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Prechronic inhalation toxicity studies of isobutyl nitrite
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
19, 169- 175 ( 1992)
Prechronic Inhalation Toxicity Studies of lsobutyl Nitrite’ C. L. GAWORSKI,*,~ C. ARANYI, **3 A . HALL III,? B. S. LEVINE,* C. D. JACKSON,?~ AND K. M. ABDOY *IIT Research Institute, 10 West 35th Street, Chicago, Illinois 60616: tpathology Associates, Inc., Chicago, Illinois 60616; SUniversity of Illinois at Chicago, Chicago, Illinois 60612; §National Center for Toxicological Research, Jefferson, Arkansas 72079: and YNational Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park, North Carolina 27709 Received September 23, 199 1; accepted February 17, I992
Prechronic Inhalation Toxicity Studies of Isobutyl Nitrite. GAWORSKI, C. L., ARANYI, C., HALL, A., III, LEVINE, B. S., JACKSON, C. D., AND ABDO, K. M. (1992). Z&dam. Appl. Toxicol. 19, 169-175. Isobutyl nitrite (IBN) is a volatile liquid that hasbecomeincreasinglypopular asan inhaled recreationaldrug. To investigate short-term toxic effects and establishexposure parametersfor chronic inhalation studies,F344/N rats and B6C3FI mice were exposedto IBN vaporson a 6 hr/day + t90,5 days/weekschedule. Twelve exposureswere administeredat concentrationsof 0, 100, 200,400,600, and 800 ppm IBN. This exposure seriesresulted in mortality in rats exposedto a600 ppm and mice exposedto 800 ppm. Animals exposed at the lower concentrations developed hyperplasia of the bronchiolar and nasalturbinate epithelium (rats and mice) and lymphocytic atrophy in the spleenand thymus (mice). Longer term, 13-week, subchronic exposures wereconducted at concentrations of 0, 10,25,75, 150, and 300 ppm IBN. Exposure to 300 ppm IBN reduced the body weight gainsin both sexesof rats and in female mice. IBN-related clinical pathology changesincluded reduced RBC counts accompanied by moderateincreasesin meancorpuscular volume and reticulocyte counts,increasedWBC counts,and mildly increased methemoglobin concentration. Bone marrow hyperplasia was observedin all groups of IBN-exposed rats, while in mice only females at 2150 ppm IBN displayed this change. Excessive splenicpulp hematopoiesiswasnoted in miceat all IBN exposure levels. Respiratory system changes included increased lung weights in rats and female mice at 300 ppm, hyperplasia of the nasal mucosa(male rats at 275 ppm and female rats at 2150 ppm), and hyperplasiaof the lung epithelium (male miceat 3150 ppm and female mice at 275 ppm). The results suggestedthat a concentration of 150ppm could he usedasthe highestexposure level for subsequentchronic inhalation tea.. Q 1992Society of Toxicology.
Isobutyl nitrite (IBN) is a volatile organic nitrite with chemical properties similar to amyl and butyl nitrite. Volatile nitrites are often included in certain commercially ’ Presented at the 28th Annual Meeting of the Society of Toxicology, Atlanta, GA, February 1989. ’ Present address: Lorillard Tobacco Co., 420 English Street. P. 0. Box 2 1688, Greensboro, NC 27420. 3 To whom correspondence should be addressed.
available over-the-counter “room odorize? preparations and have become substances of abuse used to alter consciousness,stimulate dancing, and intensify the sexual experience (Sigell et al., 1978). The principle pharmacologic action of the volatile nitrites is relaxation of the involuntary muscles,including the blood vessels.Although previous use of IBN was primarily by male homosexuals, direct inhalation of IBN vapor hasbecome increasingly popular among high school recreational drug users seeking the euphoric effects resulting from vasodilation of the cerebral blood vessels(Israelstam et al., 1978; Jaffee et al., 1983; Schwartz and Page, 1986). Toxic effects of IBN vapor exposure may include dizziness, headache, nausea,flushing of the neck, and decreasedblood pressure (Haley, 1980; Lowry, 1980; Newell et al., 1984). Ingestion of IBN may be lethal, while vapor inhalation has been reported to cause significant methemoglobinemia and severe tracheobronchitis (Covala et al., 1981; Dixon et al., 1981; Guss et al., 1985). Animal studiesconducted with mice exposed for 7 hr/day for 60 days to 400 ppm IBN resulted in 20% mortality, decreasedweight gain for the first half of the exposure period, decreasedliver microsomal glucosedphosphatase activity, and increased kidney, liver, lung, and spleenweights (McFadden and Maickel, 1985). Longer-term studies using mice exposed to IBN at concentrations up to 300 ppm for aslong as 18 weeks produced decreasedthymus and liver weight, decreasedwhite blood ceil counts, elevated methemoglobin concentrations, and mild focal hyperplasia and vacuolization of bronchial and bronchiolar epithelium (Lynch et al., 1985). IBN is a direct mutagen in the Ames assay and has a chemical structure that may allow reaction with biological amines to form n-nitroso compounds (Quinto, 1980; Newell et al., 1984; Osterloh and Goldfield, 1984). In vitro immunotoxicity studies with IBN have shown suppressionof human leukocyte proliferative responseto mitogens and cellmediated cytotoxicity reactions (Hersh et al., 1983). In vivo exposure in mice resulted in a significant depression of endogenous splenic and peripheral natural killer cell cytotoxicity potential (Lotzova et al., 1984). No apparent detrimental effect of lymphocytic proliferative response,delayed hypersensitivity response, or relative numbers of T-cells and T-
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0272-0590192 $5.00 Copyright Q 1992 by the Society of Toxicology. All rights of reproduction in any form reserved.
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cell subsets was observed in mice following inhalation of 300 ppm IBN for up to 18 weeks (Lewis et al., 1985). Since the use of aliphatic nitrites, other than amyl nitrite, is not strictly regulated, large quantities of these compounds are readily available. The increasing popularity of IBN as a drug and its potential for immunotoxic and/or carcinogenic activity enhance the need for information concerning the long-term toxic effects. The studies reported here were conducted to select dose levels for 2-year chronic investigations and were designed to identify target organs, differences in sensitivity between sexes, and dose-response relationships. METHODS Test Material IBN (molecular formula C,HgN02) is a clear yellow liquid with a boiling point of 67°C. Identification and purity (>97%) were established by combined infrared/ultraviolet, nuclear magnetic resonance, and gas chromatography techniques. IBN is a labile material, hydrolyzing slowly in the presence of water vapor to isobutanol and nitrous acid. The latter is a very reactive and unstable material at ambient temperature in the presence of oxidizing and reducing agents. The test article was stored under a nitrogen headspace in sealed, amber glass containers at -20°C. Prior to use, the chemical was warmed to room temperature and gently shaken to mix the contents. Test Atmosphere Generation IBN vapor atmospheres were generated using glass gas sampling bubblers filled with liquid IBN. The bubbler reservoirs were thermally regulated and continuously supplied with liquid IBN through a fine metering pump. A controlled flow of nitrogen carrier gas was passed through f&ted gas inlets, submerged in the reservoirs to disperse the gas through the liquid IBN and effect an efficient vaporization. The resulting vapor-laden carrier streams were directed through heated stainless steel tubing to the inlet Venturi attachments on the inhalation chambers where the vapors were mixed at high velocity and turbulence with filtered air to produce the required exposure concentrations. The carrier gas was controlled by fine metering valves and monitored with gas rotameters. Chamber concentration adjustments were made by altering the carrier gas flow rates, which directly varied the amount of vapor delivered to the chambers. For the lowest IBN chamber concentration a dilution air slip stream was installed at the gas outlet of the bubbler to dynamically remove a controlled portion of the vapor/carrier gas mixture prior to introduction to the chamber inlet. IBN in the generator reservoirs of the high and low concentration chambers was analyzed by gas chromatographic peak comparison for isobutanol changes in the liquid phase at the beginning and the end of a 6-hr exposure period. On the basis of these results a fresh sample of IBN was used each day in the test atmosphere generator reservoirs. Test ‘4tmosphere Monitoring Zsobutyl nitrite and isobutanol. IBN test atmosphere concentrations and concentrations of isobutanol were determined by either a Hewlett-Packard Model 5880 (Hewlett-Packard, Norwalk, CT) or a Perkin-Elmer Sigma 300 (Perkin-Elmer, Norwalk, CT) gas chromatograph (GC) equipped with a flame ionization detector. Both GCs were operated at oven temperatures of 50°C and injector and detector temperatures of 200°C using 1.8 m X 4mm i.d. glass columns packed with 20% SP-2 100/O. 1% Carbowax 1500 on 100/120 mesh Supelcoport and nitrogen as the carrier gas. All chamber atmospheres were monitored at least once per hour during each exposure period by sampling from a representative port near the breathing zone of the animals with a gas-tight syringe. The gas chromatograph was regularly calibrated by injecting certified gas standards of IBN and isobutanol. The
ET AL. gas standards were checked against liquid standards prepared from the IBN test article and analytical grade isobutanol, respectively. Single concentration point verifications of the calibrations were made daily. The time to reach 90% of the target concentration in the inhalation chamber (80) was established during prestudy investigations and was approximately 10 min for each chamber. Nitrous acid. Levels of nitrous acid were analyzed monthly in the high and low concentration IBN chambers. Samples of chamber air were passed through glass midget impingers containing 1% KOH, and the nitrite ion content was determined by high-pressure liquid chromatography (Waters Model 6000A HPLC Pumps, Waters Chromatography Division, Milford, MA) using a reverse-phase PBondapak Cl8 column and Waters Pit A (low uv) reagent diluted to half strength as the eluent. The nitrite ion was monitored at 2 14 nm using a Schoeffel variable wavelength Model 870 ultraviolet detector (Schoeffel/McPherson, Action, MA). Aerosol production. The high concentration chamber was sampled in the beginning of the study with a quartz crystal microbalance-based cascade impactor (QCM, California Measurements Inc., Sierra Madre, CA) to ensure that IBN was delivered to the chamber as a molecular vapor and not as an aerosol. Animals and Animal Care Male and female F344/N rats and B6C3FI mice, approximately 6 to 7 weeks of age at exposure initiation, were obtained from Simonson Laboratories (Gilroy, CA). Animals were maintained in stainless steel wire-mesh cages in 2-m3 inhalation chambers (Lab Products, Inc., Maywood, NJ), with a 2-week quarantine period prior to initiation of the exposures. Cage units were rotated on a weekly basis within the chambers. Exposure chambers were maintained at 75 + 3°F and 55 f 15% relative humidity, with air flows of 15 f 2 changes per hour. NIH-07 open formula diet (Zeigler Bros., Inc., Gardners, PA) was available ad libitum, except during exposure periods. Filtered city of Chicago drinking water was supplied ad lib&urn via automatic watering systems.A 12-hr light/dark cycle (6 AM to 6 PM light) was provided. At terminal necropsy of the subchronic study, serum samples were obtained from five rats and five mice of each sex housed in the control chamber for analysis of antibody titers (rats: KRV, H-l, CARB, Sendai, PVM, RCV/ SDA; mice: K, Poly., MHV, Ectro., GDVII, M. Ad., Reo3, LCM, MVM, CARB Sendai and PVM). All titers were negative for the agents screened. Experimental Design ll-Day repeated dose study. Groups of five animals/sex/species were exposed at target concentrations of 100, 200, 400, 600, and 800 ppm IBN on a 6 hr/day + t90, 5 day/week basis, for a total of I2 exposures. Three or 4 consecutive exposures were given immediately prior to scheduled termination for rats and mice, respectively. A control group of five animals/sex/ species was exposed to filtered air. 13-Week subchronic study. Groups of 10 animals/sex/species were exposed at target concentrations of 10,25,75, 150, and 300 ppm IBN on a 6 hr/day + t90, 5 day/week basis, for 13 weeks (rats) or 14 weeks (mice). At least two consecutive exposures were given immediately prior to scheduled termination. A control group of 10 animals/sex/species was exposed to filtered air. Observation and body weight. Animals were observed twice each day for mortality/moribundity, with clinical observations performed daily during the 14-day study and weekly during the I3-week subchronic study. Individual animal body weights were measured at exposure initiation, weekly thereafter, and at necropsy. Clinical pathology. Blood samples were collected at the terminal necropsy of the 13-week subchronic study via the retroorbital sinus from nonfasted animals anesthetized with 70% COz. Because. of the limited blood sample available from the mice, each group was randomly divided in half, and the samples were designated for either hematology or clinical chemistry evaluation. Hematology parameters examined included: hemoglobin and hematocrit; leucocyte count and differentials; erythrocyte count and indices;
ISOBUTYL
NITRITE
INHALATION
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TABLE 1 1CDay Inhalation Toxicity Study of Isobutyl Nitrite: Summary of Results Mortality IBN Target Concn (ppm)
Body weights
Male
Female
Male
Female
O/5” 015 O/5 O/5 515
015 015 O/5 115
Ob 3 -2 -26*
0 -2 1 -13*
515 515
e e
c c
Target organs
Rat 0 100 200 400 600 800 Mouse 0 100
200 400 600 800
515 015
015
0
0
015 015 015 015 315
015 015 015 015 415
-3 3 2 -14* -2o*
-3 -1 -5 -16* -2ld
Lung Lung, nasal epithelium, liver Lung, nasal epithelium. liver Liver Liver -
Lung Lung, Lung, Lung, Lung,
spleen spleen, thymus, liver spleen, thymus, liver, nasal epithelium spleen, thymus, nasal epithelium
’ Number of mortalities/number initially on test. b Values represent the percentage difference from control at termination. ’ Termination body weights not available. d Too few animals for statistical comparison. * Significantly different from control group (p G 0.05) by Dunnett’s t test.
reticulocyte count; platelet evaluation; Howell-Jolly body and Heinz body examination; and methemoglobin determination. Clinical chemistry tests included alanine aminotransferase, alkaline phosphatase, and total bile acids. Hematologic determinations were performed with a Baker 9000 hematology analyzer, and the clinical chemistry tests were performed with a Baker Centrifichem 500 automated analyzer (Serono-Baker, Allentown, PA). Necropsy, organ weights, and histopathology. Animals were anesthetized with COz and killed by exanguination by severing the axillary vessels.All animals received a complete necropsy and were examined for gross lesions. Brain, heart, right kidney, lungs, liver, right testes, and thymus weights were measured on all animals surviving at terminal necropsy. The following tissues were collected for histopathologic examination: gross lesions and tissue masses,lymph nodes (bronchial, mediastinal, mandibular, and mesenteric), mammary gland with adjacent skin, thigh muscle, salivary gland, femur including marrow, rib (costochondral junction), nasal cavity, and turbinates, tongue, larynx, pharynx, and trachea, right lung and mainstem bronchi, heart and aorta, thymus, thyroids, parathyroids, esophagus, stomach, large and small intestine, liver, gall bladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, preputial or clitoral glands, prostate, testes, epididymides, seminal vesicles, scrotal sac, vagina, ovaries, uterus, brain and pituitary, spinal cord, sciatic nerve, eyes, and Zymbol’s glands. After weighing, lungs were perfused with 10% neutral-buffered formalin using a syringe equipped with a blunt-tipped needle (20 gauge for rats and 23 gauge for mice). The lungs were perfused to approximate normal inspiratory volume (I to 2 ml in mice and 4 to 8 ml in rats), with the trachea being ligated with fine suture as the needle was withdrawn. Following removal of the soft tissues from the head, the lower jaw was removed and the nasal cavity was slowly flushed with 10% neutral-buffered formalin via the nasopharyngeal duct. After fixation the head was decalcified with dilute nitric acid. Three sections of the nasal cavity were prepared (1, level of incisorposterior edge; 2, midway between incisors and first molar; 3, middle of second molar). All tissues were fixed in 10% neutral-buffered formalin, trimmed, embedded, sectioned, and stained with hematoxylin and eosin. The lung slices were prepared from whole mount specimens, which included an entire co-
ronal (perpendicular to a sagittal plane and parallel to the long axis of the body) section of all lung lobes, including the primary bronchus and other major airways. A complete histopathologic evaluation inclusive of gross lesions was conducted on all control animals and all animals in the highest concentration group with at least 60% survivors at the time of necropsy, plus all animals in higher dose groups. Tissues demonstrating chemically related lesions (target organs) were identified, and these organs plus gross lesions were examined in lower doses until a no-observed chemical effect level was determined. Statistics
Organ weights, calculated organ weight/body weight ratios, and quantitative clinical pathology data were analyzed by one-way (by sex) analysis of variance (ANOVA) followed by a Dunnett’s t test when a significant F ratio was obtained (RS/Explore software, version 1.I, Serial No. V-658, BBN RS/ Expert Limited Partnership, BBN Software Products Corp., Cambridge, MA). The level of significance wasp d 0.05.
RESULTS 14-Day Repeated-Dose Study
All rats exposed to 600 or 800 ppm IBN died during the initial exposure period, with one female rat exposed to 400 ppm IBN euthanized in a moribund condition following the firsL 6-hr exposure (Table 1). Hepatocellular cytoplasmic vacuolization was noted in these animals, with multiple round red areas on the pleural surface of the lung observed at necropsy and lung hemorrhage seen microscopically. Rats exposed to 200 or 400 ppm IBN were lethargic and displayed rough coats and hunched posture. Decreased body weight gains occurred in rats exposed to 400 ppm IBN, with reduced
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GAWORSKI
thymus weights seen at terminal necropsy. Rats surviving the full exposure series had minimal to mild hyperplasia of the bronchiolar epithelium, minimal hyperplasia of the epithelium of the nasal turbinates, and hepatocellular cytoplasmic vacuolization. Mortalities in mice occurred only at the 800 ppm IBN exposure level, with decreased body weight gains at exposure concentrations of 2600 ppm (Table 1). Rough coats, hunched posture, and transient lethargy were noted in mice exposed at concentrations of 3400 ppm. Organ weight changes attributed to IBN exposure included increased lung weight and decreased thymus weight. Lymphocytic atrophy manifested by reduced size of splenic lymphoid follicles and thymic cortical atrophy was seen with increased severity at the higher exposure levels. Other, less significant lesions developing in mice exposed to IBN included epithelial hyperplasia of the bronchioles, peribronchiolar inflammation, hepatocellular cytoplasmic vacuolization, and alteration in the epithelium lining the nasal cavity. 13- Week Subchronic Study Chamber atmosphere analysis. The daily average IBN exposure concentrations and overall study means were all within 10% of the targeted values, with relative standard deviations also within 10%. Analyses of generator reservoir contents demonstrated a significant increase in isobutanol level during the course of a daily exposure period. However, despite this increase, the isobutanol concentration relative to IBN in the chamber atmospheres sampled during the exposures was ~3.1%. Only the 300, 150, and 75 ppm IBN chambers could be analyzed for isobutanol, since the levels in the 10 and 25 ppm chambers were below the 1.0 ppm detection limit of the gas chromatograph method. This demonstrated that the test atmospheres were not significantly affected by the isobutanol increase in the generator reservoirs when the generators were filled with fresh IBN at the beginning of each exposure day. Mean nitrous acid levels, calculated as volume/volume concentrations for direct comparison with other test atmosphere components, averaged 0.056 ppm at the 10 ppm IBN target concentration and 0.11 ppm at the 300 ppm IBN level. These concentrations were approximately two to three orders of magnitude lower than the concentrations of IBN in the chambers at either of the sampled target concentrations, and thus considered negligible. Chamber spatial homogeneities calculated as percentages relative to the mean target IBN concentration, were within 5%. Chamber atmosphere samples tested for aerosol formation were repeatedly at, or below, the limit of background sensitivity of the QCM instrument (5 pg/m3), demonstrating that aerosol was not formed in the generation process. Clinical observations and body weights. There were no mortalities in rats during the subchronic study. Ruffled fur was seen in rats exposed to the higher concentrations of IBN.
ET
AL.
Additionally, during the first 6 weeks of the study, the female rats were often noted to be hyperactive following the daily IBN exposure. These signs were primarily evident at the 300 ppm exposure level, but were occasionally noted in the group exposed to 150 ppm. Rats exposed to 300 ppm IBN had decreased body weight gains compared to controls, in which weight gains were slightly more pronounced in males than in females (Table 2). The body weight gains of rats exposed to 150 ppm IBN, or less, were not greatly affected by exposure, with the relative percentage gains of these groups within 10% of the controls. Mortalities in the mice were limited to the females exposed to either 150 or 300 ppm IBN (one at each level). Clinical observations noted in the mice during the study were considered incidental to IBN exposure. The body weight gain of male mice was not adversely affected by exposure to IBN; however, a dose-related trend toward decreased body weight gain was evident in the female mice (Table 2). Clinical pathology. Selected clinical pathology parameters are shown in Table 3. The principle hematologic change noted in animals exposed to IBN for 13 weeks was a decrease in red blood cell counts, which was generally accompanied by a moderate increase in the mean corpuscular volume and an increase in reticulocyte population. Methemoglobin concentration was mildly increased in animals exposed to the higher IBN concentrations. Although the data are not presented, exposed animals tended to have a non-dose-related increase in the number of Howell-Jolly bodies, but no significant elevation in Heinz body formation. WBC counts were increased in animals exposed to 300 ppm. Differential counts indicated an increased number of lymphocytes in these groups. No substantive changes were noted in hematocrit percentage or hemoglobin concentration, and no significant changes were noted in the clinical chemistry parameters measured (data not shown). Necropsy/organ weights. Gross necropsy observations recorded in rats and mice at necropsy appeared to be incidental findings occurring without any dose-response relationship. Increased lung/body weight ratios were noted in both sexes of rats, and female mice exposed to 300 ppm IBN (male rats + 17%, female rats + 18%, and female mice +36%). Weight changes noted in the other organs examined in rats or mice were considered to be incidental to exposure. Histopatholog!,. The most significant histopathologic changes seen in rats exposed to IBN were hematopoietic hyperplasia of bone marrow and hyperplasia of the anterior nasal mucosa (Table 4). Bone marrow hematopoietic hyperplasia appeared as an increased amount of normal hematopoietic tissue in the epiphyseal marrow of the distal femur at the expense of adipose tissue found there. The mixture of the two types of marrow was not uniform, with zones of marrow containing hematopoietic or adipose tissue seen. The cellular hematopoietic elements appeared identical in normal and hyperplastic marrow. Diaphyseal hematopoietic
ISOBUTYL
NITRITE
INHALATION
173
TOXICITY
TABLE 2 13-Week Inhalation Study of Isobutyl Nitrite: Body Weight Changes in Rats and Mice IBN target concn (ppm) Group
0
10
25
75
150
300
Male rats Initial wt. (g) Final wt. (g) Relative percentage gainb
118.0 345.0 -
115.6 339.1 -1.5
118.4 365.4 +8.8
113.0 329.3 -4.1
116.3 327.5 -7.0
116.9 296.1 -21.1
Female rats Initial wt. (g) Final wt. (g) Relative percentage gain
92.0 194.2 -
90.0 196.5 +4.2
90.2 199.8 +7.2
88.7 193.9 +2.9
88.7 187.2 -3.6
87.8 179.0 -10.8
Male mice Initial wt. (g) Final wt. (g) Relative percentage gain
23.9 34.8 -
23.1 35.1 +4.6
23.9 35.3 +4.6
23.2 35.0 +8.3
23.5 35.6 +11.0
23.2 34.6 +4.6
Female mice Initial wt. (g) Final wt. (g) Relative percentage gain
19.9 33.7 -
20.2 32.9 -8.0
19.9 32.8 -6.5
19.6 32.0 -10.1
19.8 31.6 -14.5
18.2 28.4 -26.1
a Values are the mean body weights of 9 to 10 animals/group. Wt. change (exposed) - Wt. change (control) x loo, b Relative percentage gain = Wt. change (control)
marrow constituents were variable and difficult to assess. Unfortunately, the epiphysis was occasionally trimmed away at necropsy, and these sections were not counted as examined even though abundant hematopoietic tissue was seen in the diaphyses. The incidence of this change was dose-related in
13-Week Inhalation
both sexes, with a modest increase in the average degree of severity as the exposure concentration increased for females. Nasal mucosa hyperplasia was characterized by an increase in the thickness (number of cells) of the simple cuboidal nasal mucosa in the anterior region of the nose. The change
TABLE 3 Toxicity Study of Isobutyl Nitrite: Selected Clinical Hematology Results in Rats and Mice Male
IBN target concn (ppm)
RBC (X 106/pl)
MCV (fl)
Reticulocyte (X 1O’/rU
Female WBC (X 10’//4
Methemoglobin (%I
RBC (X l@/d)
MCV UN
Reticulocyte (X 1O’/PU
WBC w 10’//.4
Methemoglobin (%)
Rat 0 10 25 75 150 300 Mouse 0 10 25 75 150 300
8.20 8.16 8.25 7.76 7.84 7.83
f f + f + +
0.29” 0.32 0.45 0.49* 0.17 0.12
9.28 f 0.27 9.00 k 0.83 9.08 + 0.48 8.73 f 0.51 8.83 f 0.21 8.13+0.52*
52 52 51 52 54 58
f 2 f 1 f I zk I ?c0* + l*
48 50 48 48 48 51
f 1 f 4 f 1 f 1 + 1 +2
13.8 14.7 20.0 17.8 34.3 74.6
f 2 + + + f
10.7 7.8 15.7 8.1 28.9 7.9 9.4 9.1 23.8 9.6 40.5* 10.4
3.7 2 5.0 0 +o 0 *o 0 &O 0 LO 8.7 + 12.4
f f f f + +
1.6 2.2 1.3 1.4 1.4 1.4*
1.5 + 1.9 f 1.7 + 2.3 + 2.6 + 2.6 f
1.4 0.6 0.5 0.5 0.8* 1.4*
8.01 1.62 7.14 1.26 7.18 1.24
+ 0.76 rt 0.46 Ik 0.53 + 0.49* f 0.27* 2 O.SO*
54+ 54k 55+ 57 f 51+ 61 +
1 9.3 f 5.7 9.0 i 1.7 8.8 + 2.1 1 8.4 z!z 6.7 1 10.8 f 11.8 9.9 * 1.4 2* 7.4 f 5.2* 10.0 * I.0 1* 9.3 * 9.4 10.4 i 2.4 1* 24.8 f 13.l* 12.1 F 1.9*
1.O zk 0.6 1.7 f 0.4 2.0 f 1.7 1.3 + 1.3 1.7 + 1.2 2.3 f 1.4
2.6 f 0.3 2.2 f 0.5 3.0 ‘- 0.9 4.1 + 0.8 4.1 It 1.2 5.6 zk 2.1*
1.8 + 1.8 f 1.4 + 2.1 f 2.3 + 4.5 f
0.4 0.6 0.6 0.8 0.3 0.8*
9.79 9.63 9.28 9.56 9.38 8.42
f 0.27 + 0.79 ?z 0.56 f 0.25 + 0.63 + 0.21*
47 f 48 f 47 + 48 f 48 + 50*
0 0 +o 2 1.8 + 4.0 1 1.7 f 3.8 I 2.0 f 4.4 1 2.6 f 5.1 1* 13.6 f 14.1
1.7 + 0.6 2.4 2 0.1 2.3 +- 0.1 2.2 + 0.4 3.2 + 1.0* 4.6 -c 0.7*
a Values represent the mean f SD of 9 to 10 rats or 3 to 4 mice/group. * Significantly different from control group (p d 0.05) by Dunnett’s t test.
4.0 f 5.5 f 5.5 + 4.1 f 3.5 + 7.1 +
0.9 1.3 0.4 0.9 0.8 1.2*
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GAWORSKI
ET AL.
TABLE 4 13-Week Inhalation Toxicity Study of Isobutyl Nitrite: Summary of Significant Histopathology Observed in Rats and Mice
Changes
IBN target concn (ppm) Males Species Rat Mouse
Tissue/lesion
0
Bone marrow hyperplasia Nasal mucosa hyperplasia
1/v o/10
Lung hyperplasia Nasal mucosa hyperplasia Spleen hematopoiesis Bone marrow hyperplasia
O/IO l/10 l/10 o/10
Females
25
15
150
300
0
10
25
75
150
300
4/l
b
317 o/10
719 2110
415 IO/10
W
O/8 o/10
3/g b
l/8
IO/IO
3/10 O/IO
719 7110
IO/IO
b
o/10
3110
b
b
3110
4110
o/10 b l/IO
b
3110 b
O/IO o/10 l/10
9/10
b
lO/lO 6/lO lO/lO
lO/lO
b
9110 o/10 9/10
5110
b
3/10
6110
9110
b
b
O/8
b
b
O/8
l/8
10
b
O/IO
b
w IO/10 l/l0 9/10 619
a Values represent number with lesions/number of tissues examined. b Not examined.
was most pronounced at the edges of the nasal turbinates and on the lateral walls of the nasal cavity. A solitary focus of pulmonary adenomatosis was seen in one male rat exposed to 300 ppm IBN. This lesion is uncommon, especially in young rats. The most striking lesion seen in mice exposed to IBN was a hyperplastic piling up and occasional dysplasia of the lining epithelium (mucosa) of the bronchial and bronchiolar tree (Table 4). The lesion was most pronounced in the bronchioles, and the severity was generally dose-related. Other, less significant, respiratory system changes included minimal hyperplastic changes in the mucosal epithelium of the anterior nasal cavity in male mice in the 300 ppm IBN group and a modest non-dose-related increase in fresh lung hemorrhage in female mice exposed to IBN. Extensive hematopoiesis was seen in the red pulp of the spleen in both sexes of mice exposed to IBN (Table 4). This change was generally more severe in the animals exposed to the higher IBN concentrations. Hyperplasia of hematopoietic elements of bone marrow was seen in females exposed to 150 or 300 ppm IBN. The lesion was diagnosed by using the absence of subepiphyseal adipose fatty marrow as the criteria. The average degree of severity was minimal. Male mice characteristically lack subepiphyseal adipose cells. DISCUSSION IBN exposure levels and use patterns experienced by humans are difficult to quantify, but could reasonably be expected to range from limited acute incidents to longer-term repeated exposures. It is estimated that actual use may vary widely from as little as 0.2 ml for the individual recreational user to the spraying of larger quantities over the dance floor of discotheques (Sigell et al., 1978). Acute inhalation of IBN at high vapor concentrations may be lethal, as demonstrated by the mortality in animals receiving a single 6-hr exposure
to 600 or 800 ppm IBN at initiation of the 14-day study. This effect occurred in a concentration range that is consistent with the reported 1-hr LC50 value of approximately 1000 ppm (McFadden et al., 198 1). Repeated exposure of rats and mice for a longer period of time (13 weeks) at reduced IBN concentrations failed to cause significant mortality, but did produce retarded weight gain, hematopoietic system alterations, and hyperplasia of the epithelium lining of the respiratory system. Hyperactivity was seen in the female rats following the first few daily exposures of the subchronic study. This response appears to be consistent with the known stimulating effects of IBN in humans, and the selective presence of this response in the female rats suggests a greater sensitivity of this sex to IBN stimulation. Interestingly, the incidence of hyperactivity diminished during the later half of the exposure period, suggesting the development of tolerance. Tolerance to amyl nitrite has previously been reported after repeated use by humans (Crandall et al., 193 1). Significantly increased methemoglobin concentration is a characteristic of nitrite exposure and has previously been noted in laboratory animals and in humans following exposure to IBN (Dixon et al., 1981; McFadden et al., 1981; Guss et al., 1985; Lynch et al., 1985). Although the methemoglobin levels determined in the animals exposed to IBN in the present study were somewhat lower than might be expected from previous reports, they were generally elevated over controls. The low methemoglobin levels in exposed animals were attributed to the delay between exposure termination and blood sampling ( 16 to 19 hr), which undoubtedly allowed sufficient time for methemoglobin reduction to occur. Mild anemia in IBN-exposed animals, characterized by a non-dose-related decrease in RBC counts, was presumably due to the decreased erythrocyte lifetime resulting from continuously increased methemoglobin. Typical compensatory responses including reticulocytosis and macrocytosis were noted in both species. Additionally, microscopic evaluation
ISOBUTYL
NITRITE
of bone marrow indicated an increase in the amount of normal hematopoietic tissue in the epiphyseal tissue in both species tested, as well as extensive hematopoiesis in the spleens of female mice exposed to IBN. Although WBC counts were significantly increased in both species exposed to 300 ppm IBN, no significant histopathologic changes were identified in the lymphoid tissue of either species. Microscopic examination of the nasal, bronchial, and pulmonary tissues obtained from rats and mice following exposure to IBN indicated changes consisting primarily of hyperplasia of the mucosal or epithelial cells in various areas of the respiratory system. Tissue changes of this type are often associated with irritant gasses, and the presence of hyperplasia in the bronchioli indicates that IBN is not totally removed in the nasal region and may penetrate into the deep lung. Lung weights were also increased in both species exposed at the higher IBN concentrations during the subchronic study. These effects are consistent with previous reports of lung weight increase and respiratory epithelial hyperplasia following IBN exposure (Covala et al., 198 1; McFadden et al., 198 1; Lynch et al., 1985). Alveolar cell hyperplasia has been suggested as a possible step preceding the development of bronchiolar neoplasia by promoters or weak carcinogens (Mohr and Dungworth, 1988), and the presence of a solitary focus of pulmonary adenomatosis in one male rat exposed at 300 ppm is notable, since this lesion is not commonly observed in younger rats. In conclusion, the results of the 13-week inhalation study with F344/N rats and B6C3Fl mice indicated the respiratory tract, bone marrow, and spleen as potential targets of IBN toxicity. Changes in the bone marrow and spleen appeared to be secondary effects associated with increased destruction of red blood cells. The results suggested that a concentration of 150 ppm could be used as the highest exposure level for subsequent chronic inhalation studies. ACKNOWLEDGMENTS This study was supported by funds from the National Toxicology Program, National Institute of Environmental Health Sciences, under Contract No. NOS-ES-65143. The authors are indebted to Ms. J. N. Bradof, Ms. M. A. Cahill, Dr. J. B. Harder, Mr. R. W. Lange, Mr. J. Raymond, Mr. P. B. Senese, and Mr. W. O’Shea for their excellent technical assistance.
REFERENCES Covala, J. R., Strimlan, C. V., and Lech, J. G. (198 1). Severe tracheobronchitis from inhalation of an isobutyl nitrite preparation. Drug Intel. C/in. Pharm. 15,5 l-52. Crandall, L. A., Leake, C. D., Loevenhart. A. S.. and Muehlberger, C. W. (1931). Acquired tolerance to and cross tolerance between the nitrous
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and nitric acid esters and sodium nitrite in man. J. PhQrmaco/. Exp. Ther. 41, 103-I 19. Dixon, D. S.. Reisch, R. F., and Santinga, P. H. (198 1). Fatal methemoglobinemia resulting from ingestion of isobutyl nitrite, a “room odorize? widely used for recreational purposes. J. Forensic Sci. 26, 587-593. Guss, D. A., Normann. S. A., and Manoguerra, A. S. (1985). Clinically significant methemoglobinemia from inhalation of isobutyl nitrite. Am. J. Emerg. Med. 3, 46-47. Haley, T. J. (1980). Review of the physiological effects of amyl, butyl, and isobutyl nitrites. Clin. Touicol. 16, 3 17-329. Hersh, E. M., Reuben, J. M., Bogerd, H., Rosenblum, M., Bielski, M., Mansell. P. W. A., Rios, A., Newell, G. R., and Sonnenfeld, G. (1983). Effect of the recreational agent isobutyl nitrite on human peripheral blood leukocytes and on in vitro interferon production. Cancer Res. 43, 13651371. Israelstam, S., Lambert, S., and Oki, G. (1978). Use of isobutyl nitrite as a recreational drug. Br. J. Addict. 73, 3 19-320. Jaffe, H. W., Choi, K.. Thomas, P. A., Haverkos, H. W., and Auerbach. D. M. (1983). Nation case-control study of Kaposi’s sarcoma and Pneumocysstiscarirzii pneumonia in homosexual men, Part I, Epidemiologic Results. Ann. Intern. Med. 99, 145-I 5 1. Lewis, D. M., Keller. W. A., Lynch, D. W., and Spira, T. J. (1985). Subchronic inhalation toxicity of isobutyl nitrite in BALB/c mice. II. Immunotoxicity studies. J. Toxicol. Environ. Health 15, 835-846. Lotzova, E., Savary. C. A., Hersh, E. M., Khan, A. A., Rosenblum, M. (1984). Depression of murine natural killer cell cytotoxicity by isobutyl nitrite. Cancer Immunol. Immunother. 17, 130- 134. Lowry, T. P. (1980). Neurophysiological aspectsof amyl nitrite. J. Psychedelic Drugs 12, 73-74. Lynch, D. W., Moorman. W. J., Burg, J. R., Phipps, F. C., Lewis, T. R.. Khan, A., Lewis, D. M., Chandler, F. W.. Kimbrough, R. D., and Spira, T. J. (1985). Subchronic inhalation toxicity of isobutyl nitrite in Balb/c mice. I. Systemic toxicity. J. To.ricol. Environ. Health 15, 823-833. McFadden. D. P., Carlson. G. P., and Maickel, R. P. (1981). The role of methemoglobin in acute butyl nitrite toxicity in mice. Fundam. Appl. To,yicol. 1, 448-45 I. McFadden, D. P., and Maickel, R. P. ( 1985). Subchronic toxicology of butyl nitrites in mice by inhalation. J. Appl. Toxicol. 5, 134-139. Mohr, U., and Dungworth, D. L. (1988). Relevance to humans of experimentally induced pulmonary tumors in rats and hamsters. In Inhalation Taricologq? The Design and Interpretation of Inhalation Studies and Their CJsein Risk Assessment (U. Mohr, Ed.), pp. 209-232. Springer-Verlag, New York. Newell, G. R., Adams, S. C., Mansell, P. W. A., and Hersch, E. M. (1984). Toxicity, immunosuppressive effectsand carcinogenic potential of volatile nitrites: Possible relationship to Kaposi’s sarcoma. Pharmacotherapy 4, 284-29 1. Osterloh, J., and Goldfield. D. (1984). Butyl nitrite transformation in vitro. chemical nitrosation reactions, and mutagenesis. J. Anal. Toxicol. 8, 164169. Quinto. 1. (1980). Mutagenicity of alkyl nitrites in the salmonella test. BON. Sot. Biol. Sper. 56, 8 16-820. Schwartz, R. H., and Page, M. (1986). Abuse of isobutyl nitrite inhalation (RUSH) by adolescents. To,xicology 25, 308-310. Sigell, L. T., Knapp, F. T., Fusaro, G. A., Nelson, E. D., and Falck, R. S. ( 1978). Popping and snorting volatile nitrites: A current fad for getting high. Am. J. Psychiatry 135, 1216-1218.
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19, 169- 175 ( 1992)
Prechronic Inhalation Toxicity Studies of lsobutyl Nitrite’ C. L. GAWORSKI,*,~ C. ARANYI, **3 A . HALL III,? B. S. LEVINE,* C. D. JACKSON,?~ AND K. M. ABDOY *IIT Research Institute, 10 West 35th Street, Chicago, Illinois 60616: tpathology Associates, Inc., Chicago, Illinois 60616; SUniversity of Illinois at Chicago, Chicago, Illinois 60612; §National Center for Toxicological Research, Jefferson, Arkansas 72079: and YNational Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park, North Carolina 27709 Received September 23, 199 1; accepted February 17, I992
Prechronic Inhalation Toxicity Studies of Isobutyl Nitrite. GAWORSKI, C. L., ARANYI, C., HALL, A., III, LEVINE, B. S., JACKSON, C. D., AND ABDO, K. M. (1992). Z&dam. Appl. Toxicol. 19, 169-175. Isobutyl nitrite (IBN) is a volatile liquid that hasbecomeincreasinglypopular asan inhaled recreationaldrug. To investigate short-term toxic effects and establishexposure parametersfor chronic inhalation studies,F344/N rats and B6C3FI mice were exposedto IBN vaporson a 6 hr/day + t90,5 days/weekschedule. Twelve exposureswere administeredat concentrationsof 0, 100, 200,400,600, and 800 ppm IBN. This exposure seriesresulted in mortality in rats exposedto a600 ppm and mice exposedto 800 ppm. Animals exposed at the lower concentrations developed hyperplasia of the bronchiolar and nasalturbinate epithelium (rats and mice) and lymphocytic atrophy in the spleenand thymus (mice). Longer term, 13-week, subchronic exposures wereconducted at concentrations of 0, 10,25,75, 150, and 300 ppm IBN. Exposure to 300 ppm IBN reduced the body weight gainsin both sexesof rats and in female mice. IBN-related clinical pathology changesincluded reduced RBC counts accompanied by moderateincreasesin meancorpuscular volume and reticulocyte counts,increasedWBC counts,and mildly increased methemoglobin concentration. Bone marrow hyperplasia was observedin all groups of IBN-exposed rats, while in mice only females at 2150 ppm IBN displayed this change. Excessive splenicpulp hematopoiesiswasnoted in miceat all IBN exposure levels. Respiratory system changes included increased lung weights in rats and female mice at 300 ppm, hyperplasia of the nasal mucosa(male rats at 275 ppm and female rats at 2150 ppm), and hyperplasiaof the lung epithelium (male miceat 3150 ppm and female mice at 275 ppm). The results suggestedthat a concentration of 150ppm could he usedasthe highestexposure level for subsequentchronic inhalation tea.. Q 1992Society of Toxicology.
Isobutyl nitrite (IBN) is a volatile organic nitrite with chemical properties similar to amyl and butyl nitrite. Volatile nitrites are often included in certain commercially ’ Presented at the 28th Annual Meeting of the Society of Toxicology, Atlanta, GA, February 1989. ’ Present address: Lorillard Tobacco Co., 420 English Street. P. 0. Box 2 1688, Greensboro, NC 27420. 3 To whom correspondence should be addressed.
available over-the-counter “room odorize? preparations and have become substances of abuse used to alter consciousness,stimulate dancing, and intensify the sexual experience (Sigell et al., 1978). The principle pharmacologic action of the volatile nitrites is relaxation of the involuntary muscles,including the blood vessels.Although previous use of IBN was primarily by male homosexuals, direct inhalation of IBN vapor hasbecome increasingly popular among high school recreational drug users seeking the euphoric effects resulting from vasodilation of the cerebral blood vessels(Israelstam et al., 1978; Jaffee et al., 1983; Schwartz and Page, 1986). Toxic effects of IBN vapor exposure may include dizziness, headache, nausea,flushing of the neck, and decreasedblood pressure (Haley, 1980; Lowry, 1980; Newell et al., 1984). Ingestion of IBN may be lethal, while vapor inhalation has been reported to cause significant methemoglobinemia and severe tracheobronchitis (Covala et al., 1981; Dixon et al., 1981; Guss et al., 1985). Animal studiesconducted with mice exposed for 7 hr/day for 60 days to 400 ppm IBN resulted in 20% mortality, decreasedweight gain for the first half of the exposure period, decreasedliver microsomal glucosedphosphatase activity, and increased kidney, liver, lung, and spleenweights (McFadden and Maickel, 1985). Longer-term studies using mice exposed to IBN at concentrations up to 300 ppm for aslong as 18 weeks produced decreasedthymus and liver weight, decreasedwhite blood ceil counts, elevated methemoglobin concentrations, and mild focal hyperplasia and vacuolization of bronchial and bronchiolar epithelium (Lynch et al., 1985). IBN is a direct mutagen in the Ames assay and has a chemical structure that may allow reaction with biological amines to form n-nitroso compounds (Quinto, 1980; Newell et al., 1984; Osterloh and Goldfield, 1984). In vitro immunotoxicity studies with IBN have shown suppressionof human leukocyte proliferative responseto mitogens and cellmediated cytotoxicity reactions (Hersh et al., 1983). In vivo exposure in mice resulted in a significant depression of endogenous splenic and peripheral natural killer cell cytotoxicity potential (Lotzova et al., 1984). No apparent detrimental effect of lymphocytic proliferative response,delayed hypersensitivity response, or relative numbers of T-cells and T-
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0272-0590192 $5.00 Copyright Q 1992 by the Society of Toxicology. All rights of reproduction in any form reserved.
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cell subsets was observed in mice following inhalation of 300 ppm IBN for up to 18 weeks (Lewis et al., 1985). Since the use of aliphatic nitrites, other than amyl nitrite, is not strictly regulated, large quantities of these compounds are readily available. The increasing popularity of IBN as a drug and its potential for immunotoxic and/or carcinogenic activity enhance the need for information concerning the long-term toxic effects. The studies reported here were conducted to select dose levels for 2-year chronic investigations and were designed to identify target organs, differences in sensitivity between sexes, and dose-response relationships. METHODS Test Material IBN (molecular formula C,HgN02) is a clear yellow liquid with a boiling point of 67°C. Identification and purity (>97%) were established by combined infrared/ultraviolet, nuclear magnetic resonance, and gas chromatography techniques. IBN is a labile material, hydrolyzing slowly in the presence of water vapor to isobutanol and nitrous acid. The latter is a very reactive and unstable material at ambient temperature in the presence of oxidizing and reducing agents. The test article was stored under a nitrogen headspace in sealed, amber glass containers at -20°C. Prior to use, the chemical was warmed to room temperature and gently shaken to mix the contents. Test Atmosphere Generation IBN vapor atmospheres were generated using glass gas sampling bubblers filled with liquid IBN. The bubbler reservoirs were thermally regulated and continuously supplied with liquid IBN through a fine metering pump. A controlled flow of nitrogen carrier gas was passed through f&ted gas inlets, submerged in the reservoirs to disperse the gas through the liquid IBN and effect an efficient vaporization. The resulting vapor-laden carrier streams were directed through heated stainless steel tubing to the inlet Venturi attachments on the inhalation chambers where the vapors were mixed at high velocity and turbulence with filtered air to produce the required exposure concentrations. The carrier gas was controlled by fine metering valves and monitored with gas rotameters. Chamber concentration adjustments were made by altering the carrier gas flow rates, which directly varied the amount of vapor delivered to the chambers. For the lowest IBN chamber concentration a dilution air slip stream was installed at the gas outlet of the bubbler to dynamically remove a controlled portion of the vapor/carrier gas mixture prior to introduction to the chamber inlet. IBN in the generator reservoirs of the high and low concentration chambers was analyzed by gas chromatographic peak comparison for isobutanol changes in the liquid phase at the beginning and the end of a 6-hr exposure period. On the basis of these results a fresh sample of IBN was used each day in the test atmosphere generator reservoirs. Test ‘4tmosphere Monitoring Zsobutyl nitrite and isobutanol. IBN test atmosphere concentrations and concentrations of isobutanol were determined by either a Hewlett-Packard Model 5880 (Hewlett-Packard, Norwalk, CT) or a Perkin-Elmer Sigma 300 (Perkin-Elmer, Norwalk, CT) gas chromatograph (GC) equipped with a flame ionization detector. Both GCs were operated at oven temperatures of 50°C and injector and detector temperatures of 200°C using 1.8 m X 4mm i.d. glass columns packed with 20% SP-2 100/O. 1% Carbowax 1500 on 100/120 mesh Supelcoport and nitrogen as the carrier gas. All chamber atmospheres were monitored at least once per hour during each exposure period by sampling from a representative port near the breathing zone of the animals with a gas-tight syringe. The gas chromatograph was regularly calibrated by injecting certified gas standards of IBN and isobutanol. The
ET AL. gas standards were checked against liquid standards prepared from the IBN test article and analytical grade isobutanol, respectively. Single concentration point verifications of the calibrations were made daily. The time to reach 90% of the target concentration in the inhalation chamber (80) was established during prestudy investigations and was approximately 10 min for each chamber. Nitrous acid. Levels of nitrous acid were analyzed monthly in the high and low concentration IBN chambers. Samples of chamber air were passed through glass midget impingers containing 1% KOH, and the nitrite ion content was determined by high-pressure liquid chromatography (Waters Model 6000A HPLC Pumps, Waters Chromatography Division, Milford, MA) using a reverse-phase PBondapak Cl8 column and Waters Pit A (low uv) reagent diluted to half strength as the eluent. The nitrite ion was monitored at 2 14 nm using a Schoeffel variable wavelength Model 870 ultraviolet detector (Schoeffel/McPherson, Action, MA). Aerosol production. The high concentration chamber was sampled in the beginning of the study with a quartz crystal microbalance-based cascade impactor (QCM, California Measurements Inc., Sierra Madre, CA) to ensure that IBN was delivered to the chamber as a molecular vapor and not as an aerosol. Animals and Animal Care Male and female F344/N rats and B6C3FI mice, approximately 6 to 7 weeks of age at exposure initiation, were obtained from Simonson Laboratories (Gilroy, CA). Animals were maintained in stainless steel wire-mesh cages in 2-m3 inhalation chambers (Lab Products, Inc., Maywood, NJ), with a 2-week quarantine period prior to initiation of the exposures. Cage units were rotated on a weekly basis within the chambers. Exposure chambers were maintained at 75 + 3°F and 55 f 15% relative humidity, with air flows of 15 f 2 changes per hour. NIH-07 open formula diet (Zeigler Bros., Inc., Gardners, PA) was available ad libitum, except during exposure periods. Filtered city of Chicago drinking water was supplied ad lib&urn via automatic watering systems.A 12-hr light/dark cycle (6 AM to 6 PM light) was provided. At terminal necropsy of the subchronic study, serum samples were obtained from five rats and five mice of each sex housed in the control chamber for analysis of antibody titers (rats: KRV, H-l, CARB, Sendai, PVM, RCV/ SDA; mice: K, Poly., MHV, Ectro., GDVII, M. Ad., Reo3, LCM, MVM, CARB Sendai and PVM). All titers were negative for the agents screened. Experimental Design ll-Day repeated dose study. Groups of five animals/sex/species were exposed at target concentrations of 100, 200, 400, 600, and 800 ppm IBN on a 6 hr/day + t90, 5 day/week basis, for a total of I2 exposures. Three or 4 consecutive exposures were given immediately prior to scheduled termination for rats and mice, respectively. A control group of five animals/sex/ species was exposed to filtered air. 13-Week subchronic study. Groups of 10 animals/sex/species were exposed at target concentrations of 10,25,75, 150, and 300 ppm IBN on a 6 hr/day + t90, 5 day/week basis, for 13 weeks (rats) or 14 weeks (mice). At least two consecutive exposures were given immediately prior to scheduled termination. A control group of 10 animals/sex/species was exposed to filtered air. Observation and body weight. Animals were observed twice each day for mortality/moribundity, with clinical observations performed daily during the 14-day study and weekly during the I3-week subchronic study. Individual animal body weights were measured at exposure initiation, weekly thereafter, and at necropsy. Clinical pathology. Blood samples were collected at the terminal necropsy of the 13-week subchronic study via the retroorbital sinus from nonfasted animals anesthetized with 70% COz. Because. of the limited blood sample available from the mice, each group was randomly divided in half, and the samples were designated for either hematology or clinical chemistry evaluation. Hematology parameters examined included: hemoglobin and hematocrit; leucocyte count and differentials; erythrocyte count and indices;
ISOBUTYL
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TABLE 1 1CDay Inhalation Toxicity Study of Isobutyl Nitrite: Summary of Results Mortality IBN Target Concn (ppm)
Body weights
Male
Female
Male
Female
O/5” 015 O/5 O/5 515
015 015 O/5 115
Ob 3 -2 -26*
0 -2 1 -13*
515 515
e e
c c
Target organs
Rat 0 100 200 400 600 800 Mouse 0 100
200 400 600 800
515 015
015
0
0
015 015 015 015 315
015 015 015 015 415
-3 3 2 -14* -2o*
-3 -1 -5 -16* -2ld
Lung Lung, nasal epithelium, liver Lung, nasal epithelium. liver Liver Liver -
Lung Lung, Lung, Lung, Lung,
spleen spleen, thymus, liver spleen, thymus, liver, nasal epithelium spleen, thymus, nasal epithelium
’ Number of mortalities/number initially on test. b Values represent the percentage difference from control at termination. ’ Termination body weights not available. d Too few animals for statistical comparison. * Significantly different from control group (p G 0.05) by Dunnett’s t test.
reticulocyte count; platelet evaluation; Howell-Jolly body and Heinz body examination; and methemoglobin determination. Clinical chemistry tests included alanine aminotransferase, alkaline phosphatase, and total bile acids. Hematologic determinations were performed with a Baker 9000 hematology analyzer, and the clinical chemistry tests were performed with a Baker Centrifichem 500 automated analyzer (Serono-Baker, Allentown, PA). Necropsy, organ weights, and histopathology. Animals were anesthetized with COz and killed by exanguination by severing the axillary vessels.All animals received a complete necropsy and were examined for gross lesions. Brain, heart, right kidney, lungs, liver, right testes, and thymus weights were measured on all animals surviving at terminal necropsy. The following tissues were collected for histopathologic examination: gross lesions and tissue masses,lymph nodes (bronchial, mediastinal, mandibular, and mesenteric), mammary gland with adjacent skin, thigh muscle, salivary gland, femur including marrow, rib (costochondral junction), nasal cavity, and turbinates, tongue, larynx, pharynx, and trachea, right lung and mainstem bronchi, heart and aorta, thymus, thyroids, parathyroids, esophagus, stomach, large and small intestine, liver, gall bladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, preputial or clitoral glands, prostate, testes, epididymides, seminal vesicles, scrotal sac, vagina, ovaries, uterus, brain and pituitary, spinal cord, sciatic nerve, eyes, and Zymbol’s glands. After weighing, lungs were perfused with 10% neutral-buffered formalin using a syringe equipped with a blunt-tipped needle (20 gauge for rats and 23 gauge for mice). The lungs were perfused to approximate normal inspiratory volume (I to 2 ml in mice and 4 to 8 ml in rats), with the trachea being ligated with fine suture as the needle was withdrawn. Following removal of the soft tissues from the head, the lower jaw was removed and the nasal cavity was slowly flushed with 10% neutral-buffered formalin via the nasopharyngeal duct. After fixation the head was decalcified with dilute nitric acid. Three sections of the nasal cavity were prepared (1, level of incisorposterior edge; 2, midway between incisors and first molar; 3, middle of second molar). All tissues were fixed in 10% neutral-buffered formalin, trimmed, embedded, sectioned, and stained with hematoxylin and eosin. The lung slices were prepared from whole mount specimens, which included an entire co-
ronal (perpendicular to a sagittal plane and parallel to the long axis of the body) section of all lung lobes, including the primary bronchus and other major airways. A complete histopathologic evaluation inclusive of gross lesions was conducted on all control animals and all animals in the highest concentration group with at least 60% survivors at the time of necropsy, plus all animals in higher dose groups. Tissues demonstrating chemically related lesions (target organs) were identified, and these organs plus gross lesions were examined in lower doses until a no-observed chemical effect level was determined. Statistics
Organ weights, calculated organ weight/body weight ratios, and quantitative clinical pathology data were analyzed by one-way (by sex) analysis of variance (ANOVA) followed by a Dunnett’s t test when a significant F ratio was obtained (RS/Explore software, version 1.I, Serial No. V-658, BBN RS/ Expert Limited Partnership, BBN Software Products Corp., Cambridge, MA). The level of significance wasp d 0.05.
RESULTS 14-Day Repeated-Dose Study
All rats exposed to 600 or 800 ppm IBN died during the initial exposure period, with one female rat exposed to 400 ppm IBN euthanized in a moribund condition following the firsL 6-hr exposure (Table 1). Hepatocellular cytoplasmic vacuolization was noted in these animals, with multiple round red areas on the pleural surface of the lung observed at necropsy and lung hemorrhage seen microscopically. Rats exposed to 200 or 400 ppm IBN were lethargic and displayed rough coats and hunched posture. Decreased body weight gains occurred in rats exposed to 400 ppm IBN, with reduced
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thymus weights seen at terminal necropsy. Rats surviving the full exposure series had minimal to mild hyperplasia of the bronchiolar epithelium, minimal hyperplasia of the epithelium of the nasal turbinates, and hepatocellular cytoplasmic vacuolization. Mortalities in mice occurred only at the 800 ppm IBN exposure level, with decreased body weight gains at exposure concentrations of 2600 ppm (Table 1). Rough coats, hunched posture, and transient lethargy were noted in mice exposed at concentrations of 3400 ppm. Organ weight changes attributed to IBN exposure included increased lung weight and decreased thymus weight. Lymphocytic atrophy manifested by reduced size of splenic lymphoid follicles and thymic cortical atrophy was seen with increased severity at the higher exposure levels. Other, less significant lesions developing in mice exposed to IBN included epithelial hyperplasia of the bronchioles, peribronchiolar inflammation, hepatocellular cytoplasmic vacuolization, and alteration in the epithelium lining the nasal cavity. 13- Week Subchronic Study Chamber atmosphere analysis. The daily average IBN exposure concentrations and overall study means were all within 10% of the targeted values, with relative standard deviations also within 10%. Analyses of generator reservoir contents demonstrated a significant increase in isobutanol level during the course of a daily exposure period. However, despite this increase, the isobutanol concentration relative to IBN in the chamber atmospheres sampled during the exposures was ~3.1%. Only the 300, 150, and 75 ppm IBN chambers could be analyzed for isobutanol, since the levels in the 10 and 25 ppm chambers were below the 1.0 ppm detection limit of the gas chromatograph method. This demonstrated that the test atmospheres were not significantly affected by the isobutanol increase in the generator reservoirs when the generators were filled with fresh IBN at the beginning of each exposure day. Mean nitrous acid levels, calculated as volume/volume concentrations for direct comparison with other test atmosphere components, averaged 0.056 ppm at the 10 ppm IBN target concentration and 0.11 ppm at the 300 ppm IBN level. These concentrations were approximately two to three orders of magnitude lower than the concentrations of IBN in the chambers at either of the sampled target concentrations, and thus considered negligible. Chamber spatial homogeneities calculated as percentages relative to the mean target IBN concentration, were within 5%. Chamber atmosphere samples tested for aerosol formation were repeatedly at, or below, the limit of background sensitivity of the QCM instrument (5 pg/m3), demonstrating that aerosol was not formed in the generation process. Clinical observations and body weights. There were no mortalities in rats during the subchronic study. Ruffled fur was seen in rats exposed to the higher concentrations of IBN.
ET
AL.
Additionally, during the first 6 weeks of the study, the female rats were often noted to be hyperactive following the daily IBN exposure. These signs were primarily evident at the 300 ppm exposure level, but were occasionally noted in the group exposed to 150 ppm. Rats exposed to 300 ppm IBN had decreased body weight gains compared to controls, in which weight gains were slightly more pronounced in males than in females (Table 2). The body weight gains of rats exposed to 150 ppm IBN, or less, were not greatly affected by exposure, with the relative percentage gains of these groups within 10% of the controls. Mortalities in the mice were limited to the females exposed to either 150 or 300 ppm IBN (one at each level). Clinical observations noted in the mice during the study were considered incidental to IBN exposure. The body weight gain of male mice was not adversely affected by exposure to IBN; however, a dose-related trend toward decreased body weight gain was evident in the female mice (Table 2). Clinical pathology. Selected clinical pathology parameters are shown in Table 3. The principle hematologic change noted in animals exposed to IBN for 13 weeks was a decrease in red blood cell counts, which was generally accompanied by a moderate increase in the mean corpuscular volume and an increase in reticulocyte population. Methemoglobin concentration was mildly increased in animals exposed to the higher IBN concentrations. Although the data are not presented, exposed animals tended to have a non-dose-related increase in the number of Howell-Jolly bodies, but no significant elevation in Heinz body formation. WBC counts were increased in animals exposed to 300 ppm. Differential counts indicated an increased number of lymphocytes in these groups. No substantive changes were noted in hematocrit percentage or hemoglobin concentration, and no significant changes were noted in the clinical chemistry parameters measured (data not shown). Necropsy/organ weights. Gross necropsy observations recorded in rats and mice at necropsy appeared to be incidental findings occurring without any dose-response relationship. Increased lung/body weight ratios were noted in both sexes of rats, and female mice exposed to 300 ppm IBN (male rats + 17%, female rats + 18%, and female mice +36%). Weight changes noted in the other organs examined in rats or mice were considered to be incidental to exposure. Histopatholog!,. The most significant histopathologic changes seen in rats exposed to IBN were hematopoietic hyperplasia of bone marrow and hyperplasia of the anterior nasal mucosa (Table 4). Bone marrow hematopoietic hyperplasia appeared as an increased amount of normal hematopoietic tissue in the epiphyseal marrow of the distal femur at the expense of adipose tissue found there. The mixture of the two types of marrow was not uniform, with zones of marrow containing hematopoietic or adipose tissue seen. The cellular hematopoietic elements appeared identical in normal and hyperplastic marrow. Diaphyseal hematopoietic
ISOBUTYL
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TABLE 2 13-Week Inhalation Study of Isobutyl Nitrite: Body Weight Changes in Rats and Mice IBN target concn (ppm) Group
0
10
25
75
150
300
Male rats Initial wt. (g) Final wt. (g) Relative percentage gainb
118.0 345.0 -
115.6 339.1 -1.5
118.4 365.4 +8.8
113.0 329.3 -4.1
116.3 327.5 -7.0
116.9 296.1 -21.1
Female rats Initial wt. (g) Final wt. (g) Relative percentage gain
92.0 194.2 -
90.0 196.5 +4.2
90.2 199.8 +7.2
88.7 193.9 +2.9
88.7 187.2 -3.6
87.8 179.0 -10.8
Male mice Initial wt. (g) Final wt. (g) Relative percentage gain
23.9 34.8 -
23.1 35.1 +4.6
23.9 35.3 +4.6
23.2 35.0 +8.3
23.5 35.6 +11.0
23.2 34.6 +4.6
Female mice Initial wt. (g) Final wt. (g) Relative percentage gain
19.9 33.7 -
20.2 32.9 -8.0
19.9 32.8 -6.5
19.6 32.0 -10.1
19.8 31.6 -14.5
18.2 28.4 -26.1
a Values are the mean body weights of 9 to 10 animals/group. Wt. change (exposed) - Wt. change (control) x loo, b Relative percentage gain = Wt. change (control)
marrow constituents were variable and difficult to assess. Unfortunately, the epiphysis was occasionally trimmed away at necropsy, and these sections were not counted as examined even though abundant hematopoietic tissue was seen in the diaphyses. The incidence of this change was dose-related in
13-Week Inhalation
both sexes, with a modest increase in the average degree of severity as the exposure concentration increased for females. Nasal mucosa hyperplasia was characterized by an increase in the thickness (number of cells) of the simple cuboidal nasal mucosa in the anterior region of the nose. The change
TABLE 3 Toxicity Study of Isobutyl Nitrite: Selected Clinical Hematology Results in Rats and Mice Male
IBN target concn (ppm)
RBC (X 106/pl)
MCV (fl)
Reticulocyte (X 1O’/rU
Female WBC (X 10’//4
Methemoglobin (%I
RBC (X l@/d)
MCV UN
Reticulocyte (X 1O’/PU
WBC w 10’//.4
Methemoglobin (%)
Rat 0 10 25 75 150 300 Mouse 0 10 25 75 150 300
8.20 8.16 8.25 7.76 7.84 7.83
f f + f + +
0.29” 0.32 0.45 0.49* 0.17 0.12
9.28 f 0.27 9.00 k 0.83 9.08 + 0.48 8.73 f 0.51 8.83 f 0.21 8.13+0.52*
52 52 51 52 54 58
f 2 f 1 f I zk I ?c0* + l*
48 50 48 48 48 51
f 1 f 4 f 1 f 1 + 1 +2
13.8 14.7 20.0 17.8 34.3 74.6
f 2 + + + f
10.7 7.8 15.7 8.1 28.9 7.9 9.4 9.1 23.8 9.6 40.5* 10.4
3.7 2 5.0 0 +o 0 *o 0 &O 0 LO 8.7 + 12.4
f f f f + +
1.6 2.2 1.3 1.4 1.4 1.4*
1.5 + 1.9 f 1.7 + 2.3 + 2.6 + 2.6 f
1.4 0.6 0.5 0.5 0.8* 1.4*
8.01 1.62 7.14 1.26 7.18 1.24
+ 0.76 rt 0.46 Ik 0.53 + 0.49* f 0.27* 2 O.SO*
54+ 54k 55+ 57 f 51+ 61 +
1 9.3 f 5.7 9.0 i 1.7 8.8 + 2.1 1 8.4 z!z 6.7 1 10.8 f 11.8 9.9 * 1.4 2* 7.4 f 5.2* 10.0 * I.0 1* 9.3 * 9.4 10.4 i 2.4 1* 24.8 f 13.l* 12.1 F 1.9*
1.O zk 0.6 1.7 f 0.4 2.0 f 1.7 1.3 + 1.3 1.7 + 1.2 2.3 f 1.4
2.6 f 0.3 2.2 f 0.5 3.0 ‘- 0.9 4.1 + 0.8 4.1 It 1.2 5.6 zk 2.1*
1.8 + 1.8 f 1.4 + 2.1 f 2.3 + 4.5 f
0.4 0.6 0.6 0.8 0.3 0.8*
9.79 9.63 9.28 9.56 9.38 8.42
f 0.27 + 0.79 ?z 0.56 f 0.25 + 0.63 + 0.21*
47 f 48 f 47 + 48 f 48 + 50*
0 0 +o 2 1.8 + 4.0 1 1.7 f 3.8 I 2.0 f 4.4 1 2.6 f 5.1 1* 13.6 f 14.1
1.7 + 0.6 2.4 2 0.1 2.3 +- 0.1 2.2 + 0.4 3.2 + 1.0* 4.6 -c 0.7*
a Values represent the mean f SD of 9 to 10 rats or 3 to 4 mice/group. * Significantly different from control group (p d 0.05) by Dunnett’s t test.
4.0 f 5.5 f 5.5 + 4.1 f 3.5 + 7.1 +
0.9 1.3 0.4 0.9 0.8 1.2*
174
GAWORSKI
ET AL.
TABLE 4 13-Week Inhalation Toxicity Study of Isobutyl Nitrite: Summary of Significant Histopathology Observed in Rats and Mice
Changes
IBN target concn (ppm) Males Species Rat Mouse
Tissue/lesion
0
Bone marrow hyperplasia Nasal mucosa hyperplasia
1/v o/10
Lung hyperplasia Nasal mucosa hyperplasia Spleen hematopoiesis Bone marrow hyperplasia
O/IO l/10 l/10 o/10
Females
25
15
150
300
0
10
25
75
150
300
4/l
b
317 o/10
719 2110
415 IO/10
W
O/8 o/10
3/g b
l/8
IO/IO
3/10 O/IO
719 7110
IO/IO
b
o/10
3110
b
b
3110
4110
o/10 b l/IO
b
3110 b
O/IO o/10 l/10
9/10
b
lO/lO 6/lO lO/lO
lO/lO
b
9110 o/10 9/10
5110
b
3/10
6110
9110
b
b
O/8
b
b
O/8
l/8
10
b
O/IO
b
w IO/10 l/l0 9/10 619
a Values represent number with lesions/number of tissues examined. b Not examined.
was most pronounced at the edges of the nasal turbinates and on the lateral walls of the nasal cavity. A solitary focus of pulmonary adenomatosis was seen in one male rat exposed to 300 ppm IBN. This lesion is uncommon, especially in young rats. The most striking lesion seen in mice exposed to IBN was a hyperplastic piling up and occasional dysplasia of the lining epithelium (mucosa) of the bronchial and bronchiolar tree (Table 4). The lesion was most pronounced in the bronchioles, and the severity was generally dose-related. Other, less significant, respiratory system changes included minimal hyperplastic changes in the mucosal epithelium of the anterior nasal cavity in male mice in the 300 ppm IBN group and a modest non-dose-related increase in fresh lung hemorrhage in female mice exposed to IBN. Extensive hematopoiesis was seen in the red pulp of the spleen in both sexes of mice exposed to IBN (Table 4). This change was generally more severe in the animals exposed to the higher IBN concentrations. Hyperplasia of hematopoietic elements of bone marrow was seen in females exposed to 150 or 300 ppm IBN. The lesion was diagnosed by using the absence of subepiphyseal adipose fatty marrow as the criteria. The average degree of severity was minimal. Male mice characteristically lack subepiphyseal adipose cells. DISCUSSION IBN exposure levels and use patterns experienced by humans are difficult to quantify, but could reasonably be expected to range from limited acute incidents to longer-term repeated exposures. It is estimated that actual use may vary widely from as little as 0.2 ml for the individual recreational user to the spraying of larger quantities over the dance floor of discotheques (Sigell et al., 1978). Acute inhalation of IBN at high vapor concentrations may be lethal, as demonstrated by the mortality in animals receiving a single 6-hr exposure
to 600 or 800 ppm IBN at initiation of the 14-day study. This effect occurred in a concentration range that is consistent with the reported 1-hr LC50 value of approximately 1000 ppm (McFadden et al., 198 1). Repeated exposure of rats and mice for a longer period of time (13 weeks) at reduced IBN concentrations failed to cause significant mortality, but did produce retarded weight gain, hematopoietic system alterations, and hyperplasia of the epithelium lining of the respiratory system. Hyperactivity was seen in the female rats following the first few daily exposures of the subchronic study. This response appears to be consistent with the known stimulating effects of IBN in humans, and the selective presence of this response in the female rats suggests a greater sensitivity of this sex to IBN stimulation. Interestingly, the incidence of hyperactivity diminished during the later half of the exposure period, suggesting the development of tolerance. Tolerance to amyl nitrite has previously been reported after repeated use by humans (Crandall et al., 193 1). Significantly increased methemoglobin concentration is a characteristic of nitrite exposure and has previously been noted in laboratory animals and in humans following exposure to IBN (Dixon et al., 1981; McFadden et al., 1981; Guss et al., 1985; Lynch et al., 1985). Although the methemoglobin levels determined in the animals exposed to IBN in the present study were somewhat lower than might be expected from previous reports, they were generally elevated over controls. The low methemoglobin levels in exposed animals were attributed to the delay between exposure termination and blood sampling ( 16 to 19 hr), which undoubtedly allowed sufficient time for methemoglobin reduction to occur. Mild anemia in IBN-exposed animals, characterized by a non-dose-related decrease in RBC counts, was presumably due to the decreased erythrocyte lifetime resulting from continuously increased methemoglobin. Typical compensatory responses including reticulocytosis and macrocytosis were noted in both species. Additionally, microscopic evaluation
ISOBUTYL
NITRITE
of bone marrow indicated an increase in the amount of normal hematopoietic tissue in the epiphyseal tissue in both species tested, as well as extensive hematopoiesis in the spleens of female mice exposed to IBN. Although WBC counts were significantly increased in both species exposed to 300 ppm IBN, no significant histopathologic changes were identified in the lymphoid tissue of either species. Microscopic examination of the nasal, bronchial, and pulmonary tissues obtained from rats and mice following exposure to IBN indicated changes consisting primarily of hyperplasia of the mucosal or epithelial cells in various areas of the respiratory system. Tissue changes of this type are often associated with irritant gasses, and the presence of hyperplasia in the bronchioli indicates that IBN is not totally removed in the nasal region and may penetrate into the deep lung. Lung weights were also increased in both species exposed at the higher IBN concentrations during the subchronic study. These effects are consistent with previous reports of lung weight increase and respiratory epithelial hyperplasia following IBN exposure (Covala et al., 198 1; McFadden et al., 198 1; Lynch et al., 1985). Alveolar cell hyperplasia has been suggested as a possible step preceding the development of bronchiolar neoplasia by promoters or weak carcinogens (Mohr and Dungworth, 1988), and the presence of a solitary focus of pulmonary adenomatosis in one male rat exposed at 300 ppm is notable, since this lesion is not commonly observed in younger rats. In conclusion, the results of the 13-week inhalation study with F344/N rats and B6C3Fl mice indicated the respiratory tract, bone marrow, and spleen as potential targets of IBN toxicity. Changes in the bone marrow and spleen appeared to be secondary effects associated with increased destruction of red blood cells. The results suggested that a concentration of 150 ppm could be used as the highest exposure level for subsequent chronic inhalation studies. ACKNOWLEDGMENTS This study was supported by funds from the National Toxicology Program, National Institute of Environmental Health Sciences, under Contract No. NOS-ES-65143. The authors are indebted to Ms. J. N. Bradof, Ms. M. A. Cahill, Dr. J. B. Harder, Mr. R. W. Lange, Mr. J. Raymond, Mr. P. B. Senese, and Mr. W. O’Shea for their excellent technical assistance.
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INHALATION
TOXICITY
175
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