Acute and repetitive human exposure to isobutane by

S'l.'EWART, R. D., HERRM.A.NN', A. A., EARE'l'TA, E. n:, FORSTER, H. V .. SIKORA, :r. J., NEWTON', P. E. :;md SOTO, R. J. Acute repetjtive human exposure to isobutane. ScaTtd. j. wo.-k emliron. & health 3 (1977) 234-.243. Eight adult volunteers of both sexe!' -wero: exposed. to isobutanc in a controlled-environment !!hambet" for the purpose ot mcmitorlnR their physiological responses to a :.;C!I'Ies of gas concenlratton5 rangi~_lnun..251l....iu...l100.Lp.pm. First, the response to exposure periodol of 1 min, 1ffiiil, 1 h, 2 h, and B h wer~. studied. There being no untoward re5ponses to these acute··exposur·e~;-·tne···erglif_v_ofunJcei·s were exposed repetitively to isobutane at conci!ntrutionl! of 500 ppm, 1, ~ or B h per day, five duys per week for two weeks. Then exposures to two m1~W.t:~_!; ___ ~! j_sol;JUtariE: and prcr~nrrrctc\"t-r;-:nn··· s·h pc1· day for two (iayli werestudied. During the lrivesHgaUon all subjects were kept under comprehensive medical sur"eillance. No untoward "ubjective re&ponses or abnormal physiological responses occurred during or following these exposures. Special emphasis was placed on evllluating the cardiac :mel pulmonary response to these exposures through the use o! continuol.lS ECG t£lcmetry and serial computerized spirometric measurements. The !ollowing serial ll'lboratory studie!l were unaltered by the exposures: complete blood count, urinalysis, serum alkal.il'lc phosphatase, SGOT, LDH, serum bilirubin, blood sugar, serum calcium, serum p)'losphorus, BUN', spontaneous electroencephalogram, vitiual evolc;ed response, a battery of cognitive tests, and an ACTH stimulation t~r.

The increased use of isobutane and propane as substitutes for fluorocarbons in aerosol produ-cts raises the question of the potential <lf these gases to injure humans.The abuse of fluorocarbon propellants and organic oolvents to obtain a "high" has resulted in the sudden death of approximately 300 American teenagers, presumably because of epinephrine sensitization of the heart and the development of fatal cardiac arrhythmia (2, 3,6,7,8,9,12,15,24).However, few persons were concerned with the possible health hazard to consumers in normal situations until after Zuskin and Boyhuys suggested that aerosol propellants might be responsible f-or the transient increase in airway rP.sista•nce observed after the use o! hair sprays (25).Any remaining complacency was shattered by Speizer, Wegman, and Ramir•ez, who reported that brief exposures to fluorocarbon-22 in the 300 ppm ra.nge resulted in the development of severe palpitation in pathology residents in Boston and suggested that exposure to "normal-use'' concentrations of certain aerosol propellants might pose health problems not previ~;>usly recognized (16).

Th~: from~
The paucity of information regarding the human inhalation toxicology of isobutane at concentral.ions encountered both ir.home-use situations and in the industrial setting prompted this inve~tigation.

EXPERIMENTAL PROCEDURE
Eight healthy volunteers of both sexes were exposed to isobutane or a mixture of isobufane and propane in concentrations ranging from those encou.ntered in the home environme-nt to those: permitted in the industrial setting (1).First, a series of single exposures to 250, 500, and 1,000 ppm fot periods from 1 min to 8 h were conducted in a controlled-environment chamber (19).There being no untoward health effects, the subjects were e;><posed repetitively to 500 ppm of isobutane five days per week for two weeks.Then exposures to two mixtures of isobutane and propane :for 1, 2, or 8 h per d01y for two days were studied.These• experiments were designed so that the absorption, excretion and physic~ logical effect of isobutane inhalation could be studied.Special emphasis was placed on the monitoring of cardiac and pulmonary performance.The exposure schedule is presented in table 1.
The investigation was performed with strict adherence to the ethical and technical requirements for hurnan inhalation experimentation previously detailed (17,22), and an informed col:'lsent was obtained from each subject after the nature of the procedure had been fully explained.

Subject$
The subjects were selected from the Caucasian, middleclass, college student population.Each subject who completed the study received $ 2.50 per hour spent at the laboratory.
The ages of the four male subjects ranged from 20 to 22 years, height from 178 to 187 em, a.nd weight from 70.0 to 81.5 kg.None of them was obese.Two of these subjects were assigned to group I (8h exposure), one to group II (2-h e,:posure), and one to group III (1-h exposure).
The ages of the four females ranged from 20 to 21 years, height .from155 to 174 em, and uteight !rom 57.2 to 72.9 kg.The --------division of the subjects into groups was identical to that for the study with male subjects.
All su'pjects were cautioned to abstain from the usc of drugs and to limit their use of alcohol to very moderate amounts.Subjects vrho were smokers were not allowed to smoke during their .stay in the controlled-environment chamber.Subjects who underwent behavioral testing (2 h and 8 h) were asked to refrain from consuming any caffeine prior to the end of each day's study {1-h poste:l!Cposure).

Exposnn schedule
Table 1 lists the e:;.c:posure sequence, the number of subjects, the gas ('oncen'traUon • investigated, and the duration of each exposure.

Zxposu.re chamber
The e;x:periments were conducled in a controlled-environment chamber.It had a 6 x 6 x .2.4 m testing room with an attached shielded room and an attached toilet facility.The air flow through the suite of rooms to the exhaust was appro:leimately 42 m~/min, which created a slight negative pressure within the chamber.The ambient temperature within the chamber was maintained at 22-23°C, while the relative humidity ranged between 45-55 °/n.The propellant gas was mixed with the air supplying the chamber, entering through four dif~usors in the ceiling o! the testing room.For the desired concentration, the gas was metered from a ('ylinder into the return 01ir duct of the air conditioner.

Analysis of exposure chambeT atmosphere
The gases used in these exp~riments were purchased from the Phillips Petroleum Company.The isobutane had a boiling point o! -11.73'~C, a vapor pressure o:r 3,733 mm Hg (about 37.7°C), a vapor density of 2.068 (about 15.5"C and 60 mm Hg), and a specific gravity of 0.563 (about 15.5/ 15.5 •q. Th::: propane had a boiling point of -42.49;C, a vapor pre::;sure of 6,612 mrn Hg, a vapor density of 1.549, and a specific gravity of 0.509 (15.5/l5.5"C).

Number of subjects
...     Two independP.ntsystems were used to monitor the chamber atmosphere.In both cases, air was withdrawn :from the chamber through a polyethylene tube (inner diameter 0.64 em) at approximately 7 1/ min, through or past the analytical device, to a small diaphragm pump that discharged back into the chamber.
The concentration of the gases in the chamber atmosphere was recorded continuously by a Wilks MIRAN-I in!rared spectrometer equipped with a 20-m pathlength gas cell.The absorbance at 3.4 fL was measured.The voltage output was connected to a strip-chart.recorder, and a voltage proportional to the pen position of that r~corder ,.ras conducted to the analog-to-digital input of a PDP-12 (DEC) computer.The computer sampled the pen position voltage each ~econd, averaged those volt~ges every 30 s, recorded the averagE: on magnetic tape, and used the average to write on a cai:hode ray tube the concentration over that 30-s interval and the cumulative or time-weighted average concentration since the beginning of the exposure session.
Gas chromatography was the second and the most sensitive tnetbod of chamber air analysis employed.A Varian Aerograph Se:rie:5 2700 gas chromatograph was equipped with a column packed with Porapak Q operated at 87'•c.Nitrogen was used a~; the carrier gas to a hydrogen flame detector operated at 1B5°C.An automatic device injected a .sample of air into th£ gas chromatograph every 170 s.Output of For the standards, saran bags were filled with room air pumped in sequence through a charcoal column, wet test meter, a Drierit~ column, and a type N all-service gas rnask canister.After a bag was iilled with a known amount o! clean, dry ai:r, a known volume of i:sobutane and/or propane was injected into the bag, For the calibration o! the analytical devices the saran bag standard was attached to the sarnpling probe within the chamber.At least three standards were analyzed before subjects were allowed to enter the chamber each day, and then standards were analyzed at approximately 1-h intervals throughout the day.

Clinical testing
All e,cposures with a duration o.r: one or more hours were conducted according to a double-blind format. •~ Prior to commencing the actual exposures, the subjects underwent a training program in the cont.rolled-E:nvironmentchamber; during this time they became ac~ customed to the chamber setting and the testing procedures.
The subjects were given a repeat physical examination prior to each exposure.At this time each of them completed a "~ymp tom checklist."This form had designated spaces tot noting the presence of headache; nau~ea; dizziness; abdominal pain; eye nose, throat irritation; or other subjective symptoms.Each .subjectreviewed this list o:£ symptoms immedi•ately upon entering the chamber and each hour during and for 5 h !allowing each e:xpo!'ure.The adjectives "mlld, moderate, and strong" appeared on the sheet as cue words, and the phrase "only abnormalities recorded" was prominently t.yped at the bottom.The home telephone numbers of each of the department physicians appeared on the form, and the subjects were encouraged to phone if they became ill while away from the la-boratory.
Prior to a:nd following the exposu.re:s,the following laboratory determinations were made: complete blood count, urinalysis, alkaline phosphatase, SGOT, LDH, bilirubin, blood sugar, calcium, phos~ phorus, :SUN.Elood and alveolar breath sam•ples were collected for hydrocarbon gas analysis.The following studies completed the preexposure evaluation: computerized spirometry, 12-lead ECG, ~nd a modified V::, ECG rhythm strip by telemetry.
After entering the environmental chamber, the subjects were under continual visual surveillance by medical personnel and all important chamber activities were videotaped by closed circuit TV.The subjects immediately performed a modi!ied Romberg test followed by a heel-to-toe test.These tests were :first performed with the eyes open and then repeated with the eyes closed.Then, each subject completed his subjective symptom checklist as previously discussed.Fi"e minute~ prior to exiting from the exposure chamber, each subject: repeated the modified Romberg test and the heel-to-toe test.Lead V.; telemetry hard copy was obtained irom each subject after 30 min of exposure and hourly thereafter.An additional telemetry 236 strip was obtained each time an ECG change was observed.
During the final 40 min of exposure subjects exposed for 2 and B h performed the following: computerized spirometry measurements, maximum midexpiratory flow rate, the Flimagan.coordination test, the Flanagan arithmetic test, the Marquette time estimation test (21), and the random number inspection test.Dur~ng the repetitive ~tudies these tests were performed twice a week during the final 2 h of exposure.
Spontaneous electroencephalograms (EEG) and visual evoked responses {VER) were recorded four times each Monday, Wednesday, and Friday of the two-month p1uiod of exposure.All recordings were obtained while the subjects were seated in a comfortable upholstered chair in the shielded room in which the hydrocarbon concentrations were identical to those in the co-ntrolled-environment chamber (11,23).The time required to perform the EEG and VER precluded studying more than four subjects per day.
Alveolar breath samples were obtained daily from each subject prior to entry into lhe environmental chamber, and serially following each exposure.These samples were each collected in 5-l saran bags with the technique pn:viously described in detail (HI, 19).
Blood samples for hyrocarbon analysis were obtai-ned.:from an antecubital vein of each subject before exposure, 15 min before exit from the chamber, and 15 min after exposure in Vacutainer IJj) tubes with edetic acid anticoagulant.The preexit sample was obtained from the exposed subject's arm after it had been stuck through an armport in the chamber wall into the uncontaminated adjacent laboratory.

Electroencephalography
Gold-plated silver disk electrodes were oriented on the scalp according to thE: 10-20 International Electrode System (13).The pastefilled disk electrode at the inion was cemented with collodion to the scalp to prevent shifting.An eight channel Grass polygraph fitted with EEG amplifiers was utilized for recording.EEG !g) 006/011 •ivit:y -w-as recordt ac..
Visual evoked 1"esponse A. complete description and illustration of the EEG-VER monitoring system ca-n bE round in a previous publication (11).The VER was recorded from the electrode at the inioD referred to the left ear.An EEG channel was used to amplify the VER, and the output was fed to an on-line averaging computer (Nicolet, 1074).The VER was triggered by a strobe flash (3 l's) at the rate of 1 per second for 128 s.Tbe strobe was operated to deliver 18 million beam candles at 1m from the subject's eyes, which were closed throughout the period o.t strobe fla$hing.Analysis time was 400 ms.The flash delay from t.he synchronizing pulse which initiated \he computex-sweep was 50 ms.The computer averaged the response to the 128 flashes, and the resultant VER was recorded on an X-Y plotter for analysis.
It has been shown that VER amplitude might be altered by varying levels of atte-ntion, cortical desynchroniza tion, and sleep (4,10,14).Accordingly, standardized conditions were used throughout each exposu1•e day and immediately preceding the actual recordings.I\ rigid schedule for food intake, physical activity, and additional testing procedures was followe-d.ln addition, alter entering the booth, the subject was always allowed 3-5 min to achieve a relaxed state; and then imrnedi•ately prior to initiating the strobe flash, the subject was asked to clap his hands live times slowly and forcibly to insure a wakeful, attentive state.
The most prominent repruducible portions of the VER complex are the third, fourth, and fifth waves (designation by Gastaut) (5,10,11).The analysis was thus restricted to thrae waves.Wave 3 was ide-ntified as proceeding in a positive direction B0-120 ms after the initiation of the strobe flash.Waves 4 and 5 were the succeeding negative and positive segments of the VE:R.Analysis involved: (a) measuring the amplitude of these waves and (b) measuring whether cha-nges had occurred in the lateney and wave form of the VER complex.

AdrenocoTtical function
Upon the completion of the repetitive exposures the subjects underwent the standard 2-day ACTH stimulation test :for the assessme.nto! the ability oi the adrenal gland to respond to stress.The reason for this testing was the observation that the organ with the highest hydrocarbon concentration following e:l(posure is the adrenal cortex.
Analysis of ambient aiT, e:rpiTed bTea.th and blood Air and breath samples for hydrocarbon analysis were injected directly onto a Porapak Q column of a Varian Aerograph Series 2.700 gas chromatograph equipped, with a hydrogen flame ionization detector.The column and operating conditions were; r:olumn; 45.72 cm X 0.32 em 0.0.stainless steel; column packing: Porapak Q, So-BO mesh; oven tempera~ ture: 87 ~ C; injector temperature: 150° C; detector temperature; 1B5L' C; carrier gas; nitrogen, 20 rnl/min.
Air standards were prepared by direct injection of the appropriate quantity of isobutane or propane into a saran bag containing a measured volume of air.
A headsp-ace sampling technique was utilized for measuring the concentration oi the hyr;iroca rbons in blood.Blood standards were prepared by the direct ad-dition of an appropriate quantity of an air sta::J~ dard containing 1,000 ppm propane or isobutanc to a Vacutainer tube containing a known quantity of blood.The freshly drawn anticoagulated blood sample was allowed to equilibrate with the hydrocarbon by agitation.The headspace sample was injected directly into a gas chromatograph lor analysis.The technique was far superior to the sol"ent extraction methods used previously.• -----------------•  ------------e,.:posurephysical examialations and clinical laboratory determinations were ob• tained <:~ccOJ:ding to the schedule previously detailed.
All subjects were placed under dose medical surveillance following each exposure.A resting 12-le<Jd ECG was obtained 15-30 min after e:~<posure.All of the pre~ exposure clinical studies were repeated on a weekly basis, and the subjects were kept under surveillance for one year follo..,.ring these exposures.
:Following the last exposure of the sequence, each sul:Jject wa$ given a comprehensive medical examination including a complete history and physical examination WJth the following laboratory studies; complete blood count, urinalysis, complete panel of clinical chemistries (23 values plus 2 calculated), and a 12-Jead ECG.Then the health of each subject was monitored for one year by the investigators.

RESULTS
Analysis of e;;r;posure chamber a~Tnosphere The daUy time-weighted :average concentrations of isobutane or propane in the controlled-environment chamber for each of the exposure conditions are found in t.able L The actual concentrations were within :a few per c:ent o! those desired.

Medical sun;eillance
No untoward subjective symptoms or ol;ljective signs oi illness were noted dur-ing exposure or in the surv-eilla•nce period which followed each exposure.Pre-and postexposure comprehensive me-dical examination:> revealed that all subjects remained in good health during the study (20) ..l\.11 of the clinical hematologies &nd chemistries remained within the limits of normal (20).When a Iaborat.orymeasurement varied mo:re than two standard deviations from the normal mea•n value (5 ~;. of all measurements), a repeat determination was obtained.to confirm that a significant change had not occurred.
Isobutane was present in the blood and expired breath of the subjects during and following exposure (20).The isobu tane blood levels a-re listed in table 2. The isobutane breath data have been presented elsewhere (20).

Effects ot exposure on the heart
None of the subjects experienced any untoward signs o-r symptoms referable to the heart during exposure or in the post• expol!ure period of surveillance.No change from the preexposure control ECG traci:ng was observed i'n the postexposure .5tanda.rd 12-lead ECGs or in the modified lead V 5 monitored continuously by telemetry during the different e;~Cposure:;, None of the subjects developed arrhythmia.

Pulmonary function studies
The functional integrity of the pulmonary airways, as monitored by the pulmonary function tests, did not appear to be af.rected by either the acute or the repetitive series of exposures (20).A summary of the spirometric data is listed in table 3 (20).Time constraints precluded the obtaining of a com~ plete EEG, and thus limited th2 value of these data.

Visuot evoked. re~;po11ose
During the final wE:ek of • e:xposure a definite reduction jn the amplitudes of the 3, 4, and 5 waves was observed.The significance of this observation is uncertain and merits further investigation.(20).The mean test performances under control and e-"posure conditions vrere plotted for each control and e;ll:posure day.Then a linear regression line with 75 11 /u confidence limits "-'a:S drawn through the 0 ppm data.Arter adjustment for the tr!l!nd through the 0 ppm data, t tests were performed to determine if the e;l(pcsure data were significantly different from the regression line.
On two occasions arithmetic scores were higher than anticipated, but in the absence of a consistent change in test performance or a dose•related response, these results are interpreted as showing no effect of exposure.

DISCUSSION
Acute e;:ocposures to isobutane in concentrations of 250, 500, or 1,000 ppm for peri-ods of 1 min to B h did not produce any untoward physiological effect as mcni tared by the method:; employed.Repetitive exposures to jsobutane 500 ppm.for 1, 2 or B h, Iive days a week for ten exposures .,verealso without any measurable untoward physiolcg•ical dfect.Mixtures oi the two gases in the concentrations investigated exerted no untoward physiological effects.
Of particular importance wa~ the obsrn•• vation that none: of the subjects showed any decrement in pulmonary function or nlteration in cardiac rhythm as the result of exposure to concentrotions of the gases permitted in the occupational setting, c:oncentrations generalty higher and oi much greater duration than would occur during the normal use of aerosol products in the home.Thus it would seem that short-term exposure to isubutane not exceeding the threshold limit value for American in• dustry does not have the potential to affect normal hearl or lungs adversely.
The reduction in the VER wave amplitude recorded during the second week of repetitive exposure to isobutane is trouble• some becal,lse this type of reduction can be 24.2 due to central n-ervous depression and has been observed prior to the: de-veloprnen l of overt signs of neurological impairment (23).This Iinding merits further investigation.
The analysis of expired breath or blood for isobutane in the early postexposure period provides a feasible diagnostic test of exposure.The use of gas chromatography permits the detection of isobutane in expired breath for at least 5 h after two ur more hours of e,.-:posure.ACKNOWLEDGMENTS This investigat.ion was .supported in part from funds collected by the Ad Hac Aerosol Committee, representatives of major trade associations and industrial companies with interests in aerosol safety.
The expertise of Michael J. Haske, Ph.D., Department of Pharmacology, and Gregory J. Harrington, M.D., Department of Neurology, in reviewing and interpreting the electroencephalograms and visu~:~l evoked response measurements is gratefully acknowledged.The authors thank the members of the task force of the Inter-Industry Aerosol Safety Committee fer their many helpful suggestions and their review of the manuscript.
Medical surveillance'the subjects were ui'Ider continuous visual surveillance by a physician and by nursing personnel during each e:Kposure.The pre-239

Table 1 .
-Exposure o:r human 5ubjects to isobutane and propane.