Y: - -, .J•. Selected Aliphatic Amines and Related Compounds: An Assessment of the Biological and Environmental Effects Board on Toxicology and Environmental Health Hazards Assembly of Life Sciences National Research Council ------- Selected Aliphatic Amines and Related Compounds: An Assessment of the Biological and Environmental Effects Committee on Amines Board on Toxicology and Environmental Health Hazards Assembly of Life Sciences National Research Council National Academy Press Washington, D.C. 1981 ------- NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their competences and with regard for appropriate balance. This report has been reviewed by a group other than the authors according to procedures approved by a Report Review Committee consisting of members of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The National Research Council was established by the National Academy of Sciences in 1916 to associate the broad community of science and technology with the Academy’s purposes of furthering knowledge and of advising the federal government. The Council operates in accordance with general policies determined by the Academy under the authority of its congressional charter of 1863, which establishes the Academy as a private, non—profit, self—governing membership corporation. The Council has become the principal operating agency of both the National Academy of Sciences and the National Academy of Engineering in the conduct of their services to the government, the public, and the scientific and engineering communities. It is administered jointly by both Academies and the Institute of Medicine. The Academy of Engineering and the Institute of Medicine were established in 1964 and 1970, respectively, under the charter of the National Academy of Sciences. At the request of and funded by the U.S. Environmental Protection Agency, Contract No. 68—01—4655 ii ------- List of Participants COMMITTEE ON AMINES DAVID B. CLAYSON, University of Nebraska Medical Center, Omaha, Nebraska, Chairman GEORGE T. BRYAN, University of Wisconsin, Center for Hea:Lth Sciences, Madison, Wisconsin DAVID H. FINE, New England Institute for Life Sciences, Waltham, Massachusetts CHARLES C. IRVING, Veterens Administration, Center for Health Sciences, Memphis, Tennessee CHARLES M. KING, Michigan Cancer Foundation, Detroit, Michigan RICHARD MONSON, Harvard School of Public Health, Boston, Massachusetts JACK L. RADOMSKI, University of Miami, Miami, Florida DONALD H. STEDMAN, University of Michigan, Ann Arbor, Michigan STEVEN R. TANNENBAUM, Massachusetts Institute of Technology, Cambridge, Massachusetts SNORRI S. THORGEIRSSON, National Cancer Institute, Bethesda, Maryland JOHN H. WEISBU1 ER, Naylor Dana Institute, Valhalla, New York ERROL ZEIGER, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina Consultants EMERICH FIALA, Naylor Dana Institute, Valhalla, New York MALCOLM C. BOWMAN, National Center for Toxicological Research, Jefferson, Arkansas National Research Council Staff ROBERT J. GOLDEN, Project Director FRANCES M. PETER, Editor AGNES E. GASKIN, Secretary EPA Project Officer ALAN CARLIN, Office of Research and Development, U.S. Environmental Protection Agency, Washington, D.C. iii ------- BOARD ON TOXICOLOGY AND ENVIRONMENTAL HEALTH HAZARDS RONALD W. ESTABROOK, University of Texas Medical School (Southwestern), Dallas, Texas, Chairman THEODORE CAIRNS, DuPont Chemical Co. (retired), Greenville, Delaware VICTOR COHN, George Washington University Medical Center, Washington, D.C. JOHN W. DRAKE, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina ALBERT M. FREEMAN, Bowdoin College, Brunswick, Maine RICHARD HALL, McCormick & Company, Hunt Valley, Maryland RONALD W. HART, National Center for Toxicological Research, Jef ferson, Arkansas PHILIP LANDRIGAN, National Institute for Occupational Safety and Health, Cincinnati, Ohio MICHAEL LIEBERMAN, Washington University School of Medicine, St. Louis, Missouri BRIAN MacMAHON, Harvard School of Public Health, Boston, Mas sachusetts RICHARD MERRILL, University of Virginia, Charlottesville, Virginia ROBERT A. NEAL, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina IAN NISBET, Massachusetts Audubon Society, Lincoln, Massachusetts CHARLES R. SCHUSTER, JR., University of Chicago, Chicago, Illinois GERALD WOGAN, Massachusetts Institute of Technology, Cambridge, Massachusetts ROBERT G. TARDIFF, National Research Council, Washington, D.C., Executive Director iv ------- CONTENTS PAGE Executive Sunimary....................,,,,, ,.,,...,.,..,,.,.... 1 CHAPTER 1 — Nitrosation of Amines and Their Control. .. ........ . 21 CHAPTER 2—GeneralAna lyticMethods.........,...........,.... 45 CHAPTER 3 — Epidemiology.. • • . . . . . • . . • . . • • e . . . . . . . . . 57 CHAPTER 4 — Triethanolamine............,.....,.....,....,..,. . 67 CHAPTER 5 — Morpholine..............................,,.. . ..... 92 CHAPTER 6 — 2—Nitropropane........ ............,.............. 143 V ------- EXECUTIVE SUMMARY In 1977, the U.S. Environmental Protection Agency (EPA) contracted with the National Academy of Sciences to prepare a series of reports on pollutants that were believed to be associated with deleterious effects in humans and the environment. As part of this effort, the agency asked the Academy to assess the health and environmental effects of selected aromatic and aliphatic amines. In response, the Committee on Amines was established within the Board on Toxicology and Environmental Health Hazards, Assembly of Life Sciences, National Research Council. The Committee on Ainines decided that the very different properties of the two classes of amines would necessitate division of the study into two separate components: one devoted to aromatic amines and related compounds and another to aliphatic aminea and nitro compounds. This resulted in the preparation of two separate reports. The task was extended to related chemicals in order to include those compounds resulting from the metabolic conversion of the amines, especially those of the aromatic group. Such compounds may be formed from nitro or azo compounds and in turn give rise to hydroxylamine derivatives. The greatest potential of these chemicals for the induction of acute and long—term toxic effects arises from their ability to be nitrosated to nitrosamines that are genotoxic with potential for mutagenicity, teratogenicity, and carcinogenicity, among other ------- toxic effects. Risk assessments for nitrosamines have not been attempted since the rate of nitrosatfon depends on the nature and concentration of the nitrosating agent and the presence of catalysts in the environment. The problem is further complicated by the fact that there are large differences in carcinogenic potency among the nitrosamines. Because aliphatic amines generally have a low level of toxicity their odor and potential for the induction of temporary respiratory tract irritation serve as warnings against exposure. Furthermore, there is virtually no dose—response Information on the related potentially carcinogenic nitrosamines. Such data could be used to estimate risk with current methodology. Consequently, risk assessment has not been attempted for these agents. The committee was unable to adequately address the environmental aspects of exposure after an intensive search of the literature revealed a general lack of information on this subject for the compounds selected. Each report contains introductory chapters providing an overview of some general information on the amines under discussion followed by more in—depth considerations of specific substances. This report contains chapters on triethanolamine, morpholine, and 2—nitropropane. Triethanolamine is used in industry and in cosmetic preparations. It is also converted by —2— ------- nitrosating agents to N—nitrosodiethaflOlamifle, which has been known f or some time to induce cancer in rodents. Morpholine is an industrially important secondary amine that is nitrosated to N—nitrosomorpholine, which is carcinogenic in animals. 2—Nitropropane was selected because of its dispersion into the environment resulting from its use in paints and other coatings. NITROSATION OF AMINES AND THEIR CONTROL N—Nitroso compounds are formed by the interaction of nitrosating agents with a variety of amines or amides. Primary, secondary, or tertiary amines may be nitrosated to nitrosamines under acidic, neutral, or alkaline conditions. Nitrosation may be brought about by nitrous acid derived from nitrite ion, the oxides of nitrogen, nitro compounds, or by transnitrosation. Nitrosamines may be detected wherever aliphatic amines are present. The oxides of nitrogen ——dinitrogen tetraoxide (N 2 o 4 ), dinitrogen trioxide (N 2 0 3 ), and nitrogen dioxide (N0 2 )——nitrosate much more rapidly than nitrous acid and lead to nitrosamines, to deaminated products, and, sometimes, to nitramines, especially in the presence of catalysts such as iodine, iodides, or metal salts. The human environment is contaminated by such nitrosamines. The highest exposures have been reported for tannery workers, especially those concerned with wet tanning, for whom exposures ranging from 23 to 47 pg/rn 3 have been observed. Nitrosomorpholine has —3— ------- been measured at levels of 0.5 to 27 pg/rn 3 , leading to human exposures between 50 to 250 pg daily. In a rocket fuel factory, dirnethylnitrosamine, an intermediate in the manufacture of l,l—dimethylhydrazine, resulted in daily exposure of workers to levels between 10 and 50 pg. Nitrosamlnes are also a component of tobacco smoke. A person who smokes 20 cigarettes per day inhales several nitrosainines in an amount totalling approximately 16.8 pg daily. There have been extensive studies of the ingestion of nitrosamines in food, especially in preserved meat and fish products, and the formation of these compounds from the interaction of nitrites and amines in the stomach. Until recently, the maximum exposure from this source has been attributed to beer. The consumption of three cans of one brand of beer has been reported to result in the ingestion of approximately 8.4 pg of dimethylnitrosamine. The nitrosamine levels in beer have now been reduced due to changes in the manufacturing process Average human exposure from all known sources is estimated to be approximately 280 ng of mixed nitrosamines per kilogram of body weight daily. Cigarette smoking is by far the largest single component of daily exposure totalling 240 ng/kg/day. The formation of nitrosamines, especially in industrial products, has been successfully controlled, once their potential health effects were identified. For example, the amount of —4— ------- nitrosodipropylamine in the pesticide trifluralin, which has been measured at levels as high as 195,000 ppb (0.195Z), was reduced by changes in the manufacturing process. The nitrosodimethylamine impurity in beer arose during drying of the moist malt. Reductions in this compound have been achieved by instituting the use of gas burners with lower nitrogen oxide content and by increasing the acidity of the malt. Other problems have been solved by similar manipulation of production conditions. The formation of nitrosamines in bacon and other meat or fish products preserved by the addition of nitrites is inhibited by adding ascorbic acid or other nitrite sinks to the food to reduce the level of nitrite available for nitrosation. Recommendation The most effective way to control potentially carcinogenic nitrosamines would be the widespread dissemination of information on nitrosamine formation. Manufacturers and users of amines and nitrosating agents should be made aware that certain processes and products probably result in the formation of nitrosamines, but that the extent of contamination can be minimized by the use of appropriate technology. GENERAL ANALYTIC METHODS Aliphatic amines are generally analyzed by gas, paper, or thin—layer chromatography. Recent studies have used gas —5— ------- chromatography following derivatization, which among other advantages prevents peak tailing or “ghost’ effects due to Interactions of the basic amine with active sites in the column. The selection of derivative is determined by the type of gas chromatographic detector used and whether the purpose of the study Is to separate primary, secondary, or tertiary amines. For example, picogram quantities of amino compounds have been measured using heptafluorobutyryl derivatives and electron capture detection. Similar principles apply for thin—layer chromatography, but the methodology is less sensitive. Thus, for example, detection of microgram amounts is possible using colored 4 ‘—nltroazobenzyl—4—amide derivatives. EPIDEMIOLOGIC STUDIES Since there have been only Infrequent reports of adverse reactions to aliphatic amines used in Industry, few epidemiologic studies have been conducted. In contrast, nitroparaf fins such as 2—nitropropane, which exhibit greater toxicity, have been examined in greater depth, but the results of even this study remains equivocal. Certain aliphatic amines such as hexamethyleneamine or triethanolamine are apparently related to dermatologic conditions. Caution In accepting these associations is necessary since the individual worker Is exposed to a wide variety of agents rather than to a single amine. —6— ------- Although there is no direct evidence that the nitrosamine products of the aliphatic amines induce cancer in humans, their widespread, though low level, occurrence in the environment combined with their carcinogenicity in a wide spectrum of laboratory animal species, suggests that they may also be carcinogenic in humans. There are two situations in which nitrosamine—induced human cancer is suspected, although not established. In Cali, Colombia, drinking water contains high levels of nitrate, which is reduced by bacteria to nitrite in the body, thereby becoming a potential nitrosating agent. These elevated high nitrate levels are associated with a high rate of human ga8tric cancer. In a region of China where there is a high esophageal cancer rate, elevated levels of nitrite and specific nitrosamines are present in the food supply. Recommendations The effects of exposure to nitrate, nitrite, and nitrosamine on the incidence of human cancer urgently need intensive epidemiologic study. Investigators conducting such studies should be equipped with a knowledge of possible concurrent exposure to other potential carcinogens, the dietary regimen and smoking habits of exposed and control populations, and the concentrations of each nitroso compounds to which the study populations are or have been exposed. —7— ------- TRIETHANOLAMINE Triethanolamine (2,2’ ,2”—trihydroxytriethylamlne) is a relatively nonvolatile substance (vapor pressure less than 0.01 mm Hg at 20°C) that boils at 335.4°C and melts at 21.1°C. It is produced in large volumes. In 1978, 52,000 mt were manufactured at several sites in the United States by the reaction of ethylene oxide and aqueous ammonia at 50 to 100°C. The mixture of mono—, di—, and triethanolamine is separated by distillation. This chemical has a wide range of uses in soaps and detergents, textile specialities, cosmetics, agricultural products, and many other products. The figures are far greater for the use of this compound than those reported for yearly production (122,000—146,000 mt) in the United States. Part of this difference may result from doubts about its use in gas purification, and the remainder may be imported. There is no information about the release of triethanolamine into the environment during production or conversion to other products. Food and cosmetic uses present the greatest potential for human exposure. Since triethanolamine may be nitrosated to N—nitrosodiethanolamine, the toxicity of both compounds must be considered. Triethanolamine in complex mixtures can be analyzed by —8— ------- extracting organic matter with methylene chloride and subsequent evaporation to dryness on a steam bath. Acidic and alkaline contaminants may also be removed. The residues may be analyzed directly by high performance liquid chromatography or, after alkylation or esterification to give volatile derivatives, by gas chromatography. Few metabolic studies of either triethanolamine or its nitrosation product have been initiated. Most of the ingested N—nitrosodiethanolaniine is excreted unchanged in the urine by rats, the proportional excretion level being independent of dose over the range of 10 to 1,000 mg/kg. This nitrosamine has also been shown to be absorbed through human and shaved rat skin. This observation is important in view of the fact that the exposure of humans to cosmetic preparations containing triethanolamine, and in some cases its nitrosation product, is dermal. The acute toxicity of triethanolamine was studied in aquatic protozoa and invertebrates. Its toxicity was less than that of diethanolamine or monoethanolamine. Acute effects were observed to result from exposures greater than 100 mg/liter, whereas chronic effects occurred above 1 mg/liter. In mammals, all three ethanolamines are weakly toxic; no LD 50 ’s were observed at levels less than 700 mg/kg for any species or route of administration. Russian studies indicate —9— ------- that 13% triethanolamine solutions penetrated rat skin and led to changes in liver and the central nervous system. Topically applied triethanolamine in mice was not carcinogenic or cocarcinogenic, although it inhibited the cocarcinogenic properties of one detergent with which it was applied. In humans, triethanolamine acts as a sensitizer to contact allergens. Workers handling cutting fluids and other mixtures containing triethanolamine, may develop derinatoses as a result of this exposure. The nitrosation product, N—nitrosodiethanolamine, has been tested at high levels in rats and hamsters. Rats given 600—1,000 mg/day in drinking water (total dose, 150—300 g/kg) all developed hepatocellular carcinomas between 242 and 325 days after treatment started. Hamsters receiving N—nitrosodiethanolamine by subcutaneous injection on two schedules experienced a high incidence of nasal cavity tumors, papillary tumors of the trachea, and hepatoceflular adenomas. These tumors were not observed in untreated control hamsters. The high doses used in rats and hamsters give no indication of the possible responses to lower doses. Further information is required in this area. There appear to be no evidence for mutagenic activity of triethanolainine in Salmonella or in the Allium cepa niitosis test. Triethanolamine in the presence of nitrite was, however, —10- ------- mutagenic to Bacillus subtilis in the absence, but not in the presence of rat liver S—9 fraction. Nitrosodiethanolamine Is mutagenic to Salmonella typhimurium and Escherichia coil . A metabolizing system was shown to be required by some, but not all test systems. Recommendations 1. Suspicion that triethanolamine of unknown purity might be carcinogenic is suggested by one study. There Is a need to repeat this study using a pure sample of triethanolamine under strictly defined conditions. 2. N—nltrosodiethanolamine, the nitrosation product of triethanolamine, is definitely carcinogenic in rats and hamsters at high exposure levels. There is a need for additional information at lower dose levels In another species to confirm these observations. 3. Mutagenicity data on N—nitrosodiethanolamine are inadequate and, to some extent, discordant. There Is a need for further studies to determine the best microbial strains to be used for this purpose and whether a metabolizing fraction should be present. Studies with mammalian cells should also be conducted. 4. There is an absence of published data on the release of triethanolamine into the environment during production or during its many uses. This subject and its environmental persistence and fate should be investigated, especially since trlethanolaniine is readily nitrosated. —11— ------- MORPHOL INE Morpholine (tetrahydro—l,4—oxazine) is a colorless, hygroscopic liquid (nip —4.9°C, bp 128.9°C) with an appreciable vapor pressure (8.0 mm Hg at 20°C). It is completely miscible in water. It is produced by reacting diethyleneglycol, ammonia, and a small amount of hydrogen over a hydrogenation catalyst at 150—400°C and 30—400 atm. Approximately 11,000 nit of morpholine is produced annually in the United States. The largest single use of the compound is in the manufacture of rubber. It Is also used as a corrosion Inhibitor, and the products of its interaction with fatty acids are used to form household soaps or waxes and polishes for motor vehicles. Some morpholine derivatives are present in pharmaceuticals. Estimates of morpholine emissions into the environment do not appear to be supported by direct analytic data. It has been assumed that all morpholine used as a corrosion inhibitor (2,700 mt/yr) and in waxes or polishes (1,000 mt/year) will be emitted into the environment. This may be an overestimate. There is a need for analytically based determinations of morpholine release and persistence in the environment, especially in view of the ready nitrosation of this substance. Morpholine may be analyzed by gas chromatography either —12— ------- directly or after derivatization with toluenesulfonyl choloride. Colorimetric analysis is possible using the dialkyldithiocarbamate complex that may be extracted into chloroform. Air monitoring of morpholine has been achieved by trapping the aliphatic amine and nitrosating it with nitrous acid to form nitrosomorpholine. Morpholine itself has low toxicity except for its irritant effects but it is converted through nitrosation to N—nitrosomorpholine, a potent carcinogen In animals. The toxicity of morpholine and its nitroso derivative is reviewed in this report. The LC 50 of morpholine is 2,250 ppm for male and female rats and 1,450 ppm for male and 1,900 ppm for female mice following inhalation. High levels of inhalation (12,000—18,000 ppm) for 8 h led to eye Irritation, hemorrhage of the lungs, and congestion of the liver and kidneys. In humans, 1 to 1.5 minutes of exposure to 12,000 ppm morpholine led to irritation of the nose and to coughing. Exposures of 50 to 100 ppm N—ethylmorpholIne for 2 to 3 minutes led to upper respiratory tract irritation. Industrial exposure to morpholine at unspecified levels led to nasal and bronchial irritation in workers. There are divergent views on the lowest toxic level of morpholine. One author suggests that the only effect of an atmospheric concentration of 25 ppm morpholine is the ammonia—like odor; another reports that the irritant effects were —13— ------- observed within 1 minute at 16 ppm. Russian studies in rats indicated that morpholine may be more toxic than is indicated in the above—mentioned reports. For example, an enhancement of thyroid activity measured by uptake was observed. When the rats were exposed to morpholine at 70 or 8 mg/m 3 for 4 h/day, 5 days/week for 4 months, destruction of the lymphoid structure of the spleen was recorded. Undiluted morpholine applied to the skin of rabbits and guinea pigs for 1 to 13 days proved fatal. Affected tissues included liver, spleen, and kidney. Neutralizedsolutions (morpholine salts) were non—toxic. In assessing the significance of these results, it would be helpful to know the relative purity of the morpholine samples used. However, the need for further work on the acute and sub—chronic toxicity of morpholine in animals is clearly indicated. Chronic studies of morpholine toxicity have been conducted mainly as a control in investigations of the carcinogenicity resulting from the coadininistration of morpholine and nitrite or nitric oxide or from the nitrosatlon product, N—nitrosomorpholine. In one study, morpholine alone or In the presence of low levels of nitrite slightly, but not significantly, enhanced the incidence of liver tumors in rats. This is most probably due to in vivo nitrosation by endogenous or added nitrite. It is unlikely to be a direct effect of morpholine. —14— ------- Morpholine in the presence of substantial amounts of nitrite leads to formation of liver tumors in rats and hamsters and to lung tumors in mice. Investigators studying rats and hamsters followed the two—generation protocol in which the chemicals were fed to the mother during pregnancy and then to the offspring after parturition. Cofeeding sodium ascorbate (Vitamin C) with morpholine and sodium nitrite inhibits the induction of tumors. The ascorbate competes with the amine for the available nitrite, thereby inhibiting the formation of N—nitrosomorpholine. Inhibition of tumor formation may be indicated by either a reduced number of tumors or a lengthened time to tumor occurrence. Of particular importance to the human environment is the demonstration that ingestion of morpholine combined with the inhalation of nitric oxide has been shown to lead to N-nitrosomorpholine formation in vivo and to enhance the incidence of lung tumors in mice. These observations should be confirmed and studies designed to demonstrate their applicability to the levels in the human environment. Morpholine is not mutagenic in the absence of nitrosating agents. This has been established in studies using Salmonella typhimurium tester strains, by chromosome studies in —15— ------- micronucleus tests, by mammalian embryo cell resistance to azaguanine or ouabain, or in cell transformation assays. In contrast, N—nitrosomorpholine was mutagenic in all of these tests and in several others. Recommendations 1. Analytical surveys should be conducted to determine the amount of morpholine that escapes Into the environment from different sources. Such information is necessary if exposure to adventitiously formed N—nitrosomorpholine Is to be avoided. 2. The acute and sub—chronic toxicity of inorpholine should be reinvestigated using defined, pure samples of the test chemical to ensure that this toxicity is due to morpholine itself and not to an occasional impurity. 3. Further studies should be conducted on the production of N—nitrosomorpholine by the action of atmospheric nitric oxide on ingested morpholine. Test concentrations of both compounds should approximate those to which humans may be exposed. 2-NITROPROPANE 2—Nitropropane is a moderately volatile liquid (vapor pressure: 13 mm Hg at 20°C) that Is manufactured by only one U.S. producer. It is produced by reacting propane and nitric acid at 370—450°C and Is separated from the accompanying —16— ------- 1—nitropropane, nitroethane, and nitromethane by fractional distillation. Since 2—nitropropane is used widely as a solvent in paints and other coatings, it may be expected to have a wide environmental distribution. Emissions from the manufacture of 2—nitropropane have not been reported, but since 3,500 metric tons (mt) are used as a solvent in paints and coatings, much of this material may be released into the atmosphere. Exposure of individuals Is probably greatest in industrial/commercial settings such as those involving painting, printing, and ship repairing. Traces exist in some food packaging materials. Overall, the National Institute for Occupational Safety and Health (NIOSH) estimates that 100,000 workers may be exposed to 2—nitropropane. Metabolism in rats results in some 2—nitropropane being excreted unchanged and some being converted to acetone and nitrite or nitrate. Subsequent methemoglobinemia, which probably results from the interaction of nitrite and hemoglobin, has been observed in acute and, to a lesser extent, chronic exposure studies. Most of the adverse health effects of 2—nltropropane have been studied after inhalation, the route of most human exposure. There is a considerable species variation in the lowest fatal level of 2—nitropropane after a 4.5—h exposure: cat, 714 ppm; rat, 1,510 ppm, rabbit, 2,380 ppm; and guinea —17— ------- pig, approximately 4,620 ppm. Rats, guinea pigs, rabbits, and a single monkey tolerated repeated exposure to 325 ppm or 83 ppm. Higher concentrations led to dyspnea, cyanosis, prostration, convulsions, and death. Most chronic toxicity studies have used rats and, to a lesser extent, rabbits. In one study in rats, exposure to 400 ppm 2—nitropropane for 7 h/day led to the death of many animalsby the third day. A lower dose, 207 ppm, 7 h/day, 5 days/wk, induced either hepatocellular adenoma or carcinoma in all 10 rats sacrificed after 6 months exposure. Rats exposed for 3 months exhibited only hepatocellular hypertrophy, hyperplasia, and necrosis. In another study, no neoplasms were observed in Sprague—Dawley rats that were sacrificed after 6 months of exposure. In a study designed to resolve the differences in response to the chronic inhalation of 2—nitropropane, Sprague—Dawley rats were exposed to 200, 100, and 25 ppm of 2—nitropropane 7—h/day, 5 days/wk for 18 months. Male, but not female, rats exhibited an increase in liver nodules compared to untreated controls. Although there is no peer reviewed, published information concerning the pathology for this series of experiments, a Health Hazard Alert published by NIOSH/OSHA (Occupational Safety and Health Administration) in October 1980 concludes that 2—nitropropane induced liver cancer in rats dosed at 200 and 100 ppm but not at 25 ppm. —18— ------- In humans, four fatalities and one near fatality resulting from exposure to high doses 2—nitropropane have been recorded. The levels of exposure in these cases are unknown. The deaths occurred 6 to 10 days after exposure. Post—mortem examination revealed fatty degeneration of the liver In one case and liver necrosis in the three other cases. Lower levels of exposure to 2—nitropropane (20—45 ppm) to lead to a variety of nonspecific symptoms, especially of the digestive tract. An epidemiologic study of the entire 1,815—person workforce of a plant manufacturing 2—nitropropane failed to associate any cause of death with exposure to 2—nitropropane. This conclusion should be viewed cautiously since only 180 deaths were recorded and only 22 years had elapsed since 2—nitropropane production was initiated. A longer study is clearly needed to ensure that 2—nitropropane Is not carcinogenic in humans. In addition to the carcinogenicity of 2—nitropropane in the rat, mutagenicity tests suggest that this chemical Is also mutagenic. Positive microbial tests involving Salmonella typhimurium and Saccharomyces cerevisiae and mammalian micronucleus tests have been reported. The Health Hazard Alert published by OSHA suggests that 2—nitropropane should be handled In the workplace as a potential carcinogen in humans. OSHA’s present Permissible Exposure Limit for that compound Is 25 ppm or 90 mg/rn 3 . -19— ------- over an 8—h day on a time—weighted average. Because of the evidence for carcinogenicity in animals, It recommends that occupational exposures should be reduced to the lowest possible levels. Ways to achieve this goal are detailed in the OSHA publication. Recommendations 1. The potential for 2—nitropropane, or the nitrite liberated from it, to form N—nitroso compounds in vivo urgently needs attention. 2. There is a need to consolidate present understanding of the carcinogenlcity of 2—nitropropane obtained through experimentation by studying its effect in species other than the rat and by determining whether it is effective at doses of 25 ppm or lower. 3. The epidemiologic study of workers producing 2—nltropropafle should be continued to ensure that a sufficient number of exposed persons have survived long enough to develop all tumors that may be induced by this chemical. 4. The chronic toxicity studies conducted for the sole manufacturer of 2—nitropropane should be peer reviewed and published —20— ------- Chapter 1 NITROSATION OF ANINES AND THEIR CONTROL Although there is concern about the adverse health effects caused by exposure to aliphatic amines, their greatest potential for damage derives from their respective nitrosamines. As noted in later chapters, the pure amines have strong tendencies to form these more toxic substances both in vivo and in vitro , as well as in the environment and there is considerable uncertainty concerning the carcinogenicity and mutagenicity of these derivatives. Consequently, in addition to the general occurrence and control of the amines themselves, this chapter addresses certain aspects of their nitrosation. Occurrence of and exposure to the parent compounds are discussed in the chapters on the individual amines. OCCURRENCE N—Nitroso compounds form readily from a variety of amine— and amide—type compounds and nitrosating agents. The amines can be primary (Scanlan, 1975; Tannenbaum etal., 1979), secondary (Mirvish, 1975; Scanlan, 1975), or tertiary (Lijinsky et al., 1972; Ohshima and Kawabata, 1978). The nitrosating species can be derived from nitrite salts or titrous acid, oxides of nitrogen——nitric oxide, nitrogen dioxide, dinitrogen trioxide, and dinitrogen tetraoxide (Challis and Kyrtopoulos, 1979; Challis etal., 1978), or nitro compounds(O—NO 2 , N—NO 2 , C—NO) (Fan etal., 1978), or by —21— ------- transnitrosation from nitroso compounds (0—NO, N—NO, and C—NO) (Burgiass etal., 1975). Depending on the reactants and the presence of catalysts, N—nitroso compound formation can occur at acidic, neutral, or alkaline pH or in organic media. Because N—nltroso compounds can be formed so readily from such a variety of widely distributed precursors, it is not surprising that low levels of N—nitroso compounds are ubiquitous in the environment. In fact, nitrosamines should be expected wherever amines are present. Rapid Nitrosation of Aiuines with Oxides of Nitrogen Primary, secondary, and tertiary amines are more readily nitrosated by nitrogen oxides such as nitric oxide (NO), nitrogen dioxide (NO 2 ), and dinitrogen tetroxide (N 2 O 4 ) under mild nonacidic conditions than by aqueous nitrous acid (HNO 2 ). Deaminated products such as N—nitrosamines and, in some circumstances, N—nitramines are thus produced. Several oxides of nitrogen, e.g., nitric oxide, nitrogen dioxide, dinitrogen trioxide (N 2 O 3 ), and dinitrogen tetroxide, react with amines under mild conditions to form deaminated products, N—nitrosamines, and, in some instances, N—nitroamines. Generally, these reactions occur much more readily than do conventional nitrosations of amines involving nitrous acid. The weakest reagent is nitric oxide, which reacts slowly with secondary amines under anaerobic solutions to produce N—nitrosamines (Challis and KyrtopoulOs, 1979; Drago etal., l961).These reactions are —22— ------- catalyzed, however, by oxygen (Dragoetal., 1961), iodine (Challis and Outram, 1979), hydrogen iodide (Challis eta].., 1978), metal salts such as zinc iodide, cuprous chloride, cupric chloride, ferrous chloride, ferric nitrate (Challis etal., 1978), and silver nitrate (Challis and Outram, 1978). In many instances the reactions are complete in less than 20 minutes. The catalysis by oxygen involves nitrogen dioxide formation, and that by iodine, hydrogen iodide, and with metal lodides the formation of nitrosyl iodide. Catalysis by other metal salts, however, can involve oxidation of the amine to either a radical (R 2 N) or radical cation (R 2 N+), which then combines rapidly with nitric oxide (Challis et al., 1978). This mechanism has been proven for silver iodide salts. Amine nitrosations by nitrogen dioxide N 2 O 4 ) and nitric oxice plus nitrogen dioxide (4N 2 0 3 ) are very rapid and probably proceed upon encounter (Challis and Kyrtopoulos, 1978). Such reactions have been examined in the gas phase in connection with smoking (Neurath at a].., 1976; Spincer and Westcott, 1976) and atmospheric pollution (Bretschnelder and Matz, 1973; Hanst eta].., 1977; Pftts et al., 1978; Tuazon at al., 1978). The reactions also have application in chemical synthesis (Lovejoy and Vosper, 1968; White, 1955; White and Feldman, 1957). More recent work has demonstrated that dinitrogen trioxide and dinitrogen tetroxide are also powerful nitrosating agents in aqueous solution; a wide range of primary and secondary amines form high yields of deaminated and 1—nitroso products, respectively, In —23— ------- approximately 3 minutes when reacted with gaseous dinitrogen tn— and tetroxide under nonacidic conditions (Challis and Kyrtopoulos, 1977; Drago et al., 1961). With dinitrogen tetroxide, small amounts of N—nitramine form concurrently. These reactions suggest that both dinitrogen trioxide and dinitrogen tetroxide exist in two tautomeric forms of different reactivity, With dilute (2—500 ppm) nitrogen dioxide, N—nitroamine formation becomes as significant as N—nitrosamine formation (Challis and Goff, 1981, personal communication). These reactions are strongly catalyzed by s—substituted alcohols such as ethylene glycol, alkanolamines, and carbohydrates (Challis etal., 1980). Industrial Exposure . The people with the largest daily exposure to preformed N—nitrosamines are factory workers in a variety of industries. The highest level of chronic exposure to N—nitrosodimethylamine (NDMA) is incurred by tanners (Rounbehler et al., 1979), especially by those who work in the wet tanning area. NDMA has been found in five of five tanneries studied, at levels varying from a low of 23 pg/rn 3 to high of 47 pg/ni 3 . The daily human exposure from this source can be as high as 440 pg. In addition, N—nitrosomorpholine (NMOR) has been shown to be present at a level of 2.0 pg/rn 3 in the finishing area where the surfaces of the hides are chemically doped. The curing and extrusion areas of rubber tire factories also have been shown to contain NNOR, at levels of 0.5 to 27 pg/rn 3 (Fajen et al., 1979). The daily human exposure at these levels is 50—250 pg. —24— ------- NMOR probably arises as a trace contaminant in bismorpholincarbarnyl— sulfenanilde, which is used as an accelerator. N—nitrosodiphenylarnine (NDPhA) has also been found at the 0.2—47.0 pg/rn 3 level in a factory that manufactures it for use in tires. NDMA was reported at the site of a rocket fuel factory where unsymmetrical dimethyihydrazine was being manufactured from NDMA (Fine etal., 1976; l977a,c). There, levels of NDMA in air varied from 2 to 36 pg/rn 3 . The average daily NDMA intake of workers ranged from 10 to 50 p8. Nitrosamiries have also been found in pesticides (Cohenetal., 1978; Fine et al., 1977b), industrial wastewater (Cohen and Bachman, 1978; Fine etal., 1977a), and deionized water (Cohen, 1977; Fiddler et al., 1977). Inhalation . Tobacco smoke has been shown to contain a variety of nitrosamines, including N—nitrosonornicotine (NNN), N—nitrosoanatabine (NALB), 4—(N—methyl—N—nitrosamino)—1(3—pyridyl)lbutaflofle (NNK), NDMA, and N—nitrosopyrrolidlne (NPYR). Substances identified in a commercial filter cigarette include the following (average levels are provided): NNN — 310 ng/cigarette, NALB — 150 ng/cigarette, NNK — 370 ng/cigarette, NDNA — 5.7 ng/cigarette, and NPYR — 5.1 ng/cigarette (Brunnemann and Hoffmann, 1978; Hoffmannetal., 1980). The total nitrosamine body intake for a person smoking 20 cigarettes per day is approximately 16.8 pg. —25— ------- Volatile nitrosamines, including NDMA, NMOR, and N—nitrosodiethylamine (NDEA), have been found in the interiors of new automobiles (Rounbehler etal., 1980). Average levels were NDM —0.3 pg/rn 3 , NMOR — 0.67 pg/rn 3 , and NDEA — 0.11 pg/rn 3 . The total body burden after a 60—minute exposure has been estimated to be 0.6 pg (Fine et al., 1980). Ingestion . Ingestion is the most widely studied route of human exposure to N—nitrosamines, and studies are well documented for a variety of foodstuffs including bacon, cured meat, meatloaf, salami, ham, tinned meat, sausage, poultry, smoked and cooked fish, shellfish, cheese, and yogurt (International Agency for Research on Cancer, 1978). Only in cooked bacon and ham does the nitrosarnine level usually exceed 1 pg/kg. Until recently, the largest exposure to ingested nitrosarnines came from beer (Spiegelhalder etal., 1979). A study of beer from Australia, France, Greece, Holland, Ireland, Japan, Mexico, the Philippines, the United Kingdom, and the United States showed an average NDMA content of 2.8 pg/liter (Goff and Fine, 1979). A person drinking three cans of beer was therefore exposed to approximately 8.4 pg of NDMA. Of course, a person drinking a beer containing more than the average amount of NDMA was exposed to proportionally more of the substance. Webb and Gough (1980) in the United Kingdom and Stephany and Schuller (1980) in the Netherlands have shown that more than 80% of the ingested nitrosarnines in their countries came from beer. —26— ------- Scotch whisky contains 0.3—2.0 jg/liter of NDMA; a single 20—mi drink contains only 0.03 jig. Wines, liqueurs, gins, brandies, vodkas, and rums have not been found to contain volatile nitrosamines (Goff and Fine, 1979). Dermal Exposure . N—Nitrosodiethanolamine (NDE1A) has been found In cutting fluids (Fanetal., 1977b), cosmetics, shampoos, and lotions (Fan et al., 1977a). Edwards et al. (1979) showed that NDE1A was present in the urine of a person wearing an NDE1A—contamlnated cosmetic purchased over the counter. Approximately 1.7% of the NDE1A applied to the skin for 8 hours appeared as NDE1A In urine over a 21—hour period. Recent studies by the Food and Drug Administration (Wenninger, 1979) indicated that many U.S. cosmetics are still contaminated with NDE1A. In vivo. N—Nitrosamines, especially NDMA, have been observed in vivo in humans (Fine et al., 1977b; Kowaiski et al., 1980; Tannenbaum, 1980). Approximately 30% of the people tested had NDMA present for some of the day. The data are based on the analysis of a few milliliters of blood, urine, or feces with typical NDMA levels of 0.01 to 0.1 1 .ig/llter. Analyses at these low levels are extremely difficult because of the problem of false artifact formation during sample preparation. Tannenbaum (1980) has estimated that daily in vivo exposure to NDMA could be as high as 1,000 pg assuming that current analytical methods are valid. —27— ------- Assessment of Relative Exposures . Fine (1980) compared the average human intake of preformed nitrosamines, in terms of relative exposure of the U.S. population, by estimating body intake and then by adjusting that amount for the number of years a population would be exposed and the number of people who would be exposed. Table 1—1 shows cigarette smoking to be at the top of the list, followed by four exposure sources of approximately equal importance: new car interiors, beer, cosmetics, and bacon. Scotch whisky is at the bottom of the list. If in vivo formation were included, an individual’s exposure could be as high as 1,000 pg. In principle, exposure should also take into account the type of nitrosamine, its route of exposure, its relative potency, and other factors. Thus, although both NDMA and NMOR have been shown to be potent carcinogens at low doses in animals (International Agency for Research on Cancer, 1978; Moiseev and Benemansky, 1975; Shank and Newberne, 1976), much of the biological data needed to make an extrapolation to humans are unavailable. Even if there were such data, the question of how to extrapolate them from animals to humans would still be the subject of intense scientific debate. Nevertheless, it is important to appreciate just how significant the8e unknown factors may be. In rats, for example, if proper account were taken of the exposure route and if the relative potency of NPYR and NDE1A versus NDMA were known (and even this minor extrapolation is subject to dispute), then the relative rankings of the importance of exposure to car interiors versus exposure to cosmetics would differ by a hundredfold. —28— ------- A Comparison Table 1—1 of Average Human Intake of Nitrosamines in Terms of Relative Exposure of the U.S. Population Average Prime Body Number Population Relative Intake of Exposure Average Measured Intake, of Years Exposed, Exposed Population Exposure Nitrosamlne* Route Concentration ng/kg/day Exposed 10 0 Arbitrary Unit Smoking! NNN Inhalation 310 og/cigarette NALB Inhalation 150 ng/cigarette NNK InhalatIon 370 ngfcigarette 240 40 50 1,500 NDMA Inhalation 5.7 rig/cigarette NPYR Inhalation 5.1 rig/cigarette New Car Interior. NDM& J Inhalation 0.3 pg/rn 3 NNOR Inhalation 0.67 pg/rn 3 9.2 23 200 130 NDEA Inhalation 0.11 pg/rn 3 Beers NDMA Ingestion 2.8 pg/liter 15 40 50 90 Cosmetics. NDE1A Dermal 11 pg/g 6.3 50 70 70 Cooked bacon!. NPYR Ingestion 5 pg/g 2.4 70 100 50 Scotch whisky! NDMA Ingestion 0.97 pg/liter 0.4 40 20 1 *NN)J = N—nitrosonornicotine NALB — N—nitrosoanatabine NNI( 4—(N--methyl—N—nitro santino)—l( 3—pyridyl)--l—butanone NOMA — N—nitrosodimethylamine NPYR N—nitrosopyrro lidine NMOR N—nitrosomorpho line NOE1A — N—nitrosodiethanolamine. ------- Average NNN, NALB, and NNK levels for a commercial cigarette with filter were obtained from Hoffmann et al. (1979). NMDA and NPYR levels were obtained from Brunneman a Hoffmann (1978). The Surgeon General’s Report on Smoking and Health (U.S. Department of Health, Education, and Welfare, 1979) was used to estimate that 33—35% of the U.S. population over the age of 18 smoke cigarettes. The average levels of nitrosamines in new. car interiors was: NDMA, 0.30 pg/rn 3 ; NDEA 0.11 g/rn 3 ; and NMOR, 0.67 pg/rn 3 (Rounbehier et al., 1980). It was assumed that the average duration of exposure per day is 60 minutes for 200 x 106 people and that a new car is purchased every 3 years. The average NDMA content for U.S. beer was calculated by averaging the data from 18 samples analyzed by Goff and Fine (1979). The average U.S. beer consumption was assumed to be 132.475 liters/yr, for an average NDMA intake of 15 ng/kg/day. It was assumed that 50 x 106 people drink this quantity of beer daily. The average NDEIA level in cosmetics was calculated by averaging the seven data points in Fanetal. (1977a). If a woman uses 2 g of cosmetics per day, then 22,000 ng of NDE1A would be applied to the skin, 2% of which would penetrate the skin (Edwards et al., 1979). The average daily intake of NDE1A would therefore be 6.3 ng/kg/day. The 1979, u.s. production of bacon was 2.85 kg/person. The USDA requires that cooked bacon shall contain less than 10 pg/kg of NPYR; it was assumed that the average NPYR level in U.S. bacon is half this amount, or 5 pg/kg. It was assumed that half the u.s. population consumes bacon, and that these people eat bacon for their entire lives. The average NDMA content of scotch whisky was calculated by averaging the data from seven samples analyzed by Goff and Fine (1979). It was assumed that 20 x 106 people drink 30 ml per day, producing an average NDMA intake of 0.4 ng/kg/day. —30— ------- CONTROL Among the compounds evaluated in this report and In Its companion report on aromatic amines, triethanolamlne, morpholine, trifluralin, and oryzalin have all been shown to be contaminated with nitrosamines (Fine, 1980). When trifluralln was first tested for the presence of N—nltrosamlnes, the impurity N—nltrosodipropylamifle was found in concentrations of 195,000 ppb (Fine, 1980). The contamination occurred during manufacture where ring nitration with nitric and sulfuric acids was followed by the addition of dipropylamine. The manufacturing process was modified as soon as the manufacturer realized that the product was contaminated; the nitrosamine impurity was quickly reduced to below 1,000 ppb. NDMA was found at levels as high as 640,000 ppb in the dimethylaniine salts of the herbicide 2,3,6—trichlorobenzoic acid (Fine, 1980). The manufacturer eliminated the problem by switching the packaging from metal (sodium—nitrite--treated) cans to plastic—lined cans. The U.S. Environmental Protection Agency now requires manufacturers to provide information on the nitrosainine levels in pesticide products. Shortly after publication of data showing that cutting fluids were contaminated with as much as parts per hundred impurities of NDE1A, the National Institute for Occupational Safety and Health (1976) issued a Current Intelligence Bulletin . Within a few months, the Industry began advertising “nitrosamine—free ” cutting fluids in its —31— ------- trade journals. The problem was solved by ensuring that the product did not contain both sodium nitrite and triethanolamine in the same formulation. The NDMA impurity in beer was found to arise from N—nitrosation by nitrogen oxides (NO ) during drying of malt. Regulatory pressure from the U.S. Food and Drug Administration (FDA) to reduce the NDMA level in beer to less than 5 ppb (5 pg/liter) has forced the industry to develop short—term solutions. These include the use of gas burners with lower NO output and an increase in the acidity of the malt by sulfuring (burning solid sulfur and sweeping the malt beds with sulfur dioxide gases). Sulfuring may not be a viable long—term solution in view of sulfur dioxide emission regulations. In the tire industry, human exposure to NMOR in some factories has been reduced tenfold by increasing the ventilation capacity tenfold. NMOR is derived from various accelerators, which decompose to release morpholine. The industry is investigating the use of non—morpholine—based accelerators. The nitrosamine levels in pork products, especially in cooked bacon, have been lowered over the past several years because of pressure from the U.S. Department of Agriculture (USDA) and the FDA to lessen the amount of nitrite added. Ascorbate has thus been added to the bacon cures. USDA currently limits the NPYR level in cooked bacon to less than 10 ppb. —32— ------- During the past few years, it has been demonstrated that reductions in nitrosamine contamination can be achieved at minimum cost, as manufacturers become aware of the problem. Thus, the most effective control technology, is the widespread dissemination of accurate knowledge about the formation of nitrosamines. Manufacturers and users of amines and nitrosating agents should be made aware that their processes and products are probably contaminated but that the extent of the contamination can be lessened. —33— ------- References Bretschneider, K., and J. Matz. 1974. Nitrosan ines in the atmospheric air and in the air at the places of employment. Arch. Geschwulstforsch. 43:36—41. (in English) [ Chem. Abs. 81:110761d, 1974] Brunnemann, K.D., and D. Hoffman. 1978. Chemical studies on tobacco smoke LIX. Analysis of volatile nitrosamines in tobacco smoke and polluted indoor environments. Pp.343—356 in E.A. Walker,’ N. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects of N—Nitroso Compounds. IARC Scientific Publication No. 19. international Agency for Research on Cancer, Lyon. Buglass, A.J., B.C. Challis, and M.R. Osborne. 1975. TransnitrosatiOfl and decomposition of nitrosamines Pp. 94—100 in P. Bogovski and E.A. Walker, eds. N—Nitroso Compounds in the Environment. IARC Scientific Publication No. 9. International Agency for Research on Cancer, Lyon. Challis, B.C., and S.A. KyrtopOulos. 1977. Rapid formation of carcinogenic N—nltrosamifles in aqueous alkaline solutions. Br. J. Cancer 35:693—696. —34— ------- Challis, B.Ce, and S.A. Kyrtopoulos. 1978. The chemistry of nitroso compounds. Part 12. The mechanism of nitrosation and nitration of aqueous piperidine by gaseous dinitrogen tetraoxide and dinitrogen trioxide in aqueous alkaline solutions. Evidence for the existence of molecular isomers of dinitrogen tetraoxide and dinitrogen trioxide. J. Chem. Soc. Perkin Trans. 2(12):1296—l302. Challis, B.C., and S.A. Kyrtopoulos. 1979. The chemistry of nitroso—compounds. Part 11. Nitrosation of amines by the two—phase interaction of amines in solution with gaseous oxides of nitrogen. J. Chest. Soc. Perkin Trans. 1(2):299—304. Challis, B.C., and J.R. Outrani. 1978. Rapid formation of N—nitrosamines from nitric oxide in the presence of silver (I) salts. J. Chem. Soc. Chem. Comniun., No. 16:707—708. [ Chest. Abs. 90:38228b, 1979] Challis, B.C., and J.R. Outram. 1979. The chemistry of nitroso compounds. Part 15. Formation of N—nitrosamines in solution from gaseous nitric oxide in the presence of iodine. J. Chest. Soc. Perkin Trans. 1, No. 11:2768—2775. [ Chest. Abs. 92:180240j, 1980] —35— ------- Challis, B.C., A. Edwards, R.R. Hunma, S.A. Kyrtopoulos, and J.R. Outram. 1978. Rapid formation of N—nitrosamines from nitrogen oxides under neutral and alkaline conditions. Pp. 127—142 in E.A. Walker, M. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects of N—Nltroso Compounds. IARC Scientific Publication No. 19. International Agency for Research on Cancer, Lyon. Challis, B.C., J.R. Outram, and D.E.G. Shuker. 1980. New pathways for the rapid formation of N—nitrosamines under neutral and alkaline conditions. In E.A. Walker, M. Castegnaro etal., eds. N—Nitroso Compounds: Analysis, Formation and Occurrence. IARC Scientific Publication No. 31. International Agency for Research on Cancer, Lyon. Cohen, J.B. 1977. Reconnaissance of environmental levels of nitrosamines in the central United States, U.S. Government Report No. EPA—330/l—77—OO1. U. S. Environmental Protection Agency, Office of Enforcement, National Enforcement Investigations Center, Denver, Cob. Cohen, J.B., and J.D. Bachman. 1978. Measurement of environmental nitrosamines. Pp. 357—372 in E.A. Walker, M. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects on N—Nitroso Compounds. IARC Scientific Publication No. 19. International Agency for Research on Cancer, Lyon. —36— ------- Cohen, S.Z., G. Zvseig, M. Law, D. Wright, and W.H. Bontoyan. 1978. Analytical determination of N—nitroso compounds in pesticides by the United States Environmental Protection Agency —— A preliminary study. Pp. 333—342 in E.A. Walker, M. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects of N—Nitroso Compounds. LARC Scientific Publication No. 19. International Agency for Research on Cancer, Lyon. Drago, R.S., R.O Ragsdale, and D.P. Eyman. 1961. A mechanism for the reaction of diethylamine with nitric oxide. J. Am. Chem. Soc. 83:4337—4339. Edwards, G.S., J.G. Fox, P. Policastro, U. Goff, W.H. Wolf, and D.H. Fine. 1979. Volatile nitrosamine contamination In laboratory animal diets. Cancer Res. 39:1857—1858. Fajen, J.M., G.A. Carson, D.P. Rounbehier, T.Y. Fan, R. Vita, E.U. Goff, M.H. Wolf, C. S. Edwards, D.H. Fine, V. Reinhold, and K. Biemann. 1979. N—nitrosamines in the rubber and tire Industry. Science 205:1262—1264. Fan, T.Y., U. Goff, L. Song, D.H. Fine, G.P. Arsenault, and K. Biemann. 1977a. N—Nitrosodiethanolamine in cosmetics, lotions and shampoos. Food Cosmet. Toxlcol. 15:423—430. —37— ------- Fan, T.Y., J. Morrison, D.P. Rounbehier, R. Ross, D.H. Fine, W. Miles, and N.P. Sen. l977b. N—nitrosodiethanolamine in synthetic cutting fluids: A part—per—hundred impurity. Science 196:70—71. Fan, T.Y., R. Vita, and H.H. Fine. 1978. C—nitro compounds: A new class of nitrosating agents. Toxicol. Lett. 2:5—10. Fiddler, W., J.W. Pensabene, R.C. Doerr, and C.J. Dooley. 1977. The presence of dimethyl and diethyl—nitrosamineS in deionized water. Food Cosmet. Toxicol. 15:441—443. Fine, D.H. 1980. N—Nitroso compounds in the environment. Pp. 39—123 in J.N. Pitts, Jr. and R. Metcalf, eds. Advances in Environmental Science and Technology, Vol. 10. Wiley & Sons, New York. Fine, D.H., D.P. Rounbehler, E.D. Pellizzari , J.E. Bunch, R.W. Berkeley, J. McCrae, J.T. Bursey, E. Sawicki, K. Krost, and G.A. DeMarrais. 1976. N—nitrosodlmethylamlfle in air. Bull. Environ. Contain. Toxicol. 15:739—746. Fine, D.H., R. Ross, D.P. Rounbehier, A. Silvergleid, and L. Song. 1977a. Formation in vivo of volatile N—nitrosamines in man after ingestion of cooked bacon and spinach. Nature (London) 265:753—755. —38— ------- Fine, D.H., D.P. Rounbehier, T. Fan, and R. Ross. 1977b. Human exposure to N—nitroso compounds in the environment. Pp. 293—307 in H.H. Hiatt, J.D. Watson, and J.A. Winsten, eds. Origins of Human Cancer, Book A. Cold Spring Harbor Conferences on Cell Proliferation, Vol. 4. Cold Spring Harbor, N. Y. Fine, D.H., D.P. Roundbehler, A. Rounbehier, A. Silvergleid, E. Sawicki, E. Krost, and G.A. DemMarrais. 1977c. Determination of dimethylnitrosamine in the air, water, and soil by thermal energy analysis: Measurements in Baltimore, Md. Environ. Sci. Technol. 11:581—584. Goff, E.IJ., and D.H. Fine. 1979. Analysis of volatile N—nitrosamines in alcoholic beverage8. Food Cosmet. Toxicol. 17:569—573. Hanst, P.L., J.W. Spence, and M. Miller. 1977. Atmospheric chemistry of N—nitroso dimethylamine. Environ. Sc!. Technol. 11:403—405. Hoffmann, D., J.D. Adams, J.J. Piade, and S.S. Hecht. 1980. Analysis of volatile and tobacco specific nitrosamines in tobacco products. In E.A. Walker, M. Castegnaro etal., eds. N—Nitroso Compounds: Analysis, Formation and Occurrence. IARC Scientific Publication No. 31. International Agency for Research on Cancer, Lyon. —39— ------- International Agency for Research on Cancer. 1978. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Volume 17. Some N—nitroso compounds. International Agency for Research on Cancer, Lyon. 365 pp. Kowaiski, B., C.T. Miller, and N.P. Sen. 1980. Studies on the in vivo formation of nitrosamines in rats and humans after ingestion of various meals. In E.A. Walker, M. Castegnaro etal., eds. N-Nitroso Compounds: Analysis, Formation and Occurrence. IARC Scientific Publication No. 31. International Agency for Research on Cancer, Lyon. Lljinsky, W., L. Keefer, E. Conrad, and R. van de Bogart. 1972. The nitrosation of tertiary amines and some biological implications. J. Natl. Cancer Inst. 49:1239—1249. Lovejoy, D.L., and A.J. Vosper. 1968. Dinitrogen trioxide. Part VI. The reactions of d1n1rro en trioxide with primary and secondary amines. J. Chem. Soc. A.:2325—2328. Mirvish, S.S. 1975. Formation of N—nitroso compounds: Chemistry, kinetics, in vivo orcurrence. Toxicol. Appi. Pharmacol. 31:325—351. —40— ------- Moiseev, G.E., and V.V. Benemanskii. 1975. Carcinogenic activity of low concentrates of nitrosdimethylamine in inhalation. Vopr. Onkol. 21(6):l07—109. [ Cheni. Abs. 83:173618z, 1975J National Institute for Occupational Safety and Health. 1976. Current Intelligence Bulletin 15: Nitrosamines in Cutting Fluids. U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, Rockville, Md. October 6, 1976. Neurath, G.B., M. Dunger, and F.G. Pein. 1976. Nitrosation of nornicotine and nicotine in gaseous mixtures and aqueous solutions. Pp. 227—236 in E.A. Walker, P. Bogovski, and L. Griciute, eds. Environmental N—Nitroso Compounds: Analysis and Formation. IARC Scientific Publication No. 14. International Agency for Research on Cancer, Lyon. Ohshima, H., and T. Kawabata. 1978. Mechanisms of N—nitrosodiniethylamine formation from trimethylamine and trimethylaminoxide. Pp. 143—153 In E.A. Walker, N. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects of N—Nitroso Compounds. IARC Scientific Publication No. 19. International Agency for Research on Cancer, Lyon. —41— ------- Pitts, J.N., Jr., D. Grosjean, K. van Cawvenberghe, J.P. Schmid, and D.R. Fitz. 1978. Photooxldation of aliphatic amines under simulated atmospheric conditions: Formation of nitrosamines, nitramines, amides, and photocheinical oxidant. Environ. Sd. Technol. 12:946—953. Rounbehier, D.P., I.S. Krull, E.U. Goff, K.M. Mills, J. Morrison, G.S. Edwards, D.H. Fine, J.M. Fajen, G.A. Carson, and V. Reinhold. 1979. Exposure to N—nitrosodimethylamine in a leather tannery. Food Cosmet. Toxicol. 17:487—491. Rounbehier, D.P., J. Reisch, and D.H. Fine. 1980. Nitrosamines in new motor cars. Food Cosmet. Toxicol. 18:147—151. Scanlan, R.A. 1975. N—nitrosamifles in foods. Crit. Rev. Food Technol. 5:357—402. Shank, R.C., and P.M. Newberne. 1976. Dose—response study of the carcinogenicity of dietary sodium nitrite and morpholine in rats and hamsters. Food Cosmet. Toxicol. 14:1—8. Spiegeihalder, B., G. Eisenbrand, and G. Preussmann. 1979. Contamination of beer with trace quantities of N —nitrosodizuethylamifle. Food Costuet. Toxicol. 17:29—31. —42— ------- Spincer, D., and D.T. Westcott. 1976. Formation of nitrosodimethylamine in smoke from cigarettes manufactured from different tobacco types. Pp. 133—139 in E.A. Walker, P. Bogovski, and L. Griciute, eds. Environmental N—Nitroso Compounds: Analysis and Formation. IARC Scientific Publication No. 14. International Agency for Research on Cancer, Lyon. Stephany, R.W., and P.L. Schuller. 1980. Daily dietary intakes of nitrate, nitrite and volatile N—nitrosamines in The Netherlands using the duplicate portion sampling technique. Oncology 37:203—210. Tannenbaum, S.R. 1980. A model for estimation of human exposure to endogenous N—nitrosodimethylamlne. Oncology 37:232—235. Tannenbaum, S.R., J.S. Wishnok, J.S. Hovis, and W.W Bishop. 1978. N—Nitroso compounds from the reaction of primary amines with nitrite and thiocyanate. Pp. 155—159 in E.A. Walker, M. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects of N—Nitroso Compounds. IARC Scientific Publication No. 19. International Agency for Research on Cancer, Lyon. Tuazon, E.C., A.M. Winer, R.A. Graham, J.P. Schinid, and J.N. Pitts, Jr. 1978. Fourier transform infrared detection of nitramines in irradiated amine —NOr systems. Environ. Sd. Technol. 12:954—958. —43— ------- U.S. Department of Health, Education, and Welfare. 1979. Smoking and Health: A Report of the Surgeon General. DHEW Pub. No. (PHS) 7—50066. U.S. Department of Health, Education and Welfare, Public Health Service, Office of the Assistant Secretary for Health, Office on Smoking and Health, Bethesda, Md. 1,136 pp. Webb, K.S., and T.A. Gough. 1980. Human exposure to preformed environmental N—nitroso compounds in the U.K. Oncology 37:195—198. Wenninger, J.A., 1979. FDA Progress Report — Nitrosamine Contamination of Cosmetic Products, March 20, 1979. Food and Drug Administration, Washington, D.C. White, E.H. 1955. The chemistry of N—alkyl —N—nitrOsamideS I. Methods of preparation. J. Amer. Chem. Soc. 77:6008—6010. White, E.H., and W.R. Feldman. 1957. The nitrosatlon and nitration of amines in alcohols 1th nitrogen tetroxide. J. Am. Chem. Soc. 79:5832—5833. —44— ------- Chapter 2 GENERAL ANALYTIC METHODS A variety of procedures for analyzing aliphatic amines are described in the literature. Among them are thin—layer chromatography (TLC), paper chromatography, gas chromatography (GC), and spectrophotometric procedures. TLC procedures are described by Churaceketal. (1972), Gruger (1972), Schwartz and Brewington (1967), and Wick etal. (1967). Paper chromatography of free amines is described by Slaughter and Uvgard (1971), and of various derivatives by Churacek et al. (1972). Much recent CC work on free amines has been performed by Gruger (1972), Jones (1963), Miller etal. (1973), Preston and Prankratz (1970), Thombropoulos (1979), and Wick etal. (1967). Other researchers (Golovnya, 1976; Kannetal., 1976; Knapp, 1979; Mosier etal., 1973; Neurathetal., 1966 and Singer and Lijinsky, 1976 a,b) have performed work on various derivatives. Spectrophotonietric procedures have been used by Burenko et al. (1977), Karweik and Meyers (1979), and Zalnierius (1974). Fong and Chang (1976 a,b) identified secondary amines by determining the increases in N—nitrosamine content following drastic nitrosation with 0.145 M sodium nitrite at pH 3. Although this listing is by no means complete, it is indicative of the wide variety of procedures that have been used —45— ------- to analyze aliphatic amines. The most widely applied procedures Involve either GC or TLC. Gas Chromatography Gas chromatography has been used In much of the recent analytic work on aliphatic amines . Direct GC separation and determination of amines without formation of derivatives Is generally unsatisfactory. Interactions between strong bases and active sites on the supports frequently cause peak tailing; even “ghost” effects are sometimes observed. Coating with alkali, which Is often recommended, is not entirely satisfactory. The use of graphitized carbon black, thermally treated in a hydrogen stream and coated with basic compounds such as tetraethylene pentamine, Is claimed to eliminate adsorption effects and to give symmetrical peaks with amines (DiCorcia and Samperi, 1974). This method was successfully used to determine the presence of aliphatic amines In aqueous solutions. Miller etal. (1972) utilized the technique to identify dimethyl— and trimethylamines In fish, using an alkali flame—ionization detector (AFID). The main progress in the GC analysis of amlnes has been through the development of suitable derivatives (Knapp, 1979). Using GC and a flame—ionization detector (FID), the trifluoroacetamides have been used to detect primary and secondary amines In fresh vegetables, preserves, mixed pickles, fish and fish products, bread, cheese, stimulants, and surface waters (Neurath et al., 1977). —46— ------- Walle and Ehrsson (1970) have used heptafluorobutyryl derivatives in combination with electron—capture methods to detect picogram quantities of amino compounds. Heptafluorobutyryl— imidazole has been recommended 88 a reagent for amines by Staab and Waither (1960). With the object of restricting analysis to the naturally occurring nitrosatable amines, Singer and Lijinsky (1976a,b) chose the classic Hinsberg method of forming the —to1uenesu1fony1 derivatives to separate the secondary amities from their accompanying primary and tertiary amities. Tertiary amities do not react, and the products of primary amities are soluble in alkali; thus, the secondary amine derivatives can be isolated easily. The .R-toluenesulfonamides are readily separated by GC and have characteristic mass—spectrometric fragmentation patterns that facilitate their identification. Numerous other derivatization methods have been proposed. For example, Gejvall (1974) analyzed the urethanes formed by reaction of amities with diethyl pyrocarbonate, using CC with AFID. The reaction of different isocyanates with amities from N,N’—di— and NIN’, N’—trisubstituted ureas was studied by Nitsehe etal. (1974) who found the tert—butyl and the 3—trifluoromethyiphenylureas to be useful derivatives for CC analysis of primary and 8ecoudary amines.Electron—capture and nitrogen detectors, as well as mass spectrometry, have also been used with these derivatives. -47- ------- Long—chain primary amines have been analyzed by GC, after conversion to their dimethylamine derivatives (Metcalfe and Martin, 1972); excellent separation was reported on silicone oil capillary columns. Thin—Layer Chromatography Thin—layer chromatography has been applied to the analysis of amines, often to a derivative selected for Its particular properties, such as color or fluorescence. A few of the more important techniques are mentioned below. Hydrochlorides of primary, secondary, and tertiary amines have been separated on buffered silica gel by Teichert et al. (1960) and could be detected in amounts ranging from 0.1 to 10.0 ig. Grasshoff (1965) carried Out a similar separation on magnesium silicate layers. Ninhydrin has been used as a general reagent for the detection of primary amines, sodium nitroprusside for secondary amines, and Dragendorff reagent for tertiary ainines. Good separation and high sensitivity on silica gel plates were achieved by Neurath and Doerk (1964), using the red—colored 4’—nitroazobenzyl—(4)amides, which permitted detection of less than 1 g of amine. Colorimetry generally detects the acylated derivatives of primary and secondary amines more reliably than it does the parent —48— ------- amines. Seller and Welchmann (1965, 1967) were the first to take advantage of the fluorescing properties of the 1—dimethylaminonaphthalefle-5—8UlfOflamideS (dansyl derivatives) on silica gel. Under ultraviolet light (365 nm), as little as mo] is detectable. Kliinisch and Stadler (1974) described microquantitative determination of aliphatic amines by the formation of fluorescent derivatives with 7—chloro—4—nitrobenzo—2oxa—l, 3—diazole. One advantage of this method is that the reagent itself does not fluoresce. The derivatives were separated by TLC on polyamide—il foils. The methods described above have been applied to a variety of matrices, including biological samples such as blood and urine (Karweik and Meyers, 1979; Tombropoulos, 1979); industrial chemicals (Zalnlerius, 1974); aIr monitoring (Burenko etal., 1977; Jones, 1963); tobacco and tobacco condensates (Neurath eta]., 1966; Singer and Lljlnsky, 1976a,b); and various food products including fresh and saltwater fish, milk, tea, beer, wine, ham, frankfurters, cheese, and bread (Golovyna, 1976; Gruger, 1972; Kann etal., 1976; Miller etal., 1972; SInger and Lljinsky, 1976a). Analysis of the specific chemicals discussed in this report will be found in the chapters on those compounds. —49— ------- REFERENCES Burenko, T.S., E.G. Zhuravlev, and T.A. Miklashevich. 1977. Determination of morpholine in air. Gig. Tr. Prof. Zabol. No. 3:55—56. [ Chem. Abs. 86:194286s, 1971J Churacek, J., H. Pechova D. Tockste1nova and Z. Zikova. 1972. Separation and Identification of primary and secondary aliphatic amines as p_(N,N_dimethylamlno)—beflZefle p’—azobenzanildes by paper and thin layer chromatography. J. Chromatogr. 72:145—152. DiCorcia, A., and K. Samperi. 1974. Gas chromatographic determination at the parts—per—million level of aliphatic amines in aqueous solutions. Anal. Chem. 46:977—981. Fong, Y.Y., and W.C. Chan. 1976a.. Effect of ascorbate on amine—nitrite carcinogenicitY. Pp. 461—464 In E.A. Walker, P. Bogovski, and L. Griciute, eds. Environmental N—NitrosO Compounds: Analysis and Formation. IARC Scientific publication No. 14. International Agency for Research on Cancer, Lyon. —50-- ------- Fong, Y.Y., and W.C. Chan. 1976b. Reduction of nitrosodimethylauiine content of Cantonese salt fish. Pp. 465—471 in E.A. Walker, P. Bogovski, and L. Griciute, eds. Environmental N—Nitroso Compounds: Analysis and Formation. IARC Scientific Publication No. 14. International Agency for Research on Cancer, Lyon. Gejvall, T. 1974. Gas chromotographic analysis of amines separated as urethane derivatives. J. Chroniatogr. 90:157-161. Golovnya, R.V. 1976. Analysis of volatile amities contained in foodstuffs as possible precursors of N—nitroso compounds. Pp. 237—245 in E.A. Walker, P. Bogovski, and L. Griciute, eds. Environmental N—Nitroso Compounds: Analysis and Formation. IARC Scientific Publications No. 14. International Ager y for Research on Cancer, Lyon. Grasshoff, H. 1965. Dtlnnschicht—Chromatographie von Aminen. J. Chromatogr. 20:165—167. Gruger, E.H., Jr. 1972. Chromatographic analyses of volatile amities in marine fish. J. Agric. Food. Chem. 20:781—785. Jones, L.R. 1963. The determination of 2—nitropropane in air. Am. md. liyg. Assoc. J. 24:11—16. —51— ------- Kann, J., 0. Tauts, K. Raja, and R. Kalve. 1976. Nitrosamines arid their precursors in some Estonian foodstuffs. Pp. 385—394 in E.A. Walker, P. Bogovski, and L. Griciute, eds. Environmental N—Nitroso Compounds Analysis and Formation. IARC Scientific Publication No. 14. International Agency for Research on Cancer, Lyon. Karweik, D.H., and C.H. Meyers. 1979. Spectrophotometric determination of secondary amines. Anal. Chem. 51:319—320. Klimisch, H.—J., and L. Stadler. 1979. Mikroquantitative Bestimmung von Aliphatischen Aminen mit 7—Chlor—4—nitrobenzo— 2—oxa—1,3—diazol. J. Chromatogr. 90:141—148. Knapp, D.R. 1979. Handbook of Analytical Derivatization Reactions. Wiley and Sons, New York. 741 pp. Metcalfe, L.D., and R.J. Martin. 1972. Gas chromatography of positional isomers of long chain amines and related compounds. Anal. Chem. 44:403—405. Miller, A., III, R.A. Scanlan, J.S. Lee, and L.M. Libbey. 1972. Quantitative and selective gas chromatographic analysis of dimethyl— and trimethylatnine in fish. J. Agric. Food Chem. 20:709—711. —52— ------- Miller, A., 111, R.A. Scanlan, L.M. Libbey, H. Petropakis, and A.F. Anglemier. 1973. Quantitative determination of dimethyl— and trimethylamine in fish protein concentrate. J. Agric. Food Chem. 21:451—453. Mosier, A.R., C.E. Andre, and F.G. Viets, Jr. 1973. Identification of aliphatic amines volatilized from cattle feedyard. Environ. Sd. Technol. 7:642—645. Neurath, C., and E. Doerk. 1964. Identifizierung und quantitative Bestimmung elnzelner primarer und sekundarer Amine aus Gemischen als 4 ’—Nitro—azobenzoj.carbonsaure— (4)—amide. Chem. Ber. 97:172—178. Neurath, G.B., M. Dunger, J. Gewe, W. Luttich, and H. Wichern. 1966. Untersuchung der Fluchtigen Basen des Tabakrauches. Beitr. Tabakforsch. 3:563—569. Neurath, G.B., M. Dunger, F.G. Pein, D. Ambrosius, and 0. Schreiber. 1977. Primary and secondary amines in the human environment. Food Cosmet. Toxicol. 15:275—282. Nitsehe, I., F. Selenka, and K. Ballschmiter. 1974. Zum Metabolismus von Dialkyldithiocarbamaten. 1. Mitt. Bestimmung der beim Abbay entatehenden Amine durch Umsetzung mit Isocyanaten und gaschroinatographische Identifizierung der gebildeten Harnstoffderivate. J. Chromatogr. 94:65—73. —53— ------- Preston, S.T., Jr., and R. Pankratz. 1970. A Guide to the Analysis of Amines by Gas Chromatography. Polyscience Corporation, Niles, Ill. Schwartz, D.P ., and C.R. Brewington. 1967. Methods for the isolation and characterization of constituents of natural products. V. Separation of 2,6,dinitrophenylhYdraZofle pyruvaniides into classes and resolution of the individual members. Mlchrocheni. J. 12:547—554. Seller, N., and M. Wiechmann. 1965. Zum Nachweiss von Aminen im 1& 0 —Mol—Mass—stab. Trennung von 1—Dime thy1amlnonaphthalifl_5_Su1f0fl5auream en auf DunflsChiCht chromatograiflmefl. Experientia 21:203—204. Seller, N., and M. Wiechmann. 1967. Zur chromatographie einiger auf Kieselgel G—Schichtefl. J. Chromatogr. 28:351—362. Singer, G.M., and W. Lljinsky. 1976a. Naturally occurring nitrosatable compounds. I. Secondary amines in foodstuffs. J. Agric. Food Chem. 24:550—553. Singer, G.M., and W. Lijiflsky. 1976b. Naturally occurring nitrosatable amines; II. Secondary amines in tobacco and cigarette smoke condensate. J. Agric. Food Chem. 24:553—555. —54— ------- Slaughter, J.C., and A.R.A. Uvgard. 1971. Volatile amines of malt and beer. J. Inst. Brew. London 77:446—450. [ Chem. Abs. 76:12811x, 1972J Staab, H.A., and C. Waither. 1960. N—(Trifluoroacetyl)— imidazole. Angew. Chem. 72:35. Teichert, K., E. Mutschler , and H. Rochlmeyer. 1960. Beitrage zur analytischen Chromatrographie. (2. Mitteilung). Dtsch. Apoth. Ztg. 100:283—286. Tombropoulos, E.G. 1979. Micromethod for the gas chromatographic determination of morpholine in biological tissues and fluids. J. Chromatogr. 164:95—99. Walle, T., and H. Ehrsson. 1970. Quantitative gas chromatographic determination of picogram quantities of amino and alcoholic compounds by electron capture detection. I. Preparation and properties of the heptafluorobutyryl derivatives. Acta Pharm. Suec. 7:389—406. [ Chem. Abs. 73:123556a, 19701 Wick, E.L., E. Underriner, and E. Paneras. 1967. Volatile constituents of fish protein concentrate. J. Food. Sci. 3 2: 365—3 70. —55-. ------- Zalnierius, J. 1974. Photometric determination of triethanolamine in aqueous solutions. Nauch. Konf. Khim .—Anal. Pribalt Reap. B. SSR [ Tezisy Dokl.], 1st, p. 125—128. [ Chem. Abs. 86:21401x, 1977] —56— ------- Chapter 3 EPIDEMIOLOGY There is much less epidemiologic evidence pertaining to the adverse health effects caused by aliphatic amines than exists for aromatic amines. Although an early association was established for the induction of bladder cancer and exposure to certain aromatic amines, no such association has been shown for aliphatic amines, perhaps because the aliphatics are much less commonly used or are less toxic. The most concern about the danger of aliphatic amines derives from the ease and rapidity with which they react to form various N—nitrosamines. Chapter 1. contains discussions about the reactions themselves; this chapter reviews what little is known about the epidemiologic aspects of exposure to aliphatic amines and their associated N—nitrosamines. 2—Ni tropropane On the basis of data from studies in animals, the National Institute for Occupational Safety and Health (NIOSH) recommended in 1976 that 2—nitropropane (2—NP) be regarded as if it were a carcinogen in humans. A retrospective cohort study of mortality among persons who manufacture 2—NP was conducted by the International Minerals & Chemical Corporation (1979). On the basis of the data provided by the company, the investigators concluded that —57— ------- there was not “any unusual cancer or other disease mortality pattern among this group of workers, either before or after the beginning of 2—NP production in 1955. However, both because the cohort is small and because the period of latency (the time between first exposure and observation) is for most relatively short, one cannot conclude from these data that 2—NP is non—carcinogenic in humans.” Among this group of 1,815 workers, 180 were deceased and seven had died from “sarcomatous” cancers. Because the sarcomas occurred at a number of sites and involved a variety of tissue types, no comparison was possible. Furthermore, because none of these cases of cancer occurred among workers judged to be directly or indirectly exposed to 2—NP, it seems unlikely that 2—NP was related to the disease. Hexame thylenete tramine For several decades, hexamethylenetetramine has been reported to cause “rubber itch,” a dermatitis in rubber workers; (Heyhyrst and Kober, 1924). The substance also appears on NIOSH’s list of suspected carcinogens (National Institute for Occupational Safety and Health, 1976). Among a group of rubber tire manufacturers, 12 skin cancers occurred, as compared to 1.9 expected (Monson and Fine, 1978). On the basis of these data, it is not possible to determine —58— ------- whether the amine caused the 8km cancers since the skin of industry workers comes Into contact with uncured rubber containing many compounds——including aromatic amines. N—Nitrosamines Many, if not most, N—nitrosamines are known to be carcinogenic in laboratory animals. Humans are exposed to N—nitrosamines by a variety of routes (Fine, 1978)——food, tobacco, air, water, the workplace, and cosmetics. In addition, N—nitrosamines form through in vivo nitrosation. Despite the known carcinogenicity of N—nitrosamines in animals and the known exposure of humans, there are no data indicating similar carcinogenicity in humans (International Agency for Research on Cancer, 1978a,b). Nor do data suggest that N—nitrosamines are not carcinogenic in humans. This lack of epidemiologic data on the health effects of N—nitrosamines in humans Is due to the difficulty in identifying populations with known exposures to high levels of these substances. Exposure, when it occurs, is at relatively low levels. It is likely that) if adverse effects do occur, their rate Is quite low. Furthermore, it may not be possible to separate the adverse effects of N—nitrosamlnea from those of other substances. Two human health situations exist In which N—nitrosaniines are suspected of being related to cancer. In Colombia, high rates of stomach cancer have been observed In an area where there arehigh nitrate concentrations (110mg/liter) in well water, together with a —59— ------- high rate of nitrate excretion by inhabitants (Cuello et al., 1976). Because of this association, it has been hypothesized that in vivo nitrosation increases the incidence of stomach cancer (Correaetal., 1975; Tannenbaumetal., 1977). In the Peoples Republic of China, high rates of esophageal cancer occur both In humans and in poultry in an area where relatively high levels of nitrosamines and nitrites are present in food (Co—ordinating Group for Research on the Etiology of Esophageal Cancer in North China, 1974). Nitrosamines have also been detected in alcoholic beverages (Goff and Fine, 1979; Walker etal., 1979). It Is generally accepted that alcohol is causally related to cancers of the pharynx, larynx, esophagus, and liver, primarily among heavy consumers of alcoholic beverages (Robinette etal., 1979; Rothinan, 1975; Schottenfeld, 1979; Williams and Horin, 1977). Breslow and Enstroui. (1974), have reported a positive correlation between mortality rates for rectal cancer and heavy consumptions of alcohol as evidenced by tax receipts for the purchase of alcoholic beverages. The specific agent in alcoholic beverages that is responsible for the increased cancer incidence Is unknown, but the possibility that nitrosamines might lead to the increased rate cannot be ruled out. Two papers partially evaluated this possibility among Irish and Danish brewery workers (Dean et al., 1979; Jensen, 1979). Both groups of workers receive free beer as part of their compensation. —60— ------- At the time of those studies, the Irish beer contained low levels of nitrosamines; the Danish beer contained more (Gaff and Fine, 1979; D.F. Fine, Personal communication). Comparative data from these two groups of brewery workers are available only for cancers of the gastrointestinal tract. As seen in Table 3-1, an increased incidence of rectal cancer was found only among the Irish workers, who presuniabl.y consumed less nitrosamines than did the Danish workers; among Danish workers, only esophageal cancer appears in markedly increased incidence relative to the Irish workers. Inasmuch as esophageal cancer has repeatedly been found more frequently among alcoholics, the deficit among the Irish brewery workers seems to be the more atypical finding. At present, it is not known whether nitrosamines have caused any cancer in humans. Because of the ubiquitous but low—level occurrence of these substances, it will be difficult to obtain epidemiologic evidence regarding the postulated association. However, as with all chemicals, it Is prudent to lessen the potential for human exposure. —6 1— ------- TABLE 3—1 Observed Gastrointestinal Cancers and Observed/Expected Ratios for Selected Cancers Among Irish and Da sh _ B wery Workers. Observed Obs./Exp. Ratios Cancer Site Irish Danish Irish Danish Esophagus 10 41 0.63 2.09 Stomach 40 92 0.94 0.88 Large intestine 32 87 1.17 1.07 Rectum 32 85 1.76 1.02 Liver 7 29 1.27 1.51 Pancreas 17 44 1.21 1.09 Data abstracted from Deanetal., 1979 and Jensen, 1979. Irish numbers are deaths from cancer; Danish numbers are Incidence rates of cancer. c Expected numbers based on Dublin County Borough death rates (Irish) and from Danish national cancer morbidity rates. —62— ------- REFERENCES Breslow, N.E., and J.E. Enstrom. 1974. Geographic correlations between cancer mortality rates and alcohol—tobacco consumption in the United States. J. Nati. Cancer Inst. 53:631—639. Co—ordinating Group for Research on the Etiology of Esophageal Cancer in North China. 1975. The epidemiology of esophageal cancer in North China and preliminary results in the investigation of its etiological factors. Sd. Sin. 18:131—148. Correa, P., W. Haenszel, C. Cuello, S. Tannenbaum, and M. Archer. 1975. A model for gastric cancer epidemiology. Lancet 2:58—60. Cuello, C., P. Correa, W. Haenszel, G. Gordillo, C. Brown, M. Archer, and S. Tannenbaum. 1976. GastrIc cancer in Colombia. I. Cancer risk and suspect environmental agents. J. Nati. Cancer Inst. 57:1015—1020. Dean, C., R. MacLennan, H. McL.oughlin, and E. Shelley. 1979. Causes of death of blue—collar workers at a Dublin brewery, 1954—73. Br. J. Cancer 40:581—589. —63— ------- Fine, D.H. 1978. An assessment of human exposure to N—nitroso compounds. Pp. 267—278 in E.A. Walker, M. Castegnaro, L. Griciute, and R.E. Lyle, eds. Environmental Aspects of N—Nltroso Compounds. IARC Scientific Publication No. 19. International Agency for Research on Cancer, Lyon. Goff, E.IJ., and D.H. Fine. 1979. Analysis of volatile N—nitrosamines in alcoholic beverages. Food Cosmet. Toxicol. 17:569—573. Hayhurst, E.R., and G.M. Kober. 1924. Poisonings in the rubber industry. Pp. 535—545 In G.M. Kober, and E.R. Hayhurst, eds. Industrial Health. P. Blakiston’s Son & Co., Philadelphia. International Agency for Research on Cancer. 1978a. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man. Volume 16. Some Aromatic Amines and Related Nitro Compounds——Hair Dyes, Colouring Agents and Miscellaneous Industrial Chemicals. International Agency for Research on Cancer, Lyon. 400 pp. International Agency for Research on Cancer. 1978b. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Volume 17. Some N—Nitroso Compounds. International Agency for Research on Cancer, Lyon. 365 pp. —64— ------- International Minerals & Chemical Corporation. 1979. 2—NP Mortality Epidemiology Study of the Sterlington, La. Employees 1—1—46 through 6—30—77, by M.E. Miller and G.W. Temple. International Minerals & Chemical Corporation, Mundelein, Ill. [ 44] PP. Jensen, O.M. 1979. Cancer morbidity and causes of death among Danish brewery workers. mt. J. Cancer 23:454—463. Hanson, R.R., and L.J. Fine. 1978. Cancer mortality and morbidity among rubber workers. J. Nati. Cancer Inst. 61:1047—1053. National Institute for Occupational Safety and Health. 1976. Suspected Carcinogens, 2nd Edition. A Subfile of the NIOSH Registry of Toxic Effects of Chemical Substances. H.E. Christensen and E.J. Fairchild, eds. HEW Publication No. (NIOSH) 77—149. U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, Cincinnati. 251 pp. Robinette, C.D., Z. Hrubec, and J.F. Fraumeni, Jr. 1979. Chronic alcoholism and subsequent mortality in World War II veterans. Amer. J. Epidemiol. 109:687—700. Rothman, K.H. 1975. Alcohol. Pp. 139—150 in J.F. Fraumeni, Jr., ed. Persons at High Risk of Cancer; An Approach to Cancer Etiology and Control. Academic Press, New York. —65— ------- Schottenfeld, D. 1979. Alcohol as a co—factor In the etiology of cancer. Cancer 43:1962—1966. Tannenbaum, SR., M.C. Archer, J.S. Wishnok, P. Correa, C. Cuello, and W. Haenszel. 1976. Nitrate and the etiology of gastric cancer. Pp. 1609—1625 in H.H. Hiatt, J.D. Watson, and J.A. Winsten, eds. Origins of Human Cancer. Book C. Human Risk Assessment. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Walker, E.A., M. Castegnaro, L. Garren, G. Toussaint, and B. Kowalski. 1979. Intake of volatile nitrosamines from consumption of alcohol. J. Nati. Cancer Inst. 63:947—951. Williams, R.R., and J.W. Horm. 1977. Association of cancer sites with tobacco and alcohol consumption and socioeconomic status of patients: Interview study from the Third National Cancer Survey. J. Natl. Cancer Inst. 58:525—547. —66— ------- Chpater 4 TRIETHANOLAMINE HO—CH 2 —CH . ,CH 2 —CH 2 —OH CH 2 —C l 2 —OH Triethanolamine (2,2’,2”—nitrilotriethanol) is a pale yellow compound that solidifies at approximately room temperature. It has a melting point of 21.1°C and a boiling point of 335.4°C. Its vapor pressure is less than 0.01 mm Hg at 20°C. All three ethanolamines—--moflO—, di —, and triethanolamine——are produced by the reaction of ethylene oxide and aqueous ammonia at 50—100°C; the products are separated by distillation. PRODUCTION Four U.S. producers currently manufacture the three ethanolamifles Olin Corporation operates a plant in Brandenburg, Ky., with a capacity of 11,000 mt/year. Jefferson Chemical Co., Inc., a subsidiary of Texaco, Inc., increased the capacity of its Port Nechea, Tex. plant from 36,000 to 68,000 mt/year in late 1979, and the Union Carbide Corporation plant in Seadrift, Tex. has a design capacity of 105,000 mt/year. Dow Chemical U.S.A. began operating a plant with 57,000—mt/year capacity in Plaquemine, La. in 1978. At that time, Dow also put a plant with 23,000—mt/year capacity at Freeport, Tex. on standby and converted another plant —67— ------- with a 23,000—mt/year capacity in Midland, Mich. to produce isopropanolamines. Allied Chemical Corporation has had a plant with 9,100—mt/year capacity on standby at Orange, Tex. since December 1973 (Stanford Research Institute International, 1976, 1977, 1978, 1979a,b). U. S. production of triethanolatnine, as reported to the U.S. International Trade Commission (1976—1979), was as follows: 1976, 47,575 nit; 1977, 47,611 nit; and 1978, 52,000 nit. USE S Information on the consumption pattern for triethanolamine alone is not available; however, the consumption of all ethanolamines was reported for 1975 (Kirk—Othmer, 1978) and for 1979 (Chemical Marketing Reporter, 1979; U.s. International Trade Commission, 1979). These figures are shown in Table 4—1. Triethanolamine is used principally as a chemical intermediate for anionic surfactants in the form of salts of rosin acids, fatty acids, alkyl benzene sulfonates, and alkyl sulfates. These in turn are used in household detergents, textile processing, cosmetics and toiletries, and metal—working compounds. The two most widely used triethanolamines are the dedecyl sulfate (triethanolamine lauryl sulfate) and the salt of —68— ------- TABLE 4—i Ethanolamine Consumption Patterns, i975. . and 1979. Quantity Percentage of Uses (103 nit) Total Consumed 1975 Soaps and detergents 37.7 31.0% Gas purificatiOii . 25.9 21.0 CosmeticS 10.5 8.6 Textile specialties 13.6 11.0 Agricultural products 4.5 3.6 Emulsion polishes 1.8 1.5 Construction 5.9 4.8 Metals 6.8 5,5 Chemical intermediateS 10.9 8.9 Other 5.0 4.0 Total 122.6 100.0 1979 DetergentS 58.2 40.0% Gas conditioning and petroleum use.2. 36.4 25.0 Other (including agricul- ture and construction) 29.1 20.0 Export 21.8 15.0 Total 145.5 100.0 Kirk—Othmer, 1978. Chemical Marketing Reporter, 1979. .2. The triethanolamine isomer is not used in gas purification and does not contribute to the consumption of ethanolamines in this application. —69— ------- dodecylbenzenesulfonic acid (U.S. International Trade Commission, 1979). U.s. production of the dodecyl sulfate was 3,290 mt in 1978 and production of the salt of dodecylbenzenesulfonic acid in 1978 was 2,820 nit, according to the same source. Other triethanolamine salts reported to be produced commercially in the United States in 1978 include the salts of (1) oleic acid, by Emkay Chemical Co., Elizabeth, N.J., and Diamond Shamrock Corporation, Harrison, N.J.; (2) stearic acid, by Glyco Chemicals, Inc., Willianisport, Pa., and Sybron Corporation, Weilford, S.C.; (3) undecylbenzenesulfonic acid, by Henkel Corporation, Hawthorne, Calif., and Witco Chemical Corporation, Houston, Tex.; (4) tallow acids, by Andrew Jergens Company, Saginaw, Mich.; and (5) mixed linear alcohols (sulfated), by Bofors Lakeway, Inc., Muskegon, Mich., and Henkel Corporation, Hawthorne, Calif. (Stanford Research Institute International, 1979). In addition to the use of triethanolamine to make the above surface—active agents, emulsions are commonly prepared in situ, with triethanolainine and fatty acids (Kirk—Othmer, 1978). Other non—surface—active salts of triethanolamine commercially produced in the United States include (1) the phosphate (used as a corrosion inhibitor) and the monosulfate, both manufactured by Emkay Chemical Company, Elizabeth, N.J.; (2) the salicylate, produced by Norda, Inc., Boonton, N.J., and R.S.A. Corporation, Ardsley, —70— ------- N.Y.;and (3) the hydrochloride, produced by Eastman Kodak Company, Rochester, N.Y., and R.S.A. Corp., Ardsley, N.Y. (Stanford Research Institute international, 1979). Other principal uses of triethanolamine are in agriculture and construction. The substance is used as an intermediate in the production of 2,4,5-.trichloropheflOxYaCetiC acid, a triethanolauiine salt, by Dow Chemical, U.S.A. at Midland, Mich. This chemical is registered for use as a herbicide by the U.S. Environmental Protection Agency (1974). Triethanolamifle is also used in the production of a triethanolamlfleCOPPer complex, marketed under the trade name A&V—70 Algaecide, by A&V Inc., Pewaukee, Wise. This chemical is registered for use as an aquatic herbicide by the U.S. Environmental Protection Agency (1974). In the construction industry, low levels of triethanolamine or its salts are added to cement clinkers to increase the efficiency of the grinding mill by reducing particle agglomeration (Chemical Marketing Reporter) 1979; Kirk-Othmer, 1978). EXPOSURE There is no information on the release of triethanolamine during either its production or its conversion to other products. Such emissions would be limited to the production sites mentioned above. —71— ------- However, triethanolamine is used in the production of other chemicals at more than 60 sites, some in heavily populated areas, thus increasing the potential for exposure by atmospheric emissions. The greatest potential for humans to be exposed to triethanolamine is probably through its food and cosmetics applications. The Food and Drug Administration (FDA) has approved the use of triethanolamine (1) in the formulation of adhesives for articles used in packaging or holding food; (2) in the formulation of resinous and polymeric coatings on food—contacting surfaces of items used In the processing, preparation, packaging, and holding of food; (3) in adjusting the pH during the manufacture of amino resins used as components of paper and paperboard in contact with aqueous and fatty foods; (4) as a component of coated or uncoated food—contacting surface of paper or paperboard used for dry food; (5) as a defoaming agent used in coatings of food—contacting surfaces or in the manufacture of paperboard or paper articles used in processing, preparation, packaging, and holding of food; (6) as a curing agent for polyurethane resins used on food—contacting surfaces; (7) as an activator in rubber articles intended for repeated use in food processing, packaging, and preparation; (8) as a component of textiles and textiles fibers used in articles Involved in processing, preparation, and packaging of food; (9) in surface lubricants used In the manufacture of metallic articles that contact food; and (10) as a chemical used in washing or to assist in the lye peeling of fruits and vegetables (21 CFR 173). Neither the extent of contamination of foods by triethanolamine, nor even the —72— ------- degree of use in such applications, is known. The use of triethanolamine in skin cleansers, lotions, and cosmetics is apparently widespread. The FDA lists triethanolamine as a component of shaving cream, shampoo, hair tonic, hair tint and dye, cleansing cream, foundation cream, hand cream, suntan lotion, lotion makeup base, rouge and blush makeup, mascara, eye shadow, cuticle removers, depilatories, and several other toiletries (Food and Drug Administration, 1980). Because of its properties as a wetting agent, the pure form of triethanolanilne may be added to cosmetics, especially to many creams and lotions, but It probably combines in situ with fatty acids to form triethanolamine fatty acid salts. The Occupational Safety and Health Administration has not established standards for occupational exposures to triethanolamine. In the National Occupational Hazard Survey, which was conducted by the National Institute for Occupational Safety and Health (1977), exposures to triethanolamine derivatives (e.g., triethanolamine dodecylbenzenesulfonate, triethanolamine oleate) were reported in a number of industries. However, no exposures to triethanolamine itself were reported. —73— ------- ANALYTIC METHODS Because of the widespread uses of triethanolamine, it is often necessary to conduct analyses to determine its presence in waxes, oils, cosmetics, soaps, cutting fluids, and other organics. In all these analyses the first step is generally to remove the organic fraction by repeated extraction with methylene chloride. The residue is dried by evaporation on a steam bath and further cleaned up to remove acidic or alkaline fractions. The three hydroxyl groups render triethanolainine nonvolatile, and concentration is therefore readily carried out without loss by evaporation. If the sample can be purified sufficiently, final quantitation can generally be achieved by weighing the residue and ensuring its identity by infrared spectroscopy (Wisneski, 1977). More conventional procedures call for quantitation by gas chromatography, following volatilization by means of a suitable derivative. Triethanolamifle is readily derivatized at its three hydroxyl groups, thereby increasing the volatility so that it may be separated by gas chromatography. The following examples are some of the derivatives that may be used (Knapp, 1979). o Methyl ether derivatives are prepared by reacting the triethanolamine with diazomethane in the presence of fluorboric acid. o Acetyl derivatives are prepared by dissolving the compound in a 1:1 solution of acetic anhydride and pyridine. An alternative procedure involves the use of acetyl methanesulfOflate in the presence of phosphorus pentoxide. —74— ------- o Pentafluorobenzoyl derivatives are prepared by reaction of the parent amine with pentafluorobenzoyl chloride. The derivative is not only volatile, but can be analyzed using an electron capture detector on the gas chromotograph, thereby enhancing both selectivity and sensitivity. o Esters are prepared by using the appropriate acid anhydride in the presence of toluenesulfonic acid. o Trimethylsily] . derivatives are prepared by reacting the compound with hexamethyldisilazane (or any other suitable trimethylsilyl derivatizing agent). Triethanolamine can be readily analyzed without derivatization by high pres sure liquid chromatography. ...7 5... ------- HEALTH EFFECTS Metabolism No data were available on the metabolism of triethanolamine and little is known of the metabolism of its N—nitroso derivative NDE1A, although it apparently requires metabolic activation to exert its mutagenic effects. When given to rats by gavage, lOX is excreted in urine as unchanged NDE1A within 24 hours, possibly explaining the substance’s rather weak carcinogenicity (Preussmannetal., 1978). This excretion level was independent of the administered dose range of 10—1000 mg/kg. Because much human contact with NDE1A is dermal, several studies have focused on this exposure route. Preussman and Spiegeihalder, (personal communication) reported that 70—80Z of undiluted NDE1A was absorbed through the shaved skin of rats. NDE1A has also been shown to cross excised human skin (Bronaugh etal., in press) and the skin of a volunteer wearing a contaminated cosmetic (Edwards etal., 1979). An ancillary finding in the latter study was that the urinary excretion in the volunteer implied a half—life in humans of 12 hours. Acute and Chronic Toxicity In aquatic protozoa and invertebrates, the chronic and acute toxicity of triethanolamine was studied by determining the survival time following exposure (Apostol, 1975). Triethanolamine was less —76— ------- toxic than diethanolamine, which was less toxic than monoethanolamine. Chronic effects occurred at concentrations above 1 mg/liter; acute effects were observable at doses of more than 100 mg/liter. For mammals, all three compounds are weak toxins. No LD 50 less than 700 mg/kg has been reported for any species or route of administration (National Institute for Occupational Safety and Health, 1977). Kostrodymova et a].. (1976) showed that 13% trlethanolamine solutions readily penetrated the skin of rat, causing changes in liver and central nervous system, which they claimed was indicated by increased levels of alanine aminotransferase and a decreased level of cholinesterase in blood. Topically applied triethanolamine did not exert carcinogenic or cocarcinogenic (with 3—methyleholanthrene) activity in mouse (Kostrodymovaetal., 1976). When administered with synthenol DS—lO (a detergent used in the USSR), the substance inhibited the cocarcinogenic properties of the detergent. Triethanolamine has been implicated as a source of occupational disorders in workers handling cutting fluids and other mixtures that contain the chemical. Calas etal. (1978) found evidence that triethanolainine was a sensitizer for contact allergens. Occupational dermatoses due to triethanolamine in cutting fluids (Selisskii eta].., 1978) and in textile and finishing plant workers (Venediktova and Gudina, 1976) have also been reported. Carcinogenicity Hoshino and Tanooka (1978) recently reported that triethanolanilne —77— ------- produced malignant tumors in mice. Male and female ICR/JCL mice were fed a diet containing 0.03% or 0.3% triethanolamine for their lifetimes. In the females, there was a significant increase in the total number of malignant tumors in both the 0.03% and 0.3% groups as compared to the controls (P<0.Ol). There was a slight, but not statistically significant dose response noted. Predominant tumor types were thymic and nonthymic lymphomas in the females; both sexes also had tumors in several other sites. No hepatomas were observed. Because the purity of the material first fed to the animals was not thoroughly established, and because the nature of any products formed as a result of mixing the triethanolamine with the diet was not studied, the identity of the carcinogenic material remains in doubt. No other references were found regarding the possible carcinogenicity of triethanolamine. NDE1A, has been shown to be present as an Impurity in triethanolamine (Fine, unpublished data, 1978), in many products that contain triethanolamine, such as cosmetics (Fan et al., 1977a), and in cutting fluids (Fanetal., l977b; Zingmark and Rappe, 1977). The carcinogenicity of NDE1A has been studied in both rat and hamster. A group of 20 BD rats was given NDE1A in drinking water at concentrations equivalent to 600—1,000 mg/kg/day, the total dose was 150—300 g/kg. All 20 animals developed hepatocellular carcinomas between 242 and 325 days after the start of treatment; four rats also had renal adenomas (Druckrey etal., 1967). In Syrian golden hamsters, two groups of 15 males and 15 females received either seven twice—weekly —78— ------- subcutaneous injections each of 2,260 mg/kg NDE1A (one—fifth of the LD 50 ) or 27 inJections of 565 mg/kg (one—twentieth of the LD 50 ) over 45 weeks. For the latter group, several injection—free intervals of 1—2 weeks were required because of local necrosis at the injection site. Total doses were approximately 15 g/kg. All surviving animals were killed at 78 weeks. In the first group, 28 of 30 animals were still alive at the appearance of the first tumor (33 weeks); 20 developed tumors——including 10 adenocarclnomas of the nasal cavity, 8 papillary tumors of the trachea, and 3 hepatocellular adenomas. In the second group, 27 of 30 animals survived 33 weeks; 19 developed tumors——including 12 adenocarcinomas of the nasal cavity, 8 papillary tumors of the trachea, and 3 fibrosarcomas at the injection site. Of 27 effective controls, 3 animals developed 1 thyroid carcinoma, 1 hemangioendothelioma of the spleen, and 2 adenomas of the adrenal gland (Hilfrich etal., 1978). Mutagenicity of Triethanolamine Triethanolamine has not been well studied; however, the studies that have been performed show no evidence of mutagenic activity in bacteria. Bacteria . Hoshino and Tanooka (1978) exposed Bacillus subtilis TKJ5211 (uvr) to analytical—grade triethanolamine in the presence and absence of sodium nitrate. An increase in the frequency of his+ revertants was seen when triethanolamine was mixed with an equal amount of sodium nitrite and allowed to react for 8 hours. This activity was greater than that observed with sodium nitrite alone. In the presence of rat liver S—9, the mutagenic activity was abolished. Triethanolamine —79— ------- alone or with rat liver S—9 was not mutagenic. The same triethanolamjne—sodium nitrite mixture, following autoclaving, was more toxic to B. subtills wild type strain than to the corresponding DNA repair—deficient strains. However, these strains were not isogenic. The mutagenic activity observed only in the absence of S—9 argues against NDE IA as the mutagen; however, the study did not test NDE1A, so the possibility that it was the mutagen formed cannot be excluded. Triethanolamine was also tested for mutagenicity in Salmonella through the Environmental Mutagenesis Test Development Program of the National Institute for Environmental Health Sciences (NIEHS). The test system used was a preincubation modification of the Salmonella/microsome test using Aroclor—induced rat and Syrian hamster liver S—9 and strains TA98, TA100, TA1535, and TA1537. No mutagenic activity was observed at dosage levels of up to 3.3 mg/plate (W. Speck, personal communication). Plants . Triethanolamine, at a concentration of 0.125 mol/liter, did not induce multipolar mitoses in Allium cepa cells after 4 hours of treatment (Barthelmess and Elkabarity, 1962). Mutagenicity of N—Nitrosodiethanolaniine The results obtained with NDE1A are summarized In Table 4—2. The only available mutagenicity data on NDE1A were obtained from studies using Salmonella, EscherichIa coli , and B. subtilis . The reported results are mixed, showing both positive and negative results in Salmonella . Some authors report that liver S—9 Is required; others find no requirement for liver S—9. —80— ------- TABLE 4—2 Summary of Mutagenicity Tests with N—Nltrosodiethanolainine (NDE1A ) Species/Strain S. typhimurlum TA1535 S. typhlmurlum GA46, TA100 S. typhimurium TA1535, C3076 TA1537, D3052, TA1538, TA98 S. typhimurium TA100, TA1535 S. typhimurium TA98, TA1538 E. coil WP2, WP2uvrA ! Streak test pró ocoi. . With rat liver S—9 only. .E . Both with and without mouse liver S—9. Results Negative Po si tivea,b Negative! Positivec Negative Posltlve!’ References Rao et a]., 1979 McMahon et al., 1979 McMahon et al., 1979 Hesbert eta]., 1979 Hesbert et al., 1979 McMahon et a]., 1979 —81— ------- Bacteria . Rao et al. (1979) tested NDE1A In Salmonella TA1535 at concentrations up to 2,000 pg per plate in the standard plate test and up to 2,000 pg/mi in the preincubation modification test. All tests were run both with and without phenobarbital—induced rat liver S—9. No positive responses were observed. NDE1A was reported to be mutagenic for both TA1535 and TA100 (Hesbert etal., 1979), both with and without S—9 activation, on a standard plate test at concentrations up to 10 mg/plate. A positive response was observed at 2 mg/plate with TA1535, which appears to contradict the negative report from Raoetal. (1979). In another study, McMahonetai. (1979) used a gradient plate test. In this procedure, a concentration gradient (ranging from 1 to 10 mg/mi in the agar) was formed in a Petri dish, and the microbial tester strain was streaked across the gradient. NDE1A was tested against a series of Salmonella and E. coil tester strains, both with and without Aroclor—induced rat liver S—9. NDE1A produced positive results only with S—9 in Salmonella strains G46 and TA100 and E. coil strains WP2 and WP2uvrA. No mutagenicity was observed either in TA1535 or any of the Salmonella frameshift—specific strains used. This result appears to be in agreement with the negative results produced by Rao et al. (1979). However, strains G46, TA100, and TA1535 all measure reversion at the same locus. G—46 is just his; TA1535 Is strain G46 with an additional rfa mutation and uvrB bio deletion. TA100 is TA1535 containing the plasmid pKN1O1, which enhances its sensitivity and —82— ------- decreases its specificity. B. coil WP2 and WP2A detect base—pair, substitution—inducing substances. There is rio obvious explanation for the negative results produced by TA1535 and the positive results produced by G46 and TA100. In a different study (Hoshino and Tanooka, 1978), NDE1A was not studied directly, but the mixture of triethanolamine and sodium nitrite at pH 3.5 (whIch is supposed to produce NDE1A) was mutagenic for a B. subtilis mutant along with a series of isogenic repair—deficient strains. A mutagen formed during the triethanol—sodium nitrite reaction was active without S—9. It lost its activity in the presence of Aroclor—induced rat liver S—9. Triethanolamine alone was not mutagenic; sodium nitrite alone showed some mutagenicity, but significantly less than that of the mixture. The repair—deficient strains were more sensitive to killing and mutagenesis than was the wild—type strain. The investigation concluded that the mutagerl formed in the reaction mixture was not NDE1A because the mutagen was active only in the absence of S—9. However, since no NDE1A control was run, the conclusion was not substantiated. Teratogenicity No data were available to evaluate the potential teratogenicity or reproductive toxicity of triethanolamine. —83— ------- CONCLUSIONS Triethanolamine does not induce point mutation in bacteria, and its ability to induce point or chromosomal mutations in eukaryotic cells has not been tested. The chemical is also of interest because of its ability to be nitrosated to form NDE1A. In vitro and in vivo mutagenicity studies of NDE1A formation, coupled with analyses of NDE1A yield, must be performed. From the results described here, it is obvious that more studies of NDE1A will have to be performed to resolve the apparent discrepancies. There Is no agreement as to the strains that are mutated or their requirement for liver S—9 fraction. Certainly, NDE1A is a mutagen for Salmonella , but the conditions under which it is mutagenic still have to be determined. When mutagenicity was observed, It usually occurred after exposure to high doses, bringing Into question the purity of the NDE1A tested and possible impurities present in the sample. No studies have been performed to evaluate the induction of chromosomal mutation. Therefore, in addition to further testing with microbial systems, studies should also be performed in vitro in mammalian systems. Studies should be conducted to determine human exposure levels to both triethanolamine and to NDE1A and to further assess their potential noncarcinogenic toxicity, teratogenicity and effects on reproduction. —84— ------- REFERENCES Production, Uses, Exposure Chemical Marketing Reporter. 1979. Chemical profiles: Ethanolamines. Chem. Mark. Rep. 2l5(15):9, April 9. Schnell Publishing Co., New York. Code of Federal Regulations. 1980. Title 21, Part 173. Secondary direct food additives permitted in food for human consumption. Office of the Federal Register, National Archives and Records Service, General Services Administration, Washington, D.C. Food and Drug Administration. 1980. Information received under 21 CFR Part 720 by the Division of Cosmetic Technology, Food and Drug Administration, Washington, D.C. Kirk—Othmer Encyclopedia of Chemical Technology. 1978. Third Edition, Vol 2. Martin Grayson, exec. ed. and David Eckroth, assoc. ed. A Wiley—Interscience Publication, John Wiley & Sons, New York. 1,036 pp. Stanford Research Institute, Chemical Information Services. 1976. 1976 Directory of Chemical Producers: United States of America. Stanford Research Institute, Menlo Park, Calif. 1,039 pp. Stanford Research Institute, Chemical Information Services. 1977. 1977 Directory of Chemical Producers: United States of America. Stanford Research Institute, Menlo Park, Calif. 1,060 pp. —85— ------- Stanford Research Institute International, 1978. 1978 Directory of Chemical Producers: United States of America. Stanford Research Institute International, Menlo Park, Calif. 1,127 pp. Stanford Research Institute International, 1979a. 1979 Directory of Chemical Producers: Western Europe. 2 volumes. Stanford Research Institute International, Menlo Park, Calif. 2,100 pp. Stanford Research Institute International, 1979b. 1979 Directory of Chemical Producers: United States of America. Stanford Research Institute International, Menlo Park, Calif. 1,122 pp. U.S. Environmental Protection Agency. 1974. EPA Compendium of Registered Pesticides, Volumes I—V. U. S. Environmental Protection Agency, Washington, D.C. U. S. International Trade CommissIon. 1976. Synthetic Organic Chemicals, U. S. Production and Sales, 1974. USITC Publication 776. U.S. International Trade Commission, Washington, D.C. 256 pp. U. S. International Trade Commission. 1977a. Synthetic Organic Chemicals, Ti. S. Production and Sales, 1975. USITC Publication 804. U.S. International Trade Commission, Washington, D.C. 246 pp. U. S. International Trade Commission. 1977b. Synthetic Organic Chemicals, United States Production and Sales, 1976. USITC Publication 833. U.S. International Trade Commission, Washington, D.C. 357 pp. —86— ------- U. S. International Trade Commission. 1978. Synthetic Organic Chemicals, U. S. Production and Sales, 1977. USITC Publication 920. U.S. International Trade Commission, Washington, D.C. 417 pp. U. S. International Trade Commission. 1979. Synthetic Organic Chemicals, U. S. Production and Sales, 1978. USITC Publication 1001. U.S. International Trade Commission, Washington, D.C. 369 pp. —87— ------- Analytic Methods Knapp, D.R. 1979. Handbook of Analytical Derivatization Reactions, Wiley and Sons, New York , 741 pp. Wisneski, H.H. 1977. Analysis of cold wave solutions. Pp. 78—82 in A.J. Senzel, ed. Newburger’s Manual of Cosmetic Analysis. 2nd Edition. Association of Official Analytical Chemists, Inc., Washington, D.C. —88— ------- Health Effects Apostol, S. 1975. Ethanolamine toxicity to aquatic invertebrates. Stud. Cercet. Biol. 27:345—351. [ Chein. Abs. 85 : 7 3051q, 19761 Barthelmess, A., and A. Elkabarity. 1962. Chemically induced multipolar mitosis. III. Protoplasma 54:455—475. [ Chem. Abs. 57:3891a, 1962j Bronaugh, R.L., E.R. Congdon, and R.J. Scheupleiri. in press. The effect of cosmetic vehicles on the penetration of N—nitrosodiethanolamine through excised human skin. J. Invest. De rma tol. Calas, E., P.Y. Castelain, and A. Piriou. 1978. Epidemiologie des dermatoses de contact a marseille. Ann. Dermatol. Venero]. 105:345—347. (English abstract) [ Cumul. Did. Medicus 19:1640, 1978j Druckrey, H., R. Preussinann, S. Ivankovic, and D. Schmaehl. 1967. Organotrope carcinogenic effects of 65 different N—nitroso compounds on BD—rats. Z. Krebsforsch. 69:103—201. [ Chem. Abs. 67:32277v, 1967J. Edwards, G.S., M. Peng, D.H. Fine, B. Spiegeihalder, and J. Kann. 1979. Detection of N—nitrosodiethanoj.ainjne in human urine following application of a contaminated cosmetic. Toxicol. Lett. 4:217—222. —89— ------- Fan, T.Y., U. Goff, L. Sing, D.H. Fine, G.P. Arsenault, and K. Biemann. 1977a. N—Nitrosodiethanolamine in cosmetics, lotions and Shampoos. Food cosmet. Toxicol. 15:423—430. Fan, T.Y., J. Morrison, D.P. Rounbehier, R. Ross, D.H. Fine, W. Miles, and N.P. Sen. 1977b. N—Nitrosodiethanolamine in synthetic cutting fluids: A part—per—hundred Impurity. Science 196:70—71. Hesbert, A., N. Lomonnier, and C. Cavelier. 1979. Mutagenicity of nitrosodlethanolamine on Salmonella typhimurium. Mutat. Res. 68:207—210 Hilfrich, J., I. Schineltz, and D. Hoffmann. 1978. Effects of N—nitrosodiethanolami.fle and 1,1—diethanoihydrazine in Syrian golden hamsters. Cancer Lett. 4:55—60. Hoshino, H., and H. Tanooka. 1978. Carcinogenicity of triethanolainine in mice and its mutagenicity after reaction with sodium nitrite in bacteria. Cancer Res. 38:3918—3921. Kostrodymova, G.M., V.M. Voronin, and N.N. Kostrodymov. 1976. The toxicity (in complex action) and the possibility of cancerogenic and cocancerogenic properties of tri—ethanolamines. Gig. Sanit., No. 3:20—25. [ in Russian, English summary] McMahon, R.E., J.C. Cline, and C.Z. Thompson. 1979. Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Cancer Res. 39: 682—693. —90— ------- National Institute for Occupational Safety and Health. 1977. Registry of Toxic Effects on Chemical Substances. 2 Vols. DHEW Publication Nos. (NIOSH) 78—104—A and B. U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, Cincinnati. [ GPO Monthly Catalog No. 1006:192, entry 17630, Sept. 1978] Preussmann, R., G. Wurtele, G. Eisenbrand, and B. Spiegelhalder. 1978. Urinary excretion of N—nitrosodiethanolamine administered orally to rats. Cancer Lett. 4:207—209. Rao, T.K., J.A. Young, W. Lijinsky, and J.L. Epler. 1979. Mutagenicity of aliphatic nitrosamines in Salmonella typhimurluin . Mutat. Res. 66:1-7. Selisskii, G.D., A.S. Obukhova, A. Anton’ev, B.A. Somov, N.y. Shaparenko, and L.V. Alchangyan. 1978. Prophylaxis of occupational dermatoses caused by inhibitors of atmospheric metal corrosion. Vestn. Dermatol. Venerol., No. 9:36—39. [ Chem. Abs. 9 0:173844q, 1979] Venediktova, K.P., and R.V. Gudina. 1976. Clinical—immunological characteristics of allergic dermatitis and eczema in textile workers. Vest. Dermatol. Venerol., No. 10:32—37. Chem. Abs. 87:28220s, 19771 Zingmark, D.A., and C. Rappe. 1977. On the formation of N—nitrosodiethano lamine in a grinding fluid concentrate after storage.Ambio 6:237—238. —91— ------- Chapter 5 MOR.PHOLINE Morpholine (tetrahydro—2H—l,4—oxazifle) is an oxygen—containing cyclic amine. It is a colorless, hygroscopic liquid with a melting point of 4.9°C and a boiling point of 128.9°C. It Is completely miscible with water in all proportions. Its vapor pressure is 8.0 mm Hg at 20°C. MorpholIne Is produced by reacting dIethylene glycol, ammonia, and a small amount of hydrogen over a hydrogenation catalyst at 150—400°C and 30—400 atm. The morphollne product is recovered by fractional distillation. Among the byproducts are 2_(2—aminoethoxy)ethaflOl and N—alkylmorphollnes. PRODUCTION Currently, the major U.S. producer of morpholine is the Jefferson Chemical Co., a subsidiary of Texaco, Inc. Its Port Neches, Tex. plant has a capacity of 12,700 mt/year (Stanford Research Institute InternatIonal, 1975). Jefferson also has a plant with a capacity of 6,800 mt/year at Conroe, Tex., which has been on standby status since 1976 (Anonymous, 1980). In addition, Dow —92— ------- Chemical Co. has had a plant with a capacity of 2,300 mt/year on standby at Midland, Mich. since 1972. Total morpholine production has been stable at about 11,000 mt/year since 1974. In addition to this domestic production by Jefferson Chemical, BASF Wyandotte imports an estimated 900 nit of morpholine per year (Anonymous, 1980). In October 1979, RASP Wyandotte announced that it had begun engineering studies for a plant to produce 8,200 mt/year at a site in Ceismar, La. Another potential producer is Air Products and Chemicals Co., which is considering building a plant of unspecifjed capacity at Pace, Fla. The company will use a new, low—pressure process (Anonymous, 1980). USE S Morpholine has a multiplicity of uses. The largest single use is as an intermediate in the production of rubber chemicals, principally delayed—action rubber accelerators, stabilizers against heat—aging effects, and bloom inhibitors in butyl rubber vulcanization. Because of the similarity in vapor pressures of morpholine and water, and because of morpholine’s neutralizing effects on carbonic acid, the chemical is also used extensively as a corrosion inhibitor in steam boiler systems (Kirk—Othmer, 1978). Morpholine reacts with fatty acids to form soaps with excellent emulsifying properties. These products find use in household and automotLve waxes and polishes. The morpholine salt of stearic acid —93— ------- is produced by Imoco—Gateway Corporation in Baltimore, Md. Other miscellaneous uses of niorpholine Itself Include formulations in cosmetic products. The chemical 18 used in eye shadow, eyeliner, and mascara, in percentages ranging from 0.1 to 5.0% (Food and Drug Administration, 1980). It is also used in the production of other derivatives. N—Morpholyl formamide, used for benzene, toluene, and xylene extraction, is produced by Fike Chemical Company, Nitro, W. Va. Morpholine hydroperiodide Is used as a pharmaceutical and as a disinfectant. According to the U.S. International Trade Commission (1978), commercially significant morpholine derivatives used as accelerators include 4—morphollnyl—2—benzothiaZYl disulflde [ N, 2—morpholinothio)— benzothiazole), produced by Goodyear Tire and Rubber Co., Akron, Ohio; N_oxydiethy1ene 2—benzothiazOle5Ulfeflamide [ 2— (4_morpholinothiO)beflZOthiazo 1 .e], produced by American Cyanamid Co., Bound Brook, N.Y., The Goodyear Tire and Rubber Co., Akron, Ohio, Pennwalt Corp., Wyandotte, Mich., and B. F. Goodrich Co., Henry, Ill.; and N_(2,6_dimethylmorphOliflO)beflZ0thiaZ0le8Ulfem e, produced until 1978 by Monsanto Company, Nitro, W. Va., and currently produced by Uniroyal Chemical Co., Naugatuck, Conn.; bis(niorpholinOthiOCarbOflYl) disulfide is produced by American Cyananiid Co., Bound Brook, N.Y. N,N’—DithiodlfllOrPhOlifle, produced by Monsanto Co., Nitro, —94— ------- W. Va., and R.T. Vanderbilt Co., Inc., Bethel, Conn., is the only commercially significant morpholine derivative used to stabilize rubber against heat—aging effects. (U.S. International Trade Commission, 1978). Diniorpholine polysulfide is used as a bloom inhibitor in butyl rubber vulcanization. 4,4 ‘—Bis [ ( 4 —analino—4—morpholino—l,3,5—triazin—2y1)amino]stjlbene— 2,2’—disulfonic acid, disodium salt, also known as C.I. Fluorescent Brightening Agent 260, is used as an optical brightener for cellulose and soaps and detergents. Two short—chain alkyl morpholines, 4—ethylmorpholine and 4—methylmorpholine, are used as catalysts in the manufacture of polyurethanes (Kirk—Othmer, 1978). They are used with other catalysts to give a balanced foaming system for polyurethane foam production. These compounds are produced by Lonza, Inc., Mapleton, Ill.; Jefferson Chemical Co., Conroe, Tex.; and Union Carbide Corp., South Charle8ton, W. Va. A number of long—chain alkyl niorpholines are also produced. N—(Coconut oil alkyl)morpholine and N—n—dodecyl morpholine are produced by Lonza, Inc., in Mapleton, Ill. The compounds are believed to be used primarily as intermediates for the corresponding amine oxides, which are used as surfactants. Two other long—chain alkyl morpholines, N—N—hexadecylmorpholine and N—(soybean oil alkyl) morpholine are used as intermediates for the corresponding quaternary ammonium salts, N—ethyl—N—hexadecylmorpholinium ethyl —95— ------- sulfate and N—ethyl—N—(soybeafl oil alkyl) morpholirtium ethyl sulfate, which are cationic surfactants. All four are produced by ICI America, Inc., in New Castle, Del. 6—Morpholino—4 , 4—diphenyl—3—heptanofle hydrochloride (phenadoxone hydrochloride) is used as a pharmaceutical. Morpholifle salts of acylated sulfonamides are used as bactericides. The morpholine salt .a_toluenesulfofic acid, used as a catalyst to produce epoxy resin coatings for articles that come into contact with food, is produced by American Bio—Synthetics Corp., Milwaukee, Wisc. Morpholine borane is produced by Alfa Products, Inc. in Denvers, Mass., a subsidiary of Thiokol Corporation. The various uses of morpholine, based on total consumption of 11,000 mt/year, are summarized in Table 5—1. EXPOSURE Because of the numerous uses of morpholine, there are many potential routes of human exposure. Releases to the atmosphere occur primarily during the production of morpholine and its derivatives. Atmospheric morpholine releases were estimated in a study conducted for the Environmental protection Agency (EPA) by Science Applications, Inc. (1980). Emissions from morpholine production were based on analogy with emissions from ethylene oxide production —96— ------- TABLE 5—1 Uses of Morpho1ine . Amount Uses ( mt/yr) Percentage of Total Used Rubber Chemicals 3,600 33 Corrosion inhibitors 2,700 25 Optical brighteners 1,100 10 Alkyl morpholines 1,100 10 Waxes and polishes 900 8 Exports 800 7 Miscellaneous uses 800 7 . . From Chemical Marketing Reporter, 1974, with permission. —97— ------- in the absence of any actual emissions data. Total production—related emissions were estimated to be 6,200 kg/year for a production level of 11,000 mt/year. The exposed population would include only those persons living in Port Neches, Tex. (population 11,000) and the surrounding area. In the same study (Science Applications, Inc., 1980), the emissions of morpholine from the various uses listed in Table 5—1 were also estimated. For the production of rubber chemicals and optical brighteners, an emission factor of 0.001 kg per kilogram of morpholine used was assumed. The emissions were assumed to be equally distributed among 96 (unspecified) sites where rubber accelerators are produced, and 128 (unspecified) sites where optical brighteners are produced. The total annual emissions from these sources were estimated at 5,100 kg/year. Miscellaneous morpholine uses were assumed to have the same emissions factor, for a total emission rate of 900 kg/year, distributed in proportion to the U.S. population. The study assumed that all morpholine used as a corrosion inhibitor (2,700 mt/year) and in waxes and polishes (1,100 mt/year) would be emitted to the atmosphere. However, such an assumption appears to result in a substantial overestimate of atmospheric emissions. The use of morpholine as a boiler additive to inhibit corrosion involves adding the chemical to the boiler feedwater, and the boiler water—stream system is a closed system involving little if any release to the atmosphere. However, a portion of the boiler —98— ------- water is occasionally discharged (blown down) to lower the buildup of salts and chemicals in the system. Therefore, the ultimate fate of morpholine in the system is in the boiler blowdown stream. The discharge of morpholine in blowdown streams from industrial and utility boilers Is a potential source of contamination of rivers and streams. In a study of toxic chemicals in power plant discharges (McCaIn and Peck, 1976), the concentration of morpholine was measured in three Hawaiian power plants known to use morpholine as a boiler feedwater additive. The morpholine concentrations detected in various power plant discharge streams ranged from no detectable amount to a maximum of 0.008 ppm. If these figures are typical, then the potential for human exposure, given the further diluting effect of the receiving water bodies, appears to be very small. In soaps, polishes, and waxes, morpholine is typically used in the form of salts of fatty acids. Therefore, it is unlikely that any pure morpholine is released to the atmosphere through evaporation. The Food and Drug Administration (FDA) has approved the use of morpholine and its derivatives in various applications, Including protective coatings applied to fruit8 and vegetables, boiler water additives for steam generation used for food preparation, adhesives for food packaging, and defoaming agents used in paper and paperboard manufacture (21 CFR 172). Possibly as a result of such uses, morpho].Ine has been found In a number of food products (Singer and LIjinaky, 1976). The results of the analyses are summarized in Table 5—2. —99— ------- TABLE 5—2 Concentrations of Morpholine in Various Food Products ! Morpholine Concentration Substance ( ppm ) Canned tuna <0.7 Frozen ocean perch 10.0 Frozen cod <0.3 Spotted trout 7.0 Small mouth bass <0.8 Salmon 1.2 Baked ham 0.5 Frankfurters 0.4 Evaporated milk 0.2 Coffee 1.0 Tea <0.1 Canned beer 0.4 Bottled beer <0.2 Wine <0.7 . From Singer and LijinSky, 1976, with permission. —100— ------- The ubiquitous occurrence of morpholine in the diver8e types of food products examined leads to the suspicion that inorpholine is present in parts—per—million quantities in many different types of foodstuffs and that this presence constitutes a rather wide8pread route of exposure. Another route of human exposure derives from the widespread use of morpholine in cosmetics, primarily in eyeliner and mascara, although a large percentage of conunercial formulations contain morpholine. The exposure route is inhalational (as well as) dermal, because of the relatively high vapor pressure of morpholine. Finally, occupational exposures may be significant. A National Occupational Hazard Survey conducted by the National Institute for Occupational Safety and Health (NIOSH) detected worker exposure to morpholine In 283 different industries (as specified by the Standard Industrial Classification four—digit code). Worker exposure to morpholine Is currently regulated by the Occupational Safety and Health Administration (OSHA). The OSHA standard for niorpholine in air is a time—weighted average of 20 ppm for 8 hours (Occupational Safety and Health AdministratIon, 1980). —101— ------- ANALYTIC METHODS Singer and Lijinsky’s (1976a,b) procedure for foodstuffs such as fish, beer, wine, tea, coffee, milk, water, frankfurters, tobacco, and tobacco condensates is based on conversion to p—toluenesulfonamide, and then gas chromatography—mass spectrometry (GC—MS) of the derivatized amine. The first step in the analysis procedure is acidification to pH 1, mixing in a blender with water, and then adjustment to pH 10 with sodium and barium hydroxide. The alkaline mixture is then steam distilled into acid, extracted with ether to remove neutral compounds, concentrated to 3 ml, and then derivatized by refluxing with an alkaline solution of —toluenesulfonyl chloride. The derivative is further purified and then analyzed by GC—MS; carbon—14—labelled amine is used as an internal recovery standard. The sensitivity of the method is approximately 0.3 ppm (by weight). Morpholine is found to be present in most samples analyzed. TombropoulOs (1979) has developed a much simplified procedure, in which the morpholine is analyzed directly by GC using a Chromosorb 103 column and a flame Ionization detector. Better specificity is obtained if a nitrogen—selective CC detector, such as an alkaline flame ionization detector or a Hall detector, is used. The method has been applied to blood, urine, and other biological samples. —102— ------- Karweik and Meyers (1979) have developed a method that utilizes diakyldithiocarbamate copper complex, which is formed from the reaction of the aliphatic amine with carbon disulfide in the presence of ammonia. The copper bis(dithiocarbamate) complex is then extracted with chloroform and its concentration determined spectrophotometrically at 434 nm. Airborne morpholine in industrial atmospheres where morpholine— based cutting fluids are used has been monitored by trapping the morpholine in a 0.025% aqueous solution of methyl orange, followed by colorimetry (Burenkoetal., 1977). Fajenetal. (1979) determined airborne morpholine in chemical and tire factories by trapping in 1—N potassium hydroxide solution, acidifying to pH 3, nitrosating with sodium nitrite, and then measuring the increase in the N—nitrosomorpholine concentrations. —103— ------- HEALTH EFFECTS Acute Toxicity The LC 50 of morpholine (continuous exposure) has been reported to be 2,250 ppm for male and female rats and 1,450 ppm and 1,900 ppm for male and female mice (Lam and Van Stee, 1978). Shea (1939) exposed rats and guinea pigs at 18,000 ppm (63,000 mg/rn 3 ) continuously for 8 hours or intermittently for up to 42 of the eyes and nose, experienced irritation of the nose and coughing. In a related study (Pennsylvania Department of Health, 1967—1969), humans exposed to N—ethylrnorpholine for 2—3 minutes at 50—100 ppm (175—350 mg/m 3 ) experienced upper respiratory tract irritations and complained of an ammonia—like odor. At 25 ppm (87.5 mg/rn 3 ), the only finding was detection of a noticeable odor. In a survey of a worksite where N—ethylrnorpholine was being used, workers described a visual “halo” effect during a 2—hour exposure to airborne concentrations of 6—22 ppm (21—77 mg/rn 3 ). This effect lasted for up to 2 hours after hours. Some deaths resulted; irritation hemorrhaging in lungs, and congestion of liver and kidneys were also reported. In other studies, rats exposed to 450 ppm of morpholine for 6 hours/day, 5 days/week for 8 weeks exhibited a decrease in food consumption and body weight gain as well as an increased organ—to—body—weight ratio for lungs and kidneys. Changes to sensory areas such as the eyes and nose were also noted (Lam and Van Stee, 1978). Shea (1939) reported that, after exposing himself to morpholine at 12,000 ppm (42,000 mg/rn 3 ) for 1.0—1.5 minutes, he —104— ------- exposure ended. As a result of these findings, the Pennsylvania Department of Health recommended a short—term exposure limit (15 minutes) of 20 ppm f or both morpholine and N—ethylmorpholine. The American Conference of Governmental Industrial Hygienists (1974) reported that the primary effects of exposure to airborne morpholine are nasal and bronchial irritation and liver damage. They compared morpholine’s action to that of ammonia but, because of greater potential for systemic effects, suggested that a somewhat lower environmental limit for morpholine was appropriate. However, the threshold—limit value/time—weighted average (TLV—TWA) concentration limit of 20 ppm (70 mg/rn 3 ) was still recommended on the basis of protection against Irritation and damage to the eyes. According to Ivanov and Germanova (1973), men exposed to morpholine at a concentration of 16 mg/rn 3 complained of irritation after exposure of only 1 minute. The authors thus characterized that concentration as the threshold for irritating action in humans. Results from other short—term inhalation exposures to morpholine are shown in Table 5—3. A series of Russian studies has resulted in reports of other effects after exposure to airborne levels of morpholine considerably lower than those previously mentioned. In one experiment (Ivanov et al., 1973), rats were exposed for 4 hours to morpholine at 260, 40, or 3 mg/rn 3 . Morpholine toxicity was evaluated by measuring respiratcry rate, lung weight, and uptake of stain by lung tissue. —105— ------- ‘-a 0 0 ’ Table 5—3 Effects of Short—Term Inhalation Exposure to Morpholine Concentration Time Species mg/rn 3 ( hours) Effects References Rat 8,497 29,740 8 1/6 deaths International Labour Office, 1972 Rat 8,000 28,000 8 No deaths Smyth etal., 1954 Rat 6,734 23,569 4 Signs of irritation International Labour Office, 1972 Rat 6,285 22,000 1 Lacrimation, rhinitis, inactivity Industrial Bio—Test Laboratories, Inc., 1970 Mouse 1,391 4,869 ? LC 50 Zaeva etal., 1968 ------- To measure the degree of lung staining, a 1% solution of neutral red stain was injected into the tall vein after the animals were exposed to morpholine. The rats were killed 1 and 10 minutes later, and then ; 0.5—g sections of the lungs were removed to extract the stain. Measurements at 1 and 10 minutes corresponded to points of maximum accumulation and secretion of the dye, based on observations in preliminary experiments. In undamaged cells the dye was taken up, stored as a granule, and then removed. Damaged cells lost the ability to collect the stain in a granule; instead, the nucleus and cytoplasm became stained by diffusion. At 260 mg/rn 3 , the investigators observed an increase in respiratory rate, but no effect on lung weight. The exposedrats also retained a greater amount of stain than did controls after 1 and 10 minutes. Rats exposed to a morpholine dosage of 40 mg/rn 3 had eliminated less stain from lung tissue after 10 minutes than had controls. No changes in respiratory rate or lung weight were observed at dosages of 40 or 3 mg/rn 3 . The investigators reported the test for stain removal from lung tissue to be the most sensitive indicator of irritation. Because 40 mg/rn 3 was the lowest tested airborne concentration that caused a change in removal of stain from lungs, that amount was characterized as the threshold limit for Irritation in rats. Grodetskaya and Kararnzlna (1973) evaluated thyroid function as another indicator of toxicity. They measured the uptake of lodine-13l. Male rats were exposed to 80 mg/rn 3 of morpholine 4 hours/day for 2, 4, or 8 days and then administered iodine —131. —107- ------- Thyroid gland uptake was measured over 48 hours. Exposed rats accumulated a larger amount of iodine—l3l than did controls, Indicating increased thyroid gland activity. Microscopic examination of the thyroid gland showed hypersecretion by thyroid cells. Rats and guinea pigs were exposed to 70 or 8 mg/rn 3 of morpholine for 4 hours/day, 5 day/week for 4 months (Migukina, 1973). The investigators examined peripheral blood, lungs, liver, and kidneys, measured nervous system activity (by an undefined summary—threshold Index), arterial pressure, and respiratory rate, and looked for chroniosomal aberrations In bone marrow cells in the anaphase and telophase. The results of the study are summarized in Table 5—4. Morpholine had its most damaging effect on the spleen. Exposure to 70 mg/rn 3 of morpholine resulted in destruction of the lymphoid structure. This effect was not reversible 1 month after exposure to morpholine ended. Based on indications of some mutagenic effect after exposure of 8 mg/rn 3 , the study used a somewhat arbitrary safety factor to recommend a maximum permissible concentration of 0.5 mg/rn 3 . Morpholine is a highly irritating compound. Small amounts can burn the skin and eyes. Shea (1939) reported that application of undiluted niorpholine to the fingertips produced an intense stinging sensation and cracking in the areas around the fingernail; even a 2% solution was not tolerated. —108— ------- TABLE 5—4 Effects of Exposure to Morpholine on Rat and Guinea Pig Concentration Index of Effect Species Effect 70 mg/rn 3 Nervous system Rat Initial increase, followed by return activity to control levels Guinea pig Initial decrease; increase by end of 4 months Arterial pressure Rat Initial increase; decrease by 2nd month Peripheral Rat Increase in hemoglobin and red blood blood cell count; decrease in in leukocytes at 1st and 4th months Guinea pig Decrease in hemoglobin and leukocyte Electrocardiogram Rat No change Organ function Rat No changes in liver, kidneys, and testes Guinea pig No changes in kidneys and testes; change in liver function Morphology Rat, guinea Swelling of alveoli and atrophy pig of respiratory lymphatics; atrophy of lymphoid elements of the spleen even in animals killed 1 month after exposure ended Mutagenesis Rat Increase in number of chroniosomal aberrations resulting from fragmentation 8 mg/rn 3 Nervous system Rat Increase through 1st month of activity exposure Arterial Rat Decrease by 2nd month pressure Peripheral Rat Decrease in lymphocytes at 2nd blood month Rat, guinea No changes in liver function pig Organ function Rat No changes in liver functionn Morphology Rat, guinea Decrease in size of lymph nodes pig of spleen; thus effect not observed in animals killed 1 month after exposure ended Mutagenesis Rat Increased in number of chromosomal aberrations although not significantly greater than spontaneous rate ! Frorn Migukina, 1973, with permission. —109— ------- Experiments (Shea, 1939) with rabbits and guinea pigs demonstrated the local as well as systemic effects from skin application of morpholine. Undiluted, unneutralized morpholine applied to the shaven skin of rabbits and guinea pigs caused deaths after 1 to 13 daily applications. Morpholine produced skin burns, necrosis, inflammation, and edematous derma. Systemic effects included congestion of the liver and spleen, fatty degeneration and necrosis of the liver, and necrosis of the kidney tubules. A diluted aqueous solution of morpholine produced similar systemic effects and deaths. In contrast, after 30 daily applications, an liluted, neutralized morpholine solution has caused only a ening of the derma. nic Toxicity Carcinogenicity Shank and Newberne (1976) investigated the carcinogenic properties of morpholine through long—term feeding studies in rats and hamsters. Pregnant rats and hamsters were fed sodium nitrite, morpholine, and N—nitrosomorpholine (NMOR) in the diet from the time of conception until delivery. Offspring of both sexes were then randomly selected for the long—term studies, which also included studies of second—generation rats. The experiments were ended at 125 weeks for rats and 110 weeks for hamsters. The dietary levels and incidence of cancer for the variou8 experimental groups are summarized in Table 5—5. In groups 3, 7, —110— ------- TABLE 5—5 Dietary Levels of Compounds Given to Rats and Hamsters and Incidence of Cancer ! Incidence (%) Dietary levels (ppm) Rat Hamster Liver Liver Lung Liver Liver Test Sodium N—Nitroso— cell anglo— anglo— cell anglo— Grou Nitrite Morpholine morpholine cancer sarcoma sarcoma cancer sarcoma 1 0 0 0 0 0 0 4 0 2 1,000 0 0 1 0 0 0 3 3 0 1,000 0 3 0 2 0 0 4 1,000 1,000 0 97 14 23 31 0 5 1,000 50 0 59 5 6 0 0 6 1,000 5 0 28 12 8 0 0 7 50 1,000 0 3 2 1 0 5 8 5 1,000 0 1 2 1 0 0 9 50 50 0 2 1 1 0 3 10 5 5 0 1 2 2 0 0 11 0 0 5 58 15 9 0 0 12 0 0 50 93 21 20 6 6 ! From Shank and Newberne, 1976, with permission . Number of animals per group ranges from 94—172; F 1 and F 2 generations combined. ------- and 8, morpholine was the predominant additive in the standard diet. Rats had a small but higher incidence of liver cell cancer and angiosarcomas than did controls. These data, especially for group 3, might suggest that morpholine by itself may be a weak carcinogen. The investigators speculated, however, that tumor production in this group may have resulted from an unknown source of nitrate combining with morpholine to form the potent NMOR carcinogen, as was suspected to be the case in the other groups. A similar study was performed by Greenblatt et al. (1971), who investigated the chronic toxicity of NMOR in addition to the potential in vivo nitrosation of morpholine by sodium nitrite. Male and female Swiss mice (20 per group) were given 6.33 g/kg of morpholine concurrently with 1.0 g/kg of sodium nitrite indrinking water. Control animals received either morpholine alone, sodium nitrite alone, or were not treated. After 40 weeks, all survivors were killed. Animals treated with both morpholine and sodium nitrite showed a 57 (20 of 35) incIdence of lung adenomas Animals treated with either morpholine alone or sodium nitrite alone did not show different results than the untreated controls. Further studies are clearly needed to clarify the carcinogenic potential of morpholine. The same study demonstrated the ability of dietary sodium ascorbate (vitamin C), given concurrently with morpholine and sodium nitrite, either to diminish the incidence of tumor formation or to —112— ------- increase the induction period. When 5.75, 11.5, or 23.0 g/kg of dietary sodium ascorbate was administered to A strain mice that also received 6.33 g/kg of morpholine and 2.0 g/kg of sodium nitrite in their drinking water, there was a 72—89% inhibition of adenoma formation (Greenblatt etal., 1971). These observations were confirmed by Mirvish etal. (1976) with male Wistar rats. Groups of 40 rats were treated for 2 years with 10 g/kg of dietary morpholine and 3 g/liter of sodium nitrite in drinking water. In additon, one group of rats also received 22.7 g/kg of sodium ascorbate in their diet. The presence of ascorbate resulted in a longer tumor induction period (93 versus54 weeks) and a slightly lower tumor incidence (49% versus 65%). No pulmonary metastases were reported in the animals that received morpholine plus sodium nitrite. A major concern is the ease by which morpholine can be nitrosated to form N—nitrosomorpholine (NMOR), which has been shown to be carcinogenic in rats, mice, and hamsters. After oral administration, NMOR produces both benign and malignant tumors of the liver and lungs in mice, of the liver, kidneys, and blood vessels in rats, and of the liver of hamsters. This subject has been extensively reviewed by the International Agency for Research on Cancer (1978). To date there is no evidence that NMOR is carcinogenic in humans. —113— ------- A recent study by Iqbal et al.. (1980) has shown a relationship between exposure to morpholine and nitrogen dioxide and the in vivo formation of NMOR. Mice (three or four per group) were gavaged with 2 mg of morpholine. The animals were then exposed to nitrogen dioxide at concentrations of 0.2 to 50 ppm for up to 4 hours in inhalation chambers. At various intervals during exposure, the mice were frozen and pulverized in liquid nitrogen. The powder was then analyzed for the presence of NMOR. There was a time—dependent NMOR yield relative to nitrogen—dioxide exposure——ranging from 370 ±. 12.5 ng/mouse after 0.5 hours to 2,230 ± 138.6 ng/mouse after 4 hours. There was also a dose—dependent NMOR biosynthesis as a function of nitrogen dioxide exposure level (0.2—50 ppm). A 4—hour exposure of 0.2 ppm nitrogen dioxide resulted in 56 ± 6 ng NMOR per mouse, a 4—hour exposure of 50 ppm of nitrogen dioxide resulted in NMOR biosynthesis of more than 1,000 ng/mouse. Control levels of NMOR in mice gavaged with morpholine or distilled water and then exposed to air were less than 5.0 ng of NMOR per mouse. This study demonstrates the in vivo nitrosating potential of nitrogen oxides interacting with amines such as morpholine. The results of this study were extended by Van Stee et al. (1980), who demonstrated excess tumor formation in male CD—i mice. They exposed 35 animals per group to nitrogen dioxide (1—2 ppm) by inhalation while receiving 0.1% v/v morpholine In drinking water. After exposure for 30 weeks, the animals (and all the appropriate controls) received deionized water and room air until moribund or dead. The group that received the morpholine and nitrogen dioxide —114— ------- had an Increased Incidence of lung adenoma relative to controls (P — 0.056, 21.2%). Various other statistical tests all Indicated a significant difference between the experimental and control groups, even with the relatively small sample sizes tested. The results of these studies provide evidence that, exposure of mice to nitrogen dioxide and a secondary amine (morpholine) can lead to In vivo nitrosamine formation as well as to an Increased incidence of tumor formation. Although the extrapolation to humans of the results of such studies 18 fraught with difficulties, these relatively low exposures give the results more relevance. Further work should be done to confirm these observations in other species, with larger sample sizes, and with simultaneous inhalation exposure to both morpholine and nitrogen dioxide. In addition, with the increased detection sensitivity of modern analytic methods, It should be possible to monitor humans exposed to similar conditions. Evidence of In vivo nltrosamlne formation in humans exposed to morpholine and nitrogen dioxide would provide strong confirmation of the animal model and make major aspects of the extrapolation unnecessary. Although NI4OR Is not known to be manufactured commercially, there Is a potential for human exposure through the in vivo nltrosatlon of morpholine, although this route has not yet been demonstrated. However, Rounbehler et al. (1980) have shown the presence of several volatile nitrosamines, including NMOR, In the Interiora of 1979—model automobiles. NMOR was detected at levels —115— ------- between a trace and 2.5 1 g/m 3 (mean of 0.65 ug/m 3 ) in the interiors of 16 of 38 automobiles tested. This nitrosamine, as well as several others detected, probably account for the “new car sme11.’ Mutagenicity of Morpholine Horpholine has been tested in Salmonella mutagenicity systems, primarily the host—mediated assay, and in a transpiacental mutagenicity assay. it is nonmutagenic in the absence of a nitrosating agent. Bacteria . Given to mice in the host—mediated assay with Salmonella , morpholine was not mutagenic. Zeiger and Legator (1971) administered up to 500 mg/kg of morpholine by gavage or intramuscularly to male mice and gave intraperitoneal injection of Salmonella typhimurium G—46 as the indicator organism. No mutagenic activity was observed. Braun et a].. (1977) treated male mice with morpholine at 1.45 to 2.90 ininol/kg by gavage in the intraperitoneal host—mediated assay. No mutagenic activity was seen in the indicator organism, S. typhimurium TA195O. In the same study, administration of equiniolar levels of sodium nitrite (2.175 and 2.90 nunol/kg) produced a mutagenic response. This response was observed only when the sodium nitrite was administered along with the morpholine or 10 minutes later. Administration of sodium nitrite 10 minutes before administration of morpholine produced no response. At levels up to 2.9 mmol/kg, sodium nitrite by itself did not produce mutagenic —116— ------- activity. Using the intrahepatic host—mediated assay, Edwards et al. (1979) demonstrated the nonmutagenicity of morpholine in S. typhimurlum TA1530 recovered from the livers of female mice receiving 4 mg/kg of morpholine by gavage. When 4 mg/kg of morpholine was administered immediately before administration of 120 mg/kg of sodium nitrite, a mutagenic response was observed. The authors estimated the relative conversion of morpholine to NMOR at 12.3—19.8%, using morpholine levels doses ranging from 4 to 40 mg/kg and sodium nitrite levels of 120 mg/kg and comparing the niutagenic responses to that obtained from pure NMOR. Sodium thiocyanate (120 mg/kg), a catalyst of nitrosation, enhanced NMOR formation as measured by mutagenicity; doses of 4 or 20 mg/kg did not affect the response. Ascorbic acid, an inhibitor of nitrosation, inhibited the mutagenic response of 40 mg of morpholine and 120 mg of sodium nitrite per kilogram at levels of 120 and 360 mg/kg, but not at the 40—mg/kg level. practical—grade morpholine was tested in vitro for mutagenicity in Salmonella at EG&G Mason Research Institute in Rockville, Md. through the Environmental Mutagenesis Test Development Program of the National Institute for Environmental Health Sciences (NIEHS). The investigators used a preincubation modification of the Salmonella/microsome test using Aroclor—induced rat and Syrian hamster liver S—9 and Salmonella strains TA9B, TA IOO, TA1535 and TA1537. No mutagenicity was observed either with or without S—9 at doses ranging up to 10.0 mg/plate (S. Haworth, personal communicatiOn, 1980). —117— ------- Mammals . Pregnant golden hamsters were administered morpholine by gavage, with and without sodium nitrite (Inul etal., 1979). Morpholine alone, at doses of 500 nig/kg, did not induce chromosomal aberrations, micronuclet, 8—azaguanine— or ouabain—resistant mutants, or transformation in cells from exposed embryos. The results from the combination of morpholine and sodium nitrite are discussed below. Mutagenicity of N—Nitrosomorpholine The results of genetic studies with NMOR are summarized in Table 5—6. The substance was found to induce gene mutations in all bacterial and mammalian cell culture systems by using a source of exogenous metabolism. NMOR is presumably the mutagen detected in the host—mediated assay in animals given both morpholine and sodium nitrite. Chromosomal aberrations were also observed in cultured mammalian cells. NMOR is active in whole animal systems, producing gene and chromosomal mutations in Drosophila . It is also responsible for DNA damage, as measured by the production of single— and double—strand DNA breaks in liver and the alkylation of nucleotides in DNA. There are, however, no adequate studies on the induction of chromosomal mutations or heritable effects in mammals. Bacteria (In Vitro) . Only in the presence of liver homogenate is NMOR mutagenic In vitro for Salmonella and Escherichia coil . Base—pair substitution mutations are induced with no evidence of induction of frame—shift mutations. NMOR was active in Salmonella TA1535 and TA153O in both plate and suspension tests, using —118— ------- Table 5—6 Results of Genetic Studies with N—nitrosomorpholine Test System Species/Strain or Cells Tested Result Reference S. typhimurium TA1535 S. typhimurlum TA1535 S. typhimurium TA153O S. typhimurlum TA195O S. typhimurium G—46 (host—mediated assay) S. typhimurium TA1530 (host—mediated assay) S. typhimurium TA195O (host—mediated assay)a S. typhimurium TA153O (host—mediated assay)j S. typhlmurium TA1530 (host—mediated assay) S. typhliuurium TA1535 E. coli B/R E. coil SD-R(TC) E. coil WU3610 E. coil WU3610 E. coil C600, A58 BHK—2l cells (8AGR) V79 cells (8AGR) V79 cells (IAGR ouqR) V79 cells (6TGR) Drosophila (sex—linked recessive lethal) Drosophila (sex—linked recessive lethal) Golden hamster embryo (8AGR, ouaR) BHK—21 cells Rat bone marrow Rat lymphocytes in vivo Mouse dominant lethal Drosophila (11/111 translocatlons) Golden Hamster embryo (aberrations) Golden Hamster embryo (micronuclei) Zeiger and Sheldon, i978 Gomez et al., 1974 Bartsch et al., 1976 Fonshtein et al., 1976 Zeiger and Legator, 1971 Zeiger, 1973, 1975 Fonshtein etal., 1976 Braun et al., 1977 Edwards et al., 1979 Charnley and Archer, 1977 Henke et al., 1964 Nakajima et al., 1974 Elespuru and Lijinsky, Elespuru, 1978 Neale, 1972 Kimble et al., 1973 Kuroki et al., 1977 Drevon er AT., 1977 Jones and Huberman, 1980 Henke et al., 1964 Vogel, 1977 Inui et al., 1979 Kimble etal., 1973, 1975 Sauro et al., 1973 Newton et al., 1977 Parkin et al., 1973 Henke et al., 1964 Inui et al., 1979 Inul et al., 1979 Gene mutation Chromosomal mutation I -I + + + + + + + + +b +. + + +. —c + + + + + +e + +1 _h + +e +e 1976 ------- Plants Arabidopsis thaliana Veleminsky and Gichner, 1968 Other nuclear damage Rat, in vivo (7—MeG excretion) Rat, in vivo (DNA strand breaks in liver) Rat, in vivo (DNA alkylation) Chang liver cells (BUdR incorporation) BRK—21/C 13 cells (UDS) Rat, in vivo (DNA alkylation) Mouse, in vivo (DNA strand breaks in liver) Rat primary hepatocytes (TdR incorp.) Rat, in vivo (liver mitotic abnormalities) + Weyland et al., 1972 Stewart and Farber, 1973 Stewart etal., 1974 Kimble et al., 1974 Kimble et al., 1975 Kleihues and Margison, Schwarz et al., 1979 Williams and Laspia, 1979 Romen and Bannasch, 1972 . Following administration of NMOR plus sodium nitrite. . Deuterated NMOR. .E Without an exogenous metabolic activation system. Using a feeder cell layer for metabolism. !. Transplacental. ! Inconsistent results. &. Weak, questionable positive. . Decreased fertility. DNA damage/repair + +c +— + + + Q 1976 ------- uninduced or phenobarbital—induced rat and mouse liver (Bartsch et al., 1976; Gomez et al., 1974; Zeiger and Sheldon, 1978) and in E. coli in suspension using Aroclor—induced or uninduced rat S—9 (Elespuru, 1978; Elespuru and Lijinsky, 1976; Nakajima etal., 1974). No inutagenicity was seen for E. coli (Henke etal., 1964) or induction of X—phage from E. coil in the absence of exogenous metabolic activation. In a comparison of uninduced rat and mouse S—9 in suspension and on the standard plate test using TA1535, mouse S—9 produced a consistently higher mutagenic response than did rat liver (Zeiger and Sheldon, 1978). Phenobarbital—induced rat liver S—9 produced a higher response in the plate test than did human liver S—9; rat and human lung S—9 produced no mutagenic activity (Bartsch at al., 1976). NMOR niutagenicity for E. coli WU3610 and S. typhimurium TA1S35 was depressed when the alpha position of NMOR was deuterated, leading to the conclusion that an enzymatic attack on the alpha position is necessary for the activation of NMOR (Charnley and Archer, 1977; Elespuru, 1978). Bacteria (Host—Mediated) . NMOR is mutagenic for Salmonella c46 and TA 1530 in the intraperitoneal host—mediated assay in the mouse (Fonshteinetal., 1976; Zeiger, 1975; Zeiger and Legator, 1971) when administered by intramuscular injection or by gavage. Alterations in both the diet and amino acetonitrile pretreatment affected the inutagenic response. Maintenance of the mice on a complete semisynthetic diet depressed the mutagenicity of NMOR as —121— ------- compared with results from the corresponding chow diet control. Mice maintained on the semisynthetic diet and then put on a protein—free diet for 8 days had a further depressed mutagenic response. Starvation or an all—casein diet f or 24 hours produced an enhanced mutagenic response (Zeiger, 1975). Anhinoacetonitrile, an inhibitor of liver microsomal metabolism, administered in a dosage of 20 to 200 mg/kg, apparently produced a dose—related depression of NMOR mutagenicity (Zeiger, 1973). The formation of NMOR from morpholine and sodium nitrite has been successfully followed, using both the intraperitoneal and intrasanguineouS host—mediated assays with S. typhimurium TA195O and TA1530. When mice were treated with equimolar concentrations of morpholine and sodium nitrite by gavage, mutagenicity was observed with morpholine administered with or before the administration of sodium nitrite. Morpholine administered 10 minutes after sodium nitrite produced no mutagenicity (Braun et al., 1977). In the intrasanguineoUs host—mediated assay, mutagenicity was seen in TA1530 recovered from the livers of mice treated by gavage with morpholine and sodium nitrite. Ascorbic acid, which inhibits nitrosation, decreased the mutagenic response; thiocyanate, which enhances the nitrosation reaction, enhanced mutagenicity (Edwards etal., 1979). Mammalian Cells in Culture . NMOR induces gene mutations in cultured mammalian cells. Equivalent cytotoxicities were observed (Kuroki etal., 1977) in V79 cells treated with NMOR with or —122— ------- without an S—9 fraction from phenobarbital—treated rats. However, 8—azaguanine resistance was produced only in the presence of S-9. In another study (Jones and Huberman, 1980), hepatocytes from phenobarbitone— or benz(a)anthracene—induced rat liver were used for metabolic activation by cocultivation with V79 cells. Both ouabain— and 6—thiouguanine—resistant mutants were induced at nontoxic levels In BHK 21 cells, NMOR induced 8—azaguanine resistance and chromosomal aberrations, predominantly dicentrics (Kimble et a]., 1973). An exogenous source of metabolic activation was not used in this study. Mammalian Cells In Vivo . Single oral doses of NMOR given to rats produced inconsistent levels of chromatid breaks and gaps In aspirates from bone marrow. Chronic dietary administration of morpholine and sodium nitrite produced no detectable cytogenetic effects In bone marrow (Sauro eta]., 1973). Administration of NMOR In drinking water resulted in mitotlc abnormalities and an increased mitotic Index in rat liver (Romen and Bannasch, 1972). After rats were given 200 to 300 mg/kg of NMOR, their lymphocytes were removed, cultured, and examined for chromosomal aberrations (Newton et a]., 1977). Increases In abnormal metaphases were observed at 250 mg/kg in rats killed at 20 hours and at 300 mg/kg in rats killed at 3 or 20 hours. These aberrations were primarily gaps and break8. The increases in single chromatid or isolocus breaks were not consistent with dosage or with each other within the same dosage. Two chromatid —123— ------- exchanges were seen in all treatment groups. In a mouse dominant lethal test, administration of 50 and 100 mg/kg of NMOR reduced mating incidence 2 and 3 weeks after treatment. No dominant lethal effects were observed 3 to 8 weeks after treatment. A dosage of 35 mg/kg resulted in normal mating and no dominant lethal effects during the first 3 weeks after treatment (Parkinetal., 1973). Intraperitoneal treatment of rats with 400 mg/kg of NMOR produced a disruption of liver nuclear structure and a breakdown of nucleolar structure (Stewart etal., 1975). In a recent study by Inul etal. (1979), 500 mg of morpholine and 500 mg/kg of sodium nitrite were administered to golden hamstfers by gavage on day 11 or 12 of pregnancy. The levels of NMOR recovered from the stomachs of these animals ranged from a high of 1.94 mg after 1 hour to 0.59 mg 24 hours after treatment. Approximately 5.6 pg of N—nitrosodiethylamine (NDEA) was recovered from animals treated only with sodium nitrite. Twenty—four hours after treatment, the embryos were excised and the cells grown and analyzed for chromosomal aberrations, micronuclei, 8—azaguanine and ouabain resistance, and transformation. Cells from animals treated with the combination of morpholine and sodium nitrite had increased numbers of chromatid gaps and breaks (which the authors concluded were significant) and increased numbers of micronuclel. There were also increased —124— ------- 8—azaquanine— and ouabain-resistant cell frequencies and an increased rate of morphologic transformation. Cells from the transformed colonies produced tumors when inoculated into cheek pouches of young golden hamsters. In all end points, sodium nitrite alone induced increases that were lower than, but in the same range as, the responses induced by the substance in combination with morpholine. The administration of sodium nitrite was considered to produce positive results for all end points. Morpholine alone produced negative results for all end points. NMOR produced positive results for all end points. However, sodium nitrite in doses of 100 to 200 mg/kg produced a higher response than did NMOR of 100 mg/kg for all end points except chromosomal aberrations, and a higher response than did 200 mg/kg of NMOR in the micronucleus and cell transformation tests. The morpholine—plus—NMOR response was higher than that produced by NMOR alone at 200 mg/kg for micronucleus and cell transformation tests, and was equivalent for ouabain resistance. These comparisons are difficult to interpret. The total dose of NMOR given to those animals receiving the combination of morpholine plus sodium nitrite cannot be calculated, but it is difficult to conceive that it approached 100—200 mg/kg. Yet, the response obtained suggests that if this level of NMOR was formed, further study is necessary. Also, the strong responses seen with sodium nitrite alone call for further explanation and more study —125— ------- because the results imply that sodium nitrite is a more potent mutagen in vivo than is NMOR. All other data indicate that sodium nitrite has little) if any, genetic activity toward anything other than microorganisms. DNA Damage . Rats receiving 400 mg/kg carbon—l4—labeled NMOR intraperitoneally (IP) were found to have labeled 7—(2—hydroxyethyl)--guaflifle plus five other labeled adducts in liver DNA (Stewart etal., 1974). Urine of rats administered a single dose of NMOR (120 mg/kg, IP) did not contain 7—methylguanine when sampled for up to 21 days (Weyland et al., 1972). Partially hepatectomized rats were treated with carbon—14—thymidine to label their liver DNA, then injected intraperitoneally with NMOR at 100 mglkg. Four hours after treatment, there was a shift in the sedimentation of DNA on alkaline gradients consistent with the introduction of single—strand DNA breaks. Reduction of the dosage to 10 mg/kg resulted in only a slight change in sedimentation rate as compared to results produced by the larger dose. The DNA breaks were repaired gradually over a period of 14 days. Double—strand breaks were also induced, as measured by neutral sucrose gradients. This type of break was not seen following administration of NDEA. Morpholine, itself, produced no measurable DNA damage (Stewart and Farber, 1973). —126— ------- Intravenous administration of 50 mg/kg of NNOR produced a small increase over background of 0 6 —methylguanine in rat liver DNA, or a lesion that was recognized by the 0 6 —methylguanlne excision system (Kleihues and Margison, 1976). Mouse liver DNA strand breakage was also observed via akaline elution technique 4 and 12 hours after treatment with 100 mg/kg of NMOR by intraperitoneal injection (Schwarz etal., 1979). In vitro studies with Chang liver cells and BHK—21 cells in culture resulted in an increase in DNA repair synthesis after treatment with NMOR at 100 pg/ml or less (Kimble et al., 1974, 1975). UsIng primary rat hepatocytes in culture, Williams and Laspia (1976) showed an Increase in DNA repair synthesis at i0 and mci. NMOR. Drosophila . NMOR is capable of inducing sex—linked recessive lethal mutations in Drosophila melanogaster sperm and spermatids after feeding (Vogel, 1977). Sex—linked recessive lethaj.s and Il/Ill translocations occur after injection of NNOR (Renke, etal. 1964). Plants . Treatment of Arabidopsis thaliana seeds with NMOR for 24 hours did not induce either sterility or mutations, expressed as the presence of M 1 siliquae (Velemiasky and Gichner, 1968). Using these treatment conditions, a number of aliphatic nitrosamines in the same study were strongly mutagenic. —127— ------- TeratogenicitY There were no data from which to evaluate the potential. teratogenicity or reproductive toxicity of morpholine. CONCLUSI ONS The carcinogenic potential for morpholine alone is somewhat equivocal because it is not possible to know the extent to which morpholine is nitrosated in vivo. What does appear more certain is that the carcinogenicity of morpholine is greatly enhanced when it is administered concurrently with a source of nitrite (e.g. sodium nitrite or nitrogen dioxide), which allows nitrosation to NMOR to occur. That nitrosation in vivo occurs is demonstrated by the inhibition of the carcinogenic effects by the presence of an antioxidant (such as sodium ascorbate) in the diet. The potential for this in viva nitrosation of morpholine (or of any other amine) needs further and immediate attention. In addition to research on experimental animals, human exposure should be carefully monitored. Individuals occupationally exposed to niorpholine should be studied to determine If concurrent exposure to nitrogen dioxide and other nitrosating agents results in an increased cancer incidence or perhaps in an elevated level of serum NMOR. In addition, more data on noncarcinogenic toxicity, teratogenicitY, and reproductive effects should be obtained. —128— ------- Because NMOR can be formed in vivo from ingested morpholine and nitrite, morpholine for this reason should be considered as hazardous as NMOR. The roles of tobacco smoke and diet as secondary nitrite sources should also be investigated. This may also provide a unique opportunity to study and (perhaps to quantify) the effects of synergism in humans. Properly designed and imaginative epidemiologic studies of this kind may shed light on the current confused situation concerning occupational exposure to chemicals and cancer incidence. Although morpholine has produced negative results in the few studies in which it was examined by itself, the primary concern remaining is its ability to be nitrosated readily to form the mutagen/carcinogen NI4OR. In vivo studies have demonstrated the formation of a mutagen, presumably NMOR, following the administration of morpholine plus sodium nitrite. NMOR is well—established as a mutagen. It induces point mutations in cells in vitro and in Drosophila as well as chromosomal aberrations in cultured cells. This latter effect i8 observed even in the absence of an S—9 preparation. In rodents, NMOR induces DNA damage in liver and low levels of chromosomal aberrations in bone marrow. It does not appear to reach the testes in an active form, although the results of the single negative rat dominant lethal test are not sufficient to conclude that NMOR cannot induce heritable effects in rats. —129— ------- REFERENCES Production, Uses, Exposure Anonymous. 1980. 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Dannenberg. 1972. Urinary 7—methyl guanine excretion of the rat after a single application of different carcinogens. Z. Krebsforsch. Klin. Onkol. 77:141—149. [ in Germanj [ Chem. Abs. 77 :57403c, 1972J Williams, G.M., and M.F. Laspia. 1979. The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test. Cancer Lett. 6:199—206. [ Cumul. md. Med. 20:4695, 1979] —14 1— ------- Zaeva, G.N., L.A. Tiinofievskaya, L.A. Bazarova, and N.V. Migukina. 1968. Comparative toxicity of a group of cyclic imino compounds. Toksikol. Nov. Prom. Khim. Vestchestv. No. 10:25—35. [ Chem. Abstr. 71:47804w 1969] Zeiger, E. 1973. Some factors affecting the host—mediated assay response. Environ. Health Perspect., Experimental Issue No. 6:101—109. Zeiger, E. 1975. Dietary modifications affecting the mutagenicity of N—nitroso compounds in the host—mediated assay. Cancer Res. 35:1813—1818. Zeiger, E., and M. Legator. 1971. Mutagenicity of N—nitrosomorpholine in the host—mediated assay. Mutat. Res. 12:469—4 71. Zeiger, E., and A.T. Sheldon. 1978. The mutagenicity of heterocyclic N—nitrosamlnes for Salmonella typhimurium . Mutat. Res. 57:1—10. —142— ------- Chapter 6 2-NITROPROPANE CH 3 — H—CH 3 NO 2 2—Nitropropane (2—NP) is a colorless liquid with a moderately high vapor pressure (13 mm Hg at 20°C). It remains liquid over a wide range of temperatures. The melting point is —93°C; the boiling point, 120°C. The compound is produced commercially through the reaction of propane and nitric acid at 370—450°C and 8—12 atm. The end products (including 1—nitropropane) are separated in fractionation towers. PRODUCTION The sole commercial producer of 2—NP worldwide is the International Minerals and Chemicals Corporation. Its main plant (in Sterlington, La.) has a capacity of 38,547 mt/year of basic nitroparaff ins (Stanford Research Institute International, 1979). The four nitroparaf fins (nitromethane, nitroethane, 1—nitropropane, and 2—NP) are produced in fixed weight ratios by the manufacturing process. Nitromethane comprises 28Z of the products; nitroethane, 8%; 1—nitropropane, 18%; and 2—NP, 46% (Anonymous, 1976). —143— ------- The National Institute for Occupational Safety and Health (1977) has estimated that 14,000 mt of 2—NP were produced in the United States in 1977. The International Minerals and Chemicals Corporation’s annual production figure for basic nitroparaffins, as stated in its annual report for 1977, was 34,000 mt. If one assumes that 46% was 2—NP, then 16,000 mt would have been produced in 1977. The company did not publish production figures for basic nitroparaffins in its 1978 annual report, but the 1979 annual report gave a figure of 30,000 tnt of basic nitroparaffins for 1979. If one assumes that 46% was again 2—NP, then 14,000 nit of 2—NP were produced in 1979. USES 2—NP is used both as a solvent and as a chemical intermediate. Its largest reported single use of is as a solvent in paints (vinyl, epoxy, nitrocellulose, and chlorinated rubber) in concentrations ranging from 57. to 25%. It contributes such desirable properties as Improved drying time, blush retardation, and greater wetting ability (International Minerals and Chemicals Corp., 1980; National Institute for Occupational Safety and Health, 1977). In 1977, 2—NP was reportedly used in the formulation of most of automotive paints (National Institute for Occupational Safety and Health, 1977). It is al8o used as a solvent in the production of vinyl resin—based clear varnishes for coatings on metal sheets produced for the manufacture of cans in the food industry, especially for beer cans, and In high—quality printing inks and in paint—removers (National —144— ------- Institute for Occupational Safety and Health, 1977). The compounds is used as an intermediate in the manufacture of a variety of chemicals, many of which are produced by International Minerals and Chemicals at its main plant in Sterlington, La., and In two other plants——at Terre Haute, md. and in Cologne, Federal Republic of Germany. One of the important derivatives is 2—nitro—2—inethyl—l—propanol, which is used as an adhesive additive in the tire industry and In plastic and wood glues. It also functions as a bactericide and fungicide in cutting oils under the trade name Bioban P——1487 (Chemical Solvents Corporation, 1974; Anonymous, 1976. 2—NP is also used as a chemical intermediate in the production of 1—chloro—2—nitropropane. This chemical Is registered with the U.S. Environmental Protection Agency (1974) for use as a soil fumigant for fruits and cotton crops. EXPOSU RE No Information is available on 2—NP released during production. One source (Science Applications, Inc., 1980) reports that Louisiana State files on air emissions Indicate that “no 2—nitropropane emissions result from its production.” This seems unlikely given the volatility of the compound; nevertheless, because production is confined to a single site, where the population is less than 5,000, emissions from production would be localized. —145— ------- The most widespread source of exposure is the 2—NP used as a solvent. It can be assumed that all of this material is released into the atmosphere. Exposures to higher concentrations of 2—NP should occur in industrial and commercial settings. A National Occupational Hazard Survey conducted by the National Institute for Occupational Safety and Health detected worker exposure to 2—NP in 217 different industries. These included industrial construction and maintenance, printing (rotogravure and flexographic inks), highway maintenance (traffic markings), shipbuilding and maintenance (marine coatings), furniture, food packaging, and plastic products. Overall, National Institute for Occupational Safety and Health, (1977) estimated that 100,000 workers have the potential for exposure to 2—NP. The Food and Drug Administration (FDA) has allowed the use of 2—NP in the formulation of adhesives for articles intended for use in packaging, transporting, or holding food (21 CFR 175). In December 1978, the FDA proposed to delete provisions for such uses of 2—NP, having concluded that the substance is carcinogenic in animals. In January 1979, the FDA extended the comment period for the proposed ruling and, as of April 1980, no final ruling has been Issued. Exposure to 2—NP In the workplace is regulated by the Occupational Safety and Health Administration (OSHA). The time—weighted average exposure over 8 hours is not to exceed 25 ppm —146— ------- of 2—NP in air. However, as a result of carcinogenicity tests in animals, National Institute for Occupational Safety and Health (1977) recommended that 2—NP in the workplace be considered as a carcinogene in humans. OSHA has issued a Health Hazard Alert on 2—NP, which may serve to reduce the extent of industrial exposure (Occupational Safety and Health Administration, 1980). —147— ------- ANALYTIC METHODS Meyer and Locher (1975) were the first to recognize the characteristic reaction of secondary nitro compounds with nitrous acid to form blue pseudonitroles. Treon and Dutra (1952) used the reaction to determine airborne 2—NP that has been trapped in isopropyl alcohol. The absorbance at 277.5 nm was, however, not satisfactory for low concentrations. Jones (1963) extended the approach by trapping the 2—NP In concentrated sulfuric acid. When heated, the sulfuric acid decomposes 2—NP into nitrous acid, which is detected by its deep red—blue color reaction with resorcinol, usually measured at 560 nm • The technique was extensively evaluated by Jones (1963). The only compounds known to interfere were other substituted secondary nitro compounds. Primary nitroparaf fins decompose to hydroxylamine under the conditions of the test and do not interfere with the determination of 2—NP. The method was sensitive to 3 — 5 kg of 2—NP. Glaser (1978) developed a gas chromatographiC (GC) method for 2—NP, sensitive at the 3—36 mg/ tn 3 level, utilizing a 3 liter air sample. A known amount of air was drawn through a sorbent tube containing 100 mg of Chromosorb 106, 60—80 mesh. After collection, the Chromosorb 106 was transferred to a stoppered container and desorbed with ethyl acetate. An aliquot of the ethyl acetate was analyzed by gas chromatography using a flame ionization detector. The claimed sensitivity of the method was 3 mg/rn 3 at —148— ------- standard temperature and pressure, or 9 1 ig of 2—NP. Although easy to use, the method has two disadvantages. First, the absorbent is easily overloaded, and backup cartridges are required in order to check for overload. Second, the selectivity is poor when using a flame ionization detector. Presumably, this could be improved by utilizing a more specific GC detector such as a nitrogen—specific - alkali bead flame ionization or a Hall detector, or a nitro—nitroso—specific system such as the thermal energy analyzer (TEA). —149— ------- HEALTH EFFECTS Metabolism Some 2—NP is expired unchanged and some is converted to acetone and nitrite or nitrate by both animals and plants. Dequidt etal. (1973) reported finding considerable levels of nitrite in the heart, lung, kidney, spleen, and liver of rats given acute lethal or repeated nonlethal doses of 2—NP by intraperitoneal injection. The potential for 2—NP or the nitrite liberated from It to form N—nitroso compounds in vivo urgently needs attention. Methemoglobinemla, presumably due to the Interaction of nitrite and hemoglobin, was observed in the studies of acute exposure, but to a much lesser extent in the chronic exposure studies. Acute Toxicity in Animals Treon and Dutra (1952) reported on the inhalation toxicity of 2—NP in four species——rats, cats, rabbits, and guinea pigs. Two parameters were assessed: the highest atmospheric concentration the animals could tolerate without noteworthy immediate effects or after—effects and the lowest concentrations to induce mortality in a similar standard time exposure. These values are summarized in Table 6—1. The variation in toxicity between cats and guinea pigs is large——approximately sevenfold. —150— ------- Table 6—1 Toxicity of 2—NP in Four Species after Exposure of 4.5 Hours Highest “Nontoxic” Lowest Fatal Species Level (ppm) Level (ppm ) Cat 328 714 Rat 714 1,510 Rabbit 1,400 2,380 Guinea pig 2,380 4,620k. ! From Treon and Dutra, 1952, with permission. . 3.5 hours exposure. —151— ------- Rats, guinea pigs, and rabbits that inhaled air containing 2—NP at 325 ppm for 7 hours on 130 occasions (five per week) all survived. Similarly, the same species and one monkey survived 130 7—hour exposures to air containing 2—NP at 83 ppm spread over 190 days. Sufficient concentrations of 2—NP induced dyspnea, cyanosis, prostration and convulsions, and, finally, coma and death. There were also lacrimation, gastric regurgitation, and salivation. Widespread pathologic changes resulted from exposure to 2—NP at 2,350 ppm. Endothelial cells appeared to be most affected. Machle etal. (1940) reported that an oral 2—NP dose of 0.25—0.50 g/kg was lethal to rabbits. Chronic Toxicity in Animals The first study reported was performed in rats and rabbits (Huntlngdon Research Centre, 1977). Inhalation exposure of 50 male Sprague—Dawley rats to 2—NP at 400 ppm for 7 hours/day led to the death of 10 of the rats by the end of the second day and of 20 by the end of the third day. This group of rats was replaced by another, younger group, which was given 2—NP at 207 ppm. The replacement rats were considerably younger (weanhing versus young adult) than those used in the rest of the experiment. Groups of 50 rats and 15 rabbits were exposed by inhalation to 207, 27, or 0 ppm of 2—NP for 7 hours/day, 5 days/week. Ten rats —152— ------- and three rabbits were killed after 2 and 10 days and after 1, 3, and 6 months. All 10 high—dose rats killed after 6 months had liver tumors (hepatocellular adenoma or hepatocellular carcinoma); none of the other treated or untreated rats or rabbits showed this effect. Rats exposed for 3 months demonstrated hepatocellular hypertrophy, hyperplasia, and necrosis. This experiment was criticized in Current Intelligence Bulletin #17 (National Institute for Occupational Safety and Health, 1977) which stated that the tumor—bearing animals started treatment at an earlier age than did either the low—exposure or control groups. This factor is probably of marginal importance to the assessment of the significance of the result. The induction of liver tumors in rats 6 months after the start of treatment indicates that 2—NP is a potent carcinogen in rats. Confirmation of this finding, using longer exposure times and lower levels of exposure, is clearly needed if meaningful risk assessments are to be formulated. Additional experiments are needed, for example, to exclude the possibility that there are metabolic or other thresholds with 2—NP that might lead to different tumor rates at high (as compared to low) exposures (Gehring and Blau, 1977). International Minerals and Chemicals Corporation commissioned the Albany Medical College, N.Y. (Alamagordo Division) to perform further studies. Male and female Sprague—Dawley rats were exposed to 2—NP at a level of 200 ppm, 7 hours/day for 5 days/week. Animals were killed after 10 days and 1, 3, and 6 months. Ten animals —153— ------- treated for 3 months were removed from treatment and held for another 3 months in an atmosphere free of the test compound. Animals exposed for 6 months showed a variety of nonneoplastic liver changes; these included fatty metamorphosis and hepatocellular hypertrophy. Rats exposed for 3 months and allowed to recover showed evidence of regeneration. In another study, male and female Sprague—Dawley rats were exposed to an atmospheric concentration of 2—NP at 100 ppm for 7 hours/day, 5 days/week for 18 months. Liver nodules were observed in 22 of 23 males and in 4 of 30 females, as compared to an incidence of 1 of 63 and 2 of 67, respectively, in unexposed animals. The histological diagnosis of these lesions does not seem to be available (International Minerals and Chemical Corporation, 1979b). In an ongoing study by the same group, male and female Sprague—Dawley rats are being exposed by inhalation to 2—NP at 25, 100 and 200 ppm for 7 hours/day, 5 days/week. After 12 months, no benign or malignant hepatocellular neoplasms were reported after gross or microscopic examination. Completion and release for peer review of the Albany Medical College’s findings on the toxicity and carcinogenicity of 2—NP are urgently needed. A Health Hazard Alert by the Occupational Safety and Health Administration (1980) gives further information on this study and indicates that 2—nitropropane at 200 and 100 ppm induces liver cancer in these rats, whereas 25 ppm failed to do so by 22 months of exposure. —154— ------- Acute Toxicity in Humans Skinner (1947) described the toxic effects in humans exposed to 2—NP in paints. He noted that five or six workers were exposed to 2—NP in concentrations between 20 and 45 ppm. Those least exposed experienced symptoms of severe headache. Those exposed to the higher concentrations experienced anorexia, nausea, vomiting, and diarrhea. When methyl ethyl ketone was substituted for the 2—NP, the above symptoms were no longer reported. Several workers intermittently exposed to 10—30 ppm were not adversely affected. These latter exposures did not exceed 4 hours/day or more than 3 days/week. Hine et al. (1978) detailed the clinical symptoms and macroscopic and microscopic findings In four fatal cases believed to have resulted from overexposure to 2—NP. Two of four men were using vinyl paints; one was using a coal tar surface—coating; the fourth was using a polyester—based resin. Death occurred 6 to 10 days after exposure; post mortem findings indicated liver necrosis In three cases and fatty degeneration of the liver in the fourth. A fifth, nonfatal case Involved a printer who spilled 2—NP, which he had used as a solvent, on the floor. In no case was there any information on the level of exposure to 2—NP or exposure to other solvents. Gaultler etal. (1964) described one fatality and one recovery —155— ------- from exposure to high levels of 2—NP. The men had been painting the inside of a tank. Both suffered liver damage. Lower levels of 2—NP exposure (20—45 ppm) led to nausea, vomiting, and diarrhea in workers at one plant (International Minerals and Chemicals Corporation, 1979b). In addition, Williams et al. (1974) reported an Increased incidence of toxic hepatitis among construction workers applying epoxy resins to the walls of a nuclear power plant; however, the hepatotoxicity could have been due to exposure to methylenedianiline, a well—documented potent hepatotoxin, rather than to exposure to 2—NP. Exposure of humans to toxic levels of 2—NP does not appear to be frequent or particularly well documented. Data for each of the examples discussed in this section are incomplete, leaving open the question of whether the outcome was dependent on other environmental contaminants, acting either alone or in conjunction with 2—NP. Mutagenicity Only one published and three unpublished mutagenicity studies of 2—NP exist. It is a mutagen for Salmonella in all studies, but failed to induce micronuclei. in polychromatic erythrocytes in mice. Bacteria . Hite and Skeggs (1979) showed that 2—NP induced mutations in Salmonella typhimurium strains TA92, TA98, TA100, and TA1537 in the standard plate test, without metabolic activation, and —156— ------- in the presence of Aroclor— and phenobarbital—induced rat liver S—9. The responses attained with S—9 were higher than the corresponding responses without S—9. No differences were observed between Aroclor and phenobarbital induction. The presence of s—9 on the plate appeared to decrease the toxicity induced by 2—NP. The source and purity of the 2—NP used were not described. 1—Nitropropane was not mutagenic in this study. Technical—grade 2—NP (Metheson/Coleman and Bell 2—nitropropane, 93.1%; 1—nitropropane, 6.7Z; acetone, 0.2%) was tested in two laboratories (Stanford Research Institute International and Case Western Reserve University) through the Environmental Mutagenesis Test Development Program of the National Institute for Environmental Health Sciences. The test system used was a preincubation modification of the Salmonella/mlcrosome test, using Aroclor—induced rat and Syrian hamster liver S—9 and Salmonella strains TA98, TA100, TA1535, and TA1537. 2—NP was mutagenic for TA98 and TA100 in one laboratory; TA98, TA100, and TA1535 demonstrated mutagenicity in the second. Again, mutagenicity was observed both with and without S—9, but the response without S—9 was weaker. Subsequent studies with nitroreductase—deficient Salmonella have shown that bacterial reduction of the nitro group does not appear to be responsible for 2—NP’s mutagenic activity (Mortelmans and Speck, personal communication, 1980). 2—NP of undefined purity was tested for Mobil Chemical Company by Litton Bionetics, Inc. (1977). The Salmonella plate test with —157— ------- TA98, TA100, TA1535, TA1537, and TA1538 was used. Also, the yeast Saceharomyces cerevisiae D4, which is used to detect mitotic gene conversion, was used in a plate—test protocol identical to that used for Salmonella. A positive mutagenic response was reported only for TA98 at levels of 10 and 20 p1 (equivalent to approximately 10 and 20 mg/plate) with Aroclor—induced rat liver S—9. The other Salmonella strains and the yeast were tested at a concentration of 5 p1/plate. It is difficult to interpret the negative response observed with yeast because the protocol employed was a departure from published techniques and had not been demonstrated to be effective with standard iuutagens. A parallel test series with l—nitropropane yielded negative results. Mammals. For the micronucleus test in CD—l mice, 2—NP was administered orally, twice daily, at 0.1, 0.2, and 0.3 mg/kg/day. (The 14—day oral LD 50 was 0.40 mg/kg). Bone marrow erythrocytes (polychromatic and normochromatic) were examined for micronuclei; significant effects were reported (Hite and Skegga, 1979). Teratogenicity There were no data from which to evaluate the potential teratogenicity on reproductive toxicity of 2—NP. —158— ------- Mortality Study in Humans International Minerals and Chemicals Corporation (IMC), the sole commercial U.S manufacturer of 2—NP, started work in Louisiana in 1946. 2—NP production was introduced there in 1955. INC (1979 a,b) reported on the mortality experience of all 1,815 persons who had, at one time or another, been employed at the plant between 1946 and 1977. A record file was established to continue surveillance of this population. By June 30, 1977, 180 of these employees had died. Mortality was compared, by cause, for different subpopulations of the work force. Overall, the 1,066 white male employees showed a standardized mortality ratio (SMR) of 85Z as compared to an equivalent age—, race—, and sex—matched segment of the total U.S. population. Such a value is usual for occupational work groups which, through normal hiring practices, exclude the physically unfit. Accidents (motor vehicle, for example) had occurred more frequently than expected. There was no evidence of an increased incidence of liver cancer in this group. There were two lymphatic tumors; only 0.8 were expected, but the finding was not statistically significant. Among the 208 black males, the SMR was 67Z. There was no statistically significant increase in mortality from any cause, although lymphatic tumors again were at increased incidence (two observed vs 0.2 expected). —159— ------- The total study population was then divided into people who had been (1) directly, (2) indirectly, and (3) not exposed to 2—NP. The increased incidence of lymphatic neoplasia, in both black and white males, was confined to those not exposed to 2—NP. Analysis failed to show that 2—NP exposure was associated with any particular cause of death. Similarly, there was no evidence of an increased mortality rate when the duration of exposure to 2—NP was considered. It must be concluded that employment at this large—scale, 2—NP manufacturing plant did not increase cancer mortality in the work population; however, maximum exposure to 2—NP lasted only 22 years and some occupational tumors have a longer latency. Second, levels of 2—NP in the environment were monitored; with few exceptions (such as drum fillers), the present occupational standard for 2—NP was not exceeded. Third, although the total population exposed for 15 or more years was not reported, it appears to be a small fraction of the total. Thus, this survey, so far, is reassuring about the potential human hazard of manufacturing 2—NP under adequate environmental conditons. However, it is still too early to be certain that 2—NP does not lead to the induction of cancer in the population exposed to it. Thus, the company should be encouraged to continue environmental and epidemiologic surveillance for several more decades. —160— ------- CONCLUSIONS At high atmospheric concentrations, 2—NP is hepatocarcinogenic in rats. There is a latency of 6 months. Rats are the only experimental species tested adequately. Results of inhalation testing at 25 ppm have not yet been reported. The results of the chronic toxicity study performed by the Albany Medical College for the International Minerals and Chemical Corporation must be peer reviewed and published before that data can be used for any kind of risk assessment. 2—NP is niutagenic in Salmonella , producing both frame—shift and base—pair substitution mutations, both with and without added liver S—9. There is no evidence on 2—NP’s ability to induce point mutations in mammalian cells. The negative results of the micronucleus test in mice are not sufficient to label 2—NP as nonclastogenic; in vitro and in vivo cytogenetic assays are needed. In addition, there is a need for more data on potential carcinogenicity, other toxicity, teratogenicity, and other reproductive effects. The one epidemiologic study reported failed to demonstrate that 2—NP was carcinogenic in humans, but additional evaluation over a longer period is required before it can be accepted that 2—NP is not carcinogenic in humans under the conditions at the Sterlington plant. The volatility of 2—NP causes it to attain high concentrations in the work environment. Because of its acute —161— ------- toxicity at high concentrations and its reported carcinogenicity in rats, the use of 2—NP requires caution in the occupational and general environment. Moreover, the levels to which humans are currently exposed should be carefully monitored. The appropriateness of the presently permitted threshold limit value/time weighted average adapted by the American Conference of Governmental Industrial Hygienists (1980) is still debatable. However, the major hazard associated with 2—NP appears to be the consequences of its release into a confined, unventilated space from paints, resins, and similar coating materials in which it is used as a solvent. —162— ------- REFERENCES Production, Uses, Exposure Anonymous. 1976. Grande Paroisse looks for growth in nitroparaffins market. Eur. Chem. News 29:23—24,30, August 6. Chemical Solvents Corporation. 1974. Buyer’s. Chemical Solvents Corporation, New York. Code of Federal Regulations. 1980. Title 21, Part 175. Indirect food additives: Adhesive coatings and components. Office of the Federal Register, National Archives and Records Service, Ceneral Services Administration, Washington, D.C. International Minerals and Chemicals Annual Reports. 1977, 1978, 1979. International Minerals and Chemicals Corp., Northbrook, Ill. International Minerals and Chemicals Corp. 1980. Technical Bulletin. Des Plaines, Ill. National Institute for Occupational Safety and Health. 1977. 2—Nitropropane. 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