by other routes (Varma, 1987). A toxic gas was released from an agricultural plant, shortly after midnight on December 3, 1984, in Bhopal. Approximately 50,000 lb of material escaped into the atmosphere from an overheated tank used to store methyl isocyanate. A fog-like cloud spread over an approximate 60-km2 area; approximately 200,000 people were exposed to it. Estimates of deaths and injuries vary, but the Indian Government numbered fatalities at about 2000 with tens of thousands seriously injured (Heylin, 1985; Lepkowski, 1985). Consequently, the Indian Government established a commission, “Scientific‘ Commission for Continuing Studies on Effects of Bhopal Gas Leakage on Life Systems,” at New Delhi and asked the authors to observe the effect of MIC on the life systems of rats. The results of this study are given in this paper. MATERIALS
AND
METHODS
In the acute toxicity study, two sets of two Charles Foster rats, obtained from CDRI Breeding Colony, were exposed to 3.52 and 35.32 ppm doses of MIC in two separate experiments for 10 min each in MIC chambers prepared in this Institute. The purity of MIC was established using gas chromatography and was above 98%. Changes in general behavior were observed, animals were autopsied immediately after death, and viscera were taken for histopathological examinations. In the subacute toxicity study, four groups of 10 animals each of the same strain and sex were used. Group I was used as control while Groups II, III, and IV were 0147-6513189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
68
TOXICITY OF INHALED METHYL ISOCYANATE
69
exposed to 0.2 12 ppm (low dose), 0.265 ppm (high dose), and 0.349 ppm (toxic dose) doses of MIC, respectively, 30 min daily for 6 days. General behavior was observed. After 6 days of exposure, treated rats were observed for 90 days. During this time, body weights were recorded at weekly intervals. Morbidity and mortality were recorded. Hemogram and biochemistry were done at the termination of experiment. After 90 days, animals were sacrificed. Absolute and relative organ weights of animals were noted. Tissues were fixed in 10% formal saline solution. Serial paraffin sections of 5 pm were stained with hematoxylin and eosin for microscopic examinations. OBSERVATIONS
AND
RESULTS
ACUTETOXICITYSTUDY
General Behavior A few seconds after exposure, dyspnea, congestion in eyes, lachrymation, and nasal secretion were observed. The lachrymatic fluid was slightly blood stained. Within 4 min of exposure to 35.32 ppm, group animals (E,RI and E1R2) became unconscious and died. Rats exposed to the 3.52 ppm dose survived during the exposure. However, one died after 39 min (E2, R,) and another (E2 ,R2) after 293 min. Gross and Microscopic Observations Gross examinations of the viscera at the time of necropsy revealed multiple lung hemorrhage, dialated and congested trachea, pulmonary edema, and brain edema, and rest of the organs appeared congested. Histopathological findings are given in Table 1. SUBACUTETOXICITYSTUDY
General Behavior During exposure, animals had congestion in eyes, lachrymation, nasal secretion and dyspnea, progressively increasing ataxia, immobility, and uncoordinated movement systems. Body Weights See Fig. 1 for results on body weight. Hematology Platelets. Variable changes in the treated groups were observed, but were mostly within the limits of normalcy. The first counts were above the normal range in rats probably because at the time of the first count, the animals were observed to have hemoconcentration, being too sick to drink and eat properly. Total leukocyte count. A fall in all groups was registered. Dzj2rential leukocyte count. These counts were variable, but, surprisingly, the lowdose and high-dose groups had an increase in the polymorph percentage at the end of the study. Toxic dose group had a high polymorph at the first instance, being much above the expected range in most of the animals. It is possible that these animals had acquired some pulmonary infection when these were estimated.
70
SETHI,
DAYAL, TABLE
AND
SINGH
1
PATHOL~GICALFINLXNGSOFANIMALSADMINISTERED MIC ATDOSE LEVELSOF35.32 AND 3.52ppm Dose group (ppm)
Animal
35.32
E,Rr
Lung
EI&
Kidneys Testis Lung
Tissue
Large intestine Kidney 3.52
EzRl
Lung
Heart Liver Spleen Large intestine Kidneys Testis
EzRz
Trachea Esophagus Lung
Heart Liver Kidney Testis
Description Septa thickened. Vesselsuniformly congested and dilated. Alveolar ducts and sacspresent. Bronchial mucosa necrosed and sloughed off. Bronchial lumina filled with blood and mucus. Congestion and cloud swelling. Functioning. Thick edematous septa. Vesselsuniformly dilated and congested. Some alveoli filled with edema fluid. Little inflammatory exudation of cells. Bronchial mucosa necrosed and sloughing. Bronchial lumina filled with blood and mucus. Peribronchial edema. Necrosed and sloughing in epithelial surface. Inflammatory cells in subepithelial zone. Mucus-like material in lumen. Congestion and cloudy swelling. No abnormality detected in heart, brain, spleen, or small intestine. Patent alveolar sacsand ducts (over inflation at places). Uniformly dilated and congested vessels. Necrosed and sloughed off bronchial mucosa. Blood and mucus in bronchial lumina. Focal necrosis. Chronic venous congestion. Follicular hyperplasia with mild congestion. Sloughing in epithelial line. Subepithelial inflammatory exudate. Mucus in lumen. Conaested Fun&ioning No abnormality detected in brain, skin, small intestine, or eyes. Necrosed epithelium. Sloughed in the lumen. Subepithelial edema and inflammation. Sloughing of the epithelium. Exudates in lumen. Subepithelial inflammation in cells. Thick septa. Patent alveoli. Dilated and congested vessels. Necrosed and sloughing in bronchial epithelium. Peribronchial edema. Small number of inflammatory cells. Congested vesselsin the epicardium. Central venous congestion. Focal necrosis. Congestion and cloudy swelling Functioning No abnormality was detected in spleen, large intestine, pancreas, stomach, small intestine, eye or brain.
TOXICITY
OF INHALED
METHYL
71
ISOCYANATE
250-
200-
A--+ CI -
Control Grow Law dose Grau~ High dose Group TOVIC do= Group
50-
0 7
,L
21
28
35
42
L9
,6
63
70
77
8L
91
. Days
FIG. 1. Subacute toxicity study. Weekly body weight gain in different groups of rats exposed to methyl cyanate.
Hemoglobin and hematocrit. Both parameters registered a fall at the end of the study. Mean corpuscular volume. The MCV increased in almost all the animals of groups exposed to the gas. Gross and Microscopic Examinations Gross examination of the viscera at the time of necropsy revealed that the lungs in the MIC-treated animals were heavy and had a rubbery consistency compared to the sponge-like feel and lighter weights of the lungs in the control groups. Upon microscopic examination of the viscera, pathological findings confined to bronchial tree, lung parenchyma, liver, and kidneys were observed. Inflammatory lesions of bronchopulmonary tissue were present uniformly in all the MIC-treated animals and the extent of damage and destruction was found to follow a rising trend from low to toxic dose groups. Incidences of bronchopneumonia and lung abscesses, together with damage to the bronchial mucosa, were fairly high in high- and toxicdose groups. It is possible that the lung tissue damaged and weakened due to the effect of MIC became more prone to infections in accordance with the dose of the gas inhaled by the animals. The findings in liver and kidneys were nonspecific in nature and could be explained by the high incidence of pulmonary infection, leading to a rise in body temperature in the MIC-treated animals. It may, however, be noted that the pathology in these two organs also tended to demonstrate a dose relationship in incidence and severity. DISCUSSION Kimmerle and Eben (1964) demonstrated the acute toxic and lethal effects of MIC vapors in mice, rats, rabbits, and guinea pigs. They reported the deaths of mice during
72
SETHI,
DAYAL,
AND
SINGH
exposure for 1 hr to 4 g of MIC evaporated in a 400-liter chamber; deaths of rats occurred after removal from the chamber. The clinical signs of restlessness, eye irritation, and respiratory distress during exposures were seen (Kimmerle et al., 1964). Methyl isocyanate is a highly reactive chemical capable of forming a variety of reaction products with water, cellular and extracellular constituents (Union Carbide Corp., 1976; Pozzani and Kinkead, 1966; Smith, 1975; Lee, 1976; Kann et al., 1974). The reaction with water is exothermic and yields carbon dioxide and several ureas and biurets, including 1,3-dimethyl urea and 1,3,5-trimethyl biuret. MIC will carbamoylate amines, compounds containing hydroxyl groups, and sulfphydryls. It causes severe burns and necrosis when applied to the skin of rabbits (Pozzani and Kinkead, 1966). Congestion in eyes, dyspnea, lachrymation, and nasal secretions in acute exposure to the gas and also uncoordinated movements indicating effects on the nervous system were observed in this subacute toxicity study. Salmon et al. (1985) exposed the rats to MIC concentrations of 11 to 65 ppm for 2 hr and emphasized an observed sedative or necrotic MIC effect on animals’ behavior during the exposures, which implied a direct CNS effect of MIC or a reaction product. To confirm a specific CNS effect, the experiments were carried out (Dayal, 1987, unpublished data in our laboratory). MIC causes severe necrosis of the mucosae lining the respiratory tract. The nasal passages, larynx, trachea, and larger conducting airways in the lungs underwent necrosis when rats, mice, or guinea pigs were exposed to concentrations as high as 20.4 ppm following an acute dynamic exposure of 6 hr duration (Fowler and Dodd, 1986). The effects of methyl isocyanate on the respiratory tract of rats were studied (Nemery et al., 1985; Till and Ward, 1986). In this study, very little gain in body weight was observed in gas-exposed rats. Similarly, Fowler and Dodd (1986) also observed statistically significant decreases (P < 0.05) in body weight throughout the postexposure period in male and female mice exposed to 20.4, 10.5, 5.4, and 2.4 ppm MIC; also the body weights of male and female rats exposed to 20.4, 10.5, or 5.4 ppm of MIC were significantly (P < 0.001) lower than those of rats of the control group throughout the postexposure period. The eyes of male and female Fischer 344 rats were evaluated immediately after a 2-hr exposure to 0, 3, 10, or 30 ppm MIC, and periodically thereafter, during a 9 lday recovery period. During exposure to 10 ppm and higher concentrations, rats kept their eyes partially closed. Copious lacrymation and occasional frothy nasal discharge were evident (Gupta et al., 1986). In these studies the animals kept their eyes closed throughout the exposure period and copious lachrymation was observed. A large number of clinical investigations were done at Bhopal by examining the MIC-exposed populations to determine the mechanisms, principal site of damage, etc. (Misra et al., 1987; Kamat et al., 1987; Sethi et al., 1987; Deo et al., 1987; Kanhere et al., 1987; Daniel et al., 1987). Isocyanate exposure has been reported to lead to obstructive breathing problems associated with hypersensitivity in a car factory (White et al., 1980). A large percentage of the populations had persistent dyspnea, muscular weakness, tremors, poor memory, and concentration (Kamat et al., 1987). The respiratory abnormalities declined with the passage of time to some extent, but the neuropsychiatric score was found to have increased at the subsequent assessment. Psychiatric morbidity following exposure to the gas was assessed (Sethi et al., 1987). Their data revealed clearly an overwhelming majority of adult psychiatric patients suffering from neurotic disorders and conditions which can be grouped into a single classic syndrome character-
TOXICITY
OF
INHALED
METHYL
ISOCYANATE
73
ized by such symptoms as anxiety, recurrent nightmares, numbing of responsiveness, insomnia, impaired concentration, irritability, autonomic hypersensitivity, and depressive symptoms (Andreasen, 1985). CONCLUSIONS Thus, it can be concluded that the lungs were the principal organ sites damaged on exposure to the gas. The lachrymation, nasal secretion, dyspnea, congestion in eyes, etc., observed in the study were comparable to the findings of others (Salmon et al., 1985). ACKNOWLEDGMENTS The methyl isocyanate for inhalation toxicity used in this study was prepared in the Division of Biopolymers, Central Drug Research Institute, Lucknow, by Dr. K. B. Mathur and M. Haq. One of us (R. Dayal) is thankful to the Scientific Commission for Continuing Studies on Effects of Bhopal Gas Leakage on Life Systems, Cabinet Secretariat, Government of India, New Delhi, for financial assistance to carry out the study.
REFERENCES ANDREASEN, N. C. (1985). Post-traumatic stress disorder. In Comprehensive Text Book of Psychiatry (H. I. Kaplan and B. J. Sadock, Eds.), p. 9 18. Williams&Wilkins, Baltimore. DANIEL, C. S., SINGH, A. K., SIDDIQUI, P., MATHUR, B. B. L., DAS, S. K., AND AGRAWAL, S. S. (1987). Preliminary report on the spermatogenic function of male subjects exposed to gas at Bhopal. Indian J. Med. Res. 86(Suppl), 83-86. DAYAL, R. (1987). 1st annual progress report of project “Toxicological Aspects on MIC” submitted to Scientific Commission for Continuing Studies on Effects of Bhopal Gas Leakage on Life Systems,” Cabinet Secretariat, New Delhi- 11000 1. DEO, M. G., GANGAL, S., BHISEY, A. N., SOMASUNDARAM, R., BALSARA, B., GULWANI, B., DARBARI, B. S., BHIDE, S., AND MARU, G. B. (1987). Immunological, mutagenic & genotoxic investigations in gas exposed population of Bhopal. Indian J. Med. Res. 86(Suppl), 63-76. FOWLER, E. H., AND DODD, D. E. (1986). Acute inhalation studies with methyl isocyanate vapor. Part II. Respiratory tract changes in guinea pigs, rats and mice. Fundam. Appl. Toxicol. 6,756-77 1. GUPTA, B. N., STEFANSIU, S. A., BUCHER, J. R., AND HALL, L. B. (1986). Effect of methyl isocyanate (MIC) vapor on the eyes of Fisher-344 rats. National Toxicology Program, National Institute of Environmental Health Sciences and Northrop Services Inc. (2), Research Triangle Park, NC. HEYLIN, M. (Feb. 11,1985). BhopalChem. Eng. News, 14-15. KAMAT, S. R., PATEL, M. H., KOLHATKAR, V. P., DAVE, A. A., AND MAHASHUR, A. A. (1987). Sequential respiratory changes in those exposed to the gas leak at Bhopal. Indian J. Med. Res. 86(Suppl), 20-38. KANHERE, S., DARBARI, B. S., AND SRIVASTAVA, A. K. (1987). Morphological study ofplacentae ofexpectant mothers exposed to gas leak at Bhopal. Indian J. Med. Res. 86(Suppl), 77-82. KANN, H. E., KOHN, K. W., WIDERLITE, L., AND GULLION, D. (1974). Effects of 1,3-bis(2-chloroethyl)1nitrosourea and related compounds on nuclear RNA metabolism. Cancer Res. 34, 1982- 1988. KIMMERLE, G., AND EBEN, A. (1964). Zur toxicitat von methylisocyanat und dessen quantitiver bestimmung in der luft. Arch. Toxicol. 20,235-241. KIMMERLE, G., POZZANI, U. C., AND KINKEAD, E. R. (1964). Animal and human response to methyl isocyanate. Arch. Toxicol. 20,235. LEE, C. K. (1976). Methyl isocyanate as an antisickling agent and its reaction with haemoglobin S. J. Biol. Chem. 251,6226-623 I. LEPKOWSKI, W. (Feb. 11, 1985). People of India, struggle towards appropriate response to tragedy. Chem. Eng. News, 16-26. MISRA, N. P., PATHAK, R., GAUR, K. J. B. S., JAIN, S. C., YESIKAR, S. S., MANORIA, P. C., SHARMA, K. N., TRIPATHI, B. M., ASTHANA, B. S., TRIVEDI, H. H., SHARMA, V. K., MALHOTRA, Y., VARMA, A., BHARGAVA, D. K., AND BATNI, G. (1987). Clinical profile of gas leak victims in acute phase after Bhopal episode. Indian J. Med. Res. 86(Suppl), 1 1- 19.
74
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DAYAL,
AND
SINGH
NEMERY, B., DINSDALE, D., SPARROW, S., AND RAY, D. (1985). Effects of methyl isocyanate on the respiratory tract of rats. Brit. J. Ind. Med. 42,799-805. POZZANI, U. C., AND KINKEAD, E. R. (1966). Animal and human response to methyl isocyanate. Presented at the Annual Meeting of the American Industrial Hygiene Association in Pittsburgh, PA, May 16-20. SALMON, A. G., KERR MUIR, M., AND ANDERSSON, N. (1985). Acute toxicity of methyl isocyanate: A preliminary study ofthe dose response for eye and other effects. Brit. J. Znd. Med. 42,795-798. SETHI, B. B., SHARMA, M., TRIVEDI, J. K., AND SINGH, H. (1987). Psychiatric morbidity in patients attending clinics in gas affected areas in Bhopal. Indian J. Med. Rex 86(Suppl), 45-50. SMITH, R. G. (1975). Effects of methyl isocyanate on the oxygen affinity of haemoglobin S. Texas Rep. Biol. Med. 33,605. TILL, G. O., AND WARD, P. A. (1986). Systemic complement activation and acute lung injury. Fed. Proc. 45, 13-18. Union Carbide Corp., Methyl isocyanate, F. 41443A, Union Corp., Danbury, CT (1976) 41 pages. VARMA, D. R. (1987). In Hazard Assessment of Chemicals (J. Saxena, Ed.), Vol. 5, p. 252. Hemisphere Publishing Corp., Washington. WHITE, W. G., SUGDEN, E., MORRIS, M. J., AND ZAPATA, E. (1980). Isocyanate induced asthma in a car factory. Lancet 1,756.
AND
ENVIRONMENTAL
SAFETY
(1989)
l&68-74
Acute and Subacute Toxicity Study of Inhaled Methyl lsocyanate in Charles Foster Rats N. SETHI, R. DAYAL, Division
of Toxicology,
Central
Drug
Received
AND R. K. SINGH
Research September
Institute,
Lucknow
226001,
India
24, I988
Charles Foster male and female rats were exposed only once to 3.52 and 35.32 ppm doses of methyl isocyanate in separate experiments for 10 min each in an acute toxicity study, while in subacute toxicity experiments, they were exposed to 0.2 12,0.265, and 0.349 ppm doses for 30 min daily, for 6 days, by inhalation. Clinical signs, mortality, body and organ weights, and changes in hematology and clinical pathology were routinely monitored to determine the principal organ sites damaged on exposure to the gas. During exposure, animals were observed to have congestion in eyes, lachrymation, nasal secretion and dyspnea, progressively increasing ataxia, and immobility. Uncoordinated movements were also observed, indicating effects on the nervous system. Upon microscopic examination of the viscera, pathological findings confined to bronchial tree, hmg parenchyma, liver, and kidneys were observed. o 1989 Academic press, IIIC. INTRODUCTION
Methyl isocyanate (CH,N=C=C=O) is the smallest of all industrially used members of the group and perhaps the most toxic. MIC is toxic following inhalation, ingestion, or topical application although, as with other isocyanates, its toxicity is much greater following inhalation than after exposure by other routes (Varma, 1987). A toxic gas was released from an agricultural plant, shortly after midnight on December 3, 1984, in Bhopal. Approximately 50,000 lb of material escaped into the atmosphere from an overheated tank used to store methyl isocyanate. A fog-like cloud spread over an approximate 60-km2 area; approximately 200,000 people were exposed to it. Estimates of deaths and injuries vary, but the Indian Government numbered fatalities at about 2000 with tens of thousands seriously injured (Heylin, 1985; Lepkowski, 1985). Consequently, the Indian Government established a commission, “Scientific‘ Commission for Continuing Studies on Effects of Bhopal Gas Leakage on Life Systems,” at New Delhi and asked the authors to observe the effect of MIC on the life systems of rats. The results of this study are given in this paper. MATERIALS
AND
METHODS
In the acute toxicity study, two sets of two Charles Foster rats, obtained from CDRI Breeding Colony, were exposed to 3.52 and 35.32 ppm doses of MIC in two separate experiments for 10 min each in MIC chambers prepared in this Institute. The purity of MIC was established using gas chromatography and was above 98%. Changes in general behavior were observed, animals were autopsied immediately after death, and viscera were taken for histopathological examinations. In the subacute toxicity study, four groups of 10 animals each of the same strain and sex were used. Group I was used as control while Groups II, III, and IV were 0147-6513189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
68
TOXICITY OF INHALED METHYL ISOCYANATE
69
exposed to 0.2 12 ppm (low dose), 0.265 ppm (high dose), and 0.349 ppm (toxic dose) doses of MIC, respectively, 30 min daily for 6 days. General behavior was observed. After 6 days of exposure, treated rats were observed for 90 days. During this time, body weights were recorded at weekly intervals. Morbidity and mortality were recorded. Hemogram and biochemistry were done at the termination of experiment. After 90 days, animals were sacrificed. Absolute and relative organ weights of animals were noted. Tissues were fixed in 10% formal saline solution. Serial paraffin sections of 5 pm were stained with hematoxylin and eosin for microscopic examinations. OBSERVATIONS
AND
RESULTS
ACUTETOXICITYSTUDY
General Behavior A few seconds after exposure, dyspnea, congestion in eyes, lachrymation, and nasal secretion were observed. The lachrymatic fluid was slightly blood stained. Within 4 min of exposure to 35.32 ppm, group animals (E,RI and E1R2) became unconscious and died. Rats exposed to the 3.52 ppm dose survived during the exposure. However, one died after 39 min (E2, R,) and another (E2 ,R2) after 293 min. Gross and Microscopic Observations Gross examinations of the viscera at the time of necropsy revealed multiple lung hemorrhage, dialated and congested trachea, pulmonary edema, and brain edema, and rest of the organs appeared congested. Histopathological findings are given in Table 1. SUBACUTETOXICITYSTUDY
General Behavior During exposure, animals had congestion in eyes, lachrymation, nasal secretion and dyspnea, progressively increasing ataxia, immobility, and uncoordinated movement systems. Body Weights See Fig. 1 for results on body weight. Hematology Platelets. Variable changes in the treated groups were observed, but were mostly within the limits of normalcy. The first counts were above the normal range in rats probably because at the time of the first count, the animals were observed to have hemoconcentration, being too sick to drink and eat properly. Total leukocyte count. A fall in all groups was registered. Dzj2rential leukocyte count. These counts were variable, but, surprisingly, the lowdose and high-dose groups had an increase in the polymorph percentage at the end of the study. Toxic dose group had a high polymorph at the first instance, being much above the expected range in most of the animals. It is possible that these animals had acquired some pulmonary infection when these were estimated.
70
SETHI,
DAYAL, TABLE
AND
SINGH
1
PATHOL~GICALFINLXNGSOFANIMALSADMINISTERED MIC ATDOSE LEVELSOF35.32 AND 3.52ppm Dose group (ppm)
Animal
35.32
E,Rr
Lung
EI&
Kidneys Testis Lung
Tissue
Large intestine Kidney 3.52
EzRl
Lung
Heart Liver Spleen Large intestine Kidneys Testis
EzRz
Trachea Esophagus Lung
Heart Liver Kidney Testis
Description Septa thickened. Vesselsuniformly congested and dilated. Alveolar ducts and sacspresent. Bronchial mucosa necrosed and sloughed off. Bronchial lumina filled with blood and mucus. Congestion and cloud swelling. Functioning. Thick edematous septa. Vesselsuniformly dilated and congested. Some alveoli filled with edema fluid. Little inflammatory exudation of cells. Bronchial mucosa necrosed and sloughing. Bronchial lumina filled with blood and mucus. Peribronchial edema. Necrosed and sloughing in epithelial surface. Inflammatory cells in subepithelial zone. Mucus-like material in lumen. Congestion and cloudy swelling. No abnormality detected in heart, brain, spleen, or small intestine. Patent alveolar sacsand ducts (over inflation at places). Uniformly dilated and congested vessels. Necrosed and sloughed off bronchial mucosa. Blood and mucus in bronchial lumina. Focal necrosis. Chronic venous congestion. Follicular hyperplasia with mild congestion. Sloughing in epithelial line. Subepithelial inflammatory exudate. Mucus in lumen. Conaested Fun&ioning No abnormality detected in brain, skin, small intestine, or eyes. Necrosed epithelium. Sloughed in the lumen. Subepithelial edema and inflammation. Sloughing of the epithelium. Exudates in lumen. Subepithelial inflammation in cells. Thick septa. Patent alveoli. Dilated and congested vessels. Necrosed and sloughing in bronchial epithelium. Peribronchial edema. Small number of inflammatory cells. Congested vesselsin the epicardium. Central venous congestion. Focal necrosis. Congestion and cloudy swelling Functioning No abnormality was detected in spleen, large intestine, pancreas, stomach, small intestine, eye or brain.
TOXICITY
OF INHALED
METHYL
71
ISOCYANATE
250-
200-
A--+ CI -
Control Grow Law dose Grau~ High dose Group TOVIC do= Group
50-
0 7
,L
21
28
35
42
L9
,6
63
70
77
8L
91
. Days
FIG. 1. Subacute toxicity study. Weekly body weight gain in different groups of rats exposed to methyl cyanate.
Hemoglobin and hematocrit. Both parameters registered a fall at the end of the study. Mean corpuscular volume. The MCV increased in almost all the animals of groups exposed to the gas. Gross and Microscopic Examinations Gross examination of the viscera at the time of necropsy revealed that the lungs in the MIC-treated animals were heavy and had a rubbery consistency compared to the sponge-like feel and lighter weights of the lungs in the control groups. Upon microscopic examination of the viscera, pathological findings confined to bronchial tree, lung parenchyma, liver, and kidneys were observed. Inflammatory lesions of bronchopulmonary tissue were present uniformly in all the MIC-treated animals and the extent of damage and destruction was found to follow a rising trend from low to toxic dose groups. Incidences of bronchopneumonia and lung abscesses, together with damage to the bronchial mucosa, were fairly high in high- and toxicdose groups. It is possible that the lung tissue damaged and weakened due to the effect of MIC became more prone to infections in accordance with the dose of the gas inhaled by the animals. The findings in liver and kidneys were nonspecific in nature and could be explained by the high incidence of pulmonary infection, leading to a rise in body temperature in the MIC-treated animals. It may, however, be noted that the pathology in these two organs also tended to demonstrate a dose relationship in incidence and severity. DISCUSSION Kimmerle and Eben (1964) demonstrated the acute toxic and lethal effects of MIC vapors in mice, rats, rabbits, and guinea pigs. They reported the deaths of mice during
72
SETHI,
DAYAL,
AND
SINGH
exposure for 1 hr to 4 g of MIC evaporated in a 400-liter chamber; deaths of rats occurred after removal from the chamber. The clinical signs of restlessness, eye irritation, and respiratory distress during exposures were seen (Kimmerle et al., 1964). Methyl isocyanate is a highly reactive chemical capable of forming a variety of reaction products with water, cellular and extracellular constituents (Union Carbide Corp., 1976; Pozzani and Kinkead, 1966; Smith, 1975; Lee, 1976; Kann et al., 1974). The reaction with water is exothermic and yields carbon dioxide and several ureas and biurets, including 1,3-dimethyl urea and 1,3,5-trimethyl biuret. MIC will carbamoylate amines, compounds containing hydroxyl groups, and sulfphydryls. It causes severe burns and necrosis when applied to the skin of rabbits (Pozzani and Kinkead, 1966). Congestion in eyes, dyspnea, lachrymation, and nasal secretions in acute exposure to the gas and also uncoordinated movements indicating effects on the nervous system were observed in this subacute toxicity study. Salmon et al. (1985) exposed the rats to MIC concentrations of 11 to 65 ppm for 2 hr and emphasized an observed sedative or necrotic MIC effect on animals’ behavior during the exposures, which implied a direct CNS effect of MIC or a reaction product. To confirm a specific CNS effect, the experiments were carried out (Dayal, 1987, unpublished data in our laboratory). MIC causes severe necrosis of the mucosae lining the respiratory tract. The nasal passages, larynx, trachea, and larger conducting airways in the lungs underwent necrosis when rats, mice, or guinea pigs were exposed to concentrations as high as 20.4 ppm following an acute dynamic exposure of 6 hr duration (Fowler and Dodd, 1986). The effects of methyl isocyanate on the respiratory tract of rats were studied (Nemery et al., 1985; Till and Ward, 1986). In this study, very little gain in body weight was observed in gas-exposed rats. Similarly, Fowler and Dodd (1986) also observed statistically significant decreases (P < 0.05) in body weight throughout the postexposure period in male and female mice exposed to 20.4, 10.5, 5.4, and 2.4 ppm MIC; also the body weights of male and female rats exposed to 20.4, 10.5, or 5.4 ppm of MIC were significantly (P < 0.001) lower than those of rats of the control group throughout the postexposure period. The eyes of male and female Fischer 344 rats were evaluated immediately after a 2-hr exposure to 0, 3, 10, or 30 ppm MIC, and periodically thereafter, during a 9 lday recovery period. During exposure to 10 ppm and higher concentrations, rats kept their eyes partially closed. Copious lacrymation and occasional frothy nasal discharge were evident (Gupta et al., 1986). In these studies the animals kept their eyes closed throughout the exposure period and copious lachrymation was observed. A large number of clinical investigations were done at Bhopal by examining the MIC-exposed populations to determine the mechanisms, principal site of damage, etc. (Misra et al., 1987; Kamat et al., 1987; Sethi et al., 1987; Deo et al., 1987; Kanhere et al., 1987; Daniel et al., 1987). Isocyanate exposure has been reported to lead to obstructive breathing problems associated with hypersensitivity in a car factory (White et al., 1980). A large percentage of the populations had persistent dyspnea, muscular weakness, tremors, poor memory, and concentration (Kamat et al., 1987). The respiratory abnormalities declined with the passage of time to some extent, but the neuropsychiatric score was found to have increased at the subsequent assessment. Psychiatric morbidity following exposure to the gas was assessed (Sethi et al., 1987). Their data revealed clearly an overwhelming majority of adult psychiatric patients suffering from neurotic disorders and conditions which can be grouped into a single classic syndrome character-
TOXICITY
OF
INHALED
METHYL
ISOCYANATE
73
ized by such symptoms as anxiety, recurrent nightmares, numbing of responsiveness, insomnia, impaired concentration, irritability, autonomic hypersensitivity, and depressive symptoms (Andreasen, 1985). CONCLUSIONS Thus, it can be concluded that the lungs were the principal organ sites damaged on exposure to the gas. The lachrymation, nasal secretion, dyspnea, congestion in eyes, etc., observed in the study were comparable to the findings of others (Salmon et al., 1985). ACKNOWLEDGMENTS The methyl isocyanate for inhalation toxicity used in this study was prepared in the Division of Biopolymers, Central Drug Research Institute, Lucknow, by Dr. K. B. Mathur and M. Haq. One of us (R. Dayal) is thankful to the Scientific Commission for Continuing Studies on Effects of Bhopal Gas Leakage on Life Systems, Cabinet Secretariat, Government of India, New Delhi, for financial assistance to carry out the study.
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