National Toxicology Program

National Toxicology Program

Abstract from Report IMM20602 on 2,3-Butanedione

Report on the Assessment of Contact Hypersensitivity of 2,3-Butanedione (Diacetyl) in Female BALB/c Mice (CAS No. 431-03-8)

Report Date: August 2012

The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of the NTP or the U.S. Government.


2,3-Butanedione, also known as diacetyl, is a natural by-product of secondary or malolactic fermentation that can be found in bay oils, beer, butter, coffee, vinegar, and other food products (Escamilla-Hurtado et al., 1996). BTD has been used as an artificial flavoring to impart the characteristic flavor most commonly associated with butter, cream, and butterscotch (Escamilla-Hurtado et al., 1996; Midje et al., 2000; Monnet et al., 2000). However, exposure to diacetyl may produce adverse effects to the skin, eye, mucous membranes, and the respiratory system (Morgan et al., 2008; Palmer et al., 2011). Studies in rats have indicated that inhalation of butter flavoring vapors containing 285-371 parts per million diacetyl for 6 hours was associated with epithelial injury in the nasal passages and pulmonary airways when the rats were necropsied one day after exposure (Hubbs et al., 2002). Kreiss et al. (2002) have demonstrated that some workers directly involved in the production of microwave popcorn displayed skin problems and shortness of breath upon exertion. The predominant aerosolized compounds to which these workers were exposed included diacetyl, methyl ethyl ketone, acetoin, 2-nonanone, and acetic acid. Diacetyl has been reported to be present in the air of popcorn plants at concentrations ranging from below detectable limits to 98 ppm (Akpinar-Elci et al., 2004; Kullman et al., 2005).

The objective of this study was to determine the sensitizing potential of BTD when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the combined local lymph node assay and irritancy assay. BTD was prepared daily in acetone:olive oil (4:1), and the highest concentration of BTD used for the LLNA was 25% (v/v). In the LLNA, BTD at concentrations of 1%, 5%, 10%, and 25% produced significant increases in lymph node cell proliferation when compared to the vehicle controls. BTD was not an irritant at concentrations up to 25%, as demonstrated by a lack of significant changes in the ear thickness 24 hours following the last exposure.

Two Mouse Ear Swelling Test studies were subsequently conducted to further investigate the potential contact hypersensitivity of BTD. In the sensitization phase, two concentrations of BTD (10% and 25%) were used, and 25% BTD was applied during the challenge phase. In the first MEST study, treatment with the positive control (2,4-dinitrofluorobenzene) failed to produce a significant change in the percent ear swelling at 48 hours post-challenge when compared to the positive control challenge only group. DNFB is well-documented as a strong sensitizer, and the lack of an appropriate response in the positive control resulted in the study being rejected. Thus a second MEST study was conducted. Consistent with the results of the LLNA, mice that were sensitized with 10% - 25% BTD and challenged with 25% BTD exhibited statistically significant increases in percent ear swelling at 48 hours post-challenge when compared to the vehicle irritancy control in the repeat study.

Overall, the results from these studies in BALB/c mice have demonstrated that BTD at concentrations ranging from 10% - 25% produced significant increases in lymph node cell proliferation that were above the three-fold threshold when compared to the vehicle controls. Furthermore, BTD was not an irritant at the concentrations tested. In the MEST studies, exposure to BTD at 10% or 25% for sensitization and 25% for challenge produced positive responses, which reached the level of statistical significance at the high dose level.

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