Rapid Report pubs.acs.org/crt
Is Diacetyl a Respiratory Sensitizer? A Reconsideration Using QSAR, QMM, and Competition Experiments J. J. Dworak,† D. W. Roberts,‡ M. A. Calter,† C. A. Fields,§ and J. Borak*,∥ †
Department of Chemistry, Wesleyan University, Hall-Atwater Laboratories, 52 Lawn Avenue, Middletown, Connecticut 06459-0180, United States ‡ School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, England § Yale School of Public Health, Yale University, 234 Church Street, New Haven, Connecticut 06514, United States ∥ Yale School of Public Health and School of Medicine, Yale University, 234 Church Street, New Haven, Connecticut 06514, United States S Supporting Information *
ABSTRACT: Concerns have been raised that diacetyl (DA) might be a respiratory sensitizer based on its LUMO energy similar to that of the respiratory allergen toluene-2,4-diisocyanate (TDI) and results of a local lymph node assay (LLNA) that reported an EC3 of 1.9%. To better understand the concerns, we performed a systematic literature review and experimental competition reactions between DA and TDI. The experimental evidence demonstrates that DA is at least 400-fold less reactive than TDI. The literature review finds evidence that the EC3 for DA is actually >11%. We conclude that DA is unlikely to have significant respiratory sensitization potential.
iacetyl (2,3-butanedione, CAS 431-03-8) is an α,βdiketone encountered frequently in foods and food flavoring.1,2 Concerns have been raised that diacetyl (DA) inhalation causes lung disease. Such concerns are based on high-dose animal studies3,4 and studies of exposed workers.5−7 The reactivity of DA, like that of other α,β-diketones, is almost certainly due to the electrophilicity of its two carbonyl groups.8 Presumably, inflammation results from reactions with primary amine groups of hard nucleophile units (e.g., lysine, arginine) in proteins, which lead to protein inactivation and cellular injury. It has been suggested, based on two concerns, that DA might act as a respiratory sensitizer. The first concern is based on analogy between DA and toluene-2,4-diisocyanate (TDI), a potent respiratory sensitizer.9 That analogy was proposed because the lowest unoccupied molecular orbital (LUMO) energy of DA (−0.51 eV) is comparable to that of TDI (−0.70 eV). Thus, it could be inferred that DA has similar reactivity to that of TDI and might be similarly potent. The second stems from results of a local lymph node assay (LLNA) that reported an EC3 for DA of 1.9%.2,10 It could be argued that since LLNA potency tends to be correlated, at least within mechanistic domains, with reactivity, an EC3 value of 1.9 could suggest relatively high reactivity and a probability of being a respiratory sensitizer.11 The first line of reasoning, based on the similarity of LUMO energies of DA and TDI, assumes that similarity in LUMO energies implies similarity in reactivity. This assumption is
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© 2013 American Chemical Society
weakened by the fact that DA and TDI belong to different reaction mechanistic domains: DA is a Schiff base, while TDI belongs to the Acyl transfer domain.12,13 Thus, transition states for reactions of the two compounds will not be similar: the relationships between LUMO energy and reactivity cannot be assumed to be the same. The second line of reasoning assumes that the reported EC3 value of 1.9% accurately represents the skin sensitization potency of DA. There is reason to question this assumption. Modeling of potency for respiratory sensitization is less advanced than for skin sensitization, but recent QSAR14 and mechanistic chemistry12,15,16 studies have yielded useful insights. Chemicals able to cause respiratory sensitization can be assigned to the same electrophilic mechanistic domains as skin sensitizers13,17 with the domains corresponding mainly to reactivity with relatively hard nucleophiles. Whether a chemical will be a sensitizer depends on whether it is able to modify appropriate proteins to a sufficient extent; it appears that the protein modification threshold is higher for respiratory than for skin sensitization. This threshold can be achieved by chemicals with high intrinsic electrophilicity or lower degrees of electrophilicity together with cross-linking ability. TDI has both high electrophilicity and cross-linking ability, so its high potency as a respiratory sensitizer is easily rationalized. Received: March 8, 2013 Published: April 10, 2013 631
dx.doi.org/10.1021/tx400097v | Chem. Res. Toxicol. 2013, 26, 631−633
Chemical Research in Toxicology
Rapid Report
resonance (NMR) spectroscopy; NMR spectra were recorded on a 400 MHz Varian instrument. n-Butylamine was distilled before use. All other reagents were used as received. All reactions were performed at room temperature (see Supporting Information for additional details). By analysis of the reaction mixture, the extent to which each competing compound has reacted is determined, from which the relative reactivity, expressed as a ratio of rate constants, can be calculated. In TDI, the two NCO groups react independently; after the first NCO group has reacted, the second one is still highly reactive, although less so than originally. Therefore, for kinetics it is appropriate to consider the rate of disappearance of NCO groups (−d[NCO]/dt). By contrast, in DA the two CO groups are not independent; when one reacts, the other becomes less reactive. In this case we consider the rate of disappearance of DA, (−d[DA]/dt). The rate equations are:
To better understand the respiratory sensitization potential of DA in light of the lines of reasoning discussed above, we performed a literature review and analytical experiment. (1) Reviews of the National Library of Medicine Medline and Toxline databases were performed for [Local Lymph Node Assay and 431-03-8], [QSAR and 431-03-8], and [Structure Activity and 431-03-8]. In addition, we performed hand searches of the references cited in identified studies as well as other LLNA- and QSAR-related studies. We identified three published studies, in addition to the Anderson report,10 which specifically considered the results of LLNA testing of DA. In 1999, Roberts et al.8 reported an EC3 for DA of 11.3, nearly 10fold greater than the corresponding value reported by Anderson. Later, Roberts and colleagues utilized LLNA data in a series of QSAR and QMM (quantitative mechanistic modeling) studies that focused on aldehydes and ketones of the Schiff base mechanistic domain.13,18,19 Using LLNA results for 12 Schiff base electrophiles (not including DA), they determined the following QMM relationship for skin sensitization by Schiff base sensitizers: p EC3 = 1.12 ∑ σ * + 0.41log P − 0.60
−d[NCO]/dt = kNCO[NCO][BA]
(1)
−d[DA]/dt = kDA[DA][BA]
(2)
Dividing eq 1 by eq 2 and integrating, we get k NCO/k DA = ln([NCO]f /[NCO]0 )/ln[DA]f /[DA]0 )
where pEC3 = log (molecular weight ÷ EC3); ∑σ* = the sum of the Taft σ* values of the two groups bonded to the reactive carbonyl group; and P = octanol/water partition coefficient. Using that relationship, they predicted EC3 values for six Schiff base sensitizers (including DA), and those predicted values were compared to experimentally determined EC3 values.19 The QMM-predicted EC3 values were nearly identical to the experimentally derived values, thus validating the results of the LLNA assays. Thus, there is independent support for the DA EC3 value reported in 1999 by Roberts et al.8 It is interesting to consider possible reasons for the nearly 10fold difference between the Roberts and Anderson EC3 results for DA. Anderson proposed that it might be due to the use of differing mouse strains:10 Roberts et al. used CBA/J mice, while Anderson et al. used BALB/c mice. Others have raised concerns about strain-specificity of LLNA results.20,21 However, other data suggest that this was not the case. The Anderson et al. study also reported LLNA results for glyoxal (CAS 107-22-2), another α,β-diketone, that had been previously studied by Patlewicz et al. using the same methods and mouse strain as those used by Roberts et al.22 The EC3 results from the Anderson and Patlewicz studies are very similar (0.74 vs 1.4); EC3 values are usually considered to be reproducible within a factor of 2.19,23 If strain differences explained the disparate LLNA results for DA, then we would have expected similar disparities in the parallel glyoxal results. Accordingly, we suggest that the EC3 value for DA reported by Anderson et al. was spuriously low, likely reflecting an unexpected contaminant. This suggestion is supported by a preliminary NTP report that the EC3 of DA in BALB/c mice ranged from “10%−25%.” 29 (2) In the analytical experiment, DA and TDI were reacted with n-butylamine, a model nucleophile, first independently and then in two competition reactions. The principle of competition experiments24,25 is to allow the compounds whose reactivity is to be compared (in this case TDI and DA) to compete for a deficiency of the reaction partner (in this case butylamine). The use of n-butylamine as a model for protein nucleophiles is well established.26−28 The reaction products were determined by means of 1H nuclear magnetic
(3)
The subscript 0 indicates the initial concentration. The subscript f indicates the final concentration. In our study, the TDI was completely consumed to afford a mixture of mono- and bis-adducts. As the monoadducts each contain one NCO group, in eq 3 we replaced [NCO]f by [monoadduct]f. Also, as it was easier to quantify the amount of DA reaction products than to detect a small change in the concentration of DA, we replaced [DA] by ([DA]0 − [DA reaction products]f). The choice of concentration units and base of logarithms do not affect the value of kNCO/kDA. If the competing compounds are expected to be similarly reactive, it is appropriate to choose a 1:1 mole ratio for their initial concentrations. If one compound is expected to be significantly more reactive than the other, it is appropriate to start with the less reactive compound in excess. In view of the LUMO energy prediction of similar reactivity, we carried out an initial experiment with equal initial amounts of NCO groups and DA (see Supporting Information for experimental results). There was no evidence for any DA adducts. Based on the detection limit of 1.25 μmol, we can estimate a lower limit value for kNCO/kDA based on the assumption of DA adduct formation just below the detection limit, putting [DA]0 − 1.25 as the [DA]f value in eq 3 as kNCO/kDA > 190 (mean of 3 experiments, SD = 10). Clearly, TDI is much more reactive than DA. A second competition experiment was performed with a 1:2.5 ratio of NCO groups to DA. This gave the result shown in Table 1. There was still no evidence for any DA adducts. The revised lower limit value for kNCO/kDA is kNCO/kDA > 410 (mean of 3 experiments, SD = 10). Thus, TDI is more than 400 times as reactive as DA. The results of the competition experiments are consistent with LLNA findings. In summary, experimental evidence demonstrates that DA is less reactive than TDI by a factor of at least 400. The LLNA EC3 value of 11% for DA is supported by the QMM evidence, and on that basis, DA is a weak sensitizer (about 500 times less potent than TDI in the LLNA 11). We therefore conclude that 632
dx.doi.org/10.1021/tx400097v | Chem. Res. Toxicol. 2013, 26, 631−633
Chemical Research in Toxicology
Rapid Report
(6) Lockey, J. E., Hilbert, T. J., Levin, L. P., Ryan, P. H., White, K. L., Borton, E. K., Rice, C. H., McKay, R. T., and Lemasters, G. K. (2009) Eur. Respir. J. 34, 63−71. (7) van Rooy, F. G., Rooyackers, J. M., Prokop, M., Houba, R., Smith, L. A., and Heederik, D. J. (2007) Am. J. Respir. Crit. Care Med. 176, 498−504. (8) Roberts, D. W., York, M., and Basketter, D. A. (1999) Contact Dermatitis 41, 14−17. (9) Egilman, D. S., Schilling, J. H., and Menendez, L. (2011) Int. J. Occup. Environ. Health 17, 122−134. (10) Anderson, S. E., Wells, J. R., Fedorowicz, A., Butterworth, L. F., Meade, B. J., and Munson, A. E. (2007) Toxicol. Sci. 97, 355−363. (11) ECETOC (2008) Document No. 46: Potency Values from the Local Lymph Node Assay: Application to Classification, Labelling and Risk Assessment, European Centre for Ecotoxicology and Toxicology of Chemicals, Brussels, Belgium. (12) Enoch, S. J., Roberts, D. W., and Cronin, M. T. (2010) Chem. Res. Toxicol. 23, 1547−1555. (13) Roberts, D. W., Patlewicz, G., Kern, P. S., Gerberick, F., Kimber, I., Dearman, R. J., Ryan, C. A., Basketter, D. A., and Aptula, A. O. (2007) Chem. Res. Toxicol. 20, 1019−1030. (14) Jarvis, J., Seed, M. J., Elton, R., Sawyer, L., and Agius, R. (2005) Occup. Environ. Med. 62, 243−250. (15) Enoch, S. J., Roberts, D. W., and Cronin, M. T. D. (2009) Chem. Res. Toxicol. 22, 1447−1453. (16) Enoch, S. J., Seed, M. J., Roberts, D. W., Cronin, M. T., Stocks, S. J., and Agius, R. M. (2012) Chem. Res. Toxicol. 25, 2490−2498. (17) Aptula, A. O., and Roberts, D. W. (2006) Chem. Res. Toxicol. 19, 1097−1105. (18) Roberts, D. W., and Patlewicz, G. (2002) SAR QSAR Environ. Res. 13, 145−152. (19) Roberts, D. W., Aptula, A. O., and Patlewicz, G. (2006) Chem. Res. Toxicol. 19, 1228−1233. (20) National Toxicology Program (1999) The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds, National Institute of Environmental Health Sciences, Research Triangle Park, NC. (21) National Toxicology Program (2011) ICCVAM Test Method Evaluation Report: Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans (NIH Pub. # 11-7709), National Institute of Environmental Health Sciences, Research Triangle Park, NC. (22) Patlewicz, G. Y., Wright, Z. M., Basketter, D. A., Pease, C. K., Lepoittevin, J. P., and Arnau, E. G. (2002) Contact Dermatitis 47, 219− 226. (23) Basketter, D. A., Blaikie, L., Dearman, R., Kimber, I., Ryan, C. A., Gerberick, G. F., Harvey, P., Evans, P., White, I. R., and Rycroft, R. J. (2000) Contact Dermatitis 42, 344−348. (24) Lee, E., and Roberts, D. W. (1973) J. Chem. Soc., Perkin Trans. 2, 437−444. (25) Ward, R. S., Diaper, R. L., and Roberts, D. W. (2001) J. Surfactants Deterg. 4, 263−270. (26) Roberts, D. W., Goodwin, B. F. J., Williams, D. L., Jones, K., Johnson, A. W., and Alderson, C. J. E. (1983) Food Chem. Toxicol. 21, 811−813. (27) Franot, C., Roberts, D. W., Smith, R. G., Basketter, D. A., Benezra, C., and Lepoittevin, J. P. (1994) Chem. Res. Toxicol. 7, 297− 306. (28) Franot, C., Roberts, D. W., Basketter, D. A., Benezra, C., and Lepoittevin, J.-P. (1994) Chem. Res. Toxicol. 7, 307−312. (29) National Toxicology Program (1999) Report on the Assessment of Contact Hypersensitivity to 2,3,Butanedione (Diacetyl) in Female BALB/c Mice, http://ntp.niehs.nih.gov/?objectid=DCB7D684-B6083DD5-5BD8E5D509D39068 (accessed Mar, 2013).
Table 1. Competition between TDI and DA: 1:2.5 Ratio of NCO Groups to DA component BAo (μmol) BAf (μmol) DA0 (μmol) DAf (μmol) TDI monoadduct0 (μmol) TDI monoadductf (μmol) NCOo (μmol) NCOf (μmol) DA reaction product0 (μmol) DA reaction productf (μmol)
run 1
run 2
run 3
81 0 93 92 (>93−1.25) 0
81 0 93 92 (>93−1.25) 0
81 0 93 92 (>93−1.25) 0
9.7
10.7
9.7
92 9.7 0
92 10.7 0
92 9.7 0
none observed (detection limit 1.25 μmol)
none observed (detection limit 1.25 μmol)
none observed (detection limit 1.25 μmol)
TDI is not a good read-across reference for DA, which is much less reactive and reacts by a different mechanism, and that, it being only a weak sensitizer in the LLNA, it is unlikely to have significant respiratory sensitization potential.
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ASSOCIATED CONTENT
S Supporting Information *
Detailed experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): M.C., D.W.R., J.D., and C.F. declare no competing interests. J.B. served as a paid External Peer Reviewer for the NIOSH Draft Criteria Document on Diacetyl and 2,3Pentanedione and as a paid consultant in diacetyl-related litigation.
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ABBREVIATIONS BA, n-butylamine; DA, diacetyl; EC3, effective concentration for a SI of 3 in proliferation of lymph node cells; LLNA, local lymph node assay; LUMO, lowest unoccupied molecular orbital; NCO, isocyanate; QMM, quantitative mechanistic modeling; QSAR, quantitative structure−activity relationship; TDI, toluene-2,4-di-isocyanate
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REFERENCES
(1) Chuang, L. F., and Collins, E. B. (1968) J. Bacteriol. 95, 2083− 2089. (2) National Institute for Occupational Safety and Health (2011) Criteria for a Recommended Standard... Occupational Exposure to Diacetyl and 2,3-Pentanedione (DRAFT), U.S. Department of Health and Human Services, Washington, DC. (3) Hubbs, A. F., Goldsmith, W. T., Kashon, M. L., Frazer, D., Mercer, R. R., Batelli, L. A., Kullman, G. J., Schwegler-Berry, D., Friend, S., and Castranova, V. (2008) Toxicol. Pathol. 36, 330−344. (4) Palmer, S. M., Flake, G. P., Kelly, F. L., Zhang, H. L., Nugent, J. L., Kirby, P. J., Foley, J. F., Gwinn, W. M., and Morgan, D. L. (2011) PLoS One 6, e17644. (5) Kreiss, K., Gomaa, A., Kullman, G., Fedan, K., Simoes, E. J., and Enright, P. L. (2002) N Engl J Med 347, 330−338. 633
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