Abstract

Trimethylsilyldiazomethane (TMSD) is a methylating reagent widely used in organic chemistry. TMSD is structurally related to the compound diazomethane, which is a known lethal respiratory toxicant in humans and in animal models. TMSD is less reactive (with lower explosive potential) than diazomethane and is considered a safer, less toxic alternative. Few toxicity data are available to support this claim, however, and TMSD is readily available commercially from chemical suppliers. Concern over the inhalation toxicity of TMSD originates from reports of the death of two chemists resulting from lung injury and acute respiratory distress syndrome following exposure to TMSD in the workplace. Other concerns include the known inhalation toxicity of diazomethane and the absence of inhalation toxicity data for TMSD. The National Toxicology Program (NTP) conducted this study to evaluate the acute inhalation toxicity of TMSD in vivo.

Groups of eight male Sprague Dawley (Hsd:Sprague Dawley^® SD^®) rats and eight male B6C3F1/N mice were exposed by nose-only inhalation to targeted vapor concentrations of 0 (air), 0 (hexanes), or 10 ppm TMSD (in hexanes) for 1 day (30 minutes) with or without a 9-day recovery period following exposure (1-day exposure or 1-day exposure – recovery). Groups of eight male rats and eight male mice were also exposed to 0 (air), 0 (hexanes), 0.3, 1, 3, or 10 ppm TMSD (in hexanes) by nose-only inhalation for up to 5 days (30 minutes/day) with or without a 5-day recovery period following exposure (5-day exposure or 5-day exposure – recovery). Animals were either euthanized the day after the single or last exposure or held for 9 or 5 days postexposure, respectively, and euthanized to assess recovery and delayed effects in the absence of TMSD exposure.

All TMSD-exposed rats survived until scheduled termination. In the 5-day exposure – recovery group exposed to 10 ppm, 4/8 (50%) of the rats exhibited labored breathing. Final mean body weights for the 1-day exposure and 1-day exposure – recovery group rats were similar to those of the air control group, but they were significantly decreased in the 5-day exposure (11.7% lower) and 5-day exposure – recovery rat groups (10.4% lower) at 10 ppm TMSD relative to the air control group. Final mean body weight gains in the 5-day exposure and 5-day exposure – recovery rat groups at 10 ppm TMSD were also significantly decreased relative to the air control groups. Absolute and relative lung weights were significantly decreased in the 1-day exposure rat group at 10 ppm TMSD. Absolute and relative lung weights of rats were significantly increased in the three other exposure groups at the same or lower exposure concentrations: 1-day exposure – recovery group at 10 ppm TMSD, 5-day exposure group at 3 and 10 ppm TMSD, and 5-day exposure – recovery group at ≥0.3 ppm TMSD exposure concentrations.

No exposure-related histopathological lesions were present in the 1-day exposure and 1-day exposure – recovery rat groups at 10 ppm TMSD. In the 5-day exposure and 5-day exposure – recovery groups, increased incidences of lung histopathological lesions included chronic-active inflammation (≥3 ppm), histiocyte (alveolar macrophage) infiltration (3 ppm), interstitial fibrosis (10 ppm), pulmonary edema (10 ppm), acute hemorrhage (10 ppm), and alveolar and bronchiolar epithelial hyperplasia (10 ppm). Alveolar and bronchiolar epithelial hyperplasia were also noted at 3 ppm TMSD in the 5-day exposure group. In addition, increased incidences of histiocyte infiltration in the mediastinal lymph nodes were observed in all rats exposed to 10 ppm TMSD in the 5-day exposure and 5-day exposure – recovery groups.

Inhaled TMSD was more toxic in mice than in rats. Five mice in the 1-day exposure – recovery group and all mice in the 5-day exposure and 5-day exposure – recovery groups died or were euthanized prior to scheduled termination after only one to three exposures, and 19/24 (79%) of the 10 ppm-exposed mice exhibited labored breathing. In the 5-day exposure – recovery group exposed to 3 ppm, all mice (8/8) exhibited labored breathing. Final mean body weights for the 1-day exposure group were similar to the air control group. In the 1-day exposure – recovery group, final mean body weights of the three mice remaining at study day 9 were significantly decreased (26.8% less than the air control group); final mean body weight gain was also significantly decreased in this group. Final mean body weights were significantly decreased in the 5-day exposure group mice at 3 ppm (10.2% lower) and 10 ppm (16.5% lower) and also in the 5-day exposure – recovery group mice at 3 ppm (21.7% lower) and 10 ppm (18.4% lower). Final mean body weight gains were also significantly decreased in both the 5-day exposure group (3 ppm) and the 5-day exposure – recovery group (1 and 3 ppm).

Absolute and relative lung weights were significantly increased in the 1-day exposure and 1-day exposure – recovery mouse groups at 10 ppm TMSD, in the 5-day exposure group mice at ≥0.3 ppm, and in the 5-day exposure – recovery group mice at ≥1 ppm. Increased incidences of lung histopathological lesions in mice were similar to those observed in rats, but also included necrosis and acute inflammation and were observed at greater incidences in the 1 and 3 ppm groups. In addition, lesions occurred in the larynx of mice exposed to 3 and 10 ppm TMSD, which included squamous epithelial hyperplasia and ulceration and acute inflammation. Lung and laryngeal lesions in the 1-day exposure – recovery mouse group at 10 ppm TMSD were similar to those in the 5-day exposure – recovery mouse group but occurred at lower incidences.

This in vivo study showed that inhaled TMSD at low vapor concentrations (≤10 ppm) caused acute and progressive lung injury, as reported in human case studies (in particular, pulmonary edema), and that these effects can occur after a single 30-minute exposure to the chemical. These data provide useful information for mitigating exposure risks in the workplace and alerting chemical suppliers to the dangers of TMSD.

Contents

• Foreword
• Collaborators
• Contributors
• Peer Review
• Publication Details
• Acknowledgments
• Introduction
  • Background
  • Chemical Use and Human Exposure
  • Case Reports
  • Study Rationale
• Materials and Methods
  • Procurement and Characterization of Trimethylsilyldiazomethane
  • Vapor Generation and Exposure System
  • Vapor Concentration Monitoring
  • Chamber Atmosphere Characterization
  • Animal Source
  • Animal Welfare
  • Exposure Concentration Selection Rationale
  • Health and Safety
  • One-day and Five-day Studies
  • Statistical Methods
  • Quality Assurance Methods
• Results
  • Data Availability
  • Rats: One-day and Five-day Studies
  • Mice: One-day and Five-day Studies
• Discussion
• References
• Appendix A. Chemical Characterization and Generation of Exposure Concentrations
• Appendix B. Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration
• Appendix C. Sentinel Animal Program
• Appendix D. Supplemental Data