Determination of LCt₅₀s in anesthetized rats exposed to aerosolized nerve agents

Abstract

Nerve agents pose a threat to the respiratory tract with exposure that could result in acute compromised lung performance and death. The determination of toxicity by inhalation is important for the rational development of timely therapeutic countermeasures. This study was designed to deliver aerosolized dilute nerve agents in a dose-response manner to investigate the extent of lethality of nerve agents: soman, sarin, VX and VR. Male rats (240–270 g) were anesthetized intramuscularly with 10 mg/kg xylazine and 90 mg/kg ketamine. Following anesthesia, rats were intubated with a glass endotracheal tube (ET) and placed in a glove box. The ET was connected to a closed circuit nebulizer system (Aeroneb, Aerogen, Inc.) that delivered a particle size of < 2.0 µm and was in series between the ventilator and the ET. Nerve agents were delivered by a small animal ventilator set for a volume of 2.5 mL × 60–80 breaths/min. VX or VR were nebulized and delivered in concentrations ranging from 6.25–800 µg/kg over a 10-min exposure time period. Sarin (GB) or soman (GD), 6.5–1250 µg/kg, were delivered in a similar manner. Lethality by inhalation occurred either during the 10-min exposure period or less than 15 min after the cessation of exposure. Survivors were euthanized at 24 h postexposure. LCt₅₀ estimates (± 95% confidence intervals [CIs]) were obtained from the sequential stage-wise experiments using the probit analysis. Probit analysis revealed that the LD₅₀ for VX was 110.7 µg/kg (CI: 73.5–166.7), VR 64.2 µg/kg (CI: 42.1–97.8); soman (GD), 167 µg/kg (CI: 90–310), and sarin (GB), 154 µg/kg (CI: 98–242), respectively. Although VR is a structural isomer of VX, the compounds appear to be markedly different in terms of toxicity when delivered by aerosol. These relationships were converted to actual 10 min LCt₅₀ equivalents: VX = 632.2, VR = 367, GD = 954.3 and GB = 880 mg·min/m³. Validation of exposure was verified by the determination of blood levels of acetylcholinesterase (AChE) across doses for the agent VR.

Keywords::

• Chemical warfare nerve agents
• aerosol
• inhalation LCt₅₀ determination
• acetylcholinesterase
• anesthetized rodents

Acknowledgments

The authors wish to thank Theresa Tezak-Reid, Stephanie Sill, and Adam Wyatt from USAMRICD graphics group and Douglas Sommerville from the Edgewood Chemical and Biological Center for technical advice.

The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the United States Army or the Department of Defense.

The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense, and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, Publication No. 85-23, 1996), and the Animal Welfare Act of 1966 (P.L. 89-544), as amended.

Declaration of interest

This project was supported by the Department of Defense and the Defense Threat Reduction Agency. 3.F0007.RC.C.09. The authors report no declarations of interest.