ToxSci Advance Access originally published online on July 3, 2008
Toxicological Sciences 2008 105(2):433-434; doi:10.1093/toxsci/kfn134
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Published by Oxford University Press 2008.
Response to: Comments on Respiratory Toxicity of Diacetyl in C57Bl/6 Mice
* Respiratory Toxicology, Laboratory of Molecular Toxicology, National Toxicology Program/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
1 To whom correspondence should be addressed at Respiratory Toxicology, Laboratory of Molecular Toxicology, MD IF-00, NIEHS, Research Triangle Park, NC 27709. Fax: (919) 541-0356. E-mail: morgan3{at}niehs.nih.gov.
Received June 25, 2008; accepted June 27, 2008
We appreciate the opportunity provided by Finley et al. to highlight the rationale for the diacetyl exposure regimens used in our animal studies and their relevance to the actual worker exposure concentrations measured in the sentinel microwave popcorn packaging plant. In addition, we appreciate the opportunity to emphasize the unique respiratory toxicological profile of diacetyl in mice which includes epithelial injury and lymphocytic bronchiolitis after inhalation and fibrohistiocytic lesions in the terminal bronchioles after aspiration.
Worker exposures to diacetyl in the microwave popcorn packaging plants vary considerably, and as with any occupational exposure, it is not possible to duplicate exactly an exposure scenario for all workers. After evaluating the types of worker exposures reported by Kreiss et al. (2002)
, we designed several diacetyl exposure profiles utilizing constant or episodic exposure regimens at concentrations reported in the sentinel plant. We purposely avoided stating that the animal exposure regimens used in this study were "similar" or "represent typical worker exposures" (misquote by Finley et al.), and we stand by our statement that the exposure regimens we used are "relevant to workplace exposures".
The authors disagree with Finley et al. that the exposure concentrations we used were orders of magnitude higher than typical time-weighted average (TWA) values measured in popcorn packaging plants. The concentrations of diacetyl used in the 12-week inhalation study were based upon the 6.5- to 8-h TWA diacetyl exposures measured by Kreiss et al. (2002) at the sentinel popcorn packaging plant. These TWA concentrations ranged from 1.34 to 97.9 ppm in the mixing room with a mean of 37.8 ppm (10 samples taken). We selected diacetyl concentrations of 0, 25, 50, 100 ppm to represent this exposure range. Selection of 100 ppm diacetyl as the high concentration in this study was reasonable, because in a plant where 97.9 ppm exposures were documented, about 20% of workers had airways obstruction (Kreiss et al., 2002).
Contrary to the comment by Finley et al., nowhere in the paper do we "assert that diacetyl must be able to cause obliterative airway lesions in humans." The authors agree with Hubbs et al. (2002) that in addition to diacetyl, other components of artificial butter flavoring may contribute to respiratory disease. Diacetyl was studied in these initial studies because it is the most prevalent volatile organic compound detected in the vapors from heated artificial butter flavoring, and because a relationship between diacetyl exposures and the incidence of obstructive lung disease was reported (Kreiss et al., 2002). Whereas diacetyl did not cause obstructive airway disease in these short-term rodent studies, OB-like lesions may develop with longer exposures. It is also possible that other volatile components of artificial butter flavoring may act additively or synergistically with diacetyl to cause airway disease. Other volatile components of artificial butter flavoring are also being investigated in inhalation studies.
The respiratory tract lesions resulting from diacetyl aspiration may not be unique to diacetyl. One would expect that structurally related ketones could cause similar lesions. Differences in the site of toxicity would be influenced by individual chemical properties such as water solubility and reactivity of carbonyls. Aspiration studies with other volatile components of artificial butter flavoring are in progress. Because many of these volatile components are ketones and aldehydes structurally similar to diacetyl, it would not be surprising if their toxicity profiles were similar to that of diacetyl.
The authors agree with the statement of Finley et al. that the lymphocytic bronchitis and lymphocytic bronchiolitis caused by diacetyl are suggestive that obliterative bronchiolitis (OB) may eventually occur, but their presence cannot be assumed to represent an early form of chronic severe lung disease. We appreciate the opportunity to emphasize this point. Diacetyl exposure caused lymphocytic bronchiolitis in our 12-week exposures; however, we do not know if these lesions progress with continuing exposure. For this reason we referred to lymphocytic bronchiolitis as a "potential precursor lesion."
Obstructive airway disease has been reported after exposure to ammonia, chlorine, hydrogen sulfide, nitrogen dioxide, and phosgene; however, acute exposures to high concentrations were required. In contrast, most microwave popcorn packaging workers who developed OB had worked for at least one year with no reports of acute exposure episodes during that time (Kreiss et al., 2002). Our point in this discussion was that, unlike these other reactive gases, diacetyl and other components of artificial butter flavoring appear to cause OB after repeated and/or intermittent exposures and not just in response to an acute high concentration exposure (although the effects of acute high concentration diacetyl exposure have not been evaluated). Our intent was not to infer similarities in mechanisms of action, and we regret that Finley et al. misinterpreted the point of this discussion. Indeed the reactive gases cited do not all have the same mechanism, and the mechanism of action of diacetyl is unknown.
In our diacetyl aspiration studies we did not observe significant inflammation but observed fibrohistiocytic lesions in the terminal bronchioles. Although Finley et al. assert that inflammation is required for the development of OB and that fibrohistiocytic lesions are not consistent with OB, this assertion is based primarily upon studies of OB in the context of human lung transplantation where serial lung biopsies have shown that lymphocytic airway inflammation is a risk factor for the development of OB (Glanville et al., 2008). In response we would point out that our earlier inhalation studies produced significant lymphocytic airway inflammation, suggesting perhaps a shared pathogenesis to toxin-induced and transplant-related OB. However, it is quite plausible that toxins, such as diacetyl, could contribute to the development of OB through mechanisms that differ from transplant-related OB.
Some of the concerns expressed by Finley et al. are based upon incorrect information.
Animals were not exposed to heated diacetyl vapors during inhalation exposures, as stated by Finley et al. Although 40°C air was passed through the generation system to vaporize the liquid, the animals were exposed to diacetyl vapors in 24°C–25°C air.
Dosing of mice by oropharyngeal aspiration (Foster et al., 2001) does not involve injection. Rather 50 µl of a dose solution is deposited at the base of the tongue and is aspirated by the animal into the airways. The animals were not treated with a concentrated diacetyl solution, as stated by Finley et al. The 50 µl of dose solutions contained either 2.4 µl (100 mg/kg), 4.8 µl (200 mg/kg), or 9.6 µl (400 mg/kg) of diacetyl diluted with sterile water.
Numerous comments by Finley et al. question the National Institute of Occupational Safety and Health (NIOSH) exposure data (Kreiss et al., 2002) that we referenced as rationale for selecting animal exposure concentrations. The NIOSH studies were carefully conducted and accurately reported in a peer-reviewed journal, and we are confident in the use of these data to select animal exposure concentrations relevant to occupational exposures. Although we disagree with many of the statements made by Finley et al. concerning the NIOSH study, we do not think it is appropriate for us to address comments concerning the NIOSH data.
Finally we would again emphasize that our results using exposures relevant to human workplace conditions demonstrate that diacetyl exposure in rodents leads to significant epithelial injury and lymphocytic airway inflammation which are known to precede the development of OB in humans.
The authors have no personal financial relationships with commercial interests relevant to this publication.
REFERENCES
Foster WM, Walters DM, Longphre M, Macri K, Miller LM. Methodology for the measurement of mucociliary function in the mouse by scintigraphy. J. Appl. Physiol. (2001) 90:1111–1117.
Glanville AR, Aboyoun CL, Havryk A, Plit M, Rainer S, Malouf MA. Severity of lymphocytic bronchiolitis predicts long-term outcome after lung transplantation. Am. J. Respir. Crit. Care Med. (2008) 177:1033–1040. (Epub 2008 Feb 8).
Hubbs AF, Battelli LA, Goldsmith WT, Porter DW, Frazer D, Friend S, Schwegler-Berry D, Mercer RR, Reynolds JS, Grote A, et al. Necrosis of nasal and airway epithelium in rats inhaling vapors of artificial butter flavoring. Toxicol. Appl. Pharmacol. (2002) 185:128–135.[CrossRef][Web of Science][Medline]
Kreiss K, Gomaa A, Kullman G, Fedan K, Simoes EJ, Enright PL. Clinical bronchiolitis obliterans in workers at a microwave-popcorn plant. N. Engl. J. Med. (2002) 347:330–338.
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