Acute inhalation toxicity of aliphatic (C1–C5) nitrites in rats

FUNDAMENTALANDAPPLlEDTOXICOLOGY8,101-

106(1987)

Acute Inhalation Toxicity of Aliphatic (Cl-C,) DENNIS R. KLONNE,’

Nitrites in Rats

CHARLES E. ULRICH, JOHN WEISSMANN, AND ANDREW K. MORGAN

International Research and Development Corporation, 500 N. Main Street, Mattawan, Michigan 49071

Acute Inhalation Toxicity of Aliphatic (Ci-C,) Nitrites in Rats. KLO~, D. R., ULRICH, C. E., WEISSMANN, J., AND MORGAN, A. K. (1987). Fundam. Appl. Toxicol. 8, 10 1- 106. The 4-hr inhalation LCSO was determined for methyl-, ethyl-, n-propyl-, n-butyl-, isobutyl-, and isopentyl nitrite in Sprague-Dawley rats. LCSO values were 176, 160, 300, 420, 777, and 7 16 ppm, respectively. The dose-mortality curves were characterized by extremely steep slopes. Toxic signs observed during exposure included cyanosis, prostration, and rarely, convulsions. There were no effectsof exposure on body weight gain during a 14day postexposure observation period. Signs of pulmonary hemorrhage were apparent in rats which died during exposure but were much less prominent in rats sacrificed at study termination. No animals died after cessation of exposure, and rapid recovery was apparent after exposure. Concentration X Time (CT) relationships suggested that the actual concentration was more important than the “dose” in determining the lethal effects of inhalation exposure to nitrites. Because of the extremely steep dosemortality curves, the aliphatic nitrites are more hazardous than the LC50 values would indicate. 0 1987 Society ofTcv.icdogy

Nitrites produce smooth muscle relaxation, peripheral vasodilatation, hypotension, methemoglobinemia, and a euphoria when inhaled (Israelstam et al., 1978; Jackson, 1979; Nickerson, 1975; Sigell et al., 1978; Stokinger, 1982). Following inhalation there is a vasodilatation produced which is followed by a reflex vasoconstriction and tachycardia (Sigel1 et al., 1978). Symptoms after inhalation include nausea, headache, fainting, cyanosis, anoxia, convulsions, and death (Jackson, 1979; Stokinger, 1982; Nickerson, 1975). Symptoms pass rapidly upon cessation of inhalation (Jackson, 1979; Wood and Cox, 1981). The low-molecular-weight nitrites are highly volatile liquids that have industrial applications ranging from antifreeze intermediates to jet propellants. Amy1 nitrite has also had pharmacologic use as a vasodilator. In

addition, these chemicals have also become substances of abuse. In 1968 the sale of amyl nitrite (primarily composed of isopentyl nitrite (i-PN)) was regulated as a prescription drug (Israelstam et al., 1978) because of its nonmedical use as a “recreational” pharmaceutical (Sigell et al., 1978). However, several nitrites remain unregulated and are easily obtainable (Jackson, 1979; Sigell et al., 1978). Repeated nitrite inhalation can lead to tolerance to nitrite-induced headache more readily than to its other pharmacological effects (Nickerson, 1975). This might then allow an abuser to increase his dose. There is little information in the literature concerning the toxicity of nitrites in experimental laboratory animals. A gavage study in albino rats (Wood and Cox, 198 1) indicated that the “butyl nitrite” LD50 was 8 1 mg/kg. Death was preceded by “a grey pallor of the extremities, labored breathing, ataxia, exten’ To whom correspondence should be addressed: pres- sor rigidity and fasciculations.” There was no delayed toxicity and the slope of the mortality ent address: Bushy Run Research Center, Union Carbide curve was quite steep (value of 22). Corp., R.D. No. 4, Mellon Road, Export, Pa. 15632. 101

0272-0590/87 $3.00 Copyright 0 1987 by the Society of Toxicology. All rights of repmduction in any form resmwd.

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A study which determined the LD50 of several butyl nitrites [n-butyl nitrite (n-BN), isobutyl nitrite (i-BN), set-butyl nitrite (sBN), and tert-butyl nitrite (t-BN)] by gavage in mice found a range of values from 17 1 to 428 mg/kg (McFadden and Maickel, 1982). The potency of the nitrites was IZ- > iso> tert- > s-BN, with the most toxic being IZBN and the least toxic being s-BN. Methylene blue or toluidine blue given 15 min before the oral administration of i-BN resulted in an approximate 50% reduction in lethality (McFadden and Maickel, 1982). Treatment with these and other agents immediately after an orally administered dose of i-BN resulted in no significant reduction in mortality. When mice were exposed to atmospheres of these same four butyl nitrites for 1 hr the relative toxicities (in decreasing order) were n- > iso- > set - > t-BN (McFadden et al., 198 1). The respective LCSOs (95% confidence limits) were 567 (531-625) 1033 (843-1234), 1753 (1552-1964), and 10,852 (626- 15,408) ppm. This indicated a reversal of the order of toxicity for s-BN and t-BN observed in the gavage studies in mice (McFadden and Maickel, 1982). Similar to the gavage studies, the pretreatment of mice with methylene blue produced significant reduction in lethality following inhalation of the butyl nitrites. Additionally, it was noted that the relative inhalation toxicity corresponded with the relative potency of each nitrite in producing methemoglobin formation both in vivo and in vitro (McFadden et al., 198 1). Very steep dose-mortality curves were observed during preliminary inhalation studies on several aliphatic nitrites in rats. Investigations were then conducted on a series of aliphatic nitrites to determine the inhalation LC50 and the slope of the dose-mortality curves. Because of the steepness of the dosemortality curves and the rapidity of recovery from prostration and respiratory distress following exposure, additional studies were conducted to determine if Haber’s rule, Concentration X Time (CT) = k was applicable to acute aliphatic nitrite exposure.

ET AL.

METHODS General. Five male and five female Sprague-Dawley rats (Charles River, Portage, Mich.) fed a standard lab chow (Purina) ad libitum and maintained on a 12-hr light/dark cycle were used for each exposure. The body weight range was approximately 2 15-295 g for males and 160-220 g for females. Animals were observed daily for 14 days after exposure. At sacrifice, rats were anesthetized with sodium pentobarbital (ip), exsanguinated via the abdominal aorta and then subjected to a necropsy for examination of organs in the abdominal and thoracic cavities. Animals which died on study were also necropsied. Body weights were obtained immediately prior to exposure and on Days 7 and 14 of the postexposure observation period. The calculation of the LC50 for the combined sexes, together with the slope and 95% confidence limits were based on the method of Finney ( 1964). Chemicals. The methyl and ethyl nitrites were 98-99s pure (confidential source). The n-propyl nitrite (Catalog No. P27718) was purchased from Pfaltz and Bauer (Stamford, Conn.). The n-butyl (Catalog No. 57) and isopentyl (Catalog No. P436) nitrites were purchased from Eastman Kodak Company (Rochester, N.Y.). Isobutyl nitrite was obtained from Frank Enterprises (Columbus, Ohio). The respective molecular weights and (CAS Nos.) for methyl nitrite (MN), ethyl nitrite (EN), n-propyl nitrite (n-PN), n-BN, i-BN, and i-PN are 61 (624-9 l-9), 75 (109-95-5), 89 (543-67-9), 103 (544-16-l), 103 (542-563), and 117 (110-46-3). Inhalation exposure techniques. Exposures were conducted in 54-liter glass chambers. Animals were individually housed in stainless-steel cages during exposure. The exposure duration was 4 hr for the LC50 studies. For studies investigating Haber’s rule (CT product) a 2-hr exposure at twice the LC50 concentration or a 6-hr exposure at two-thirds the LC50 concentration was used. Exposure chamber temperature and relative humidity were monitored throughout the exposure period and maintained at 7 1 + 3°F and 50 + 10% RH, respectively. Exposure atmospheres for methyl nitrite and ethyl nitrite were generated by metering the gas through a rotameter (Fischer-Porter) and diluting to the desired concentration with conditioned compressed air prior to the chamber inlet. Exposure atmospheres for n-PN, n-BN, iBN, and i-PN were generated by vaporization of the liquid on a glass bead column. The total chamber airflow passed through the column and diluted the nitrite to the desired concentration. The nitrite flow to the bead column was controlled by a Sage syringe pump (Model 355). Analytical techniques. Exposure atmospheres were continuously monitored by an infrared spectrophotometer (Miran IA gas analyzer) and the output was displayed on a strip chart recorder (Linear, Model 156). The ir was calibrated by serial injections of the gas (MN, EN) or liquid (n-PN, n-BN, i-BN, i-PN) into a closed-loop system.

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TABLE 1 FOUR-hr LC50 VALUES AND SLOPESFOR SEVERAL ALIPHATIC (C,-C,) NITRITES IN RATS” Nitrite

LC50 (mm)

Methyl Ethyl n-Propyl n-Butyl Isobutyl Isopentyl

176 160 300 420 777 716

95% Confidence limits 169-183

Slope 22

151-169

18

293-308 410-431 747-809 702-731

27 31 21 47

a LC50 with confidence limits and slope were calculated by a modification of the probit analysis method of Finney ( 1964). The LC5Os are for the combined sexes of Sprague-Dawley rats.

A calibration curve was performed in triplicate with each nitrite before initiating the LC50 study. Before each exposure, the calibration was verified and in all casesagreement within ?5% of the calibration curve was observed. The analytical concentrations were calculated as timeweighted average concentrations. Because of the extremely steep slope of the mortality curve, exposures where a concentration excursion (a change in concentration greater than approximately 5% of the target concentration) occurred during the 4-hr exposure period were stopped and the animals were discarded. Nominal exposure concentrations were also calculated for the liquid nitrite exposures and generally agreed within +-lo% of the measured analytical concentrations. Nominal concentrations were not calculated for MN and EN because of the necessity to keep the cylinders packed in dry ice during exposures which resulted in varying degrees of frosting on the cylinders. That variability, combined with the small amount of gas required for each exposure, relative to the total cylinder weight, prevented an accurate nominal concentration from being obtained.

RESULTS Median lethal concentration (LC50) values with 95% confidence limits for the combined sexes for the 4-hr exposure of rats to various nitrites are presented in Table 1. There was a trend for the LC50 value to increase with increasing molecular weight for the unbranched Cl-C4 aliphatic nitrites. The branched nitrites are apparently less toxic

OF ALIPHATIC

NITRITES

103

than the unbranched nitrites, with isobutyl nitrite having an LC50 value almost twice that of n-butyl nitrite. The LC50 values indicate that the materials would be considered “moderately toxic” by the classification system of Hodge and Sterner (1949), or Deichmann and Gerarde (1969). The slopes for the LC50 curves were steep, ranging from 18 to 47 (Table 1). As the slope values indicated, there was a very narrow range over which 0- 100% mortality was exhibited, less than 100 ppm for all but i-BN, which had approximately a 200 ppm range. The steepest slope was obtained for i-PN, which had a value about twice that of MN, EN, and i-BN. Analysis of data from the CT studies with n-PN, n-BN, and i-BN (Table 2) indicated that the exposure concentration of these nitrites had more effect on rodent mortality than the “dose.” Exposure durations of 2 and 6 hr were selected for evaluation, with exposure concentrations of n-PN, n-BN, and i-BN chosen such that the CT value was equal to their respective 4-hr LC50 values. Thus, approximately 50% mortality would be expected in each experiment if Haber’s rule was in effect. For the 2-hr exposures there was 100% mortality approximately half way through the exposure interval, while no mortalities were observed in the studies of 6 hr duration at approximately two-thirds the LC50 value for n-PN or i-BN. One mortality was observed immediately before termination of the 6-hr exposure to n-BN. Thus, Haber’s rule does not apply to lethality due to inhalation of aliphatic nitrites. During the exposure, the animals initially showed increased activity and blanching of the ears and feet, then gradually became cyanotic, and developed respiratory difficulties. In cases where death ensued, prostration, and, rarely, convulsions were observed after the initial signs. There were no significant differences in mortality between males and females. In no instance did an animal die after cessation of an exposure. Animals that survived

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ET AL.

TABLE 2 CONCENTRATIONXT~MERELA~ONSHIPFORACUTEEXPOSURESTONITRITESBYINHALATION

Nitrite

Desired 4-hr CT product a

n-Propyl

1208

n-Butyl

1676

Isobutyl

3116

Desired exposure duration 0-4

ACtd exposure concentration (mm)

2 6 2 6 2 6

600 187 845 285 1560 510

Time of final death (W’

Actual CT prodmY

Number of deaths (per 10 rats)

1.2 1.1 1.0 -

720 1122 930 1710 1560 3060

10 0 10 1 10 0

’ CT product = 4 hr LC50 value (ppm) X 4. ’ Time into exposure that final death was observed. ’ Exposure duration (hr) X actual concentration (ppm).

the 4 hr of exposure generally showed a marked improvement in behavior and appearance within lo-20 min of the exposure termination, even if they appeared close to death (i.e., severe respiratory difficulties, prostration) at the end of exposure. The signs of cyanosis, bluish ears and feet, persisted for up to several hours after the exposure. There were no significant effects of exposure on body weight gain during the 14-day postexposure observation period. Necropsy examination of animals which died during the study indicated that signs of pulmonary hemorrhage were the only exposure-related macroscopic observation. At the terminal necropsy, after the postexposure observation period, there were indications of mild pulmonary hemorrhage (petechiae) found in less than half the animals, DISCUSSION Consistent with the gavage study of Wood and Cox (198 I), steep slopes for the LC50 curves were observed with the inhalation of nitrites. For comparison, Table 3 presents inhalation toxicity data for several compounds with lower LC5Os for rats than the nitrites reported in this paper. The LCSOs, 95% confidence limits, and slopes were recalculated by

the same method used for these nitrite data. It is interesting to note that the slopes of the nitrite dose-mortality curves are generally much steeper (values of 18-47) than those found for these more acutely toxic chemicals. In agreement with the inhalation data of McFadden et al. (198 I), n-BN was found to be about twice as toxic as the branch-chained i-BN, although those studies were conducted with mice for 1 hr exposure durations. Based on the 1-hr LC50 values obtained for mice versus the 4-hr value for rats, mice may be more sensitive to nitrite-induced toxicity than rats. The data presented for methyl nitrite are also in good agreement with the data reported by Gage (1970) which indicated a single 4-hr exposure to 250 ppm produced 88% mortality, with paleness and gasping being observed in those rats. The cardiovascular effects of organic nitrites are considered to be the major determinant of toxicity, despite the prominent methemoglobinemia associated with their absorption (Stokinger, 1982). The vasodilatation and hypotension, however, are transient (Stokinger, 1982; Jackson, 1979; Wood and Cox, 198 1) as exemplified in this study by the rapid recovery of rats upon cessation of exposure, even when death appeared imminent. However, normal coloration of ears and feet

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OF TABLE

LC50

ALIPHATIC

105

NITRITES

3

AND SLOPE VALUES FOR SEVERAL COMFQUNDS WHOSE VAFQRS ARE MORE LETHAL THAN THE ALIPHATIC NITRITES a

Compound

Exposure duration (mitt)

Nickel carbonyl Hexachlorocyclopentadiene Perfluoroisobutylene Methyl isocyanate

30 60 2 240

LC50 (mm)

95% Confidence limits

Slope

Reference

34

28-41 4-26 82-9 1 8-17

4.5 1.6 19 3.4

Kincaid et ai., 1953 Treon et al., 1955 Smith et al., 1982 Dodd etal., 1986;

10 86

11

BRRC,

1981

a All data were recalculated by the LC50 method used for the nitrites (Finney, 1964).

did not return until much later. Recently, McFadden and Maickel (1982) and McFadden et al. (198 1) have observed that pretreatment of mice with methylene blue provides some reduction in lethality from inhaled or orally administered nitrites. This indicates that methemoglobin formation is partially responsible for nitrite-induced lethality. However, this mechanism may not be as important a determinant for lethality in rats as the cardiovascular effects produced by nitrites. For comparison, the reported 4-hr LC50 of 1807 ppm for carbon monoxide in rats (Sax, 1979), which has a very high affinity for hemoglobin and produces carboxyhemoglobin, is 2-l 1 times higher than the LC50 values found for nitrites in this study. The rapid recovery from signs such as prostration and respiratory distress, even though signs of methemoglobin formation (i.e., cyanosis) persist after cessation of exposure, together with the small concentration range over which lethality is produced and the lack of a CT versus mortality relationship suggest that once a “threshold” concentration is achieved, death occurs. Severe hypotension and cardiovascular collapse would be consistent with these findings. However, additional studies on methylene blue pretreatment and/or administration of pressor agents during exposure would be helpful in elucidating the mechanism of ali-

phatic nitrite-induced lethality. In conclusion, the hazard involved with acute inhalation abuse of these substances is greater than the LC50 values would indicate. ACKNOWLEDGMENTS The authors are grateful to Ms. Florence Wilt for her assistance in the preparation of this manuscript, and to Mr. Steve Church for his technical assistance.

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Puper, pp. l-22. FDA, NCTR, Office of Scientific Intelligence, Jefferson, Ark. KINCAID, J. F., STRONG, J. S., AND SUNDERMAN, F. W. (1953). Nickel poisoning. I. Experimental study of the effects of acute and subacute exposure to nickel carbonyl. AMA Arch. Ind. Hyg. Occup. Med. 8,48-60. MCFADDEN, D. P., CARLSON, G. P., AND MAICKEL, R. P. ( 198 I). The role of methemoglobin in acute butyl nitrite toxicity in mice. Fundam. Appl. Toxicol. 1, 448-45

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MCFADDEN, D. P., AND MAICKEL, R. P. (1982). Butyl nitrites-An example of hazardous, noncontrolled recreational drugs. Res. Commun. Subst. Abuse 3,233236.

NICKERSON, M. (1975). Vasodilator drugs. In The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, eds.), 5th ed., MacMillian, New York. SAX, N. I. (1979). General chemicals. In Dangerous

ET AL. Properties of Industrial Materials (N. I. Sax, ed.), 5th cd., p. 470. Von Nostrand-Reinhold, New York. SIGELL, L. T., KAPP, F. T., FUSARO, G. A., NELSON, E. D., AND FALCK, R. S. (1978). Popping and snorting volatile nitrites: A current fad for getting high. Amer. J. Psychiatr. 135,1216-1218. SMITH, L. W., GARDNER, R. J., AND KENNEDY, G. L. (1982). Short-term inhalation toxicity of perIIuoroisobutylene. Drug. Chem. Toxicol. 5,295-303. STOKINGER, H. E. (1982). Aliphatic nitro compounds, nitrates, nitrites. In Patty’s Industrial Hygiene and Toxicology (G. D. Clayton, and F. E. Clayton, eds.), 3rd ed., Vol. 2C. Wiley, New York. TREON, J. F., CLEVELAND, F. P., AND CAPPEL, J. (1955). The toxicity of hexachlorocyclopentadiene. AMA Arch. Ind. Health 11,459-472. WOOD, R. W., AND Cox, C. (198 1). Acute oral toxicity of butyl nitrite. J. Appl. Toxicol. 1,30-3 1.