Nitrosamines and Related N-Nitroso Compounds - ACS Publications

Chapter 3

N-Nitrosodimethylamine in Nonfat Dry Milk

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R. A. Scanlan, J. F. Barbour, F. W. Bodyfelt, and L. M. Libbey Department of Food Science and Technology, Oregon State University, Corvallis, OR 97331

In 1980, using a detection limit of 0.3 ppb, N-nitrosodimethylamine (NDMA) was found in 51 of 71 commercial nonfat dry milk samples. The range for the positive samples was from 0.3 to 6.5 ppb with a mean for all samples of 0.72 ppb. In 1992, the mean NDMA value for 56 samples of instantized nonfat dry milk samples was 1.05 ppb, the range was 0.1 to 5.3 ppb. A comparison of the N D M A content of regular nonfat dry milk powder and instantized powder showed no additional N D M A formation from instantizing. Several researchers have suggested that N D M A in nonfat dry milk forms during the direct-fire drying process while others have contended that the source was fluid milk. Eight tracking experiments in commercial processing plants detected no N D M A until the drying process began. Furthermore, most of the 62 samples of nonfat dry milk prepared by direct-fire drying contained N D M A while eight of nine samples dried by indirect-fire processes did not contain NDMA. Our results indicate that the main source of N D M A in nonfat dry milk is the direct-fire process used in its manufacture.

Hotchkiss (i) has summarized the extensive research regarding nitrosamine formation in foods and beverages that has been conducted over the past several decades. When nitrosamine precursors and nitrosating agents are present, and under certain processing conditions, nitrosamines form in foods. N-Nitrosodimethylamine (NDMA) is most commonly found. The earliest research focused on cured meats to which nitrites had been added but more recent work found that nitrosamine formation is possible when foods are direct-fire dried. Secondary and tertiary amines present in the foods react with oxides of nitrogen produced by the combustion process. For example, as early

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as 1972, Sen et al. (2) reported the presence of N D M A in fish meal that had been direct-fire dried. Widespread occurrence of N D M A in beer prompted studies to determine its origin (3). The direct-fire malting process seemed a likely source. O'Brien et al. (4) conclusively demonstrated that N D M A formed in malt during the kilning step where the products of combustion come in direct contact with the malt being dried. Because indirect-fire drying greatly reduced the amount of N D M A formed, the malting industry largely switched to indirect-fire processes for malt manufacture. N D M A levels in beer have been greatly reduced since this change (5). Because of the known link between N D M A formation and direct-fire drying processes, researchers have investigated other dried foods, including nonfat dry milk. Several studies in the early 1980s consistently found N D M A in this product. Libbey et al. (6) detected N D M A ranging from 0.4 to 4.5 ppb in six out of seven samples of nonfat dry milk and confirmed its presence in one of the samples by mass spectral analysis. Sen and Seaman (7) found N D M A in 11 samples of instant skim milk powders, ranging from 0.3 to 0.7 ppb with an average of 0.4 ppb, while Lakritz and Pensabene (8) detected N D M A in nine out of ten samples of nonfat dry milk. Additionally, these researchers (8) reported "apparent" N D M A in pasteurized milk (mean, 0.1 ppb), and, therefore, suggested fluid milk as a source of N D M A in nonfat dry milk. In 1983, Frommberger and Allmann (9) reported that of 129 samples of German milk powders, 87% contained less than 0.5 ppb NDMA. The highest sample contained 13.2 ppb. In New Zealand, with a minimum detection limit of 1 ppb, Weston (70) found no N D M A in five samples of indirect-fire dried skim milk powder, 20 samples of direct-fire dried skim milk powder, or 10 samples of direct-fire dried buttermilk powder. The next year, Havery et al. (11) reported N D M A in 84% of 57 nonfat dry milk samples, with an average of 0.6 ppb. Ten samples were greater than 1 ppb; none was detected in nine of the samples. In the late 1980s, Osterdahl (12) detected only traces of N D M A in three out of 27 samples of milk powder in Sweden. In a comprehensive report of volatile nitrosamines in foodstuffs and beverages in West Germany in 1991, Tricker et al. (13) reported occasional trace levels of N D M A in dried milk powder. The purpose of this research was to determine the N D M A content of nonfat dry milk currently produced in the United States and compare these values with amounts found 12 years ago. In addition, we felt it important to determine whether the N D M A in nonfat dry milk is simply the result of N D M A contamination in fluid milk, or whether it is formed during the drying process. Experimental In 1980, 71 commercially-produced nonfat dry milk samples were obtained through the American Dairy Products Institute (formerly the American Dry Milk Institute). The samples were collected from a number of processing plants throughout the United States. N D M A levels were determined by a vacuum distillation procedure, as described in (6). Fifty-gram samples were analyzed in order to produce a minimum detection limit of 0.3 ppb.


NITROSAMINES AND RELATED AT-NTTROSO COMPOUNDS

Raw whole milk 87% H 0 2

/

Separation

Skim milk 91%H 0

c

2

Cream

Pasteurization

I

Pasteurized skim milk 91%H 0 2

I

Preheat

Hot well skim milk 91% H 0 2

Concentration Condensed skim milk] about 60% H 0 2

I

Spray drying

Nonfat dry milk 4% H 0 2

_

,

Instantized milk powder 4% H 0 2

Figure 1. Schematic of Nonfat Dry Milk Manufacturing


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In 1992, retail samples of instantized nonfat dry milk were purchased in supermarkets in 13 U.S. cities (Boston, Chester NY, New York City, Washington DC, Atlanta GA, Wichita KS, Sioux City IA, Logan UT, Los Angeles, San Francisco, Corvallis OR, Olympia WA, and Seattle). These samples were analyzed for N D M A according to the procedure of Havery et al. (11) with a minimum detection limit of 0.1 ppb. For the tracking study, samples were obtained in 1981 from two nonfat dry milk manufacturing plants, one in California and one in the Midwest. N D M A was determined in nonfat dry milk samples according to Libbey at al. (6) with a minimum detection limit of 0.2 ppb. N D M A levels in fluid milk samples were determined by an atmospheric pressure, steam distiUation procedure (described in (14)) except that 500 g instead of 300 g samples were used, thereby lowering the minimum detection limit to 0.02 ppb. An abbreviated representation of the process by which nonfat dry milk is manufactured is depicted in Figure 1. For more information on the process, consult (J). In the United States, most, but not all, nonfat dry milk is manufactured by direct-fire, spray-drying processes. Results Survey of NDMA in Nonfat Dry Milk. Table I contains a distribution of the N D M A content of 71 commercial nonfat dry milk samples manufactured in 1980. Sixty-two were manufactured by direct-fire, spray-drying processes while nine were prepared by indirect-fire processes. The N D M A values represented in Table I were not corrected for recovery which was approximately 75%. Levels of N D M A ranged from not detectable ( 3.0 1 0.72 mean "Not detected; detection limit 0.3 ppb. The mean for all 51 positive samples is 1.01 ppb. a

b

b

Table II reports the distribution of the N D M A content of 56 instantized nonfat dry milk samples purchased at retail outlets in summer 1992. The N D M A values were not corrected for recovery which was approximately 70%. Levels of


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NTTROSAMINES AND RELATED AT-NTTROSO COMPOUNDS

N D M A ranged from 0.1 to 5.3 ppb with a mean of 1.05 ppb. The tendency for the 1992 levels to be larger than the 1980 levels was statistically significant according to the Wilcoxon rank sum test (16), in which the one-tailed P-value was 0.003. In order to eliminate a potential bias introduced by different detection limits, all values below 0.3 ppb in the 1992 data set were considered as nondetectable for the Wilcoxon rank sum test. Table II. N-Nitrosodimethylamine in 56 Samples of Instantized Nonfat Dry Milk, 1992 Number of Samples Range (ppb) 0 nd 37 0.1-1.0 14 1.1-2.0 3 2.1-3.0 >3.0 2 1.05 mean Not detected; detection limit 0.1 ppb. a

a

Comparison of NDMA Content of Regular and Instantized Nonfat Dry Milk Powder. This experiment was designed to determine if there was any significant difference between N D M A levels in five samples of regular powders and in their corresponding instantized powders. Results, reported in Table III, show that generally the levels were very similar. Only one sample showed an increase in the level of N D M A when instantized. These results strongly indicate that the amount of N D M A in nonfat dry milk is not further increased during the instantizing process.

Table III. N-Nitrosodimethylamine in Regular and Instantized Nonfat Dry Milk Powder Instantized powder (ppb) Sample # Regular powder (ppb) 1 0.5 0.4 0.3 2 0.4 3 0.6 0.7 3.0 4 1.8 nd 5 nd» Not detected; detection limit 0.3 ppb. a

Comparison of NDMA Content of Nonfat Dry Milk Dried by Direct-Fire and by Indirect-Fire Processes. Sixty-two of the 71 nonfat dry milk samples


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N-Nitrosodimethylaminein Nonfat Dry Milk

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manufactured in 1980 were dried by the direct-fire process; nine by an indirect-fire method. A comparison of their respective N D M A contents is made in Table IV. Most, but not all, of the samples dried by direct-fire contained NDMA. However, eight of nine samples dried by indirect-fire did not contain NDMA. This result strongly suggests that when N D M A is found in nonfat dry milk it is formed by the direct-fire drying process. Table IV. N-Nitrosodimethylamine in Direct- and Indirect-Fire Nonfat Dry Milk Samples Direct-fire Indirect-fire Number of Samples N D M A Number of Samples NDMA 1 positive (0.6 ppb) 50 positive 12 8 nd nd Not detected; detection limit 0.3 ppb a

a

The Tracking Study. The results from eight separate tracking experiments conducted in two commercial processing plants are reported in Table V. N D M A was detected in all nonfat dry milk samples. None of the fluid milk samples from which the nonfat dry milk was made contained N D M A at the detection limit of 0.02 ppb.

Table V. N-Nitrosodimethylamine Levels in Tracking Studies Midwestern plant California plant ppb Run # 8 1 5 6 7 2 3 4 Raw whole milk nd nd nd" nd nd nd nd nd „b Pasteurized milk nd nd nd Hot well skim milk Condensed milk NDM^beginning N D M , middle M D M , end

nd

nd

nd

nd nd nd 0.6 0.6 0.5 0.5 0.5 0.7 0.5 0.6 0.5 Not detected; detection limit 0.02 ppb. Not sampled. °Nonfat dry milk.

- nd- nd-

nd

nd

-

nd

nd 0.7 1.0 0.8

nd 1.6 1.0 0.8

nd 0.6 0.6 0.7

nd 0.7 0.6 0.6

nd 0.8 0.7 0.6

a

b

When referring to the data in Table V, it is worth noting the significance of the minimum detection limits for N D M A in the fluid milk (0.02 ppb) and in


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N TROSAMINES AND RELATED JV-NTTROSO COMPOUNDS

the nonfat dry milk (0.2 ppb samples. Fluid milk is concentrated approximately tenfold when manufactured into nonfat dry milk. Therefore, nonfat dry milk samples that contain 0.2 ppb or more N D M A would have to have been manufactured from fluid milk containing 0.02 ppb N D M A or more, if the source of the N D M A in the dry milk was from the fluid milk. Conversely, if there is no detectable N D M A in the fluid milk (< 0.02 ppb), finding N D M A at 0.2 ppb or greater in the nonfat dry milk would strongly suggest that the N D M A was formed during drying rather than by concentration from the fluid milk. Thus, the data in Table III strongly suggest that the N D M A in the nonfat dry milk samples was formed during drying and was not concentrated from N D M A in the fluid milk. Discussion Previous researchers have speculated on the sources of N D M A in nonfat dry milk. Several have proposed that N D M A is formed during the direct-fire drying processes of dry milk manufacture (7). Others have suggested that N D M A in nonfat dry milk is concentrated from that already contained in fluid milk (7). However, distinguishing between these possible sources has been difficult-partly due to the insufficiently low detection limits used. Our results demonstrate that N D M A found in nonfat dry milk is not due to concentration of a contaminant already present in fluid milk. No N D M A was detected in the tracking experiment until the drying stage. Further, N D M A was detected in most of the samples dried by the direct-fire process but no N D M A was detected in eight of nine samples dried by indirect-fire methods. We conclude that the direct-fire process is the main source of N D M A in nonfat dry milk. A comparison of the results from the 1980 and 1992 surveys indicate that N D M A levels have not decreased in this product. Since N D M A formation occurs as a result of direct-fire drying, the results from this work suggest that N D M A formation in nonfat dry milk could be ameliorated by conversion from direct- to indirect-fire drying processes. Acknowledgements The authors express appreciation to the American Dairy Products Institute, Inc., for funding support and help in obtaining samples. This research was also supported in part by Grant No. C A 25002, awarded by the National Cancer Institute, DHHS. The authors are also indebted to Carole Nuckton for help in preparing the manuscript. Literature Cited 1. 2. 3.

Hotchkiss, J. H . Cancer Surveys. 1989, 8, pp. 295. Sen, N . P.; Schwinghamer, L. A.; Donaldson, B. A.; Miles, W. F. J. Agric. Food Chem. 1972, 20, pp. 1280. Spiegelhalder, B.; Eisenbrand, G.; Preussmann, R. In Proc. VI Inter. Sym. N-Nitroso Compds. Walker, E.A.; Gricuite, L.; Castegnaro, M.; Borzsonyi,


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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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M ; Davis, W. Eds; International Agency for Research on Cancer. Public. No. 31; Lyon, France, 1980, pp. 467-477. O'Brien, T. J.; Lukes, B. K.; Scanlan, R. A . Mast. Brew. Assoc. Tech. Quart. 1980, 17, pp. 196. Scanlan, R. A.; Barbour, J. F.; Chappel, C. I. J. Agric. Food Chem. 1990, 38, pp. 442. Libbey, L. M.; Scanlan, R. A.; Barbour, J. F. Fd. Cosmet. Toxicol, 1980, 18, pp. 459. Sen, N . P.; Seaman, S. J. Assoc. Off. Anal. Chem. 1981, 64, pp. 1238. Lakritz, L; Pensabene, J. W. J. Dairy Sci. 1981, 64, pp. 371. Frommberger, R.; Allmann, H . In Das Nitrosamin-Problem, Preussmann, R. Ed.; Verlag Chemie GmbH, Weinheim, 1983, pp. 58-63. Weston, R. J. J. Sci. Food Agric. 1983, 34, pp. 893. Havery, D. C.; Perfetti, G.A.; Fazio, T. J. Assoc. Off.Anal.Chem.1984, 67, pp. 20. Österdahl, B. -G. Food. Addit. and Contam. 1988, 5, pp. 587. Tricker, A . R.; Pfundstein, B.; Theobald, E.; Preussmann, R.; Speigelhalder, B. Food Chem. Toxic. 1991, 29, pp. 729. Scanlan, R. A.; Barbour, J. F.; Hotchkiss, J. H.; Libbey, L. M. Food Cosmet. Toxicol. 1980, 18, pp. 27. Campbell, J. R.; Marshall, R. T. The Science of Providing Milk for Man, McGraw Hill, Inc., New York, 1975, pp. 687-720. Snedecor, G. W.; Cochran W. G. Statistical Methods, 7th edition; The Iowa State University Press: Ames, IA, 1980; pp. 144. Ellen, G. In The Significance of N-Nitrosation of Drugs; Eisenbrand, G.; Bozler, G.; Nicolai, H . v. Eds.; G.F. Verlag, Stuttgart, 1990, pp. 19-46.

RECEIVED June 29,1993


Nitrosamines and Related N-Nitroso Compounds - ACS Publications

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