Abstract

Acetoin and 2,3-pentanedione are highly volatile components of artificial butter flavoring (ABF). Concerns over the inhalation toxicity of these compounds originate from the association between occupational exposures to ABF and adverse fibrotic lung effects, specifically obliterative bronchiolitis (OB) in the distal airways. 2,3-Pentanedione has been used as a replacement for 2,3-butanedione (diacetyl) in some ABF due to concerns about the respiratory toxicity of 2,3-butanedione. However, 2,3-pentanedione is structurally similar to 2,3-butanedione and has been shown to exhibit potency similar to 2,3-butanedione regarding airway toxicity following acute inhalation (whole-body) exposure. This report describes a series of studies to evaluate the 2-week inhalation toxicity of acetoin and the 3-month inhalation toxicity of acetoin and 2,3-pentanedione.

In the 2-week studies, groups of five male and five female Wistar Han [Crl:WI(Han)] rats and B6C3F1/N mice were exposed via whole-body inhalation to acetoin vapors at concentrations up to 800 ppm for 6 hours per day, 5 days per week, for 2 weeks (plus 2 to 3 days). In the 3-month study of acetoin, groups of 10 male and 10 female rats and mice were exposed via whole-body inhalation to vapor concentrations up to 800 ppm for 6 hours per day, 5 days per week, for 13 to 14 weeks. There were no significant exposure-related adverse effects in rats or mice exposed to acetoin for either 2 weeks or 3 months.

In the 3-month 2,3-pentanedione study, groups of 10 male and 10 female rats and mice were exposed via whole-body inhalation to vapor concentrations up to 100 ppm for 6 hours per day, 5 days per week, for 13 to 14 weeks. All rats and mice survived until scheduled euthanasia. Clinical observations in rats and mice exposed to 50 or 100 ppm included abnormal breathing, eye abnormality, and sneezing. There were no significant exposure-related changes in terminal mean body weights of rats, and mean body weights remained within 8% of the control group for the duration of the study. Absolute and relative lung weights and relative heart weights were significantly increased in female rats exposed to 100 ppm compared to those of the control group.

In male and female mice exposed to 50 or 100 ppm 2,3-pentanedione for 3 months, terminal mean body weights were significantly decreased compared to control mice. Relative lung weights of male mice exposed to ≥50 ppm and female mice exposed to 100 ppm were significantly increased, whereas absolute lung weights were significantly decreased in female mice exposed to ≥50 ppm 2,3-pentanedione. Absolute heart, kidney, and liver weights of male and female mice and absolute spleen weights of female mice exposed to ≥50 ppm, as well as absolute spleen weights of male mice and relative spleen weights of female mice exposed to 100 ppm, were significantly decreased. Relative heart and spleen weights of male mice and relative kidney weights of female mice exposed to 100 ppm were significantly increased.

On day 23 and at the end of the 3-month 2,3-pentanedione study, the white blood cell, lymphocyte, monocyte, and/or neutrophil counts were significantly increased in various exposed male and female rats but most consistently at 50 and 100 ppm. The one exception was a significant decrease in the lymphocyte count in the 100 ppm male group at study termination. The leukocyte increases were consistent with inflammation, and the decrease in lymphocytes was most likely due to chronic stress. In addition, at study termination, globulin concentration was significantly increased, and albumin concentration was significantly decreased in the 100 ppm female rats. Related to these changes, the albumin/globulin (A/G) ratio was significantly decreased in male rats exposed to 50 or 100 ppm and in female rats exposed to 100 ppm. These changes were also consistent with an exposure-related proinflammatory response.

Exposure to 2,3-pentanedione, but not acetoin, for 3 months caused adverse nonneoplastic histopathological effects in the nose, larynx, trachea, and lungs of rats and mice.

Significant effects in the nose with exposure to ≥50 ppm 2,3-pentanedione included, among others, an increase in the incidences of suppurative inflammation, respiratory metaplasia of the olfactory epithelium, and hyperplasia and squamous metaplasia of the respiratory epithelium in male and female rats and mice (≥25 ppm for hyperplasia in male rats and squamous metaplasia in male and female mice). Significant effects in the larynx with exposure to 50 and/or 100 ppm included an increase in the incidences of hyperplasia of the squamous epithelium in male and female rats, hyperplasia and squamous metaplasia of the respiratory epithelium in male rats, and chronic active inflammation and squamous metaplasia of the respiratory epithelium (≥25 ppm) in female rats. In male and female mice with exposure to 50 and/or 100 ppm, there were significant increases in the incidences of suppurative inflammation, atypical squamous metaplasia of the respiratory epithelium, and atypical hyperplasia of the squamous epithelium.

Significant effects in the trachea with exposure to 50 and/or 100 ppm 2,3-pentanedione included an increase in the incidences of hyperplasia and regeneration of the epithelium (and squamous metaplasia by positive trend only) in male and female rats and inflammation, regeneration, and atypical squamous metaplasia of the epithelium in male and female mice (and atypical hyperplasia in females by positive trend only). Significant effects in the lung with exposure to 100 ppm 2,3-pentanedione included an increase in the incidences of inflammation and hyperplasia of the bronchial and bronchiolar epithelium and bronchial epithelial regeneration in male and female rats (and squamous or goblet cell metaplasia of the bronchial epithelium by positive trend only). In mice with exposure to 50 and/or 100 ppm 2,3-pentanedione, there were significant increases in the incidences of inflammation, atypical squamous metaplasia, and regeneration of the bronchial epithelium in males and females, atypical hyperplasia of the bronchial epithelium in males, and hyperplasia of the bronchiolar epithelium in males.

Exposure of female rats to 50 and/or 100 ppm 2,3-pentanedione caused an increase in the incidence of acute inflammation in the cornea and ciliary body of the eye. Acute inflammation in the cornea was also observed in male rats and female mice (by positive trend only).

Acetoin was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, and TA1535 when tested with and without 10% or 30% induced rat or hamster liver S9. Results of the peripheral blood micronucleus tests with acetoin and 2,3-pentanedione were negative in male and female rats and mice following 3 months of exposure via whole-body inhalation. In the acetoin study, an exposure concentration-related increase in the percentage of immature erythrocytes (% PCE) was observed in male, but not female, rats, suggesting a mild perturbation in erythropoiesis in male rats. In the 2,3-pentanedione study, a small exposure concentration-related increase in % PCE was seen in female rats but not in male rats. No alterations in % PCE values were observed in mice in either study.

Under the conditions of this inhalation study, there were no significant exposure-related adverse effects in rats or mice exposed to acetoin for 2 weeks or 3 months. Exposure to 2,3-pentanedione via whole-body inhalation for 3 months caused significant adverse effects primarily in the respiratory tract, but also in the eyes, of rats and mice. These airway findings attributed to 2,3-pentanedione included exposure-related inflammation, injury (degeneration), regeneration, squamous metaplasia, and/or hyperplasia of the tracheal, bronchial, and bronchiolar epithelium. Interestingly, the hyperplasia and squamous metaplasia of the tracheal and bronchial epithelium observed in the mice were considered atypical and therefore potentially preneoplastic. The no-observed-effect level (NOEL) for the bronchial and bronchiolar adverse effects in the lung was 25 ppm in rats and 12.5 ppm in mice after 3 months of exposure to 2,3-pentanedione. These lesions are most relevant because the distal bronchi/bronchioles are the target sites for OB, and hyperplasia/squamous metaplasia could accompany or be precursors to fibrotic lesions (e.g., if the tested exposure concentration was higher) and the morphology of the bronchial/bronchiolar lesions is similar to OB. Regarding eye toxicity, inflammation and degenerative lesions of the cornea were observed, which extended into other ocular compartments in some animals, including the anterior chamber, ciliary body, sclera, conjunctiva, and iris. The NOEL of 2,3-pentanedione for this study overall was 12.5 ppm on the basis of adverse respiratory tract effects in rats and mice. These 3-month inhalation exposure data, including NOELs for adverse respiratory tract effects, can inform regulatory agencies to help mitigate exposure risks to 2,3-pentanedione vapors in the workplace.

Contents

• Foreword
• Collaborators
• Contributors
• Peer Review
• Publication Details
• Acknowledgments
• Abstract
• Overview
• Introduction
  • Chemical and Physical Properties
  • Production, Use, and Human Exposure
  • Regulatory Status
  • Absorption, Distribution, Metabolism, and Excretion
  • Toxicity
  • Reproductive and Developmental Toxicity
  • Carcinogenicity
  • Genetic Toxicity
  • Study Rationale
• Materials and Methods
  • Procurement and Characterization of Acetoin and 2,3-Pentanedione
  • Vapor Generation and Exposure System
  • Vapor Concentration Monitoring
  • Chamber Atmosphere Characterization
  • Animal Source
  • Animal Welfare
  • Exposure Concentration Selection Rationale
  • Two-week Studies
  • Three-month Studies
  • Statistical Methods
  • Quality Assurance Methods
  • Genetic Toxicology
• Results
  • Data Availability
  • Acetoin
  • 2,3-Pentanedione
  • Genetic Toxicology
• Discussion
• References
• Appendix A. Chemical Characterization and Generation of Chamber Concentrations
• Appendix B. Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration
• Appendix C. Sentinel Animal Program
• Appendix D. Genetic Toxicology
• Appendix E. Supplemental Data