Y:
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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

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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

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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
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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.
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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
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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
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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

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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
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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
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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
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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
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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.
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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.
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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
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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
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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,
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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.
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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
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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
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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.
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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
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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
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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
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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.
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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 .
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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
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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
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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
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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—

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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.
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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.
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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—

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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.
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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—

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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.

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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—

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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—

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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—

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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—

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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.
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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.
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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.
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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.
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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.
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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
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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).
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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]
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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
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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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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
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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.
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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,
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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.
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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
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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.
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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.
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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.
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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
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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
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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
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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
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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.
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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
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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
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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,
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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
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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
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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.
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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
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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.
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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.
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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).
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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.
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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.
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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
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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.
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‘-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

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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.
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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.
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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.
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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—

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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.

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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—

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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—

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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—

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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—

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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—

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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—

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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—

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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

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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

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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—

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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—

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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—

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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
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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
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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).
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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.
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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.
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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.
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REFERENCES
Production, Uses, Exposure
Anonymous. 1980. Air products may become morpholine producer.
Chem. Eng. News 58(8):28.
Chemical Marketing Reporter. 1974. Chemical profiles:
Morpholine. Chem. Mark. Rep. 205(22):9, June 3. Schnel].
Publishing Co., New York.
Code of Federal Regulations. 1980. Title 21, Part 172. Food
additives permitted for direct addition to food for human
consumption. Office of the Federal Registrar, 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—Intersclence Publication. John Wiley &
Sons, New York. 1,036 pp.
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McCain, J.C., and J. M. Peck. 1976. The Toxicity of Selected
Chemicals Used in Power Generating Stations to Hawaiian Fishes.
University of Hawaii, Honolulu.
Occupational Safety and Health Administration. 1980. Toxic and
Hazardous Substances. 29 CFR 1910.1000. Office of the Federal
Register, Washington, D.C.
Science Applications, Inc. 1980. Human Exposure to Atmospheric
Concentrations of Selected Chemicals. Draft Report No. EF—l56R
to U. S. Environmental Protection Agency, March, 1980.
Singer, G.M., and W. Lijinsky. 1976. Naturally occurring
nitrosable compounds. I. Secondary ainines in foodstuffs. J.
Agri. Food Chem. 24:550—553.
Stanford Research Institute International . 1975. Directory of
Chemical Producers—U.S.A. Supplement, Stanford Research
Institute International, Menlo Park, Calif.
U.S. International Trade Commission. 1978. Synthetic Organic
Chemicals: United States Production and Sales, 1977. USITC
Publication 920. U.S. International Trade Commission,
Washington, D.C. 417 pp.
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Analytic Methods
Burenko, T.S., E.G. Zhuravlev, and T.A. Miklashevich. 1977.
Determination of morpholine in the air. Gig. Tr. Prof. Zabol. No.
3:55—56. [ Chem. Abs. 86:194286s, 1977J
Fajen, J.M., G.A. Carson, D.P. Rounbehier, T.Y. Fan, K. Vita, U.E.
Goff, M.H. Wolif, G.S. Edwards, D.H. Fine, V. Reinhold, and K.
Bietuann. 1979. N—Nitrosalnines in the rubber and tire industry.
Science 205:1262—1264.
Karweik, D.FI., and C.H. Meyers. 1979. Spectrophotoinetric
determination of secondary amines. Anal. Chein. 51:319—320,
Singer, G.M., and W. LijinskY. 1976a. Naturally occurring
nitrosatable compounds. I. Secondary amines in foodstuffs. J.
Agric. Food Chem. 24:550—553.
Singer, G.M., and W. LijinSkY. 1976b. Naturally occurring
nitrosatable amines. II. Secondary amines in tobacco and
cigarette smoke condensate. J. Agric. Food Chem. 24:553—555.
TombropOulos, E.G. 1979. Micromethod for the gas chromatographic
determination of morpholine in biological tissues and fluids. J.
Chromatogr. 164:95—99.
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Health Effects
American Conference of Governmental Industrial Hygienists. 1974.
Pp. 175—176 in Documentation of the Threshold Limit Values for
Substances in Workroom Air with Supplements for those Substances
Added or Changed since 1971. American Conference of Governmental
Industrial Hygienists, Third Edition, Second Printing.
Cincinnati.
Bartsch, H., A. Camus, and C. Malaveille. 1976. ComparatIve
mutagenicity of N—nltrosamines in a semisolid and in a liquid
incubation system In the presence of rat or human tissue
fractions. Mutat. Res. 37:149—162.
Braun, R., J. Schoneich, and D. Ziebarth. 1977. In vivo formation
of N—nitrosO compounds and detection of their mutagenic activity
in the host—mediated assay. Cancer Res. 37:4572—4579.
Charnley, C., and M.C. Archer. 1977. Deuterium isotope effect in
the activation of nitrosomorpholine into a bacterial mutagen.
Mutat. Res. 46:265—268.
Drevon, C., T. Kuroki, and R. Montesano. 1977.
Microsotne—mediated mutagenesis of a Chinese hamster cell line by
various chemIcals. Pp. 207—213 in D. Scott, B.A. Bridges, and
F.H. Sobels, eds. Progress in Genetic Toxicology.
Elsevier/NOrth—Holland, New York.
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Edwards, G., w.Z. Whong, and N. Speciner. 1979. Intrahepatic
mutagenesis assay: A sensitive method for detecting
N —nitromOrphOlifle and in vivo nitrosatlon of morpholine. Mutat.
Res. 64:415—423.
Elespuru, R.K. 1978. Deuterium isotope effects in niutagenesis
by nitroso compounds. Mutat. Res. 54:265—270.
Elespuru, R.K., and W. Lijinsky. 1976. MutagefliCity of cyclic
nitrosamineS in Escherich.ia coli . following activation with rat
liver microsomes. Cancer Res. 36:4099—4101.
Fonshtein, L.M., S.K. Abilev, A.M. Zekhnev, and A.A. Shapiro.
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Gomez, R.F., M. Johnston, and A.J. Sinsky. 1974. Activation of
nitrosonjorpholifle and nitrosopyrrolidine to bacterial mutagens.
Mutat. Res. 24:5—7.
Greenblatt, M., S. Mirvish, and B.T. So. 1971. Nitrosamine
studies: Induction of lung adenomas by concurrent administration
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Cancer Inst. 46:1029—1034.
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Grodetskaya, N.S., and N.M. Karamzina. 1973. Initial reactions
by the organism to the effects of industrial substances in
minimal effective concentrations (Limac, LimCh). Pp. 9—18 in
A.A. Letavet and I.V. Sanotskiy, eds. The Toxicology of New
Industrial Chemical Substances, Issue No. 13. USSR Academy of
Medical Sciences, Meditsina, Moscow. [ Translated from the
Rus8ianj
Henke, R., C. Hohne, and H.A. Kunkel. 1964. Uber die mutagene
Wirkung von Rontgenstrahlen, N—nitroso—N—methyl—urethafl and
N—nitroso—morpholin bel Drosophila melanogaster. Biophysik
1:418—421.
Industrial Blo—Test Laboratories, Inc. 1970. Bio—Fax Data
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Northbrook, Ill.
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.
International Labour Office. 1972. Pp. 915—916 in Encyclopaedia
of Occupational Health and Safety, Volume II. International
Labour Office, Geneva.
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Inui, N., Y. Nishi, N. Taketomi, M. Mori, M. Yamamoto, T. Yamada,
and A. Tanimura. 1979. Transpiacental niutagenesiS of products
formed in the stomach of golden hamsters given sodium nitrite
and morpholine. mt. J. Cancer 24:365—372.
Iqbal, Z.M., K. Dahi, and S.S. EpsteIn. 1980. Role of nitrogen
dioxide in the biosynthesis of nitrosamines in mice. Science
207:1475—1477.
Ivanov, N.G., and A.L. Cermanova. 1973. Comparative sensitivity
of animal and man to the effects of Irritant toxins. Pp. 36—41
in A.A. Letavet and I.V. Sanotskiy, eds. The Toxicology of New
Industrial Chemical Substances, Issue No. 13. USSR Academy of
Medical Sciences, Meditsifla, Moscow. [ Translated from the
Russian]
Ivanov, N.G., A.L. GermanoVa, A.M. Klyackina, G.G. Maksimov, and
V.S. pozdnyakov. 1973. Comparative evaluation of the methods
of finding the irritating action of industrial toxins and the
calculations of their maximum permissible concentrations in the
air of a work zone. Pp. 1928 In A.A. Letavet and I.V.
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Meditsifla, Moscow. [ Translated from the Russian]
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Jones, C.A., and E. Huberman. 1980. A sensitive
hepatocyte—mediated assay for the metabolism of nitrosamines to
mutagens for mammalian cells. Cancer Res. 40:406—411.
Kimble, C.E., A.J. Sinsky, and P. Gorczyca. 1973. Mutagenesis
of BI-LK—21 cells by N—nltroso compounds and ultraviolet light.
In Vitro 8:442—443, abstract no. 149.
Kimble, C.E., P.A. Gorczyca, and A.J. Sinskey. 1974. Repair of
lesions induced in liver cell DNA by N—nitroso compounds as
measured by the bromodeoxyuridine photolysis method. Mutat.
Res. 24:35—39.
Kimble, C.E., P.A. Gorczyca, and R.G. Reynolds. 1975.
N—Nitrosomorpholine and N—nitrobutylamine—stimulated DNA
synthesis in BHK—21/C13 cells. Mutat. Res. 31:153—161.
Kleihues, P., and C.P. Margison. 1976. Exhaustion and recovery
of repair excision of 0 6 —niethylguanine from rat liver DNA.
Nature 259 :153—155.
Kuroki, T., C. Drevon, and R. Montesano. 1977.
Microsome—mediated mutagenesis in V79 Chinese hamster cells by
various nitrosamines. Cancer Res. 77:1044—1050.
Lam, H.F., and E.W. Van Stee. 1978. A re—evaluation of the
toxicity of morpholine. Fed. Proc. 37:679, abstract no. 2459.
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Migukina, N.V. 1973. Evaluation of the danger of morpholine
during chronic action. Pp. 87—94 in A.A. Letavet and I.V.
Sanotskiy, eds. The Toxicology of New Industrial Chemical
Substances, Issue No. 13. USSR Academy of Medical Sciences,
Meditsina, Moscow. [ Translated from the Russian]
Mirvish, S.S., A.F. Pelfrene, H. Garcia, and P. Shubik. 1976.
Effect of sodium ascorbate on tumor induction in rats treated with
morpholine and sodium nitrite, and with nitrosomorpholine. Cancer
Lett. 2:101—108.
Nakajima, T., A. Tanaka, and K.I. Tojyo. 1974. The effects of
metabolic activation with rat liver preparations on the
mutagenicity of several N—nitrosamines on a streptomycin—dependent
strain of Escherichia coli . Mutat. Res. 26:361—366.
Neale, S. 1972. Effect of pH and temperature on the
nitrosamide—induced mutation in Escherichia coli. Mutat. Res.
14:155—164.
Newton, M.F., B. Bahner, and L.J. Lilly. 1977. Chromosomal
aberrations in rat lymphocytes treated in vivo with
l—phenyl—3 ,3—dimethyltriazene and N—nltrosomorpholine. A further
report on a possible method for carcinogenicity screening. Mut.
Res. 21: 155—161.
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Parkin, H., H.B. Waynforth, and P.N. Magee. 1973. The activity
of some nitroso compounds in the mouse dominant lethal assay.
I. Activity of N—nitroso—N—methylurea, N—methyl—N—nitroso—
N’—nitroquanidine and N—nitrosomorpholine. Mutat. Res.
21: 155—161.
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Romen, W., and P. Bannasch. 1972. Karyokinesis and nuclear
morphology during hepatocarcinogenesis. I. Mitoses and mitotic
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Rounbehier, D.P., J. Reisch, and D.H. Fine. 1980. Nitrosamines
In new motor—cars. Food Cosmet. Toxicol. 18:147—151.
Sauro, F., L. Friedman, and S. Green. 1973. Biochemical,
inutagenle and pathological effects of nitrosamines in rats.
Toxicol. Appl. Pharmacol. 25:449, abstract no. 27.
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Schwarz, M., J. Huniniel, K.E. Appel, R. Richart, and W. Kunz.
1979. DNA damage induced in vivo evaluated with a
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Cellular injury and carcinogenesis. Evidence for the
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N—nitrosoinorpholine. Z. Krebsforsch. 82:1—12.
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Stewart, B.W., R.M. Hicks, and P.N. Magee. 1975. Acute
biochemical and morphological effects of N—nitrosomorpholine in
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Van Stee, E.W., G.A. Boorman, and J.C. Haseman. 1980. Pulmonary
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nitrosamines in Arabidopsis thaliana . Mutat. Res. 5:429—431.
Vogel, E. 1977. Identification of carcinogens by mutagen testing
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damage measured. Pp. 1483—1497 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.
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[ in Germanj [ Chem. Abs. 77 :57403c, 1972J
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Zaeva, G.N., L.A. Tiinofievskaya, L.A. Bazarova, and N.V. Migukina.
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Zeiger, E. 1973. Some factors affecting the host—mediated assay
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Zeiger, E. 1975. Dietary modifications affecting the mutagenicity
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Res. 57:1—10.
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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).
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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
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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.
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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
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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).
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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
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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).
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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.
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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.
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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
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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
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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.
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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
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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
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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
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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.
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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).
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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.
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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
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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.
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Analytic Methods
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