SCIENTIFIC AND TECHNICAL
ASSESSMENT REPORT
ON NITROSAMINES
U.S. Environmental Protection Agency
Office of Research and Development
Washington, D.C. 20460
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EPA-600/6-77-001
November 1976
SCIENTIFIC AND TECHNICAL
ASSESSMENT REPORT
ON
NITROSAMINES
Program Element No. 1AA601
Assembled by
Environmental Research Center
Research Triangle Park, North Carolina
for
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Program Integration
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental Protection Agency, have
been grouped into series. These broad categories were established to facilitate further development and
application of environmental technology. Elimination of traditional grouping was consciously planned to
foster technology transfer and a maximum interface in related fields. These series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
9. Miscellaneous Reports
This report has been assigned to the SCIENTIFIC AND TECHNICAL ASSESSMENT REPORTS (STAR)
series. This series assesses the available scientific and technical knowledge on major pollutants that would be
helpful in possible EPA regulatory decision-making regarding the pollutants or assesses the state of
knowledge of a major area of completed study. The series endeavors to present an objective assessment of
existing knowledge, pointing out the extent to which it is definitive, the validity of the data on which it is
based, and uncertainties and gaps that may exist. Most of the reports will be multi-media in scope, focusing
on a single medium only to the extent warranted by the distribution of environmental insult.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and Development, EPA, and approved for
publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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NOTICE
This is a special preprint issued for those who have
an immediate interest in, and need for, information on
nitrosamines in the environment. The document has been
submitted for publication in the STAR series. It will
be available through the normal EPA distribution channels,
or through the U.S. Government Printing Office, in early
1977.
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PREFACE
This document was prepared by a task force directed by Dr. F. G.
Hueter, Director, Criteria and Special Studies Office, Health Effects
Research Laboratory, U.S. Environmental Protection Agency (EPA),
Research Triangle Park (RTP), N. C. The objective of the task
force was to review and evaluate current knowledge on nitrosamines
in the environment as related to possible deleterious effects on
human health and welfare. The document is not an exhaustive scientific
review of the subject but is an attempt to place the subject in
perspective relative to the need for control of nitrosamines.
The following persons served on the task force:
From Health Effects Research Laboratory, EPA (RTP):
F. G. Hueter, Chairman
Jean French Susan Parker
J. H. B. Garner James R. Smith
C. Kommineni Beverly E. Til ton
From Environmental Sciences Research Laboratory, EPA (RTP):
Ronald L. Bradow James Mulik
Basil Dimitriades Eugene Sawicki
Philip L. Hanst John Sigsby
Kenneth Krost
From Industrial Environmental Research Laboratory, EPA (RTP):
David Oestreich
m
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From Health Effects Research Laboratory, EPA (Cincinnati):
Richard Enrione Nancy Ulmer
John Garner
From Office of Air Quality Planning and Standards, EPA:
John Bachmann Donald Lokey
From Industrial Environmental Research Laboratory, EPA (CincinnatiJ.-
Joseph Lasonaro Peter Lederman
From Eastern Regional Research Center, U.S. Department of Agriculture:
A. E. Wasserman
From the 6570th Aerospace Medical Research Laboratory, Department
of the Air Force:
A. A. Thomas
Contributions were made through personal communications with many
other persons within Federal agencies and the scientific community.
Comments by W. Lijinsky, Oak Ridge National Laboratory; and by
Hans Popper, Mount Sinai School of Medicine of the City University
of New York, have been most helpful.
Writing and editing contributions were made by J. Nduaguba,
Wright State University School of Medicine, Dayton, Ohio; and by
M. Quigley, Ventre Associates, Bethesda, Maryland.
IV
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The substance of the document was reviewed by an Ad Hoc Study Group of
the EPA Science Advisory Board in public session. Members of the Group
were:
Martin Alexander - Cornell University, Chairman
Jack G. Calvert - Ohio State University
George B. Hutchison - Howard University
Peter Magee - Temple University
John Mitchell - DuPont Company
Darrell Nelson - Purdue University
Ronald C. Shank - University of California
Aaron E. Wasserman - U. S. Department of Agriculture
Thomas D. Bath - SAB Staff Officer
The document was also reviewed by representatives of the Department of
Health, Education, and Welfare (NCI, NIEHS, NIOSH, and FDA), Department
of Commerce, Department of Agriculture, Occupational Safety and Health
Agency, U. S. Air Force, Health Departments of the State of Maryland and
City of Baltimore, West Virginia Air Pollution Control Board, Texas Air
Pollution Control Board, and members of the scientific community at
large.
All comments and criticisms have been reviewed and incorporated in the
document where deemed appropriate.
iva
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CONTENTS
Page
LIST OF FIGURES .viii
LIST OF TABLES viii
LIST OF ABBREVIATIONS AND SYMBOLS x
ABSTRACT xiv
1. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS 1
1.1 SUMMARY 1
1.1.1 Effects 1
1.1.2 Physical Properties, Chemistry, and Metabolites . 3
1.1.3 Concentrations and Human Exposure 5
1.1.4 Measurement Technology 8
1.1.5 Sources and Uses . 9
1.1.6 Control Technology 12
1.2 CONCLUSIONS 12
1.3 RECOMMENDATIONS 14
1.3.1 Biological Studies 15
1.3.2 Studies of Population Exposure Patterns 17
1.3.3 Epidemiological Studies 18
1.3.4 Studies on Environmental Distribution, Transfor-
mation, and Removal of Nitroso Compounds 18
1.3.5 Studies on Control Technology 19
2. INTRODUCTION 21
3. EFFECTS 22
3.1 EPIDEMIOLOGY 22
3.2 TOXICOLOGY 25
3.2.1 Introduction 25
3.2.2 Acute Toxicity 26
3.2.3 Carcinogenicity 28
3.2.4 Mutagenicity and Teratogenicity 40
3.2.5 Mechanism of Action 42
3.3 HUMAN HEALTH HAZARD 44
3.4 ECOLOGICAL EFFECTS 45
3.4.1 Plants and Microorganisms 45
3.4.2 Domestic Animals 47
3.5 REFERENCES FOR SECTION 3 50
4. CHEMISTRY AND BIOCHEMISTRY OF N-NITROSO COMPOUNDS 60
4.1 INTRODUCTION 60
4.2 FORMATION OF N-NITROSO COMPOUNDS 67
4.2.1 Kinetics of Nitrosation 67
4.2.2 Factors That Influence Nitrosation 74
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Page
4.3 DEGRADATION OF N-NITROSO COMPOUNDS ~78
4.3.1 Decomposition 78
4.3.2 Metabolism 80
4.4 ATMOSPHERIC CHEMISTRY OF NITROSAMINES 84
4.5 WATER AND SOIL CHEMISTRY OF NITROSAMINES 90
4.6 FOOD CHEMISTRY OF NITROSAMINES -90
4.7 REFERENCES FOR SECTION 4 92
5. SAMPLING AND ANALYTICAL TECHNIQUES FOR NITROSAMINES 99
5.1 AIR 99
5.2 WATER 106
5.2.1 Collection and Preservation of Samples 106
5.2.2 Analytical Techniques 106
5.3 FOOD 108
5.4 REFERENCES FOR SECTION 5 108
6. ENVIRONMENTAL CONCENTRATIONS AND HUMAN EXPOSURE Ill
6.1 AIR Ill
6.2 WATER * 112
6.3 FOOD AND DRUGS 116
6.3.1 Nitrosamines in Processed Foods 116
6.3.2 Nitrosamines in Plants and Fruits 126
6.3.3 Nitrosamines in Drugs, Pesticides, and Natural
Products 127.
6.4 RELATIVE EXPOSURE 130
6.5 POPULATIONS AT RISK . 133
6.6 REFERENCES FOR SECTION 6 134
7. SOURCES T4Q"
7.1 NATURAL OCCURRENCE 140
7.1.1 Formation of Nitrogen Compounds in the
Nitrogen Cycle 140
7.1.2 Nitrogen Compounds in Soil 144
7.1.3 Nitrogen Compounds in Aquatic Habitats T50
7.1.4 Nitrogen Compounds in Plants 155
7.1.5 Nitrosamines in Air 162
7.2 OCCURRENCE IN WASTE WATER 162
7.3 INDUSTRIAL SOURCES OF OCCURRENCE . 163
7.3.1 Industrial Processes in Which Nitrosamines Occur
as Primary Product or Intermediate 163
7.3.2 Industrial Sources in Which Nitrosamines Occur
Incidentally 165
7.3.3 Industrial Sources of Nitrosamine Precursors. . . 167
7.4 MOBILE SOURCES . . . 181
7.4.1 Bureau of Mines Studies 183
7.4.2 Southwest Research Institute (SWRI) Studies . . . 185"
7.4.3 Exxon Studies 185
7.4.4 University of Michigan Study 185
7.4.5 Penn State Study 186
7.5 REFERENCES FOR SECTION 7 186
VI
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Page
8. CONTROL TECHNOLOGY 192
8.1 CONTROL TECHNOLOGY FOR STATIONARY SOURCES OF
NITROSAMINES 192
8.1.1 Introduction 192
8.1.2 Control Technology for Air Emissions of
Nitrosamines "192
8.1.3 Control Technology for Nitrosamines in
Industrial Waste Water 196
8.1.4 Ultimate Disposal Methods for Nitrosamine-
Containing Wastes 201
8.1.5 Control of Plant Location 203
8.2 TECHNOLOGY FOR CONTROL OF AIR EMISSIONS OF
NITROSAMINE PRECURSORS 203
8.2.1 Control Technology for Amines 204
8.2.2 Control Technology for Nitrogen Oxides 206
8.2.3 Control Technology for Particulates 207
8.3 REFERENCES FOR SECTION 8 208
BIBLIOGRAPHIC DATA SHEET 210
vn
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LIST OF FIGURES
Figure Page
4-1 Structures of Ammonia and of Primary, Secondary, and
Tertiary Amines . 61
4-2 Reactions of Various Amines with Nitrous Acid 63
4-3 Structures of Various N-Nitrosamines 64
4-4 Structures of Various N-Nitrosamides 65
4-5 General Structures of Some Amides That Can Undergo
Nitrosation. . 66
4-6 Nitrosation of Aminopyrine to Give Dimethylnitrosamine
and l-bikefobutyryl-l-phenyl-2-methyli-2-nitrosohydrazide . 71
4-7 Formaldehyde-Catalyzed Nitrosation of Secondary Amines
at pH 6 to 11 77
4-8 Possible Pathway for the Metabolism of Dialkyl-
nitrosamines, Using Dimethylnitrosamine As an Example. . . 82
5-1 Schematic of GLC-TEA Interface 102
6-1 Concentration of Dimethyl nitrosamine in Fish Broth
after Incubation at 37°C 119
7-1 Main Portions of the Nitrogen Cycle 145
7-2 Disappearance of Three Nitrosamines from Williamson
Silt Loam 151
7-3 Nitrate Concentrations in the Kaskaskia and Sangamon
Rivers 153
7-4 Structures and Diverse Sources or Uses of Pyrrole and
Related Compounds 174
LIST OF TABLES
Table Page
3-1 Acute Toxicity of Some N-Nitroso Compounds 27
3-2 Histopathology of Liver Tissue From Mice Given
Dimethyl nitrosamine Orally 32
vi i i
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Table Page
3-3 Sites of Tumors Produced by Some N-Nitroso Compounds ... 39
4-1 Rate Constants 1C] and K£ (Equations 4-3 and 4-4) for the
Nitrosation of Amines at Optimum pH and 25°C 69
4-2 Rate Constant Ke (Equation 4-8) for the Nitrosation of
Amides at 25°C and pH 2 73
5-1 Protocol and Results of Comparative Study of TERC and
RTI Methods for Measurement of Nitrosamines in
Ambient Air in Baltimore, Md 105
6-1 Nitrosamine Concentrations in Ambient Air 113
6-2 Estimated Average Daily Ingestion of Nitrate and
Nitrite by U.S. Resident 125
6-3 N-Nitrosamines in Weed Killers 129
7-1 Budget for the Nitrogen Cycle 146
7-2 Effect of Lime and Nitrogen on Nitrite Accumulation. . . . 148
7-3 Nitrate-Nitrogen Content, as Percentage of Dry Matter,
of _0at Varieli.es GTOWJOL .for Hay under Jrjri^gation _and
. . . _
Fertilization (Laramie) and under Dry Land Conditions
(Archer) in Wyoming 157
7-4 Distribution of Nitrate-Nitrogen in Dent Corn Plants,
Expressed as Percentage of Air-Dry Weight 158
7-5 Content of Total Nitrogen and Nitrate-Nitrogen in
Leaves of Fodder Sugar Beets, Expressed as Percentage
of Dry Weight 159
7-6 Distribution of Nitrate-Nitrogen in 2-inch Segments of
Leaves on a Shoot of Pennisetum purpureum. . 160
7-7 Summary of Nitrogen Oxides Emissions in the United
States, 1970 168
7-8 Drugs, Pesticides, and Naturally Occurring Amines That
Have Experimentally Produced N-Nitroso Compounds In Vivo
or In^ Vitro 176
7-9 Producers of Amines in the United States 178
7-10 Amine Production in the United States, 1972 180
7-11 Actual Production or Production Capacity of Some Amine
Producers in the United States "181
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LIST OF ABBREVIATIONS AND SYMBOLS
B.W.
°C
14C
cn)3
C02
CoA
(CH3)2NH
(CH3)2N-NO
C-N=0
CaS04
[(CH3)2N-CS-S]2Zn
E(CH3)2N-CS-S]3Fe
DEN
DMA
DMN
DNA
EPA
°F
Fe
ft
9
GC
Benzene, or phenyl group
Body weight
Degrees Celsius
Radioisotope of carbon
Cubic centimeter
Carbon dioxide
Coenzyme A
Dimethyl amine (DMA)
Dimethylnitrosamine (DMN)
C-nitroso; a nitroso group attached to a
carbon atom
Calcium carbonate
Calcium sulfate
Ziram (a fungicide)
Ferbam (a fungicide)
Di ethylni trosami ne
Dimethyl amine
Dimethylnitrosamine
Deoxyribonucleic acid
U.S. Environmental Protection Agency
Degrees Fahrenheit
Iron
Foot
Gram
Gas ehromatography
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G.I. Gastrointestinal
GLC Gas-liquid chromatography
ha Hectare
HC1 Hydrochloric acid
HCN Hydrogen cyanide
HN02 Nitrous acid
H2N02+ Nitrous acidium ion
HONO Nitrous acid
H20 Water
HPLC High-pressure liquid chromatography
hv Used to denote photon or photolytic energy
in. Inch
K Rate constant for a reaction (also K-J, K2, etc.)
kg Kilogram
km Kilometer
KOH Potassium hydroxide
LD50 Dose lethal to 50% of recipients
m Meter
M Molar
m3 Cubic meter
mg Milligram
Mg Megagram
min. Minute
ml Milliliter
MS Mass spectrometry
MT Metric ton
N or N2 Nitrogen
15
N Isotope of nitrogen
xi
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NADPH Reduced (hydrogenated) nicotinamide adenine
dinucleotide phosphate
NaN02 Sodium nitrite
NCS~ Thiocyanate ion
ng Nanogram
NH2 Used to denote amines or ami no group
NH3 Ammonia
Ammonium ion
Ammonium sulfate
nm Nanometer
N-6-MI N-nitrosoheptamethyleneimine
N-N=0 N-nitroso; nitroso group attached to a nitrogen atom
NO Nitric oxide
N=0 Nitroso group
N02 Nitrogen dioxide
N02~ Nitrite ion
NOs" Nitrate ion
N20 Nitrous oxide
N203 Dinitrogen trioxide (nitrous anhydride)
NOBr Nitrosyl bromide
NOC1 Nitrosyl chloride
N03-N Nitrate nitrogen
NOX Nitrogen oxides
NOX Nitrosyl halide
NPip N-nitrosopiperidine
NPro N-nitrosoproline
NPyr N-nitrosopyrrolidine
xi i
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03 Ozone
ON'NCS Nitrosyl thiocyanate ion
o.d. Outside diameter
pg Picogram .
pH Log of the reciprocal of the hydrogen ion
concentration; a measure of acidity
Ph Phenyl group or radical
pKa Log of the reciprocal of the dissociation
constant for an acid
ppb Part per billion
ppm Part per million
ppt Part per trillion
psi Pound per square inch
R Functional group (also R', R-\, R2, etc.)
RNA Ribonucleic acid
RTI Research Triangle Institute; RTP, N.C.
RTP Research Triangle Park, N*C.
S Sulfur
scfm Standard cubic foot per minute
sec Second
TEA "Thermal energy analysis"
TERC Thermo Electron Research Center; Waltham, Mass,
ug Mi crogram
UMDH Unsymmetrical dimethylhydrazine
UV Ultraviolet radiation
wt Weight
yr Year
Zn Zinc
Zn° Metallic zinc
xiii
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ABSTRACT
This report is a review and evaluation of the current knowledge of N-
nitrosanrines in the environment as related to possible deleterious
effects on human health and welfare. Sources, distribution, measure-
ment, and control technology for NA and their precursors are also
considered. Nitrosamines (characterized by the N-N=0 group) are
formed by the reaction of amines with nitrous acid. Nearly 70 percent
of all N-nitroso compounds studied have been found to be carcinogenic,
with a wide range in potency, in all species of laboratory animals
tested via all routes of administration. The experimentally produced
carcinogenesis appears to be caused by metabolites rather than by
nitrosamines themselves. Epidemiological studies to date do not
show a direct relationship between exposure to NA and cancer in man.
Nitrosamines can exist in the gaseous, liquid, or solid state, depending
on molecular weight. They can be emitted directly to the atmosphere
or can enter the environment as a solute in water or hydrocarbons.
Because they are rapidly photolyzed by UV light and are rapidly
metabolized in animals, no accumulation of nitrosamines is expected
to occur in the atmosphere or in the human body.
Ambient air concentrations of NA of up to 36 vg/m have been found
near an emission source; and of up to 0.2 yg/m^ in major population
centers. The relative contributions of natural and man-made sources
are indeterminable at present. Average dietary intake of NA is not
xiv.
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likely to exceed a few yg/day. Intake of NA in municipal drinking
water would probably be much less than 1 yg/day. NA in cigarettes
range from 0 to 180 ng/cigarette.
xv
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1. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
1.1 SUMMARY
1.1.1 Effects
In all species tested in laboratory animal studies, approxirately 70 per-
cent of all N-nitroso compounds studied were found to be carcinogenic,
with a wide range of potency. There is no direct evidence, however, that
nitrosamines have caused cancer in humans, although a few cases of acci-
dental, or occupational, exposure to dimethylnitrosamine are known to
have resulted in liver damage. Nitroso compounds have been cited as a
possible cause of high rates of cancer in rubber workers, but these
workers are also exposed to other chemicals shown to produce cancer in
animals. Metabolic studies in vitro using rat and human liver slices
have shown that the pathway of biochemical changes in the two are simi-
lar to some degree. Epidemiological studies to date do not, however,
show a direct relationship between exposure to nitroso compounds and
cancer in man. It can be concluded, then, that in the few studies in
which N-nitrosamines are cited as a possible cancer link the association
is implied rather than tested. Thus, no causal relationship between
human cancer and nitroso compounds has been established; however, there
is no reason to assume that the human species should be immune in view
of the available data from animal studies.
The toxic and carcinogenic properties of nitrosamines in experimental
animals are well established, with the N-nitrosamines considered to be
among the most potent and versatile of all chemical carcinogenic agents.
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In laboratory studies, these compounds have produced cancer in over 20
species tested via all routes of exposure in essentially all vital
organs. One compound, diethylnitrosamine, has exhibited a toxic effect
in rat, mouse, hamster, guinea pig, pig, rabbit, dog, rainbow trout,
aquarium fish, grass parakeet, and monkey. The nature of the toxic
response apparently is related to the chemical characteristics of the
compound administered. The site of activity appears to depend on the
compound, the diet, age of animal, species, dosage level, route of
administration, and rate of exposure.
Inhalation studies are limited; however, in those conducted to date, the
production of tumors of the nasal cavity and other neoplasms in the
experimental animals has been reported. In an inhalation study in which
rats and mice were exposed to 5 ug DMN/m for a time equivalent to
greater than two-thirds of the lifespan, there was no increase in the
incidence of tumors in experimental as opposed to control animals. Oral
administration is an effective method of dosage, with small daily doses
(a few mg/kg of body weight) over an extended period being more effective
than large single doses in producing tumors. Tumors have been induced,
though, with a single dose of nitrosamine.
The level of dose required to induce tumors increases with the length of
the carbon chain of the alkyl group (27 to 40 mg/kg body weight for
dimethylnitrosamine to 1200 mg/kg body weight for dibutylnitrosamine).
There appears to be a causal relationship between the level of oral dose
administered daily and the time of induction or appearance of tumors.
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At very low doses, the time required for induction might not fall within
the normal life span of the animal.
No experimental data are available on the effects in domestic animals of
exposure to nitrosamines or their precursors. Nitrites, which are pre-
cursors, are formed in the gastrointestinal tract of animals and may be
absorbed into the blood, producing methemoglobi hernia, especially in rumi-
nants and horses. Nevertheless, no evidence of cancer in domestic animals
from the in vivo formation of nitrosamines from this and other precursors
has been reported.
1.1.2 Physical Properties, Chemistry, and Metabolites
The simple aliphatic nitrosamines are yellow or yellow-green non-hygro-
scopic liquids that boil without decomposition at 150° to 200°C. The
compounds are partially soluble in organic solvents, with solubility in
water varying inversely with molecular weight. The simple aromatic
nitrosamines are low-melting solids or yellowish oils that are insoluble
in water and undergo decomposition at atmospheric pressure. The density
3
of nitrosamines ranges from 0.9 to 1.2 g/cm , increasing with molecular
weight.
The chemistry of nitrosamines relative to the problem of human exposure
and biological effects is not well known. It is known, however, that
nitrosamines are rapidly photolyzed 1n the presence of ultraviolet light
and are rapidly metabolized 1n animals. Therefore, one would not expect
a persistent buildup of this specific compound in the atmosphere or in
the human body. Various studies have been made to assess the potential
for the in vivo formation of nitrosamines, but the results are not yet
3
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conclusive. A large number of variables are involved in the biochemical
processes, many of which have not been determined quantitatively in a
biochemical system representative of the human environment. Nitrosation
in the systems studied has been found to depend on the concentration of
nitrite and secondary amines, the pH of the medium, the presence or
absence of promoters or inhibitors (ascorbate has been shown to be an
effective inhibitor), temperature, time of reaction, the basicity of the
amines, and the presence of nitrosating organisms (nitrosation may occur
in the intestine or in an infected urinary tract). The possibility of
nitrosamine formation in vivo resulting from the inhalation of oxides of
nitrogen (NCL) and/or nitrates and amines has not been thoroughly
studied. In one study with rats, nitrosamines were found when a mixture
containing 15 percent NOp was bubbled through lung homogenate; but no
nitroso compounds were found in lung tissue when the animals inhaled an
atmosphere containing 0.01 to 0.5 mg N02 per liter.
Early laboratory work demonstrated that nitrosamines are rapidly metabo-
lized, so that even very large doses are removed within 24 hours.
Further, the evidence indicates that dimethylnitrosamine is not accumu-
lated in any one organ of the body. The general consensus is that the
related carcinogenesis is caused by some active metabolite rather than
by the nitrosamine itself. Alkylating agents, such as diazomethane or
a carbonium ion, have been suggested as possible active metabolic prod-
ucts of nitrosamines; other suggested intermediates include the corre-
sponding aldehyde, nitrous acid, a hydroxlamine derivative, and a
hydrazine derivative (all known mutagens).
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1.1.3 Concentrations and Human Exposure
The relative risk of nitroso-compound-induced cancer in humans can be
discussed only in a qualitative sense, based on hypothetical reasoning.
Given a reasonable qualitative estimate of relative exposure, it is
still not possible to translate this to an effective biological burden;
i.e., the dose-response relationship at the concentration to which the
human population is exposed is not known. If we define relative expo-
sure simply in terms of total dose over time, then it might be divided
into two components: (1) direct exposure to nitrosamines via inhalation,
ingestion, and smoking; and (2) exposure to precursors (nitrogen com-
pounds, including amines) via inhalation, ingestion, and smoking.
There are no reliable estimates of the average daily dietary intake of
nitrosamines; however, based upon the available measurements, it is not
likely to exceed a few micrograms per day. Various nitroso compounds,
including N-dimethylnitrosamine, have been identified in a variety of
vegetables, fruits, and meats. The concentrations reported vary widely
(from non-detectable to mg/kg); however, the validity of the analytical
methodology used in many cases is now under question. The long-
established practice of using nitrates or nitrites for the curing and
processing of meats, and for the control of Clostridium botulinum (the
organism responsible for botulism), may contribute to the nitrosamine
levels in these products; again, the concentrations vary widely and
results are not always consistent. Further, the available information
indicates that nitrite levels are being reduced in these food products.
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The direct intake from drinking water from municipal water supplies
probably would be much less than 1 yg per day. At present there are
very limited data regarding the presence of nitrosamines in water, and
results to date are not conclusive. Recent studies indicate the presence
of N-nitroso derivatives of pesticides in some water samples. Analysis
for nitrosamines in a few well-water supplies in the United States, char-
acterized by high nitrate levels and coliform counts, revealed results of
£ 15 pg/g. Nitroso compounds, as well as nitrates and amines, are found
in a variety of pharmaceutical products, constituting another potential
for human exposure. Reported values of nitroso compounds in cigarettes
range from 0 to 180 ng/cigarette. A one-pack-per-day smoker may then be
exposed to a few micrograms per day. Nitrosamines in ambient air at
3
levels up to 36 yg/m have been reported near an emission source, while
3
concentrations as high as 0.2 yg/m have been reported in major popula-
3
tion centers. Assuming 20 m of air inhaled per day, exposure via inhala-
tion again might be a few micrograms per day. It then seems reasonable
to assume that direct exposure to nitrosamines via each of the primary
routes may be of the same order of magnitude.
The second component of exposure; i.e., the exposure to precursors
(nitrogen compounds, including amines) again may be via inhalation,
ingestion, and smoking. In this case, the daily intake of both nitrates
and amines via ingestion of food and water may be on the order of a few
hundred milligrams per day. Nitrosamine precursors are commonly found
in foodstuffs and water at much higher concentrations than the nitroso
compounds themselves. Nitrite formation in human saliva, particularly
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among smokers, has been estimated at about 6 to 10 nig/day. Human saliva
in smokers also has been found to contain thiocyanate ion, which has been
shown to catalyze the reaction of nitrite and secondary amines to form
nitrosamines. Tobacco smoke contains several secondary amines and oxides
of nitrogen. Nitrate intake via inhalation (ambient air) may be on the
order of several hundred micrograms per day in some areas, such as near
point sources of emissions. Data are not available on amine intake.
Although the reaction between ingested amines and nitrites appears to
produce nitrosamines in the digestive tract of animals, almost no studies
have assessed the results of inhaling potential nitrosamine precursors.
Since the reaction has been found to be entranced in acidic media, it
might be hypothesized that nitrosamine formation in the neutral environ-
ment of the lung would be less efficient than under the acidic conditions
found in the stomach. Furthermore, preliminary estimates suggest that
exposure to precursor amines and nitrogen oxides from food, water, and
smoking is probably greater than that through inhalation. Thus, until
further evidence is available, risks from inhaled nitrosamine precursors
can be viewed as theoretically possible but likely to be of lower order
than that from direct nitrosamine inhalation.
Concentrations of nitrosamines in the nanogram to microgram per unit
volume or mass (cubic meter, liter, or gram) range now have been mea-
sured in air, water, soil, plants, and foodstuff. The environmental
loading may consist of both natural and anthropogenic components. Avail-
able data do not permit a reasonable estimate of the relative contribu-
tions from natural and man-made sources. Further, data are not
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sufficient to describe the distribution of concentrations in time and
space.
If one considers the potential for exposure to nitrosamines or their
precursors, then it must be concluded that the entire human population
is subject to exposure. Direct exposure to nitrosamines is probably
greatest among certain occupational personnel and populations living in
the vicinity of primary nitrosamine emission sources, because of addi-
tional exposure via inhalation.
The most important gap in our present data involves the biological
response in the human population in relation to the potential exposure
via all routes. Except for possible occupational or accidental exposure,
the relatively equivalent human exposure is well below the levels used
in animal experiments. The problem lies in determining the extent of
the environmental hazard and some measure of risk/benefit. This will
require a well-organized research effort over an extended period of time.
Recommendations for such a research program are outlined in Section 1.3.
1.1.4 Measurement Technology
Technology with which to detect and measure nitrosamines has been devel-
oped primarily for the assessment of food and biological samples. Many
analytical procedures have been devised. Recently, an improved method
has been developed that involves gas chromatography followed by mass
spectrometry and a catalytic chemiluminescence technique; the method has
sensitivity in the ppt (pg/g) range for food and biosamples. In view of
this improved capability, many of the earlier findings of nitrosamines
8
-------
in food are considered questionable. The recent development represents
a significant improvement in analytical capability; however, the instru-
mentation is still in the development phase. A standardized monitoring
technique suitable for wide field use is still not available.
1.1.5 Sources and Uses
N-nitrosodimethylamine has been used in the past as an industrial solvent
(a use now abandoned) and is currently used in one facility as an inter-
mediate in the synthesis of the rocket fuel, 1,1-dimethylhydrazine.
Patents have been issued covering the potential use of various nitro-
samines as solvents in the fiber and plastic industry, antioxidants in
fuels, additions to fertilizers, softeners for copolymers, synergists to
self-extinguishing agents used in expanded polymer materials, insect
repellants, insecticides, fungicides, bactericides, and lubricating oils.
The major industries involved in the manufacture or use of nitrosamines
are rubber processing and the manufacture of organic chemicals and
rocket fuel. Several nitroso compounds are available commercially, pri-
marily for sale to the scientific community for research. Some are pro-
duced on a large scale, but annual production figures are not available.
Diphenylnitrosamine (which to date has not been found to be carcinogenic)
is used as a vulcanizing retarder, and dinitrosopentamethylenetetramine
is used as a blowing agent in the production of microcellular rubber.
One of the main liquid rocket fuels has been 1,1-dimethylhydrazine. A
high-yield procedure for preparing unsymmetrical hydrazines involves
reduction of the corresponding nitrbsamine intermediate.
-------
A wide range of possibilities exists either for the direct emission of
nitrosamines into the environment or for the emission of precursors that
may result in the formation of nitroso compounds in the environment.
Since detailed emission inventories have not been prepared, it is impos-
sible at this time to provide quantitative estimates of environmental
loading.
A number of studies have been conducted to determine the emission of
nitrosamines from automobiles. Using the best analytical techniques
available presently, neither amines nor nitrosamines have been detected
in automobile exhaust. Spiking studies strongly indicate that if such
compounds are found in combustion they are destroyed by the exhaust
environment. Automobile exhaust samples in light-proof bags were spiked
with nitrosamines at levels several times the detection limit. After
30 minutes, less than 30 percent of the original amounts remained.
Application of analytical procedures developed for the determination in
ambient air of N-nitrosodimethylamine indicates that this compound is
not present in motor vehicle exhaust at the 30 parts per trillion by
volume level.
Nitrosamines, if present at all, would be further diluted by ambient
air by a factor of at least 100 near roadways and at least 1,000 off
roadways. Therefore, it is estimated that auto exhaust contributes
either no nitrosamines to the environment, or that potential ambient air
levels from such contributions range from below 30 parts per hundred
trillion on roadways.
loT
-------
Theoretical considerations and limited chamber experiments indicate that
nitrosamines may be formed (primarily during hours of darkness) in a
polluted atmosphere containing secondary amines and nitrous acid. The
same evidence indicates that the concentrations resulting from such for-
mation should decrease rapidly toward zero soon after sunrise. Available
ambient measurements do not fully support this concept. Logically, it
might be assumed that concentrations observed in the ambient air during
daylight would be a consequence of direct emissions of nitrosamines from
anthropogenic sources; emissions of secondary amines in the presence of
sufficient nitrogen oxides concentrations (formation rate would need to
exceed destruction rate in sunlight); or direct emissions of nitrosamines
from soil or water. These possibilities have not been confirmed. In
any event, it is not likely that concentrations in the ambient air would
exceed a fraction of a part per billion except very near sources of
direct emissions.
Laboratory studies have shown that nitroso compounds can be formed in
soil, water, and sewage. Based on these studies, it appears that forma-
tion may take place at neutral pH in the presence of colloidal material.
Recent laboratory studies indicate that nitrosamines may move rapidly
through the soil by leaching into ground water or being taken up directly
by plants. Sources from which nitrosamines may be leached include feed-
lots, which are known to produce a variety of secondary amines at con-
centrations well above the present detectable level (ppt).
11
-------
Amines and nitrates which are readily converted to nitrite by microbial
actions are ubiquitous in the environment from both natural and anthro-
pogenic sources. Nitrosamines are synthesized by a combination reaction
of nitrite or nitrous acid with secondary amines. Hence the necessary
precursors for formation of nitroso compounds are ever present.
1.1.6 Control Technology
Control technology has not been adequately developed for nitrosamines.
Nitrosamines as a class of compounds can exist in the gaseous, liquid, or
solid state, depending on molecular weight. They can be emitted to the
environment in any of the phases, as well as entering the environment as a
solute in water or hydrocarbons. Available technologies, such as wet
scrubbing, incineration, catalytic reduction, and ultraviolet irradia-
tion, should be reasonably efficient in controlling emissions to the
atmosphere. Only limited data are available regarding the control of
nitrosamines in water (including industrial waste water) and other waste.
Again, a wide range of technologies exists from which to select tech-
niques that singly or in combination might prove to be practical for each
particular case.
1.2 CONCLUSIONS
In spite of the wide gaps in knowledge of nitroso compounds in the
environment and of their impact on human health, a few conclusions appear
valid at this time:
• N-nitroso compounds or their metabolites are carcinogenic and
mutagenic in a wide range of animal species. Tumors can be
12
-------
induced in essentially all vital organs. N-nitroso compounds
are one of the most potent groups of carcinogens, but potency
varies over a wide range and depends upon chemical character-
istics. These compounds are carcinogenic in experimental animals
via all routes of administration and generally act systemically,
although some have produced tumors at the site of injection. The
tumors induced depend on the route of administration, level of dose,
and frequency and length of treatment. Optimum effectiveness in
tumor inducement appears to be in small doses given over long periods
of time. Administration to experimental animals by inhalation
has produced tumors in the nasal cavity and in other organs.
t There is no direct evidence that nitrosamines have produced can-
cer in humans.
• Nitroso compounds are readily metabolized by animals.
• Nitroso compounds can be formed by the reaction of secondary,
and some tertiary, amines and nitrites. The reaction, under
suitable conditions, may occur in air, water, soil, food, ani-
mal stomach or intestines, or in the urinary tract (where it
occurs through microbial action).
t Nitroso compounds, or their precursors, are widespread in all
media of the environment as the result of contributions of both
natural and anthropogenic sources.
13
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0 The human population may be continuously exposed to m'troso com-
pounds or their precursors. The consequence of this exposure in
terms of human cancer is unknown.
• Although detailed emission inventories have not been prepared,
it appears that man-made sources contribute to the environmental
loading of nitroso compounds and their precursors and hence may
pose a potential health hazard. The extent of man-made contribu-
tions is unknown; prudence dictates that adequate control be
exercised consistent with economic and social constraints.
1.3 RECOMMENDATIONS
In assessing the need for regulatory or control action relative to
environmental pollutants, it is apparent that the research approach in
most cases has failed to provide essential information relative to real-
istic exposure patterns. This is particularly true with toxic or car-
cinogenic compounds that may be present in very low concentrations from
both natural and man-made sources; nitrosamines are such compounds.
Since the impact of realistic exposure conditions upon human health or
welfare has not been evaluated, the tendency is to adopt the unrealistic
position that the exposure level should always be zero. While animal
toxicological research provides an excellent basis for identifying
potential problem areas, it has not provided effects data at realistic
doses. The primary objective in animal experiments has been to obtain
positive results, so that doses have not been representative of environ-
mental exposure levels. Occupational studies, when available, provide
14
-------
realistic data for occupational environments but still do not provide
definitive information needed for assessing community exposure problems.
Consistent results from community studies over long periods of time can
provide a valuable body of data, if adequate exposure assessment and
experimental design are carefully applied. From a regulatory and con-
trol point of view, the research approach should be oriented toward
determining exposure conditions that do not produce significant adverse
effects. The initial objective would be to establish a level at which
observed biological effects are vanishingly small statistically. It
would then be necessary to describe a realistic total environmental
exposure pattern and compare this to the observed adverse-effects level.
Conditions under which the exposure pattern equals or exceeds the no-
observed-effects level, in time and space, would be identified. Control
strategies and technology would then be developed to handle problem areas.
A recommended research approach is described below.
r i ^ . ' ' _ .
1.3.1 Biological Studies
Biological studies should be designed specifically to determine the dose-
response relationship that can be related to current human exposure
levels. The program should consist of two phases: (1) direct exposure
to nitrosamines; and (2) exposure to precursors that might result in the
formation of nitrosamines in the body, both in the respiratory system
and the gastrointestinal tract.
(
1.3.1.1 Lifetime Animal Nitrosamine Exposure—The primary objective is to
determine a no-observed-effects level for direct exposure to nitrosamines
15
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via inhalation and ingestion. Specific outputs should include: (1) iden-
tification of active metabolites and their uniqueness to nitrosamine
metabolism; (2) mean distribution and standard deviation of the dose-
response curve at low dosages; (3) a time-effect relationship model that
might be used for simulation studies and the prediction of cancer in
humans resulting from nitrosamine exposure; and (4) the biological
effects of nitrosamines in the presence of suspected co-carcinogens or
precarcinogens. This program will require a repository of standard ref-
erence agents.
1.3.1.2 Nitrosamine Precursor Exposure—The basic objective is to deter-
mine the significance of in vivo formation of nitrosamines in humans
resulting from exposure via inhalation and ingestion to Nitrogen com-
pounds and amines at levels normally found in the environment, and in
the presence or absence of inhibitors or promoters.
No data are available on the possible formation of nitrosamines in the
lung from the inhalation of precursors. The likelihood of nitrosamine
formation in the lung from nitrites and amines is considerably reduced
by two facts: (1) the pH of the lung is not normally favorable to
nitrosamine formation from these precursors; and (2) nitrite readily
combines with the hemoglobin of the blood, and, in a highly vascularized
organ such as the lung, would probably be unavailable as a reactant in
nitrosation. The possibility of nitrosamine formation in the lung from
precursors other than nitrites, in reactions not involving nitrite inter-
•
mediates, is yet unknown. If it is possible for precursors in the
16
-------
ambient air, such as NCL and nitrates, to be transformed to nitrosamines
in the respiratory tract, then exposure via inhalation becomes a greater
problem. Specific questions that should be studied include:
t Is the respiratory tract capable of reducing inhaled nitrates
to nitrite?
• Does the respiratory tract provide the other necessary precursors
for the formation of nitrosamines in the respiratory tract?
• What are the various metabolic phases of nitrosamines in vivo?
• What enzymes are necessary for this metabolism to occur and are
all these enzymes found in man?
e Do salivary nitrite levels vary with differences in.exposure to
N02 and nitrates in ambient air, i.e., to what extent does inha-
lation of N02 and nitrates contribute to nitrite levels found in
saliva?
• To what extent does ingestion of precursors in food and water
contribute to in vivo formation of nitrosamines?
1.3.2 Studies of Population Exposure Patterns
There is a need for much more definitive information concerning popula-
tion exposure patterns via all exposure routes. Actual exposure will
depend on: (1) nitrosamine (and precursor) concentrations in food,
water, air (indoor and outdoor), and tobacco; (2) diet; (3) smoking
habits; and (4) activity patterns. These studies will require as a
17
-------
prerequisite adequate measurement capability and environmental monitor-
ing for the specific population under study.
1.3.3 Epidemiological Studies
In the final analysis, evaluation of the potential threat to human health
due to nitrosamines in the environment will require epidemiological
studies. The nature of the chemical carcinogen problem makes it extremely
difficult to find exposure patterns that permit the separation of effects
of nitrosamines in the presence of a variety of chemicals. Both occupa-
tional and community studies will be required. Health indicators should
be developed to provide intermediate as well as end-point information.
The final output should be a risk/benefit model.
1.3.4 Studies on Environmental Distribution, Transformation, and Removal
of Nitroso Compounds
The primary objective is to determine the extent to which nitrosamine
and other nitroso compounds may be formed, transported, and accumulated
in the environment. The question of the possible formation of nitro-
samine in polluted atmospheres should be resolved, as well as the pos-
sible accumulation in water supplies.
These studies should cover other similar compounds such as pesticide
derivatives. Atrazine, for example, is a principal herbicide used in
the U.S. (approximately 90 million pounds used per year). This compound
contains two secondary amine groups per molecule and could theoretically
form nitroso derivatives from nitrites. A research study should be
established to determine, for example, whether or not nitrosatrazine
18
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derivatives are present in surface waters (particularly the Mississippi/
Missouri River System) and their persistence and fate in water purifica-
tion plants. This study should include other nitroso derivatives and
should cover such possible sources as sewage treatment plants, feedlots,
and certain industries.
1.3.5 Studies on Control Technology
Control methodology for nitrosamines may be approached from two direc-
tions, i.e., control of the compounds as they are emitted from chemical
processing, or control of the precursors.
Dimethylnitrosamine is the principal nitrosamine of current concern.
Water scrubbing of dimethylnitrosamine, followed by proper incineration
techniques, can effectively limit its emission from industrial sources
to the atmosphere.
Amine emissions can be controlled by one or more different techniques
that depend on the source of the emission. Methodology employed will
vary according to the emission source: manufacture, industrial use,
refining, incineration, combustion, and sewage treatment. The use of
only limited controls for NO and other nitrogenous compounds is possi-
ble, however, because these control are not absolute, and they will
probably not have as great an impact as will the control of amines.
Although limited controls are presently available, there is still an
ongoing research effort to develop catalysts for the control of NO emis-
^
sions from the automobile.
19
-------
Commitment of resources to the development of technology for controlling
higher-moleeular-weight nitrosamines or nitrosamine precursors appears
questionable at this time for the following reasons:
• Secondary and tertiary amines may react with nitrites in the
gastrointestinal (G.I.) tract to form nitrosamines. It is
almost impossible to eliminate the ingestion of amines because
the amine functional group occurs in drugs, foods, and pollutants.
The only possibility of preventing the formation of nitrosamine
in the G.I. tract would be to eliminate the intake of NO ,
J\
nitrate, and nitrite.
• The cost of developing and implementing control technology for
all possible sources of nitrosamines and their precursors would
be quite high.
• Until target emission levels which are based on good dose-response
health effects data can be specified, the cost-benefit relation-
ship cannot be established.
2.0'
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2. INTRODUCTION
Nitrosamines have been reported recently in the ambient air in the
vicinity of probable sources and in a limited number of water samples.
This summary report was prepared to assess the impact of these findings
on human health and welfare. The document is not an exhaustive scien-
tific review, with complete bibliography, but a summary of our current
knowledge on nitrosamines in the environment that can serve as a basis
for regulatory and control decisions. There is an extensive body of
literature on the biological activity of nitroso compounds used in
laboratory animal studies, but an extremely limited amount of data on
these compounds in the air and water. We present information for mak-
ing prudent decisions on the regulatory issues involved and identify
the areas where research is urgently needed.
The concern for nitrosamines in the environment is based primarily upon
possible human health effects; therefore, this subject is treated first
in the document. For those readers not familiar with the chemistry of
nitrosamines, it may be helpful to begin with Section 4.
21
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3. EFFECTS
3.1 EPIDEMIOLOGY
Although biochemical studies have shown that human tissues can metabolize
nitrosamines into an active ultimate carcinogen, there is very little
evidence that exposure to dimethylnitrosamine, or similar nitrosamines
carcinogenic in animals, has led to cancer in man. The epidemiologic
evidence for the association of nitrosamines with human cancer is very
limited. In the few studies in which nitrosamines are cited as a possi-
ble link to the observed cancers, the association is implied rather than
tested.
p
Weisburger and Raineri have postulated that the appreciable decline in
gastric cancer in the United States in the last 40 years may be due, in
part, to a better means of preserving foodstuffs at low temperatures.
It is believed that during the earlier practice of storing leftover
foods at room temperature the nitrate present in the food was reduced to
nitrite, and that the nitrite then reacted with naturally occurring
substrates such as methylguanidine to form a nitroso derivative which
may be carcinogenic. The authors support their argument by pointing out
a high correlation between gastric cancers in Chile, Colombia, Eastern
3-5
Europe, England, and Japan and exposure to environmental nitrates.
Possible factors contributing to high clusters of cancer of the esopha-
gus in certain sections of Africa have been investigated. Ingestion of
an alcoholic beverage known to contain detectable levels of dimethyl-
nitrosamine was once thought to be a major factor in these high esophageal
22
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cancer rates. However, subsequent analysis of the suspect alcoholic
samples, based on mass spectrometry, showed that none of the samples
contained nitrosamines in excess of 0.005 mg/kg. It was thus concluded
that nitrosamines probably could not be implicated in the observed
<
cancers.
There is a very high incidence of esophageal cancer, including cancer of
the gastric cardia, in certain areas of North China, where in some communi-
ties it ranks as the leading cause of death. Large-scale epidemiologic
surveys have been conducted in high- and low-incidence areas to identify
geographically related etiologic factors. Food samples in the high-
incidence area contained significantly higher levels of diethylnitro-
samine, dimethylnitrosamine, and methylphenylnitrosamine than those in
the low-incidence communities. A fungus, Geotrichum candidum, which has
been shown in the laboratory to act as a co-carcinogen with nitrosamines,
was also found in samples of pickled vegetables used as a common food in
the high-incidence areas. Although these North China studies strongly
suggest a relationship between ingestion of food containing nitrosamines
and a fungus which may serve as a co-carcinogen with cancer of the
esophagus, the data are limited by the absence of a case-control study.
Analyses of gastric juice of noncancerous patients in the high-incidence
communities showed the presence of nitrosamines and their precursor com-
pounds, viz., secondary amines, nitrites, and nitrates.
A study of the high incidence of uterine cervical cancer in certain
regions of South Africa has revealed the presence of dimethylnitrosamine
23
U.S EPA Headquarters Library
Mai! code 3404T
1200 Pennsylvania Avenue NW
Washington, DC 20460
202-566-0556
-------
in discharges from the vaginal vault of pregnant women living in the
region.
Exposure of rubber workers to an array of nitroso compounds from both
inhalation and skin contact has been cited as a possible cause of the
high rates of cancer of the gall bladder, bile ducts, and salivary
9
glands observed in that occupational group. However, these workers are
also exposed to other chemical agents, such as 3,3'-dichlorobenzidine,
which are also known to cause similar tumors.
Several cases of accidental dimethylnitrosamine poisoning have been
reported. Two men employed by an automotive production facility where
dimethyl nitrosamine was used as a solvent were accidentally exposed. One
of the men recovered after exhibiting signs of liver damage. The other
died and at necropsy a cirrhotic liver with regenerating nodules was
revealed. Another report of the hepato toxicity of dimethylnitrosamine
involved three men using the nitrosamine for 10 months as a solvent in a
British industrial research laboratory. Two of the three men reported
bronchial pneumonia and signs of liver injury, one of which was confirmed
as cirrhosis upon necropsy after death. The other technician, who had
experienced a hard liver with an irregular surface, recovered after
termination of exposure.
Studies by Nicholson et al. on various occupational groups exposed to
inhalation of nitrosamines thus far have failed to show any association
between such exposure and an excess risk of cancer. However, there has
24
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been insufficient time since exposure to permit an appropriate latency
period.
The lack of strong epidemiologic evidence for an association of human
cancer with exposure to nitrosamines may well be a reflection of the
limited epidemiologic studies that have been conducted on nitrosamines
thus far.
3.2 TOXICOLOGY
3.2.1 Introduction
N-nitroso compounds manifest multipotent biological activity in many
species of animals. The biological actions of nitroso compounds include
the induction of acute and chronic toxicity, carcinogenicity, mutageni-
city, and teratogenicity. Interest in the toxicology of N-nitroso com-
12-13
pounds was stimulated when N-nitroso-N-methyl urethane and
14 15
dimethylnitrosamine ' were recognized as industrial hazards. In an
American automobile factory, where dimethylnitrosamine was used as a
solvent, two men were accidentally poisoned. One man recovered after
signs of liver damage; the other died in a clinical accident and a
14
necropsy revealed a cirrhotic liver with regenerating nodules. Two
of three men in a British industrial research laboratory, working with a
solvent over a period of 10 months, showed signs of liver injury. One
died of bronchopneumonia and a necropsy showed liver cirrhosis. The
other technician developed a hard liver with an irregular surface but
chc
.16
recovered after exposure to the solvent was terminated. In charac-
terizing the toxicity of dimethylnitrosamine, Magee and Barnes
25
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discovered its carcinogenic action in rats. The toxicology of N-nitroso
17 18
compounds has been reviewed by Magee and Barnes, Druckrey et al.,
19 20 21
Magee, Magee and Swann and, more recently, by Shank.
3.2.2 Acute Toxicity
The potency of N-nitroso compounds in causing acute tissue injury and
death varies considerably (Table 3-1). Acute toxicities of N-nitro-
samines and N-nitrosamides expressed as single-dose oral LDj-Q values in
adult rats range from a low of about 20 mg/kg to a high of more than
5000 mg/kg, with many compounds having an LD,-Q between 150 and 500 mg/
kg. The more reactive compounds produce hemorrhagic destructive lesions
at the site of contact; others result in hepatic damage, pulmonary
lesions, and convulsions. Spills have led to irritation of eyes, lungs,
and skin. The histopathology of acute poisoning from dimethyl- and
diethylnitrosamine has been well studied. Dimethyl- and diethylnitro-
samine are hepatotoxins causing centrilobular necrosis with hemorrhages
in 24 to 48 hours; death occurs in 3 to 4 days or the animals survive
and recover completely in approximately 3 weeks. Manifestations of N-
nitrosamine hepatotoxicity include "blood cysts"--areas of destruction
of the parenchyma filled with recently extravasated erythrocytes—and
necrosis of the endothelium of the central and sublobular veins with
extrusion of necrotic hepatic parenchyma! cells into the lumen. The
kidney lesions are limited exclusively to the convoluted renal tubules.
Rats treated with sublethal doses of dimethylnitrosamine developed veno-
31
occlusive lesions in the liver. Veno-occlusive lesions have also been
32
seen in mink treated with dimethylnitrosamine. Typical acute liver
26
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Table 3-1. ACUTE TOXICITY OF SOME
N-NITROSO COMPOUNDS
Compound
Dimethyl ni trosami ne
Diethylni trosamine
Di -n-propyl ni trosamine
Di -n-butyl ni trosamine
Di -n-amyl ni trosami ne
Methyl -n-butyl ni trosami ne
Methyl -t-butyl ni trosami ne
Ethyl -n-butyl ni trosami ne
Ethyl -t-butyl ni trosami ne
Ethyl -2-hydroxyethyl ni trosami ne
Di -2-hydroxyethyl ni trosami ne
Methyl phenyl ni trosami ne
Methyl benzyl ni trosami ne
Nitrosomorphol ine
Methyl ni trosourea
Methyl ni trosourethane
Ni trosohexamethyl enei mi ne
Ni trosoheptamethy 1 eneimi ne
Nitrosooctamethyl enei mine
aLD(j0Uinits: mg/kg body weight,
male rats.
b •
.. ^i. f
LD50a
27 to 41
216
400b
1200
1750
130
700
380
1600
7500
5000
200
18
282
180
240
336
283
566
single oral dose,
Reference
22
22
23
24
25
22
22
26
26
26
27
22
26
25
25
28
29
30
30
adult
29 33
changes have been induced in rats by the heterocyclic nitrosamines. '
The N-nitrosamides are extremely irritating locally; for example, N-
methyl-N-nitrosourethane causes severe necrotic stomach lesions after
18
gavage. N-methyl-N-nitrosourea given systemically has induced acute
27
-------
34 20
effects on the bone marrow of hamsters and mice. N-N1trosam1des,
such as N-methyl-N-nitrosourea and N-methyl-'N-nltrosourethane, have
moderate LD$Q values.
A great deal of work remains to be done in correlating the structure
and the acute toxicity of N-nitrosoamines. It does seem, according to
21
Shank, that acute toxicity decreases with chain length of dialkyl-
nitrosamines. The predominant effect of liver damage caused by
dialkylnitrosamines seems to be best explained by the assumption that
these compounds require metabolism for their tissue-damaging action and
that the adversely active molecular species is a metabolite of the
parent nitrosamine. This hypothesis would explain the relatively
selective action on the liver since, generally speaking, the enzymes
responsible for the metabolism of drugs and other foreign compounds are
present in highest amount in the liver. Detailed studies of the acute
toxicity of the N-nitroso compounds as a class have not been common
because the striking carcinogenicity of many of these compounds has
commanded such intense interest.
3.2.3 Carcinogenicity
The N-nitrosamines are highly potent and versatile carcinogens. Since
the first report of hepatic cancer induced in rats by dimethylnitro-
samine, many more alkylnitrosamines have been found to induce tumors
in various species in a wide range of organs. While the induction of
cancer in the rat from nitrosamines has been studied most frequently,
cancer has been detected also in the mouse, hamster, rabbit, guinea pig,
28
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dog, monkey, and fish. Among the organs affected have been the liver
(most often), kidney, lung, stomach, esophagus, bladder, nose, fore-
stomach, intestine, and tongue. Since large doses of these compounds
result in extensive liver damage and toxic effects, cancer studies have
been conducted in most cases by feeding animals daily doses, generally
between 5 pg and 100 yg, of nitroso compounds. Not only has it been
produced by oral administration, but cancer has also been produced by
injection, inhalation, and skin application.
The route and time of exposure affect the total dosage of nitrosamine
required for development of cancer. The smaller the daily dose, the
smaller is the cumulative total dose administered by the time of appear-
ance of cancer. With smaller doses, the median time to appearance of
35
cancer is lengthened. The lowest dose required to produce a carcino-
genic response is not known with any degree of certainty. At a dietary
oc
concentration of 2 mg DMN/kg of feed, Terracini et al. found liver
tumors after 104 weeks in 1 of 26 surviving rats. Tumor formation
occurred after the same length of time in 8 of 76 when the dietary con-
centration was 5 mg DMN/kg of feed. The lowest dose at which tumors
were observed corresponded to a daily dose of 0.12 mg DMN/kg body
weight (B.W.) for an average rat (0.25 kg). In a similar study,
18
Druckrey et al. reported that at the lowest dose level they used,
0.075 mg/kg B.W. per day, 20 animals survived for more than 600 days.
Of this group, 11 had benign or malignant tumors of the liver and of
the esophagus and all 4 rats that survived more than 940 days developed
tumors. This is the lowest level of any nitrosamine yet reported to
29
-------
produce a carcinogenic response. However, an important unknown factor
in these ingestion experiments is the possibility of in vivo formation
of nitrosamines.
37
A study was initiated by Thomas to test the effects of chronic inha-
lation of unsymmetrical dimethylhydrazine (UMDH). Dimethylnitrosamine
(DMN) is an intermediate in the synthesis of UMDH and has been shown to
be a contaminant of the UMDH used in this study. Overt signs of toxicity
were nonexistent in mice, hamsters, rats, and dogs exposed for 6 months
to 6.0 ppb (plus 5.0 ppm UMDH} and 0.6 ppb (plus 0.5 ppm UMDH) N-nitroso-
dimethyl amine, respectively. Significant exposure effects at 6 months
were limited to slight-to-moderate hepatotoxicity in dogs exposed to the
higher level of UMDH plus N-nitrosodimethylamine, with indications of
partial recovery apparent by the end of 4 weeks post-exposure. Hepato-
toxicity was measured by the assay of serum glutamic-pyruvic transaminase
levels and by bromsulphalein retention.
This study was expanded to include an examination of the dose-response
3fi
relationship between oral DMN administration and hepatotoxicity. Oral
doses were determined by calculating them as equivalents of doses
received from 6-hour inhalation exposures to DMN; the standard minute
volume for 25-g mice was used in the calculations and the assumption
was made that absorption of DMN was 100 percent for both routes of
exposure. Male mice were dosed with 5, 10, 50, 500, 1000, and 5000 yg/kg
B.W. Mice given the lowest two levels, 5 and 10 yg/kg B.W., were dosed
5 days/week for 60 calendar days. Mice given the higher levels were
30
-------
dosed for 28 calendar days. Five mice from each group (including a
control group) were sacrificed at 7, 9, 14, 21, and 28 days. All sur-
vivors of the four higher doses were sacrificed at 28 days. Livers
were removed from animals that died or were sacrificed and were exam-
ined for gross and histopathology. Each group of mice contained extra
animals to compensate for losses from toxicity or from the gastric intu-
bation procedure itself. Of the 327 mice tested, 25 died during the
•
experiment, most of them from pneumonia. Only 1 of the 25—a mouse
receiving the highest dose--died from chemical toxicity; necropsy showed
severe, massive liver necrosis.
Grossly, some enlargement of one or more lobes of the liver was seen in
all groups except the control group. In addition, mice receiving the
highest dose showed subcutaneous edema, ascites, and roughening of the
liver capsule. Histological examination showed vacuolization of hepa-
tocytes in all groups (including controls), where it was-thought to be
an artifact formed during anesthesia prior to sacrifice.
Results of histological examinations are summarized in Table 3-2.
In a recent inhalation study by Moiseyev and Benemanskii,50 mice and
rats were subjected to continuous exposure of low levels of dimethyl-
nitrosamine (DMN) for 17 months. Dose levels were 0.2 mg/m , 0.005 mg/
3 3
m , and 0 mg/m . Significant increases in both benign and malignant
3
tumors were noted for groups exposed to 0.2 mg/m , with tumors occur-
ring earlier than in the controls and in a variety of organs. Tumors
3
developed in 42 of 51 rats (82 percent) exposed 'to 0.2 mg/m as compared
31
-------
Table 3-2. HISTOPATHOLOGY OF LIVER TISSUE
FROM MICE GIVEN DIMETHYLNITROSAMINE ORALLY
38
Oral
dose,
ug/kg
0
5
10
50
500
1000
5000
Inhalation
equivalent
(6-hr), ppb
0
5
10
50
500
1000
5000
Hepatic
response
Vacuolization
Vacuolization
Vacuolization
Vacuolization
Vacuolization
Vacuolization
Vacuolization and
multi focal hepatic
necrosis
No. animals
with response/
total
no. animals
40/61
48/61
53/59
38/38
33/36
35/36
35/36
34/36
Percent of
animals
with response
65
78
90
100
91
97
97
94
to tumors in 22 of 75 (29 percent) controls. Animals (78 rats and 68
mice) inhaling 0.005 mg/m of DMN did not differ from controls. The
3
0.2 mg/m dose'level corresponds to 0.170 mg/kg body weight per day for
rats. Wistar rats were more susceptible to neoplasm development than
Balk/G mice.
Diethylnitrosamine (DEN) has been shown to exhibit an organotropic toxic
and carcinogenic activity in the respiratory system, including the nasal
40 41
cavity of many species of animals. ' Some of the neoplasms of the
nasal cavity of these animals induced by DEN are histologically and
42
biologically similar to those seen in humans. Inhalation of nitro-
43
samines produced tumors of the upper respiratory tract of hamsters.
Neoplasms have also developed in the offspring of female mice and
32
-------
AA 'AC — -• — — - _..— .. — _ . - — -
hamsters and rats45 treated during pregnancy with several nitroso com-
pounds. The transfer of DEN through rat milk has been shown. Lac-
tating rats 5 days post-partum were given 130 mg/kg body weight of DEN
by gavage. The stomach contents (mainly curdled milk) of the suckling
pups of these rats were analyzed for the presence of DEN and were found
to contain 5, 16, and 36 mg/kg DEN at 2, 4, and 6 hours, respectively,
after they started ingesting mother's milk. No DEN was detected in the
mother's milk 49 hours post-gavage. These concentrations may not reflect
exact amounts of DEN transferred through mother's milk, however, as the
young might have contained some milk without DEN from previous helping
and, most important, there may be active process(es) of diffusion and
digestion of DEN from the stomach of the young. This would mean that
each pup probably received 20 \ig of DEN within the time that the DEN was
present in mother's milk. Suckling young rats that received DEN through
mother's milk subsequent to several administrations of DEN (130 mg/kg)
47
to the nursing mothers developed multiple neoplasms. These observa-
tions show that the induction of neoplasms in the offspring may have
been due at least in part to the ingestion of unaltered DEN through the
mother's milk.
Both nitrosamides and nitrosamines appear to induce neoplasms trans-
placenta! ly. N-nitrosoethylurea given to pregnant rats on the 15th day
48
of gestation produced brain and spinal cord tumors in the offspring;
exposure during days 10 through 21 of gestation have resulted in renal
4Q
tumors in the offspring several months after treatment. Diethylnitro-
samine given subcutaneously to pregnant rats on days 9 through 15 of
33
-------
gestation induced tracheal papillomas 1n almost half the offspring within
50
25 weeks of the first administration.
Recently, hamsters have been shown to develop neuroepithelial neoplasms
(esthesioneuroepitheliomas) of the nasal cavity when dimethylnitrosamine
(DMN) was given subcutaneously and ferric oxide (an inert participate
material) installed intratracheally; DMN alone did not induce nasal
cavity neoplasms in this experiment or in others. This is an example
of modification of the neoplastic expression of a known carcinogen by
respirable particulate material. The intragastric administration of
methylcholanthrene and the. intraperitoneal administration of DMN to
Swiss mice resulted in an increased incidence and decreased latency
52
period of neoplasms, compared with the mice treated with either compound.
Rats have been shown to develop sarcomas at the site of subcutaneous
injections of the N-nitroso derivative of the pesticide, carbaryl (N-
methyl-1-naphthyl carbamate). In studies of the carcinogenic activity of
N-nitrosocarbaryl, 14 of 16 Wistar rats developed tumors at the site of a
single subcutaneous dose (1000 mg/kg B.W.) and died within 450 days after
injection. No other tumors or toxic effects were found. In 37 Wistar
rats given a single oral dose of N-nitrosocarbaryl (200 to 1500 mg/kg
B.W.), no tumors or other toxic effects had occurred at the end of 21
months, or about 638 days.
Several cyclic nitrosamines induce neoplasms of the liver and other
54
organs. A high incidence of neoplasms of the liver, tongue, and
esophagus was seen in rats given N-nitrosohexamethyleneimine in drinking
34
-------
29
water. N-nitrosoheptamethyleneimine (N-6-M1) produced squamous neo-
plasms of the lung and esophagus in rats. Subsequently, Lijinsky and
5ff
his colleagues induced a high incidence of squamous neoplasms of the
.j-'
lung in rats fed heptamethyleneimine together with nitrite. While some
nitroso compounds are equally carcinogenic independent of the route of
application, the N-6-M1 was shown to be less effective when given sub-
Sir
cutaneously. However, recent results show that a single subcutaneous
injection of N-6-M1 induced a high incidence of respiratory tract neo-
11''
plasms in hamsters and mice. " In an attempt to evaluate respiratory
infection as a cofactor in the development of respiratory tract neo-
plasia, germ-free, specific-pathogen-free and infected (chronic murine
pneumonia) rats were given N-6-M1 in drinking water for 22 weeks. Rats
were sacrificed 2 weeks post-treatment. The incidence of lung neoplasms
indicated that chronic respiratory infection enhanced the neoplastic
58'
response of the lungs to a systemic carcinogen. These data show that
cyclic, non-alkylating nitroso compounds induce neoplasms similar to
43 59
those induced by most noncyclic nitrosamines ' and by alkylating
60
cyclic nitrosamines. Aminopyrine has been shown to yield dimethyl-
nitrosamine in vitro and to induce malignant neoplasms of the liver
55
on continued simultaneous administration with nitrite. Sprague-Dawley
rats given 1 g/kg of solution or 0.25 g/kg of solution of aminopyrine
and sodium nitrite for 30 and 50 weeks in drinking water, respectively,
developed hemangioendotheliomas of the liver. The current permissible
sodium nitrite level in processed meats in the U.S. is 0.2 g/kg.
35
-------
The possibility that nitrosamines are formed from nitrite and secondary
amines under the acidic conditions of the stomach was hypothesized '
and shown to be a reality. * Recently, lung adenomas were induced
in Swiss mice chronically fed nitrite plus morpholine, piperazine, or
N-methylaniline. The continued administration of morpholine or methyl-
benzylamine simultaneously with nitrite to rats in their drinking water
induced the tumors expected from the known carcinogenic effects of the
ga
corresponding nitrosamines. However, no neoplasms were induced in
rats by feeding secondary ami no acids--p>oTTne, hydroxyproline, and argi-
./ggf
nine--concurrently with nitrite. An extension of the above hypoth-
Gtf~R&
esis ' is to study the formation of nitrosamides from corresponding
~7n
alkylureas and nitrite under acid conditions. Sander and Burkle "
induced a spectrum of neoplasms in rats by feeding nitrite together with
N, N"-dimethylurea, methylurea, ethylurea, or 2-imidazolidone (a cyclic
urea). Neurogenic neoplasms in the offspring of female rats fed nitrite
and ethylurea during pregnancy were seen. These neurogenic neoplasms
are similar to those induced in the offspring of rats given nitrosamides
48
during the latter half of pregnancy. Recently, nitrosamide formation
in vivo has been shown in Swiss mice fed nitrite and methylurea and
72
ethylurea concurrently.
18
Druckrey et al. conducted an extensive study of 65 nitroso compounds
to show carcinogenic activity and to correlate potency and type of can-
cer with chemical structure. All symmetrically substituted dialkyl-
nitrosamines produced carcinomas of the liver. The only exception was
the dibutyl compound, which on subcutaneous injection also produced
36
-------
bladder cancer. The diamyl derivative resulted selectively in pulmonary
cancer. Four other symmetrical nitrosamines proved noncarcinogenic:
ally!, cyclohexyl, phenyl, and benzyl. Asymmetrical dialkylnitro-
samines, especially when one of the alkyl groups was a methyl group,
produced carcinomas of the esophagus; methylallylnitrosamine induced
tumors of the kidney. Cyclic nitrosamines, synthesized for example
from nitrite and pyrrolidine or morpholine (nitrosopiperidine or nitroso
morpholine), produced cancer of the liver. Nitrosopiperidine also
resulted in carcinoma of the esophagus. Nitrosamines with other chemi-
cal functional groups in the structure produced malignant tumors as well.
Several acylalkylnitrosamines gave carcinoma of the forestomach on oral
application but only local tumors at the site if the material was
injected. Of the approximately 100 N-nitroso compounds that have been
studied, more than 80 have been shown to be carcinogenic in test
animals.
i~±-
74
In a study still in progress, four groups of Sprague-Dawley rats were
given N-nitrosopyrrolidine daily in the drinking water "at concentra-
tions ensuring the required dosage." A fifth group served as controls.
All animals were carefully observed until they died or became moribund,
the rats being sacrificed in the latter case. Of 24 rats treated with
10 mg/kg B.W.-day, all had died by day 600: 3 by day 322 without tum-
ors, 5 with benign tumors, and 10 with malignancies, 9 of which were
hepatocellular carcinomas. Mean tumor induction time was 436 days and
the mean total dose applied was 4033 mg/kg B.W. A second group of 39
rats was treated with 3 mg/kg B.W.-day. Of these, 35 developed tumors,
37
-------
34 of which were in the liver. Mean induction time was 515 days and
the mean total dose was 1442 mg/kg B.W. Doses given to the third and
fourth groups, 1.0 and 0.3 mg/kg B.W.-day, respectively, had not led
to the induction of typical liver tumors after about 660 days of treat-
ment, with 50 percent of the animals still living at that time. Among
the control rats, pneumonia was the main cause of death, though two
controls had malignancies and two had benign tumors. The medium life
74 "/
expectancy of the controls was about 550 days.
In the same study, the nitroso derivatives of two pesticides were
administered to rats by stomach tube. Two groups of rats were given
aqueous solutions containing 20 mg/kg.and 5 mg/kg, respectively, of N-
nitrosobenzthiazuron once each week. At the higher dose, 51 of 62 rats
developed tumors, with a mean induction time of 448 days and a mean
total dose of 1156 mg/kg B.W. The tumors were.localized mainly in the
forestomach (31), kidney (17), and liver (11). At the lower dose, 24
of 64 rats were still living at the end of 600 days of treatment. Seven
of the 40 animals that died before day 600 had tumors, including one
each in the liver and the forestomach.
The second nitrosated pesticide given was N-nitrosocarbaryl, which was
administered in a vegetable oil suspension by stomach tube in doses of
130"mg/kg B.W. twice weekly. Mean tumor induction time was 167 days,
with tumors induced in 17 of 32 rats. The forestomach was again the
target organ.
38
-------
Table 3-3 lists some of the N-nitroso compounds and some sites where
they induce tumors.
Table 3-3. SITES OF TUMORS PRODUCED BY SOME
N-NITROSO COMPOUNDS
Site
Skin
Nose
Nasal sinus
Tongue
Esophagus
Stomach
Duodenum
Colon
Lung
Bronchi
Liver
Pancreas
Kidney
Urinary bladder
Brain
Spinal cord
Thymus
Lymph nodes
Blood vessels
Compound
Methylnitrosourea
Di ethy 1 ni trosami ne
Di methyl ni trosami ne
Ni trosohexamethy 1 enei mi ne
Nitrosoheptamethyleneimine
Ethyl butyl ni trosami ne
Methyl ni trosourea
Cycasin
Di ethy 1 n i trosami ne
Di ethy 1 n i trosami ne
Di methyl ni trosami ne
Ni trosomethyl ure thane
Dimethyl ni trosami ne
Di buty 1 ni trosami ne
Methylnitrosourea
Ni trosotrimethyl urea
Nitrosobutylurea
Ethylni trosourea
Nitrosomorpholine
Reference
76
77
78
29
30
17
79 \-
80 :
44
44
16
81 -!
82 '
83 f
84 " •
85 '
86
87
88 '"
89.
There is good correlation between metabolism of nitrosamines and tumor
induction. Dimethylnitrosamine is readily metabolized by rat liver,
less so by rat kidney and lung; liver tumors are the most frequent
39
-------
neoplasms obtained with dimethylnitrosamine administration; renal tumors
are much less frequent; and lung tumors are seen only under special
experimental conditions. Diethylnitrosamine is readily metabolized
by hamster lung but much less so by hamster liver, and its administra-
tion to hamsters results in more lung tumors than liver tumors. The
nitrosamides, presumably because of their instability in neutral and
alkaline solutions, produce tumors at the site of administration or
where decomposition is favored.
3.2.4 Mutagenicity and Teratogenicity
Many carcinogenic nitroso compounds are mutagenic, and a few have
been found to be teratogenic.
The nitrosamides, but not the nitrosamines, are mutagenic in in vitro
bacterial systems. The nitrosamines appear to require metabolic activa-
tion by mammalian enzyme systems before they can exert a mutagenic
9(T
effect. This was demonstrated by Popper et al., who added dimethyl-
nitrosamine (DMN) directly to cultures of the Bacillus subtil is 168 ilv
auxotroph and found that DMN by itself was not mutagenic at concentra-
tions as high as 300 mM. DMN became mutagenic for this auxotroph when
DMN and the bacteria were incubated with mouse liver microsomes in the
QT '
presence of an NADPH-generating system. Czygan et al. subsequently
found that the oxidative demethylation of DMN and its transformation to
a mutagen are both dependent on the cytochrome P-450 content of micro-
somes. Cytochrome P-450 is the terminal oxidase in the microsomal
40
-------
biotransformation system that metabolizes steroids, drugs, and carcino
gens and other foreign compounds.
In a subsequent experiment, Cyzgan et al. studied the capacity of
human liver microsomes (from patients undergoing abdominal surgery) to
alter the mutagenicity of DMN. They found in humans, as in mice, that
DMN mutagenicity was activated by liver microsomes in the presence of
an NADPH-generating system and that the resulting mutagenicity was pro
portional to the cytochrome P-450 content of the microsomes. In the
livers of the 29 patients examined, the microsomal cytochrome P-450
varied over a twelvefold range. The microsomes showed a proportional
differential ability to alter the mutagenicity of DMN in the in vitro
assay system. Czygan et al . stated that the observed variations in
cytochrome P-450 content in the livers "may be related to diseases,
therapy, or environmental pollutants."
"95^
Czygan et al". also investigated the effects of dietary protein and
choline levels on the ability of isolated rat hepatic microsomes to alter
the mutgenicity of DMN. They found a positive correlation between die-
tary protein and choline content and microsomal protein and cytochrome
P-450 content. They concluded that the status of the microsomal bio-
transformation system that activates the mutagenicity of DMN can be
influenced by nutritional factors.
Chromosomal changes have been obtained with sodium nitrite by Fahmy
94
et al., who suggested that more than one mechanism could be at work
in mutagenicity, depending on pH: action by nitrite at low pH or by an
41
-------
alkylating agent at higher pH. It was further pointed out that metaboli-
cally produced aldehydes and their reduction products, such as hydroxyl-
amine or hydrazines, could be the active mutagens since such compounds
~qT • •
are mutagenic in some systems.
N-nitroso compounds have also been shown to be potent teratogens. N-
nitrosomethylurea given to rats on the 13th or 14th day of gestation
results in fetal deaths and resorption and deformities in those that
QC ' '
reach term. When N-nitrosoethylurea is given to rats before the 12th
day of pregnancy, the compound is not carcinogenic but is a powerful
96
teratogen.
3.2.5 Mechanism of Action
The mechanism of acute toxicity and carcinogenesis has been the object
of a great deal of research. A few basic features are well agreed on:
nitrosamides are easily decomposed to products that apparently damage
any cells that they contact, hence give rise to cancer at the site of
exposure. Nitrosamines are moce stable, require enzymatic action, and are
therefore active only in organs that provide the proper enzyme system.
The active intermediate agent, the proximate carcinogen, can be one of
several chemical species, including nitrite, hydroxy1 amine, a hydrazine
derivative, or an alkylating intermediate, possibly a diazoalkane or
carbonium ion. Alkylation of the 7 position of guanine in nucleic
acids, which has been observed in somatic organs and which may later
produce tumors, is essentially the same asthatwhich occurs with DNA of
germ cells and is thought to be responsible for genetic mutations.
Methylation of messenger RNA resulting from dimethylnitrosamine poisoning
42
-------
97 98
effectively inhibits translation in protein synthesis ' by inactivat-
Tg~g,
ing guanine in the genetic code. Ludlum • has shown, however, that
polyribonucleotides containing 7-methyl-guanylic acid serve as normal
templates for RNA polymerase. Incorporation of 3-methylcytosine in
polycytidylic acid, however, not only lowered template efficiency in
producing polyguanylic acid, but also produced incorrect polymers of
guanylic and uridylic acids. This suggests that alkylation of cytosine
at the 3 position may be more closely related to carcinogenesis than
alkylation of the 7 position of guanine.
Some maintain that denaturation of cellular macromolecules involved in
_ _ ; n ;
101
metabolic control is an alternative path of carcinogenesis. This
idea is partly based on shortcomings of the alkylation hypothesis. For
example, the heterocyclic nitrosomorpholine cannot form a diazoalkane
or other alkylating intermediate (there is no experimental proof that
the ring can be opened metabolically) , yet it is carcinogenic. Simi-
larly, phenyl methyl nitrosamine, which has caused cancer of the esophagus
in the rat, does not form methyl diazoni urn ion.
Furthermore, there are a number of instances in which there is poor
correlation between the formation of 7-methyl guanine and a compound's
ability to induce tumors in specific tissues. Methyl nitrosourea is a
potent brain carcinogen and its administration produces approximately
the same level of 7-methyl guanine in brain DNA as does methyl methane-
sulfonate, which is only a weak brain carcinogen. ' Methylnitro-
sourea treatment produced 0 -methyl guanine, which was not detected in
brain DNA from methylmethanesulfonate-treated rats. Loveless has
43
-------
suggested that this minor base may be more Important 1n mutagenesis and
carcinogenesis than 7-methylguanine.
Whatever the initial step in carcinogenesis, it seems plausible that
some change in the heritable properties of the transformed cells must
occur. The result of alkylation of DNA, the genetic material in most
organisms, is thought to involve a change in base sequence or the loss
of one or more base pairs.
3.3 HUMAN HEALTH HAZARD
A causal relationship of nitrosamines to human cancer is suggested by
the following: (T) All mammals studied have been shown to be susceptible
to tumor induction by at least one nitrosamine; (2) nitrosamines (or their
metabolites) are highly effective carcinogens by any route of administra-
tion—inhalation and oral intake included; (3) some of the animal neo-
plasms induced by nitrosamines are predominantly epithelial and their
organotypic and histologic features resemble those seen in humans;
(4) nitrosamines are metabolized in vitro similarly by human, guinea pig,
and rat tissue; (5) nitrosamines occur in the human environment—cured
meat and fish products, tobacco and tobacco smoke, and ambient air; and
(6) evidence is conclusive that nitrosamines or nitrosamides can be
formed in vivo, most likely in the mammalian stomach or possibly in the
lower gastrointestinal tract, through interaction of secondary and ter-
tiary amines ingested as food or drugs with nitrite and as a result of
microbial metabolism.
44
-------
The cause and effect relationship between certain environmental and
pharmaceutical chemicals, and the development of certain human neo-
plasms, has been shown. The incidence of "cancer" in man is quite high,
and the distribution of "cancers" of certain types in some segments of
the population seems to reflect well-defined exposures to carcinogens.
/
Although the daily exposure of man to nitrosamines is unknown, nitro-
samine precursors are ubiquitous in the environment. Continued, chronic
exposure to these nitrosamine precursors could conceivably result in the
formation of nitroso compounds; and, even if such nitroso compounds were
present at very low levels, unremitting insults from these compounds
over several decades might contribute to human cancer. Moreover, the
__L_
distinct possibility that other noxious or etiologic agents could act
synergistically with nitrosamines to modify their biological effects
should be considered. It should be noted in this discussion, however,
that only a few nitrosatable precursors, and their respective nitro-
samine derivatives, have been shown to induce neoplasms in laboratory
animals.
3.4 ECOLOGICAL EFFECTS
3.4.1 Plants and Microorganisms
The presence of nitrates, nitrites, or nitrosamines in the environment
does not usually present a hazard to plants or microorganisms. Nitrates
are an integral part of the life cycle of plants and many microorga-
nisms. " Nitrates are of great importance in plant nutrition and
constitute the chief form of nitrogen in soils available to plants.
45'
-------
As indicated later (Section 7). nitrates are formed, assimilated, and
transformed by microorganisms during the nitrogen cycle.
Nitrites in^small amounts are not hazardous to plants or microorga-
106-108.110 .... . , ... . . . .
msms. Nitrites in low concentrations may be taken up by
plants, while in high concentrations they are injurious to plant roots.
Plants vary in their sensitivity to nitrides. Legumes are more suscep-
tible than grasses. For example, when 376 kg/ha of sodium nitrite was
applied to pasture grasses, no visible damage occurred and corn (Zea
mays) grown in water cultures was able to tolerate up to 200 mg/liter of
110"
nitrite nitrogen.
112
No nitrosamines were detectable in spinach containing nitrite. It
was hypothesized that the weakly alkaline medium in spinach and low
concentrations of secondary amines were important factors in limiting
the synthesis of nitrosamines even when high nitrite levels were
present.
Microorganisms are extremely sensitive to nitrite in high concentrations
although nitrite is formed at low concentrations by microbial activity.
Nitrite accumulation inhibits soil-borne fungi, such as Fusarium oxys-
porium, as well as many bacteria. Nitrites have also been shown to
be mutagenic in some fungi and bacteria.
Microorganisms^appear to play an important role in the formation of
nitrosamines. " Recent evidence indicates that nitrosamines in
soil and water are stable and are not readily degraded by microorganisms
46
-------
present in the environments where they are formed. Nitrosamines
have not been shown to be toxic to microorganisms. '
Plants are capable of assimilating nitrosamines from the soil and from
water cultures. ' They are also capable of forming nitrosamines
vi a" '
by metabolic activity. The presence of nitrosamines in the leaves,
stems, and fruits of plants appears to have no detrimental effect on the
Ills""'
plants. The nitrosamines absorbed by plant roots do not accumulate
to any extent within the plant but appear to be metabblically broken
down and to disappear quite rapidly from the plant. Therefore, decreas-
ing concentrations of nitrosamines in the growth media result in a
decrease in plant uptake and a rapid decrease in the concentrations in
plants due to metabolic breakdown. The mechanism by which plants break
down nitrosamines is not known at present. The metabolites formed from
TTfT-
nitrosamine breakdown by plants also are not known.
The nitrosamine, 4-(nitrosomethylamino}-benzaldehyde, was found in a
culture medium in which the fungus, Clitocybe suaveolens, was grown;
however, it is not known whether this is a de novo synthesis of the
nitrosamine or whether the fungus provided a medium in which the syn-
thetic reaction could take place.
3.4.2 Domestic Animals
Nitrates become hazardous to animals when they accumulate in food plants
and are ingested by animals. "Nitrate toxicity" is in reality
"nitrite toxicity" and occurs following the reduction of nitrate to
~~i QC 120^ 122 "
nitrite within the gastrointestinal tract of animals. ' " Nitrite
47
-------
moves into the blood stream, oxidizes the ferrous iron of hemoglobin to
ferric iron, producing methemoglobin, which is incapable of transporting
oxygen to body tissues. ' Methemoglobinemia is the term used to
designate this condition.
Horses and ruminants, because of their anatomy and the microflora of
their digestive tracts, are most susceptible to the reduction of
nitrates to nitrites prior to their absorption into the circulatory
i
system. 0&>1Z Monogastric animals are, for the most part, not easily
H ^ ,
poisoned by nitrates. They are, however, susceptible to nitrites. '
Nitrate toxicity may be acute or chronic. ' Acute toxicity results
in death within a few hours. Chronic toxicity, caused by the consump-
tion of low levels of nitrate over a period of time, results in a general
debilitation of the animals.106'121'122 The debilitation may be the
result of vitamin deficiency, for nitrite appears capable of destroying
121
carotene and vitamin A. Thyroxine, a hormone which controls the
rate of metabolism and which is produced by the thyroid gland, may be
affected by nitri.te. Abortion in cattle may be another effect of the
i p ] 122
chronic ingestion of nitrate. ' ' Clinical signs associated with
chronic nitrate poisoning in animals include dyspnea, grinding of teeth,
uneasiness, vasodilation, lowered blood pressure, reduction of milk
. i .
123
secretion, abortion, avitaminosis A, and thyroid dysfunction.
Animals normally susceptible to "nitrate toxicity" can adapt to the
intake of higher levels of nitrate than usual. Experiments in which
pregnant heifers were fed 440 to 660 mg of nitrate per kg body weight
48
-------
over a long period showed that the heifers adapted to a deficiency of
oxygen in their blood by increasing the number of circulating erythro-
cytes. Other experiments have shown cattle and sheep capable of
adapting to high levels of nitrate.
For many years, farmers in South Africa have known that ruminants could
tolerate higher levels of nitrate when fed molasses. Because of the
South African information, livestock feeders are aware of the importance
of feeding high-energy feeds when nitrates are present in appreciable
amounts. High-energy nutrients are thought to produce a more intense
reducing environment in the rumen, so that nitrates are reduced beyond
the hazardous nitrite and hydroxylamine intermediates to usable ammonia.
Vitamin A supplements have had a protective effect against nitrate tox-
1 1
icity in lambs but not in adult sheep." Ascorbic acid has also been
shown to have a protective action against nitrate toxicity. In addition,
ruminants are better able to tolerate nitrates when urea is added to the
The effects of nitrosamines and their formation within the gastrointes-
tinal tract have been studied chiefly in laboratory animals (see Section
3.2.3). No information is available regarding the effects of nitrosamines
on or their formation within the intestinal tracts of domestic animals
such as cattle, hogs, horses, or sheep.
49
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3.5 REFERENCES FOR SECTION 3
1. Montesano, R. and P.N.~Magee. Comparative metabolism* in "vitro"of
nitrosamines in various animal species including man. In: Chemi-
cal Carcinogenesis Essays. Lyon, France. IARC Scientific Publi-
cation No. TO. 1974. p. 39-56.
2. Weisburger, J. H. and R. Raineri. Assessment of human exposure
and response to N-nitroso compounds: A new view on the etiology
of digestive tract cancers. Toxicol. and Appl. Pharmacol. 31:
369-374, 1975.
3. Berg, J. W., W. Haenzel, and S. S. Devesa. Epidemiology of gastro-
intestinal cancer. In: Seventh National Cancer Conference Pro-
ceedings. Philadelphia, American Cancer Society, Inc. and Lippin-
cott. 1973. p. 459-464.
4. Weisburger, J. H. Chemical carcinogenesis in the gastro-intestinal
tract. In: Seventh National Cancer Conference Proceedings.
Philadelphia, American Cancer Society, Inc. and Lippincott. 1973.
p. 466-473.
5. Hill, M. J., 6. Hawksworth, and 6. Tattersall. Bacteria, nitro-
samines, and cancer of the stomach. Brit. J. Cancer. 28:562-567,
1974.
6. McGashan, H. C., C. L. Walters, and A. E. M. McClean. Nitrosamines
in African alcoholic spirits and esophageal cancer. Lancet.
2.:1017, 1968.
7. The Coordinating Group for Research on Etiology of Esophageal Can-
cer in North China. A Preliminary Report. Chinese Med. J. j_(3):
167-183, 1975.
8. Harrington, J. S., J. R. Nunn, and L. Inwig. Dimethylnitrosamine
in the human vaginal vault. Nature. 241_:49-50, 1973.
9. Fox, A. J., D. C. Lindars, and R. Owen. A survey of cancer in the
rubber and cable making industries. Results of five year analyses,
1967-71. Brit. J. Ind. Med. 3J_:140, 1974.
10. Shank, R. C. Toxicology of N-nitroso compounds. Toxicol. and
Appl. Phamacol. 31_:361-368, 1975.
11. Nicholson, W. Identification of human populations at risk due to
exposure to N-nitroso compounds. Prepared under EPA Contract.
U.S. Environmental Protection Agency. Research Triangle Park,
N.C. 1975.
50
-------
12. Watrous, R. M. Health hazards of the pharmaceutical industry.
Brit. J. Ind. Med. 4^111-125, 1947.
13. Wrigley, F. Toxic effects of nitrosomethylurethan. Brit. J. Ind.
Med. 5_:26-27, 1948.
14. Hamilton, A. and H. L. Hardy. Industrial Toxicology, 2nd Ed. New
York, Paul B. Hoeber, Inc. 1949.
15. Barnes, J. M. and P. N. Magee. Some toxic properties of diemthyl-
nitrosamine. Brit. J. Ind. Med. 1J_:167-174, 1954.
16. Magee, P. N. and J. M. Barnes. The production of malignant primary
hepatic tumors in the rat by feeding dimethylnitrosamine. Brit. J.
Cancer. 10:114-122, 1956.
17. Magee, P. N. and J. M. Barnes. Carcinogenic nitroso compounds.
Adv. Cancer Res. Kh 163-246, 1967.
18. Druckrey, H., R. Preussmann, S. Ivankovic, and D. Schma'hl. Organo-
trope carcinogene Wirkungen bei 65 verschiedenen N-nitroso-
verbindungen an BD-Ratten. Z. Krebsforsch. 69_:103-201, 1967.
19. Magee, P. N. Toxicity of nitrosamines: their possible human health
hazards. Fd. Cosmet. Toxicol. 9_:207-218, 1971.
20. Magee, P. N. and P. F. Swann. Nitroso compounds. Brit. Med. Bull.
25:240-244, 1969.
21. Shank, R. C. Toxicology of N-nitroso compounds. Toxicol. and Appl.
Pharmacol. 31_:361-368, 1975.
22. Heath, D. F. and P. N. Magee. Toxic properties of dialkylnitro-
samines and some related compounds. Brit. J. Ind. Med. 19:276-282,
1962.
23. Pour, P, F. W. Kruger, A. Cardesa, J. Althoff, and U. Mohr. Car-
cinogenic effect of di-n-propylnitrosamine in Syrian golden ham-
sters. J. Nat. Cancer Inst. 51_: 1019-1027, 1973.
24. Druckrey, H., R. Preussmann, S. Ivankovic, C. H. Schmidt, H. D.
Mennel, and K. W. Stahl. Selective induction of bladder cancer in
rats with dibutyl- and N-butyl-N-(4-hydroxybutyl)-nitrosamine. Z.
Krebsforsch. 66_:280-290, 1964. [Chem. Abstr. 63:4785f.]
25. Druckrey, H., R. Preussmann, D. Schma'hl, and M. Miiller. Chemische
(Constitution und carcinogene Wirkung bei Nitrosaminen.
Naturwissenschaften. 48:134-135, 1961.
51
-------
26. Druckrey, H., R. Preussmann, G. Blum, S. Ivankovic, and J. Afkham.
Erzeugung von Karzinomen der Speiserohre durch unsymmetrische
Nitrosamine. Naturwissenschaften. 5J):100-101, 1963.
27. Schmahl, D. Enstehung, Wachstum und Chemotherapie maligner Tumoren.
Arzneimittel-Forsch. 13, 1963.
28. Druckrey, H., R. Preussmann, J. Afkham, and 6. Blum. Erzeugung von
Lungenkrebs durch Methylnitrosourethan bei intravenoser Gabe an
Ratten. Naturwissenschaften. 49_:451-452, 1962.
29. Goodall, C. M., W. Lijinsky, and L. Tomatis. Tumorigenicity of N-
nitrosohexamethyleneimine. Cancer Res. 28:1217-1222, 1968.
30. Lijinsky, W., L. Tomatis, and C. E. M. Wenyon. Lung tumors in rats
treated with N-nitrosoheptamethyleneimine and N-nitrosooctamethyle-
neimine. Proc. Soc. Exptl. Biol. Med. 130:945-949, 1969.
31. McLean, E., G. Bras, and A. E. M. McLean. Venous occlusions in the
liver following dimethylnitrosamine. Brit. J. Exptl. Path. 46:367-
369, 1965.
32. Carter, R. L., W. H. Percival, and F. J. C. Roe. Exceptional sensi-
tivity of mink to the hepatoxic effects of dimethylnitrosamine. J.
Path. 97.:79-88, 1969.
33. Lijinsky, W., K. Y. Lee, L. Tomatis, and W. Butler. Nitrosazetidine-
a potent carcinogen of low toxicity. Naturwissenschaften. 54:518,
1967.
34. Herrold, K. M. Carcinogenic effect of N-methyl-N-nitrosourea
administered subcutaneously to Syrian hamsters. J. Path. Bact. 92:
35-41, 1966.
35. Walker, P., J. Gordon, L. Thomas, and R. Ouellette. Environmental
Assessment of Atmospheric Nitrosamines. Prepared by Mitre Corpora-
tion, McLean, Virginia, under Contract No. 68-02-1495. U.S.
Environmental Protection Agency. Research Triangle Park, N.C.
February 1976.
36. Terracim", B., P. N. Magee, and J. M. Barnes. Hepatic pathology in
rats on low dietary levels of dimethylnitrosamine. Brit. J. Cancer.
2J_: 559-565, 1967.
37. Thomas, A. A. A 6-month chronic inhalation exposure of animals to
UMDH to determine its oncogenic capacity. U.S. Air Force. Wright-
Patterson AFB, Ohio. Status Report. 1976.
38. Thomas, A. A. Letter to James R. Smith, Criteria and Special
Studies Office, HERL, U.S. Environmental Protection Agency, Research
Triangle Park, N.C. June 18, 1976.
52
-------
39. Moiseev, 6. E. and V. V. Benemanskii. Carcinogenic activity of low
concentrations of nitrosodimethyl amine in inhalation. Vopr. Omkol.
2j[(6) :107, 1975 (Russian). [Chem. Abstr. 83:173618Z, 1975 (English).]
40. Thomas, C. Zur Morphologic der Nasenhb'hlentumoren bei der Ratte.
Z. Krensforch. 67;1-10, 1965.
41. Montesano, R. and U. Saffiott. Carcinogenic response of the respira-
tory tract of Syrian golden hamster to different doses of diethyl-
nitrosamine. Cancer Res. 28_:2197-2210, 1968.
42. Herrold, K. M. Epithelial papillomas of the nasal cavity; experi-
mental induction in Syrian hamsters. Arch. Path. (Chicago). 78:
189-195, 1964.
43. Dontenwill, W. and U. Mohr. Carcinome des Respirationstractus nach
Behandlung von Goldhamstern mit Diathylnitrosamin. Z. Krebsforsch.
64:305-312, 1961.
44. Mohr, U. Diaplacenture Cancerogenese in Experiment. Langenbeck
Arch. Klin. Chir. 321_:273-279, 1968.
45. Thomas, C. and R. Bollmann. Untersuchungen zur diaplacentaren Kreb-
serzeugenden Wirkung des dia'thylnitrosamins an Ratten. Z. Krebs-
forsch. 71_: 129-134, 1968.
46. Schoental, R., T. A. Gaugh, and K. S. Webb. Carcinogens in rat
milk: transfer of ingested diethylnitrosamine into milk by lactat-
ing rats. Brit. J. Cancer. 30:238-240, 1974.
47. Schoental, R. and E. C. Appleby. The development of tumors in a
female rat and her offspring following administration of diethyl-
nitrosamine to the mother during nursing. Brit. J. Cancer. 28:84,
1973.
48. Ivankovic, S. and H. Druckrey. Transplazentare Erzeugung maligner
Tumoren des Nervensystems. I. Athylnitroso- hams toff an BD .IX-
Ratten. Z. Krebsforsch. 71_: 320-360, 1968.
49. Wrba, H., K. Pielsticker, and U. Mohr. Die diaplazentarcarcinogene
Wirkung von Diathylnitrosamine bei Ratten. Naturwissenschaften.
54:47, 1967.
50. Mohr, U., J. Althoff, and A. Authaler. Diaplacental effect of the
carcinogen diethylnitrosamine in the Syrian golden hamster. Cancer
Res. 26:2349-2352, 1966.
51. Stenback, F., A. Ferrero, R. Montesano, and P. Shubik. Synergistic
effect of ferric oxide on dimethylnitrosamine carcinogenesis in the
Syrian golden hamster. Z. Krebsforsch. 79:31-38, 1973.
53
-------
52. Cardesa, A., P. Pour, and M. Rustia. The syncarcinogenic effect of
methylcholanthrane and dimethylnitrosamine in Swiss mice. Z.
Krebsforsch. 79_:98-107, 1973.
53. Eisenbrand, G., 0. Ungerer, and R. Preussmann. The reaction of
nitrite with pesticides. II. Formation, chemical properties and
carcinogenic activity of the N-nitroso derivative of N-methyl-1-
naphthyl carbamate (carbaryl). Fd. Cosmet. Toxicol. 13:365-367,
1975.
54. Garcia, H. and W. Lijinsky. Tumorigenicity of five cyclic nitro-
samines in MRC rats. Z. Krebsforsch. 77:257, 1972.
55. Lijinsky, W., H. W. Taylor, C. Snyder, and P. Nettesheim. Possible
link between incidence of lung and liver tumors and ingestion of
amines with nitrite. Nature. 244:176, 1973.
56. Schmahl , D. Zur .Carcinogenen Wirkung von N-nitrosohexamethylenimin.
Naturwiscenschaften. 55_:653, 1968.
57. Althoff, J., P. Pour, A. Cardesa, and U. Mohr. Comparative studies
of neoplastic response to a single dose of nitroso compounds. Z.
Krebsforsch. 78:78-81, 1972.
58. Schreiber, H., P. Nettesheim, W. Lijinsky, C. B. Richter, and H. E.
Wai burg. Induction of lung cancer in germfree, specific-pathogen-
free and infected rats by N-nitrosoheptamethyleneimine. Enhance-
ment by respiratory infection. J. Nat. Cancer Inst. 49:1107-1114,
1972. ~~
59. Althoff, J., F. W. Kruger, U. Mohr, and D. Schmahl. Dibutylnitro-
samine carcinogenesis in Syrian golden and Chinese hamsters. Proc.
Soc. Exptl. Biol. (New York) J136_: 168- 173, 1971.
60. Lijinsky, W. and A. E. Ross. Alkylation of rat liver nucleic
acids not related to carcinogenesis by N-nitrosamines. J. Nat.
Cancer Inst. 42^:1095-1100, 1969.
61, Lijinsky, W. and M, Greenblatt. Carcinogen dimethyl ami ne produced
In vivo from nitrate and ami no pyHne. Nature New B1ol, 236:177-
~
62. Taylor, H. W. and W. Lijinsky. Tumor Induction 1n rats by feeding
aminopyrine or oxytetracycllne with nitrite. Intl. J. Cancer. 16:
211-215, 1975. ~~
54
-------
63. Sander, J. Kann nitrit in der Menschlichen Nahrungursache einer
Krebsentstehung durch nitrosamin—bildung seln? Arch. Kyg. Bakt.
151:22-28, 1967.
64. Druckrey, H., D. Steinhoff, H. Bentherner, H. Schneider, and P.
Klarner. Screenijig of nitrate for chronic toxicity in rats.
"Arzneimittel-Forsch. J3.: 320-323 i 1963.
65. Sander, 0., G. Burkle, L. Flohe, and B. Aeikens. Untersuchungen
in vitro u'ber die Moglichbeit einer bildung cancerogener nitro-
samide in Magen. Arzneimittel-forsch. 2_1/411-414, 1971.
66. Mirvish, S. S. Kinetics of nitrosamide formation from alkylureas,
N-alkylurethans, and alkylguanidines: Possible implications for
the etiology of human gastric cancer. J. Nat. Cancer Inst. 46:
1183-1193, 1971.
67. Greenblatt, M., S. Mirvish, and B. T. So. Nitrosamine studies:
Induction of lung adenomas by concurrent administration of sodium
nitrate and secondary amines in Swiss mice. J. Nat. Cancer Inst.
46:1029-1034, 1971.
68. Sander, J. and G. Burkle. Induction maligner tumoren bei ratten
durch gleichzeitige verfutterung von nitrit und sekundaren aminen.
Z. Krebsforsch. 73_: 54-66, 1969.
69. Greenblatt, M., C. Kommineni, and W. Lijinsky. Null effect of
concurrent feeding of sodium nitrite and ami no acids to MRC rats.
J. Nat. Cancer Inst. 50:799-802, 1973.
70. Sander, J. and G. Burkle. Induction maligner tumoren bei ratten
durch orale gabe von imidazolidinon und nitrit. Z. Krebsforsch.
75_:301, 1971.
71. Ivankovic, S. and R. Preussmann. Transplanzentare erzeugung
maligner tumoren.. Naturwissenschaften. 57^:460, 1970.
72. Mirvish, S. S., M. Greenblatt, and C. Kommineni. Nitrosaiinde
formation iji vivo: induction of lung adenomas in Swiss mice by
concurrent feeding of nitrite and methyl urea or ethyl urea. J.
Nat. Cancer Inst. 48:1311-1315, 1972.
73. Preussmann, R. Toxicity of nitrite and N-nitroso compounds. In:
Proc. Intl. Symp. Nitrite Meat Prod. Zeist. Pudoc, Wageingen,
Germany; Centre for Agricultural Publishing and Documentation.
1974.
74. Preussmann, R., G. Eisenbrand, and D. Schmahl. Carcinogenicity
testing of N-nitrosopyrrolidine and of N-nitrosobenzthiazuron and
N-nitrosocarbaryl in rats. Z. Krebsforsch. [In Press.]
55
-------
75. Montesano, R. M. and P. N. Magee. Metabolism of nltrosamlnes by
rat and hamster tissue slices in vitro. Proc. Amer. Assoc. Cancer
Res. 12:14, 1971.
76. Graffl, A. and F. Hoffmann. A strong carcinogenic effect of
methylnitrosourea on the mouse skin in the drop test. Acta Biol.
Med. Ger. 1_6:K1-K3, 1966. [Chem. Abstr. 65:4388h.]
77. Herrold, K. M. Induction of olfactory neuroepithelial tumors in
Syrian hamsters by diethylnitrosam.ine. Cancer. 17:114-121, 1964.
78. Druckrey, H., S. Ivankovic, H. D. MenneT, and R. Preussmann.
Selective production of carcinomas of the nasal cavity in rats by
N,N'-dinitrosopiperazine, nitrosopiperidine, nitrosomorpholine,
methylallylnitrosamine, dimethylnitrosamine, and methyl vinylnitro-
samine. Z. Krebsforsch. 66:138-150, 1964. [Chem. Abstr. 61:
12436f.]
79. Druckrey, H., D. Steinhoff, R. Preussmann, and S. Ivankovic.
Krebserzeugung durch ein analige Dosis von Methylnitroharnstoff
und verschiedenen Dialkylnitrosaminen. Naturwissenschaften. 50:
735, 1963.
80. Laqueur, G. The induction of intestinal neoplasms in rats with
the glycoside cycasin and its aglycone. Virchows Arch. Path.
Anat. 340:151-163, 1965.
81. Druckrey, H., S. Ivankovic, J. Biicheler, R. Preussmann, and C.
Thomas. Erzeugung von Magen- und Pankreas-Krebs beim Meerschwein-
chen durch Methylnitrosoharnstoff und -urethan. Z. Krebsforsch.
71:167-182, 1968.
82. Magee, P. N. and J. M. Barnes. The experimental production of
tumors in the rat by dimethylnitrosamine (N-nitrosodimethylamine).
Acta Union Intl. Contra Cancrum. 1_5_:187-190, 1959.
83. Druckrey, H., R. Preussmann, D. Schmahl, and M. Muller. Erzeugung
von Blasenkrebs an Ratten mit N-N'-Dibutylnitrosamin. Natur-
wissenschaften. 49_:19, 1962.
84. Druckrey, G., S. Ivankovic, and R. Preussmann. Selektiv Erzeugung
maligner Tumoren im Gehirn und Riickenmark von Ratten durch N-
methyl-N-nitrosoharnstoff. Z. Krebsforsch. 66_:389-408, 1965.
85. Ivankovic, S., H. Druckrey, and R. Preussmann. Induction of tumors
of the peripheral and central nervous system by trlmethylnitro-
sourea in the rat. Z. Krebsforsch. 66:541-548, 1965. [Chem.
Abstr. 63:4787c.] ~~
.56
-------
86. Yokoro, K., N._Imamura, S. Takizawa, H. Nishihara, and E.
Nishihara. LeukemogenTc and mammary tumorigenie effects of
N-nitrosobutylurea in mice and rats. GANN. 61:287-289, 1970.
87. Vesselinovitch, S. D., N. Mihailovich, K. V. N. Rao, and L.
Itze. Perinatal carcinogenesis by urethane. Cancer Res. 31;
2143-2147, 1971.
88. Bannasch, P. and H. A. Mueller. Lichtmikkorkopische Untersuchingen
Liber die Wirkung yon^ N-Nitrosomorpholin. auf die Leber von Ratten
und Maus. ArzneimitteT-Forsch. ^805-814, 1964.
89. Newberne, P. M. and R, C. Shank. Induction of liver and lung
tumors in rats by the simultaneous administration of sodium
nitrate and morpholine. Fd. Cosmet. Toxicol. 'Vl_:819-825, 1973.
90. Popper, H., P. Czygan, H. Greim, F. Schaffner, and A. J. Garro.
Mutagenicity of primary and secondary carcinogens altered by
normal and induced hepatic mircosomes. Proc. Soc. Expt. Biol.
Med. 142 (3):727-729, 1973.
91. Czygan, P., H. Greim, A. J. Garro, F. Hutterer, F. Schaffner,
H. Popper, 0. Rosenthal, and D. Y. Cooper. Microsomal metabolism
of dimethylnitrosamine and the cytochrome P-450 dependency of its
activation to a mutagen. Cancer Res. ,33_:2983=2986, 1973.
92. Czygan, P., H. Greim, A. J. Garro, F. Hutterer, J. Rudick, F.
Schaffner, and H. Popper. Cytochrome P-450 content and the
ability of liver microsomes from patients undergoing abdominal
surgery to alter the mutagenicity of a primary and a secondary
carcinogen. J. Nat. Cancer Inst. 5_1_ (6):1761-1764, 1973.
93. Czygan, P., H. Greim, A. Garro, F. Schaffner, and H. Popper. The
effect of dietary protein deficiency on the ability of isolated
hepatic mircosomes to alter the mutagenicity of a primary and a
secondary carcinogen. Cancer Res. 34_: 119-123, 1974.
94. Fahmy, 0. G., M. J. Fahmy, and J. Massasso. Differential muta-
genicity of the amine and amide derivatives of nitroso compounds
in Drosophila melanogaster. Mutat. Res. 2:201-217, 1966.
95. VonKreybig, T. Effect of a carcinogenic dose of methylnitrosourea
on the embryonic development of the rat. Z. Krebsforsch. 67:46-50,
1965. [Chem. Abstr. 63:12098h.]
96. Napalkov, N. P. and V. A. Alexandrov. On the effects of blastomo-
genic substances on the organism during embryogenesis. Z.
Krebsforsch. 71:32-50, 1968.
57
-------
97. Villa-Trevino, S. A possible mechanism of inhibition of protein
synthesis by dime thy! nitrosamine. Biochem. J. 1_05: 625-631 , 1967.
98. Shank, R. C. Effect of dimethylnitrosamine on enzyme induction
in rat liver. Biochem. J. J08:625-631, 1968.
99. Wilhelm, R. C. and D. B. Ludlum. Coding properties of 7-
methylguanine. Science. Ijtt: 1403- 1405, 1966.
100. Ludlum, D. B. The properties of 7-methylguanine-containing tem-
plates for ribonucleic acid polymerase. J. Biol . Chem. 245:477-
482, 1970.
101. Argus, M. F. , J. C. Arcos, and C. Hochligeti. Studies on the _
carcinogenic activity of protein denaturing agents : Tlepatocar-
cinogenicity of dioxane. J. Nat. Cancer I ns t. 3_5_: 949-958, 1965.
102. Kleihues, P. and P. N. Magee. Alkylation of rat brain nucleic
acids by N-methyl-N-nitrosourea and methylmethane-sulphonate. J.
Neurochem. 20:59.5-606, 1973.
103. Swann, P. F. and P. N. Magee. Induction of rat kidney tumors by
ethyl methane-sulphonate and nervous tissue tumors by methyl
methane-sulphonate and ethyl methane-sulphonate. Nature (London).
223:947-948, 1969.
104. Loveless, A. Possible relevance of 0 -alkylation of deoxyguano-
sine to the mutagenicity and carcinogenicity of nitrosamines and
nitrosamides. Nature (London). 223:206-207, 1969.
105. Brookes, P. and P. D. Lawley. Alkylating agents. Brit. Med.
Bull. 210:91-95, 1964.
106. Wright, M. J. and K. L. Davidson. Nitrate accumulation in crops
and nitrate poisoning in animals. Adv. in Agron. 16:197-247,
1964.
107. Alexander, Martin. Microbial Ecology. New York, John Wiley.
1971. 511 p.
108T Alexander, Martin. Soil Microbiology. New York, John Wiley.
1961. 472 p.
109. Brock, Thomas D. Biology of Microorganisms. Englewood Cliffs,
New Jersey, Prentice-Hall. 1970. 737 p^
110. McKee, H. S. Nitrogen Metabolism in Plants. Oxford, Clarendon
Press. 1962. 728 p.
58
-------
111. Shirley, R. L. Nutritional and physiological effects of nitrates,
nitrites, and nltrosamlnes. B1oSc1. 25.:789-794, 1975.
112. Foster, J. W. Chemical Activities of Fungi. New York, Academic
Press. 1949. 648 p,
113. Mills, A. L. Nitrosation of Secondary Amines by Axenic Cultures
of Microorganisms and 1n Samples of Natural Ecosyterns. PhD. Thesis.
Ithaca, New York, Cornell Univ. 1976. 95 p.
114. Ayanaba, A., W. Verstraete, and M. Alexander. Formation of
dimethylnitrosamine, a carcinogen and mutagen, in soils treated
with nitrogen compounds. Soil Sci. Soc. Amer. Proc. 37:565-568,
1973. ~~~
115. Tate, R. L., Ill, and M. Alexander. Stability of nitrosamines in
samples of lake water, soil, and sewage. J. Nat. Cancer Inst. 54:
327-330, 1975.
116. Wasserman, A. E. and C. N. Huhtanen. Nitrosamines and the inhibi-
tion of Clostridia in medium heated with sodium nitrite. J. Food
Sci. 37:785-786, 1972.
117. Dean-Raymond, D. and M. Alexander. Plant uptake and leaching of
dimethylnitrosamine. Ithaca, New York, Cornell Univ. 1976.
118. Sander, J., M. Ladenstein, J. LaBar, and F. Schweinsberg. Experi-
ments on the degradation of nitrosamines in plants. In: N-Nitroso
Compounds in the Environment. Lyon, France, IARC Proceedings,
October 17-20, 1973, p. 205-210.
119. Smith, T. A. The occurrence, metabolism and functions of amines
in plants. Biol. Rev. 46:201-241, 1971.
120. Wolff, I. A. and A. E. Wasserman. Nitrates, nitrites, and nitro-
samines. Science. 1_77^:15-19, 1972.
121. Emerick, R. J. Consequences of high nitrate levels in feed and
water supplies. Fed. Proc. 33:1183-1187, 1974.
122. Accumulation of nitrates. National Academy of Sciences, Washington,
D.C.
123. Ridder, W. E., F. W. Oehme, and D. C. Kelley. Nitrates in Kansas
ground waters as related to animal and human health. Toxicol. 2:
397-405, 1974.
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4. CHEMISTRY AND BIOCHEMISTRY OF
N-NITROSO COMPOUNDS
4.1 INTRODUCTION
Organic nltroso compounds contain a nltroso group (-N = 0) that is at-
tached to a nitrogen (N-nitroso) or a carbon atom .(C-nitroso). . /
The N-nitroso compounds have been known for years to be highly
toxic chemicals, and many have been studied extensively since 1956 when
Magee and Barnes reported that N-nitrosodimethylamine (dimethylnitros-
amine) produces primary hepatic tumors in rats. In contrast, the
C-nitroso compounds have been of little interest.
The carcinogenic N-nitroso compounds can be conveniently divided into
two groups, the N-nitrosamines and the N-nitrosamides. The N-nitros-
amines consist of the dialkyl, alky Vary1, diary!, and various.cyclic
nitrosamines. N-Ttitrosamides consist of the alkyl and aryl nitro-
sami des.
Ti-Nitrosamines are formed when amines react with nitrous add. Amines
themselves are organic compounds derived from ammonia in which one or
more hydrogen atoms have been replaced by an alkyl or aryl group. Re-
placement by an alkyl group yields an aliphatic amine, while replace-
ment by an aryl group gives an aromatic amine. The structures of
ammonia and of primary, secondary, and tertiary amines are shown in
Figure 4-1.
Primary, secondary, and tertiary amines behave differently when treated
with nitrous acid. Primary aliphatic amines react to yield nitrogen
60.
-------
H
H-N-H
AMMONIA
T '
CHa-N-H
METHYLAMINE
(A PRIMARY. ALIPHATIC
AMINE)
H
/0~\-N-H
PHENYLAMINE
(A PRIMARY, AROMATIC
AMINE)
CH3
DIMETHYLAMINE
(A SECONDARY ALIPHATIC
AMINE)
<£>
CH3
N-H
METHYLPHENYLAMINE
(A SECONDARY AROMATIC
AMINE)
CH3-N-CH3
TRIMETHYLAMINE
(A TERTIARY ALIPHATIC
AMINE)
<£>
CH3 "
N-CHa
DIMETHYLPHENYLAMINE
(A TERTIARY AROMATIC
AMINE) -
Figure 4-1. Structures of ammonia and of primary, secondary, ,and tertiary amines.
•61
-------
and alcohols; secondary amines yield N-nitrosamines; and tertiary
aliphatic amines usually form unstable salts which are destroyed on
2
neutralization. However, one tertiary anrine, amlnopyrlne, yields
dimethylnitrosamlne and an open-ring compound when nltrosated. Pri-
mary aromatic amines form diazonium salts while tertiary aromatic
amines undergo ring substitution with a nitroso group (-N = 0), which
attaches generally to the para position of the aromatic ring (Figure
4-2).4 All these reactions, despite differences in final product, in-
volve the same initial step, which is an electrophilic attack by the
nitrosonium ion from nitrous acid that results in the displacement of
a hydrogen in the amine (the hydrogen on the ami no nitrogen). Ter-
tiary aromatic amines, which have no hydrogen attached to the nitrogen
atom, are attacked instead at the highly reactive aromatic ring.
Nitrosation of the amides,N-alkylureas, N-arylureas, N-acylureas,
N-alkylcarbamates, and N-alkylguanidines results in the formation of
nitrosamides.
General and specific structures of some nitrosamines are shown in
Figure 4-3 and those of nitrosamides in Figure 4-4. General structures
of some amides that can undergo nitrosatlon are shown 1n Figure 4-5.
Some of these chemicals have been used as chemical Intermediates and
reagents, and some as solvents 1n organic synthetic chemistry. The
A
chemistry of the nitroso compounds has been reported in detail. As
only the nitroso group is common to all the nitroso compounds, the
physical properties of these compounds cover a wide range. At ambient
62
-------
H0-N=0
N
H
AMINE (NaN02+HCI)
a. Primary aliphatic amine and nitrous acid.
NH2 + H0-N=0-
AMINE (NaN02+HCI)
b. Primary aromatic amine and nitrous acid.
N-H + H0-N=0 -
I*'
AMINE (NaN02+HGD
N2 + R-OH -f H20
ALCOHOL
\Co)- N = N+ + ci-1
DIAZONIUM SALT
N-NITROSAMINE
c. Secondary aliphatic amine and nitrous acid, where R and R' may be alkyl or aryl groups
or may represent carbon atoms in a ring structure.
R
R-N-R+ H0-N=0
AMINE (NaN02 + HCI)
d. Tertiary aliphatic amine and nitrous acid.
H0-N = 0
AMINE (NaN02 + HCI)
e. Tertiary aromatic amine and nitrous acid.
N02'l
SALT
JL- NITROSO COMPOUND
Figure 4-2. Reactions of various amines with nitrous acid.
63
-------
'\
/
N-N-0
CH3"
CH3'
N-N-0
OIMETHYLNITROSAMINE
CH3 CH2 - COOH
^N/
N = 0
NITROSOSARCOSIME
METHYLPHENYLNITROSAMINE
NITROSOPIPERIDINE
NITROSOPYRROLIDINE
DIPHENYLNITROSAMINE
0
N = 0
NITROSOPROLINE
•COOH
N = 0
NITROSOMORPHOLINE
Figure 4-3. Structures of various N-nitrosamines (R-j is an alkyl or aryl radical and R~2 may
be one of many other functional groups).
64
-------
CH3 : CH3
X'N-N = 0
H2N —C^ CH3CH20-C
t , 3 H
ir Q
N4IITROSO-N-METHYL UREA i N-NITROSO-N-METHYLURETHANE
H2N-C
n
NH
H2N-C
ti
NH
N-NITROSO-N-METHYLGUANIDINE
Figure 4-4. Structures of various N-nitrosamides (R-j is an alkyl or aryl radical and R2
may be one of many other functional groups).
65
-------
0
H _^—ip ~~
/C\ H
H2N NH2 H2N - C - OR
UREA (CARBAMIDE) ALKYLCARBAMATES (URETHANES)
Q
5 H
RN-C-NH2
H H2N
N-ALKYUREAS 6UAKIOIME (CARSAMIOWf)
Figure 4-5. General structures of some amides that can undergo nitrosation.
* 66 ,
V
-------
temperature, the N-nitrosam1nes are, depending on their structure,
either liquids (e.g., dimethyl nltrosanvine) or solids (e.g., dlphenyl-
nitrosamine). Except for the dimethyl and dlethyl derivatives, they
are quite insoluble in water but soluble in many organic solvents. The
boiling points of the lower n-alkylnitrosam1nes range from 150° to
to 220°C.
4.2 FORMATION OF N-NITROSO COMPOUNDS
N-nitroso compounds can be formed when a secondary amine and nitrite
react under favorable conditions.
4.2.1 Kinetics of Nitrosation
For nitrosation to occur, nitrite is usually first converted to nitrous
acid (pKa = 3.37), which explains why nitrosation 1s catalyzed by acid.
The HN02 is then converted to an active nltrosating species, e.g.,
nitrous anhydride (NzOs), nitrosyl thiocyanate (ON-NCS), nitrosyl
halide (NOX), or nitrous addium ion (H2N02*).
Most secondary amines are nitrosated according to the following
equations:
2HN02 „ N203 + H20 : (4-1)
Rl\ V
NH + N20a - - N - N = 0 + HN02 (4-2)
The nitrosating agent is nitrous anhydride ^03) produced from two
molecules of nitrous acid. The rate of nitrosation is proportional to
67 u.S EPA Headquarters Library
Mai! code 3404T
1200 Pennsylvania Avenue NW
Washington, DC 20460
202*566-0556
-------
the concentrations of the amlne and the ^03 and hence to [HNC^]^. The
rate equation can therefore be expressed as:
Rate = KI x [RgNH] x [HN02]2 (4-3)
or
Rate = K2 [total amine] x [total nitrite]2 (4-4)
where [R2NH] and [HN02] are the concentrations of the nonlonized amine
and the free nitrous acid, respectively, and hence have values dependent
on the total amine and total nitrite concentrations as well as on pH.
In equation (4-3), although the nonionized amine and free nitrous acid
both vary with pH, the KI is independent of pH. Equation (4-4) is
easier to apply, since the total concentrations of amine and nitrite
are used, irrespective of the actual species present; but here the
7 8
stoichiometric rate constant <2 varies with pH. ' . The reaction rate
q
and l<2 show maximum values at pH 3.4, the pKa for nitrous acid. The
rate constants K-| and K2 for 14 secondary amines and 1 tertiary amine
are listed in Table 4-1. The ease of nitrosation, as given by l<2,
/
increases as the basicity of the amine decreases, i.e., as pKa de-
creases. Thus, the weaker bases will be nitrosated more rapidly
because of the higher relative concentration of free amine at all
values of pH below their pKa.
Brackets indicate concentrations of reactants.
68
-------
Table 4-1. RATE CONSTANTS K] AND, K2 (EQUATIONS 4-3 AND
4-4) FOR THE NITROSATION OF AMINES AT OPTIMUM pH AND 25°C
Amine
Secondary amines
Pi peri dine3
Dime thy 1 ami nea
Pyrrol idinea
N-Methyl ethanol ami ne
N-Methyl benzyl ami ne
Prolineb
Sarcosine
Prolylglycine
Hydroxyproline3'
Prolyll eucyl glyci neami de
Morpholine
Mononi trosopi perazi ne
Piperazine
N-Methyl aniline0
Tertiary amine
Aminopyrine0
n* 23
PKa
11.2
10.72
11.27
9.5
9.54
-
-
8.97
-
8.97
8.7
6.8
f 5.57
>9.8
4.85
5.04
KF,
M"2sec"1
0.00045
0.0017
0.0053
0.010
0.013
0.037
0.23
0.25
0.31
0.38
0.42
6.7
83
250
80
KI x 10"5?
M'^ec"1
1.4
1.5
21.0
0.62
0.92
1.4
2.6
5.0
2.1
6.2
2.3
0.83
0.62
18.0
1.0
Ref.
10
7
6
6
6
11
11
11
11
11
14,10
10
10
13
12
a Naturally occurring.
No pKa given because of complex ionization.
c Examined at 0°C.
69
-------
The only tertiary amine whose kinetics have been examined is the
12
analgesic, aminopyrine, which is rapidly nitrosated to give dimethyl-
3 15
nitrosamine ' and the ring-opened compound, 1-diketobutyryl-l-phenyl-
12
2 -methyl -2-nitrosohydrazide, as shown in Figure 4-6. The optimum pH
for dimethyl nitrosamine formation was 2.0; the kinetics can be expressed
as:
Rate = K3 [HNOg]2 (4-5)
The ease of nitrosation of aminopyrine is attributed to its enamine
structure and low pKa.
Nitrosation of ureas, guani dines, and carbamates may be important be-
cause (1) at least 14 ureas and 8 guani dines are found in nature, where
the ureas probably arise by the bacterial deimidation of guani dines or
carbamylation of amines; and (2) many ureas and carbamates are used
as drugs and, often because of their anticholinesterase activity, as
insecticides. N-alkyl ureas and carbamates are rapidly nitrosated at
•i •> acT
pH 1 to 2. * For nitrosation of these and other amides (equations
4-6 and 4-7), the reaction rate increases about 10 times for each 1-
unit drop in pH from 3 to 1 , does not show a pH maximum, and follows
the rate equation expressed in (4-8) and (4-9). '
HN02 + H+ * H2N02+ (4-6)
RNH-COR' + H2N02 — RN(NO)-COR' + H20 (4-7)
Rate = K6 [RNH-COR'] [HN02] [H+] (4-8)
70
-------
CH3 CH3
CH3
AMINOPYRINE
H20
0 0 0 Ph CH3
_ J JUL_L.± __
CHa-C-C-C-N-NI-NO
-2H
OH, OH 0 Ph CH3
_!._ -1 - IJL_1'J J_
CH3-C = C-C-N-N-NO
Figure 4-6. Nitrosation of aminopyrme tp give dimethylnitrosamine.and 1-diketobutyryl
-1 phenyl-2-methyl-2-nitrosohydrazide7'2
71
-------
Rate = K7 [amide] [nitrite] [H+] (4-9)
The main nitrosating agent is probably nitrous acidium ion (H2N02"*".
protonated nitrous acid). The nitrosation rate is proportional to the
[amide] and [HN02+] (equation 4-7), which in turn is proportional to
[H+] and [HN02] (equation 4-6). Thus the rate equations (4-8) and (4-9)
apply. In equation (4-8), Kg depends on nitrite ionization and hence on
pH, but does not depend on ionization of the amides since these are
usually nonionized above pH 2; e.g., methyl urea has a pKa of 0.7. The
reaction of benzamide and acetamide with HN02 probably also proceeds
via H2N02+, which may be favored for amide ni trosation because of
1 Q
electrostatic interaction with the amidic oxygen.
Nitrosation of the 21 amides in Table 4-2, including 14 ureas, 2 carba-
mates, and 2 guanidines, followed equation (4-8). The Kg values varied
300,000 times between different amides. They were lowest for simple
n
alkyl- and arylamides and for guanidines; next lowest for the acylureas,
dihydrouracil, and hydantoin; and highest for 2-imidazalidone (ethylene
urea). Unlike for the amines, there is no simple rule relating ease of
nitrosation to other properties of the amide.
In summary, the kinetic results show that N-alkylureas, N-arylureas,
N-alkylcarbamates, secondary aromatic amines, secondary amine deriva-
tives of piperazine and morpholine, and tertiary enamines are more
readily nitrosated than simple aliphatic secondary and tertiary amines,
N-acylureas, and N-alkylguanidines. Thus, the former classes of com-
pounds are of more interest than the latter classes as potential
72
-------
precursors of human carcinogens. However, potential carcinogenicity
depends on the relative amount of compound to which man is exposed.
Table 4-2. RATE CONSTANT K6 (EQUATION 4-8) FOR THE;
NITROSATION OF AMIDES AT 25°C AND pH 2
Ami de
N-Methyl benzami de
N-Methylacetamide
Dihydrothyminek
Methylguanidineb
1 -Methyl -3-ni troguani di ne
Dihydrouracilb
Hydantoi n
Ethyl N=ethylcarbamate
Hydantoi c acidb
Ethyl N-methylcarbamate
2-Ureidoethanol
DL-Citrullineb
Phenylurea
Ethyl urea
Tri methyl urea
Ethyl cyanamide
Methyl urea
Ethoxyphenyl urea
2- ( 1 H ) -Tetrahydropyrimi di none
(propylene urea)
1 ,3-Di methyl urea
2-Imidazolidone (ethylene urea)
K6.
M"2sec"1
0.0014
0.0025*
0.0035
0.004
0.008
0.010
0.042
0.10
0.18
0.37
0.38
0.72
2.2
3.0
7.0
8.0
10.5
13
18
200
400
References
6
6
6
17
6
6
10
6
17
6
6/20
17,
6
6
T7
6
6
6
6
a Measured at pH 1.
Naturally occurring.
73
-------
4.2.2 Factors That Influence Nitrosatlon
The acidity prevailing in the mammalian stomach favors the formation of
2fl"22~
nitrosamines from amines and nitrites. Sander and his colleagues '
showed the formation of the corresponding N-n1troso compound by incu-
bating several secondary amines (e.g., diphenylamine, N-methylaniline,
and N-methylbenzyl amine) with nitrite in the presence of gastric juice
"oT
under various conditions. Sen et al. demonstrated the in vitro
formation of diethylnitrosamine and sodium nitrite in the gastric juice
OVl"'
from rats, rabbits, cats, dogs, and man. Eisenbrand et al. showed
the formation of dimethlynitrosamine in the stomachs of rats given
aqueous solutions by stomach tube of sodium nitrite and one or the other
of the fungicides, ziram ([(CH3)2N-CS-S]2Zn) and ferbam ([(CH3)2N-CS-
25
S]sFe). Sander and Seif showed the formation of nitrosodiphenylamine
(1 pg to 5 ug) in the stomachs of people given nitrite and diphenylamine.
In the simplest case of nitrosamine formation, the rate of reaction is
proportional to the square of the nitrite concentration and directly
proportional to the concentration of the unprotonated amine. This
simple situation is greatly complicated by the additional chemical and
physical components and factors in the actual reaction environment,
some of which can be listed as follows:
• Not all nitrosatable substances follow the same kinetic pat-
tern, even in simple reaction systems. For example, amides
and ureas follow a completely different rate law from other
nitrosatable compounds, and the optimum pH for nltrosatlon of
74
-------
ami no adds and peptldes 1s different from that for
amines.
• Many reactions of Interest are multlstep processes; e.g.,
26*
the formation of N-nitrososarcosine from creatine or
27 28'
N-nitrosodimethyl amine from trimethylamine. ' In each
of these cases, nitrite is a reactant in more than one step,
which leads to a complicated kinetic pattern. In other
cases, e.g., the formation of N-nitrosopyrrolidine from
spermidine, even the reaction mechanism is not yet under-
sOQr'
stood."
t A number of chemical substances can accelerate the rate of
nitrosamine formation.
Thiocyanate increases the rate of nitrosation of morpholine,
N-methylaniline, aminopyrine, and sarcosine ' because the
/ >
nitrosating species ON«NCS is produced. The kinetics follow
equations (4-10) and (4-11). The optimum pH for nitrosation
at high [NCS~] is 2.0, when nitrite becomes fully protonated.
Below pH 2, the rate falls because NCS~ becomes protonated.
Bromide and chloride have effects similar to those of NCS~,
due to the formation of NOBr and NOC1; but the rate does not
fall below pH 2. The order of activity is NCS" » Br" > Cl".8
For sarcosine nitrosation under specific conditions, addition
of NCS" increased the reaction rate by a factor of 400 at pH
0.5, 100 at pH 1.5, and 10 at pH 2.5.11 The reaction of
75
-------
nitrous add with thiocyanate proceeds as shown:
HNO£ + H+ + NCS" 5=^ ON-NCS + H20
ON-NCS + R2NH i«--J. * R2N-NO + H+ + NCS"
Rate = «4 [R2NH] [HN02] [H+] [NCS~] (4-10)
or, since [R2NH] [H+] is independent of pH,
Rate = K5 [amine] [HN02] [NCS"] (4-11)
Thus thiocyanate- and halide-catalyzed nitrosation of amines
(4-10) and (4-11) will compete favorably with the N203 mecha-
nism (4-3 and 4-4) under three conditions: (1) at pH < 2.5,
where reaction by (4-4) but not by (4-11) begins to slow down;
(2) at high [NCS"] or [halide]; and (3) at low [nitrite], where
reaction by (4-4) drops sharply because it is proportional to
[nitrite]2, whereas reaction by (4-11) is proportional only to
[nitrite].14 Since attack by N20a (ON-ONO), ON-NCS, and NOX
are similar reactions, the order of amine reactivity according
to (4-4) (Table 4-1) probably also applies to nitrosations
catalyzed by NCS" and halides.
The thiocyanate ion could affect intragastric nitrosation since
it occurs in saliva, especially that of smokers (which has
about 6 mM NCS"), and in gastric juice (0.2 to 0.7 mM NCS").31
It should therefore not be surprising if the kinetic pattern of
nitrosation in stomach contents 1s complex indeed. *3* In
76
-------
another mechanism of acceleration recently discovered, formalde-
hyde can alter the mechanism of nitrosation to enable nitrosa-
33*'
tion in neutral or basic media, conditions that should
ordinarily lead to an infinitesimally small amount of nitros-
amine. The mechanism of the formaldehyde-catalyzed nitrosation
of various secondary amines at pH 6 to 11 can be written as
shown ;v-in Figure 4-7.
R\
FT
(IMINIUMION)
N-NO + CH,= 0-» N-CH,
/ \
R I
ON-0
Figure 4-7. Formaldehyde-catalyzed nitrosation I of secondary amines at pH 6 to 11.
77
-------
Catalytic effects on nltrosation by sulfate and phosphate
have also been shown.
t Other chemical substances retard the rate of nitrosation, e.g.,
34 35-39~ -TRy
tannins, sulfhydryl compounds, and ascorbic acid. --
Nitrite can react with various secondary amines in the acidic
f>pcoc"
environment of the stomach to form m'trosamines.' '
Ascorbic acid reacts rapidly with nitrite under the same acidic
40'
conditions to form nitrosoascorbate as an intermediate.
Ivankovic et al. have shown that ascorbic acid inhibits the
42""
teratogenic and carcinogenic effects of ethylnitroso urea
formed in the gastrointestinal tract of rats. These results
35-38
confirm other studies " which have shown that ascorbic acid
given concurrently with precursor amines and nitrite prevents
the acute toxic effects consequent to the formation of N-
nitroso compounds in animals. Recent studies by Sen and
35
Donaldson have indicated that glutathione is more efficient
than ascorbic acid in inhibiting the formation of nitrosopiper-
azines from piperazine adipate and nitrite.
4.3 DEGRADATION OF N-NITROSO COMPOUNDS
4.3.1 Decomposition
Nitrosamines are chemically stable under physiological conditions and
in strong alkaline medium; but under acid conditions, they undergo
3^3 "
photodecomposition to yield nitrous acid and the parent amine. The
hydrolytic reaction for dialkylnitrosamine can be expressed as follows:
'78
-------
RN\H+ RN
N -NO ,, £ NH2+ + HNO? (4-12)
/ V /
RZ' \OH2 R2
It is assumed that water and proton act 1n a concerted manner to cleave
the N-N bond in the N-nitrosamine. Nitrous acid in an aqueous solution
is fairly stable and is a good nitrosating agent in the acidic pH
range. Thus, recombination of the parent amine with nitrous acid to
regenerate the dialkylnitrosamine also occurs at a considerable rate.
Under basic conditions, photolysis yields substantial amounts of nitrite
ion (N02~). When exposed to ultraviolet light, N-nitrosamines decompo~se7
resulting in the quantitative formation of nitrite. This decompo-
sition reaction has been used in one of the methods for estimating
44,
N-n1trosamines. -' While the N-nitrosamines are stable at alkaline pH,
the N-nitrosamides are unstable under such conditions and may decom-
pose even at neutrality to form powerful alkylating agents, the diazo-
alkanes. The half-life of the N-nitrosamide, N-nitrosomethyl urea,
for example, is 125 hours at pH 4.0, 24 hours at pH 6.5, 1.2 hours at
^45
pH 7.0, and only 0.1 hour at pH 8.0. Preussmann and Schaper-
• • XV
Druckrey have shown that in acid nitrosamides decompose to give
quantitative yields of nitrite. These differences in chemical stability
may underlie the differences in biological behavior of the nitrosamines
and the nitrosamides since there is evidence that a decomposition prod-
uct rather than the parent molecule may be responsible for the biologi-
cal action of these compounds. It has been suggested that the nitros-
amines require enzyme-catalyzed decomposition before they become
79
-------
biologically active, whereas the nltrosamldes can decompose 1n the
47"^
tissues without enzymatic activation.
4.3.2 Metabolism
N-nitroso compounds are relatively rapidly metabolized 1n whole ani-
14
mals. In the rat or mouse, for example, the administration of C-
labeled dimethylnitrosamine leads to the majority of the isotope
ap
15
appearing as C02 after 6 to 10 hours. In similar experiments with
N-labeled dimethyl amine, the isotope appeared in urea and proteins,
indicating that the nitroso group as well as the ami no group 1s trans-
formed metabolically into ammonia, which 1s in equilibrium with the
49T"~~
endogenous ammonia "pool" of the animal. • Thus the carcinogenic N-
nitroso compounds can be metabolized to a large extent by pathways which
are of importance to the normal animal.
50
Montesano and Magee have shown that dimethylnitrosamine and diethyl-
nitrosamine are metabolized to varying degrees by tissue slices of
several organs other than the liver. Of the tissue slices from the
rat, liver was the most active in metabolizing dimethylnitrosamine;
kidney showed appreciable activity; and lung and esophagus were defi-
nitely active, while the activity of small Intestine was extremely low.
A similar general pattern was seen with the diethyl compound, but the
rate of metabolism was lower in the liver slices and higher 1n the
lung than with the dimethyl compound.
With hamster tissues the pattern of dimethylnitrosamine metabolism was
similar to that found with rat tissues. Metabolism of the diethyl
80
-------
compound, however, was remarkably different 1n that hamster lung tissue
had a greater capacity than liver for metabolizing this carcinogen,
while the other organs were considerably less active.
Metabolic capacity may be correlated with carcinogenicity in kind and
degree. Diethylnitrosamine, readily metabolized by hamster lung tissue,
is a powerful carcinogen for the hamster respiratory tract, whereas
dimethylnitrosamine, less readily metabolized, shows no carcinogenicity
in hamster lung at all.
Biochemical studies with human tissues have indicated that the liver
metabolizes nitrosamines especially well. Evidence from such studies
points to the production of an active, ultimate carcinogen by means of
the metabolic degradation of the parent nitrosamine. This ultimate
carcinogen has been shown to interact with significant cellular macro-
molecules. If the carcinogenicity of nitrosamines is indeed related
to a capacity for metabolizing the parent compound, then 1t is reason-
able to believe that man would be sensitive to this class of carcino-
gens.
As the result of several years of research on the metabolism of nitros-
amines, a pathway was proposed for the metabolism of dialkyl-
nitrosamines; it is shown in Figure 4-8, using dimethyl nitrosamine as
an example.
Dialkylnitrosamines undergo an enzymatic hydroxylation at the o carbon
in one of the aliphatic chains. Further oxidation and hydrolysis
81
-------
CH3 CH2OH JiCHO
X. MARPII - '\ ^
^N - N = Q ' " % ;;« - N = 0 (FORMALDEHYDE)
/ "2 /
CH3^ CH3 /
DIALKYLNITROSAMINE H
(DIMETHYLNITROSAMINE) N _ N = 0
MONOALKYLNITROSAMINE
U -t- O U • -^ f*U II _-* O LI M *• M A U
«2 **"3 ^"™ «H2 II 1<- yrlj""!! N *~ Un
DIAZOHYDROXIDE
DIAZOMETHANE
Figure 4-8. Possible pathway for the metabolism of dialkylnitrosamines,
using dimethylnitrosamine as an example.52-54
82
-------
produce an aldehyde and the monoalkylnltrosanrine. It Is believed that
the monoalkylnitrosamlne rearranges to the corresponding dlazohydroxlde,
which becomes the diazoalkane. This diazoalkane then decomposes to
nitrogen and the alkyl carbonium 1on, the latter alkyl atlng nearby
nucleic acids and other macromolecules. No evidence of the production
of a diazoalkane in this way has been presented, although this possi-
5 2 -"SB"""
bility has been widely postulated. Recently Lijinsky and co-
workers have shown that in the case of alkylation of nucleic acids
by dimethyl nitrosamine alkylation is a transmethylation with no exchange
of hydrogen atoms involved in deriving the methonium ion. This, then,
argues against the formation of dlazomethane as an intermediate in
dimethyl nitrosamine metabolism. Ethylation of nucleic acids has been
57""
demonstrated after administration of di ethyl nitrosamine, which sup-
ports a-oxidation of one ethyl group and transethylation of the second.
Recently Kruger has provided evidence that $-ox1dation of di alkyl -
nitrosamines may also occur, in a manner that resembles e-oxidation
of fatty acids. The first step would require enzymatic dehydrogenation
between the a and 3 carbons of one of the alkyl chains. Adding water
to the double bond produces the e-hydroxylated nitrosamine, because the
nitroso group would have an inductive effect that would be analogous to
the effect the activated carbonyl group has in the s-oxidation of fatty
acids.
Oxidation of the hydroxylated nitrosamine would continue, yielding
acetyl-CoA and methyl alkyl nitrosamine; the nitrosamine might then
83
-------
undergo a-oxidation, producing either the methonlum ion or the carbonlum
Ion corresponding to the second alkyl group. Studies on the metabolism
of [ C]di-n-propyln1trosamine labeled 1n the 1 or 2 position and [1 -
C]di-n-butylnitrosamine support this pathway.
4.4 ATMOSPHERIC CHEMISTRY OF NITROSAMINES
For the assessment of possible N-nitrosam1ne pollution in ambient air,
the chemistry of major interest is that pertaining to the stability in
air of these compounds and to their presence in ambient and source at-
mospheres. Although the presence of N-nitrosamines in ambient air has
been reported by Bretschneider and Matz, Fine et al., • Pellizzari,
63
and by researchers at Dupont, the amounts of N-nitrosamines detected were
' T-
small. Information on the stability and formation of N-n1trosamines in
the gas phase, particularly in ambient atmospheres, is sparse.
The various members of the N-nitrosamine family range from high-vapor-
pressure liquids to rather non-volatile solids (at room temperature),
but they are capable of emitting vapor at concentrations sufficiently
high to exert adverse health effects.64'6^
There is no evidence of thermal instability of nitrosamines in the
gaseous phase. In the condensed phase, nitrosamines are stable 1n an
alkaline or mildly acidic environment, decomposing hydrolytically only
in prolonged contact with strong acid. Thus, although acidic aerosols
are known to exist in ambient air under normal atmospheric conditions,
they are not believed to cause significant thermal degradation of
nitrosamines.
84
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Under ultraviolet and visible light, N-n1trosam1nes undergo photo-
chemical decomposition with subsequent split-off of the nltrosyl group,
This photolytlc decomposition may be a factor limiting the lifetime of
'M9~'
nitrosamlnes 1n the atmosphere. The rate of such photochemical de-
nitrosation is known to vary from compound to compound, but quantitative
data are limited.^66
The chemical formation of nitrosamines has been the subject of numerous
studies that have recently been reviewed by Mirvish. Although most of
the reported studies have been concerned with condensed-phase reaction
systems, the formation of nitrosamine in the gas phase has been demon-
strated. ' Neurath et al. 'showed that the formation of nitrosamines
from secondary amines requires an equimolar mixture of nitrogen dioxide.
This reaction, which occurs 1n the gaseous phase, can be represented
as follows:
> •-. . '
2 R2NH + NO + N02 -2 R2NNO + H20 (4-13)
eg
Bretschneider and Matr showed that diethylamine and nitrogen dioxide
(N02) at concentrations of 50 to 100 parts per million (ppm) reacted
within seconds to form measurable levels of nitrosamine. In some cases,
as in tobacco smoke, nitrosamines can form so readilyjthat nitrosamine
top
formation can take place during the collection step.
It now appears that a much more intense look will have to be taken at
the concentration of nitrosamine precursors in polluted atmospheres and
in the background chemical environments^-of high-risk groups. Thus, from
85
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a genotoxic viewpoint, knowledge of the environmental concentrations of
nitric oxide, nitrogen dioxide, nitrous acid, nitrites, nitrates, and
primary, secondary, tertiary, and quarternary amines will be required.
In addition, knowledge of the nitrosamine cocarcinogens and the cata-
lysts favorable to nitrosamine formation will also be necessary.
xr-gj
Some work with gas-phase systems is currently being conducted by EPA.
In this study, gaseous dimethyl ami ne, ((^3)2 NH, has been shown to react
with gaseous nitrous acid, MONO, in air to yield N-nitrosodimethylamine,
(CH3)2 N-NO. This on-going research has shown that, in a humid atmos-
phere containing dimethylamine, NO, N02, and MONO at concentrations of
0.5 to 2 pptn, the amine reacted at a rate of about 4 percent per minute
yielding N-nitrosodimethylamine as the major reaction product. In the
absence of HONO and humidity, the rate was lower by a factor of four,
approximately. The N-m'trosodimethylamine decomposed in sunlight with
a half-life of about 30 minutes. Long-path infrared absorption spec-
troscopy was used to monitor these reactions at parts-per-million
levels.
Assuming a bimolecular second-order reaction, an upper-limit rate
constant of 0.1 ppm min was calculated for the reaction of di-
methylamine with HONO. Such a rate indicates that dimethyl amine re-
leased into a polluted urban atmosphere could be nitrosated during the
night. The extent of the nighttime nitrosation would depend upon the
NO, N02, and ^0 concentrations. For example, in an atmosphere con-
taining 0.18 ppm N02 and 20,000 ppm H20, the equilibrium concentration
86
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of HONO would be 0.01 ppm, and dimethy!amine would react at a rate of
about 6 percent per hour. The nltrosatlon rate and accumulated nltros-
amine concentration would diminish rapidly after sunrise as a result
of the decomposition of nitrosamine and HONO by sunlight.
The homogeneous gas-phase formation of nitrosamine has been questioned
-TO5*
by Glasson, however. Although his results do confirm that the di-
methylamine disappears at the rate observed by the EPA group, the forma-
tion rate of nitrosamine is less than 1 percent of the loss rate of di-
methylamine. This suggests that the formation of nitrosamine Is very
slow and that the reaction is not homogeneous. The observations made
by GTasson are compatible with a theoretical calculation made by EPA
of the rate constant for the formation of nitrosamine from the reaction
of dimethyl amine with HONO.
fi'f'
Recent work by Fine has indicated that under certain conditions di-
methylamine can be converted to dimethylnitrosamine. Fine found high
conversions of dimethyl amine to dimethylnitrosamine when sampling with a
cold-trap collection system under conditions of high ozone levels in
the presence of NOX and dimethyl amine. At the time of this report, it
has not been established whether Fine's observation is due to an artifact
formed within the cold-trap collection system or is the result of a
gas-phase reaction. Based, however, on theoretical calculations made
by EPA for the reaction of dimethyl amine 1n the presence of 03 and NOX,
it appears that this reaction cannot occur homogeneously.
87
-------
The bulk of available evidence indicates that the ease of nitrosation
of the amine can be influenced by amine basicity^2 substrate concen-
7 '71 8 72 • 8
tration, thiocyanate ion, chloride ion, acetate ion, formalde-
hyde, readily oxidized phenolic compounds, and pH. Assuming
optimum pH conditions do occur on the surface of ambient aerosol par-
ticles, the rate of nitrosation of dimethylamine in ambient air can be
calculated from the existing kinetic data to be:
Ratedimethyl amine = I"™** percent/hour (4-14)
where [nitrite] is the concentration (molarity) of total nitrite on the
aerosol particle surface. For more reactive amines, the rate can be
higher by more than four orders of magnitude. Because nitrate concen-
trations in ambient air are uncertain, accurate calculations of the
nitrosamine concentrations expected to form in air cannot be made.
However, the rate data presented here and the fact that ambient air
aerosol contains a significant concentration of nitrite suggest that
formation of nitrosamines in atmospheres containing acidic aerosol and
elevated secondary amine concentrations, e.g., around amine emission
sources, may be important.
To summarize the information available on atmospheric chemistry of
nitrosamines, it appears that atmospheric reactions constitute a prob-
able source of ambient nitrosamines, but they also include nitrosamine
destruction steps. The formation process may Include heterogeneous
as well as homogeneous gas-phase reaction steps; the relative importance
88
-------
of the respective steps 1s unknown. Precursors in the heterogeneous
process are the amlne and, In.all probability, nitrite, aerosol, and
catalytic agents (e.g., halldes). Also, precursors 1n the homogeneous
gas-phase process are the amlne and the nitrogen oxides or one or more
nitrogen oxide derivatives (e.g., ^03, NHOg). The main atmospheric
destruction process is probably sunlight-Induced photolysis. Overall,
the information available has only qualitative validity. The magnitudes
of the formation and destruction processes and the dependence of these
processes on the various factors are either totally unknown or are known
with very little confidence.
In the face of these uncertainties, the existing information on the
atmospheric chemistry of nltrosamines can have only vague implications
regarding the control of atmospheric nltrosamines. Thus, to decide
whether the nitrosamine problem is an ambient-atmosphere problem or an
industrial-atmosphere problem requires that the n1trosamine-to-precursor
dependencies be better known. If the problem is one of nitrosamine •
pollution in the ambient atmosphere, then the question of control be-
comes extremely complex, requiring consideration of a multitude of
factors, some of which may be of non-atmospheric nature. In addition,
deliberations regarding relative control requirements for amlne and NOX
emissions should include consideration of occurrence and relative
levels of these two precursors 1n the human body. For example, 1f
relatively high levels of amines occur in the human body, then NOX
emission control may be relatively more effective in suppressing nitros-
amine formation both in the ambient air and in the human body.
89
-------
The atmospheric chemistry studies to date are preliminary, were con-
ducted with high concentrations of the reactants, and have not assessed
whether other pollutants such as fine particulates and sulfur oxides
have any significant influence on the formation and degradation of
nitrosamines. Much additional research is required to evaluate the
significance of secondary formation mechanisms and to determine which
precursors, if any, should be controlled to reduce nltrosamine levels
most effectively.
4.5 WATER AND SOIL CHEMISTRY OF NITROSAMINES
(
Recently environmental chemists have directed their attention to the
possible formation of nitrosamines in water supplies and soils. One
proposal is that these compounds in water and soil environments may
result from the reaction of nitrite with amines, such as those derived
74
from pesticides. Tate and Alexander have reported the formation of
dimethy!amine and diethylamine in soil treated with dimethyldithio-
carbamate (dibam) and diethyldithiocarbamate. The current study of
Wolfe may provide some insight into the possible formation of nitros-
amines in drinking water.
4.6 FOOD CHEMISTRY OF NITROSAMINES
Amine precursors such as proteins, phospholiplds, and amlno adds are
present in foodstuffs and may be available for reaction with nitrites
to form nitrosamines. Nitrites are used to cure meats and fish. They
are also commonly found in vegetables such as spinach, lettuce, celery,
and beets, as well as in many water supplies. Secondary amines have
-------
been found in fish, vegetables, and fruit jutces, although the concen-
75 76 28
tratlons may vary wtdely. • Fiddler et al. found that quar-
ternary anmonlum compounds, such as chollne, acetylchollne, carnltlne,
betaine; and some tertiary amines formed trace amounts of nitrosamines.
?fi
Archer et al. found that carcinogenic nitrososarcosine can be formed
from nitrite and creatine, which are present in vegetables, milk, and
meat. Keybets et al. found significant concentrations of nitrite in
spinach after storage, but found no evidence of nitrosamines under normal
conditions. When diethyl amine was added and the pH lowered to about 3,
a trace of diethylnitrosamine was found. Nitrosation generally occurred
below pH 4.5; however, with high nitrite concentrations and a long re-
action time, nitrosamines were formed even at pH 7.6. Using a tritium-
labeled amine in buffered aqueous solutions, Mirvish found that the
formation of dimethylnitrosamine was directly proportional to the di-
methyl amine concentration and to the square of the nitrite concentration.
The maximum yield of nitrosamines occurred at pH 3.4. Based upon
hypothetical calculations, Mirvish concluded that the concentration of
dimethylnitrosamine formed would be too low to be carcinogenic.
Nitrosation depends upon the basicity of the amine, and may increase a
thousandfold as the basicity decreases from dimethylamine to aromatic
amines. Mirvish also found that ascorbate effectively inhibited the
-* 78
nitrosation of amines by nitrite. Ender and Ceh found that the amount
of nitrosamines formed increased with temperature and time of reaction,
but that significant nitrosation could occur even at -18°C.
91
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4.7 REFERENCES FOR SECTION 4
1. Magee, P.N. and J.M. Barnes. The production of malignant primary
hepatic tumors 1n the rat by feeding dimethylnltrosamlne. Br. J.
Cancer. J_0:114-122, 1956.
2. Fieser, L.F. and M. Fleser. Organic Chemistry, 3rd Ed. New York,
Reinhold. 1956.
3. Lijinsky, W., E. Conrad, and R. Van de Bogart. Carcinogenic _
nitrosamines formed by drug/nitrite Interactions. Nature (London).
239:165-167, 1972.
4. Feuer, H.(ed.). The Chemistry of the N1tro and Nltroso Groups.
New York, Intersdence Publishers, Inc. 1969.
5. Morrison, R.T. and R.N. Boyd. Organic Chemistry, 3rd Ed. Boston,
Allan and Bacon. 1974.
6. Mirvish, S.S. Formation of N-nitroso compounds—chemistry, kinet-
ics and in vivo occurrence. Toxlcol. and Appl. Pharmacol. 31;
325, 197"57
7. Mirvish, S.S. Kinetics of dimethyl amine nltrosation in relation
to nitrosamine carcinogenesls. J. Nat. Cancer Inst. 44:633, 1970.
8. Ridd, J.H. Nitro^ation. diazotization and__deami nation. Quart.
Rev. Chem. Soc. (Tondon). 15:418-441, 1961.
9. Perrin, D.D. Dissociation Constants of Organic Bases 1n Aqueous
Solution. London, Butterworth. 1965.
10. Mirvish, S.S. Kinetics of N-n1trosat1on reactions in relation to
tumorigenesis experiments with nitrite plus amines or ureas. In:
N-Nitrosp Compounds in the Environment (P. Bogovski, E.A. Walker,
and W. (tarts, edsTJ." Lyon, France. " IARC Scientific Public. No. 3.
1972.
11. Mirvish, S.S., J. Sams, T.Y. Fan, and S.R. Tannenbaum. Kinetics
of nitrosation of the ami no adds proline, hydroxyprol 1 ne and
sarcosine. J. Nat. Cancer Inst. 5J_: 1833-1840, 1973.
12. Mirvish, S.S., B. Gold, M. Eagen, and D. Arnold. Kinetics of the
nitrosation of aminopyrlne to give dimethylnltrosamlne. Z. Krebs-
forsch. 82_: 259-268, 1974.
13. Kalatzis, E. and J.H. Ridd. Nitrosation, diazotlsatlon and deaml-
nation. Part XII. The kinetics of N-n1trosat1on of N-methylan1-
line. J. Chem. Soc. (London). 19668:529-533. 1966.
92
-------
14. Fan, T.Y. and S.R. Tannenbaum. Factors Influencing the rate of
formation of nitrosomorphoHne from morphollne and nitrite: Ac-
celeration by thiocyanate and other antons. J. Agrlc. Food Chem.
21:237, 1973.
15. Lijinsky, W., L. Keefer, E. Conrad, and R. Van de Bogart. Nitro-
sation of tertiary amines and some biologic implications. J. Nat.
Cancer Inst. 49_: 1239-1249, 1972.
16. Mirvish, S.S. Studies on N-nitrosation reactions: Kinetics of
nitrosation, correlation with mouse feeding experiments, and
natural occurrence of nitrosatable compounds (ureides and guani-
dines). In: Topics in Chemical Cardnogenesis (W. Nakahara, S.
Takayama,~T. Suglmura, and S. Odashima, eds.). Tokyo, University
of Tokyo Press. 1972. p. 279-295.
17. Mirvish, S.S. Kinetics of nitrosamide formation from alkylureas,
N-alkylurethans, and alkylguanidines: Possible Implications for
the etiology of human gastric cancer. J. Nat. Cancer Inst.
46:1183-1193, 1971.
18. Sander, J. and F. Schweinsberg. Wechselbeziehungen zwischen Nitrat,
Nitrit und kanzerogenen N-N1trosoverb1ndungen. Zbl. Bakt. Hyg. B.
156_: 299-340, 1972.
19. Challis, B.C. and J.A. ChalHs. Reactions of the carboxamide
group. In: The Chemistry of Amides (J. Zablcky, ed.). London,
Interscience. 1970. p. 733-848.
20. Preussmann, R., H. Druckrey, and J. Bucheler. Carcinogene Wirkung
von Phenylnitroso-harnstoff. Z. Krebsforsch. 71_:63-65, 1968.
21. Sander, J. Kann Nitrit in der menschlichen Nahrung Ursache einer
Krebsentstehung durch Nltrosaminbildung sein? Arch. Hyg. Bakt.
1_51_: 22-28, 1967.
22. Sander J., F. Schweinsberg, and H.P. Menz. Untersuchungen iiber die
Entstehung cancerogener Nitrosamlne 1n Magen. Z. Physio!. Chem.
349:1691, 1968.
23. Sen, N.P., D.C. Smith, and L. Schwinghamer. Formation of N-nitros-
amines from secondary amines and nitrite in human and animal
gastric juice. Fd. Cosmet. Toxlcol. 7_:301-307, 1969.
24. Eisenbrand, G., 0. Ungerer, and R. Preussmann. Rapid formation of
carcinogenic N-nitrosamines by interaction of nitrite with fungi-
cides derived from dithiocarbamlc acid in vitro under simulated
gastric conditions and in vivo In the raT stomach. Fd. Cosmet.
Toxicol. 12:229-232,
-93
-------
25. Sander, J. and F. Self. Baktertelle reduction von Nltrat 1m Magen
des Menschen als Ursache etner N1trosam1nb1ldung. Arzne1m1ttel-
Forsch. 12:1091, 1969.
26. Archer, M.C. , S.C. Clark, J.E. Thllly, and S.R. Tannenbaum. En-
vironmental nitroso compounds. Reaction of nitrite with creatlne
and creatinlne. Science. 174:1341-1342, 1971.
27. Scanlan, R.A., S.M. Lohsen, D.D. Bills, and L.M. Ubbey. Formation
of dimethylnitrosamine from dimethylamlne and tr1 methyl ami ne at
elevated temperatures. 0. Agrlc. Food Chem. 22_: 149-1 50, 1974.
28. Fiddler, W., J.W. Pensabene, R.C. Doerr, and A.E. Wasserman.
Formation of _N-ni trosodimetnyl ami ne from natural ly occurri ng
_ ___
ammonium compounds arid tertiary amines. Nature (London)
236_:307, 1972.
29. Bills, D.D., K.I. Hildrum, R.A. Scanlan, and L.M. Libbey. Potential
precursors of N-nttrosopyrrolidine in bacon and other fried foods.
J. Agric. Food Chem. 21:876-877, 1973.
30. Boyland, E. and S.A. Walker. Catalysis of the reaction of amino-
pyrine and nitrite by thiocyanate. Arzne1m1ttel-Forsch. 24:1181-
1184, 1974.
31. Lane, R.P. and M.E. Bailey. Effect of pH on dimethyl nitrosamine
formation in human gastric juice. Fd. Cosmet. Toxicol. 11:851-
854, 1973.
32. Newberne, P.M. and R.C. Shank. Induction of liver and lung tumors
in rats by the simultaneous administration of sodium nitrite and
morpholine. Fd. Cosmet. Toxicol. 1_1_: 81 9-825, 1973.
33. Keefer, L.K. and P.P. Roller. N-nitrosation by nitrite ion in
neutral and basic medium. Science. 1_8]_: 1245- 1246, 1973.
34. Bogovski, P., M. Castergnaro, B. Pignatelli, and E.A. Walker. The
inhibiting effect of tannins on the formation of nltrosamlnes.
In: N-Nitroso Compounds: Analysis and Formation (P. Bogovski,
R. Preussmann. and E.A. Walker. eds^L^J-ygn , France. IARC Sci-
entific Public. No. 3. 1972. p. 127-129.
35. Sen, N. P. and B. Donaldson. The effect of ascorbic acid and
glutathione on the formation of nitrosopiperazines from piper-
azine adipate and nitrite. In: N-N1troso Compounds: Analysis
and Formation (P. Bogovski, R. Preussmann, and E.A. Walker, eds.).
Lyon, France. IARC Scientific Public. No. 9. 1973. p. 103.
94
-------
36. M1rv1sh, S.S., L. Wallcave, M. Eagen, and P. Shublk. Ascorbate-
nltrlte reaction: Possible means of blocking the formation of
carcinogenic N-nltrosp-compounds. Science. 177_:65, 1972.
37. Fiddler, W., O.W. Pensabene, I. Kushnlr, and E.G. Piotrowskl.
Effect of frankfurter cure Ingredients on N-n1trosodimethylam1ne
formation in a model system. J. Food Sc1. 38;714-715, 1973.
38. Fiddler, W., J.W. Pensabene, E.G. Piotrowski, R.C. Doerr, and A.E.
Wasserman. Use of sodium ascorbate or erythorbate to inhibit
formation of N-nitrosodimethyl amine 1n frankfurters. J. Food Sci.
38:1084-1085, 1973.
39. Epstein, S.S. In vivo studies on interactions between secondary
amines and nitrTtes or nitrates. In; N-Nitroso Compounds:
Analysis and Formation (P. BogovskTT R. Preussmann, and E.A.
Walker, eds.). Lyon, France. IARC Scientific Public. No. 3.
1971. p. 109-115.
40. Dahn, A., L. Loewe, and C.A. Bunton. Uber die oxidation von
Ascorbinsaure durch salpetrige Saure. VI. Ubersicht und Diskussion
der Ergebnisse. Helv. Chem. Acta. 43_:320, 1960.
41. Ivankovic, S., R. Preussmann, D. Schmahl, and J. Zeller. Prevention
by ascorbic acid of in vivo formation of N-nitroso compounds. In:
N-Nitroso Compounds Tn" the Environment (P. Bogovski, E.A. Walker,
and W. Davis, eds.). Lyon, France. IARC Scientific Public. No. 9.
1974. p. 101.
42. Ivankovic, S. and H. Druckrey. Transplacentare Erzeugung maligner
Tumoren bei den Nachkommen nach einmaliger injektion von Athyl-
nitrosoharnstoff an schwangere Ratten. Z. Krebsforsch. 71:320,
1968.
43. Chow, Y.L., M.P. Lau, R.A. Perry, and J.N.S. Tarn. Photochemistry
of nitroso compounds in solution. XX. Photoreduction, photoelimi-
nation, and photoaddition of nitrosamlnes. Can. J. Chem. 50:1044,
1972. ~~
44. Preussmann, R., D. Dalber, and H. Hengy. A sensitive color reaction
for nitrosamines on thin-layer chromatograms. Nature. 201:502,
1964.
45. Druckrey, H., R. Preussmann, S. Ivankovic, and D. Schmahl. Organo-
trope carciriogene Wirkungen bei 65 verschiedenen N-n1troso-Verbin-
dungen an BD-Ratten. Z. Krebsforsch. 69_: 103, 1967.
46. Preussmann, R. and F. Schaper-Druckrey. Investigation of a colori-
metric procedure for determination of nitrosamides and comparison
with other methods. In; N-Nitroso Compounds: Analysis and
95
-------
Formation {P. Bogoyskl L R_. Preussmannj and E^.A. Walter ,_eds.).
Uyon, France. lARC Scientific Public. No. 3. 1972. "p. "B
47. Magee, P.N. Possibilities of hazard from nltrosamlnes 1n Industry.
Ann. Occup. Hyg. 1_5:19, 1972.
48. Dutton, A.H. and D.F. Heath. Demethylation of dime thy Initrosamine
in rats and mice. Nature. 1_78_: 644, 1956.
49. Heath, D.F. and A.H. Dutton. The detection of metabolic products
from dimethyl nitrosamlne in rats and mice. Biochem. J. 70:619,
1958.
50. Montesano, R.M. and P.N. Magee. Metabolism of nitrosamines by rat
and hamster tissue slices in vitro. Proc. Amer. Assoc. Cancer Res.
12:14, 1971.
51. Montesano, R. and P.N. Magee. Comparative metabolism in vitro of
nitrosamines in various animal species including man. "Tnl CFemi-
Carcinogenesis Essays (R. Montesano, L. Tomatls, and W. "Davis,
eds.). Lyon, France. IARC Scientific Public. 1974. p. 39-56.
52. Magee, P.N. and M. Vandekar. Toxic liver injury. The metabolism
of dime thy! nitrosamine in vitro. Biochem. J. 7p_:600-605, 1958.
53. Rose, F.C. In: Symposium on the Evaluation of Drug Toxicity
(A.L. Wai pole and A. Spinks, eds.). London, Churchill. 1958.
p. 116.
54. Magee, P.N. and T. Hultln. Toxic liver injury and carcinogenesis.
Methylation of rat-liver slices by dimethyl nitrosamine in vitro.
Biochem. J. 83:106-114, 1962. ~
55. Venulet, J. and R.L. Van Etten. Biochemistry and pharmacology of
the nitro and nitroso groups. In: The Chemistry of Nitro and
Nitroso Groups (H. Feuer, ed.). New York, Interscience. 1970.
56. Lijinsky, W., J. Loo, and A.E. Ross. Mechanism of alkvlation of
nucleic acids by nitrosodimethylamine. Nature (London). 218;
1174-1175, 1968.
57. Magee, P.N. and K.Y. Lee. Cellular injury and carcinogenesis.
Alkylation of ribonucleic acid of rat liver by dimethyl nitrosamine
and N-butyl -methyl -nitrosamine In vivo. Biochem. J. 91:35-42,
1964. ~~
58. Kruger, F.W. New aspects in metabolism of carcinogenic nitrosamines.
In: Proc. 2nd International Symposium of the Princess Takamatsu
Cancer Research Foundation, Topics in Chemical Carcinogenesis (W.
96
-------
Nakahara, S. Takayama, T. Suglmura, and S. Odashima, eds.). Balti-
more, University Park Press. 1972. p. 213-232.
59. Bretschnelder, K. and J. Matz. Nitrosamines (NA) in the atmospheric
air and in the air in the workplace. Archiv. fur Ceschwulstfor-
schung. 42(1):36-41, 1973.
60. Fine, D.H_., D.P. Rounbehjer, J.M. Bejcherj and S.S._Epstein. N_-_
nitroso compounds in the environment. Paper presented at the Inter-
national Conference on Environmental Sensing and Assessment. Las
Vegas, Nevada, September 1975.
61. Fine, D.H. N-nitrosamines^in urban community air. Progress Report.
EPA Contract No. 68-02-2363T January 5, 197571
62. Pellizzari, E.D. Identification and estimation of N-nitrpsodimethyl-
amine and other pollutants in ambient air in the Baltimore, Maryland,
and Kanawha Valley Areas. Progress Report. EPA Contract No. 68-02-
1228. January 5, 1975.
63. El DuPont De Nemours & Company, Inc. Dimethylnitrosamine (DMN)
sampling program. December 15, 1975.
64. Magee, P.N. and J.M. Barnes. Carcinogenic nitroso compounds. In:
Advances in Cancer Research, Vol. 10 (A. Hadow and S. Weinhous,
eds.). New York, Academic Press. 1967. p. 103.
65. Scanlan, R.A. N-nitrosamines in foods. CRC Critical Reviews in
Food Technology. 5_(4):357, 1975.
66. Preussmann, R. On the significance on N-nitroso compounds as
carcinogens and on problems related to their chemical analysis.
In: N-Nitroso Compounds: Analysis and Formation (P. Bogovski,
R. Preussmann, and E.A. Walker, eds.). Lyon, France. IARC
Scientific Public. No. 3. 1972. p. 6.
67. Neurath, G., B. Pirmann, H. Luttich, and H. Wichern. Zur Frage
der N-Nitrosoverbindungen im Tabakrauch. II. Beitr. Tabakforsch.
3:251, 1965.
68. Neurath, G. Zur Frage des Vorkommens von N-Nitrosoverbindungen
im Tabakrauch. Experentia (Basel). 23:400, 1967.
69. Wolfe, N.L. U.S. Environmental Protection Agency, Athens, Georgia.
Personal communication with N.S. Ulmer of U.S. Environmental Pro-
tection Agency, Cincinnati, Ohio, October 22, 1975.
70. GTasson, W.A., General Motors Corporation, Warren, Michigan. Public
comment made at the Amer. Chem. Soc. Meeting, New York, N.Y., April
1976.
97
-------
71. Boy!and, E., E. Nice, and K. Williams. The catalysis of nitrosation
by thiocyanate from saliva. Fd. Costnet. Toxicol. 9_:639, 1971.
72. Lijinsky, W. and M. Greenblatt. Carcinogen dimethylnitrosamine
produced in vivo from nitrite and aminopyrine. Nature New Biol.
236:177-l7S,~T9T2.
73. Challis, B.C. and C.D. Bartlett. Possible cocarcinogenic effects of
coffee constituents. Nature (London). 254_:532-533, 1975.
74. Tate, R.L. and M. Alexander. Formation of dimethyl amine and diethyl-
amine in soil treated with pesticides. Soil Science. 118:317-321,
1974.
75. Preusser, E. Aliphatic amines in the seeds of fruits and germinating
plants of Vicia faba and Zea mays. Biol. Zbl. 85_: 19-21, 1966.
76. Wick, E.L., E. Underriner, and E. Paneras. Volatile constituents of
fish protein concentrate. J. Food Sci. 32_:365-370, 1967.
77. Keybets, M.J., E.H. Broot, and G.H. Keller. An investigation into
the possible presence of nitrosamines in nitrate bearing spinach.
Fd. Cosmet. Toxicol. 8:167-171, 1970.
78. Ender, F. and L. Ceh. Conditions and chemical reaction mechanisms
by which nitrosamines may be formed in biological products with
reference to their possible occurrence in food products. Z.
Lebensm. Unters. Forsch. 1_45_, 1971.
98
-------
5. SAMPLING AND ANALYTICAL TECHNIQUES
FOR NITROSAMINES
5.1 AIR
Until recently, the detection of low concentrations of nitrosamines,
such as those expected in the environment, has been difficult because
of the general properties of these compounds. For example, they do
not fluoresce, they do not have well-defined absorption spectra, and
2
they are difficult to separate from other nitrogenous compounds. Most
of the m'trosamines found in the environment are volatile at least at
steam temperature, but the majority of nitrosamines are not volatile,
3
which makes their detection difficult. Furthermore, a thorough assess-
ment of nitrosamine pollution requires not only the measurement of
nitrosamines but also of their precursors--that is, amines and nitro-
3
sating agents—for which no satisfactory methods are yet available.
Previous technology for the detection and measurement of nitrosamines
was developed primarily for the assessment of food and biological sam-
ples. Recent interest in nitrosamines in ambient air instigated the
development of methods specific for sampling and analysis of ambient
air. The main steps in these methods are sample concentration, organic
component separation, and component identification and measurement.
4-9
Fine and coworkers at the Thermo Electron Research Center (TERC) have
developed a new, specific method for detecting N-nitroso compounds that
is based upon the catalytic cleavage of the N-NO bond and the subsequent
infrared detection of chemiluminescence. The technique, called thermal
energy analysis (TEA) by its developers, is coupled with gas-liquid
99
-------
chromatography (GLC-TEA) and, for some uses, with high-pressure
liquid chromatography (HPLC-TEA). This new technique is highly specific
for heat-labile nitrosyl groups. The method appears to have the most
acute sensitivity yet reported, so that N-nitroso compounds at the level
of 1 ng/ml can be detected. In this method, air is sampled typically
for 2 hours through three successive, cooled vacuum traps at a flow rate
of about 2 liters/min. This freezes out the compound. The contents of
the three traps are combined and stored at -78°C for later analysis.
Before analysis, the combined sample is thawed and extracted three times,
each time with 8 ml of redistilled dichloromethane. The combined extracts
are concentrated by evaporation to a final volume of 0.5 ml, spiked with
an internal standard, and chromatographed. Chromatographic separation
__ _. i-
of nitrosamines is accomplished on a 21-ft by 1/8-in. o.d. stainless steel
column packed with 2 percent KOH and with 5 percent FFAP on Chromasorb
WHP 80/100 mesh or with 10 percent FFAP on Chromasorb WHP 80/100 mesh.
The column is operated isothermally at 185°C with a nitrogen carrier at
30 ml/min. Identification and measurement are accomplished by means of
thermal decomposition of the separated nitrosamines and measurement of
jue
10
4
resultant NO by a chemiluminescence technique. The method proceeds as
described below and as shown in Figure 5-1.
1. The sample is introduced into the injection port and is subsequently
separated by the chromatograph column; the TEA pyrolyzer then splits
off the nitrosyl radical from N-nitroso compounds:
100
-------
R
N'+'N=0 (5-1)
(R and R' may be any organic radical)
2. The effluent is expanded through a narrow constriction into an
evacuated reaction chamber, where the nitrosyl radical is subse-
quently reacted with ozone, yielding electronically excited nitrogen
dioxide:
•N=0 + 03 - » N0*2 + 02 (5-2)
3. The excited nitrogen quickly returns to ground state, emitting light
in the near infrared region of the spectrum:
N02* - *N02 + hv (5-3)
This light is monitored by an infrared-sensitive photomultiplier
tube; the intensity of the light is proportional to the number of
4
nitrosyl radicals present.
Calibration is linear over five orders of magnitude with GLC-TEA; there-
fore, quantitative analysis at the <3 ng/ml N-nitroso compound concen-
tration level is possible.
A practical detection sensitivity of 1 pfcomole of an N-nitroso compound
is possible, but even greater sensitivity can be realized if the solvent
is removed.
The TEA detector determines the selectivity of the method because it
requires that a compound be catalytically pyrolyzed at a low temperature
to give a nitrosyl radical that will react with ozone to produce infrared
101
-------
OZONE
TEA
GAS CATALYTIC RE
CHROMATOGRAPH PYROLYZER . . CH
CARRIER .^-^
GAS _ /f^\
J ' O
/
,L^"*'
/v . t j
1 1
INJECTION HEATED RESTRICTION
PORT TRANSFER TUBE
250° C
ACTION PHOTOMULTIPLIER
AMBER TUBE
»
^ : •••••••'..
!
1 c RED FILTER
&
VACUUM
PUMP
Figure 5-1. Schematic of GLC-TEA interface.10
: 102
-------
light. Although luminescence is also produced by the reaction of other
compounds with ozone, such emissions are in the blue or visible spectrum.
Despite the fact that other compounds or functional groups might react
with ozone to produce an infrared luminescence, none have yet been
found. In the event of such interference, the effect may be eliminated
by interposing a cold trap between the catalytic pyrolyzer and the ozone
reaction chamber.
The solvent front, which can be observed on the chromatogram, has three
distinct effects characteristics: there is an initial sharp positive
peak, followed by a broad negative peak which overshoots the baseline
in a positive direction before decaying back to the baseline. A cold
trap, placed as previously discussed and maintained at a temperature of
q
-15°C, eliminates the negative peak completely. This simplifies quan-
titation of early peaks.
Some concern over the accuracy of these methods has existed, especially
with regard to the possibility that false readings of nitrosamines may
result from the reaction of collected amines and nitrogen oxides during
collection or analysis (artifact formation). A number of experiments
conducted in the laboratory and the field have indicated that artifact
formation is not a significant problem even at concentrations higher
11 12
than those typically found in urban atmospheres. ' Furthermore,
side-by-side monitoring, with the TERC method and with one developed
by the Research Triangle Institute (discussed below), indicates that the
two completely different collection and analytical methods appear to
1112
agree rather well. '
103
-------
The HPLC-TEA method mentioned earlier is used for analysis of nonvolatile
q
nitrosamines.
A second method that is used for the analysis of nitrosamitres In-ambient
x ._ _. — ^ 11
air was developed at the Research Trianglfr: Institute' (RTI). By the RTI
method, nitrosamines are collected by passing "Srir through 3"CartHdge
packed with solid sorbent (TENAX). Sampling times range, from 1 to 5 hours.
Samples are desorbed by heating into a gas chromatographic column.
For component identification and measurement, the chromatographic instru-
ment is interfaced to a Varian Ch-7 mass spectrometer and to a Varian
620 computer for processing of the retention time and mass spectral data.
The RTI method appears to be less sensitive than the GLC-TEA method
developed by TERC but provides more reliable and more specific component
identification data.
Because of the usefulness of both the GLC-TEA and RTI methods, and
because of their differences in analytical procedure, a comparative
study of the two methods was recently sponsored by the Environmental
Science Research Laboratory—RTF of EPA to compare the two. The mea-
surements were made at several locations in Baltimore where nitrosamines
in the ambient air have been reported. The protocol and results of
this study are shown in Table 5-1. Although the same samples were not
analyzed by both techniques, the data reported from the two methods seem
to agree well.
104
-------
Table 5-1. PROTOCOL AND RESULTS OF COMPARATIVE STUDY OF
TERC AND RTI METHODS FOR MEASUREMENT
OF NITROSAMINES IN AMBIENT
AIR IN BALTIMORE, MD.
Date
Oct. 15
1975
I
Ox ^ Oct. 16
1975
Oct. 17
1975
— •*• t —
ho . —
ro^lf?
stfal a™c =
O5~ 3 CD -O
' i—i ~ . . c: k
TERC method
Nitrosamines,
Sampling location Sampling period yg/m3
, FMC E Parking Lot3 12:17 to 1:35 p.m.
2:30 to 3:30 p.m.
4:15 to 5:45 p.m.
6:00 to 7:00 p.m.
, Sewage Plant, Patapsco 11:10 to 1:10 p.m.
Bay
1:30 to 3:30 p.m.
3:50 to 5:50 p.m.
, Chesy Pier 11:00 to 1:00 p.m.
1:15 to 3:15 p.m.
3:45 to 5:45 p.m.
Food and Machinery Corporation; the other sampling
13.1
14.6)
11. 4Y
8.7J
NDb
ND )
ND j
1.8
K2|
O.lJ
locations
Sampling
11:00 to 12
3:00 to 6
10:30 to 1
2:00 to 5
9:56 to 1
2:10 to 6
were nearby
RTI method
Ni
period
:50 p.m.
:50 p.m.
:50 p.m.
:50 p.m.
:46 p.m.
:00 p.m.
•
trosamines,
yg/m3
3.2
13.4
NDb
ND
0.9
0.1
Non-detectable concentration.
-------
5.2 WATER
5.2.1 Collection and Preservation of Samples
Although there are no known reports on collection and preservation of
water samples for nitrosamines analysis, the usual precautions employed
in the collection of samples for organic analysis are recommended.
Glass sample bottles should be carefully cleaned and analyzed for con-
taminants. The pH of the sample source should be checked prior to
sample collection to see if an upward adjustment of sample pH to 8 will
be required to stabilize the sample immediately after collection. The
sample container should be filled completely with sample and sealed
with a tight-fitting lid containing a nonporous, nonreactive liner. The
sample should be protected from light, kept cold (preferably iced), and
analyzed as soon as possible after collection.
5.2.2 Analytical Techniques
The search for the role N-nitroso compounds may play in the occurrence
of human cancer initially led to a tremendous development and applica-
tion of concentration technologies such as extraction and distillation;
and of analytical technologies such as polarography, fluorimetry, photo-
metry, chromatography, and mass spectrometry for determining these com-
pounds and their precursors in food, beverages, and complex biological
mixtures. Efforts to develop appropriate analytical procedures for
determining the N-nitroso compounds in water supplies were limited, how-
ever, until 1975. Shortly thereafter Dure et al. reported the use of
one-step extraction and a gas chromatographic system along with a high-
sensitivity N-selective flame ionization detector to determine the
106
-------
levels of nitrosamines up to 100 ng/Hter with a qualitative detection
limit of 25 ng/liter.
4-9 14 15
Fine and his co-workers '»'^»'«' also reported the development and appli-
cation of both a gas chromatographic system and a high-pressure liquid
chromatographic system, individually combined with a catalytic pyrolyzer/
chemiluminescence detection system for the determination of volatile and
nonvolatile nonionic nitroso compounds, respectively, at parts-per-
trillion levels in water supplies. Briefly, the procedure consists of
(1) extraction of the N-nitroso compounds into dichloromethane either
directly from the water or after their sorption onto carbon; (2) drying
of the aqueous extracts with sodium sulfate; (3) concentration of the
dried extract on a Kuderna Danish evaporator to a volume of 0.5 ml;
(4) separation of the N-nitroso compounds on an appropriate gas or
liquid chromatographic column; (5) elution of each N-nitroso compound
individually from the column; (6) decomposition of each nitroso radical
to the nitrosyl radical; (7) reaction of the latter with ozone to form
excited. NO,,, and (8) measurement of light emitted from excited NO,,,
when it decays back to the ground state.
Recently reported developments in the application of gas chromatographic/
mass spectrometric technology to the determination of the nitrosamines are1'
encouraging. This approach may be the method of choice, for it will
afford a way to positively identify nitroso compounds. Several poten-
tial compounds of interest (e.g., N-nitrosoatrazine) do not survive gas
chromatography. It may be possible to derive some of these compounds,
but this may reduce the reliability of the analysis.
107
-------
In all of these approaches, more data are needed with which to evaluate
the efficiency of the sample concentration and clean-up systems and the
specificity and accuracy of the analytical systems. The possibility of
both nitrosamine formation and decomposition during these procedures
must also be considered.
5.3 FOOD
A variety of methods have been used to detect nitrosamines in food, such
as polarography, UV absorption, thin-layer chromatography, and gas chroma-
tography. Many of the earlier methods used for the evaluation of
extractive procedures have proved to be of doubtful value because of the
possibility of artifacts and contamination.
It appears that gas chroma tography followed by mass spectrometry is now
the most acceptable procedure for the measurement of nitrosamines in
food.
The total analytical procedure usually involves an extraction step, fol-
lowed by distillation, partitioning with solvents; a clean-up step with
column or thin-layer chromatography; and, finally, separation, detection,
and confirmation. A survey of analytical techniques used in the isolation
ion
16
and detection of nitrosamines has been published by Wassermann and
Eisenbrand.
5.4 REFERENCES FOR SECTION 5
1. Wassermann, A. E. A survey of analytical procedures for nitrosa-
mines. In.: N-Nitroso Compounds: Analysis and Formation (P.
Bogovski, R. Preussmann, and E. A. Walker, eds.). Lyon, France.
IARC Scientific Public. No. 3. 1972. p. 10-15.
108
-------
2. Lijinsky, W. and S. S. Epstein. Nitrosamines as environmental
carcinogens. Nature (London). 225_:21-23, 1970.
3. Preussmann, R. On the significance of N-nitroso compounds as car-
cinogens and on problems related to their chemical analysis. In:
N-Nitroso Compounds: Analysis and Formation (P. Bogovski, R.
Preussmann, and E. A. Walker, eds.). Lyon, France. IARC Scien-
tific Public. No. 3. 1972. p. 6-9.
4. Fine, D. H. and D. P. Rounbehler. Trace analysis of volatile N-
nitroso compounds by combined gas chromatography and thermal energy
analysis. J. Chromatography. J09:271-279, 1975.
5. Fine, D. H., F. Rufeh, and D. Lieb. ' Group analysis of volatile and
nonvolatile N-nitroso compounds. Nature (London). 247:309, 1974.
6. Fine, D. H., F. Rufeh, D. Lieb, and D. P. Rounbehler. Description
of the thermal energy analyzer (TEA) for trace determination of
volatile and nonvolatile N-nitroso compounds. Anal. Chem. 47(7):
1188-1191, 1975.
7. Fine, D. H., D. P. Rounbehler, N. M. Belcher, and S. S. Epstein.
N-nitroso compounds in the environment. Presented at International
Conference on Environmental Sensing and Assessment, Las Vegas,
Nevada, September 1975.
8. Fine, D. H., D. P. Rounbehler, N. M. Belcher, and S. S. Epstein.
N-nitroso compounds: detection in ambient air. Science. 192(4246):
1328-1330, 1976.
9. Oettinger, P. E., F. Huffman, D. H. Fine, and D. Lieb. Liquid
chromatograph detector for trace analysis of nonvolatile N-nitroso
compounds. Anal. Letters. £(6)-411-414, 1975.
10. Walker, P., J. Gordon, L. Thomas, and R. Ouellette. Environmental
Assessment of Nitrosamines. Prepared by Mitre Corporation, McLean,
Virginia, under Contract No. 68-02-1495. U.S. Environmental Pro-
tection Agency. Research Triangle Park, N.C. February 1976.
11. Pellizzari, E. D. Identification and Estimation of N-Nitrosodi-
methylamine and Other Pollutants in the Baltimore, Maryland, and
Kanawha Valley Areas. Progress Report. Prepared by Research Tri-
angle Institute under Contract No. 68-02-1228. U.S. Environmental
Protection Agency. Research Triangle Park, N.C. January 1975.
12. Fine, D. H. Nitrosamines in Urban Community Air. Progress Report.
Prepared by Thermo Electron Research Center, Waltham, Massachusetts,
under Contract No. 68-02-2363. U.S. Environmental Protection
Agency. Research Triangle Park, N.C. January 1975.
109
-------
13. Dure, G., L. Weil, and K. E. Quentin. Determination of nitrosamines
in natural water and waste water. Z. Wasser. Abwasser. Forsch.
8(6):20-30, 1975.
14. Fine, D. H., D. Lieb, and F. Rufeh. Principle of operation of the
thermal energy analyzer for the trace analysis of volatile and
nonvolatile N-nitroso compounds. J. Chromatog. 107:351-357, 1975.
15. Fine, D. H., D. P. Rounbehler, F. Huffman, A. W. Garrison, N. L.
Wolfe, and S. S. Epstein. Analysis of volatile N-nitroso com-
pounds in drinking water at the part-per-trill ion level. Bull.
Environ. Contamin. Toxicol. [In Press.]
16. Eisenbrand, G. Recent developments in trace analysis of volatile
nitrosamines: a brief review. In: N-Nitroso Compounds in the
Environment (P. Bogovski, E. A. walker, and W. Davis, eds.). Lyon,
France. IARC Scientific Public. No. 9. 1974. p. 6-11.
110
//
-------
6. ENVIRONMENTAL CONCENTRATIONS AND HUMAN EXPOSURE
6.1 AIR
The presence of N-nitrosamines in ambient air recently has been reported
1 23 4 5
by Bretschneider and Matz, Fine et al., ' Pellizzari, and DuPont.
Bretschneider and Matz found traces of dimethylnitrosamine and diethyl-
nitrosamine in commercial plants producing dimethyl and diethyl amines.
A trace of dimethylnitrosamine was found in one dust sample. Measure-
2 3
ments by Fine et al. ' were made in Baltimore, Maryland; Philadelphia,
Pennsylvania; Waltham, Massachusetts; Wilmington, Delaware; Belle, West
4
Virginia; and New York City, New York. Measurements by Pellizzari were
made in Baltimore, Maryland, and Belle, West Virginia, during the same
period as the second series of measurements by Fine et al. Measurements
5
were made by DuPont in Belle, West Virginia. Different measurement
techniques were used by each of the investigators in Baltimore and Belle.
These techniques were described in Section 5. Although the measurement
methods have not been thoroughly evaluated, the agreement between the
different reported values seems to leave little doubt concerning the
presence of N-nitrosamine in some ambient air samples at detectable
levels (above 0.01 yg/m , for example). Stack samples from the dimethyl-
hydrazine plant in Baltimore also contained N-dimethylnitrosamine.
2 3
In the initial study by Fine et al., ' dimethylnitrosamine concentra-
tions in Baltimore ranging from 0.033 to 0.960 yg/m were reported.
The reported concentrations in Belle, West Virginia, ranged from 0.014
3
to 0.051 yg/m . A trace was found in one sample taken in Philadelphia,
ill
-------
but no detectable amount was found In Delaware. Results are shown in
Table 6-1. ' Later measurements 1n Baltimore, Maryland, and 1n West
3 4 5 l
Virginia by Fine et al., Pellizzari, and DuPont revealed higher
concentrations in Baltimore and Belle at -sampling sites in the imme-
3
diate vicinity of commercial plants. Values ranged from 1 to 36
at the Food Machinery and Chemical Corporation (FMC) property in Balti-
more. These preliminary data are not sufficient for the determination
of temporal and spatial distributions.
6.2 WATER
At present, our knowledge about the occurrence of nitrosamines in water
supplies is limited to the results of only a few investigations. Shabad
and IVnitskii reported the occurrence of both nitrosamines and ben-
zo(a)pyrene in a reservoir in Russia. Unfortunately, the details of
their investigations are not available at this time.
Q
Sander et al. in 1974 analyzed tap, river, and waste water purified by
a biological treatment plant in West Germany. Although they did not
detect any nitrosamines (i.e., £0.5 parts per billion, ppb) in the
samples, they reported that the river water contained 0.6 to 4.3 ppb
dicloromethane-extractable nitrogen, 0.18 to 1.1 mg NOp'/liter, and
0.17 ppb each of dimethyl ami ne, diethylamine, arid pyrrol idine or
piperidine, amines readily converted to nitrosamines. In the purified
-^•' ' ':'
water samples, they detected 9.3 to 10.4 ppb total nitrogen, 0.64 to
0.83 mg N02~/liter, and 0 to 2.1 ppb of each of the amines. The tap
water, however, contained none of the amines and no N02~ or total
112
-------
Table 6-1. NITROSAMINE CONCENTRATIONS IN AMBIENT AIR
MEASURED IN 12 U.S. CITIES OR LOCALITIES2"4'6
Location
Baltimore, Md., area
FMC property
Within 0.6-km radius
of FMC property
Within 0.6- to 1-km radius
of FMC property
Outside 2-km radius
of FMC property
Belle, W. Va.
So. Charleston and
Charleston, W. Va.
Nitro, W. Va.
Northern New Jersey
Philadelphia, Pa,d
Wilmington, Del .
Waltham, Mass.
New York, N.Y.
Denver, Colo.
(Eisenhower tunnel)
Los Angeles, Calif.
St. Louis, Mo.
Approx.
hours of
.sampling
54
27
30
14
78
30
5
11
12
12
4
10
1
1
1
3
Concentration range, yg/m
(1- to 2-hr peaks)
DMN
1 to 36
0.03 to 7.6
^
Trace to
2.0
.0.02 to 0.13
b
ND to 0.98
NDbto 0.06
NDb
NDb
NDe
ND6
NDe
NDb to 0.016
Trace
NDe
NDe
Unknown la
h
NDu to 0.08
ND to 0.17
b
ND to 0.03
0.02 to 0.04
h
ND to 0.06
—
_
—
NDb
NDb
NDb
- .
-
-
-
Unknown
b
ND to
0.02 to
b
ND to
b
ND to
b
ND to
—
—
_
NDb
NDb
NDb
-
-
-
-
2a
2.7
0.32
0.34
0.3
0.08
-
-------
Unknowns were analyzed only by; the TEA technique. Total sampling time for unknowns in Balti-
more, Md., was about half that for DMN and in Belle, W. Va., was about one-third that for DMN.
bND = not detectable (< 0.0002 yg/m3).
A
cTrace is defined as a detectable but nonquantifiable amount >0.0002pg/m .
Sampled during initial TEA study only.
eND = not detectable (< 0.1 pg/m3).
-------
Q
nitrogen. Dure et al. investigated the occurrence of nitrosamines in
water processed at a Munich, Germany, clarification plant and in sedi-
ments from Starnberger Lake and the Main River. In each instance, the
nitrosamine level was <_ 0.1 ppb.
2
In September 1975, Fine et al. released preliminary data indicating the
possible presence of several nitrosamines in New Orleans-area drinking
water samples. N-nitrosoatrazine was suggested as the probable identity
of one of the nitroso compounds, while the concentration levels of "the
various nitrosamines were estimated to be <^0.1 ppb.
In late September 1975, the staff of the Water Quality Division of the
Health Effects Research Laboratory, EPA, Cincinnati, collected water
samples from six rural wells in Washington County, Illinois, and six in
Runnels County, Texas. The samples were characterized by both high
nitrate levels and coliform counts. The samples were analyzed for
nitrosamines by Fine and his associates at Thermo Electron Research Cen-
ter in Waltham, Massachusetts; in each of the 12 samples the concentra-
tion of volatile and nonvolatile nonionic nitrosamines was <_ 15 parts
per trillion (ppt).
3
Fine et al. reported 6000 yg/liter of dimethylnitrosamine in a rain-
water puddle near the unsymmetrical dimethylhydrazine (UDMH) plant in
Baltimore. A sample from a drainage ditch on the border of the property
contained 5.9 yg/liter, and water samples from the adjacent cover con-
tained 0.26 to 0.94 yg/liter.
115
-------
For discussion concerning the occurrence of nitrosamines in water, see
Section 7.1.3.
6.3 FOOD AND DRUGS
6.3.1 Nitrosamines in Processed Foods
The presence of the carcinogen N-nitrosodimethylamine (DMN) was demon-
strated in fish meal as a result of the reaction of sodium nitrite with
the secondary amine, dimethy!amine. Studies then followed to investi-
gate the presence of this nitrosamine in human foods treated with sodium
11 12
nitrite and sodium nitrate, especially in cured meat products. '
Data reported prior to 1970, however, are open to question because the
analytical methodology included nonspecific identification procedures
without adequate confirmation of the nitrosamine. The use of the alkali
flame-ionization detector for gas-liquid chromatography (GLC), and
several other techniques, followed by confirmation with a QIC-mass
spectrometric method, has led to the detection and identification of
nitrosamines in cured meat products in concentrations as low as 3 to
5 yg/kg.
Among more recent studies are a number of investigations on the presence
of nitrosamines in smoked and nitrite-preserved fish.
13
In a study by Howard et al., samples of nitrite-preserved and non-
nitrite-preserved smoked chub were analyzed for dimethylnitrosamine (DMN)
by means of gas-liquid chromatography (GLC), using a modified thermionic
detector. Apparent values of DMN were 1 to 2 ppb (below the GLC
116
-------
quantifiable limit but confirmed by mass spectrometry) in both nitrite-
preserved and non-nitrite-preserved samples. These results led the
authors to conclude that DMN is not produced in the flesh under the
conditions of the commercial processing treatment used.
The possible formation of DMN through nitrosation of amines in smoked
chub was studied in aqueous model systems containing methyl amines and
nitrites. Reaction conditions employed (pH, reaction time, and tem-
perature) were more severe than those occurring during the commercial
processing of nitrite-treated smoked chub. Polarographic analyses indi-
cated that no detectable amounts (1.4 x 10 mole) -of DMN were produced.
Marine fish reportedly have a higher amine content than freshwater fish
and, therefore, should have a greater potential for nitrosation reactions.
15
Dimethylnitrosamine was found by GLC and determined by mass spectro-
metry (MS) in sable samples at the following levels: raw, 4 ppb; smoked,
4 to 9 ppb; smoked nitrite-treated, 8 to 14 ppb; and smoked, nitrite- and
nitrate-treated, 23 ppb. Neither raw shad nor salmon contained DMN, but
one smoked salmon sample contained 5 ppb DMN and smoked nitrate-treated
15
salmon and shad products contained 10 to 17 ppb.
A number of foodstuffs were analyzed for nitrosamines by GLC, using an
electron-capture detector, after extraction by neutral steam distilla-
tion of samples. Diethylnitrosamine was found at 1.5 yg/kg in Cheshire
cheese, fried bacon, raw pig's liver, and stale cod (raw and fried). The
cod also contained 1.0 yg/kg DMN and 6.0 yg/kg N-nitrosopyrrolidine
(NPyr). Of 25 food samples tested, 16 (7 of which were cod samples)
117
-------
contained NPyr at levels of 1.0 (Norwegian goat's milk and fresh cod)
to 11.0 yg/kg (raw pig's liver).16
In a study by Fong and Chan, samples of marine salt fish from the
markets in Hong Kong were analyzed for DMN. Pickling and drying of such
fish are done in the open, so that the fish are subject to contamination
by bacteria. These samples contained 0.05 to 0.3 ppm DMN as determined
by GLC and confirmed in several samples by GLC-MS. Fish prepared with
crude salt contained much more DMN than fish prepared with pure sodium
chloride. Levels of nitrate were found to be appreciable in the crude
salt commonly used to prepare salt fish, amounting to 17, 18, 20, 20,
30, and 40 ppm in six samples of crude salt purchased from local retailers.
The nitrite content was no more than 1 ppm in any of these samples. The
level of DMN found varied with the species of fish. Salted white herring
contained more DMN than salted yellow croaker, and a single batch of
pomfret contained no detectable DMN.
Fong and Chan further conducted a study on the role of nitrate-reducing
I g
bacteria in the formation of nitrosamines. They demonstrated that
Q
fish broth inoculated with Staphylococcus aureus (10 cells/ml) and
untreated fish broth (unsterilized, uninoculated) showed an increase in
DMN content with time. Sterilized fish broth showed no such change in
DMN content (Figure 6-1). Content of DMN was determined by GLC-MS tech-
niques. The authors concluded that the amount of DMN present seems to
be dependent on the storage conditions, degree of contamination by
18
nitrate-reducing bacteria, and the amount of precursors present.
118
-------
Figure 6-1. Concentration of dimethylnitrosamine in fish broth after incubation at 37°C. _
(Portion A, sterilized; portion B, inoculated with10i9 Staphylococcus aureus per lot; portion
C, untreated.)18
• 119
-------
The effects of processing and storage on sablefish were studied by
19
Gadbois et al. Sablefish was treated with 0 to 1300 ppm nitrite prior
to being smoked. The meat was analyzed for the presence of N-nitroso
compounds by gas-liquid chromatography immediately after processing and
again after 2 weeks' storage at 40°F. Trace amounts of DMN (< 10 ppb)
were detected; nitrite levels in these samples ranged from 0 to 550 ppm.
Levels of DMN did not increase with higher nitrite levels, and a slight
decrease in DMN concentrations was found after storage at 40°F. The
identity of the isolated compound was confirmed by GLC as the nitramine
19
derivative and by GC-MS.
20
Havery et al. surveyed 121 food samples for the presence of N-nitro-
samines. N-nitrosopyrrolidine was confirmed in fried bacon at levels
up to 139 ppb. Dimethylnitrosamine, N-nitrosopyrrolidine, and N-
nitrosopiperidine were also confirmed in spice-cure mixtures at levels
ranging from 50 to 2000 ppb.
20
Included in this survey were Icelandic national foods processed under
atypical conditions that are theoretically conducive to nitrosamine
formation. For example, pieces of shark and skate are coated with
coarse salt and buried in the ground, where they remain until a certain
stage of fermentation is reached. The putrified fish are hung outdoors
for 1 to 3 months and then are eaten without further preparation. Other
fish, such as trout and salmon, are pickled in a salt solution contain-
ing 0.6 percent nitrite before smoking. These modes of food processing
in conjunction with the high concentrations of amines normally found in
•120
-------
fish should have provided an ideal environment for nitrosamine formation.
20
Nonetheless, no nitrosamines were found in these samples.
Conventionally cured hams studied in a number of laboratories around
the world have been found free of the nitrosamines for which they were
21
tested, except for an occasional report of a low concentration of DMN.
Only 3 of 41 samples of frankfurters tested were reported to contain
22
DMN; unpublished data on other samples of frankfurters showed the
absence of this nitrosamine. Other emulsion-type products, however,
have been reported to contain variable quantities of DMN and/or N-
nitrosopyrrolidine (NPyr); e.g., Hungarian sausage, mettwurst, salami,
dry sausage, thuringer, knockwurst, and thuringer liver sausage.
?i
Panelaks et al."~ surveyed 197 cured meat samples for DMN and found b7
positive, but the identity of the nitrosamine was not confirmed. In an
94
additional 100 samples, they found that 29 samples contained DMN,
17 contained NPyr, and 9 contained N-nitrosodiethylamine. Only five of
these positive findings, however, were confirmed by mass spectrometry.
The presence of NPyr and N-nitrosopiperi dine (NPip) in emulsion-type
products was explained with the finding of the two nitrosamines in
25
premixed cures. For convenience in handling, cure components—includ-
ing sodium chloride, sodium nitrite, sodium nitrate, spices, and other
ingredients—were premixed to the processor's formulation and stored for
several months prior to use. Sodium nitrite can react with components
in pepper to form NPip and with unknown paprika components to produce
pc
NPyr. The source of DMN has not been identified. Regulations have
121
-------
been promulgated now to forbid premixing of nitrite or nitrate salts
27
with other cure ingredients.
Nitrosamines have also been reported in jellied meat products. DMN and
28
NPyr were found in head cheese, souse, and blood and tongue sausage.
This class of meat products contains pieces of organ meat or offal
previously cured with nitrite or nitrate salts. Methods of preparation,
which differ among processors, may include the further addition of
nitrite or nitrate salts to the blood. Further studies on the sources
of nitrosamines or the mechanism of their formation have not been carried
out.
The product of greatest concern, however, is bacon. N-nitrosopyrroli-
dine has been reported in almost all samples of bacon examined after
frying and DMN has been found sporadically. These nitrosamines were not
identified in the raw, uncooked bacon; they appear only following the
29-31
application of heat. Consideration has been given to the precur-
sor(s) and the mechanism of formation of NPyr. Proline, which is present
in collagen, can be either nitrosated initially to form nitrosoproline
(NPro) with decarboxylation to NPyr on heating, or the amino acid can
32
be decarboxylated first to pyrrolidine, and then nitrosated. Putres-
cine, a diamine, can form pyrrol i dine by ring closure upon heating. The
latter compound can then be nitrosated. The addition of either proline
or putrescine and nitrite to ground pork belly resulted in an increase
in NPyr formation, although greater amounts were found with proline than
with putrescine. Nitrosoproline was identified and the identity confirmed
122
-------
33
in raw bacon but, because of the difficulties involved in analysis of
nonvolatile nitrosamines, the quantitative determination of the concen-
tration present could not be assured.
Studies on the decarboxylation of NPro showed the temperature at which
maximum conversion of NPro to NPyr occurs (365°F) approximates that
which is recommended for frying or broiling bacon by most processors.
Time-temperature studies with bacon fried to the same degree of doneness
(medium well done) showed that longer frying times at lower temperatures
produced little or no NPyr, while higher frying temperatures led to the
formation of the nitrosamine.
N-nitrosopyrrolidine was found in bacon drippings at concentrations as
high as, or higher than, those present in the edible portion of fried
bacon. ' Studies with fried, separated, lean and adipose tissue
29 34
showed that the nitrosamine is produced in the adipose tissue * and
is not present as a result of the greater solubility in lipid of NPyr
produced in the lean. The precursor for NPyr in adipose tissue has not
been identified, although it has been suggested that the collagen present
in adipose tissue is the source of proline.
In view of the potential carcinogenic nature of NPyr, reduction or
elimination of this compound from bacon is desirable. The complete
elimination of NPyr should be accomplished by the removal of nitrite or
nitrate salts from the cure, although it should be noted that nitric
oxides in smoke may be able to react with the bacon amines also. Public
health considerations with respect to inhibiting the outgrowth of
123
-------
Clostridium botulinum spores, and thus avoiding botulinus poisoning,
militate against the complete ban of nitrite 1n bacon at this time.
Reduction of the nitrite concentration, however, could result 1n lower
35
concentrations of NPyr.
The prevention or reduction of nitrosamine formation through the use of
other compounds must be considered. Sodium ascorbate (or sodium erythor-
bate), used in processing cured meats to accelerate cure color develo-
ment, has been found to prevent the formation of experimentally produced
36 37
DMN in frankfurters and NPyr in laboratory-prepared bacon. Studies
on commercially produced bacon were sponsored by the American Meat
Institute in cooperation with the Food and Drug Administration and Depart-
ment of Agriculture. Results with commercial batches of product confirmed
the reduction of NPyr concentration by ascorbate; but, for reasons still
unknown, the nitrosamine was not completely eliminated. Studies are in
progress to determine the effects of decreasing concentrations of nitrite
and increasing concentrations of ascorbate.
It has been reported that phenols present in the smoke used in processing
38 39
react with nitrite, ' thus potentially reducing the concentration of
nitrite available for nitrosation of the amines.
The Expert Panel on Nitrite and Nitrosamines, appointed by the Secretary
of Agriculture, has recommended the complete elimination of nitrate from
cured meat products (except, temporarily, in dry-cured and fermented
products) and the reduction of residual nitrite to various concentrations
that would depend on the class of meat product.
124
-------
Since nitrite is a chemical and possibly a physiological precursor of
nitrosamines, and since nitrate is easily reduced to nitrite under
physiological conditions, the levels of both nitrate and" nitrite enter-
ing the digestive tract are pertinent to any assessment of exposure to
40
nitrosamine precursors. White calculated the average daily ingestion
by a U.S. resident of nitrate and nitrite. Rather than calculate possi-
ble ingestion from hypothetical meals, White based his estimates on per
capita consumption and production statistics and on recent nitrite data
for 34 vegetables. His estimates are presented in Table 6-2.
Table 6-2. ESTIMATED AVERAGE DAILY INGESTION
OF NITRATE AND NITRITE BY
U.S. RESIDENT^
Nitrate
Vegetables
Fruits, juices
Milk and products
Bread
Water
Cured meats
Saliva
Total
mg
86.1
1.4
0.2
2.0
1 0.7
15.6
30. Oa
106.0
%
81.2
1.3
0.2
1.9
0.7
14.7
-
100
Nitrite
mg
0.20
0.00
0.00
0.02
0.00
3.92
8.62
12.76
%
1.6
0.0
0.0
0.2
0.0
30.7
67.5
100
Not included in total; see text.
According to .these data, four-fifths of dietary nitrate originates with
vegetables and only one-sixth from cured meats. Other sources are insig-
nificant. Two-thirds of the nitrite entering the stomach originates in
125
-------
saliva, and slightly less than one-third conies from cured meats. White
assumes salivary nitrate to come from food and does not include it in
the total nitrate ingested. He assumes salivary nitrite to orginate in
40
the mouth and hence includes it in intake.
White's data do not represent actual ingestion but are averages calcu-
lated from available data.
6.3.2 Nitrosamines in Plants and Fruits
Nitrosamines in the range of 0.4 to 30 yg/kg have been found in some
41 42
samples of mushrooms. Hedler and Marquardt found diethylnitrosamine
in samples of wheat plants, grain, and flour; but the method was not
quantitative and was not confirmed. Thewlis was unable to find diethyl-
44
nitrosamine in wheat flour. Kroller analyzed 30 samples of wheat flours
and was unable to detect nitrosodiethylamine at levels greater than 0.01
45
mg/kg in more than one of these samples. DuPlessis et al. found
dimethylnitrosamine in the fruit of a plant (Solanum incahum) in South
46
Africa. Cycad nuts are known to be toxic, and Laquer et al. reported
that rats fed the nuts developed liver and kidney tumors. Keybets et
47
al. examined spinach in which nitrate-reducing bacteria produced a
high nitrite level, but did not detect m'trosamines. He concluded that
the pH was too high and the concentration of secondary amines too low
to allow the formation of m'trosamines. Tobacco contains several
secondary amines and up to 1000 ppm of oxides of nitrogen; hence the
possibility for the formation of m'trosamines exists. Rhoades and John-
48
son recently identified dimethylnitrosamine in tobacco smoke
126.
-------
condensation. They also found that growing conditions greatly affect
the amount of DMN found in tobacco.
6.3.3 Nitrosamines in Drugs, Pesticides, and Natural Products
Secondary and tertiary ami no compounds used as drugs, pesticides, and
herbicides, and some natural products such as spermine and spermidine,
have been shown to react with nitrite to form N-nitroso compounds.
Lijinsky and his colleagues have pointed out that many drugs and pesti-
49-53
cides contain tertiary amino groups and may therefore be expected
to undergo nitrosation in the body. Examples of drugs that react with
nitrite in vitro to give varying yields of carcinogenic nitrosamines
are oxytetracycline (an antibiotic) and aminopyrine (an analgesic) which
yield dimethylnitrosamine; disulphiram (an antialcoholic) and nike-
thamide (a respiratory stimulant), which yield diethylnitrosamine; and
tolazamide (an oral hypoglycemic drug), which gives nitrosohexamethyl-
eneimine.
Among commonly used pesticides, several are esters of N-methyl-carbamic
acid and therefore are analogs of N-methylurethane. They react with
nitrous acid to form N-nitroso derivatives; for example, nitroso-
carbaryl. A study of the in vitro nitrosation of carbaryl under
simulated gastric conditions showed that 10 M carbaryl reacted with a
fivefold molar excess of nitrite to form 1.7 percent of theoretical
55
yield of N-nitrosocarbaryl after 60 minutes. Such derivatives could
be formed in the environment or in vivo from nitrite and residues of
the carbamates in crops; for example, nitrocarbaryl, both carcinogenic
127
-------
and mutagenie. This Is a less potent carcinogen than nitrosomethylure-
thane but a more potent mutagen.
Widely used agricultural chemicals such as zinc dimethyldithiocarbamate
(ziram); 1-naphthyl-N-methylcarbamate (propoxur); N-(2-benzothiazolyl)-
N'-methyl urea (benzthiazuran); 2-chloro-4,6-bisethylamino-1,3,5-triazine
(simazin); and 2-chloro-4-ethylamine-6-isopropylamino-l,3-5-tr1az1ne
(atrazine) have been found to nltrosate easily. Dimethylnitrosamine
(DMN) has been shown to be the product of the In vitro and in vivo (in
the rat) reactions of ziram with sodium nitrite. The in Vitro reaction
of ferbam (iron dimethyldithiocarbamate) with sodium nitrite also pro-
cp
duced DMN. Atrazine has been shown to react in vitro with nitrite
(at pH 2.0) about two orders of magnitude faster than dimethyl amine and
about three orders of magnitude faster than carbaryl.
59
A recent investigation by Fine and Ross of six commercial weed
killers revealed nitrosamines at levels ranging from 0.3 mg DMN/liter
in a home lawn-care product to 640 mg DMN/liter in an industrial
herbicide. Dipropylm'trosamine (DPN) was found at 154 mg/liter in
Treflan, a major agricultural herbicide. These findings are summarized
59
in Table 6-3. The DMN detected in Trysben 200 is apparently a
product of the reaction between dimethyl amine, a Trysben 200 component,
and sodium nitrite used as a corrosion inhibitor in 1-gallon cans of
the herbicide. (The DMN is 0.1 percent by weight; the minimum level
regulated by the Occupational Safety and Health Administration is
1 percent by weight.)
128
-------
Table 6-3. N-NITROSAMINES IN WEED KILLERS
59
Product
Sears Broad
Leaf Weed
Unico Turf
Treeter T
Use
Concn.,
N-N1trosam1ne mg/liter
Black Leaf Home
Weed Killer lawn care
OMN
a
Home
lawn care
DMN
Home
lawn care
DMN
0.3
Active Ingredients
12.72% 2,4-Dichloro-
phenoxyacetic acid as
dimethyl amine salt
4.07% 2,4,5-Trichloro-
phenoxypropionic acid
as dimethyl amine salt
12.00% 2,4-Dichloro-
phenoxyacetic acid as
dimethyl amine salt
2.40% 3,6-Dichloro-o-
anisic acid as dimethyl -
amine salt
10.59% 2-(2-Methyl-4-
chlorophenoxy) propionic
acid as dimethylamine
salt
3.23% 2,4-Dichlorophenoxy-
acetic acid as dimethyl-
amine salt
1.28% 3,6-Dichloro-o-
anisic acid as dimethyl -
amine salt
Du Pont
Trysben 200
Weed Killer
Benzac 1281
Industrial
herbicide
Industrial
herbicide
DMN
187-195
DMN
640
Treflan E.G. Agricultural DPN
herbicide
154
26.10% 2,3,6-Trichloro-
benzoic acid and related
polychlorobenzoic acids
as dimethylamine salt
26.10% 2,3,6-Trichloro-
benzoic acid and related
polychlorobenzoic acids
as dimethylamine salt
44.50% a,a,a-Tr1fluoro-
2,6-dinitro-N,N-d1propyl-
p-toluidine
Less than 0.05 nig/liter.
129
-------
Because the agricultural herbicide, Treflan, is used on food crops,
the investigators sampled air and irrigation water from tomato fields
in the Sacramento Valley, California, before, during, and after application
CO
of Treflan in May 1976. No DPN was found in any of the ambient air
o
samples (detection limit = 1 ng/m ) or in any of the water samples
(detection limit = 20 ng/liter). Preliminary analyses of tomatoes
picked prematurely (September is harvest-time) showed no detectable
amount of DPN. Tests on Treflan by its manufacturer have not shown
the product to be carcinogenic. Fine and Ross postulated that the
incomplete removal of nitric acid used in the synthesis of the product
could result in nitrosation of dimethyl amine, also used in the manufacturing
59
process.
6.4 RELATIVE EXPOSURE
Nitroso compounds, or their precursors, are widely distributed in the
environment, resulting from both natural and anthropogenic processes.
Exposure may be via inhalation, ingestion, and, for the human species,
smoking of tobacco. Exposure may be direct by these routes, or nitro-
samines (as well as other nitroso compounds) may be formed in vivo
from nitrogen and amine compounds. Therefore, the potential for
exposure to very low levels of carcinogenic nitroso compounds is ever-
present.
It is not possible at this time to provide a meaningful estimate of
either total or relative exposure via each route of intake. Nitrosamines
130
-------
have been found in a variety of foodstuffs in varying concentrations,
but many reported values are doubtful because of sampling and analyti-
cal problems. Nitrosamines have been found in the ambient air in the
vicinity of an anthropogenic source at concentrations of a few parts
per billion. Theoretical considerations suggest that nitrosamines
might be found in polluted ambient atmospheres (possibly in the parts-
per-trillion range) and therefore may be a potential source of direct
exposure via inhalation.
There is a substantial body of evidence indicating that nitrosamines
(and other carcinogenic nitroso compounds) can be formed in vivo from
nitrates and amines (precursors). Exposure to these precursors again
61
may be via ingestion, inhalation, and smoking. It has been estimated
that a single meal may contain as much as 100 mg of secondary amines,
with an average daily intake of 20 to 30 mg. The average nitrite
production in human saliva has been estimated at 6 to 10 mg/day. A
single cigarette may contain 0.5 mg nitrite.
The intake of nitrate nitrogen from drinking water could be as much as
100 mg/day in dry areas where farm wells contain over 50 mg/liter of
nitrate nitrogen. (The EPA standard is 10 nig/liter nitrate nitrogen.)
Using a standard respiratory volume per day, and assuming an exposure to
nitrate concentrations of 3 to 5 yg/m3 for 12 hours per day, the calculated
inhaled nitrate would be 21.5 yg per day. Peak atmospheric nitrate levels
_ CO '
in some local areas may exceed 100 yg/w*. Kant found that nitrosamines
131
-------
were formed in rat lung homogenates after a gas mixture containing 15 per-
cent nitrogen dioxide was bubbled through the homogenates. (It 1s known
that nitrites are produced when nitrogen dioxide 1s bubbled through water.)
However, when rats Inhaled an atmosphere containing 0.01 to 0.5 mg nitrogen
oxides per liter, no nitrogen oxide compounds were found in the lung
tissue. No data are available regarding in vivo formation of nitro-
samines in humans as a result of inhalation of nitrogen dioxide. Fur-
ther, no estimates of amine concentrations in the atmosphere are
available.
It is apparent that under normal living conditions there is a potential
for the formation of nitrosamines in vivo, though meaningful estimates
of the total burden cannot be made. It seems evident, however, that
exposure to precursors via inhalation is relatively small compared to
that from food, water, and smoking.
Again, a reliable daily dietary intake of nitrosamines is not available;
but, based on measured concentrations in foodstuffs and water, the direct
daily intake is not likely to exceed a few micrograms per day. In the
vicinity of a nitrosamine emission source, reported values indicate that
the average daily intake via inhalation may reach a few micrograms per
o
day. Assuming 20 m. of air are breathed per day, the daily intake via
inhalation would therefore be comparable to that from ingestion. For
populations not living in the vicinity of emission sources, the daily
intake via inhalation would likely be less than 1 pg/day.
132
-------
6.5 POPULATIONS AT RISK
Our present knowledge does not permit us to estimate the risk of tumor
induction in the human population resulting from exposure to nitro-
samines. This would require much more definitive information than is
now available concerning dose-response relationships in humans and
exposure patterns over time. Though evidence.of carcinogenic potential
from animal experiments seems conclusive, there is at present no defini-
tive information that nitroso compounds have caused cancer in humans
under any conditions.
With our present state of knowledge, risk of exposure can be discussed
only in a qualitative manner. Data indicate rather clearly, however,
that the entire human population is continuously exposed to very low
levels (ppb or ppt) of nitroso compounds via ingestion. Exposure to
nitrosamines via drinking water does not appear to be as widespread as
via food, and is probably very small. Detectable quantities have been
found in only a few water supplies.
Nitrosamines have been measured in ambient air in the vicinity of emis-
sion sources in the ppb range. In addition, chamber experiments and
theoretical considerations suggest that nitrosamines may be formed in
polluted air. Concentrations in the ppt or the low ppb range may occur,
at least under conditions favorable to nitrosamine formation, in all
major industrial areas. Therefore, there is a potential risk of direct
exposure via inhalation to low concentrations among major segments of
the population.
133
-------
6.6 REFERENCES FOR SECTION 6
1. Bretschneider, K. and J. Matz. Nitrosanrines (NA) in the atmospheric
air and in the air at the workplace. Archiv. fur Geschwulst-forsch.
42:36-41, 1973.
2. Fine, D. H., D. P. Rounbehler, N. M. Belcher, and S. S. Epstein.
N-nitroso compounds in the environment. Paper presented at the
International Conference on Environmental Sensing and Assessment,
Las Vegas, Nevada, September 1975.
3. Fine, D. H. N-Nitrosamines in Urban Community Air. Progress Report.
Prepared by Thermo Electron Research Center, Waltham, Massachusetts,
under Contract No. 68-02-2363. U.S. Environmental Protection Agency.
Research Triangle Park, N.C. January 1975.
4. Pellizzari, E. D. Identification and Estimation of N-Nitrosodi-
methylamine and Other Pollutants in Ambient Air in the Baltimore,
Maryland, and Kanawha Valley Areas. Progress Report. Prepared by
Research Triangle Institute under Contract No. 68-02-1228. U.S.
Environmental Protection Agency. Research Triangle Park, N.C.
January 1975.
5. E. I. DuPont de Nemours & Company, Inc. Dimethylnitrosamine Sam-
pling Program. December 1-5, 1975.
6- ; Fine, D. H. Report of Three Days of Monitoring for N-Nitroso Com-
i pounds in the State of New Jersey. Progress Report. Prepared by
j Thermo Electron Research Center, Waltham, Massachusetts, under Con-
j tract No. 68-02-2363. U.S. Environmental Protection Agency.
Research Triangle Park, N.C. July 1976.
7. Shabad, L. M. and A. P. Il'nitskii. Contamination of reservoirs
by carcinogenic hydrocarbons. VOP Profil Zagrejazneniya Uhnesh.
Sredy, Chastnosti Vodocmov, Kanterogen Veshchesto. 1972:5-13.
8. Sander, J. E., E. Schweinsberg, M. Ladenstein, and F. Schweinsberg.
Toxicological evaluation of some important nitrogen-containing
substances in water. Zentrabl, Bakteriol, Parasitenk, Infek-
tionskr. Hyg. Abstr. l_:0rig., Reihe A, 227(1/4):71-80, 1974.
9. Dure, G., L. Weil, and K. E. Quentin. Determination of nitrosamines
in natural water and waste water. Z. Wasser Abwasser Forsch. 8{1):
20-30, 1975. [Chem. Abstr. 83(4):32828F.]
10. Ender, F., G. Havre, A. Helgebostad, N. Koppang, R. Madsen, and L.
Ceh. Isolation and identification of a hepatotoxic factor in her-
ring meal produced from sodium nitrite preserved herring. Natur-
wissenschaften. 51_(4) :637-638, 1964. [Chem. Abstr. 62_:7035A. ]
134
-------
11. Ender, F. and L. Ceh. In: Proceedings of Second Conference on
Tobacco Research. FreiBUrg, Germany. 1967. p. 83.
12. Freimuth, U. and E. Glaser. Occurrence of nitrosamlnes in foods.
Die Nahrung. HC5):357-361, 1970.
13. Howard,_J_._W., T. Fazio,, and J. 0,., Watts. Extraction and gas
chromatographic determination of N-nitrosodimethylamine in smoked
fish: Application to smoked nitrite-treated chub. J. Assoc. Off.
Anal. Chem. 53:269-274, 1969.
14. Malins, D. C.', W. T. Roubal, and P. A. Robisch. The possible nitro-
sation of amines in smoked chub. J. Agric. Food Chem. 18:740-741,
1970.
15. Fazio, T., J. N. Damico, J. N. Howard, R. H. White, and J. 0. Watts.
N-nitrosodimethylamine in smoke-processed marine fish. J. Agric.
Food Chem. l_9:250-253, 1971.
16. Alliston, T. G., G. B. Cox, and R. S. Kirk. The determination of
steam-volatile N-nitrosamines in foodstuffs by formation of electron-
capturing derivatives from electrochemically derived amines. Analyst.
97:915-920, 1972.
17. Fong, Y. Y. and W. C. Chan. Dimethylnitrosanrine in Chinese marine
salt fish. Fd. Cosmet. Toxicol. 1^:841-845, 1973.
18. Fong, Y. Y. and W. C. Chan. Bacterial production of dimethylnitro-
samine in salted fish. Nature. 243_:421-422, 1973.
19. Gadbois, D. F., E. M. Ravesi, R. C. Lundstrom, and R. S. Maney.
N-nitrosodimethylamine in cold-smoked sablefish. J. Agric. Food
Chem. 231:665-668, 1975,
20. Havery, D. C., D. A. Kline, E. M. Miletta, F. L. Joe, Jr., and T.
Fazio. Survey of food products for volatile N-nitrosamines. J.
Assoc. Off. Anal. Chem. 59_:540-546, 1976.
21. Fazio, T., R. H. White, and J. W. Howard. Analysis of nitrite-and/
or nitrate-processed meats for N-nitrosodimethylamine. J. Assoc.
Off. Anal. Chem. 54:1157-1159, 1971.
22. Wasserman, A. E., W. Fiddler, R. C. Doerr, S. F. Osman, and J. C.
Dooley. Dimethylnitrosamine in frankfurters. Fd. Cosmet. Toxicol.
JO:681-684, 1972.
23. Panalaks, T., J. R. lyengar, and N. P, Sen. Nitrate, nitrite, and
dimethylnitrosamine in cured meat products. J. Assoc. Off. Anal.
Chem. 56_:621-625, 1973.
135
-------
24. Panalaks, T., J. R. lyengar, B. A. Donaldson, W. F. Miles, and
N. P. Sen. Further survey of cured meat products for volatile N-
nitrosamines. J. Assoc. Off. Anal. Chem. 57^:806-812, 1974.
25. Sen, N. P., W. F. Miles, B. A. Donaldson, T. Panalaks, and J. R.
lyengar. Formation of nitrosamines in a meat curing mixture.
Nature. 245:104-105, 1973.
26. Sen, N. P., B. A. Donaldson, C. Charbonneau, and W. F. Miles.
Effect of additives on the formation of nitrosamines in meat curing
mixtures containing spices and nitrite. J. Agric. Food Chem. 22:
1125-1130, 1974.
27. 9 CFR 121.13. Revised in Federal Register. 38(221):31679,
November 16, 1973.
28. Fiddler, W., J. I. Feinberg, J. W. Pensabene, A. C. Williams, and
C. J. Dooley. Dimethylnitrosamine in souse and similar jellied
cured-meat products. Fd. Cosmet. Toxicol. 13:653-654, 1975.
29. Fazio, T., R. H. White, L. R. Dusold, and J. W. Howard. Nitro-
sopyrrolidine in cooked bacon. J. Assoc. Off. Anal. Chem. 56:
919-921, 1973.
30. Pensabene, J. W., W. Fiddler, R. A. Gates, J. C. Fagan, and A. E.
Wasserman. Effect of frying and other cooking conditions on nitro-
sopyrrolidine formation in bacon. J. Food Sci. 39_:314-316, 1974.
31. Sen, N. P., B. A. Donaldson, J. R. lyengar, and T. Panalaks.
Nitrosopyrrolidine and dimethylnitrosamine in bacon. Nature. 241:
473-474, 1973.
32. Huxel, E. T., R. A. Scanlan, and L. M. Libbey. Formation of N-
nitrosopyrrolidine from pyrrolidine-ring-containing compounds at
elevated temperatures. J. Agric. Food Chem. 22_:698-700, 1974.
33. Kushnir, I., J. I. Feinberg, J. W. Pensabene, E. G. Piotrowski,
W. Fiddler, and A. E. Wasserman. Isolation and identification of
nitrosoproline in uncooked bacon. J. Food Sci. 40_:427-428, 1975.
34. Fiddler, W., J. W. Pensabene, J. C. Fagan, E. J. Thome, E. G.
Piotrowski, and A. E. Wasserman. A research note. The role of
lean and adipose tissue on the formation of nitrosopyrrolidine in
fried bacon. J. Food Sci. ^9:1070-1071, 1974.
35. Sen, N. P., J. R. lyengar, B. A. Donaldson, and T. Panalaks. Effect
of sodium nitrite concentration on the formation of nitrosopyrroli-
dine and dimethylnitrosamine in fried bacon. J. Agric. Food Chem.
22:540-541, 1974.
136
-------
36. Fiddler, W., E. G. Piotrowski, J. W. Pensabene, and A. E. Wasser-
man. Proceedings of the 18th Meeting of Meat Research Workers,
University of Guelph, Canada. 1972. p. 416.
37. Herring, H. K. Proceedings of the Meat Industry Research Confer-
ence. 1973. p. 47.
38. Challis, B. C. Rapid nitrosation of phenols and its implications
for health hazards from dietary nitrites. Nature. 244:466, 1973.
39. Knowles, M. E., J. Gilbert, and D. J. McWeeny. Nitrosation of
phenols in smoked bacon. Nature. 249:672-673, 1974.
40. White, J. W., Jr. Relative significance of dietary sources of
nitrate and nitrite, J. Agric. Food Chem. 23;886-891, 1975.
41. Ender, F. and L. Ceh. Occurrence of nitrosamines in foodstuffs for
human and animal consumption. Fd. Cosmet. Toxicol. 6:569-571,
1968.
42. Hedler, L. and P. Marquardt. Occurrence of diethylnitrosamine in
some samples of food. Fd. Cosmet. Toxicol. 6^:341-348, 1968.
43. Thewlis, B. H. Nitrosamines in wheat flour. Fd. Cosmet-. Toxicol.
6_:822-823, 1968.
44. ((roller, E. Detection of nitrosamines in tobacco smoke and food.
Deut. Lebensm. Rundsch. 63^:303-305, 1967.
45. DuPlessis, L. S., J. R. Nunn, and W. A. Roach. Carcinogen in a
Transkeian Bantu food additive. Nature. 222;1198-1199, 1969.
46. Laquer, G. L., 0. Mickelson, M. G. Whiting, and L. T. Kurland.
Carcinogenic properties of nuts from Cycas circinalis indigenous
to Guam. J. Nat. Cancer Inst. 31:919-951, 1963.
47. Keybets, M. J., E. H. Broot, and G. H. Keller. An investigation
into the possible presence of nitrosamines in nitrite-bearing
spinach. Fd. Cosmet. Toxicol. *3:167-171, 1970.
48. Rhoades, J. W. and D. E. Johnson. N-dimethylnitrosamine in tobacco
smoke condensate. Nature. 236_:307-308, 1972.
49. Lijinsky, W. and S. S. Epstein. Nitrosamines as environmental
carcinogens. Nature. 2215:21-23, 1970.
50. Lijinsky, W. Reaction of drugs with nitrous acid as a source of
carcinogenic nitrosamines. Cancer Res. 34:255-258, 1974.
137
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51. Elespuru, R. K. and W. Lijinsky. The formation of carcinogenic
nitroso compounds from nitrite and some types of agricultural
chemicals. Fd. Cosmet. Toxicol. Vh807-817, 1973.
52. Lijinsky, W. In: Regulation of Food Additives and Medicated
Animal Feeds. House of Representatives, 92nd Congress. U.S.
Government Printing Office. Washington, D.C. No. 5270 1144.
1971. p. 8-167.
53. Lijinsky, W., E. Conrad, and R. Van de Bogart. Carcinogenic nitro-
samines formed by drug/nitrite interactions. Nature. 239:165-167,
1972.
54. Lijinsky, W. and R. K. Elespuru. Mutagenicity and carcinogenicity
of N-nitroso derivatives of carbamate insecticides. Presented at
4th Meeting of International Agency for Cancer Research, Estoniz,
U.S.S.R., October 1-2, 1975.
55. Eisenbrand, G., 0. Lingerer, and R. Preussman. The reaction of
nitrite with pesticides. II. Formation, chemical properties,
and carcinogenic activity of the N-nitroso derivative of N-methyl-
1-naphthyl carbamate (carbaryl). Fd. Cosmet. Toxicol. 13:365-367,
1975. ~
56. Wolfe, N. L., R. G. Zepp, J. A. Gordon, and R. C. Finches. N-
nitrosamine formation from atrazine. Southeast Environmental
Research Laboratory, U.S. Environmental Protection Agency. Athens,
Georgia. 1975.
57. Eisenbrand, G., 0. Ungerer, and R. Preussmann. Rapid formation
of carcinogenic N-nitrosanrines by interaction of nitrite with
fungicides derived from dithiocarbamic acid in vitro under simu-
lated gastric conditions and in vivo in the rat stomach. Fd.
Cosmet. Toxicol. J2.:229-232,• T974.
58. Wolfe, N. L., R.. G. Zepp, J. A. Gordon, and R. G. Fincher. N-
nitrosamine formation from atrazine. Bull. Environ. Contamin.
Toxicol. Ji(3):342-347, 1976.
59. Fine, D. H. and D. Ross. Presented at American Chemical Society
Meeting, San Francisco, September 1976. Cited in: Chem. and Eng.
News. September 20, 1976, p. 33 and 34.
60. Pesticide Chem. News. September 22, 1976, p. 12-15.
138
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61. Eisenbrand, G., 0. Ungerer, and R. Preussman. Fonnation of N-
nitroso compounds from agricultural chemicals and nitrite. In:
N-Nitroso Compounds in the Environment. Proceedings of lARCTRbrk-
ing Conference, October 17-20, 1973. Lyon, France. IARC Scien-
tific Publication No. 9. 1975. p. 71-74.
62. Kant, V. Formation of nitrosamines (in lung tissue) after inhala-
tion of nitrogen oxides. Cezk. Hyg. 1970:213-215. 1970.
139
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7. SOURCES
7.1 NATURAL OCCURRENCE
Nitrates, nitrites, and amines, which are precursors in the formation
of nitrosamines, are chemical compounds that occur naturally in food,
water, soil, and air. Unlike many man-made chemical compounds currently
causing environmental problems, the above-named chemical compounds have
probably been in existence throughout the evolutionary history of man.
Nitrites are in themselves toxic to man, other animals, and plants.
However, the nitrosamines that may be formed in soil, water, air, food,
and in the gastrointestinal tract through the chemical and microbial
nitrosation of secondary and tertiary amines are the greater hazard to
man.
7.1.1 Formation of Nitrogen Compounds in the Nitrogen Cycle
Natural occurrence of nitrates, nitrites, and secondary and tertiary
amines is the result of their formation during the nitrogen cycle.
Except for man-made fertilizers, all nitrogen necessary for the exist-
ence of terrestrial or aquatic organisms becomes available through the
nitrogen cycle. Nitrogen in the form of nitrates or ammonia is taken
up by plants and incorporated into proteins or nucleic acids. When
plants are eaten by animals, the proteins and nucleic acids become
available to the animals for use in forming their own nitrogen compounds.
In the environment, through the breakdown of animal excreta and the decay
of dead plant and animal tissues by microorganisms, complex nitrogen-
containing compounds are ultimately converted into ammonia. Should
140
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a portion of the decay process occur without free oxygen, amines may be
2
formed. These transformations of nitrogen are mediated almost entirely
2-7
by microorganisms and involve organic, inorganic, and volatile com-
pounds. In general outline, the nitrogen cycle is identical in terres-
trial and aquatic habitats; only the microorganisms that mediate the
3 6
various transformations are different. * Though termed a cycle, the
transformations of nitrogen occur simultaneously and involve its being
2
shuttled back and forth into different forms.
8-10
The largest source of elemental nitrogen is the atmosphere. ~ For
nitrogen to become available for use by plants and animals, it must be
transformed or fixed into organic nitrogen. Small amounts of nitrogen
are fixed in the atmosphere and come down to earth in rainwater as
p c_o ,
ammonia and nitrate salts, ' ~ but biological fixation is the princi-
pal way in which nitrogen becomes available to life on earth.
^a
7.1.1.1 Biological Nitrogen Fixation—Biological fixation results in the
transformation of nitrogen gasxinto nongaseous nitrogen compounds. The
transformation is carried out by a variety of microorganisms, some of
which live symbiotically in the root nodules of plants (chiefly legumes)
while others are free-living in the soil. The process may be accom-
plished under aerobic or anaerobic conditions.
7.1.1.2 Organic Nitrogen Formation (Assimilation)—Fixed nitrogen, as
either ammonia or nitrates, is assimilated by microorganisms and plants
and converted into plant protein, nucleic acids, and various other
organic complexes. Plants are eaten by animals and animals by other
141
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animals. Nitrogen is immobilized in the many nitrogen-containing com-
pounds until the organisms die or, in the case of animals, until certain
products are excreted.
7.1.1.3 Mineralization (Ammonif ication) — In transformation by minerali-
zation, or ammonification, a portion of the large reservoir of organic
complexes in the soil is decomposed and converted into inorganic ammonia.
Microbial action results in the breakdown of protein, polypeptides,
nucleoproteins, nucleic acids, and aromatic compounds. From the degra-
dation of protein-rich materials, if the transformation proceeds
2
anaerobically, the final products can be ammonia and amines.
The ammonia formed during mineralization may be assimilated by aquatic
plants, terrestrial plants, or by microorganisms; may be bound by clay
particles in the soil; may escape into the air; or may be converted by
microorganisms into nitrates in the nitrification process. Under 4*!-
anaerobic conditions, ammonia accumulates because nitrification cannot
occur.
7.1.1.4 Mi tri f i cation—Mi tri f i cation is the microbial conversion of
ammonia to nitrites and nitrates. Nitrification occurs in the soil, in
2
manure piles, during sewage processing and in marine environments.
The first step in the conversion is the oxidation of ammonia to nitrite
ion by Nitrosomonas bacteria. These bacteria use ammonia as their sole
source of energy. Nitrite in turn is rapidly oxidized by Nitrobacter
organisms usually found in association with Nitrosomonas. In some cul-
tures, nitrite may accumulate if the Nitrobacter cannot oxidize all the
142
-------
257
nitrites produced by Nitrosomonas. ' ' Nitrite is usually converted
to nitrate so rapidly that it seldom accumulates in the soil; in alka-
line soils, however, nitrite accumulation may occur if levels of ammonium
are high.
Nitrates—whether added to the soil in fertilizers or formed by
nitrification—may be assimilated by plants; washed downward through
the soil into ground water or through surface runoff into streams,
rivers and oceans; may be transformed into atmospheric nitrogen during
microbial denitrification; or may be reduced by microorganisms to
ammonia.
7.1.1.5 Nitrate Reduction—Microorganisms can utilize nitrates to build
organic nitrogen compounds just as plants do, or they can utilize some
or all of the nitrate for its oxygen content if local conditions are
anaerobic. The second case is denitrification (see below). Incomplete
reduction may result in an accumulation of nitrite.
7.1.1.6 Denitrification—When certain bacteria are deprived of oxygen,
they will turn to nitrates, N0~,as a source of oxygen. The first step
is the reduction of nitrate to nitrite. Many bacteria are capable of
reducing nitrite to N« or N2<) gases, again under local anaerobic condi-
tions. If the two types of reduction are not in balance, nitrites may
accumulate in the medium. Further, nitrites may accumulate in alkaline
2
soils. Only local anaerobic conditions are necessary for denitrifica-
tion and significant denitrification may occur in the center of aerated
floes of bacteria or at the base of slime layers. The ratio of N2 to
143
U.S EPA Headquarters Library
Mail code 3404T
1200 Pennsylvania Avenue NW
Washington, DC 20460
202-566-0556
-------
NpO produced probably depends on pH, temperature, and other environ-
mental factors.
Figure 7-1 is a diagrammatic presentation of the movement of nitrogen
in the biosphere. The transformation and movement of nitrogen as
explained in the foregoing pages relate to the biogeochemical circula-
tion of nitrogen. The circulation of nitrogen is a long-term process.
Q
Turnover times for the three largest "pools" of nitrogen are 3 x 10
years for atmospheric nitrogen; 2500 years for nitrogen in the seas
when nitrates and organic compounds are counted together; and less than
1 year for nitrates and nitrites in the soil. A more detailed account
of the distribution and annual transfer rates is shown in Table 7-1.
Because the nitrogen cycle is so complex and has so many exchange rates,
it is difficult to estimate with a great degree of accuracy. Table 7-1
lists the estimates with probable errors. Thus the transfer rates can
be estimated only within broad limits. ' The only two quantities of
nitrogen known with any degree of accuracy are the amount of nitrogen
in the atmosphere and the rate of industrial fixation.
7.1.2 Nitrogen Compounds In Soil
The levels of nitrate and nitrite nitrogen in the soil are in a constant
state of flux. Nitrites exist in the soil for only short periods of
time. Under normal conditions, nitrite is rapidly converted to nitrates,
nitrous oxide, and nitrogen; or undergoes spontaneous decomposition to
235
nitric oxide. ' ' Nitrite does accumulate in alkaline soils when
p
ammonium levels are high, however. The accumulation results because
144
-------
ANIMALS
GASEOUS LOSS
N-FIXATION
i
FERTILIZER
AND RAIN
RESIDUES,
MANURES
AND WASTES
Figure 7-1. Main portionsj)f the nitrogen cycle. _ Additions of chemical : fertilizer make up an
increasingly important source of this element.?
145
-------
Table 7-1. BUDGET FOR THE NITROGEN CYCLE8'11
CTt
Land
Input
Biological nitrogen fixation
Symbiotic-31
Non symbiotic3^ ~,
Atmospheric nitrogen fixation 3,
Industrially fixed nitrogen fertilizer
N-oxides from combustion
Return of volatile nitrogen compounds
in rain 3,
River influx 31
N£ from biological denitrifi cation
Natural NO?
Volatilization (HN3)
Total
Storage ,1
Plants'31
Animals3^ 3,
Dead organic matter
Inorganic nitrogen3^
Dissolved nitrogen3^
Million
MT/yr
14
30
4
30
14
?
__
--
_—
—
>92
12,000
200
760,000
140,000
Error?
25
50
100
5
25
—
__
—
__
—
30
30
50
' 50
Sea
Million
MT/yr
10
--
4
—
6
?
30
__
—
50
800
170
900,000
100,000
20,000,000
Atmosphere
Error, Million
% MT/yr
50
—
100
—
25 20
__
50
83
?
?
>103
50
50
100
50
10
Error,
'
—
—
—
25
—
__
100
__
—
—
__
--
__
Nitrogen gas31 — — — -- 3,800,000,000 3
NO + NH42?7 - -- — -- •
-------
Table 7-1 (continued). BUDGET FOR THE NITROGEN CYCLE8'11
Loss 3,
Den itrifi cation
Volatilization
River runoff^ »32 (includes enrichment
from fertilizers)
Sedimentation^l
N2 in all fixation processes
N&3 in rainl?
N02 in rain
NpO in rain
'-Total
Land
Million Error,3
MT/yr %
43
30 50
73
Sea
Million Error,
MT/yr %
40 100
0.2 50
40.2
Atmosphere
Million Error,
MT/yr %
92 50
< 40 50
>132
aThe error columns list plus-or-minus probable errors as a percentage of the estimate.
-------
the nitrite formed is not metabolized further to nitrate. The effect on
nitrite accumulation of the interaction of alkalinity and ammonium is
illustrated in Table 7-2. The accumulation of nitrite in calcareous
soils is proportional to the rate of ammonium addition; under constant
nitrogen fertilization the effect increases as the hydrogen ion concen-
2
tration decreases. With decrease in pH or ammonium levels, the suppres-
sion of nitrate formation is relieved. The sensitivity to ammonium salts
exhibited by microorganisms (Nitrobacter group) that transform nitrite to
nitrate appears to be the factor in nitrite buildup.
Table 7-2. EFFECT OF LIME AND NITROGEN ON
NITRITE ACCUMULATION2
Nitrate nitrogen, ppm
Treatment Soil 1 Soil 2 Soil 3 Soil 4
None 0000
Calcium carbonate +
calcium sulfate
[CaC03 + CaSo4], 1% 54 59 5 5
Ammonium sulfate
[(NH4)2S04], 500 ppm N 3 0 0 0
Calcium carbonate +
ammonium sulfate
[CaC03 + (NH4)2S04] 255 218 260 310
Through microbiological action, nitrates in the soil are constantly being
formed and also being lost. Losses can be caused by assimilation by
higher plants or microorganisms, loss through drainage and runoff water,
or loss through transformation into gaseous nitrogen compounds. A meas-
ure of nitrate concentrations in soil is, therefore, only an indication
148
-------
2357
of short-term concentrations. ' ' ' To be available to plants and
12
microorganisms, nitrates must be in the soil solution. Bowen lists
the total nitrogen content of the soil as ranging from 200 to 2500 ppm,
while the nitrate-nitrogen in soil solution is considered to range from
2 to 800 ppm. High soil nitrate levels have been found chiefly in areas
of low rainfall as a result of nitrification and absence of leaching
through runoff.
As mentioned in the previous section, amines are formed in soil during
the degradation of protein-rich organic matter, as in crop residues and
in green and farm manures. Further, dimethyl amine itself is found in
pres
2,7
urine and feces and would be present in manures. Humus is also believed
to contain ami no combinations.
Ayanaba et al. have noted the formation of dimethylamine in soil from
trimethylamine. It forms readily at pH values of 5,8 and 6.5 but slowly
at a soil pH of 3.8.
Ayanaba et al. have shown that microorganisms in soil may participate
in the formation of nitrosamines, such as dimethylnitrosamine (DMN), in
one of three ways: (1) by converting tertiary amines or other nitro-
«
genous compounds to secondary amines; (2) by forming nitrite through the
reduction of nitrate or oxidation of ammonium, the latter leading to
appreciable nitrite accumulation in alkaline environments; and (3) by
causing an enzymatic reaction between-nitrite and the secondary amine.
149'
'f
-------
Mills has noted the formation of nitrosamines in soil and water at
neutral pH's in the presence of colloidal material. He also reported
that the formation of nitrosamines in soil need not be mediated by
microbial action.
Tate and Alexander reported the persistence of nitrosamines when
Williamson silt loam was amended with N-nitrosodimethylamine, N-nitroso-
diethylamine and N-nitrosodi-n-propylamine at three different levels
(Figure 7-2). However, in the event of rainfall, nitrosamines may be
18
rapidly leached from the soil.
The precursors of nitrosamine formation are unquestionably present in
soil, but the extent to which nitrosamines are actually formed in the
soil and therefore represent a potential health hazard to man is not
known.
7.1.3 Nitrogen Compounds in Aquatic Habitats
Aquatic habitats contain the precursors of nitrosamines, just as soil
does. In the discussion on the natural occurrence of nitrosamines, men-
tion was made that the nitrogen cycle in terrestrial and aquatic habi-
tats is the same in general outline, with only the microorganisms that
mediate the transformations being different. ' Nitrates, nitrites, and
amines are all found in aquatic habitats. »I9»^° Nitrates are not only
formed in the nitrogen cycle, but are frequently present at high levels
in runoff waters in agricultural areas. A study made in Illinois indi-
cates that nitrate concentrations in the Kaskaskia and Sangamon Rivers
peaked in May and June, the time of heavy chemical fertilizer
150
-------
N-NITROSODIMETHYLAMINE
NITROSODIETHYLAMINE
NITROSODIPROPYLAMINE
TIME, days
Figure 7-2. Disappearance of tTiree nTtrosamines from Williamson silt'ioam.T8
151
-------
21
application. This was in contrast to nitrate levels in the Skillet
Fork River which drains an area not heavily fanned. The Skillet Fork
showed no May-June peak (Figure 7-3). Another study made in the area of
the Sangamon River watershed using nitrogen-15 led to the estimate that
at the time of peak nitrate concentration in the spring a minimum of 55
to 60 percent of nitrate-nitrogen found in surface waters of the water-
22
shed was from fertilizer nitrogen.
Precipitation, sewage, and nitrogenous wastes from farms and packing
houses, as well as the use of organic chemicals such as pesticides, are
other sources of nitrogen that can lead to increases of nitrates, nitrites,
21
and amines in water.
Ground water receives nitrates from many of the same sources as surface
21 -25
waters; fertilizers, runoff, and percolation from feedlots are among
the main contributors. Nitrites are frequently found in low concentra-
23
tions because they can be readily oxidized to nitrates.
Specific concentrations of nitrates in streams or particularly in lake
waters will depend on the sources of the nitrates. The concentration
of any nitrogen compound in water is the net result of the rates of nitro-
gen immobilization (assimilation by aquatic organisms), mineralization
(decomposition of organic complexes), nitrification (formation of
o
nitrates), and denitrification (production of gaseous nitrogen). These
levels vary during the year with the N03-nitrogen and NO^-nitrogen levels
in lakes being high in the spring and minimal during middle to late sum-
mer. The NhL-nitrogen levels, on the other hand, are usually highest in
152
-------
(9
H
== I
in =
Ul
o
t-
t±-*—^~*"
OKASKASKIA, 1945-1950
O KASKASKIA, 1956 -1968
• SKILLET FORK, 1945 -1950
• SKILLET FORK, 1945-1961 ,
T:
JA~N. FEB. MAR. APR. MAY JUN. JUL. AUG. SEPT. OCT. NOV. DEC.
Figure 7-3. Nitrate concentrations in the Kaskaskia and Sangamon Rivers.22
153
-------
20
surface waters in the fall and highest in deep waters during summer.
The levels are influenced by biological utilization and/or denitrifica-
20
tion. The effects of biological activity may also be seen in many
lakes that exhibit a N03-nitrogen distribution pattern in which levels
are low in surface and bottom waters and at a maximum in intermediate
depths. In surface waters, the nitrate is immobilized, and in bottom
20
water, denitrified. Not all aquatic plants utilize NCL-nitrogen.
Some take up NH,-nitrogen preferentially over NCk-nitrogen. The "bloom"
of aquatic organisms has been associated chiefly with high N03-nitrogen
levels.
— 13-15 22
Ayanaba, Alexander, and their coworkers ' have demonstrated that
nitrosamines can be formed in aquatic environments. Dimethylnitrosamine
(DMN) was formed in lake water upon the addition of dimethylamine (DMA)
and nitrite (N02~), DMA and nitrate (N03~), trimethylamine and NOp", or
tri methyl ami ne and NO.,". Mills noted the formation of DMN in sterile
and nonsterile lake water and sewage, just as had occurred in soil. On
the basis of these studies, he concluded, first, that microorganisms are
not necessary to bring about the combination of DMA and N0p~ to form DMN
even at pH values near neutral; second, that the chemical nitrosation
reaction is promoted by conditions of low pH and high concentrations of
organic matter; and, third, that in this situation the role of micro-
organisms in the formation of DMN is an indirect one and includes the
formation of secondary amine precursors, formation of N02~, and modifi-
cation of the environment through the production of organic matter and
pH alteration. Secondary amines are widespread in nature, but their
154
-------
concentration in soil and water is usually low. An accumulation of
secondary amines in water could result in spontaneous nitrosation. The
major requirement for nitrosation in water and soil is probably the
simultaneous presence of precursors.
7.1.4 Nitrogen Compounds in Plants
Nitrosamines are not commonly found in plants, but the precursors neces-
sary for their formation are. Dimethylnitrosamines, however, have been
detected in a member of the tobacco family, Solanum ihcahum, used as food
?fi
by the Transkeian Bantu.
Secondary amines and nitrites are the chief precursors needed for the
formation of nitrosamines. Secondary amines .such as DMA, diethylamine,
methylethyl amine, and methylpropyl amine have been found in tobacco
plants. Algae have been found to contain many amines, including DMA,
trimethylamine, and other methyl amines. Nitrites are found only in
27
small concentrations in plant tissues. The sources of nitrites in
I c pi O7
food are nitrates in plants which become reduced after harvest. ' '
The nitrate levels in plants are of interest, therefore, in determining
potential nitrite levels.
In most plants, nitrate is reduced to ammonia and then used in the syn-
28
thesis of protein and other organic compounds. Accumulation of nitrate
in plants may indicate that the rate of assimilation has not kept up with
the rate of uptake, but the accumulation of nitrate in such cases is only
07
temporary and diminishes as the plant ages. The stage of development
and a variety of environmental factors influence the nitrate content, so
155
-------
that variation from plant to plant, as well as within species, genus, or
27
family, may be very great. Plants that normally have low levels of
nitrate may under certain conditions accumulate it to very high levels.
Perennial forage grasses have been found to be low in nitrate in many
tests, but have been observed to accumulate nitrate in other cases.
The age of plants, plant variety, site of growth of some plant species,
soil type, and environmental factors such as moisture, temperature, pH,
and the nitrate concentration in the soil all are important in deter-
27 28
mining the plant nitrate concentration. '
High nitrate levels in plants are associated with rapid assimilation and
low rates of reduction. The nitrate content of oats, corn, sugar beets,
27 28
and Pennisetum (Napiergrass) are given in Tables 7-3 through 7-6. '
The plant families most often considered to be accumulators of nitrates
are: Amaranthaceae (Amaranth), Chenopodiaceae (Goosefoot), Cruciferae
(Mustard), Asteraceae (Aster), Poaceae (Grass), and Solanaceae (Night-
shade). No single family has been thoroughly sampled for nitrate accumu-
27
lation. Of the families listed, the Mustard, Grass, and Nightshade
families contain the largest number of edible plants.
Nitrate is not uniformly distributed throughout plant tissues—stems
usually contain more nitrates than leaves, and leaves more nitrate than
flower parts. Roots have not been extensively tested but appear to
contain lower levels than stems. In fodder sugar beets, the oldest
leaves were found to be highest in nitrate nitrogen.
156
-------
Table 7-3. NITRATE-NITROGEN CONTENT, AS PERCENTAGE OF
DRY MATTER, OF OAT VARIETIES GROWN FOR HAY UNDER
IRRIGATION AND FERTILIZATION (LARAMIE) AND UNDER
DRY LAND CONDITIONS (ARCHER) IN WYOMING1!
Stages3
La ramie
Variety
Improved Garry
Swedish Select
12- Variety
average
1
1
1
1
.22
.36
.32
0.
1.
1.
2
87
06
00
3
0.78
0.84
0.81
4
0.65
0.87
0,00
1
0.40
1.00
0 = 58
Archer
2
0.41
0.64
0,48
3
0.20
0.42
0.33
4
0.18
0.19
0.20
Overall
mean
0.59
0.80
0.67
•Stages: 1—25% of heads flowering; 2—50% of heads in milk; 3—50% of
heads in soft dough; 4—50% of heads in hard dough.
157
-------
Table 7-4. DISTRIBUTION OF NITRATE-NITROGEN IN
DENT CORN PLANTS, EXPRESSED AS PERCENTAGE
OF AIR-DRY WEIGHT11
Nodal
position
from
root to
tassel
1
2
3
4
5
6
7
8
9
10
11
12
13
14
NOg-nitrogen, % of
Leaf
lamina
0.049
0.034
0.024 •
0.014
0.007
0.007
0.006
0.007
0.006
0.008
0.006
0.007
0.008
0.003
Leaf
midrib
0.081
0.063
0.052
0.048
0.031
0.031
0.001
0.004
-
-
-
-
-
_
Leaf
sheath
0.126
0.060
0.060
0.043
0.032
0.024
0.021
0.012
0.012
0.010
0.024
0.020
0.006
0.008
Internode
0.293
0.269
0.202
0.139
0.120
0.097
0.092
0.092
0.080
0.031
0.063
0.066
0.095
_
air- dry
Shank
-
0.085
0.046
0.049
0.027
0.020
0.015
--.-
-"'
-
-
-
-
_
wt
Ear
(fertile)
-
-
-
-
0.008
0.011
0.010
-
-
-
-
-
-
_
Tassel
'
-
-
-
- •
-
-
-
-
-
-
-
-
0.020
158
-------
Table 7-5. CONTENT OF TOTAL NITROGEN AND NITRATE-
NITROGEN IN LEAVES OF FODDER SUGAR BEETS,
EXPRESSED AS PERCENTAGE OF DRY WEIGHT11
Sample
Petioles
Total N
N03-Na
N03-N, % of total
Blades
Total N
N03-N
N03-N, % of total
Total
Center
3.80
0.23
6.0
5.70
0.07
1.2
and nitrate nitrogen, % of dry wt
Leaf p
Inner
2.03
0.45
22.1
4.80
0.06
1.4
osition
Middle
1.73
0.74
42.5
4.15
0.10
2.4
Outer
1.89
1.28
67.5
3.60
0.18
5.0
Weighted
mean
1.87
0.83
44.2
4.10
0.12
3.0
)3-N is nitrate-nitrogen.
159
-------
Table 7-6. DISTRIBUTION OF NITRATE-NITROGEN IN
2- inch SEGMENTS OF LEAVES ON A SHOOT OF
1 Pennisetum purpureum^
Segment,
inches
Sheath
0-2
2-4
4-6
Blade
0-2
2-4
4-6
6-8
8-10
10-12
12-14
14-16
--
Approximate
Outside (lowest)
leaf
0
0
0
0
-
-
-
-
-
-
-
nitrate nitrogen,
Fourth
leaf
16
16
2
1
0
0
0
0
0
-
-
ppma
Eighth
leaf
concealed
concealed
concealed
1
>6
>6
2
2
0
0
0
Rough quantitative analysis with dimethy1 amine.
160
-------
The site of nitrate reduction in plants is not definitely known. It has
been suggested that in woody plants nitrate is reduced in the roots; in
27 28
herbaceous plants, in the leaves. McKee, however, thinks that suffi-
cient information does not exist for determining the site of reduction
with any degree of certainty.
The activity of the enzyme, nitrate reductase, has been said to influence
nitrate levels in plants. Energy is required for nitrate reduction, with
carbohydrate the energy source. High levels of nitrogen appear to stimu-
late plants to utilize stored carbohydrate for energy with which to reduce
the nitrate via nitrate reductase. As a result of rapid nitrate assimila-
tion and rapid carbohydrate utilization, the level of plant nitrate
increases and the carbohydrate level decreases, so that a plant high in
27
carbohydrate is likely to be low in nitrate.
Nitrogen uptake by plants is also influenced by the nitrogen content of
the soil and by soil moisture, with potassium nitrate appearing to be
28
more rapidly taken up than either calcium or sodium nitrate.
Molybdenum and manganese deficiencies have been shown to be associated
with accumulations of nitrate in both plants and microorganisms. Molyb-
denum is the metallic component of the nitrate reductase enzyme.
Moisture-dependent processes are involved in the transformation and trans-
location of nitrate. Microbial activity* which releases nitrate from com-
plex organic compounds, requires moisture. The nitrate released in this pro-
cess also requires moisture to move through the soil to the plant roots
161
-------
and move across the cell membranes. Fertilizer nitrogen also requires
moisture if a plant is to utilize it. Thus, plants that have been under
a moisture-shortage stress may accumulate high levels of nitrate in a
very few days. The moisture shortage results in a disturbance of assimi-
latory processes. As a result, a drop in nitrate reductase activity may
occur while the plant continues to assimilate nitrates. Attempts to
duplicate nitrate accumulation through experimental moisture stress have
27
been few and not very successful.
A variety of other factors, such as light, herbicides, temperature, soil
type, and parasitization by diseases and insects, have all been said to
influence nitrate concentrations in plants.
Studies exist which indicate that spinach and lettuce can assimilate
nitrosamines when they are present in the soil.
7.1.5 Nitrosamines in Air
Theoretically, nitrosamines can be formed in the ambient air when sec-
ondary amines, nitrogen oxides, and sulfur dioxides are present in the
air.29
•
Sufficient data are not now available to determine the extent to which
nitrosamines are formed in polluted or unpolluted atmospheres.
7.2 OCCURRENCE IN WASTE WATER30
Since N-nitroso compounds are used as intermediates in certain industrial
processes, N-nitrosamines may be found in waste water and sewage. An
162
-------
investigation in Baltimore, Maryland, and in Belle, West Virginia, has,
in fact, shown the presence of nitrosamines in waste water.
Waste water samples taken from DuPont in the West Virginia study showed
the presence of DMN at levels of 4.2, 2.3, and 2.4 yg/liter. A waste
water sample from Fike Chemical Company in Nitro, West Virginia, also
contained DMN at the 3.0 yg/liter level. It is suspected that DMN levels
in waste water arise from either DMN impurities in the amine process or
subsequent nitrosation of the amine in the waste treatment facility.
A water sample taken from a sewage treatment facility adjacent to the
Food Machinery and Chemical Corporation (FMC) plant in Baltimore con-
tained 2.7 yg/liter of DMN. The plant produces unsymmetrical dimethyl-
hydrazine (UMDH). On the FMC property, close to the UDMH plant, a rain-
water puddle contained over 6000 yg/liter of DMN. A mud sample in the
same area contained over 200 yg/liter. A water sample from a drainage
ditch on the border of the FMC property with an adjacent firm (British
Petroleum) contained 5.9 yg/liter. Water taken directly from an adja-
cent cove contained DMN levels in the 0.26 to 0.94 yg/liter range.
7.3 INDUSTRIAL SOURCES OF OCCURRENCE
7.3.1 Industrial Processes in Which Nitrosamines Occur As Primary Prod-
duct or Intermediate,
Although a large patent literature exists on nitrosamines, these com-
pounds do not appear to have been used widely. Patent applications
reveal potential use in the manufacture of rubber, dyestuffs, gasoline
additives, lubricating oils, explosives, insecticides, fungicides,
dielectric fluids, acrylonitrile, plasticizers, industrial solvents
163
U.S EPA Headquarters Library
Mai; code 3404T,
1200 Pennsylvania Avenue NW
Washington, DC 20460
202-566-0556
-------
31
and hydrazine. Apart from the variety of potential applications
described in the patent literature, three major industries are involved
in the handling of nitrosamines today, organic chemicals manufacturing,
rubber processing, and rocket fuel manufacturing.
32
A review of Chem Sources-U.S.A. reveals that a large variety of nitro-
samines (including dimethyl-, diethyl-, dipropyl-, dibutyl-, and
dipentylnitrosamine) are available commercially. The only nitrosamines
produced in quantities greater than 1000 pounds per year are diphenyl-
nitrosamine and dimethylnitrosamine.
Thus far, diphenylnitrosamine has not been shown to be carcinogenic, and
consequently is of little interest here. The rubber industry requires
chemicals to modify the basic elastomers to produce the desired improved
qualities in rubber goods. Such chemicals, including monomers, amount
33
to more than 5.5 billion pounds per year. Of the variety of different
types of chemicals used, two types are pertinent to this discussion,
vulcanizing retarders and blowing agents. Diphenylnitrosamine (not
carcinogenic) is a well-known vulcanizing retarder and dinitrosopenta-
methylenetetramine is used as a blowing agent in the production of micro-
31
cellular rubber.
One of the main liquid rocket fuels has been 1,1-dimethylhydrazine. A
high-yield procedure for preparing unsymmetrical hydrazines involves the
reduction of the corresponding nitrosamine precursor. In the commercial
process, 1,1-dimethylhydrazine is prepared in a 73 percent yield by
34
reducing DMN with zinc and acetic acid.
164
-------
(7-1)
The FMC plant in Baltimore is a commercial source that produces DMN as an
35
intermediate in the production of 1 ,1-di methyl hydrazine.
7.3.2 Industrial Sources in Which Nitrosamines Occur Incidentally
Di methyl nitrosamine and N-4-dinitrosomethylaniline have been suspected to
be related to the higher-than-normal mortality from cancer in rubber
oc
factory workers. However, the use of these two compounds per se in
rubber processing is doubtful; it is more likely they originated from the
corresponding amines, which may have been used as antioxidants.
Lower-molecular-weight nitrosamines are volatile at room temperature while
higher-molecular-weight species are steam volatile. Because of possible
loss of these compounds from complex reaction mixtures by volatility and
also because of the diversity of the nitrogen-based organic chemical
industry, it is reasonable to expect that many more sources of nitro-
samines to the atmosphere will be identified.
Nitrosamines have been demonstrated to be present in the following prod-
ucts and processes, and, as a result, may contribute to ambient air con-
tamination during their manufacture or use. Listed also are examples of
industries where the occurrence of nitrosamines has not been documented
but could be expected.
• Combustion of rocket fuel. There is some evidence that nitro-
samines are formed during the burning of 1 ,1 -dimethyl hydrazine
37
as a rocket fuel .
165
-------
• Fish meal processing. The presence of 30 to 100 ppm of DMN has
been found in fish meal, which also contains relatively large
38
amounts of secondary and tertiary amines.
• Soya bean oil. DMN has been found also to the extent of 0.38
to 0.45 ppm in one source of soya bean oil. '
• Tobacco. Nitrosation of the secondary amine nornicotine (its
ratio to nicotine in tobacco is about 1:20) may contribute to
the occurrence of NO-nornicotine in tobacco smoke and in cured,
41 42
unsmoked tobacco. ' There are some data to suggest that smoke
from tobacco grown in soil treated with high levels of nitrogen
is more likely to contain nitrosamines and to contain them in
higher concentrations than smoke from tobacco grown in the fields
with a low nitrogen content. Evidence for the presence of DMN,
nitrosopyrrolidine, methyl butylnitrosamine, and nitrosopiperi dine
43-46
in tobacco smoke has been obtained.
• Explosives manufacture. High explosives contain various organic
nitrates as the main component of the explosive. However, a
multitude of various additives is also used to impart specific
properties to the explosives. Pertinent to the discussion here
are stabilizers (secondary amines, e.g., diphenylamine); binders
(natural or synthetic nitrogenous materials); compatibility
agents and softening point depressants (nitrosophenols and their
esters); and fuel sensitizers for ammonium nitrate (amines, amides,
urea).47
166
-------
• Power plants. It has been suggested that amine additives used
to modify fly-ash resistivity might possibly react with flue gas
NO to form nitrosamines. An unpublished report,* however, cities
A
evidence to the contrary. No nitrosamine was detected in tests
at a power plant at which diethy1amine sulfate was used as the
additive (the detection limit was calculated to be 5 parts per
trillion). The sampling method employed a TENAX adsorber, and
sampling volume was approximately 50 cubic feet. Organics were
desorbed from TENAX using pentane, and aliquots were analyzed
by gas chromatography-mass spectrometry after concentration by
Kuderna-Danish evaporation.
7.3.3 Industrial Sources of Nitrosamine Precursors
g
7.3.3.1 Nox Sources—An estimated 20.69 x 10 kg of nitrogen oxides were
emitted from industrial, commercial, and domestic sources in the United
States during 1970. Table 7-7 shows the contribution to this total from
48
each source category.
About 1 percent of the total man-made NO emitted to the ambient air is
A
formed by chemical processes, mainly related to the manufacture and use
of nitric acid. Use of nitric acid in military ordnance works for
explosives manufacture is a major source of NO emissions, with total
A
NO emissions from the Volunteer Ordnance Works equal to total emissions
X
48
from all U.S. non-military uses of nitric acid.
*Harris, D.B. Test Results of the Effect of Flyash Conditioning Agent on
Coal-Fired Power Plant Emissions. Industrial Environmental Research
Laboratory. U.S. Environmental Protection Agency. Research Triangle
Park, N.C. Unpublished Report. 1976.
167 "
-------
Table 7-7. SUMMARY OF NITROGEN OXIDES
EMISSIONS IN THE UNITED STATES, 197048
Source
Transportation
Motor vehicles
Gasoline
Diesel
Aircraft
Railroads
Vessels
Non-highway use of motor fuels
Fuel combustion in stationary sources
Coal
Fuel oil
Natural gas
Liquid petroleum gas and kerosene
Wood
Industrial processes
Solid waste disposal
Miscellaneous
Forest fires
Structural fires
Coal refuse
Agricultural
Total
109 kg/yr
10.. 6
8.3
7.1
1.2
0.36
0.09
0.18
1.7
9.1
3.5
1.2
4.1
0.18
0.09
0.18
0.36
0.45
0.18
Negligible
Negligible
0.27
20.69
Percent
of total
51.3
39.9
34.2
5.7
1.8
0.4
0.9
8.3
43.8
17.1
5.7
19.7
0.9
0.4
0.9
1.8
2.2
0.9
Negligible
Negligible
1.3
100.0
168
-------
Nitrogen oxides emissions from organic nitrations are shown in Table 7-7
under industrial processes.
7.3.3.2 Secondary Amine Sources—A detailed review of all the specific
unit operations and processes involving the use or generation of second-
ary amines is beyond the scope of this report. The two sources described
below may be expected to emit secondary amines.
• Feedlots. Dimethyl amine has been identified in the air from the
decomposition of livestock and poultry manure. At least eight
amines (methyl amine, dimethyl amine, trimethylamine, ethyl amine,
triethyl amine, n-propylamine, n-butylamine, and n-hexylamine)
have been identified in the air from decomposition of livestock
49
and poultry manure. A survey of two Texas panhandle feedlots
indicated the presence of the following amines in a 20.6-liter
sample:
Compound Wt collected, yg
Ethylamine 2.7
Dimethy1 amine 13.0
n-Propylamine 1.5
n-Butylamine 1.2
n-Hexylamine 0.7
The concentration of dimethy1 amine in air above these feedlots
(at 18 m) is calculated to be 632 yg/m .
The secondary amines found in feedlots are of primary interest in the for-
mation of nitrosamines. Tertiary amines can degrade to secondary amines;
169
-------
however, this adds another reaction step and slows the kinetics of nitro-
samine formation from tertiary amines. There are two routes possible to
nitrosamine formation in feedlots, (1) reaction of amines with nitrite in
wastes and subsequent emission by volatilization or on dust; and (2)
atmospheric reaction of feedlot-generated amines with NOX. Certainly
more work needs to be done to establish feedlots as a possible source of
nitrosamines. The recent explosive growth of feedlots in some areas of
the U.S. of up to 18 percent per year reinforces the need for
investigation.
Pertinent to the problem of wastes in feedlots, nitrosamines were found
after incubating dimethylamine and nitrite with microorganisms derived
from sewage and soil with whole acidic soil and sewage. Under these
conditions, trimethylamine was also converted to dimethylamine. Amines
and high concentrations of nitrites are commonly found in soils and
sewage.51'52
• Rendering plants. Animal matter not suitable as food for either
humans or pets is rendered into salable products. Ground animal
matter is conveyed to cookers which may handle as much as
40,000 pounds in batch processes. Temperatures of 300°F for 1
to 4 hours are required to digest bones, hooves, hides, and hair,
with most of the moisture being evaporated and exhausted. This
exhaust steam contains extremely odorous gases. Amines that have
been identified in the exhaust gases are: trimethylamine,
ethylamine, diethylamine, triethylamine, putrescine (
170
-------
CO
and cadaverine (H2N(CH2)5NH2). It has not been established
whether exhaust gases contain m'trosamines. Since the tissues of
animals contain dimethylamine and trimethyl amine almost ubiqui-
tously as metabolic or bacterial degradation products of nitrogen
metabolism, and since m'trosamines have been shown to form from
amines and nitrite in the alimentary tract of animals, the pres-
ence of m'trosamines in the offal of animals is a possibility.
At the high cooking temperatures, many m'trosamines would be
volatilized and practically all m'trosamines are steam-
distillable.54
The manufacture of the following products or the products themselves may
be sources of nitrosatable secondary amines.
• Antioxidants. Dimethylamine is used in liquid tetrafluoroethylene.
Ethylamine is injected in the air in which rubber products are
fl i
56
55
stored to inhibit degradative oxidation. Various aryl and alkyl
amines are used to inhibit the oxidation of lubricants.'
• Vulcanization accelerators. Large quantities of amines are used
in the preparation of rubber vulcanization accelerators. Sec-
ondary amines are used in the preparation of thiuram sulfides
(R2N-CS-SS-CS-NR2) and metallic dialkyldithiocarbamates
(R2NCSSNa).57
• Pharmaceuticals. The applications of aliphatic amines in the
preparation of Pharmaceuticals are too numerous to list. Some
examples are: (1) ephedrine [(CH(OH)CH(CH3)NHCH3L an adrenergic
171
-------
drug used as a bronchodilator; (2) diethylamine, used in the
preparation of chloroquine; (3) di-n-propylamine, used in the
preparation of Benemide, an adjunct to penicillin therapy; and
(4) amines used in germicidal preparations.
• Self-polishing waxes. Morpholine, together with fatty acids, is
57
used as an emulsifying agent for self-polishing waxes. Mor-
pholine evaporates as the film dries.
• Synthetic detergents. Dimethylamine is a contaminant in
CQ
detergents.
• Pesticides. Secondary amines are used extensively in the prepara-
57
ation of herbicides, fungicides, and insecticides. Many pesti-
cides contain secondary and tertiary amine groups and might be
expected to form nitroso derivatives. Discussion has been given
earlier in this report concerning nitrosation of pesticides (see
Sections 3.2.3, 4.4, 5.2, 5.3.3).
Pesticides may be discussed in terms of a number of generic groups,
The groups which are most interesting from the standpoint of con-
taining possible nitrosamine precursors include: (1) triazines
(e.g., atrazine); (2) carbamates (e.g., Sevin); (3) thiocarbamates
(e.g., Addicarb); (4) hydrazides (e.g., maleic hydrazide); (5)
urea-based compounds (e.g., Bromicid); (6) nitrated hydrocarbons
(e.g., Trifluralin); and (7) amides (e.g., Diphenamid).
172
-------
• Solvents. An important solvent for acrylonitrile polymers and
copolymers, dimethylformanride, is made from methyl formate and
dimethyl amine. About 14 million pounds of dimethyl amine are
57
used annually.
t Uranium oxide. Amines such as dilaurylamine are used in the sol-
59
vent extraction of uranium oxide from ore-leach-liquors.
• Wet-strength paper. Pol ye thy Ten inline is used in paper to improve
its wet strength.
t Corrosion inhibitors. Morpholine is used as a corrosion
inhibitor.
• Animal glues. Amines are emitted during cooking and drying
operations.
• Photographic products. Amines are used as developing agents,
33
e.g., p-methylaminophenol.
• Leather. Dimethylamine is used as an accelerator in the liming
operation involved in the tanning of leather.
The above list of amines which may be precursors to nitrosamine formation
is by no means complete. The number of permutations on a fundamental
organic structure is immense, as illustrated by the group of related com-
pounds shown in Figure 7-4. It should also be mentioned that chlorophyll
and heme contain the basic pyrrole structure.
173
-------
p
H
PYRROLE
(POSSIBLY PRESENT
IN COAL TAR)
_ _
PYRROLINE
r'V-i
L>Q
PYRROLIDONE
(USED TO MAKE 'NYLON 4
AND POLYVINYL PYRROLIDONE.
A CONSTITUENT OF HAIR SPRAYS)
SKATOLE
(FOUND IN
FECES)
PYRROLIDINE
(FOUND IN
BACON)
INDOLE
(USED IN
PERFUMES)
Figure 7-4. Structures and diverse, sources or uses of pyrrole and related compounds^
174
-------
The discussion of pyrrole-type compounds was given to point out that no
exhaustive list of possible amine precursors to nitrosamlnes can be given
in this report. The report can, at best, cite examples of possible
precursors and note those compounds important in commerce that should be
examined to determine whether they could be important sources of nitro-
samines to the environment.
Table 7-8 gives a list of amine compounds that have been found to yield
nitroso derivatives. A partial list of United States amine producers
is given in Table 7-9. Given in Table 7-10 is a selected list of some
of the larger-volume amines which are produced along with production
figures. The list of production figures is not complete and conse-
quently is of limited value, but it does offer some perspective. It is
impossible, with the present data base, to develop an understanding of
the importance of amine manufacturing as a source of nitrosamines pre-
cursors compared to other sources in which amines are incidental to the
existence and purpose of the source.
All of the companies listed in Table 7-9 were contacted in an effort to
procure data from each indicating quantity of production. At this time^
only three of the responding companies have made production figures
available, as shown in Table 7-11. Using the 1975 Directory of Chemical
Producers, the production capacities for several companies were procured
(also given in Table 7-11).
175
-------
Table 7-8. DRUGS, PESTICIDES, AND NATURALLY
OCCURRING AMINES THAT HAVE EXPERIMENTALLY
PRODUCED N-NITROSO COMPOUNDS JJ. VIVO
OR IN VITRO61»a
Compound
C1assb
Product
Secondary amines
Morpholine D,
Piperazine D
Atrazine P
Simazine P
Phenmethrazine D
Ethambutol D
Zi ram P
Thiram P
Ferbam P
Tertiary amines
Aminopyrine D
Oxytetracycline D
Chlorpromazine D
Dextropropoxyphene D
Chlorpheniramine D
Methadone D
Methapyrilene D
Quinacrine D
Lucanthone D
Tolazamide D
Cyclizine D
Trimethylamine N
2-Dimethylaminoethanol N
N,N-Dirnethylglycine methyl ester
N,N-Dimethylglycine N
Triethanolamine
Nitrilotri acetic acid
N,N-Dimethyldodecylamine
Nicotine N
Pyribenzamine D
Carbaryl P
Propoxur P
Benzthiazuron P
Quaternary amines
Tetramethy1 ammonium chloride
Neurine chloride N
Acetyleneline N
Choline N
NO-derivative
MNP.DNP
NO-derivative
NO-derivative
NO-derivative
NO-derivative
DMN
DMN
DMN
DMN
DMN
DMN
DMN
DMN
DMN
DMN
DEN
DEN
NO-hexamethyleneimine
DNP
DMN
DMN
DMN
DMN,NO-sarcosine
NO-diethanolamine
NO-iminodiacetic acid
DMN,NO-N-methyldode-
cylamine
NO-nornicotine
NO-derivative
NO-carbaryl
NO-propoxur
NO-benzthiazuron
DMN
DMN
DMN
DMN
176
-------
Table 7-8 (continued). DRUGS, PESTICIDES, AND
NATURALLY OCCURRING_AMINES THAI HAVE
" EXPERIMENTALLY PRODUCED"N-NITROSO
COMPOUNDS IN VIVO OR IN VITRO61>a
Compound Classb Product
Betaine N DMN
Carnitine N DMN
Trimethylamine-N-oxide N DMN
Tribenzylamine-N-oxide - Dibenzylnitrosamine
aAdapted from S. S. Mirvish. Formation of N-nitroso compounds:
chemistry, kinetics and in vivo occurrence. Tox. and Appl.
Pharmacol. 31_t 1975.
D = drug; P = pesticide; and N = naturally occurring.
cNO-derivative of the compound nitrosated.
177
-------
Table 7-9. PRODUCERS OF AMINES IN THE UNITED STATES
61
ACCTO CHEMICAL COMPANY
Charlestadt, New Jersey
AIR PRODUCTS & CHEMICALS, INC.
Pensacola, Florida
ALDRICH CHEMICAL COMPANY, INC.
Milwaukee, Wisconsin
AMES LABS
Mil ford, Connecticut
BASF WYANDOTTE CORPORATION
Parsippany, New Jersey
CELANESE CORPORATION
Bay City, Texas
COMMERCIAL SOLVENTS CORPORATION
Terra Haute, Indiana
DOW CHEMICAL COMPANY
Freeport, Texas
EASTMAN KODAK COMPANY
Kingsport, Tennessee
Rochester, New York
E. I. DUPONT de NEMOURS, INC.
Belle, West Virginia
Houston, Texas
EL PASO PRODUCTS
Odessa, Texas
ELI LILLY & COMPANY, INC.
Lafayette, Indiana
GAF CORPORATION
Calvert City, Kentucky
Linden, New Jersey
Rensselaer, New Jersey
HILTON-DAVIS CHEMICAL COMPANY
Cincinnati, Ohio
JEFFERSON CHEMICAL COMPANY
Austin, Texas
Conroe, Texas
Port Neches, Texas
MILES LABORATORY
Elkhart, Indiana
MILLMASTER ONYX CORPORATION
Beaumont, Texas
MONSANTO
Luling, Louisiana
NALCO CHEMICAL COMPANY
Chicago, Illinois
NEASE CHEMICAL CORPORATION
State College, Pennsylvania
PENNWALT CORPORATION
Wyandotte, Michigan
PIERCE CHEMICALS, INC.
Rockford, Illinois
R.S.A. CORPORATION
Ardsley, New York
REILLEY TAR & CHEMICAL CORPORATION
Indianapolis, Indiana
ROHM & HAAS
Philadelphia, Pennsylvania
Deerpark, Texas
SHELL CHEMICAL COMPANY
Martinez, California
STAUFFER CHEMICAL COMPANY
Edison, New Jersey
STERLING DRUG COMPANY
Cincinnati, Ohio
178
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Table 7-9 (continued). PRODUCERS OF AMINES IN THE
UNITED STATES61
UNION CARBIDE CORPORATION
Institute, West Virginia
South Charleston, West Virginia
Taft, Louisiana
Texas City, Texas
UNI ROYAL
Naugatuck, Connecticut
VIRGINIA CHEMICALS
Portsmouth, Virginia
WARNER LAMBERT
Holland, Michigan
179
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Table 7-10. AMINE PRODUCTION IN THE
UNITED STATES, 1972^1
Production, tons
Amine (104)
Diethanolamine 4.7
Hexamethyleneamine 30.65
N-(l,4-dimethylpentyl)-N'- 1.0
pheny 1-p-pheny1enedi ami ne
Aniline 19.9
Triethanolamine 4.15
Ethanolamine 11.0
Dimethyl amine 4.75
Diary1arylenediamines, mixed 1.0
Hexamethylenetetramine 4.75
Melamine 3.4
Ethylene diamine 3.1
Toluene, 2,4-diamine 6.65
Monomethy1 ami ne 1.65
Trimethylamine 1.4
Dipropylamine 1.35
Di-n-butylamine 0.19
Diethyl amine 0.55
All other ethyl amines 2.2
Total butyl amines 0.95
Atrazine 4.5
180
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Table 7-11. ACTUAL PRODUCTION OR PRODUCTION CAPACITY
OF SOME AMINE PRODUCERS IN THE UNITED STATES
Actual production (AP)
or production
Producer Compound capacity (PC)
Aldrich Chemical dl-a-Methylbenzylamine 90 to 175 kg/yr
Company, Inc. each of last 5 yr (AP)
Ames Laboratories p-Methy1 benzyl amine 20 to 50 Ib/yr
each of last 5 yr (AP)
Virginia Chemicals, Inc. Dipropylamine 11,100,000 Ib (AP)
Tripropylamine 42,000 Ib (AP)
Dibutyl amine 1,523,000 Ib (AP)
Tributylamine 150,000 Ib (AP)
(1974 figures)
GAP Corporation Mono-, di-, and 10,000 Ib (PC)
trimethylamine
E. I. DuPont Mono-, di-, and 165,000,000 Ib (PC)
de Nemours, Inc. trimethylamine
Commercial Solvents Mono-, di-, and 18,000,000 Ib (PC)
Corporation trimethylamine
Air Products & Mono-, di-, and 50,000,000 Ib (PC)
Chemicals, Inc. trimethylamine
7.4 MOBILE SOURCES
Studies have been conducted on nitrosamines in exhaust. The exhaust from
three 1972 model cars and two engines operated on dynamometer stands was
analyzed for a variety of nitrogen-containing compounds, including
co
dimethylnitrosamine, in a fuel-additive study. A study of the impact
on emissions of six fuel additives in five 1973 and 1974 model cars
CO
involved analysis for amines and nitrosamines. A method of analysis
(N-specific gas chromatography) for amines and nitrosamines was developed
181
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64
as a part of a program on fuel additive effects, and this technique was
used in fuel additive product analysis in flat-flame burner experi-
ments. ' A study of the composition of particulate matter emitted
from diesel engines involved analysis for absorbed amines and nitrosamines
using a variety of fuels and additives. Another fuel additive program
used gas chromatographic and mass spectrometric techniques to search for
N-nitrosamines from engines and combustion bombs, using simple fuels and
fn
high levels of simple nitrogen-containing additives. Sensitive spot
tests for nitrosamines were used in a study of the particulate emissions
from four monolithic oxidation catalysts, three pelleted oxidation cata-
lysts, and two NO reduction catalysts.
/\
Thus far, analyses for nitrosamines and amines have been carried out as
major parts of seven research programs involving many automobiles, diesel
engines, emission control systems, fuels, and fuel additives. No evi-
dence for either amine or nitrosamine emissions has been developed from
any tested sources in any of the studies found to date.
Analysis for nitrosamines in low concentrations in source emissions is a
task of high complexity. N-nitroso compounds are relatively unstable
species at source effluent temperatures, and there are many compounds of
potential concern. In most cases, surveys of source emissions have
searched only for N-nitrosodimethylamine, the lowest member of the
homologous series of N-nitrosated secondary amines. Most workers have
used nitrogen-specific detectors in conjunction with gas chromatography
to determine this compound.
182
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Since emitted amines can be N-nitrosated in the ambient air, analysis for
the potentially complex variety of potential amine emission products is
also important. Again, the most commonly used tool has been gas chroma-
tography with N-specific effluent detection. This class of compounds has
been one of the most difficult of all organic species to handle. Extra
precautions, including all-glass gas handling systems, super-inert
chromatographic substrates, and relatively high-temperature separations,
have all been necessary to avoid serious losses of model compounds in
standardization experiments. In one study, new methods of detection,
handling, and analysis have been extensively explored and the experi-
menters' efforts have met with moderate success. However, analysis for a
variety of amines and nitrosamines from model combustion systems has been
entirely negative.
It should be recognized that the initial publication of the N-specific
detector system used in the recent work of Fine and Epstein on nitrosamines
64
in ambient air resulted from work done under an EPA grant.
Further discussion of source emission studies is given below:
7.4.1 Bureau of Mines Studies
The gaseous emissions from three 1972 Chevrolet automobiles and two sta-
tionary 1972 Chevrolet engines were analyzed for nitrogen compounds using
both clear gasoline and gasoline containing Chevrolet additive F-310, an
amine-type carburetor detergent. Compounds analyzed included ammonia,
pyridine, Cp to C. aliphatics, N-nitrosamines, aniline and toluidine N-
nitrosamines, cyanogen, hydrogen cyanide, and nitromethane. Detection
183
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methods used included electron capture, alkali flame-ionization, micro-
coulometry, and electrolytic conductivity. This last method had the best
overall selectivity and sensitivity and was used for the major analytical
effort. Detection limits for auto exhaust for compounds of interest
ranged from 20 ppb for aromatic amines to 150 ppb for nitrosamines. Of
the compounds searched for, only HCN (1.0 to 1.5 ppm) and nitromethane
(0.2 to 0.3 ppm) were confirmed as present in automobile exhaust. All
others were absent or were present at levels below detection limits.
In later work at the Bureau, six additives, including F-310 in normal and
high aromatic base fuels, were examined in 1973-1974 model cars (a Ford,
a Chevelle, a Volkswagen, and two Mazda rotary engine cars), using the
same techniques. Detection limits were extended to about 25 ppb for all
compounds of interest. Compounds found to be present in ppb quantities
included HCN, cyanogen, and alkyl and arylnitro compounds. Further, experv
ments were conducted in which automobile exhaust was spiked with known
concentrations of ammonia, alkyl and aryl amines, nitrites, and nitro-
samines. Auto exhaust samples in light-proof bags were spiked with the
compounds of interest to several times the detection limit. After 30
minutes, less than 30 percent of the original amounts remained. The
results of all these vehicle experiments showed that all this latter class
of compounds are unstable and undetectable in auto exhaust. None of the
nitrogen compounds found were elevated with use of N-containing additives.
184
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7.4.2 Southwest Research Institute (SWRI) Studies66'67
Experiments at Southwest Research Institute sought nitrogen compounds from
catalyst-equipped engines. Using techniques similar to those developed
by the Bureau of Mines, nitrogen compounds were sought in the exhaust of
non-air-pumped pelleted catalysts with two base gasolines and two N-
containing additive packages. No nitrogen compounds were found at 25 ppb
detection limit.
Further experiments were conducted at SWRI in an effort to identify N-
compounds possibly adsorbed on diesel particulate emissions. Tested
were both two-stroke and four-stroke cycle diesel engines, using three
widely different diesel base fuels and two additives, one of them an
organic nitrite ester used as an ignition promoter. Again, no nitrogen-
containing products were detected.
7.4.3 Exxon Studies
Particulate emissions from eight catalyst systems were characterized by
Exxon Research. Particulate samples were examined by spot-plate tests
for nitrosamines and amines.
fid fiR
7.4.4 University of Michigan Study04'00
Under a study at the University of Michigan, a super-sensitive N-
sensitive detection system involving chemiluminescence detection has been
developed and reported. ' Chromatographic techniques for the analysis
of a wide variety of amines and nitrosamines were also developed and
carefully standardized. Studies of partial combustion products from
185
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amine antioxidants were performed on a simplified combustor, but no N-
containing compounds were found.
7.4.5 Penn State Study68
A gas chromatograph-mass spectrometry study of the composition of auto
exhaust with and without very high levels of amine antioxidants was
carried out at the Pennsylvania State University. No N-containing prod-
ucts were identified.
7.5 REFERENCES FOR SECTION 7
1. Wolff, A. and A. E. Wasserman. Nitrates, nitrites, and nitro-
samines. Science. J_77:15-19, 1972.
2. Alexander, M. Soil Microbiology. New York, John Wiley and Sons.
1961. 472 p.
3. Alexander, M. Microbial Ecology. New York, John Wiley and Sons.
1971. 511 p.
4. Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. The Microbial
World, 3rd Ed. Englewood Cliffs, New Jersey; Prentice-Hall, Inc.
1970. 873 p.
5. Brock, Thomas D. Biology of Microorganisms. Englewood Cliffs,
New Jersey; Prentice-Hall, Inc. 1970. 737 p.
6. Delwich, C. C. The nitrogen cycle. Sci. Amer. 223_:137-147, 1970.
7. Buckman, H. 0. and N. C. Brady. The Nature and Property of Soils,
7th Ed. New York, MacMillan Company. 1969. 653 p.
8. Smith, R. L. Ecology and Field Biology, 2nd Ed. New York,
Harper and Row. 1974. 850 p.
9. Whittaker, Robert H. Communities and Ecosystems, 2nd Ed. New
York, MacMillan Company. 1975. 387 p.
10. McLaren, A. D. and J. Skujins. Soil Biochemistry, Vol. 2. New
York, Marcel Dekker. 1971. 527 p.
186
-------
11. Inger, R. F., A. D. Hosier, F. H. Bormann, and W. F. Blair. Man
in the Living Environment. Report of the Workshop on Global
Ecological Problems. University of Wisconsin Press. Madison,
Wisconsin. 1972. 288 p.
12. Bowen, J. J., Jr. Trace Elements in Biochemistry. London, Academic
Press. 1966. 241 p.
13. Ayanaba, A. and M. Alexander. Transformations of methylamines and
formation of a hazardous product, dimethylnitrosamine, in samples
of treated sewage and lake water. J. Environ. Qual. 3^83-89, 1974.
14. Ayanaba, A., W. Verstraete, and M. Alexander. Formation of dimethyl-
nitrosamine, a carcinogen and mutagen, in soils treated with nitrogen
compounds. Soil Sci. Soc. Amer. Proc. 37^565-568, 1973.
15. Ayanaba, A., W. Verstraete, and M. Alexander. Possible microbial
contribution to nitrosamine formation*in sewage and soils. J. Nat. •
Cancer Inst. 5£:811-813, 1973.
16. Mills, Aaron L. Nitrosation of secondary amines by axenic cultures
of microorganisms and in samples of natural ecosystems. Ph.D. Thesis,
Cornell University. Ithaca, New York. 1976. 95 p.
17. Tate, R. L. and M. Alexander. Stability of N-nitrosamines in samples
of lake water, soil, and sewage. J. Nat. Cancer Inst. 54:327-330,
1975.
18. Dean-Raymond, D. and M. Alexander. Plant uptake and leaching of
dimethylnitrosamine. Department of Agronomy, Cornell University.
Ithaca, New York. 1976.
19. Chen, R. L., D. R. Kenney, and J. A. Konrad. Nitrification in sedi-
ments of selected Wisconsin lakes. J. Environ. Qual. J_:151-154,
1972.
20. Kenney, D. R. The nitrogen cycle in sediment water systems. J.
Environ. Qual. 2_: 15-29, 1973.
21. Commoner, Barry. Threats to the integrity of the nitrogen cycle.
In: Global Effects of Environmental Pollution (F. S. Singer, ed.),
New York, Springer-Verlag. 1970. p. 70-95.
22. Kohl, D. H., G. B. Shearer, and B. Commoner. Fertilizer nitrogen:
contribution to nitrate in surface water in a Corn Belt watershed.
Science. ]_74:1331-1334, 1971.
23. Ridder, W. E., F. W. Oehme, and D. C. Kelley. Nitrates in Kansas
ground water as related to animal and human health. Toxicol. 2:
397-405, 1974.
187
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24. Stewart, B. A., F. G. Viets, Jr., G. L. Hutchinson, and W. D. Kemper.
Nitrates and other water pollutants under fields and feedlots.
Environ. San. and Tech. £: 736-739, 1967.
25. Verstraete, W. and M. Alexander. Microbial involvement in the for-
mation of dimethylnitrosamine in nature. J. Appl. Bacteriol. 34: 1973.
26. DuPlessis, L. S., J. R. Nunn, and W. A. Roach. Carcinogen in a
Transkeian Bantu food additive. Nature. 222:1198-1199, 1969.
27. Wright, M. and K. L. Davison. Nitrate accumulation in crops and
nitrate poisoning in animals. Adv. in Agron. _^6:197-247, 1964.
28. McKee, H. S. Nitrogen Metabolism in Plants. Oxford, Clarendon
Press. 1962. 728 p.
29. Bretschneider, K. and J. Matz. Nitrosamine (NA) in der Atmospharis-
chen und in der Luft am Arbeitsplatz. [Nitrosamines (NA) in the
atmospheric air and in the air at the workplace.] Archiv. fur
Geschwulstforsch. 42_:36-41, 1973.
30. Fine, D. H. N-Nitrosamines in Urban Community Air. Prepared by
Thermo Electron Corporation, Waltham, Mass., under EPA Contract
No. 68-02-231214. Progress Report. U.S. Environmental Protection
Agency. Research Triangle Park, N.C. January 1975.
31. Magee, P. N. Possibilities of hazard from nitrosamines in industry.
Ann. Occup. Hyg. 1_5:19-22, 1972.
32. Chem Sources-U.S.A. Flemington, New Jersey; Directories Publishing
Company. 1975.
33. Shreve, R. N. Chemical Process Industries, 3rd Ed. New York,
McGraw-Hill. 1967.
34. Hatt, H. H. Organic Syntheses, Vol. II. New York, John Wiley and
Sons, Inc. 1943. p. 208.
35. David Oestreich. Industrial Environmental Research Laboratory, EPA,
Research Triangle Park, N.C. Personal communication, October 23,
1975.
36. Mancuso, T. F. and M. J. Brennan. Epidemiological considerations of
cancer of the gallbladder, bile ducts, and salivary glands in the
rubber industry. J. Occup. Med. J_2:333-341, 1970.
37. Simoneit, B. R. and A. L. Burlingame. Organic analyses of selected
areas of Surveyor III recovered on the Apollo 12 mission. Nature
(London). 234:210-211, 1971.
-------
38. Ender,F.,G. Havre, A. Helgebostad', N. Koppange, R. Madsen, and L. Ceh.
Isolation and identification of a hepatotoxic factor in herring meal
produced from sodium nitrite preserved herring. Naturwissenschaften.
5]_:637-638, 1964. [Chem. Abstr. 62_:7035A.]
39. Hedler, L. A possible method for the detection of nitrosamines in
fats and oils. J. Am. Oil Chem. Soc. 48:329A, 1968.
40. Marquardt, P. Presented at IARC/OKF2 Meeting on Nitrosamine Analysis,
Heidelberg, Germany, October 1971.
41. Hoffman, D., C. Rathkamp, and Y. Y. Lin. Chemical studies on tobacco
smoke. Hydrazine in cigarette smoke. In: N-Nitroso Compounds in
the Environment (P. Bogovski, E. A. Walker, and W. Davis, eds.).
Lyon, France. IARC Scientific Publication No. 9. 1974.
42. Rhodes, J. W. and D. E. Johnson. N-dimethylnitrosamine in tobacco
smoke condensate. Nature (London). 236:307-308, 1972.
43. KroHer, E. Detection of nitrosamines in tobacco smoke and food.
Deut. LeTiensm. Rundsch. 63_:303-305, 1967.
44. Neurath, G. B. In: N-Nitroso Compounds: Analysis and Formation
(P. Bogovski, R. Preussmann, and E. A. Walker, eds.). Lyon, France.
IARC Scientific Publication No. 3. 1972.
45. Neurath, G. B., B. Pirmann, and H. Wichern. N^Nitroso compounds in
tobacco smoke. Beitr. Tabakforsch. 2_: 311-319, 1964. [Chem. Abstr.
61:938E.]
46. Serfontein, W. J. and P. Hurter. Nitrosamines as environmental
carcinogens. II. Evidence for the presence of nitrosamines in
tobacco smoke condensate. Cancer Res. 2_6:575-579, 1966.
47. Lieping, R., R. W. Handy, and J. W. Harrison. Characteristics of
non-military explosives. Defense Documentation Center. Alexandria,
Va. AD778207. February 1974.
48. Control Techniques for Nitrogen Oxides Emissions from Stationary
Sources. The Committee on Challenges of Modern Society, NATO.
Brussels, Belgium. October 1973.
49. Peters, J. A. and T. R. Blackwood. Source Assessment Document No. 6,
Beef Cattle Feedlots. Prepared by Monsanto Research Corporation,
Research Triangle Park, N.C., under Contract No. 68-02-1320. U.S.
Environmental Protection Agency. Research Triangle Park, N.C.
50. Ayanaba, A., W. Verstraete, and M. Alexander. Brief communication:
possible microbial contribution to nitrosamine formation in sewage
and soil. J. Nat. Cancer Inst. 50:811-813, 1973.
189
-------
51. Shuval, H. I. and N. Gruener. Epidemiological and toxicological
aspects of nitrates and nitrites in the environment. Amer. J. Pub.
Health. 62:1045-1052, 1972.
52. Tannenbaum, S. R., A J. Sinskey, M. Weisman, and W. Bishop. Nitrite
in human saliva. Its possible relationship to nitrosamine formation.
J. Nat. Cancer Inst. 5_3:79-84, 1974.
53. Mixon, Forest. Personal communication, October 27, 1975.
54. Preussmann, R. On the significance of N-nitroso compounds as car-
cinogens and on problems related to their chemical analysis. In:
N-Nitroso Compounds: Analysis and Formation (P. Bogovski, R. Preuss-
mann, and E. A. Walker, eds.). Lyon, France. IARC Scientific
Publication No. 3. 1972. p. 6.
55. Wolk, I. L. U.S. Pat. 2,363,717. November 28, 1944.
56. Stewart, W. T., A. Goldschmidt, and 0. L. Harle. U.S. Pat. 2,687,
377. August 24, 1954.
57. Astle, M. J. Industrial Organic Nitrogen Compounds. American Chemi-
can Society Monograph No. 150. New York, Reinhold Publishing
Corporation. 1961.
58. Byrd, J. F., H. A. Mills, C. H. Schellhase, and H. E. Stokes. Solv-
ing a major odor problem in a chemical process. J. Air Poll. Contr.
Assoc. J4_:509-516, 1964.
59. Coleman, C. F., K. B. Brown, J. G. Moore, and D. J. Crouse. Solvent
extraction with alkyl amines. Ind. Eng. Chem. 5_0:1756-1762, 1958.
60. Hull, W. Q. and W. G. Bangert. Animal glue. Ind. Eng. Chem. 44:
2275-2284, 1952.
61. Walker, P., J. Gordon, L. Thomas, and R. Ouelette. Environmental
Assessment of Nitrosamines. Prepared by Mitre Corporation, McLean,
Virginia, under Contract No. 68-02-1495. U.S. Environmental Pro-
tection Agency. Research Triangle Park, N.C. February 1976.
62. Hum, R. W., J. R. Allsup, and F. Cox. Effect of gasoline additives
on gaseous emissions. Part I. Prepared under interagency agreement
IAG-D4-0040 by U.S. Bureau of Mines, Bartlesville, Oklahoma. U.S.
Environmental Portection Agency. Research Triangle Park, N.C.
Publication No. EPA-650/2-75-014. 1975.
63. Hum, R. W., F. Cox, and J. R. Allsup. Effects of gasoline additives
on gaseous emissions. Part II. Prepared under interagency agreement
IAG-D4-0040 by U.S. Bureau of Mines, Bartlesville, Oklahoma. U.S.
Environmental Protection Agency. Research Triangle Park, N.C. Publi-
cation No. EPA-600/2-76-026. 1976.
190
-------
64. Steffenson, D. M. and D. H. Stedman. Optimization of the operating
parameters of chemiluminescent nitric oxide detectors. Anal. Chem.
46:1704-1709, 1974.
65. Stedman, D. H. Effects of Fuel Additives on Gasoline Engine Exhaust
Emissions. Prepared under Grant No. R-802419 by University of
Michigan, Ann Arbor, Michigan. Interim Report. U.S. Environmental
Protection Agency. Research Triangle Park, N.C. 1974.
66. Dietzman, H. E. Protocol to Characterize Gaseous Emissions as a
Function of Fuel and Additive Composition. Prepared under Contract
No. 68-02-1275 by Southwest Research Institute, San Antonio, Texas.
U.S. Environmental Protection Agency. Research Triangle Park, N.C.
Publication No. 650/2-75-048. 1975.
67. Hare, C. T. Methodology for Determining Fuel Effects on Diesel Par-
ticulate Emissions. Prepared by Southwest Research Institute, San
Antonio, Texas. U.S. Environmental Protection Agency. Research
Triangle Park, N.C. Publication No. EPA-650/2-75-056. 1975.
68. Lestz, S. S. Analysis of combustion products from nitrogenous gaso-
line additives. Prepared under Grant No. R-80425 by Pennsylvania
State University, University Park, Pa. Interim Report. U.S.
Environmental Protection Agency. Research Triangle Park, N.C. 1975.
69. Belzer, M. Particulate emissions from prototype catalyst cars.
Prepared by Exxon Research and Engineering Co., Linden, N.J. U.S.
Environmental Protection Agency. Research Triangle Park, N.C.
Publication No. EPA-650/2-75-054. 1975.
191
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8. CONTROL TECHNOLOGY
8.1 CONTROL TECHNOLOGY FOR STATIONARY SOURCES OF NITROSAMINES
8.1.1 Introduction
Nitrosamines as a class of compounds can exist in the gaseous, liquid,
or solid state, depending on molecular weight. They can be emitted to
the environment in any of the phases or as a solute in water or hydro-
carbons. They can be adsorbed in solid particles or absorbed in aero-
sols, and can also be formed in the environment by the reaction of
1 2
secondary or tertiary amines with nitrous acid or nitric oxides. '
Because of the wide range of physical properties and sources of nitrosa-
mines, control technology requirements for many types of sources are
discussed. Data are limited since nitrosamines are not widely manu-
factured. The control techniques discussed are therefore based on the
application of general principles commonly used for the control of many
types of pollutants. Limited experimental data on control of nitrosa-
mines in water are presented in detail. Because of the possibility of
in situ nitrosamine formation in air, control of the precursors is
covered briefly.
8.1.2 Control Technology for Air Emissions of Nitrosamines
Most nitrosamines are liquids having relatively high boiling points and,
as such, are not found in large quantities in the gaseous state. How-
ever, small amounts at the ppb or ppm level can exist in equilibrium
with the liquid. Because of the low vapor levels possible and the
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limited use of nitrosamines in the chemical industry, very little has
been done to explore vapor-phase control techniques. Methods suggested
are based on the known properties of the materials and on engineering
judgment.
8.1.2.1 Wet Scrubbing—The two lowest-molecular-weight nitrosamines
(dimethyl and diethyl) are soluble in water, though other compounds in
this class are insoluble. Consequently, water scrubbing can be used as
an effective control technique in the case of dimethyl- and diethylnitros-
amine. For higher-molecular-weight nitrosamines, wet scrubbing is not an
efficient control method except when the aqueous medium contains a soluble
oxidizing agent such as hydrogen peroxide, potassium permanganate, or
3 4
chlorine. ' The efficiency of removal would depend on the rate of oxi-
dation, which is a function of the oxidizing agent and the pH of the
scrubbing medium. The oxidation rate is also affected by temperature and
pressure, the presence of catalytic species, and the efficiency of utili-
zation of oxidant. Catalytic species can increase the oxidation rate or
cause premature decomposition of the oxidizing agent, resulting in poor
utilization of the oxidizing agent. Discussion given in Section 8.1.3
indicates that under drastic conditions of temperature and pressure wet-
air oxidation is more effective than oxidation with hydrogen peroxide, a
stronger oxidant. Unfortunately, there are practical limitations to the
temperature and pressure conditions that may be employed in a continuous
scrubbing process. The presence of other oxidizable species in the
effluent gas stream could result in competitive reactions that could
reduce the efficiency of the system and increase the cost of control. It
193
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is possible that scrubbing reaction products may in themselves present
environmental problems. For example, the use of chlorine as an oxidant
could result in the formation of organochloramines or chlorinated hydro-
carbons. Further work is necessary to determine the most suitable
oxidant.
The cost of control technology is to a great extent a function of the
reaction rate (which affects the size of equipment) and the required
control efficiency. These parameters have not been defined. Typical
capital costs for scrubbers used for particulate removal run from $0.25
to $3.00 per standard cubic foot per minute (scfm) capacity.
8.1.2.2 Incineration—Control of nitrosamine emissions by high-tempera-
ture oxidation would be an efficient method. Selection of its use would
depend on nitrosamine concentration, the moisture content of the effluent
gas stream, the constancy of emission, and the cost and availability of
fuel.
Capital costs for conventional incinerators without heat exchangers may
be expected to be $2.00 to $3.00 per scfm. Heat exchangers increase
capital costs by a factor of two. Operating costs may vary from $3.00
to $30.00 per hour over the capacity range of 5,000 to 38,000 scfm.
It may be possible to reduce the operating temperature, and thus the
costs of fuel, for catalytic incineration. Work is being done to develop
better catalysts, and their successful development could make this a
practical method of control at some future date. Capital costs for
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catalytic units may be expected to be about twice those for conventional
incinerators. However, operating costs for catalytic units may be less
than half those for conventional units.
8.1.2.3 Adsorption—Activated charcoal may be used to adsorb nitrosa-
mines from effluent gas streams. In instances where the moisture content
of the effluent gas is high, the temperature of the gas is also usually
high and a condenser must then be used to reduce the gas temperature (and
moisture content) since adsorption is inefficient at high temperatures
(>120°F). In these instances, the bulk of the nitrosamines would be
condensed with the water and the adsorber would take out the residual.
It has been estimated that adsorption preceded by condensation is as
efficient a control method as the use of an afterburner (incinerator)
preceded by a condenser. Capital costs for carbon sorption may be
expected to be $2.00 to $8.00 per scfm capacity.
8.1.2.4 Catalytic Reduction—Reduction of nitrosamines to hydrazines
is a well-documented reaction. The development of catalysts to improve
the efficiency of this reduction could make this a practical control
method. Investigations on the use of a copper-iron couple as the reduc-
ing agent may lead to useful information. This reduction system has
been successfully applied to chlorinated pesticides and polychlorinated
biphenyl compounds in manufacturing waste waters. Possibly this system
could be used in combination with a water scrubber to reduce the nitrosa-
mines captured in the scrubbers to hydrazines. Capital costs for waste
water treatment are projected to be $1,000 per gallon per minute capacity;
operating costs are projected to be negligible.
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8.1.2.5 Ultraviolet Radiation—Mi trosamines are known to degrade photo-
2
chemically both in air and in aqueous solution. It may be possible to
take advantage of this reaction to control the emission of nitrosamines.
The degradation products would be expected to be the parent compounds
(e.g., amines and nitrite). The quantum yield for this reaction is
unlikely to be high enough to allow ultraviolet radiation to be used as
an effective control technology for atmospheric emissions. The practi-
cal consideration of retaining large volumes of waste gases for the
required long irradiation times would make ultraviolet irradiation a dif-
ficult approach. However, if the quantum yield were high enough, this
method could be the simplest and most economical control technique for
removing nitrosamines from effluent gases.
8.1.3 Control Technology for Nitrosamines in Industrial Waste Water
Data concerning the control of nitrosamines in water are relatively scarce.
Many laboratory investigations relative to the chemical properties of
nitrosamines hold out several possible decontamination techniques, but to
date none has proved to be effective in practice. In fact, only in the
case of UDMH rocket-fuel production waste (U.S. Air Force) have actual
treatment studies been conducted with the goal of complete elimination of
a nitrosamine (dimethylnitrosamine). These studies, while not yet totally
successful, point out that a combination of techniques will probably have
to be utilized in order to achieve complete detoxification.
Several methods for destroying nitrosamines in water have been suggested.
These include oxidation by chlorine, hydrogen peroxide, ozone, or wet air;
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reduction by sodium hydrosulfite; photochemical degradation; and biodegra-
dation. In addition, suggested methods for separating or removing
nitrosamines from water include: (1) steam stripping/distillation, fol-
lowed by recycle and reuse of the nitrosamine; and (2) carbon adsorption,
followed by incineration or thermal regeneration of the carbon.
8.1.3.1 Oxidation by Chlorine--The data generated by the Food Machinery
and Chemical Corporation, Baltimore, Maryland, for the U.S. Air Force on
the caustic waste generated from the production of UDMH for rocket fuel
has indicated that chlorination is capable of removing from 50 to 95 per-
cent of the DMN (initial concentration, 12 percent), depending on the
amount of sodium hypochlorite used. When a sevenfold excess of sodium
hypochlorite was added to a 125-ppm DMN solution, 95 percent removal was
achieved within 30 minutes.
Gas chromatographic-mass spectrometric analysis (GC-MS) revealed the
presence of chloromethane (CH3C1) and dichloromethane (CH2C12) in chlori-
nation products of DMN; and these compounds, along with chloroform, carbon
tetrachloride, and chlorinated ethanes, in the chlorination products of
UDMH. Based on the presence of these chlorinated hydrocarbons, which,
like DMN, are suspected carcinogens, it does not appear that chlorination
is a feasible treatment method.
8.1.3.2 Oxidation by Hydrogen Peroxide—The laboratory experiments by FMC
Corporation for the U.S. Air Force have shown that in acidic solutions
40 percent of the DMN was removed by hydrogen peroxide after a 1-hour
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reflux at 100°C. Hydrogen peroxide oxidation, therefore, should not be
considered an effective treatment method.
8.1.3.3 Oxidation by Ozone—Since hydrogen peroxide does not effectively
remove DMN from aqueous solution, a stronger oxidizing agent must be
employed. However, as can be seen in the case of chlorination (where the
presence of chlorinated hydrocarbons after treatment precludes its use),
the oxidizer must also react to form innocuous products and byproducts.
Ozone can be expected to possess these critical properties, but no firm
data exist on what reaction conditions are necessary to achieve essen-
tially complete removal. Possible use of ozonation in conjunction with
ultraviolet (UV) photolysis may also be beneficial.
Since commercial waste water treatment systems are available that use
ozonation with or without UV photolysis, these options should be con-
sidered and investigated further in the control and removal of DMN from
the water environment.
8.1.3.4 Oxidation by Wet Air—Laboratory data gathered by Zimpro, Inc.,
for the Air Force have shown that wet air oxidation (wet-ox) is success-
ful in reducing the DMN concentration in waste water from 400 ppm to less
than 1 ppm within one hour at 300°C at 3000 psi. Unfortunately, no pilot-
scale or full-scale experiments have yet been conducted to assess the full
potential of this promising technique. The residence time and high pres-
sure requirements would make this approach very expensive.
8.1.3.5 Reduction by Sodium Hydrosulfite—The laboratory experiments by
FMC Corporation for the U.S. Air Force have been conducted to determine
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the extent of the reduction of DMN to dimethyl amine by sodium hydrosulfite.
Results indicate that, in alkaline solution, 60 percent of the DMN (initial
concentration, 1.5 percent) was removed after 2 hours at 50°C. This
method, therefore, is not effective enough to warrant further attention.
8.1.3.6 Photodegradation--Data on the photochemical cleavage of the
nitroso group from nitrosamines in aqueous solution indicate that for
very low concentrations (^ 25 ppm) virtually 100 percent of the nitrosa—
mine is converted to the amine by irradiation at 230 nm (UV light); but,
as the concentration of nitrosamine is increased, the extent of cleavage
o
is decreased. It appears, therefore, that photodegradation will be
effective only on wastes that have been pretreated by some other technique
to reduce nitrosamines to a very low residual concentration.
8.1.3.7 Biodegradation—The very fact that DMN is formed as a metabolic
product in many species and does not bioaccumulate leads one to suspect
that it is susceptible to bacterial degradation. Few data are available,
however, to indicate to what degree, how fast and under what conditions
a biological treatment system can remove the material from the waste
stream or what the byproducts of such removal would be. Because of this
lack of data and the nature of the DMN, it is recommended that a waste
stream containing this compound not be channeled to a municipal or joint
municipal-industrial sewage treatment plant unless pilot-scale testing of
the waste shows that the DMN is totally removed.
8.1.3.8 Stream Stripping/Disti 11 ation—One method of separating nitrosa-
mines from an aqueous waste stream is to distill the material and recycle
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it to the process. The FMC Corporation studies with DMN (for the U.S. Air
Force) showed that this method could reduce the concentration from 1180
ppm to 60 ppm when 8 percent of the waste water was allowed to distill
over with the DMN. Stream stripping of the solution was considered to be
a pretreatment method. Actual use of the technique is under way at the
FMC UDMH plant, where the DMN concentration of the waste is reduced from
the 1.5 percent range to the 400 ppm range. However, 400 ppm is probably
not an environmentally acceptable DMN concentration, so that further
removal will be necessary before disposing of the stripped waste.
8.1.3.9 Carbon Adsorption--The only available data concerning the removal
of nitrosamines from solution have been generated for caustic DMN waste by
the Air Force. Isotherms have been obtained for three different activated
_3
carbons, with capacities calculated in the range of 1 x 10 moles/g to
_3
2.4 x 10 moles/g. Column studies using 25 g of carbon have shown that
breakthrough at 1 ppm DMN occurs after 0.5 liter of waste containing 218
ppm DMN has been treated. If 1 ppm is an acceptable effluent concentra-
tion, carbon requirements would be on the order of 0.5 pound per gallon
of this waste. Activated carbon therefore may not be a logistically feasi-
ble treatment method for large volumes of DMN wastes. However, even though
hard data are lacking, nitrosamines such as nitrosodiphenylamine would be
expected to be much more efficiently removed by activated carbon. This
method should be studied further for high-molecular-weight nitrosamines.
Disposal of the contaminated activated carbon may present a problem.
Incineration of the carbon will result in destruction of the adsorbed
nitrosamine but will be extremely expensive because of fuel and carbon
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costs. Regeneration of the carbon with proper treatment of off-gases may
be possible, but no data are available on the efficiency of regeneration
or the air pollution hazard associated with this specific technique. Dis-
posal of the spent carbon in a landfill should not be considered because
it is very likely that nitrosamines would be leached out of the carbon by
rainwater.
8.1.4 Ultimate Disposal Methods for R$tr^sarnSne-€orv$ain4ng Wastes
Even if treatment techniques are successful in destroying or removing
nitrosamine in water, the remaining sludges or waste water will not be
safe enough for reuse and must be disposed of properly. Several alterna-
tive techniques include landfill disposal, deep-well disposal, ocean
dumping, and incineration.
8.1.4.1 Landfill Disposal--The nature of landfill disposal is such that
it is susceptible to leaching and rainwater runoff. Therefore, before
this type of disposal is used for toxic materials, permanent and total
detoxification and/or immobilization must be performed. The U.S. Air
Force has looked at methods of immobilizing its DMN-contaminated waste
water; but, to date, the various gelling and "chem-fixing" techniques
have not been successful in totally eliminating leaching of DMN by rain-
water. Thus, landfill disposal should not be considered until such time
as a satisfactory immobilization technique is developed.
8.1.4.2 Deep-Well Disposal--The technique of disposing of waste into a
specially drilled and cased deep well, the location of which has been
carefully selected, may be acceptable under special circumstances. In
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this technique, waste is permanently entombed in porous rock. Deep-well
injection has been used to dispose of many toxic wastes with satisfactory
results for many years. A few cases, however, have resulted in near
disasters when improper drilling techniques were used or when the well
was located in a drinking water aquifer or an aquifer that eventually
surfaced. Extreme care must be exercised, therefore, before a decision
is made to inject nitrosamine waste into a disposal well. This approach
is generally not considered acceptable in geologic fault regions.
8.1.4.3 Ocean Dumping—The ocean disposal of nitrosamine wastes, like
any other waste, is subject to many restrictions and preconditions.
Before disposal, permits must be issued and, prior to their issuance,
both public hearings and biological and treatment studies may be required.
Ultimately, an environmental impact assessment must be made that compares
the impact of ocean dumping with that of other disposal methods. Since
it is unlikely that untreated nitrosamine has no environmental impact,
pretreatment will almost certainly be required.
8.1.4.4 Incineration—The incineration of nitrosamine waste is theoreti-
cally possible. Studies have shown that nitrosamines, specifically DMN,
are decomposed in oxygen at 300°C and above. Although wastes containing
these compounds are candidates for incineration, other constituents of
the waste may preclude the use of this disposal technique. The high salt
content of UDMH rocket fuel waste, for example, may lead to breakout of
the refractory lining of an incinerator. Such factors must be considered
in selection of materials for incinerator design. It is important,
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therefore, to ascertain the exact nature of the waste before deciding to
incinerate it.
8.1.5 Control of Plant Location
Considering the possibility of nitrosamine formation from the atmospheric
reaction of NO with secondary and tertiary amines and with compounds con-
/\
taining tertiary amino groups, it might be reasonable to keep separate the
effluent streams containing these reactive components.
A control strategy might involve minimizing contact between NO -containing
/\
plumes and industrial effluent gases containing amines or nitrosamine pre-
cursors. This strategy would involve the use of appropriate industrial
zoning regulations. While this approach would not completely eliminate
the atmospheric formation of nitrosamines, it would reduce it. Since the
rate of production of nitrosamines is dependent on the concentration of
the reactants, dilution of the reactants before they come in contact will
depress the reaction rate.
8.2 TECHNOLOGY FOR CONTROL OF AIR EMISSIONS OF NITROSAMINE PRECURSORS
Secondary amines and nitrous acid or nitric oxides are precursors for the
formation of nitrosamines in the vapor phase. Organic decomposition
resulting in the amines as a byproduct can yield nitrosamines in the
presence of nitrites. Vapor-phase amines can be controlled, but the con-
trol of nitric oxides, nitrites, and fine particulates is most difficult.
Control methods for these material are discussed in great detail in the
literature, and only a summary is presented here.
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8.2.1 Control Technology for Amines
The motivation for control of amine emissions has historically been the
control of offensive odors. Because of this objective, the quantifica-
tion of amine emissions has been in terms of "odor units," and much of
the literature defines control efficiency in terms of odor units. Because
of the public reaction to offensive odors, high levels of amines have not
been emitted into the ambient air.
Wet scrubbing can be used to remove amines from an effluent gas stream.
Aqueous solutions of hydrochloric, sulfuric, or sulfamic acids, in con-
centrations of approximately 5 percent, are used as absorption media.
Reductions in the amine concentrations of 90 to 99 percent are reported
g
in a study by Dickerson and Murthy. Scrubbing is the most economical
and efficient method for amine removal from these process streams, which
produce large volumes of effluent gas with low concentrations of amines.
Amine hydrochloride salts are formed in the case of hydrochloric acid
scrubbers and there is the possibility of reaction with nitrite ions to
form nitrosamines if the nitrate-nitrite content of the water is high.
Extracted amines must be recovered from the water phase. Neutralization
and distillation, followed by either recycle or disposal by non-polluting
methods (incineration, for example), are required to make this method
acceptable.
Incineration or high-temperature oxidation (combustion) destroys amines
in the effluent gas phase. The feasibility of this control method
depends on the moisture content of the gas phase, the concentration of
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amines in the effluent gas, and the volume of effluent gas being handled.
The cost involved may be prohibitive, given current energy costs. Further-
more, the products of combustion of the effluents should not be overlooked,
for in the case of amines they would include oxides of nitrogen that would
have to be controlled.
Adsorption on activated charcoal is used effectively to remove organic
vapors. The adsorbed organic materials may be eluted with heated air,
nitrogen, or an organic solvent; then condensed and recovered. Gas streams
must be cooled, since efficient adsorption does not take place at tempera-
tures in excess of 120°F. The adsorptive capacity of activated charcoals
for low-molecular-weight amines is low, so that this method is less effec-
tive for those amines that have the capacity of forming the potentially
most active carcinogens. Gas streams containing particulate matter must
be "cleaned" before passing through the adsorber. This usually means a
scrubber-contact condenser. The cost of regeneration of the adsorbent,
the relatively low capacity for low-molecular-weight amines, and the
necessity for removing particulate matter make this method less interest-
ing than wet scrubbing except in some special circumstances.
Condensation of an organic vapor to its liquid state by cooling is used
to purify gas effluents when the effluent has a relatively low moisture
content and easily condensable organic vapors. As was mentioned above,
this method is usually used in conjunction with other control methods to
recover adsorbed organic materials and to reduce the moisture content of
the effluent gases to be incinerated. For low-molecular-weight organics,
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this method will only be of limited value since 100 percent removal cannot
be attained.
8.2.2 Control Technology for* Nitrogen Oxides
Nitrogen oxides control is difficult. Summarized, the simplest modifica-
tion is control of the type of fuel burned; but, unfortunately, the most
desirable fuel for the control of NO emissions is the least available and
/\
the most expensive. Process modifications resulting in lower flame tem-
peratures and a minimum of excess air for combustion give reduced NO emis-
/\
sions. These modifications may eventually be adopted even though the use
of minimum excess air may result in the increased emission of carbon
monoxide and particulate matter.
Catalytic reduction of NO on noble metal catalysts is feasible techni-
/\
cally but is limited by the presence of other more easily reduced species
in effluent gases, making the economics of the process unfavorable for
most stationary combustion sources. The development of non-noble metal
catalysts is being pursued and could make this a practical method of con-
trol at some future date.
Aqueous scrubbers are not effective for NO control because nitric oxide
/\
has very low water solubility. Oxidative solutions have been used but
have not resulted in an economically acceptable reduction of NO to the
/Q
desired levels. The presence of other more easily oxidized species in
the effluent gas requires excessive concentrations of oxidants and makes
the economics of this control method impractical.
206
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Exhaust recycle can provide some control for NO emissions from the
A
4
internal combustion engine, but power is reduced and operating costs
are increased. Therefore, this method has not been widely adopted.
The absorption of nitric oxide emissions in organic solvents or on poly-
meric adsorbents is currently under investigation. There are no short-
range solutions expected and the potential of a long-range solution is
uncertain.
8.2.3 Control Technology for Particulates
Many nitrosamines and their precursors have relatively high melting and
boiling points and do not exist in the gas phase. They may be omitted
in some processes as dust or as aerosols. Dusts, whether consisting of.
finely divided particles of nitrosamine precursors or of inert materials
upon which precursors are adsorbed, have the potential for reacting with
nitrogen oxides in the atmosphere. The particulates, therefore, must be
removed from the effluent gas and disposed of safely. The usual par-
ticulate removal technologies are applicable and are summarized here.
Bag filters are the most efficient method for the removal of particulates
from effluent gas. The nature and size of the particle will determine
the filter material, and the efficiency of the process will depend on the
velocity, temperature, pH, and moisture content of the stream, and the
chemical nature of the gas and particulates.
Wet scrubbing is a reasonably economical and efficient method of removing
relatively large particulates from effluent gas streams. As the particle
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size decreases, energy requirements and, therefore, cost Increase until
the cost exceeds that of fabric filtration or of electrostatic separation,
Disposal of the contaminated water presents a problem and militates
against the use of wet scrubbing.
Electrostatic precipitation of dust particles is an efficient method of
control. The initial cost is the highest of any method of control, .how-
ever, and for this reason electrostatic precipitation would not be the
control method of choice except where other methods are inapplicable for
some reason.
Inertia! separators are often used to collect particulate matter. The
efficiency of collection is not high for small particles, and the method
is not recommended for toxic particulates because total removal cannot
be attained.
8.3 REFERENCES FOR SECTION 8
1. Morrison, R. T. and R. N. Boyd. Organic Chemistry, 3rd Ed. Boston,
Allan and Bacon. 1974.
2. Smith, P. A. and R. N. Leoppky. Nitrosative cleavage of tertiary
amines. J. Amer. Chem. Soc. 89_:1147, 1961.
3. Kissinger, L. W. and M. Schwartz. Some Michael-like additions of
primary nitrosamines. J. Org. Chem. 23_:1342, 1958.
4. Industrial Pollution (N. Irving Sax, ed.). New York, Van Nostrand
Reinhold Company. 1974. Chapter 13.
5. Haring, C. H. Cost effectiveness relationships in odor control.
Ann. N.Y. Acad. Sci. 237:336, 1974.
6. Strauss, W. Industrial Gas Cleaning. New York, Pergamon Press.
1966. p. 111.
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7. Control equipment for gases and vapors. In: Air Pollution Engineer-
ing Manual, 2nd Ed. (J. A. Danielson, ed.JT 1973. p. 171.
8. Chow, Y. L., M. P. Laa, R. A. Perry, and J. N. S. Tarn. Photochem-
istry of nitroso compounds in solution. XX. Photoreduction, photo-
elimination and photoaddition of nitrosamines. Can. J. Chem. 50:
1044, 1972.
9. Dickerson, R. C. and B. N. Murthy, Odors: evaluation, utilization,
and control. Ann. N.Y. Acad. Sci. 23^:374, 1974.
10. Mitlellman, R. and B. R. Taylor. Odors: evaluation, utilization,
and control. Ann. N.Y. Acad. Sci. 237:350, 1974.
11. Billings, C. E. Handbook of Fabric Filter Technology. G.C.A. Cor-
poration. Bedford, Mass. (National Technical Information Service,
Springfield, Va. PB 200 648.)
209 ,
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TECHNICAL REPORT DATA
(Please read Jnaructions on the reverse before completing)
1. REPORT NO.
EPA-600/ 6-77-001
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
Scientific and Technical Assessment Report on
Nitrosamines
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research and Development
Criteria and Special Studies Office,
Research Triangle Park, N.C. 27711
10. PROGRAM ELEMENT NO.
1AA001
11. CONTRACT/GRANT NO.
HERL
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research and Development
Office of Program Integration
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report is a review and evaluation of the current knowledge of N-nitrosamines (NA]
in the environment as related to possible deleterious effects on human health and
welfare. Sources, distribution, measurement, and control technology for NA and their
precursors are also considered. NA (characterized by the N-N=0 group) are formed.by
the reaction of amines with nitrous acid. Nearly 70% of all N-nitroso compounds
studied have been found to be carcinogenic, with a wide range in potency, in all
species of laboratory animals tested via all routes of administration. The experi-
mentally produced carcinogenesis appears to be caused by metabolites rather than by
NA themselves. Epidemiological studies to date do not show a direct relationship
between exposure to NA and cancer in man. Ambient air concentrations of NA of up to
36 yg/m3 have been found near an emission source; and of up to 0.2 yg/m3 in major
population centers. The relative contributions of natural and man-made sources are
indeterminable at present. Average dietary intake of NA is not likely to exceed a
few yg/day. Intake of NA in municipal drinking water would probably be much less
than 1 yg/day. NA in cigarettes range from 0 to 180 ng/cigarette.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Nitrosamines
Pollution
Toxi ci ty
Carcinogenicity
Chemical analysis
Nitrogen oxides
Air
Water
Foo'd
Ecology
Ami nes
Control
Measurement
Nitrates
Nitrites
Environmental
distribution
Environmental pollution
02A
06A
06C
06F
06T
07B
07C
07D
14A
14B
13B
18. DISTRIBUTION STATEMENT
Release JUnlimited
19. SECURITY CLASS (ThisReport}
Unclassified
21. NO. OJr.PAGES
T29" . .
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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