Evaluation of the hypersensitivity potential of alternative butter flavorings

Abstract

Concern has been raised over the association of diacetyl with lung disease clinically resembling bronchiolitis obliterans in food manufacturing workers. This has resulted in the need for identification of alternative chemicals to be used in the manufacturing process. Structurally similar chemicals, 2,3-pentanedione, 2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione, used as constituents of synthetic flavoring agents have been suggested as potential alternatives for diacetyl, however, immunotoxicity data on these chemicals are limited. The present study evaluated the dermal irritation and sensitization potential of diacetyl alternatives using a murine model. None of the chemicals were identified as dermal irritants when tested at concentrations up to 50%. Similar to diacetyl (EC3=17.9%), concentration-dependent increases in lymphocyte proliferation were observed following exposure to all four chemicals, with calculated EC3 values of 15.4% (2,3-pentanedione), 18.2% (2,3-hexanedione), 15.5% (3,4-hexanedione) and 14.1% (2,3-heptanedione). No biologically significant elevations in local or total serum IgE were identified after exposure to 25-50% concentrations of these chemicals. These results demonstrate the potential for development of hypersensitivity responses to these proposed alternative butter flavorings and raise concern about the use of structurally similar replacement chemicals. Additionally, a contaminant with strong sensitization potential was found in varying concentrations in diacetyl obtained from different producers.

Keywords: Butter flavorings; Diacetyl; Hypersensitivity; LLNA.

Fig. 1

Ear swelling as a result of dermal exposure to butter flavorings. Analysis of irritation after exposure to 2,3-hexanedione (A), 3,4-hexanedione (B), 2,3-heptanedione (C), 2,3-pentanedione (D), and diacetyl (E). Bars represent means ± SE of 5 mice per group.

Fig. 2

Sensitization potential after dermal exposure to butter flavorings. Analysis of the sensitization potential of 2,3-hexanedione (A), 3,4-hexanedione (B), 2,3- heptanedione (C), 2,3-pentanedione (D), and diacetyl (E) using the LLNA. ³H-thymidine incorporation into draining lymph node cells of BALB/c mice following exposure to vehicle or concentration of butter flavoring shown above. Bars represent means ± SE of 5 mice per group. Levels of statistical significance are denoted as ^**(p ≤ 0.01) as compared to acetone vehicle.

Fig. 3

Identification of a potential diacetyl contaminant. Analysis of the sensitization potential of diacetyl produced by Aldrich & Fluka (A) and Acros & TCI (B) using the LLNA and of diacetyl products. ³H-thymidine incorporation into draining lymph node cells of BALB/c mice following exposure to vehicle or concentration of butter flavoring shown above. Bars represent means ± SE of 5 mice per group. Levels of statistical significance are denoted as ^*(p ≤ 0.05) and ^**(p ≤ 0.01) as compared to acetone vehicle. (C) Overlaid gas chromatograms of prepared 0.082 M diacetyl solutions (see text) showing the variation in contaminant concentration. All chromatograms are on the same scale.

Fig. 4

Proposed diacetyl contaminant. Possible mechanism leading to the aldol condensation product, 5-methyl-4-heptene-2,3,6-trione, between two diacetyl molecules.