Preparation and Characterization of SM
SM (Bis(2-chloroethyl) sulfide) was synthesized by proprietary methods. The identification and purity of SM was characterized by gas chromatography/mass spectroscopy (GC/MS, Agilent, Santa Clara, CA), proton nuclear magnetic resonance imaging (1H NMR, Bruker, Billerica, MA), and MiniCAMS GC/flame ionization detector (FID) (O I Analytical, Pelham, AL). The GC/MS ionization pattern in the spectrum was consistent with the structure of SM. 1H NMR analysis indicated a single, pure compound with chemical shifts consistent with the structure of SM. GC/FID also showed a single peak in an extended chromatogram intended to identify the presence of reaction byproducts or SM breakdown products; none were detected. The purity of SM was determined to be greater than 99%.
Configuration of the Vesicant Exposure Suite
The exposure suite floor plan () consists of two ante rooms located on either side of a central exposure room. The air pressure in the central exposure room was maintained at a negative pressure to the ante rooms that are maintained at a negative pressure with respect to the main hallway to ensure test agent was confined to the exposure room in the unlikely event of a spill or leak. All SM work was conducted inside the exposure room, with the ante rooms serving as procedure and storage areas. Entrance into the exposure suite is granted via key-card access limited to a maximum of 10 individuals.
Sulfur mustard exposure suite floor plan
All work is conducted in stainless steel/Lexan glove boxes maintained at negative pressure with respect to the exposure room. The glove boxes each contain a pass box to ensure the safe transfer of materials. Each glove box exhaust and all of the transit lines were directed through a charcoal filter to remove any SM present. The glove box system was configured with an audio-visual alarm system to sound if the glove box pressure increased to ambient or greater.
A central SM atmosphere generation glove box was connected by a series of valves to three other glove boxes used to expose animals by various routes of administration. All delivery systems were composed of stainless steel or anodized aluminum unless otherwise noted. Air flows were controlled and monitored using calibrated rotameters. A separate glove box was dedicated to the delivery of potential therapeutics. A limited access storage cabinet was located in the exposure room for storage of synthesized SM and precursors. The analytical equipment for monitoring exposure atmospheres and industrial hygiene was located in the exposure room.
SM Vapor Generation
SM vapors were generated with a custom built stainless steel J-tube (McClellan and Henderson, 1995
). The J-tube was filled with glass beads and blanketed with a heat jacket and a temperature controller to maintain an operating temperature of 160°C (). Nitrogen, supplied by gas cylinder, was used as the carrier gas through the J-tube at a flow rate of 2.1 L/min. Neat SM was injected into the J-tube via syringe through a rubber septum. The syringe speed was controlled by a syringe pump (Kent Scientific, Torrington, CT), with the speed determined by the desired vapor concentration.
FIGURE 2 J-tube for sulfur mustard vapor generation. Neat sulfur mustard is introduced into the stainless steel J-tube via syringe pump. The J-tube is packed with glass beads and wrapped with heat tape to control the temperature of vaporization. Vaporization is (more ...)
SM Aerosol Generation and Particle Size Determination
SM aerosol generation was attempted using two separate approaches. First, an attempt to condense SM vapors by passing them through a 90-cm countercurrent heat exchanger (condenser) that chilled the vapor to approximately 5°C was conducted. This approach was unsuccessful. SM aerosols from ethanol ultimately were generated by compressed jet nebulization with a Swirler nebulizer (Swirler, Amici Inc., Spring City, PA). Solution concentrations of SM in ethanol ranged from 0.1%–1%. Because the goal of the aerosol was to deliver SM to the deep lung, a small particle size was targeted. Previous studies have indicated that a 0.5-micron aerosol provides enhanced pulmonary deposition in rodents (~10–15%) compared with larger aerosols (Rabbe, 1982
). This targeted small particle size was implemented with a nebulizer customized to enhance pulmonary deposition through the creation of small particles. The nebulizer was operated at a pressure of 2100 cm of H2
The concentration of the SM aerosol atmospheres was controlled by the concentration of SM in the nebulizer solution. Aerosol size determination was determined by a time-of-flight analyzer (APS, TSI, Inc., Shoreview, MN). Importantly, this method of aerosol size analysis does not specifically measure the size of SM. Rather, it measures the size of all of the droplets in the aerosol, the majority of which are composed of ethanol in the formulation. An alternative approach to specifically collect and measure SM droplets by a cascade impactor was evaluated. However, spike-recovery of SM onto cascade impactor substrates showed that the agent quickly evaporated, prohibiting the ability to quantitatively (or qualitatively) assess the size and quantity of SM in the aerosol.
Nose-Only Inhalation Exposure System
A schematic depicting the nose-only inhalation exposure glove box is shown in . A 48-port nose-only inhalation chamber (In-Tox Products, Moriarty, NM) was operated at an exhaust flow rate of 20.5 L/min. The exhaust was filtered through both a HEPA and charcoal filter before returning to the main exhaust line. The chamber was maintained from 1.25–3.75 cm of water negative by the controlled addition of filtered dilution air. Oxygen and temperature were monitored.
Inhalation exposure schematic showing a nose-only inhalation system that precedes charcoal/HEPA filters that are used to remove SM from the effluent. Sample ports on either side of the exhaust filter enable evaluation of scrubbing efficiency.
Intratracheal Inhalation Exposure System
A schematic depicting the intubation exposure system is shown in , and is a more detailed description of the exposure plenum. SM vapor atmospheres were generated as previously described. This system was fabricated from stainless steel for the distribution and exposure plenums and Teflon-lined Tygon tubing (Saint Gobain, Valley Forge, PA) was used for the vapor delivery lines. Flow from the generator glove box into the intubation exposure glove box was directed through a distribution plenum. The distribution plenum was attached to four exposure lines that transited SM vapor to four individual exposure plenums. The exhaust flow through the distribution plenum was maintained at 20.5 L/min. A small plug of glass wool was placed inside the distribution plenum to help enhance the homogeneity of the vapor prior to its distribution to the exposure plenums. Flows at the exposure plenum were 700 mL/min. These flows were controlled by mass flow controllers operated remotely by a computer interface and custom LabView (version 8.0) application.
FIGURE 4 Intubation inhalation exposure system schematic. Vapors are introduced to a central distribution plenum and then diverted to individual exposure plenums that are connected directly to an intubation tube. Rodents breathe on demand from the exposure plenum. (more ...)
FIGURE 5 Exposure sled schematic illustrating the intubation sled that is used to deliver SM directly to the intubated rat. SM vapors traverse through the exposure plenum at 700 ml/min, and the rat breathes from the plenum on demand. Oxygen and SM concentration (more ...)
Dilution air to the distribution plenum was composed of a mixture of compressed air and medical grade oxygen that transited through an isoflurane (JD Medical, Phoenix AZ) vaporizer to supply a continuous anesthetic into the exposure atmosphere. The flow through the vaporizer was set to approximately 23 L/min and adjusted as needed to maintain negative 0.64-1.27 cm of water at the exposure plenum. The vaporizer output was connected to the SM intubation system and the vaporizer adjusted between 1.5–2.5% isoflurane in the system in order to maintain the breathing rates of rodents (40–50 breaths per minute) placed on the system. The oxygen concentration also was adjusted to maintain 21% oxygen at the exposure plenum. is a schematic depicting the exposure plenum/exposure sled layout.
SM Atmosphere Characterization
Samples were collected from both the nose-only inhalation system and the intubation systems directly from the breathing zone of the animal. Samples were collected by drawing a gas-tight syringe from the systems and injecting the sample onto the MiniCAMS Tenax sorbent. Samples were analyzed by sorbent desorption GC with a flame photometric detector (MiniCAMS, O I Analytical, Pelham, AL). The GC method resulted in a SM chromatographic peak at 125 s. SM vapor concentrations were calculated by comparison of the generated peak area to a standard curve generated on the day of exposure. Two five-point standard curves were created: a low level curve linear from 3.2–12.7 ng and a high level curve linear from 25.4–106 ng. Standards were created in acetone. The R2 values were greater than 0.98, and each point was calculated to be within 15% of theoretical and 20% at the curve’s sensitivity limit of 3.2 or 25.4 ng.
Animal Handling and Exposure
Female F344 rats (Charles River Labs, Wilmington MA, 11–13 weeks, 170–190 g) were quarantined for a minimum of 2 weeks prior to use. Animal studies were approved by the LRRI Institutional Animal Care and Use Committee, conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, and carried out in compliance with the Guide for the Care and Use of Laboratory Animals (NRC, 1996). During all periods animals were provided water and food (Harlan-Teklad, Madison, WI) ad-libitum. The exception was during the procedures when animals were removed from their standard housing for SM exposures. For nose-only exposure, rodents were conditioned to nose-only tube restraints. Air flow was maintained through the system during addition and removal of rodents from the nose-only exposure chamber. No anesthetic was used for nose-only inhalation. For intubation exposures, rodents were injected subcutaneously with an anesthetic cocktail consisting of acepromazine (0.79 mg/kg), ketamine (39.5 mg/kg), and xylazine (3.95 mg/kg); however, when these animals were intubated and exposed to SM, the anesthesia wore off prior to the conclusion of the exposure. Control atmospheres did not result in a similar observation. As an alternative, animals were anesthetized with isoflurane (5% induction, 2% maintenance). Anesthetized rodents were intubated with a 14-gauge Teflon catheter (approximately 5 cm in length). The catheter was inserted into the trachea to a terminal location approximately half way between the bifurcation of the trachea and the larynx. The placement and seal of the catheter was verified by observing air displacement in a ground glass gas-tight syringe during an inhalation/exhalation cycle. Intubated rodents were transferred to the exposure plenum. An airflow consisting of SM + vehicle or vehicle alone was constantly running through the exposure plenum. Rodents were intubated and exposed to SM on a single occasion and were allowed to recover for observations up to 21 days post exposure. Rodents were not fasted prior to SM exposures. The nose-only exposure system was able to accommodate ~45 rats during a single exposure. The intubation exposure system was able to accommodate 4 rats per exposure.
After exposure the animals were returned to temporary cages for off gassing. The cages were then coupled to a vacuum line to assist in increasing the air exchange to remove any residual SM that may have sorbed to their fur during exposure. SM concentration in the cages was monitored until the concentration was determined to be less than 1.6 μg/m3
, which is lower than the occupational 8-h time-weighted average concentration of 3 μg/m3
as set by the U.S. Army Center for Health Promotion and Preventive Medicine (US Army, 2004
). Off-gassing times were between 15 min for the intubation exposures and 90 min for the nose-only inhalation exposures. At the conclusion of the off-gassing period, animals were returned to their normal cages.
Necropsy and Histopathology
Rodents were euthanized with an ip injection of a barbiturate followed by exsanguination. The lungs were inflated with 10% neutral buffered formalin (NBF) via tracheal cannula until the pleura was tense and fixed further by immersion in NBF. The skulls were removed, and nasal passages were flushed with NBF. Skulls were fixed further in NBF and decalcified with formic acid. Standard transverse sections of the rostral and caudal portions of the nasal passages were obtained (4 per rat; Young, 1981
). The left, right accessory, and right caudal lung lobes were trimmed sagittally along the axial airways. Tissues were processed routinely, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin for evaluation by light microscopy.