Acute Exposure Guideline Levels
PREFACE
Under the authority of the Federal Advisory Committee Act (FACA) P.L.92-463 of 1972, the National Advisory Committee for Acute ExposureGuideline Levels for Hazardous Substances (NAC/AEGL Committee) has beenestablished to identify, review, and interpret relevant toxicologic andother scientific data and develop AEGLs for high-priority, acutely toxicchemicals.
AEGLs represent threshold exposure limits for the general public and areapplicable to emergency exposure periods ranging from 10 minutes (min)to 8 hours (h). Three levels—AEGL-1, AEGL-2, andAEGL-3—are developed for each of five exposure periods (10 and 30min and 1, 4, and 8 h) and are distinguished by varying degrees ofseverity of toxic effects. The three AEGLs are defined as follows:
AEGL-1 is the airborne concentration (expressed as parts per million ormilligrams per cubic meter [ppm or mg/m3]) of a substanceabove which it is predicted that the general population, includingsusceptible individuals, could experience notable discomfort,irritation, or certain asymptomatic, nonsensory effects. However, theeffects are not disabling and are transient and reversible uponcessation of exposure.
AEGL-2 is the airborne concentration (expressed as ppm ormg/m3) of a substance above which it is predicted thatthe general population, including susceptible individuals, couldexperience irreversible or other serious, long-lasting adverse healtheffects or an impaired ability to escape.
AEGL-3 is the airborne concentration (expressed as ppm ormg/m3) of a substance above which it is predicted thatthe general population, including susceptible individuals, couldexperience life-threatening health effects or death.
Airborne concentrations below the AEGL-1 represent exposureconcentrations that could produce mild and progressively increasing buttransient and nondisabling odor, taste, and sensory irritation orcertain asymptomatic, nonsensory effects. With increasing airborneconcentrations above each AEGL, there is a progressive increase in thelikelihood of occurrence and the severity of effects described for eachcorresponding AEGL. Although the AEGL values represent thresholdconcentrations for the general public, including susceptiblesubpopulations, such as infants, children, the elderly, persons withasthma, and those with other illnesses, it is recognized thatindividuals, subject to idiosyncratic responses, could experience theeffects described at concentrations below the corresponding AEGL.
SUMMARY
Tear gas is a white crystalline powder with a pepper-like odor. It wasfirst synthesized by Corson and Stoughton in 1928 and is, thus,abbreviated as CS (Corson andStoughton 1928; USArmy Chemical School 2005). CS was developed in the 1950s asa replacement for the chemical incapacitant, 1-chloroacetophenone (CN),because CS was a much more potent irritant than CN, but wassignificantly less toxic (WHO1970; Colgrave andCreasey 1975; Hu etal. 1989). It was adopted for use by the military, and waswidely used in the Vietnam War (WHO 1970; Hu et al.1989). It is currently used as an incapacitating agent bothby military and law enforcement personnel (HSDB 2005). Upshall (1973) reported that an aerosolconcentration of CS at 4 mg/m3 will disperse the majority ofrioters within 1 min, and at 10 mg/m3 will deter trainedtroops. With the exception of more severe cutaneous reactions, recoveryfrom exposure is generally rapid upon exposure to fresh air, generallywithin 30 min after exposure (Ballantyne 1977). CS may be manufactured through carbonylcondensation by combining o-chlorobenzaldehyde andmalononitrile (HSDB 2005).Recent production data on CS were not found.
Human studies did not identify a no-effect level for CS or effects of CSthat would be consistent with the definition of AEGL-1. The severity ofthe effects observed at the lowest tested concentrations in humans(ocular stinging and watering, and nasal, throat, and mouth irritation)exceeded those defined by AEGL-1. Therefore, AEGL-1 values for CS arenot recommended. AEGL-2 values were based on human exposure to CS at anaverage concentration of 0.75 mg/m3 for 60 min (Beswick et al. 1972). Allfive subjects tolerated the exposure, but reported ocular stinging andwatering, increased salivation, cough, and face stinging. Some subjectsalso reported throat irritation (4 subjects), nasal stinging and running(3 subjects), mouth stinging (2 subjects), chest burning (2 subjects),nausea (2 subjects), and headache (2 subjects). An intraspeciesuncertainty factor of 3 was applied because contact irritation is aportal-of-entry effect and is not expected to vary widely amongindividuals. Furthermore, the responses of volunteers with jaundice,hepatitis, or peptic ulcer or who were 50-60 years old were similar tothose of “normal” volunteers when exposed at a highlyirritating concentration of CS for short durations. The ability totolerate CS at 14-73 mg/m3 and the recovery time involunteers with a history of drug allergies, seasonal allergies, asthma,or drug sensitivity were similar to normal volunteers; although moresevere chest symptoms were reported in the people with pre-existingconditions (Gutentag et al.1960; Punte et al.1963). An interspecies uncertainty factor of 1 was appliedbecause the study was conducted in humans. A modifying factor of 3 wasalso used because the effects observed at 0.75 mg/m3 wereconsidered AEGL-2 effects. Time scaling was not performed becauseirritation is a function of direct contact with CS and is unlikely toincrease with duration of exposure at this level of severity (NRC 2001).
AEGL-3 values were based on calculated lethality thresholds for CS ateach exposure duration. Rat data from the studies by McNamara et al. (1969),Ballantyne and Callaway(1972), and Ballantyneand Swanston (1978) were used calculate LC01(lethal concentrations, 1% lethality) values for CS. Calculations wereperformed using the probit analysis-based dose-response program of ten Berge (2006). Timescaling was performed using the equation Cn × t = k,where the exponent n ranges from 0.8 to 3.5 (ten Berge et al. 1986). An empirical valuefor n of 0.70 was determined on the basis of the rat data. The 4-hAEGL-3 value was adopted as the 8-h AEGL-3 value because time scalingyielded an 8-h value inconsistent with the AEGL-2 values, which werederived from robust human data. A total uncertainty factor of 10 wasapplied. A factor of 3 was used to account for interspecies differences,because clinical signs are likely caused by a direct chemical effect onthe tissues and this type of portal-ofentry effect is unlikely to varygreatly between species. Furthermore, calculated LCt50 values fordifferent species were all well within a factor of 2 of each other(88,480 mg-min/m3 for rats, 67,200 mg-min/m3 forguinea pigs, 54,090 mgmin/m3 for rabbits, and 50,010mg-min/m3 for mice) (Ballantyne and Swanston 1978). Anuncertainty factor of 3 was used to account for intraindividualvariability because contact irritation is a portal-of-entry effect andis not expected to vary widely among individuals. As noted above insupport of the AEGL-2 values, a factor of 3 is also supported by theresults of studies by Punte etal. (1963) and Gutentag et al. (1960) in subjects with pre-existingconditions.
AEGL values for CS are presented in Table 7-1.
TABLE 7-1
AEGL Values for Tear Gas.
1. INTRODUCTION
CS is a white crystalline powder with a pepper-like odor. It was firstsynthesized by Corson and Stoughton in 1928 (thus, the abbreviation CS)(Corson and Stoughton1928; US Army ChemicalSchool 2005). It was developed in the 1950s as a replacementfor the chemical incapacitant, 1-chloroacetophenone (CN), because CS wasa much more potent irritant than CN, but was significantly less toxic(WHO 1970; Colgrave and Creasey 1975;Hu et al. 1989). CSwas adopted for use by the military, and was widely used during theVietnam War (WHO 1970;Hu et al. 1989; Smith and Greaves 2002). Itis currently used as an incapacitating agent by military and lawenforcement personnel (HSDB2005). It is reported that an aerosol concentration of 4mg/m3 will disperse the majority of rioters within 1 min,and 10 mg/m3 will deter trained troops (Upshall 1973). With the exception of moresevere cutaneous reactions, recovery from exposure is generally rapidupon exposure to fresh air, usually within 30 min after exposure (Ballantyne 1977).
Because CS is stable when heated and has a low vapor pressure, itrequires a means of dispersem*nt (Blain 2003). Different forms ofdispersem*nt include the combination of CS with a pyrotechnic compoundin a grenade or canister, generating a smoke or fog, and dispersem*nt ofa fine powder as an aerosol (WHO1970; Smith andGreaves 2002). CS1 is a micronized powder formulation of CScontaining 5% silica gel for dissemination by an explosive burst ordusting apparatus, and CS2 is the same as CS1 except that the CS1 ismicroencapsulated with silicone to improve its weather resistance andflow properties (WHO1970).
In controlled studies investigating the toxicologic properties of CSaerosol, CS was disseminated as a 2-10% solution in methylene chlorideor acetone by means of a pneumatic atomizing nozzle assembly (Gutentag et al. 1960; Owens and Punte 1963; Punte et al. 1963) or bythermal dispersion by spraying the molten chemical (Gutentag et al. 1960; Punte et al. 1962, 1963).
CS may be manufactured through carbonyl condensation by combiningo-chlorobenzaldehyde and malononitrile (HSDB 2005). Recentproduction data of CS were not available.
Hydrolysis of CS produces malononitrile ando-chlorobenzaldehyde (NTP 1990). Hydrolysis of CS is relativelyrapid, with a half-life of about 15 min at a pH 7, but CS reacts fasterwith an alkaline solution, having a half-life of about 1 min at a pH of9 (Blain 2003).
CS has a vapor pressure of 3.4 × 10-5 mm Hg; thus, atconcentrations greater than 0.35 mg/m3, it will exist invapor and aerosol forms. CS in the vapor phase will be degraded byreaction with photochemically produced hydroxyl radicals, with anestimated half-life of 110 h. CS in the particulate phase will beremoved by wet and dry deposition.
The chemical and physical properties of CS are presented in Table 7-2.
TABLE 7-2
Chemical and Physical Properties of Tear Gas.
2. HUMAN TOXICITY DATA
2.1. Acute Lethality
No human acute lethality data on CS were found.
2.2. Nonlethal Acute Toxicity
2.2.1. Experimental Studies
In a review article, Blain (2003) reported a TC50 (concentrationthat caused a perceptible effect on 50% of the populationexposed for 1 min) of 0.004 mg/m3 for ocularirritation and 0.023 mg/m3 for airway irritation. AnICT50 (concentration intolerable to 50% of the populationexposed for 1 min) was also reported. No further details werepresented.
A group of male volunteers was exposed to CS aerosol with a massmedian diameter (MMD) of 0.9 microns (94 ± 15mg/m3; 4% larger than 10 microns) or up to 60microns (85 ± 16 mg/m3; 4% smaller than 20microns) to assess differences in ocular and respiratoryresponses to different particle sizes of CS (Owens and Punte1963). Six volunteers who had the best ability totolerate CS were chosen from a group of approximately 50.Subjects wore tightly fitted goggles and a nose and mouthrespirator designed to protect against particle sizes less thanone micron, and were exposed individually in a wind tunnel witha constant air speed of 5 mph. The exposure protocol wasdesigned to restrict exposure to either the small or largeparticles to the eyes, to the respiratory system, or to both theeyes and respiratory system. The wind tunnel was elevated to aheight of 5 feet, and a rubber-lined port was installed in thebottom of the duct enabling the subject to insert his head intothe airstream of the tunnel and remove it quickly after theexposure. CS was disseminated from a 2% solution in methylenechloride by means of a pneumatic atomizing nozzle assembly. CSconcentrations were determined from air samples collected usingfilter paper placed on air sampling probes located around thehead area (one on top and one on each side at eye level),followed by extraction with ethanol and measurement withultraviolet spectrophotometry. A modified cascade impactor wasused to measure the CS aerosol containing the small particles,while the larger particles were sized microscopically, measuringand counting the various particles in the pre-ground materialbefore dissemination. Tolerance time was defined as the time atwhich a subject could no longer remain in the atmospherecontaining the compound and left the exposure chamber, andrecovery time was defined as the time after the exposure whenthe subjects were able to sort and arrange a series of 24playing cards from which the corner numbers were removed.Control values were determined before each test. The resultsindicate that small particles are more effective in rapidlyproducing ocular irritation (see Table 7-3). The onset of ocularresponse is hypothesized to be faster with small particlesbecause of they are more soluble than larger particles in ocularfluid. Once begun, however, the irritation process wouldcontinue for a longer period with the large particles comparedwith the small particles. Respiratory effects were more severewith small particles (no volunteers could withstand exposure formore than 30 seconds [sec]) and required more time for recoverythan the large particles. The difference in response is due tothe ability of smaller-sized particles to penetrate more deeplyinto the respiratory tract. When both the eyes and therespiratory system were exposed to CS, the respiratory responsepredominated with exposure to the small particles, whereas theocular response predominated with exposure to the largeparticles.
TABLE 7-3
Tolerance and Recovery Time in Humans Exposed to TearGas Particles (1-60 microns).
A group of 4-6 volunteers was exposed to CS aerosol in a windtunnel (8 × 8 × 8 feet; fixed wind speed of 5 mph)(Gutentag et al.1960; Punte etal. 1963). Volunteers were both military and civilianpersonnel. Each volunteer’s medical history was recorded,and each was given pre-exposure and post-exposure physicalexaminations. Volunteers were classified as“normal” or were placed in one of four specialcategories: those with hypertension (diastolic pressure of80-110 mm Hg or normal blood pressure reading with a history ofhypertension; pre-exposure tests included electrocardiogram,chest X-ray, liver function, and urinalysis); those with hayfever, drug sensitivity, or bronchial asthma (volunteers withasthma had normal chest X-ray before exposure); those with ahistory of jaundice, hepatitis, or peptic ulcers withoutgastrointestinal bleeding; and those that were 50-60 years ofa*ge. Subjects classified as normal were further categorized intountrained men with or without protective masks or trained menwith or without protective masks. The trained men had previousexposure to CS, whereas the untrained men did not. CS wasdispersed as a 10% solution in acetone or methylene dichloridewith a spray nozzle (MMD 3.0 or 1.0 micron, respectively) or bythermal dispersion (spraying the molten chemical; MMD 0.5micron). Airborne samples of the aerosol were collected atvarious points in the wind tunnel. Particle size wascharacterized using a 6-stage modified cascade impactor, andexposure concentrations were measured using ultravioletspectrophotometry. The subjects did not report any noticeabledifference in symptoms from the different dispersionmethods.
Groups of 3-6 untrained men without masks were exposed to CS inacetone, and tolerance times were recorded. Times ranged from 53to >120 sec at 5 mg/m3, 19-43 sec at 12mg/m3, and up to 5 sec at 442 mg/m3.When groups of 1-7 trained men were exposed, tolerance timesranged from 37 to >120 sec at 4 mg/m3, 18-41sec at 10 mg/m3, and up to 12-25 sec at 141mg/m3. To compare the effects of hyperventilationon symptoms, untrained subjects ran for approximately 100 yardsbefore exposure. Exercising subjects could not tolerate CS aswell as normally breathing subjects; groups of three subjectsexposed at 10, 13, or 39 mg/m3 could tolerate CS forup to 13, 13, and 9 sec, respectively. While ocular irritationwas minimal, chest symptoms were more pronounced and recoverytime was slightly prolonged (by 1-2 min). The reactions ofsubjects with jaundice, hepatitis, or peptic ulcer or those thatwere 50-60 years old were similar to those of normal subjects.Subjects with a history of drug allergies or sensitivities, hayfever, or asthma also tolerated exposure to CS at concentrationscomparable to those tolerated by normal subjects, but the groupwith pre-existing conditions had a higher percentage ofindividuals with more severe chest symptoms, with many of themlaying prostrate on the ground for several minutes. However, nowheezing or rhonchi were heard, and recovery was as rapid asthat seen in other exposure groups. When subjects were exposedto CS at temperatures ranging from 0-95°F, tolerance tothe chemical was slightly reduced at the high temperature of95°F. Whether the decrease in tolerance was an actualeffect of the exposure, the uncomfortable climate, or acombination of both was unclear. An increase in skin-burningsymptoms with increased temperature was attributed to anincrease in perspiration.
As part of the study described above, the potential fordeveloping tolerance to CS was investigated by exposing a groupof four subjects to CS at 1.5 mg/m3 for 90 min in a20,000-L chamber (Punteet al. 1963). No data were provided about themonitoring of the CS aerosol. During exposure, subjects wereallowed to smoke, read, and play cards. Only one subjectreported nasal irritation (after 2 min), three subjects reportedheadaches (after 45, 50, and 83 min), and all four subjectsreported ocular irritation (after 20, 24, 70, and 75 min). Inthe second part of the experiment, the four subjects wereexposed to CS at 1.5 mg/m3 for 40 min, and thenadditional CS aerosol was added to the chamber to achieve anairborne concentration of 11 mg/m3 in about 10 min.Although the subjects had not been told of the increase inconcentration, they all left within 2 min due to respiratoryirritation. The exposure concentration was estimated to be4.3-6.7 mg/m3 when the subjects left the chamber. Inthe third part of the experiment, the subjects were exposed toCS at 6 mg/m3, which was attained over 10 min.Symptoms reported by the subjects included nasal and throatirritation, chest burning, sneezing, ocular irritation andlacrimation, headache, and dermal irritation. Three of the foursubjects reported that the exposure was unbearable after 18, 20,and 29 min, with chest symptoms being the reason the subjectsleft the chamber. The remaining subject was able to tolerate theagent, and the exposure was terminated after 40 min. Theinvestigators attempted to enter the chamber without the benefitof the gradual increase in exposure concentration, and wereunable to remain in the chamber. In the fourth experiment, aconcentration of 6.6 mg/m3 was attained over 30 min.The usual signs and symptoms of CS exposure developed, but to alesser degree. One of the subjects had to leave after 2 minbecause of a violent cough, but he returned to the exposurechamber after his cough had ceased upon exposure to fresh air.He remained in the exposure chamber for the duration of the60-min exposure.
To assess the potential effect of CS exposure on ventilation,cardiac frequency, and breathing pattern, a group of 11 healthysoldier volunteers was exposed to CS aerosol (particle diameterof 1 micron) at a concentration that was progressively increasedfrom 0.2 mg/m3 to 1.3 mg/m3 (Cotes et al. 1972;Cole et al.1977). The exposure duration was not specified, butappeared to be approximately 80 min. CS aerosol was produced bysaturating the exposure chamber the evening before the exposure,followed by flushing with air to remove all of the gas exceptthat adsorbed onto the walls and equipment. During exposure,pyrotechnic generators were ignited to progressively raise theconcentration of CS throughout the exposure session. Subjectswore woolen or denim battle dress covered with cotton coveralls,boots, and gaiters. Electrocardiogram electrodes were applied tothe chest, and subjects wore a full respirator into the chamber.For the commencement of exposure, each subject removed his ownrespirator. During each exposure, each subject completed two8-min periods of exercise, which consisted of cycling at 20W upto 120W. During exercise, the subjects breathed through anoral-nasal mask and three-way valve box. Inspiration was fromthe chamber and expiration was through a 6-L capacity mixingbottle into a low resistance gas meter. Cardiac frequency wasmeasured by electrocardiograph, while a thermister in the valvebox recorded respiratory frequency. A control exposure includingexercise was conducted the day before and the day after exposureto CS. A major difference between the control and CS exposureswas that ventilation was continued throughout the controlsession but not the CS-exposure session; therefore, thetemperature was much higher during the CS-exposure sessions thanthe control session (~24° vs. 20.5°C forcontrols).
All subjects experienced intense discomfort, including cough,lacrimation, and substernal pain, when first exposed to the CSaerosol. Discomfort was severe enough that two subjects withdrew(one before and one after the first period of exercise), and twoadditional subjects were unable to complete the first period ofexercise due to coughing. Coughing coincided with ignition ofthe CS generators. The discomfort disappeared with continuingexposure. Although cardiac frequency increased during exposureto CS compared with control air, the difference was eliminatedwhen the cardiac frequency was corrected for the increasedambient temperature (corrected to the arbitrary temperature of20°C). The ventilation minute volume was reduced fromexposure to CS compared with controls. The reduction appeared tobe due to a decrease in respiratory frequency. The exposure wasrepeated using 17 volunteers (Cole et al. 1975, 1977). Exposureconditions were the same with the following exceptions: the CScandles were ignited between and not during periods of exercise,CS concentrations were slightly higher (0.92-2.15mg/m3), and the subjects were seen on fiveconsecutive half-day sessions (the first, third, and fifthsessions were for control observations and the other twosessions were allocated one each for exposure to ammonia and toCS [the order of exposure changed between the different weeks ofthe study]). Results were generally the same as those observedin the first study. The only difference was that the reductionin the ventilation minute volume was the result of a diminutionin tidal volume and occurred despite an increase in respiratoryfrequency.
To investigate the potential for developing tolerance to CS, 35healthy male volunteers were exposed for 60 min to increasingconcentrations of CS aerosol (Beswick et al. 1972). Exposures wereconducted in a 100-m3 chamber. The chamber wasgenerally saturated an hour before the exposure, followed by airbeing blown through the chamber to remove the CS not absorbed onthe walls and equipment. A number of parameters were assessedbefore and after exposure, including chest radiograph results,hematology and clinical-chemistry analysis, andrespiratory-function tests to assess peak flow, tidal volume,and vital capacity. A total of 10 exposure trials wereconducted, with no volunteers exposed more than once. Exposureconcentrations were kept relatively constant in the first threetrials: 0.53-0.86 mg/m3 in trial 1 (three subjects),0.71-0.78 mg/m3 in trial 2 (five subjects), and0.31-0.74 mg/m3 in trial 3 (six subjects). For theseven remaining trials, exposure concentrations were increasedby a factor of 2, 3, or 4 during the exposure period: 0.8-1.4mg/m3 in trial 4 (five subjects), 0.84-2.3mg/m3 in trial 5 (four subjects), 0.7-2mg/m3 in trial 6 (four subjects), 0.63-2.3mg/m3 in trial 7 (two subjects), 0.57-2.1mg/m3 in trial 8 (two subjects); 0.42-1.8mg/m3 in trial 9 (two subjects), and 0.45-1.7mg/m3 in trial 10 (two subjects). Chamberconcentrations were measured at 10-min intervals. Volunteersentered the chamber wearing full respirators and protectivecoveralls. CS was generated and allowed to mix for 3 min beforeremoval of the respirator. Symptoms from all volunteers werereported during individual interviews after exposure. Theresults of the 10 trials were consolidated into five groups:group I included trial 2 (five subjects), group II includedtrials 1 and 3 (nine subjects), group III included trial 4 (fivesubjects), group IV included trials 5 and 6 (eight subjects),and group V included trials 7, 8, 9, and 10 (eight subjects).One of the six subjects in trial 3 left the exposure chamberafter 8 min of exposure with complaints of severe stinging ofthe eyes, throat irritation, cough and dyspnea, salivation, andnausea, and one subject in group IV left after 55 min due tovomiting. All other subjects remained in the chamber for theentire 60-min exposure period. A summary of the symptoms of theexposed individuals is presented in Table 7-4. The predominant symptomsincluded excess production of mucus and saliva (34/34 subjects),ocular irritation (stinging in 32/34 subjects and lacrimation in32/34 subjects), runny nose (28/34 subjects), and face stinging(32/34 subjects). Symptoms generally resolved within 10 min ofleaving the chamber. Nausea was reported by 11/34 subjects andtwo vomited, which appeared to follow swallowing of largeamounts of saliva. The development of tolerance was assessed intwo of the trials (group IV in Table 7-4); in these trials, half ofthe subjects removed the respirator at the start of the exposure(with the CS concentration increasing with time), while theremaining subjects did not remove their respirators until thelast 5 min of exposure. The subjects that were exposed to CSthroughout the entire exposure period were able to withstand theentire 60-min exposure (concentrations increasing from 0.84 to2.30 mg/m3 and 0.70 to 2.00 mg/m3) exceptfor the one individual that had to leave the chamber at 55 minbecause of vomiting. Of the subjects that removed theirrespirators for the last 5 min of exposure, only one of eightsubjects could remain in the chamber for more than 1 min; fiveleft within 30 sec of removing their respirators. Noexposure-related changes were observed in hematology or clinicalchemistry parameters. Decreases in heart rate after exposureceased were ascribed to the sense of relief each volunteer feltat the end of an uncomfortable experience, and the increase insystolic blood pressure observed in individuals when exposurecommenced was due to the abrupt onset of discomfort; continuedexposure resulted in normal blood pressure readings. Noabnormalities were noted in measurements of respiratoryfunction, but the investigator noted that the sample size wassmall and, thus, may not be representative. It was concludedthat the main effects of CS are due to local irritation ofexposed nerve endings, and systemic changes noted are due tostress.
TABLE 7-4
Symptoms of Volunteers Exposed to Tear Gas for 60Minutes.
Three groups of volunteers were exposed to CS aerosol at variousconcentrations to investigate potential effects on visual acuity(Rengstorff1969). The first group was composed of 10 malevolunteers exposed to CS2 aerosol (CS treated withCab-o-Sil® 5 and hexamethyldisilaxane) atconcentrations of 0.1 to 1.7 mg/m3. The exposure wasconducted in a wind tunnel suspended 4.5 feet above the floor;the volunteer sat on a chair at the end of the wind tunnel andput his head through a rubber aperture in the tunnel until hecould no longer tolerate the exposure or for a maximum of 10min. A powder dispenser disseminated specific concentrations ofCS2 (MMD of 0.8 microns) into the air at a wind speed of 4.5mph. An Orthorater was used to measure the binocular far andnear visual acuity of the subjects before and after exposure.The second and third groups were exposed to CS aerosol in acircular steel chamber. CS aerosol (MMD of 0.9 micron) in amethylene dichloride solution was disseminated using a thermalgenerator, and introduced into the chamber as a uniform cloud.Subjects wore protective masks for the first 5 min in thechamber, and then removed their masks for the commencement ofexposure. The second group was composed of 34 volunteers, and anOrthorater was again used to measure the binocular far and nearvisual acuity before and after exposure. A summary of the amountof time volunteers from this group could tolerate exposure to CSis presented in Table7-5. The third exposure involved 22 volunteers whohad a baseline visual acuity of 20/20 and who could remain inthe exposure chamber for 10 min. Binocular acuity was measuredusing a Snellen visual acuity projector before, during, and afew minutes after exposure. The Snellen chart contained a row of20/30, 20/25, and 20/20 letters. No exposure-related changes invisual acuity were noted except those due to the inability ofsome subjects to keep their eyes open because of intense ocularirritation. Visual acuity returned to normal in all subjectsseveral minutes after exposure to CS ended.
TABLE 7-5
Tolerance of Humans Subjects Exposed to Tear Gas forUp to 10 Minutes in Study Evaluating VisualAcuity.
To assess the effect of CS exposure on respiration, a group ofsix volunteers (four with previous exposure to CS) were exposedto various concentrations of CS (3 micron) in a wind tunnelwhile a portable breathing device monitored respiration (Craig et al. 1960).Subjects remained in the tunnel until the exposure becameintolerable (see Table7-6). Notable coughing was observed in subjectsexposed at 15 mg/m3 for 61 sec or at 150mg/m3 for 12 sec. On the basis of the recordingsmade during exposure, it was concluded that although thebreathing pattern of the volunteers was disrupted, adequateventilation was maintained. Therefore, the incapacitation of CSis attributed to the unpleasant sensations of exposure ratherthan to any degree of respiratory failure.
TABLE 7-6
Tolerance of Human Subjects Exposed to Tear Gas inStudy Evaluating Respiratory Effects.
A group of 38 US Marines was exposed to a cloud of CS dispersedby a thermal canister as part of a training exercise to test theability and speed of the trainees in donning their gas masks(Thomas et al.2002). The exposure occurred after 6 days ofstrenuous training with minimal sleep and reduced foodconsumption, and was followed by a 1.5-mile run. Temperature andrelative humidity at the time of exposure were approximately24°C and 91%, respectively. Clinical signs and symptomsbegan to develop 36-84 h post-exposure during and after periodsof strenuous exercise (one subject became symptomatic after a1,000-meter pool swim at 36 h post-exposure; seven becamesymptomatic after a second swim consisting of a 1,000-meter openocean swim 60 h post-exposure; and one became symptomatic aftera third swimming event consisting of a 1,500-meter open oceanswim 84 h post-exposure). A total of nine subjects wereaffected, with four requiring admission into intensive care.Effects of exposure included dyspnea upon exertion, hemopytosis(ranging from frank blood to blood-tinged sputum), cough, rales,reduced arterial blood gas (range of 60-68), and infiltratesvisible on chest radiograph. Signs and symptoms resolved by 72h, and lung function before and after exercise challengereturned to normal within 1 week post-exposure. When theexposure in this study was recreated (without test subjects) andair sampling was performed, CS concentrations were found torange from less than quantifiable to approximately 17mg/m3.
McDonald and Mahon(2002) proposed that the pulmonary symptoms in theMarines described in the study by Thomas et al. (2002) were not theresult of CS exposure, but were rather the result of wateraspiration or swimming-induced pulmonary edema (SIPE). Theirconclusions were based on the observations that all subjectsbecame symptomatic immediately after swimming, that there was arapid resolution of symptoms, and that there was no evidence ofairway dysfunction. Delayed pulmonary effects of CS exposure areunusual, and there were no other reports of such symptoms eventhough approximately 200,000 Marines have been exposed to CSsince 1996 under similar field conditions.
2.2.2. Case Reports
The effects of exposure to CS are generally of an acute nature.However, reactive airways dysfunction syndrome was reported intwo individuals exposed to CS. One case involved a healthy21-year-old female exposed to CS smoke at a nightclub for 5-10min (Hu and Christiani1992). She exhibited the typical signs and symptomsof CS exposure, including tightness and burning in her chest andcoughing. Results of a physical examination and chestradiography were normal, and she was released from the hospital.She continued to experience coughing and shortness of breath,and had reduced measurements of forced expiratory volume in 1sec (68% of predicted) and forced vital capacity (78%) 4 weeksafter exposure. Cough and shortness of breath were still presentat the 2-year follow-up exam, and were made worse by exertion,cold air, and some environmental pollutants. The second casereport involved exposure to a riot-control agent containing 1%CS and 1% oleo resin capsicum (Roth and Franzblau 1996). A healthy53-year-old male was exposed for at least 30 sec, andimmediately experienced symptoms of mucous membrane irritation,cough, and chest tightness. Wheezing and shortness of breathcontinued for months after exposure, and were severe enough torequire hospitalization. Pulmonary function test resultsindicated reversible and fixed obstructive pulmonary disease.Effects of exposure to the capsicum cannot be excluded.
A 4-month-old infant exposed to CS for 2-3 h developedpneumonitis and persistent leukocytosis (Park and Giammona 1972). The infantwas exposed when a CS canister was fired into a house to subduean adult. Upon hospitalization, the infant had copious nasal andoral secretions and was sneezing and coughing. A chest X-raydemonstrated that the lungs were clear, but laboratory testingrevealed leukocytosis. The infant developed severe respiratorydistress by the second day of hospitalization, with pulmonaryinfiltrates evident on X-ray by day 7. Pulmonary infiltrationbegan to decrease on day 15, and the lungs were clear on day 17.White blood cell counts were elevated throughouthospitalization, and finally decreased when the infant wasdischarged from the hospital.
CS is a common riot-control agent in Britain; consequently,typical symptoms following exposure to CS have been describedfollowing its use in confined spaces, such as a night club(Breakell andBodiwala 1998) or bus (Karagama et al. 2003), use by policeon individuals for self-defense (Euripidou et al. 2004), or underconditions of large-scale riot control (Himsworth 1969; Anderson et al.1996). Symptoms of exposure included but were not limitedto ocular irritation, lacrimation, blurred vision, burningsensations sometimes accompanied by first degree burns, cough,headache, shortness of breath, chest pain, sore throat,retching, vomiting, and salivation (Himsworth 1969; Anderson et al.1996; Breakell andBodiwala 1998; Karagama et al. 2003; Euripidou et al. 2004). In general,the symptoms resolved rapidly; however, there were reports ofeffects lasting longer that that predicted. The hand-held spraycanisters used by police contain CS dissolved in methyl isobutylketone, an industrial solvent and denaturant (Gray 2000; Euripiouet al. 2004). It has, therefore, been proposed that the ketonecombined with the CS may result in longer lasting adverseeffects than CS preparations without the solvent.
2.3. Developmental and Reproductive Toxicity
The National Teratology Information Service collected outcome data on30 pregnant women who were exposed to CS gas: 12 women during thefirst trimester, 11 during the second trimester, and seven duringthe third trimester (McElhatton et al. 2004). Acute maternal toxicity(transient symptoms of ear, nasal, and throat irritation) wasreported by 50, 82, and 57% of the exposed women, respectively.Pregnancy outcome was not adversely affected by exposure to CS.Birth weight was within the normal range except for one female babyweighing less than 2,500 g. One infant had a congenital anomaly(hypospadia), and this anomaly has a background incidence of 1 in1,000 live born male infants. No concentration or duration exposureparameters were described.
2.4. Genotoxicity
No data on the genotoxicity of CS in humans were found.
2.5. Summary
CS is a potent irritant, with symptoms of exposure includinglacrimation, blepharospasm, erythema of the eyelids, chesttightness, coughing, nasal irritation and discharge, salivation,throat irritation, nausea, vomiting (from swallowing excess saliva),and cutaneous irritation (ranging from stinging to contactirritation or allergic dermatitis). Upshall (1973) reported that an aerosolconcentration of CS at 4 mg/m3 will disperse the majorityof rioters within 1 min, and 10 mg/m3 will deter trainedtroops. With the exception of more severe cutaneous reactions,recovery from exposure is generally rapid upon exposure to freshair, generally within 30 min after exposure (Ballantyne 1977).
Data on human tolerance to CS are summarized in Table 7-7. Many studiesinvestigated the amount of time that elapsed before subjects couldno longer remain in an atmosphere containing CS. Gutentag et al. (1960)and Punte et al.(1963) reported tolerances of 5 sec at 442mg/m3, 12-25 sec at 141 mg/m3, 9 sec at 39mg/m3, and more than 90 min at 1.5 mg/m3.Tolerance to low concentrations of CS could be increased whenexposure concentration was increased over time (Punte et al. 1963; Beswick et al. 1972). Astudy investigating the differences in respiratory and ocularresponses to different particles sizes of CS found that smallparticles are more effective than larger particles in producingocular and respiratory irritation. Recovery time for ocularirritation took longer for large particles, because the onset ofirritation was delayed due to the lower solubility of largeparticles. Recovery time for respiratory irritation took longer forsmall particles, because the smaller sized particles penetratedfurther into the respiratory tract (Owens and Punte 1963).
TABLE 7-7
Summary of Selected Human Toxicity Data on TearGas.
Pregnancy outcomes were not affected in a prospective case study of30 pregnant women who were exposed to CS gas and experiencedtransient symptoms of ear, nasal, and throat irritation (McElhatton et al. 2004).No other reproductive or developmental toxicity data of CS in humanswere available. No human data on the toxicity of repeated exposuresto CS or on the genotoxicity or carcinogenicity of CS werefound.
3. ANIMAL TOXICITY DATA
3.1. Acute Lethality
3.1.1. Monkeys
Groups of eight immature male and female Macacamulatta monkeys (3-4 kg) were exposed to a cloud ofCS dispersed via an M7A3 CS grenade in a 20,000-L chamber at anaverage CS concentration of 900 mg/m3 for 3 min,1,700 mg/m3 for 5 min, 2,850 mg/m3 for 10min, or 2,500 mg/m3 for 32 min (Striker et al. 1967). The reportstated that the cloud was sampled and measured at various times,but details were not provided. A group of eight monkeys servedas controls; they were treated similarly to the exposed monkeysexcept they were not put into an exposure chamber. Monkeys wereobserved frequently for clinical signs during the first 72 hafter exposure. Chest radiographs were taken before exposure andafter 2, 6, or 12 h or 1, 3, 7, or 30 days post-exposure.Monkeys were killed after 12 h or after 3, 7, or 30 days.Clinical signs in monkeys exposed to CS at 900 mg/m3for 3 min or at 1,700 mg/m3 for 5 min includedblinking and a “fright reaction” observedimmediately after removal from the exposure chamber, whichdisappeared within a few minutes after the monkeys were moved tofresh air. Monkeys exposed at 2,850 mg/m3 for 10 minexhibited frequent blinking, labored respiration, coughing, oraland nasal discharge, occasional vomiting, and decreased activityand response to external stimuli. One monkey also had copiousocular discharge. Clinical signs were most severe at 12 h andwere generally resolved within 72 h. Clinical signs in monkeysexposed to CS at 2,500 mg/m3 for 30 min were severeand included prostration, dyspnea, copious oral and nasaldischarge, and scleral congestion after removal from theexposure chamber. Five monkeys died; four died 3-12 h afterexposure and one died on day 4. Dyspnea was most severe at 12 h,while oral and nasal discharge and effects on the eyes were mostsevere at 24 h. Radiographic findings were present only in thisgroup; infiltrates appeared 3 h after exposure, were most severeafter 24 h, and cleared after 3 days.
Pathologic examination of the monkeys 12 h after exposure to CSat 900 or 1,700 mg/m3 revealed mild pulmonarycongestion, bronchorrhea, emphysema, and atelectasis (Striker et al.1967). These effects disappeared in the monkeys examinedon day 3, but recurred in monkeys examined on days 7 and 30.Pathologic lesions were more severe and developed earlier inmonkeys exposed at 2,850 mg/m3 for 10 min. Pulmonaryedema and congestion and bronchorrhea were found at 12 h, andprogressed to purulent bronchitis and bronchopneumonia at day 3.After 1 week, acute pleuritis and interstitial pneumonitis wereseen, and mucosal lesions and bronchopneumonia were resolving.Lesions were still present after 4 weeks, and includedemphysema, atelectasis, and focal interstitial pneumonitis.Pathologic findings in monkeys that died after exposed to CS at2,500 mg/m3 for 30 min included severe pulmonaryedema and congestion. The three surviving monkeys were killed ondays 3, 7, or 30. The monkey killed on day 3 days hadconsiderable edema, but congestion was less prominent.Examination of the monkey killed on day 7 revealed emphysemainvolving all lobes and bronchiolitis, but most of the edema hadcleared. The monkey killed on day 30 had small shrunken lungs,purulent mucoid material filling many small bronchioles, anddistinct bronchiolitis.
McNamara et al.(1969) exposed groups of four monkeys (strain and sexnot specified) to seven different CS concentration-durationcombinations. No further experimental details were available.Mortality data from this study are summarized in Table 7-8.
TABLE 7-8
Mortality Data from Studies by McNamara et al. (1969) inDifferent Species Exposed to Tear Gas.
3.1.2. Rats
Groups of 10 rats were exposed to an aerosol of CS for 25-90 min(Punte et al.1962). Animals were exposed in a dynamic inhalationchamber containing individual cages on racks. Aerosol wasgenerated by passing dry nitrogen through an aspirator. MoltenCS was maintained in a side-armed flask in an oil bath at140-150°C. The aerosol was easily generated and liquiddroplets recrystalized before entering the exposure chamber.Chamber concentrations were measured by drawing chamber airthrough filter paper for subsequent analysis byspectrophotometry. Samples for particle-size determinations werecollected by a Cascade impactor, and MMD was derived by use ofstage calibrations based on the density of the compound; theparticle size was about 1.5 microns (MMD). Observations forclinical signs were made during and after exposure. Survivinganimals were maintained for 14 days, and then killed andexamined histopathologically. Immediately after exposure began,the animals became excitable and hyperactive, and lacrimationand salivation occurred within 30 sec. Lethargy and dyspneaoccurred after approximately 5-15 min. Dyspnea persisted forapproximately an hour after exposure ceased, and all other signssubsided about 5 min after the rats were removed from thechamber. Histopathologic examinations revealed an increase inthe number of Goblet cells in the respiratory tract andconjunctiva, necrosis in the respiratory and gastrointestinaltracts only if particles had impacted the surface, and anoccasional animal with pulmonary edema and hemorrhage in theadrenal glands. The calculated LCT50 was 32,500mg-min/m3.
An unpublished report by McNamara et al. (1969) appears to provide dataadditional to those that were published by Punte et al. (1962). Specific studydetails are not provided in the unpublished report, but one setof study results is consistent with those published by Punte et al. (1962).The report includes the mortality results from tests withadditional animal species exposed by inhalation to CS, as wellas mortality data for CS dispersed by different methods. Asdiscussed above, Punte etal. (1962) reported mortality data for rats, but thevalues were reported only in terms of mg-min/m3.Specific concentrations of CS (sprayed as molten agent) withcorresponding exposure durations for these data are reported inthe unpublished study by McNamara et al. (1969) and are presented in Table 7-8.
Groups of 18 male albino SPF rats were exposed topyrotechnically-generated CS smoke in a 10-m3 chamber(Colgrave and Creasey1975). The rats were exposed to at 5,871 ± 476mg/m3 for 15 min, at 6,030 ± 590mg/m3 for 10 min, or at 6,800 ± 1,166mg/m3 for 5 min (averages and standard deviationswere calculated on the basis of the values reported by theinvestigators as 6,000, 6,000, and 6,400 mg/m3,respectively). CS was released from four CS cartridges, eachcontaining CS (12.5 g), potassium chlorate (16 g), lactose (15g), and kaolin (7.5 g). The cloud of CS in the exposure chamberwas sampled at approximately 1-min intervals for the 10- and15-min exposures and at 30-sec intervals during the 5-minexposure. The analytic method used to measure CS concentrationswas not described. Survivors were killed at times ranging from15 min to 2 days post-exposure. All animals were necropsied, andselected tissues were analyzed by both light and electronmicroscopy. Nonexposed controls were used to establish thetypical macroscopic and microscopic appearance of the tissues ofthe particular strain used. Mortality occurred in four ratsexposed at 5,871 mg/m3 for 15 min (death occurredwith 24 h), and in two rats exposed at 6,030 mg/m3for 10 min (death occurred within 24 or 36 h). All animalsexposed at 6,800 mg/m3 survived until the studyterminated after 2 days. Animals that died after exposure to CSfor 15 min developed marked pulmonary congestion with scatteredalveolar hemorrhages and patchy edema. Survivors developed lessmarked pulmonary congestion and only occasional areas of edemaand hemorrhaging. Rats that died after exposure to CS for 10 minalso developed pulmonary congestion, but the severity was muchless than that seen with the 15-min exposures. Hemorrhages andedema were occasionally seen in the lungs of survivors.Examination of rats exposed to CS for 5 min revealed mildpulmonary congestion with occasional hemorrhage up to 6 hpost-exposure. Rats killed between 12 h and 2 days post-exposurehad no pulmonary findings except for one rat with moderate andextensive pulmonary congestion. Electron microscopic examinationof the lungs from all exposed rats revealed changes in theepithelium and interstitium, with accumulation of fluid betweenthe membrane layers and collagen-containing areas of the septum.Degenerative changes of the epithelium and endothelium led torupture or dissolution of the capillary wall. The investigatorsstated that the changes were similar in all exposed rats, withthe changes varying only in the degree of severity. Damage wasevident as early as 15 min after exposure, and became moresevere after 30 and 60 min.
Ballantyne and Callaway(1972) exposed groups of male and femaleWistar-derived SPF rats to pyrotechnically-generated CS smoke atconcentrations of 750 mg/m3 for 30 min, 480mg/m3 for 1 h, or 150 mg/m3 for 2 h ina 10-m3 exposure chamber. A group of control animalswas also maintained, but no description of the treatment of thecontrols was provided to determine whether they were exposedunder similar conditions to clean air. The grenades used for theexposure contained CS (2 g), potassium chlorate (2.4 g), lactose(2.4 g), and kaolin (1.2 g). Although the report stated that theconcentration of CS in the exposure chamber was sampled at thestart of the exposure and at 6-min intervals up to and including57 min, no information was provided about the analytictechnique. Groups of animals were killed after 1, 10, and 28 or29 days. Some of the animals exposed at 480 or 150mg/m3 were retained for up to 32 months toevaluate potential lasting toxicity and pathology (Marrs et al. 1983a).Animals that died or were moribund after 1 month and thosekilled after 32 months were subjected to gross necropsy, and theheart, lungs, small intestine, liver, pancreas, spleen, kidneys,brain, gonads, and pituitary and adrenal glands were removed andprocessed for histologic examination.
All animals exposed for 30 min at 750 mg/m3 survivedto the scheduled necropsy, and histopathologic changes werefound only on post-exposure day 1 (see Table 7-9). One rat had congestion ofalveolar capillaries and a few scattered alveolar hemorrhages,while another rat had a few minute foci of renal tubularnecrosis at the inner cortex. No pathologic changes were foundin rats at post-exposure day 10 or 28. Exposure to CS at 480mg/m3 for 1 h resulted in the mortality of somerats (see Table7-9), with the majority of the deaths occurring onpost-exposure days 1 and 2. Pathologic changes in animalssurviving exposure at 480 mg/m3 were generallyconfined to post-exposure day 1; lesions were limited to minimalpulmonary congestion and hepatic congestion in one rat, minimalpulmonary hemorrhage and hepatic necrosis in another rat, andmild pulmonary congestion in a third rat. Two rats had mildpulmonary edema. A few rats killed after 10 days had healedlesions, as evidenced by binucleate liver cells aroundcentrilobular veins and immature epithelium in some renaltubules. No abnormal pathologic changes were noted at day 29.Histopathologic findings in rats that died were much more severeand included renal changes (mild-to-moderate necrosis of thecortex, moderate-to-severe necrosis of the medulla, and somemild congestion), pulmonary changes (mild-to-severe congestion,mild hemorrhage, and some mild edema), and hepatic changes(glycogen depletion in all rats and a few cases of mildcongestion and mild-to-moderate necrosis).
TABLE 7-9
Summary of Acute Toxicity Data from Studies by Ballantyne andCallaway (1972) in Hamsters and Mice Exposedto Tear Gas.
Exposure to CS at 150 mg/m3 for 2 h resulted in nomortality. Pathologic examination of the animals revealedlesions only on day 1 post-exposure. Lesions were confined tofemale animals; one rat had a few scattered alveolarhemorrhages, one had acute mucoid enteritis, and one hadpneumonic consolidation of the upper right lung lobe.
Exposure to CS at 480 mg/m3 for 1 h or at 150mg/m3 for 2 h did not affect the lifespan ofrats, and no statistically significant increases were found innon-neoplastic lesions in the exposed groups compared withcontrols (Marrs et al.1983a). Common non-neoplastic lesions in male andfemale rats included changes in the lungs (engorgement,congestion, inflammatory changes, and pulmonary edema) andpyelonephritis of the kidneys. Liver congestion was also acommon finding. No exposure-related neoplastic lesions wereevident in male rats. Female rats in the 150-mg/m3group exhibited an increased incidence of pituitary tumors;incidence was 26% in the control group, 29% in the group exposedto CS at 480 mg/m3 for 1 h, and 47% in the groupexposed at 150 mg/m3 for 2 h. The increases were notstatistically significant.
In another experiment, Ballantyne and Callaway (1972) exposed groups of 10rats for 5 to 20 min to CS at an approximate concentration of4,000 mg/m3, followed by a 14-day observation period.An anti-riot grenade containing approximately 50 g of CS wasignited in a 10-m3 static chamber and allowed to burnto completion. All animals that died and the survivors killed atthe end of the 14-day observation period were subjected to grossand histologic examinations. Clinical signs during exposurecould not be recorded because the aerosol generated in thechamber resulted in a complete lack of visibility. Upon removalfrom the chamber, animals exhibited signs of increased buccaland nasal secretions and dyspnea, particularly at the longerexposure durations. Mortality data are summarized in Table 7-10. Noanimals died during exposure. Necropsy revealed pulmonary edemaand congestion, often with multiple, variable sized areas ofhemorrhage, and mucus in the trachea and major bronchi.Histopathologic examination of these animals revealed severecongestion of the alveolar capillaries and intrapulmonary veinsand alveolar hemorrhage. Mucus was seen in some bronchi andbronchioles, and occasional areas of collapse and hemorrhagewere seen distal to a completely occluded bronchiole.Moderate-to-marked pulmonary edema was also observed in severalanimals. No evidence of acute inflammatory cell infiltrate wasobserved in any of the lungs examined, suggesting that the CSaerosol produced direct injury to the pulmonary capillaryendothelium. Circulatory failure evidenced as congestion of theliver, kidneys, and spleen and dilation of the right ventriclewas present in most of the animals that died. Animals thatsurvived to day 14 days did not have any residual pathologicchanges at necropsy.
TABLE 7-10
Summary of Mortality Data from Studies by Ballantyne andCallaway (1972) in Different Species Exposedto Tear Gas.
Groups of 20 or 21 male Porton-Wistar rats were exposed by wholebody inhalation to various concentrations of CS aerosol for10-60 min (see Table7-11) (Ballantyne and Swanston 1978). Animals were exposedin a 1-m3 dynamic flow chamber. The aerosol wasgenerated by filling a Collision spray with molten CS (heated to150°C) and passing pure nitrogen into the air stream. Theresultant aerosol was fed into the diluting air stream. Thechamber atmosphere was sampled for 1 min at 5-min intervals byaspirating air through glass fiber discs held in double conefilters. A bubbler containing hydrochloric acid in ethanol wasconnected in line to the glass filter to act as an additionaltrap. The contents of the bubbler were used to elute CS from thefilter discs, and the concentration of CS in the resultantextract was measured by absorption spectrophotometry andcompared with a prepared standard. Signs of toxicity includedincreased nasal and buccal secretions and increased rates ofrespiration when removed from the chamber; effects disappearedwithin approximately 1 h post-exposure. No animals died duringexposure; deaths generally occurred within the first 2 daysfollowing exposure. A summary of mortality data is presented inTable 7-11.Necropsy findings in animals dying within 48 h includedpulmonary congestion and edema (with some animals also havingmultiple variable sized hemorrhages) and congestion of thetrachea. Moderate amounts of mucus were also seen in thetrachea. Histopathologic examination of the lungs from theseanimals revealed moderate-to-marked congestion, inter- andintra-alveolar hemorrhaging, and excess secretions in thebronchioles and intrapulmonary bronchi. Examination of animalsdying after 48 h revealed similar findings, as well as evidenceof early bronchopneumonia. Congestion of the liver, kidneys,spleen, and small intestines were also frequently seen inanimals dying from exposure. No abnormal findings were found inanimals surviving the 14-day observation period.
TABLE 7-11
Summary of Mortality Data from Studies by Ballantyne andSwanston (1978) in Different Species Exposedto Tear Gas.
3.1.3. Mice
Groups of 20 mice were exposed to an aerosol of CS for 10-60 min(Punte et al.1962). Experimental procedures, clinical signs, andnecropsy results are similar to those described for thecorresponding study in rats (see Section 3.1.2). The calculatedLCT50 was 43,500 mg-min/m3. An unpublished report byMcNamara et al.(1969) appears to provide data additional to thosethat were published in this study. Specific study details arenot provided in the unpublished report, but one set of studyresults is consistent with those published by Punte et al. (1962).The report includes the mortality results from tests withadditional animal species exposed by inhalation to CS, as wellas mortality data for CS dispersed by different methods. Punte et al. (1962)reported mortality data for mice, but the values were reportedonly in terms of mg-min/m3. Specific concentrationsof CS (sprayed as molten agent) with corresponding exposuredurations for these data are reported in the unpublished studyby McNamara et al.(1969) and are presented in Table 7-8.
Ballantyne and Callaway(1972) exposed groups of 10 mice for 5-20 min to CSat an approximate concentration of 4,000 mg/m3,followed by a 14-day observation period. The experimentalprotocol, clinical signs, and necropsy findings were similar tothose described in the corresponding study in rats (see Section 3.1.2).Mortality data are summarized in Table 7-10.
Groups of 19-40 male albino mice were exposed by whole bodyinhalation to various concentrations of CS aerosol for 15-30 min(see Table 7-11)(Ballantyne andSwanston 1978). The experimental protocol, clinicalsigns, and necropsy findings were similar to those described inthe corresponding study in rats (see Section 3.1.2). Mortality dataare summarized in Table7-11.
3.1.4. Guinea Pigs
Groups of ten guinea pigs were exposed to an aerosol of CS forexposure durations of 5-40 min (Punte et al. 1962). Experimentalprocedures, clinical signs, and necropsy results are similar tothose described for rats in Section 3.1 2. The calculatedLCT50 is 8,300 mg min/m3. An unpublished report byMcNamara et al.(1969) appears to provide data additional to thosethat have been published. Specific study details are notprovided in this report, but one set of study results isconsistent with those published by Punte et al. (1962). The reportincludes the mortality results of additional animal speciesexposed by inhalation to CS, as well as mortality data for CSdispersed by various methods. As described above, Punte et al. (1962)reported mortality data for guinea pigs, but the values werereported only in terms of mg min/m3. Specificconcentrations of CS (sprayed as molten agent) withcorresponding exposure durations for these data are reported inMcNamara et al.(1969) and are presented in Table 7-8.
Ballantyne and Callaway(1972) exposed groups of five guinea pigs for 5 to 20min to an approximate CS concentration of 4,000mg/m3, followed by a 14-day observation period. Theexperimental protocol, clinical signs, and necropsy findings aresimilar to those described in the rat study (see Section 3.1.2).Mortality data are summarized in Table 7-10.
Groups of ten to twenty female Dunkin Hartley guinea pigs wereexposed by whole body inhalation to various concentrations of CSaerosol for durations of 10 to 45 min (see Table 7-11) (Ballantyne and Swanston1978). Experimental protocol, clinical signs, andnecropsy findings are as described for the rat study in Section 3.1.2.Mortality data are summarized in Table 7-11.
3.1.5. Rabbits
Groups of four rabbits were exposed to an aerosol of CS forexposure durations of 30-90 min (Punte et al. 1962). Experimentalprocedures, clinical signs, and necropsy results are similar tothose described for rats in Section 3.1.2, except thathyperactivity, salivation, and lachrymation were not reported.The calculated LCT50 is 17,000 mg min/m3. Anunpublished report by McNamara et al. (1969) appears to provide dataadditional to those that have been published. Specific studydetails are not provided in this report, but one set of studyresults is consistent with those published by Punte et al. (1962).The report includes the mortality results of additional animalspecies exposed by inhalation to CS, as well as mortality datafor CS dispersed by various methods. As described above, Punte et al. (1962)reported mortality data for guinea pigs, but the values werereported only in terms of mg min/m3. Specificconcentrations of CS (sprayed as molten agent) withcorresponding exposure durations for these data are reported inMcNamara et al.(1969) and are presented in Table 7-8.
Ballantyne and Callaway(1972) exposed groups of five rabbits for 5 to 20 minto an approximate CS concentration of 4,000 mg/m3,followed by a 14-day observation period. The experimentalprotocol, clinical signs, and necropsy findings are similar tothose described in the rat study (see Section 3.1.2). Mortality dataare summarized in Table7-10.
Groups of five to ten female New Zealand white rabbits wereexposed by whole body inhalation to various concentrations of CSaerosol for durations of 5 to 60 min (see Table 7-11) (Ballantyne and Swanston 1978).Experimental protocol, clinical signs, and necropsy findings areas described for the rat study in Section 3.1.2. Mortality dataare summarized in Table7-11.
3.1.6. Hamsters
Ballantyne and Callaway(1972) exposed groups of male and female goldenhamsters to pyrotechnically-generated CS smoke at concentrationsof 750 mg/m3 for 30 min, 480 mg/m3 for 1h, or 150 mg/m3 for 2 h in a 10-m3exposure chamber. The experimental protocol is the same as thatfor the corresponding study in rats (see Section 3.1.2). All animalsexposed for 30 min at 750 mg/m3 survived to thescheduled necropsy, and histopathologic changes were observedonly on post-exposure day 1 (see Table 7-11). Three hamsters had a fewscattered alveolar hemorrhages, and one also had congestion ofthe alveolar capillaries. No pathologic changes were found atpost-exposure day 10 or 28. Exposure at 480 mg/m3 for1 h killed some hamsters (see Table 7-11); the majority of the deathoccurred after 1-2 days. Pathologic changes in animals survivingexposure at 480 mg/m3 were generally found only onpost-exposure day 1. Eight hamsters had mild pulmonarycongestion, and four of them also had mild pulmonary hemorrhage.One hamster with no lung lesions had mild renal congestion andnecrosis in the medulla, and another had mild necrosis in themedulla. A few hamsters killed after 10 days had healed lesions,as evidenced by binucleate liver cells around centrilobularveins and immature epithelium in some renal tubules. No abnormalpathologic changes were found at day 29. Histopathologicfindings in hamsters that died were generally similar to thosein rats (see Section3.1.2); however, the lesions were less severe inhamsters than in rats.
Exposure to CS at 150 mg/m3 for 2 h resulted in themortality of two male hamsters (after 12 or 16 days), andnecropsy revealed bronchopneumonia. Pathologic examination ofsurviving animals revealed lesions only on day 1. Lesions werefound only in female hamsters; one had a few scattered alveolarhemorrhages and two had a few scattered foci of acute renaltubular necrosis at the inner cortex.
Exposure to CS at 480 mg/m3 for 1 h or at 150mg/m3 for 2 h did not affect the lifespan of thehamsters, and no statistically significant increases innon-neoplastic lesions were found in the exposed groups comparedwith controls (Marrs etal. 1983a). Common non-neoplastic lesions in male andfemale hamsters included changes in the lungs (engorgement,congestion, inflammatory changes, and pulmonary edema) andpyelonephritis of the kidneys. No exposure-related neoplasticlesions were evident in male or female hamsters.
3.1.7. Dogs
McNamara et al.(1969) exposed groups of four dogs (strain and sexnot specified) to eight different CS concentration-durationcombinations. No further experimental details were available.Mortality data are summarized in Table 7-8.
Following a 30-sec exposure to CS at 25 mg/m3, one dogexhibited increased blood pressure, an altered respiratorypattern, tachycardia, and increased femoral artery blood flow(Cucinell et al.1971). In another test, two dogs were exposed for 23min to CS at 2,600 mg/m3. One dog survived and theother dog died after 52 h. The investigators noted that dogsrecover partially when exposed to a lethal dose of CS but thendevelop respiratory distress and die within 48-70 h.
3.2. Nonlethal Acute Toxicity
3.2.1. Mice
An RD50 (concentration that reduces the respiratoryrate by 50%) for CS of 4.0 mg/m3 (95% confidenceinterval: 3.3-5.2 mg/m3) was reported for maleSwiss-Webster mice (Kaneet al. 1979).
3.2.2. Rabbits
To investigate whether CS exposure can cause diarrhea, fourrabbits were exposed to thermally-generated pure CS in a10-m3 chamber (Ballantyne and Beswick 1972). Theexposures involved the following: one rabbit each was exposed at58 mg/m3 for 30 min, 46 mg/m3 for 20 min,54 mg/m3 for 12 min, or 17 mg/m3 for 17min. Animals were placed singly in cages with removable trayslined with several layers of filter paper arranged to collectstool samples. The number of stool pellets passed, their totalweight, and their water content were recorded for several daybefore and after exposure. Exposure to CS did not result in anincreased incidence of diarrhea.
Two rabbits were exposed in a static chamber to the entirecontents of a 3-ounce unit containing 71.5 g of CS (Gaskins et al.1972). The unit required 20 sec to fully dispense. Bothrabbits became unconscious after approximately 2 min of exposureand were moved to fresh air. The rabbits regained their rightingreflex approximately 10-20 min after exposure and were almostcompletely recovered after 1 h (moderate ocular wetness was theonly visible effect). Gross necropsy of the rabbits performedafter 2 weeks did not reveal any abnormalities. Two otherrabbits were exposed to 23.2 g of CS during dispersion of a CSunit requiring about 10 sec to completely discharge. Thedispensed CS formed a cloud in the chamber. The rabbits tried toavoid the spray as it was dispensed, and then sat quietly withtheir eyes tightly closed for the remainder of the 5-minexposure. No abnormalities were observed in the eyes or skin ofthe rabbits.
3.3. Repeat-Dose Studies
3.3.1. Rats
Groups of five male and five female F344/N rats were exposed toCS2 at concentrations of 0, 1, 3, 10, 30, or 100mg/m3 for 6 h/day, 5 days/week for 2 weeks (NTP 1990). (CS2contains 94% CS, 1% hexamethyldisilizane, and 5%Cab-o-Sil®). All rats exposed at 30 or 100mg/m3 died before the end of the study. Rats fromall exposure groups exhibited adverse clinical signs, rangingfrom erythema and blepharospasm at the lower concentrations todacryorrhea, mouth breathing, listlessness, and mouth breathingat the higher concentrations. Rats in the 1-mg/m3group gained more weight over the exposure period than controls,but at concentrations of 3 mg/m3 and higher bodyweight was generally decreased.
Groups of 10 male and 10 female F344/N rats were exposed to CS2at 0, 0.4, 0.75, 1.5, 3, or 6 mg/m3 for 6 h/day, 5days/week for 13 weeks (NTP 1990). One male rat exposed at 6mg/m3 died, and all others survived to studytermination. Clinical signs of ocular irritation (partial orcomplete eyelid closure) were noted in all exposure groups, andrats exposed at 6 mg/m3 developed erythema of theextremities that persisted overnight. Rats exposed to CS2 at 1.5mg/m3 or higher gained significantly less weightover the study period than controls; final mean body weight was17-44% lower than that of controls for males and 10-24% lowerfor females. An approximate 46% reduction in thymus weightrelative to body weight was noted in male and female ratsexposed at 6 mg/m3. Concentration-relatedhistopathologic changes included focal erosion with regenerativehyperplasia and squamous metaplasia of the respiratoryepithelium. Acute inflammation and hyperplasia of therespiratory epithelium were also found.
One group of 56 male rats was exposed to a mean CS concentrationof 1,470 or 1,770 mg/m3 for 5 min/day for 5 days andanother group of 49 male rats was exposed at a meanconcentration of 12.5 or 14.8 mg/m3 for 80 min/dayfor 9 days to (Ballantyneand Callaway 1972). Exposures to thethermally-generated CS aerosol (MMD of 1-2 micrometers) wereconducted in a 1-m3 chamber, with chamber air sampledcontinuously throughout exposure at a rate of 1 L/min using adouble cone filter. The samples were analyzed for CS content(details not provided). Groups of three to five survivors werekilled after 1, 6, and 24 h and 2, 3, 4, 5, 7, 10, 14, and 21days, and gross and microscopic examinations were performed. Allanimals survived the 5-min exposures. Histopathologicexamination revealed minimal congestion of the alveolarcapillaries after 1 or 6 h in two of five rats and a fewscattered alveolar hemorrhages after 2 days in one of four rats.Scattered patches of bronchopneumonia were found in one of fiverats after 7 days, in one of three rats after 8 days, one ofthree rats after 10 days, and two of five rats after 18 days.Pathologic changes in control rats included scattered alveolarhemorrhages in two of 11 rats and subacute mucoid enteritis inone of 11 rats. Death occurred in five of the 49 rats exposed at12.5 or 14.8 mg/m3 for 80 min/day; one died after theseventh exposure, two after the eight exposure, and two died 5days after the final exposure. Necropsy revealed widespreadacute bronchopneumonia. Histopathologic examination of thesurviving animals revealed lesions for up to 5 days afterexposure and not thereafter.
3.3.2. Mice
Groups of five male and five female B6C3F1 mice wereexposed to CS2 at concentrations of 0, 1, 3, 10, 30, or 100mg/m3 for 6 h/day, 5 days/week for 2 weeks (NTP 1990). All miceexposed at 10 mg/m3 and greater died before studytermination. Mice from all exposure groups exhibited adverseclinical signs, ranging from erythema and blepharospasm at thelower concentrations to dacryorrhea, mouth breathing,listlessness, and mouth breathing at the higher concentrations.Mice exposed at 1 mg/m3 gained more weight over theexposure period than controls, but generally lost body weight atexposure concentrations of 3 mg/m3 and higher.
Groups of 10 male and 10 female B6C3F1 mice wereexposed to CS2 at 0, 0.4, 0.75, 1.5, 3, or 6 mg/m3for 6 h/day, 5 days/week for 13 weeks (NTP 1990). All mice exposed at 6mg/m3 died and one male and one female mouse fromthe 3-mg/m3 group died during the second week ofexposure. Closed or partially-closed eyes during exposure wereobserved in mice from all exposure groups through week 6, and inmice exposed at 3 mg/m3 during weeks 12 and 13.Concentration-related decreases in body weight compared withcontrols were found in all exposure groups; final mean bodyweights of mice in the 3-mg/m3 group were 13% lowerfor males and 9% lower for females. Exposure-relatedhistopathologic changes were observed in mice exposed at 1.5mg/m3 and higher, and included focal inflammationand squamous metaplasia (primarily in the nasal turbinates) andinflammation of the vomeronasal organ.
3.3.3. Rats, Mice, Guinea Pigs, and Rabbits
Groups of five to 10 guinea pigs, five rabbits, 10 rats, and10-20 mice were exposed to CS at approximate concentrations of30-40 mg/m3 for 5 h/day for 1-7 successive days(Ballantyne andCallaway 1972). An anti-riot grenade containing 0.5to 0.75 g of CS was ignited every 30 min in a 10-m3static chamber to maintain the nominal concentration. Theinvestigators stated that concentrations were determined bycontinuous sampling throughout the exposure, but no details wereprovided. Animals were removed to fresh air following eachexposure, and were maintained for a 14-day post-exposure period.All animals that died and the survivors killed at the end of thestudy were given gross and histologic examinations. A summary ofthe mortality data is presented in Table 7-12. The description ofclinical signs was limited to a statement that rabbits and ratsexhibited more rhinorrhea and lacrimation than did mice, whereasguinea pigs showed few clinical signs apart from occasionalsneezing during the first hour of exposure. Necropsy of animalsthat died revealed moderate-to-marked congestion of the alveolarcapillaries and intrapulmonary veins and inter- andintra-alveolar areas of hemorrhage; many of the animals thatdied also had congestion of the liver, kidneys, and smallintestine. Moderate pulmonary edema was noted in a “fewof the animals.” No residual pathologic changes werefound in animals that survived until the end of the study.
TABLE 7-12
Summary of Mortality Data in Different Species Exposed toTear Gas for 5 Hours per Day for Up to 7 Days.
3.4. Developmental and Reproductive Toxicity
Groups of 22-24 pregnant Porton strain rats or 12 pregnant NewZealand white rabbits were exposed to CS aerosol for 5 min/day ongestation days 6-15 or 6-18, respectively (Upshall 1973). CS The aerosol had aparticle size of 1-2 micrometers and was generated by melting purecrystalline CS at 120°C using a Collison spray. A preliminarystudy investigated exposure to CS at 0 or 20 mg/m3, andwas followed by a concentration-response study that evaluated CS atconcentrations of 0, 6, 20, or 60 mg/m3. Control ratswere recaged and moved out of their normal environment during thetest-group exposure, and control rabbits were exposed to asiliconized silica aerosol at 60 mg/m3. Additionalcontrol groups of pregnant rats were exposed to a particulateaerosol (60 mg/m3 of Neosil) or to water aerosol toevaluate the stress of aerosol exposure. Rats were killed ongestation day 21 and rabbits on gestation day 30. Cesarean sectionswere performed, and the fetuses were evaluated for skeletal orvisceral abnormalities. In addition, the lungs, liver, kidneys, andadrenal glands from the rabbit dams in the concentration-responsestudy were evaluated histologically. No definitive effects oftreatment were noted. In the preliminary rat study, exposed animalsexhibited a decrease in maternal weight gain compared with controls(-23%), but a clear concentration-response relationship was notobserved in the main study (-23, -12, and 15% for the 6, 20, or 60mg/m3 groups, respectively). Fetal weight appeared todecrease with increasing concentration in the main rat study (3.3,3.2, and 3.1 g, respectively, vs. 3.5 for controls), but the fetalweights were comparable those in other studies. No otherstatistically significant effects were observed. No exposure-relatedeffects were found in exposed rabbits or their offspring. Althoughthe exposure concentrations were sufficient to cause extremeirritation, clinical signs in exposed rats and rabbits were notreported.
3.5. Genotoxicity
In general, CS was not mutagenic to Salmonellatyphimurium. Mutations were not induced with or without thepresence of S9 at CS concentrations of 12.5-800 µg/plate instrains TA97a, TA98, TA100, TA102, or TA104 (Meshram et al. 1992); of up to 1.5 mg/platein strains TA98, TA100, TA1535, or TA1537 (Wild et al. 1983); ranging from 10µg/plate to 2 mg/plate in strains TA98, TA1535, TA1537, or TA1538(von Däniken et al. 1981); or at CS2 concentrations of 3.3-333µg/plate in strains TA98, TA100, TA1535, or TA1537 (NTP 1990). Equivocalresponses for CS and CS2 were reported in strain TA100 only without S9(von Däniken et al. 1981; NTP 1990), and for CS2 in strain TA97 but only with 30% S9(NTP 1990).Cytotoxicity from CS was observed starting at 200 µg/plate, butthe presence of 30% S9 generally reduced the cytotoxicity.
Other in vitro genotoxicity testing was generally positive. CS inducedsister chromatid exchanges and chromosomal aberrations in Chinesehamster ovary cells both with and without S9 at CS2 concentrations of 6µg/mL and greater (NTP1990). Trifluorothymidine resistance in mouse L5178Y lymphomacells was induced in the absence of S9 at a CS concentration of 2.5µg/mL (McGregor et al.1988; NTP1990). V79 Chinese hamster cells exposed to CS in culture at 19,38, or 75 µM for 3 h and evaluated 6 days later showed reducedsurvival (by about 20, 30, and 80%, respectively; estimated from agraph), and exhibited a concentration-related increase in the frequencyof mutants resistant to 6-thioguanine (mutations induced approximately4- to 5-fold above controls at the highest concentration) (Ziegler-Skylakakis et al.1989). Exposure to CS also increased the frequency ofmicronuclei by approximately 2-fold at 19 µM and up to 18-fold at75 µM (measured 24 h after exposure), but did not induceDNArepair synthesis as assessed using the BrdUrd density-shift method. Aconcentration-dependent increase was observed in spindle celldisturbances, particularly C-metaphases (chromosomes completelyscattered in cytoplasm and often highly contracted), when cells wereexposed to CS at 5, 9, 19, or 38 µM for 3 h (Schmid and Bauchinger 1991).The C-mitotic effect was also reflected in the appearance of a metaphaseblock and the disappearance of other mitotic figures (prophases andana-telophases). When a differential staining technique was applied toallow for visualization of the spindle apparatus and chromosomes, aconcentration-dependent increase in the number of mitoses with abnormalspindles was again observed, particularly apolar mitoses (mitoticfigures without any signs of polar spindle configurations) (Salassidis et al. 1991).Further investigation into the mechanism of CS-induced c-mitotic spindledamage found that exposure of cells to CS at 38 µM for 20 h or 3h followed by 20 h of recovery resulted in an increase in the number ofaneuploid cells and in the polyploid index (Schmid and Bauchinger 1991). The number ofaneuploid cells and the polyploid index were increased to a much greaterextent by exposure to the metaboliteo-chlorobenzaldehyde than to CS, suggesting that thismetabolite may play a role in the induction of spindle damage. Acomparison of the effectiveness of various exposure conditions revealedthat cells exposed to CS at concentrations of up to 38 µM for 20h exhibited a concentration-dependent increase in the number of S-cellsand the frequency of chromatid-type aberrations (single breaks, isolocusbreaks and exchanges, and gaps). Exposure to CS for 3 h followed by a20-h recovery period resulted in similar effects but was not aseffective. No effects were observed when cells were incubated with thesupernatant from the 3 h exposure (Bauchinger and Schmid 1992). The cell cycletime of the V79 cell line is approximately 8-10 h; therefore, the cellshad time to run through one or two S-phases.
Genotoxicity testing in vivo was generally negative. CS did not bind toDNA in the liver or kidneys of rats injected intraperitoneally withradiolabeled CS at 13 mg/kg and evaluated 8 or 75 h after dosing, butdid bind to nuclear proteins in these organs (von Däniken et al.1981). CS did not cause an increase in sex-linked recessive mutations ingerm cells of male Drosophila when administered in thefeed at concentrations ranging from 5 × 10-4 M to 2.6 ×10-3 M for 3 days (Wild et al.1983), and did not increase micronucleated polychromaticerythrocytes in the bone marrow of NMRI mice administered CS byintraperito-neal injection at 19 or 38 mg/kg or by oral administrationat 113 or 226 mg/kg (Wild et al.1983). The oral dose of 226 mg/kg killed 10 of 13 exposedmice.
3.6. Chronic Toxicity and Carcinogenicity
Groups of 50 male and 50 female B6C3F1 mice and 50 male and 50female F344/N rats were exposed to CS2 at target concentrations of 0,0.75, or 1.5 mg/m3 (mice) or 0, 0.075, 0.25, or 0.75mg/m3 (rats) for 6 h/day, 5 days/week for 105weeks (NTP 1990). Ratsexposed at 0.75 mg/m3 developed histopathologic changes inthe respiratory and olfactory epithelium of the nasal passage andinflammation and proliferation of the periosteum of the turbinate bones.No neoplastic effects were present. Lesions seen in the nasal cavity ofexposed mice included inflammation in the anterior middle portions ofthe nasal passage and focal hyperplasia and/or squamous metaplasia ofthe respiratory epithelium. No other adverse effects were noted. Femalemice exhibited a statistically significant, exposure-related reductionin the incidences of hyperplasia and adenomas of the pituitary glandpars distalis (adenoma rates in the 0-, 0.75-, and 1.5-mg/m3groups were 16/47, 5/46, and 1/46, respectively). Lymphomas in femalemice also occurred with a significant negative trend (21/50, 12/50, and8/50, respectively).
Groups of 75 male SPF Porton strain mice, 50 male Porton Wistar-derivedrats, and 50 Dunkin Hartley guinea pigs were exposed to CS at nominalconcentrations of 0, 3, 30, or 300 mg/m3 (MMD of 3-4micrometers) for 1 h/day, 5 days/week for up to 55 exposures (11 weeks)in mice and up to 120 exposures (24 weeks) in rats and guinea pigs(Marrs et al. 1983b).Exposure at the high concentration resulted in excessive mortality inmice and guinea pigs within days of exposure; therefore, tests at thehigh concentration was discontinued after three exposures in mice andafter five exposures in rats and guinea pigs (the number of deaths werenot provided) (Marrs et al.1983b). During the first month of the experiment, 17% of themice and 46% of the guinea pigs in the high-concentration groups died. Asignificant trend (p < 0.001) was found in the incidence of earlydeath in mice with concentration. The investigators also reported asignificant trend (p < 0.001) in the incidence of early deathwith concentration in guinea pigs; however, most of the mortality inguinea pigs occurred during the first month. Post-mortem examination of10 guinea pigs that died during exposure revealed acute alveolitis inseven of the animals, with mild alveolitis present in the other three.The cause of death in mice that died during exposure to CS could not bedetermined. The investigators reported that toxic signs were not usuallyobserved, and that death occurred suddenly and without warning. No causeof death could be ascribed to animals that died during the observationperiod. CS exposure did not affect the growth of rats or guinea pigs,but did result in a concentration-related decrease in the growth ofmice. No definitive, exposure-related histologic findings were observedin mice, rats, or guinea pigs at the end of the study. Noexposure-related neoplasms were found.
3.7. Summary
Clinical signs in the acute and repeated-dose animal studies suggest thatCS is highly irritating. The majority of the acute inhalation exposuredata in animals focused primarily on lethality, and death was generallycaused by pulmonary edema and congestion. Renal damage was alsooccasionally noted, but may have been secondary to anoxia. Results ofgenotoxicity tests were mixed. Results in gene mutations tests usingS. typhimurium were generally negative, as wereresults of in vivo genotoxicity assays. CS induced trifluorothymidineresistance in mouse L5178/TK lymphoma cells in the absence of S9, andinduced both sister chromatid exchanges and chromosomal aberrations inChinese hamster ovary cells in the presence and absence of S9. Nodevelopmental toxicity was found in rats or rabbits, and there was noevidence of carcinogenicity in rats or mice.
4. SPECIAL CONSIDERATIONS
4.1. Metabolism and Disposition
4.1.1. Absorption
One cat with a cannulated trachea was exposed to CS aerosol by anoral-nasal mask to assess absorption of CS by the upper respiratorytract (cannulation prevented access to the lower respiratory tract),while a second cat was exposed via a tracheal tube to assessabsorption by the lower respiratory tract (Leadbeater 1973). Bloodconcentrations of CS and its metabolites after exposure to the upperand lower respiratory tract were about 30 and 80%, respectively, ofthose measured in intact cats.
4.1.2. Toxico*kinetics
The half-lives of CS, 2-chlorobenzylmalonitrile, and2-chlorobenzaldehyde were measured in cats and rabbits (Leadbeater1973; Paradowski1979). The chemicals were administered directly into thefemoral artery via a cannula in cats and directly into the ear veinof rabbits. The half-lives of CS, 2-chlorobenzylmalonitrile, and2-chlorobenzaldehyde in cats were 5.5, 9.5, and 4.5 sec,respectively, and in rabbits they ranged from 19-25, 38-55, and38-41 sec, respectively. The in vitro half-lives of these chemicalsin the blood of cats, humans and rats were also measured. In catblood, the half-life was 5 sec for CS, 470 sec for2-chlorobenzylmalonitrile, and 70 sec for 2-chlorobenzaldehyde. Therespective half lives in humans were 5, 660, and 15 sec and in ratswere 7, 30, and 15 sec (Leadbeater 1973). The in vitro half-life ofCS in the blood of rabbits was approximately 60 sec; theinvestigators postulated that this half-life might be longer thanthose of rats, cats, and humans because of the higher concentrationof CS tested in rabbits (Paradowski 1979).
CS incubated with rat liver hom*ogenate for 5 min (ethanol-buffer; pH7.4; 37°C) resulted in a 59% decrease in the initial amountof glutathione, with 26% of the depletion occurring spontaneously(non-enzymatically) (Rietveldet al. 1986). Binding to glutathione in vivo wasconfirmed by enhanced urinary thioether excretion in rats followingintraperitoneal administration of CS (Rietveld et al. 1983, 1986). The thioether wasidentified as 2-chlorobenzylmercapturic acid.
4.1.3. Metabolism
Metabolism of CS appears to be qualitatively similar in differentspecies. In vivo, CS can be hydrolyzed to 2-chlorobenzaldehyde ormalononitrile or can be reduced to 2-chlorobenzyl malononitrile (seeFigure 7-1)(Leadbeater 1973; Paradowski1979). 2-Chlorobenzaldehyde can then be either oxidizedto 2-chlorobenzoic acid for subsequent glycine conjugation orreduced to 2-chlorobenzyl alcohol for ultimate excretion as2-chlorobenzyl acetyl cysteine or 2-chlorobenzyl glucuronic acid.Malononitrile can break down to cyanide, and be excreted asthiocyanate. The reduction of CS to 2-chlorobenzyl malononitrile isa relatively minor pathway; 2-chlorobenzyl malononitrile can beconjugated with glycine or can be hydrolyzed to 2-chlorophenyl2-cyanoproprionate.
FIGURE 7-1
Predominant metabolic pathways of tear gas in ratsproposed by Leadbeater (1973), Paradowsk (1979), and Rietveld et al.(1983).
Radiolabeled CS was administered intravenously to rats at 0.08, 0.8,and 80 µmol/kg (3H-ring labeled), 0.8 and 80 µmol/kg(14C-cyanide labeled), or 0.8 and 80 µmol/kgof (14C=C side-chain labeled) (Brewster et al. 1987). Rats were alsotreated intragastrically with CS at 80, 106, and 159 µmol/kg(14C-cyanide labeled). The major urinary metabolitesrecovered in rats up to 96 h after intravenous or intragastricadministration of CS were 2-chlorohippuric acid (49% of dose),2-chlorobenzyl glucuronic acid (10%), 2-chlorobenzyl cysteine (8%),and 2-chlorobenzoic acid (8%), and minor metabolites included2-chlorophenyl acetyl glycine (3%), 2-chlorobenzyl alcohol (1.6%),and 2-chlorophenyl 2-cyano propionate (1.6) (see Figure 7-1).
In another investigation, urinary concentrations of cyanide andthiocyanate were measured over a 24-h period in untreated rats, inrats administered CS intravenously, or in rats exposed to the CShydrolysis product malononitrile intraperitoneally orintragastrically (Brewster etal. 1987). Following CS and malononitrile administration,urinary cyanide concentrations remained at or below baseline levels,while thiocyanate concentrations generally increased with the CS ormalononitrile dose. The percentage molar conversion from CS tothiocyanate was 21.5% at an intraperitoneal dose of 212µmol/kg and 30% at an intragastric dose of 212µmol/kg. In tests with malononitrile, the percentage was 60%or more at an intraperitoneal dose of 80 µmol/kg orintragastric dose of 212 µmol/kg.
Metabolism in rabbits is similar to that in rats. The predominantbiotransformation pathway in the blood of rabbits administered highdoses of CS by intravenous injection (0.5 LD50 to theLD50) was hydrolysis of CS to 2-chlorobenzyaldehydeand malononitrile (~30-40%) (Paradowski 1979). A minor pathway involved reduction to2-chlorobenzyl malononitrile (10%) The investigators indicated thatthe remaining 50-60% of the administered CS disappeared from theblood by other means; no other explanation was provided. The liveris involved in the metabolism of CS, as demonstrated by an increasein the half-lives of CS and its metabolites in the blood of rabbitswhen the liver was excluded from the circulation. More of the CS wasaccounted for after dosing, with approximately 75% of the CShydrolyzed to 2-chlorobenzyaldehyde and 15% reduced to2-chlorobenzyl malononitrile. When the kidneys were excluded fromthe circulation, no changes were observed in CS or metabolites inthe blood.
Maximum blood concentrations of CS and its derivatives in cats wereattained 30 min after intragastric administration at 40 mg/kg(Leadbeater 1973). After 90 min, blood concentrations of2-chlorobenzylmalonitrile and 2-chlorobenzaldehyde were stillelevated, can CS concentrations had returned to zero. Whenanesthetized cats were exposed for 60 min via oral-nasal masks to CSaerosol (75 or 750 mg/m3 of pyrotechnically-generated CSaerosol, 750 mg/m3 of pure CS aerosol from molten CS, or62.5 mg/m3 of CS aerosol generated from an aqueoussuspension of micronized CS in acetic acid using a Collisonsprayer), concentrations of CS and 2-chlorobenzylmalonitrile rapidlyreached steady values, but concentrations of 2-chlorobenzaldehydecontinued to increase. Comparison of the blood concentrationsresulting from exposure to CS at 750 and 75 mg/m3 showedthat there was not a 10-fold decrease in the concentration of CS andits metabolites 2-chlorobenzylmalonitrile and 2-chlorobenzaldehyde;the concentrations were reduced by 4.5, 7.7, and 5.9, respectively.Exposure of cats to CS at 100 mg/m3 for 5 min/day for 4days, followed by exposure to CS at 75 or 750 mg/m3,resulted in reduced blood concentrations of CS and itsderivatives.
Rats receiving a single oral dose of CS at 50-500 mg/kg had lowerblood concentrations of CS and its derivatives than cats (Leadbeater1973). CS was only detected in high-dose group. Blood concentrationsof 2-chlorobenzylmalonitrile and 2-chlorobenzaldehyde in rats andcats did not increase in a dose-related manner. Rats exposed byinhalation to CS aerosol at concentrations of 14-245mg/m3 for 5 min had measurable amounts of CS and2-chlorobenzylmalonitrile in their blood immediately after exposure,but 2-chlorobenzaldehyde was detected only in rats exposed atconcentrations greater than 100 mg/m3.
Animal data suggest that CS should be absorbed by the humanrespiratory tract following inhalation exposure, and that metabolismof CS should proceed via a pathway similar to those found inlaboratory animals. However, humans are not able to tolerateconcentrations of CS as great as those tolerated by animals. Sixhealthy human males were exposed by inhalation to CS at 0.5-1.5mg/m3 over 90 min, and blood was drawn before andafter exposure to measure CS and its derivatives (Leadbeater 1973).Two men left the chamber within 20 min. CS and 2-chlorobenzaldehydewere not detected in the blood of any of the volunteers, and only atrace of 2-chlorobenzylmalonitrile was detected in the blood of oneman who remained in the chamber for the entire exposure.
4.1.4. Distribution and Elimination
To evaluate the fate of CS, radiolabeled CS was administeredintravenously (3H-ring labeled, 14C-cyanide labeled, or14C=C side-chain labeled) or intragastrically(14C-cyanide labeled) to rats, and urine, feces, andCO2 were collected for 96 h (Brewster et al. 1987). The majority ofthe administered dose was recovered in the urine (44.4 to 100%).Recovery in feces was 1.2-23.4%, and recovery in CO2 wasminimal at 0-2.1%. Comparison of recovery data for the threedifferent radiolabels after intravenous administration showed thatmore radioactivity was recovered in the feces of rats administeredthe 14C=C sidechain labeled CS (21-23%) than from ratsadministered the other two labels (4-8%).
Male mice were administered 14CN-CS by intravenousinjection, and were killed at selected intervals to evaluatedistribution by autoradiography (Brewster et al. 1987). A significantamount of radioactivity was present in the gastrointestinal tractafter 5 min. After 1 h, significant amounts of radioactivity werepresent in the gastrointestinal tract, urinary bladder, mouth, andesophagus, with lesser amounts in the blood, liver, and salivaryglands. At 24 h, most of the residual radioactivity was present inthe mouth, salivary glands, gastrointestinal tract, or urinarybladder.
4.2. Mechanism of Toxicity
CS is an SN2 alkylating agent and, therefore, reacts directly withnucleophilic compounds (Cucinellet al. 1971). Consequently, sulfhydryl-containing enzymes andother biologic compounds are prime targets. Most notably, CS reactsrapidly with the disulfhydryl form of lipoic acid, a coenzyme in thepyruvate decarboxylase pathway. In in vitro studies, CS reacted readilywith cysteine, N-acetyl L-cysteine, glutathione, dithiothreitol, andlipoic acid, and had first-order reaction constants of 0.33, 0.42, 0.85,4.88, and 10.4, respectively. Incubation of rat liver hom*ogenate with CSfor 5 min (ethanol-buffer; pH 7.4; 37°C) resulted in a 59%decrease in the initial amount of glutathione, with 26% of the depletionoccurring spontaneously (non-enzymatically) (Rietveld et al. 1986). Binding toglutathione in vivo was confirmed by enhanced urinary thioetherexcretion in rats after intraperitoneal administration of CS; thethioether was identified as 2-chlorobenzylmercapturic acid (Rietveld et al. 1983, 1986). In another study,rats administered CS intraperitoneally at a dose that was 120% of theLD50 became moribund approximately 30 min after injection(most likely due to the relatively slow generation of cyanide from themalononitrile metabolite). The role of cyanide in CS-induced lethalityis supported by the observation that administration of thiosulfateintraveneously reduced mortality by 65% compared with control rats(21/32 exposed rats survived vs. 1/11 control rats). Intravenousadministration of CS at 8 mg/kg in dogs resulted in a rapid drop in theplasma sulfhydryl concentration, which returned to normal withinapproximately 3 h (Cucinell etal. 1971).
4.3. Other Relevant Information
4.3.1. Species Variability
CS is a potent acute irritant. Ocular and pulmonary toxicity resultsfrom direct contact with CS and its associated alkylatingproperties; therefore, the mechanism of action is not expected tovary greatly between species. Ballantyne and Swantson (1978)calculated LCT50 values of 88,480 mg-min/m3 for rats,67,200 mg-min/m3 for guinea pigs, 54,090mg-min/m3 for rabbits, and 50,010mg-min/m3 for mice. These values are well within afactor of two of each other.
4.3.2. Susceptible Populations
CS is an irritant and the mechanism of toxicity is a direct contacteffect; therefore, the mechanism of action is not expected to varygreatly between individuals. The reactions in people with jaundice,hepatitis, or peptic ulcer or those that were 50-60 years old weresimilar to those of “normal” volunteers when exposedat highly irritating concentration of CS for short durations (Gutentag et al. 1960;Punte et al.1963). Subjects with a history of drug allergies orsensitivities, hay fever, or asthma also tolerated exposure to CSand were similar to normal subjects, but the group with pre-existingconditions had a higher percentage of individuals with more severechest symptoms (many of them laying prostrate on the ground forseveral minutes). However, no wheezing or rhonchi were heard, andrecovery was as rapid as that seen in other exposure groups.
4.3.3. Concentration-Exposure Duration Relationship
The concentration-exposure time relationship for many irritant andsystemically-acting vapors and gases can be described by theequation Cn × t = k, where the exponent n rangesfrom 0.8 to 3.5 (ten Berge etal. 1986). The value of n for CS was determined on thebasis of acute lethality data from studies of rats, mice, rabbits,guinea pigs, dogs, and monkeys (see Table 7-13). The dose-response software often Berge (2006)was used in the analyses (see Apprendix A for details).
TABLE 7-13
Values of the Exponent n for Tear Gas.
6. DATA ANALYSIS FOR AEGL-2
6.1. Human Data Relevant to AEGL-2
Five subjects exposed to CS at 0.71-0.78 mg/m3 (averageconcentration 0.75 mg/m3, calculated from the six intervalmeasurements) tolerated a 60 min exposure (Beswick et al. 1972). All subjects reportedocular stinging and watering, increased salivation, cough, and facestinging. Other effects reported by some of the subjects included throatirritation (4 subjects), nasal stinging and running (3 subjects), mouthstinging (2 subjects), chest burning (2 subjects), nausea (2 subjects),and headache (2 subjects). Nausea was likely due to swallowing largeamounts of saliva and the headaches were likely due to frontal sinusirritation (Beswick et al.1972). In another study, four subjects exposed to CS at 1.5mg/m3 tolerated a 90 min exposure, but experiencedclinical signs of irritation. One subject developed nasal irritationwithin 2 min, three subjects developed headache (at 45, 50, and 83 min),and all four experienced ocular irritation (at 20, 24, 70, and 75 min)(Punte et al. 1963).When the CS concentration was gradually increased over the course of 60min, most of the 30 subjects were able to tolerate exposure toconcentrations ranging from 0.31 to 2.3 mg/m3; one subjectleft at 5 min because of vomiting but returned for the duration of theexposure, another vomited at 55 min of exposure, and one subject leftafter 8 min because of irritation (Beswick et al. 1972). The two cases ofvomiting were attributed to swallowing large amounts or saliva. Clinicalsigns noted during the 60 min exposure included ocular, nasal, mouth,and throat irritation, nausea, chest discomfort, headache, and stingingof the face.
6.2. Animal Data Relevant to AEGL-2
Blinking, mild pulmonary congestion, and emphysema were observed inmonkeys exposed to CS at 900 mg/m3 for 3 min or 1,700mg/m3 for 5 min. Monkeys exposed at 2,500mg/m3 for 32 min exhibited blinking, labored respiration,coughing, oral and nasal discharge, vomiting, decreased activity,pulmonary edema, and congestion (Striker et al. 1967). Mice exposed to CS at 40mg/m3 for 5 h had rhinorrhea and lacrimation, and guineapigs exposed at 45 mg/m3 for 5 h showed occasional sneezingduring the first hour of exposure.
6.3. Derivation of AEGL-2 Value
AEGL-2 values are based on human exposure to CS at 0.75 mg/m3for 60 min (Beswick et al.1972). All five subjects tolerated the exposure, butexperienced ocular irritation, increased salivation, and coughing; somesubjects also reported nasal, mouth, and throat irritation, nausea, andheadache. An intraspecies uncertainty factor of 3 was applied becausecontact irritation is a portal-ofentry effect and is not expected tovary widely among individuals. This factor is also supported by thefinding that responses of volunteers with jaundice, hepatitis, or pepticulcer or who were 50-60 years old were similar to those of“normal” volunteers when exposed at a highly irritatingconcentration of CS for short durations. The ability to tolerate CS at14-73 mg/m3 and the recovery time in volunteers with ahistory of drug allergies, seasonal allergies, asthma, or drugsensitivity were similar to “normal” volunteers; althoughmore severe chest symptoms were reported in the volunteers withpre-existing conditions (Gutentaget al. 1960; Punte etal. 1963). An interspecies uncertainty factor of 1 wasapplied because the study was conducted in humans. A modifying factor of3 was also used because the effects observed at 0.75 mg/m3were considered AEGL-2 effects (watering eyes could impair escape). Timescaling was not performed because ocular irritation is a function ofdirect contact with the CS and is unlikely to increase with duration ofexposure at this level of severity (NRC 2001). AEGL-2 values for CS arepresented in Table 7-14,and the calculations are in Apprendix B.
TABLE 7-14
AEGL-2 Values for Tear Gas.
These values are supported by the Punte et al. (1963) study in which four subjects tolerated a90-min exposure at 1.5 mg/m3 and reported ocular and nasalirritation and headache. The values are also supported by otherexperiments conducted by Beswicket al. (1972). When an additional 30 subjects were exposedfor 60 min to CS at 0.31-2.3 mg/m3, one subject left at 5 minbecause of vomiting, but returned for the duration of the exposure, andanother vomited at 55 min of exposure. Both cases of vomiting wereattributed to swallowing large amounts or saliva. One subjectvoluntarily left after 8 min because of irritation; this subject wasexposed at 0.56-0.86 mg/m3 (AEGL-2 values are below thisrange).
7. DATA ANALYSIS FOR AEGL-3
7.1. Human Data Relevant to AEGL-3
No human studies of CS were available for deriving AEGL-3 values.
7.2. Animal Data Relevant to AEGL-3
Animal lethality data are available for rats, mice, rabbits, guinea pigs,dogs, and monkeys exposed to varying concentrations of CS for varyingdurations (McNamara et al.1969; Ballantyne andCallaway 1972; Ballantyne and Swanston 1978). Exposure durations rangedfrom 5 to 300 min and concentrations of CS ranged from 37 to 5,176mg/m3. Mortality incidences ranged from 0 to 100%,depending on concentration-duration pairings. The experimentalparameters are summarized in Tables 7-8, 7-10, 7-11, and 7-12.
7.3. Derivation of AEGL-3 Values
Using the rat, mouse, rabbit, guinea pig, dog, and monkey data sets ofMcNamara et al.(1969), Ballantyne andCallaway (1972), and Ballantyne and Swanston (1978), the lethality threshold forCS at each AEGL-3 exposure duration was calculated using the probitanalysis-based dose-response program of ten Berge (2006) (see Apprendix A). The threshold for lethalitywas set at the LC01. The rat, mouse, and rabbit data allyielded similar time-scaling values and AEGL-3 values (see Apprendix A). Largevariances in the dog and monkey data precluded calculation of 95%confidence intervals. The rat data set was used to derive AEGL-3 values,because it yielded values that were the most consistent with theavailable human data. Time scaling was performed using the equationCn × t = k, where the exponent n ranges from 0.8to 3.5 (ten Berge et al.1986). An empirical value for n of 0.70 was determined on thebasis of the rat data. The 4-h AEGL-3 value was adopted as the 8-hAEGL-3 value because time scaling yielded an 8-h value inconsistent withthe AEGL-2 values, which were derived from a rather robust humandataset. A total uncertainty factor of 10 was applied. A factor of 3 wasused to account for interspecies differences, because clinical signs arelikely caused by a direct chemical effect on the tissues and this typeof portal-of-entry effect is unlikely to vary greatly between species.Furthermore, calculated LCt50 values for different species are all wellwithin a factor of 2 of each other (88,480 mg-min/m3 forrats, 67,200 mg-min/m3 for guinea pigs, 54,090mg-min/m3 for rabbits, and 50,010 mg-min/m3for mice) (Ballantyne andSwanston 1978). An uncertainty factor of 3 was used toaccount for intraindividual variability because contact irritation is aportal-of-entry effect and is not expected to vary widely amongindividuals. As noted above in support of the AEGL-2 values, a factor of3 is also supported by the results of studies by Punte et al. (1963) and Gutentag et al. (1960) insubjects with pre-existing conditions. AEGL-3 values for CS arepresented in Table 7-15,and calculations presented in Apprendix B.
TABLE 7-15
AEGL-3 Values for Tear Gas.
The AEGL-3 values are considered protective. No mortality was noted inrats, rabbits, or mice exposed to CS at 1,802, 1,434, or 4,250mg/m3 for 10 min, respectively (Ballantyne and Swanston 1978), suggestingthat the 10-min AEGL-3 of 140 mg/m3 is appropriate.Similarly, no deaths were observed in 10 mice or five guinea pigsexposed at 40 or 44.7 mg/m3, respectively, for 5 h (Ballantyne and Callaway1972). One of 10 rats died after exposure to CS at 37mg/m3 for 5 h (Ballantyne and Callaway 1972). These data support the 4-hAEGL-3 of 1.5 mg/m3.
9. REFERENCES
ACGIH (American Conference of Government and IndustrialHygienists). o-Chlorobenzylidene Malononitrile (CAS Reg. No.2698-41-1); Documentation of the Threshold Limit Values and BiologicalExposure Indices; Cincinnati, OH. 1991. pp. 275–278.
ACGIH (American Conference of Government and IndustrialHygienists). TLVs and BEIs Based on the Documentation of theThreshold Limit Values for Chemical Substances and PhysicalAgents and Biological Exposure Indices; o-Chlorobenzylidene malononitrile (CAS Reg. No.2698-41-1); Cincinnati, OH. 2012.
AIHA (American Industrial Hygiene Association). Emergency Response PlanningGuidelines; o-Chlorobenzylidene malononitrile (CAS Reg. No.2698-41-1); Fairfax, VA. 2008.
Anderson PJ, Lau GS, Taylor WR, Critchley JA. Acute effects of the potentlacrimator. o-chlorobenzylidene malononitrile (CS) tear gas. Hum. Exp.Toxicol. 1996;15(6):461–465. [PubMed: 8793527]
Ballantyne B. Riot control agents: Biomedical and health aspects ofthe use of chemicals in civil disturbances. In: Scott RB, Fraser J, editors. Medical Annual: The Year Book of Treatment. Bristol: John Wright; 1977. pp. 7–41.
Ballantyne B, Beswick FW. On the possible relationship between diarrheaand. o-chlorobenzylidene malononitrile (CS). Med. Sci. Law. 1972;12(2):121–128. [PubMed: 5048697]
Ballantyne B, Callaway S. Inhalation toxicology and pathology of animalsexposed to. o-chlorobenzylidene malononitrile. Med. Sci. Law. 1972;12(1):43–65. [PubMed: 5024110]
Ballantyne B, Swanston DW. The comparative acute mammalian toxicity of1-chloroacetophenone (CN) and 2-chlorobenzylidene malononitrile(CS). Arch. Toxicol. 1978;40(2):75–95. [PubMed: 350195]
Bauchinger M, Schmid E. Clastogenicity of 2-chlorobenzylidene malonitrile(CS) in V79 Chinese hamster cells. Mutat. Res. 1992;282(4):231–234. [PubMed: 1379683]
Beswick FW, Holland P, Kemp KH. Acute effects of exposure to orthochlorobenzilidenemalononitrile (CS) and the development oftolerance. Br. J. Ind. Med. 1972;29(3):298–306. [PMC free article: PMC1009428] [PubMed: 5044601]
Blain, P.G. Tear gases and irritant incapacitants.1-chloroacetophenone, 2-chlorobenzylidene malononitrile anddibenz[b,f]-1,4-oxazepine. Toxicol. Rev. 2003;22(2):103–110. [PubMed: 15071820]
Breakell A, Bodiwala GG. CS gas exposure in a crowded night club: Theconsequences for an accident and emergencydepartment. J. Accid. Emerg. Med. 1998;15(1):56–57. [PMC free article: PMC1343011] [PubMed: 9475226]
Brewster K, Harrison JM, Leadbeater L, Newman J, Upshall DG. The fate of 2-chlorobenzylidene malononitrile (CS) inrats. Xenobiotica. 1987;17(8):911–924. [PubMed: 3118582]
Cole TJ, Cotes JE, Johnson GR, Martin H de V, Reed JW, Saunders MJ. Comparison of effects of ammonia and CS aerosol uponexercise ventilation and cardiac frequency in healthymen. J. Physiol. 1975;252((2)):28P–29P. [PubMed: 1107519]
Cole TJ, Cotes JE, Johnson GR, H. de Martin V, Reed JW, Saunders MJ. Ventilation, cardiac frequency and pattern ofbreathing during exercise in men exposed to. o-chlorobenzylidene malononitrile (CS) and ammonia gas inlow concentrations. Q. J. Exp. Physiol. Cogn. Med. Sci. 1977;62(4):341–351. [PubMed: 337342]
Colgrave HF, Creasey JM. Ultrastructure of rat lungs following exposureto. o-chlorobenzylidene malononitrile (CS). Med. Sci. Law. 1975;15(3):187–197. [PubMed: 1099387]
Corson BB, Stoughton RW. Reaction of alpha, beta-unsaturateddinitriles. J. Am. Chem. Soc. 1928;50(10):2825–2837.
Cotes JE, Evans LR, Johnson GR, H. de Martin V, Reed JW. The effect of CS aerosol upon exercise ventilationand cardiac frequency in healthy men. J. Physiol. 1972;222((1)):77P–78P. [PubMed: 5037111]
Craig FN, Blevin WV, Cummings EG. 1960. Breathing Patterns during Human Exposure toCS. Technical Report No. CWLR 2399. AD 318487.. US ArmyChemical Warfare Laboratories, Army Chemical Center, MD. June1960.
Cucinell SA, Swentzel KC, Biskup P, Snodgrass H, Lovre S, Stark W, Feinsilver L, Vocci R. Biochemical interactions and metabolic fate ofriotcontrol agents. Fed. Proc. 1971;30(1):86–91. [PubMed: 4924314]
Euripidou E, MacLehose R, Fletcher A. An investigation into the short term and medium termhealth impacts of personal incapacitant sprays. A follow up ofpatients reported to the National Poisons Information Service(London). Emerg. Med. J. 2004;21(5):548–552. [PMC free article: PMC1726417] [PubMed: 15333526]
Gaskins JR, Hehir RM, McCaulley DF, Ligon EW. Lacrimating agents (CS and CN) in rats and rabbits:Acute effects on mouth, eyes, and skin. Arch. Environ. Health. 1972;24(6):449–454. [PubMed: 4555642]
Gray PJ. Is CS spray dangerous? Formulation affectstoxicity. BMJ. 2000;321(7252):46–47. [PubMed: 10939810]
Gutentag PJ, Hart J, Owens EJ, Punte CL. 1960. The Evaluation of CS Aerosol as a Riot Control Agentin Man. Technical Report CWLR 2365. AD 316 686. US Army ChemicalWarfare Laboratories, Army Chemical Center, MD. April1960.
Haber FR. Five Lectures from the Years 1920-1923 [in German]. Berlin, Germany: Verlag von Julius Springer; 1924. On the history of the gas war; pp. 76–92.
Himsworth H. London: H.M.S.O.. 1969.
HSDB (Hazardous Substances Data Bank). TOXNET Specialized Information Services. US National Library of Medicine; Bethesda, MD: 2005. [Dec. 30, 2013]. 2- Chlorobenzalmalononitrile (CAS Reg. No.2698-41-1). http://toxnet
.nlm.nih.gov/ Hu H, Christiani D. Reactive airways dysfunction after exposure to teargas. Lancet. 1992;339(8808):1535. [PubMed: 1351200]
Hu H, Fine J, Epstein P, Kelsey K, Reynolds P, Walker B. Tear gasharassing agent or toxic chemicalweapon. J. Am. Med. Assoc. 1989;262(5):660–663. [PubMed: 2501523]
Kane LE, Barrow CS, Alarie Y. A short-term test to predict acceptable levels ofexposure to airborne sensory irritants. Am. Ind. Hyg. Assoc. J. 1979;40(3):207–229. [PubMed: 495461]
Karagama YG, Newton JR, Newbegin CJ. Short-term and long-term physical effects of exposureto CS spray. J. R. Soc. Med. 2003;96(4):172–174. [PMC free article: PMC539444] [PubMed: 12668703]
Leadbeater L. The absorption ofortho-chlorobenzylidenemalononitrile (CS) by the respiratorytract. Toxicol. Appl. Pharmacol. 1973;25(1):101–110. [PubMed: 4714331]
Marrs TC, Clifford E, Colgrave HF. Late inhalation toxicology and pathology produced byexposure to a single dose of 2-chlorobenzylidene malononitrile(CS) in rats and hamsters. Med. Sci. Law. 1983a;23(4):257–265. [PubMed: 6419002]
Marrs TC, Colgrave HF, Cross NL, Gazzard MF, Brown RF. A repeated dose study of the toxicity of inhaled2-chlorobenzylidene malononitrile (CS) aerosol in three speciesof laboratory animal. Arch. Toxicol. 1983b;52(3):183–198. [PubMed: 6407458]
McDonald EC, Mahon RT. US Marine Corps Amphibious Reconnaissance (Recon)students requiring hospitalization with pulmonary edema afterstrenuous exercise following exposure too-chloro-benzylidenemalonitrile. Mil. Med. 2002;167((11)):iii–iv. [PubMed: 12448609]
McElhatton PR, Sidhu S, Thomas SH. Exposure to CS gas in pregnancy. J. Toxicol. Clin. Toxicol. 2004;42(4):547.
McGregor DB, Brown A, Cattanach P, Edwards I, McBride D, Caspary WJ. Responses of the L5178Y tk+/tk- mouse lymphoma cellforward mutation assay. II. 18 coded chemicals. Environ. Mol. Mutagen. 1988;11(1):91–118. [PubMed: 3338442]
McNamara BP, Owens EJ, Weimer JT, Ballard TA, Vocci FJ. Toxicology of Riot Control Chemicals CS, CN, and DM; Edgewood Arsenal, MD.. November 1969. 1969.
Meshram GP, Malini RP, Rao KM. Mutagenicity evaluation of riot control agento-chlorobenzylidene malononitrile (CS) in the AmesSalmonella/microsome test. J. Appl. Toxicol. 1992;12(5):377–384. [PubMed: 1447485]
MSZW (Ministerie van Sociale Zaken enWerkgelegenheid). Den Haag: SDU Uitgevers. 2004. [Jan. 2, 2014]. Nationale MAC-lijst 2004:o-Chloorbenzilydeenmalonitril. http://www
.lasrook.net /lasrookNL/maclijst2004.htm. NIOSH (National Institute for Occupational Safety andHealth). Documentation for Immediately Dangerous to Life or HealthConcentrations (IDLHs): o-Chlorobenzylidenemalononitrile; Cincinnati, OH. 1994. [Dec. 30, 2013]. [online]: http://www
.cdc.gov/niosh/idlh/2698411 .html. NIOSH (National Institute for Occupational Safety andHealth). NIOSH Pocket Guide to Chemical Hazards: o-Chlorobenzylidenemalononitrile; Cincinnati, OH. 2011. [Dec. 30, 2013]. http://www
.cdc.gov/niosh/npg/npgd0122 .html. NRC (National Research Council). Guidelines for Developing Community Emergency ExposureLevels for Hazardous Substances. Washington, DC: National Academy Press; 1993.
NRC (National Research Council). Standing Operating Procedures for Developing Acute ExposureGuideline Levels for Hazardous Chemicals. Washington, DC: National Academy Press; 2001. [PubMed: 25057561]
NTP (National Toxicology Program). Toxicology and Carcinogenesis Studies of CS2 (94% o-Chlorobenzalmalononitrile Cas No. 2698-41-1) in F344/N Rats andB6C3F1 Mice (Inhalation Studies); Research Triangle Park, NC. 1990. [Dec. 30, 2013]. http://ntp
.niehs.nih .gov/ntp/htdocs/LT_rpts/tr377.pdf. [PubMed: 12692644] O’Neil MJ, Heckelman PE, Koch CB, Roman KJ, editors. The Merck Index: An Encyclopedia of Chemicals, Drugs andBiologicals. 14th. Whitehouse Station, NJ: Merck; 2006. o-Chlorobenzylidenemalononitrile; p. 352.
Owens EJ, Punte CL. Human respiratory and ocular irritation studiesutilizing o-chlorobenzilidene malononitrileaerosols. Am. Ind. Hyg. Assoc. J. 1963;24:262–264. [PubMed: 13940844]
Paradowski M. Metabolism of toxic doses of o-chlorobenzylidenemalononitrile in rabbits. Pol. J. Pharmacol. Pharm. 1979;31:563–572.
Park S, Giammona ST. Toxic effects of tear gas on an infant followingprolonged exposure. Am. J. Dis. Child. 1972;123(3):245–246. [PubMed: 4554617]
Punte CL, Weimer JT, Ballard TA, Wilding JL. Toxicologic studies on o-chlorobenzylidenemalononitrile. Toxicol. Appl. Pharmacol. 1962;4(5):656–662. [PubMed: 14489226]
Punte CL, Owens EJ, Gutentag PJ. Exposures to ortho-chlorobenzylidene malononitrile:Controlled human exposures. Arch. Environ. Health. 1963;6(3):72–80.
Rengstorff RH. The effects of the riot control agent CS on visualacuity. Milit. Med. 1969;134(3):219–221. [PubMed: 4977501]
Rietveld EC, Delbressine LP, Waegemaekers TH, Seutter-Berlage F. 2-Chlorobenzylmercapturic acid, a metabolite of theriot control agent 2-chlorobenzylidene malononitrile (CS) in therat. Arch. Toxicol. 1983;54(2):139–144. [PubMed: 6418119]
Rietveld EC, Hendrikx MM, Seutter-Berlage F. Glutathione conjugation of chlorobenzylidenemalononitriles in vitro and the biotransformation to mercapturicacids in rats. Arch. Toxicol. 1986;59(4):228–234. [PubMed: 3103585]
Rinehart WE, Hatch T. Concentration-time product (CT) as an expression ofdose in sublethal exposures to phosgene. Am. Ind. Hyg. Assoc. J. 1964;25:545–553. [PubMed: 14248960]
Roth VS, Franzblau A. RADS after exposure to a riot-control agent: A casereport. J. Occup. Environ. Med. 1996;38(9):863–865. [PubMed: 8877834]
Salassidis K, Schmid E, Bauchinger M. Mitotic spindle damage induced by 2-chlorobenzylidenemalonitrile (CS) in V79 Chinese hamster cells examined bydifferential staining of the spindle apparatus andchromosomes. Mutat. Res. 1991;262(4):263–266. [PubMed: 1708109]
Schmid E, Bauchinger M. Analysis of the aneuploidy inducing capacity of2-chlorobenzylidene malonitrile (CS) and metabolites in V79Chinese hamster cells. Mutagenesis. 1991;6(4):303–305. [PubMed: 1943720]
Smith J, Greaves I. The use of chemical incapacitant sprays: Areview. J. Trauma. 2002;52(3):595–600. [PubMed: 11901348]
Striker GE, Streett CS, Ford DF, Herman LH, Helland DR. A Clinicopathologic Study of the Effects of RiotControl Agents on Monkey: IV; o-Chlorobenzylidene Malononitrile (CS) Grenade; Edgewood Arsenal Aberdeen Proving Ground, MD. January 1967. 1967.
ten Berge WF. DoseRespBeta 2006. 2006. [July 5, 2013]. Concentration-time Response in Acute InhalationToxicity. http://home
.wxs.nl/~wtberge/doseresp .html. ten Berge WF, Zwart A, Appelman LM. Concentration-time mortality response relationship ofirritant and systemically acting vapours andgases. J. Hazard. Mater. 1986;13(3):301–309.
Thomas RJ, Smith PA, Rascona DA, Louthan JD, Gumpert B. Acute pulmonary effects fromo-chlorobenzylidenemalonitrile “tear gas”: Aunique exposure outcome unmasked by strenuous exercise after amilitary training event. Mil. Med. 2002;167(2):136–139. [PubMed: 11873536]
Upshall DG. Effects of o-chlorobenzylidene malononitrile (CS) andthe stress of aerosol inhalation upon rat and rabbit embryonicdevelopment. Toxicol. Appl. Pharmacol. 1973;24(1):45–59. [PubMed: 4734456]
US Army Chemical School. Chapter 3 in Potential Military Chemical/Biological Agentsand Compounds. 2005. [Dec. 30, 2013]. Military compounds and theirproperties. FM 3-11.9, MCRP 3-37.1B, NTRP, 3-11.32, AFTTP(I) 3.255.January 2005 [online]. Available: http://www
.fas.org/irp /doddir/army/fm3-11-9.pdf. von Däniken A, Friederich U, Lutz WK, Schlatter C. Tests for mutagenicity in Salmonella and covalentbinding to DNA and protein in the rat of the riot control agento-chlorobenzylidene malononitrile (CS). Arch. Toxicol. 1981;49(1):15–27. [PubMed: 6798951]
WHO (World Health Organization). Health Aspects of Chemical and Biological Weapons; Geneva, Switzerland. 1970. [Dec. 30, 2013]. http://whqlibdoc
.who .int/publications/24039.pdf. Wild D, Eckhardt K, Harnasch D, King MT. Genotoxicity study of CS(ortho-chlorobenzylidenemalononitrile) in Salmonella,Drosophila, and mice. Failure to detect mutageniceffects. Arch. Toxicol. 1983;54(2):167–170. [PubMed: 6418120]
Ziegler-Skylakakis K, Summer KH, Andrae U. Mutagenicity and cytotoxicity of 2-chlorobenzylidenemalonitrile (CS) and metabolites in V79 Chinese hamstercells. Arch. Toxicol. 1989;63(4):314–319. [PubMed: 2504130]
APPENDIX A. TIME-SCALING CALCULATIONS FOR TEAR GAS
The relationship between dose and time for any given chemical is a functionof the physical and chemical properties of the substance and the uniquetoxicologic and pharmacologic properties of the individual substance.Historically, the relationship according to Haber (1924), commonly called Haber’s Lawor Haber’s Rule (C × t = k, where C = exposure concentration,t = exposure duration, and k = a constant), has been used to relate exposureconcentration and duration to effect (Rinehart and Hatch 1964). This conceptstates that exposure concentration and exposure duration may be reciprocallyadjusted to maintain a cumulative exposure constant (k) and that thiscumulative exposure constant will always reflect a specific quantitative andqualitative response. This inverse relationship of concentration and timemay be valid when the toxic response to a chemical is equally dependent onthe concentration and the exposure duration. However, an assessment by tenBerge et al. (1986) of LC50 data for certain chemicals revealedchemical-specific relationships between exposure concentration and exposureduration that were often exponential. This relationship can be expressed bythe equation Cn × t = k, where n represents achemical-specific (and even a toxic end point-specific) exponent. Therelationship described by this equation is basically the form of a linearregression analysis of the log-log transformation of a plot of C vs. t. tenBerge et al. (1986) examined the airborne concentration (C) and short-termexposure duration (t) relationship relative to death for approximately 20chemicals and found that the empirically derived value of n ranged from 0.8to 3.5 among this group of chemicals. Hence, the value of the exponent n inthe equation Cn × t = k quantifies the relationshipbetween exposure concentration and exposure duration. Haber's Rule isthe special case where n = 1. As the value of n increases, the plot ofconcentration vs. time yields a progressive decrease in the slope of thecurve.
An n of 0.70 mg/m3 for CS was obtained by analysis of lethalitydata in rats (McNamara et al. 1969; Ballantyne and Callaway 1972; Ballantyneand Swanston 1978) using the software of ten Berge (2006). Thisexposure-time relationship for lethality was considered appropriate forderiving AEGL-3 values. The 4-h AEGL-3 value was adopted as the 8-h AEGL-3value because time scaling yielded an 8-h value inconsistent with the AEGL-2values that were derived from robust human data.
TABLE A-1Results of ten Berge Program (1% Lethality)
| Species | Exponent n | LC01 Point Estimate,mg/m3 (95% confidence limits) | Reference(s) | ||||
|---|---|---|---|---|---|---|---|
| 10 min | 30 min | 1 h | 4 h | 8 h | |||
| Rat | 0.704 (0.543–0.865) | 1,385 (477–2,500) | 290 (97–496) | 109 (32–196) | 15 (3.1–35) | 5.6 (-0.93–15) | McNamara et al. 1969; Ballantyne and Callaway 1972;Ballantyne and Swanston 1978 |
| Mouse | 0.701 (0.509–0.892) | 998 (208–1,899) | 208 (36–404) | 77 (11–166) | 11 (-0.86–3.2) | 4.0 (-0.23–15) | McNamara et al. 1969; Ballantyne and Callaway 1972;Ballantyne and Swanston 1978 |
| Rabbit | 0.658 (0.467–0.849) | 656 (227–1,136) | 124 (28–249) | 43 (7.0–103) | 5.2 (0.40–19) | 1.8 (0.094–8.6) | McNamara et al. 1969; Ballantyne and Callaway 1972;Ballantyne and Swanston 1978 |
| Guinea pig | 0.559 (0.018–1.099) | 3.65 (0–100) | 0.51 (0–25) | 0.15 (0–12) | 0.012 (0–3.3) | 0.0036 (0–1.8) | McNamara et al. 1969; Ballantyne and Callaway 1972;Ballantyne and Swanston 1978 |
| Dog | 0.356 (-1.464–0.751) | 349a | 7,604a | 53,150a | 2,597,000a | 18,150,000a | McNamara et al. 1969 |
| Monkey | 0.187 (-0.281–0.656) | 26a | 0.075a | 0.0018a | 0.0000011a | 0.000000028a | >McNamara et al. 1969; Striker et al. 1967 |
| Monkey | 2.123 (-21–25) | 11a | 6.6a | 4.7a | 2.5a | 1.8a | McNamara et al. 1969 |
- a
Large variances precluded estimating 95% confidencelimits.
TABLE A-2Data in Rats Used in Log Probit Model
Date: 02 October 2008 Time: 09:11:09
| Sequence No. | Concentration (mg/m3) | Minutes | Exposed | Responded |
|---|---|---|---|---|
| 1 | 560 | 25 | 10 | 1 |
| 2 | 543 | 35 | 10 | 2 |
| 3 | 489 | 45 | 10 | 3 |
| 4 | 454 | 55 | 10 | 5 |
| 5 | 500 | 60 | 10 | 2 |
| 6 | 500 | 80 | 10 | 6 |
| 7 | 500 | 90 | 10 | 8 |
| 8 | 750 | 30 | 8 | 0 |
| 9 | 150 | 120 | 8 | 0 |
| 10 | 3,950 | 5 | 10 | 0 |
| 11 | 4,760 | 5 | 10 | 0 |
| 12 | 4,250 | 10 | 10 | 1 |
| 13 | 4,330 | 10 | 10 | 1 |
| 14 | 4,150 | 15 | 10 | 0 |
| 15 | 5,176 | 15 | 10 | 7 |
| 16 | 4,000 | 20 | 10 | 9 |
| 17 | 4,300 | 20 | 10 | 8 |
| 18 | 1,802 | 10 | 20 | 0 |
| 19 | 1,806 | 45 | 20 | 8 |
| 20 | 1,911 | 45 | 20 | 9 |
| 21 | 2,629 | 60 | 21 | 20 |
| 22 | 2,699 | 60 | 20 | 20 |
| 23 | 37 | 300 | 10 | 1 |
Used Probit Equation Y = B0 + B1*X1 + B2*X2
X1 = conc mg/m3, ln-transformed
X2 = minutes, ln-transformed
ChiSquare = 50.11
Degrees of freedom = 20
Probability Model = 2.13E-04
Ln(Likelihood) = -47.54
B 0 = -9.6233E+00 Student t = -3.9484
B 1 = 1.1705E+00 Student t = 5.6748
B 2 = 1.6634E+00 Student t = 5.6382
Variance B 0 0 = 5.9402E+00
Covariance B 0 1 = -4.8485E-01
Covariance B 0 2 = -6.7032E-01
Variance B 1 1 = 4.2542E-02
Covariance B 1 2 = 4.9302E-02
Variance B 2 2 = 8.7045E-02
Estimation ratio between regression coefficients of ln(conc) andln(minutes)
Point estimate = 0.704
Lower limit (95% CL)
= 0.543
Upper limit (95% CL) = 0.865
Estimation of conc mg/m3 at response of 1%
Minutes = 10
Point estimate conc mg/m3 = 1.385E+03 for response of1%
Lower limit (95% CL) conc mg/m3 = 4.772E+02 for responseof 1%
Upper limit (95% CL) conc mg/m3 = 2.500E+03 for responseof 1%
Estimation of conc mg/m3 at response of 1%
Minutes = 30
Point estimate conc mg/m3 = 2.906E+02 for response of1%
Lower limit (95% CL) conc mg/m3 = 9.659E+01 for responseof 1%
Upper limit (95% CL) conc mg/m3 = 4.963E+02 for responseof 1%
Estimation of conc mg/m3 at response of 1%
Minutes = 60
Point estimate conc mg/m3 = 1.085E+02 for response of1%
Lower limit (95% CL) conc mg/m3 = 3.223E+01 for responseof 1%
Upper limit (95% CL) conc mg/m3 = 1.958E+02 for responseof 1%
Estimation of conc mg/m3 at response of 1%
Minutes = 120
Point estimate conc mg/m3 = 4.052E+01 for response of1%
Lower limit (95% CL) conc mg/m3 = 1.021E+01 for responseof 1%
Upper limit (95% CL) conc mg/m3 = 8.137E+01 for responseof 1%
Estimation of conc mg/m3 at response of 1%
Minutes = 240
Point estimate conc mg/m3 = 1.513E+01 for response of1%
Lower limit (95% CL) conc mg/m3 = 3.122E+00 for responseof 1%
Upper limit (95% CL) conc mg/m3 = 3.501E+01 for responseof 1%
Estimation of conc mg/m3 at response of 1%
Minutes = 480
Point estimate conc mg/m3 = 5.649E+00 for response of1%
Lower limit (95% CL) conc mg/m3 = 9.345E-01 for responseof 1%
Upper limit (95% CL) conc mg/m3 = 1.540E+01 for responseof 1%
