oEPA
United States
Environmental Protection
Agency
Office of Water
Regulations and Standards
Criteria and Standards Division
Washington DC 20460
EPA 440/5-80-064
October 1980
Ambient
Water Quality
Criteria for
Nitrosamines
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AMBIENT WATER QUALITY CRITERIA FOR
NITROSAMINES
Prepared By
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Regulations and Standards
Criteria and Standards Division
Washington, D.C.
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio
Carcinogen Assessment Group
Washington, D.C.
Environmental Research Laboratories
Corvalis, Oregon
Duluth, Minnesota
Gulf Breeze, Florida
Narragansett, Rhode Island
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DISCLAIMER
This report has been reviewed by the Environmental Criteria and
Assessment Office, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
AVAILABILITY NOTICE
This document is available to the public through the National
Technical Information Service, (NTIS), Springfield, Virginia 22161.
11
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FOREWORD
Section 304 (a)(l) of the Clean Water Act of 1977 (P.L. 95-217),
requires the Administrator of the Environmental Protection Agency to
publish criteria for water quality accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects
on health and welfare which may be expected from the presence of
pollutants in any body of water, including ground water. Proposed water
quality criteria for the 65 toxic pollutants listed under section 307
(a}(l) of the Clean Water Act were developed and a notice of their
availability was published for public comment on March 15, 1979 (44 FR
15926), July 25, 1979 (44 FR 43660), and October 1, 1979 {44 FR 56628).
This document is a revision of those proposed criteria based upon a
consideration of comments received from other Federal Agencies, State
agencies, special interest groups, and individual scientists. The
criteria contained in this document replace any previously published EPA
criteria for the 65 pollutants. This criterion document is also
published in satisifaction of paragraph 11 of the Settlement Agreement
in Natural Resources Defense Council, et. al. vs. Train, 8 ERC 2120
(D.D.C. 1976), modified, 12 ERC 1833 (D.D.C. 1979).
The term "water quality criteria" is used in two sections of the
Clean Water Act, section 304 (a)(l) and section 303 (c)(2). The term has
a different program impact in each section. In section 304, the term
represents a non-regulatory, scientific assessment of ecological ef-
fects. The criteria presented in this publication are such scientific
assessments. Such water quality criteria associated with specific
stream uses when adopted as State water quality standards under section
303 become enforceable maximum acceptable levels of a pollutant in
ambient waters. The water quality criteria adopted in the State water
quality standards could have the same numerical limits as the criteria
developed under section 304. However, in many situations States may want
to adjust water quality criteria developed under section 304 to reflect
local environmental conditions and human exposure patterns before
incorporation into water quality standards. It is not until their
adoption as part of the State water quality standards that the criteria
become regulatory.
Guidelines to assist the States in the modification of criteria
presented in this document, in the development of water quality
standards, and in other water-related programs of this Agency, are being
developed by EPA.
STEVEN SCHATZOW
Deputy Assistant Administrator
Office of Water Regulations and Standards
111
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ACKNOWLEDGEMENTS
Aquatic Life Toxicology
William A. Brungs, ERL-Narragansett
U.S. Environmental Protection Agency
David J. Hansen, ERL-Gulf Breeze
U.S. Environmental Protection Agency
Mammalian Toxicology and Human Health Effects
John Garner, HERL (author)
U. S. Environmental Protection Agency
Michael L. Dourson (doc. mgr.), ECAO-Cin
U.S. Environmental Protection Agency
Jerry F. Stara (doc. mgr.), ECAO-Cin
U.S. Environmental Protection Agency
Penelope A. Fenner-Crisp, ODW
U.S. Environmental Protection Agency
Wallace Hayes
University of Mississippi Medical Center
Elliot Lomnitz, OWPS
U.S. Environmental Protection Agency
Roy E. Albert*
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Michael C. Archer
Ontario Cancer Research Center
Patrick R. Durkin
Syracuse Research Corporation
David H. Fine
New England Institute for Life Sciences
Si Duk Lee, ECAO-Cin
U.S. Environmental Protection Agency
Technical Support Services Staff: D.J. Reisman, M.A. Garlough, B.L. Zwayer,
P.A. Daunt, K.S. Edwards, T.A. Scandura, A.T. Pressley, C.A. Cooper,
M.M. Denessen.
Clerical Staff: C.A. Haynes, S.J. Faehr, L.A. Wade, D. Jones, B.J. Bordicks,
B.J. Quesnell, P. Gray, R. Rubinstein.
*CAG Participating Members:
Elizabeth L. Anderson, Larry Anderson, Dolph Arnicar, Steven Bayard, David
L. Bayliss, Chao W. Chen, John R. Fowle III, Bernard Haberman, Charalingayya
Hiremath, Chang S. Lao, Robert McGaughy, Jeffrey Rosenblatt, Dharni V. Singh,
and Todd W. Thorslund.
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TABLE OF CONTENTS
Page
Criteria Summary
Introduction A-l
Aquatic Life Toxicology B-l
Introduction 8-1
Effects B-l
Acute Toxicity B-l
Residues B-l
Miscellaneous B-2
Summary B-2
Criteria B-3
References B-7
Mammalian Toxicology C-l
Introduction C-l
Sources of and Routes of Exposure to N-Nitroso
Compounds C-l
Ingestion from Water C-5
Ingestion from Food C-7
Inhalation C-ll
Dermal C-13
Pharmacokinetics C-17
Distribution C-17
Metabolism C-17
Effects C-19
Acute, Subacute, and Chronic Toxicity C-19
Teratogencitiy C-23
Mutagenicity C-24
Carcinogencity C-25
Criterion Formulation C-44
Existing Guidelines and Standards C-44
Current Levels of Exposure C-44
Special Groups at Risk C-45
Basis and Derivation of Criterion C-45
References C-50
Appendix C-64
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CRITERIA DOCUMENT
NITROSAMINES
CRITERIA
Aquatic Life
The available data for nitrosamines indicate that acute toxicity to
freshwater aauatic life occurs at concentrations as low as 5,850 yg/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
nitrosamines to sensitive freshwater aauatic life.
The available data for nitrosamines indicate that acute toxicity to
saltwater aauatic life occurs at concentrations as low as 3,300,000 ug/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
nitrosamines to sensitive saltwater aauatic life.
Human Health
For the maximum protection of human health from the potential carcino-
genic effects due to exposure of N-nitrosodiethylamine and all other nitros-
amines except those listed below, through ingestion of contaminated water
and contaminated aauatic organisms, the ambient water concentrations should
be zero based on the non-threshold assumption for this chemical. However,
zero level may not be attainable at the present time. Therefore, the levels
which may result in incremental increase of cancer risk over the lifetime
are estimated at 10" , 10" , and 10" . The corresponding recommended
criteria are 8.0 ng/1, 0.8 ng/1, and 0.08 ng/1, respectively. If the above
estimates are made for consumption of aauatic organisms only, excluding con-
sumption of water, the levels are 12,400 ng/1, 1,240 ng/1, and 124 ng/1,
respectively.
VI
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For the maximum protection of human health from the potential carcino-
genic effects due to exposure of N-nitrosodimethylamine through ingestion of
contaminated water and contaminated aauatic organisms, the ambient water
concentrations should be zero based on the non-threshold assumption for this
chemical. However, zero level may not be attainable at the present time.
Therefore, the levels which may result in incremental increase of cancer
risk over the lifetime are estimated at 10 , 10 , and 10. The
corresponding recommended criteria are 14 ng/1, 1.4 ng/1, and 0.14 ng/1,
respectively. If the above estimates are made for consumption of aquatic
organisms only, excluding consumption of water, the levels are 160,000 ng/1,
16,000 ng/1, and 1,600 ng/1, respectively.
For the maximum protection of human health from the potential carcino-
genic effects due to exposure of N-nitrosodibutylamine through ingestion of
contaminated water and contaminated aauatic organisms, the ambient water
concentrations should be zero based on the non-threshold assumption for this
chemical. However, zero level may not be attainable at the present time.
Therefore, the levels which may result in incremental increase of cancer
risk over the lifetime are estimated at 10" , 10~5, and 10. The
corresponding recommended criteria are 64 ng/1, 6.4 ng/1, and 0.64 ng/1, re-
spectively. If the above estimates are made for consumption of aquatic
organisms only, excluding consumption of water, the levels are 5,868 ng/1,
587 ng/1, and 58.7 ng/1, respectively.
For the maximum protection of human health from the potential carcino-
genic effects due to exposure of N-nitrosopyrrolidine through ingestion of
contaminated water and contaminated aquatic organisms, the ambient water
concentrations should be zero based on the non-threshold assumption for this
chemical. However, zero level may not be attainable at the present time.
vn
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Therefore, the levels which may result in incremental increase of cancer
risk over the lifetime are estimated at 10, 10 , and 10" . The
corresponding recommended criteria are 160 ng/1, 16 ng/1, and 1.6 ng/1, re-
spectively. If the above estimates are made for consumption of aauatic
organisms only, excluding consumption of water, the levels are 919,000 ng/1,
91,900 ng/1, and 9,190 ng/1, respectively.
For the maximum protection of human health from the potential carcino-
genic effects due to exposure of N-nitrosodiphenylamine through ingestion of
contaminated water and contaminated aquatic organisms, the ambient water
concentrations should be zero based on the non-threshold assumption for this
chemical. However, zero level may not be attainable at the present time.
Therefore, the levels which may result in incremental increase of cancer
risk over the lifetime are estimated at 10~5, 10~6, and 10~7. The
corresponding recommended criteria are 49,000 ng/1, 4,900 ng/1, and 490
ng/1, respectively. If the above estimates are made for consumption of
aauatic organisms only, excluding consumption of water, the levels are
161,000 ng/1, 16,100 ng/1, and 1,610 ng/1, respectively.
Vlll
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INTRODUCTION
The nitrosamines belong to a large group of chemicals generally called
N-nitroso compounds. Also Included in this group are the structurally-re-
lated nitrosamides. Because they frequently coexist with N-nitrosamines in
the environment, nitrosamides are addressed also in this document.
Synthetic production of N-nitrosamines is limited to small Quantities,
and the only nitrosamine produced in Quantities greater than 450 kg/yr is
N-nitrosodiphenylamine. It is used as a vulcanizing retarder in rubber pro-
cessing and in the manufacture of pesticides. The general physical proper-
ties of diphenylnitrosamine are: molecular weight, 198.24 and a melting
point of 66.5*C (Tanikaga, 1969). Other N-nitroso compounds are produced
primarily as research chemicals and not for commercial purposes (U.S. EPA,
1976).
Nitrosamines are characterized by the functional group -N-N-0 and
nitrosamides are characterized by the functional group -C-N-N-0. Depending
on the nature of the radical group, nitrosamines exist in several forms,
including symmetrical dialkyl-nitrosamines, asymmetrical dialkyl-nitroso-
amines, nitrosamines with functional groups, cyclic nitrosoamines and acyl-
alkylnitrosamines with functional groups, cyclic nitrosamines and acylalkyl-
nitrosamines or nitrosamides (Searle, 1973).
The nitrosamines vary widely in their physical properties and may exist
as solids, liauids, or gases. They are soluble in water and organic sol-
vents. Nitrosamines of low molecular weight are volatile at room tempera-
ture, and high molecular weight nitrosamines are steam volatile (U.S. EPA,
1976).
A-l
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The most significant source of N-nitrosamines and N-nitrosamides in the
environment is probably nitrosation of amine and amide precursors (Bogovski,
et al. 1972). These reactions may occur in air, soil, water, food, and ani-
mal systems, when the precursors are present simultaneously (Mysliwy, et al.
1974; Fine, et al. 1977b; Rounbehler, et al. 1977; Mills, 1976). The extent
of exposure to the general population of N-nitrosamines and N-nitrosamides
is unknown. The most significant exposures, resulting from anthropogenic
sources, are probably restricted to limited industrial areas (Fine, et al.
1977a,b,c).
A-2
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REFERENCES
Bogovski, P., et al. 1972. N-nltroso compounds, analysis and formation.
IARC Sci. Publ. No. 3. Int. Agency Res. Cancer, Lyon, France.
Fine, D.H., et al. 1977a. Human Exposure to N-nitroso Compounds in the
Environment. IrK H.H. Hiatt, et al. (eds.), Origins of Human Cancer. Cold
Spring Harbor Lab., Cold Spring Harbor, New York.
Fine, O.H., et al. 1977b. Formation in vivo of volatile N-nitrosamines in
man after ingestion of cooked bacon and spinach. Nature. 265: 753.
Fine, O.H., et al. 1977c. Determination of dimethylnitrosamine in air,
water, and soil by thermal energy analysis: Measurements in Baltimore, Mary-
land. Environ. Sci. Techno!. 11: 581.
Mills, A.L. 1976. Nitrosation of secondary amines by axenic cultures of
microorganisms and in samples of natural ecosystems. Ph.D. Thesis. Cornell
Univ., Ithaca, New York.
Mysliwy, T.S., et al. 1974. Formation of N-nitrosopyrrolidine in a dog's
stomach. Br. Jour. Cancer. 30: 279.
Roundbehler, D.P., et al. 1977. Quantitation of dimethylnitrosamine in the
whole mouse after biosynthesis j_n vivo from trace levels of precursors.
Science. 197: 917.
A-3
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Searle, C.S. 1973. Chemical carcinogens; ACS monograph. Am. Chem. Soc.,
Washington, O.C.
Tanikaga, R. 1969. Photolysis of nitrosobenzene. Bull. Chem. Soc. (Jap.)
U.S. EPA. 1976. Environmental assessment of atmospheric nitrosamines.
MTR-7512. Mitre Corp., McLean, Virginia. Contract No. 68-02-1495.
A-4
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Aquatic Life Toxicology*
INTRODUCTION
The data base is limited to three fish and two invertebrate species;
three acute tests with N-nitrosodiphenylamine were conducted using static
tests and unmeasured concentrations. Feeding studies with N-nitrosodime-
thylamine and rainbow trout demonstrated a dose-related carcinogenic re-
sponse. This response is similar to dose-related effects with mammals and
numerous nitrosamines, including N-nitrosodimethylamine. Details of these
later studies are available in the human health effects portion of this doc-
ument. An additional study with a crayfish showed extensive degeneration of
the antenna! gland and other effects after a 6-month exposure to the same
compound.
EFFECTS
Acute Toxicity
The acute value of n-nitrosodiphenylamine for Daphnia magna and the
bluegill is 7,760 ug/1 and 5,850 ug/1, respectively, (Table 1). This latter
result is significantly different from that for the mummichog, a saltwater
species, for which the 96-hour LC5Q for N-nitrosodiphenylamine is
3,300,000 ug/1 (Table 1). No explanation for this difference is apparent.
Residues
Bioconcentration of N-nitrosodiphenylamine by the bluegill (U.S. EPA,
1978) reached steady-state within 14 days and the bioconcentration factor
was 217 (Table 2). Depuration rate was rapid so that the half-life of this
compound in the tissues was less than 1 day.
*The reader is referred to the Guidelines for Deriving Water Quality Crite-
ria for the Protection of Aauatic Life and Its Uses in order to better un-
derstand the following discussion and recommendation. The following tables
contain the appropriate data that were found in the literature, and at the
bottom of each table are calculations for deriving various measures of tox-
icity as described in the Guidelines.
B-l
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Miscellaneous
Grieco, et al. (1978) fed Shasta strain rainbow trout N-nitrosodimethy-
lamine in the diet for 52 weeks (Table 3). After this time the fish were
placed on a control diet for an additional 26 weeks. No hepatocellular car-
cinomas were detected at 26 weeks after feeding began. At 52 weeks, how-
ever, a direct dose-related response of hepatocellular carcinoma occurred in
trout fed 200, 400, and 800 mg dimethylnitrosamine/kg. A greater incidence
of carcinomas was observed at 78 weeks, even though feeding was discontinued
after 52 weeks. For further information and details on mammalian carcino-
genesis of nitrosamines, the reader is referred to the human health effects
portion of this document.
Another study, by Harshbarger, et al. (1971), exposed the crayfish, Pro-
cambarus clarki i, for 6 months to N-nitrosodimethylamine under renewal pro-
cedures. Microscopical studies revealed extensive degeneration in all parts
of the antennal gland at 200,000 ug/1 and hyperplasia of the tubular cells
in the hepatopancreas at 100,000 ug/1.
Summary
Daphnia magna and the bluegill are the tested freshwater species with
acute values for N-nitrosodiphenylamine of 7,760 and 5,850 ug/1, respective-
ly. These results are auite different from that for the saltwater mummichog
for which the acute value is 3,300,000 ug/1. The bluegill bioconcentrated
the same compound to a factor of 217, but the tissue half-life was less than
one day.
Chronic feeding studies with rainbow trout and N-nitrosodimethylamine
demonstrated a dose-related response of hepatocellular carcinoma over a
feeding range of 200 to 800 mg/kg. An aqueous exposure of crayfish to the
same compound resulted in extensive antennal gland degeneration and other
effects at concentrations of 100,000 to 200,000 ug/1.
B-2
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CRITERIA
The available data for nitrosamines indicate that acute toxicity to
freshwater aquatic life occurs at concentrations as low as 5,850 ug/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
nitrosamines to sensitive freshwater aquatic life.
The available data for nitrosamines indicate that acute toxicity to
saltwater aquatic life occurs at concentrations as low as 3,300,000 ug/1 and
would occur at lower concentrations among species that are more sensitive
than those tested. No data are available concerning the chronic toxicity of
nitrosamines to sensitive saltwater aquatic life.
B-3
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Table 1. Acute values for nltrosanfnes
Species
Cladoceran,
Daphnla magna
Bluegllt,
Lepomis macrochirus
Mummlchog,
Fundulus heteroclltus
LC50/EC50
Method* Chwlcal (ug/l)
FRESHWATER SPECIES
S. U N-nltroso- 7,760
dlpheny lamina
S, U N-nltroso- 5,850
dlpheny lamina
SALTWATER SPECIES
S, U N-nltroso- 3,300.000
dlphenylamlne
Species Mean
Acute Value
(wa/D
7,760
5,850
3,300,000
Reference
U.S. EPA, 1978
U.S. EPA, 1978
Ferraro, et al.
1977
* S » static, U » unmeasured
B-4
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Table 2. Residues for nitrosanines (U.S. EPA, 1978)
Bioconcentratlon Duration
Tissue Chemical Factor (days)
FRESHWATER SPECIES
Blueglll, whole body N-nltroso- 217 14
Lepomls macrochlrus diphenylaralne
B-5
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Table 3. Other data for nltrosa»tn«s
Spec Us
Crayfish,
Procambarus clarkll
Rainbow trout,
Salmo galrdnerl
Che*lea I
dimethy Inltros-
aralne (N-nltrosodl-
methyloffline)
dimethy Inltros-
amlne (N-nltrosodI-
methy lanlne)
Duration Effect Result
FRESHWATER SPECIES
6 nos Antenna! gland 100,000-
degeneratlon and 200,000 ug/l
hyperplasla of
hepatopancreas
78 wks Dose-related Feeding In
hepatocellular diet at 200-
carcinomas 600 mgAg
Reference
Harshbarger, et al.
1971
Grleco, et al. 1976
B-6
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REFERENCES
Ferraro, A.F., et al. 1977. Acute toxicity of water-borne dimethylnitrosa-
mine (DMN) to Fundulus heteroclitus (L). Jour. Fish Biol. 10: 203.
Grieco, M.P., et al. 1978. Careinogenicity and acute toxicity of dimethyl-
nitrosamine in rainbow trout (Salmo galrdneri). Jour. Natl. Cancer Inst.
60: 1127.
Harshbarger, J.C., et al. 1971. Effects of N-nitrosodimethylamine on the
crayfish, Procambarus clarkii* In: Proceedings of the Fourth International
Colloauium on Insect Pathology, College Park, Maryland, August 25-28, 1970.
p. 425.
U.S. EPA. 1978. In-depth studies on health and environmental impacts of
selected water pollutants. Contract No. 68-01-4646, U.S. Environ. Prot.
Agency.
B-7
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Mammalian Toxicology and Human Health Effects
INTRODUCTION
The N-nitrosamines represent one group of those organic compounds char-
acterized by a m'troso group (-N=0) attached to a nitrogen (N-nitroso com-
pounds). Closely related to the N-nitrosamines are the N-nitrosamides. The
formation of both groups of compounds from precursors in the environment,
and in the animal or human body, occurs through a common mechanism (nitrosa-
tion). Both groups of compounds are typically highly toxic, again probably
through common mechanisms. It is extremely unlikely that the human popula-
tion would be exposed only to nitrosamines or only to nitrosamides since the
precursors of both generally occur together. Thus, although this document
is intended to refer specifically to N-nitrosamines, it has been considered
prudent to follow the precedent of earlier literature (in which the term
"nitrosamines" is frequently used synonymously with N-nitroso compounds) and
to include some discussion of the N-nitrosamides.
It has also not proven possible to treat the health effects of N-nitros-
amines without considering sources of both preformed N-nitrosamines and
their precursors.
Sources of and Routes of Exposure to N-nitroso Compounds
Exogenous Sources: N-nitrosamines are widespread in the environment.
Concentrations in the nanogram to microgram per unit volume or mass range
have been recorded in air, water, soil, plants, and foodstuffs (Fine, et al.
1977a). Synthetic production is limited to small quantities: N-nitrosodi-
phenylamine is the only nitrosamine produced in quantities greater than 450
kg/yr. Other N-nitroso compounds are produced primarily as research chemi-
C-l
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cals and not for commercial purposes (Walker, et al. 1976). The most prob-
able source of environmental N-nitrosamines and N-nitrosamides is nitrosa-
tion of amine and amide precursors (Bogovski, et al. 1972).
Both nitrosating agents and nitrosatable compounds are ubiquitous in the
environment from natural and man-made sources. The most widespread form of
inorganic nitrogen is nitrate. Nitrate is a common constituent of plants
and is the primary form which plants absorb from the soil. Nitrite is found
only in low concentrations because of its greater reactivity. However, ni-
trate is readily converted to nitrite by microbial reduction, and, according
to some evidence (Klubes and Jondorf, 1971), bacteria are capable of promot-
ing the synthesis of nitrosamines from a secondary amine and nitrate without
conversion of the latter to nitrite. Oxides of nitrogen may also *.ct as
nitrosating agents. It has been estimated that 20.7 x 10 kg of nitrogen
oxides were emitted from industrial, commercial, and domestic sources in the
United States during 1970 (U.S. EPA, 1977).
Nitrosatable compounds occur in great variety. Some are ubiquitous in
nature either as components of living organisms (for example, amino acids
such as proline, tryptophan, and arginine; cyclic amines such as purines and
pyrimidines) or as products of the anaerobic decay of protein-rich organic
matter (amines, ureas, etc.). Many agricultural chemicals are nitrosatable
amino compounds (for example, the antisuckering agent, dimethyldodecylamine;
the methylcarbamate insecticides). Amines are emitted from coking plants
and petroleum refineries and, together with other forms of combined nitro-
gen, including nitrates, from sewage treatment plants, etc. Industrial
amine production has been reviewed and summarized by Walker, et al. (1976).
C-2
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Nitrosation of amide or amine precursors may occur in the air, soil,
water and in some stored or preserved foods. The major requirement is prob-
ably the simultaneous presence of precursors (Mills, 1976).
Endogenous Sources: There is now conclusive evidence that nitrosation
of amines and amides even in trace concentrations occurs in the gastrointes-
tinal tract of both animals and man (Mysliwy, et al. 1974; Fine, et al.
1977b; Rounbehler, et al. 1977).
Nitrate may be ingested in the food, mainly as a preservative in cured
meats. It can originate in the body from reduction of nitrate by bacteria
containing the enzyme nitrate reductase. The major site is the oral cavity
by bacterial reduction of nitrate in ductal saliva (Tannenbaum, et al.
1974), although other sites have been demonstrated or proposed, including
the stomach, in human subjects with gastric hypoacidity (Sander and
Schweinsberg, 1972), and the infected urinary bladder (Hawksworth and Hill,
1974). Recent studies (Tannentiaum, et al. 1978a) indicate that nitrite is
also formed de_ novo in the upper portion of the human intestine, probably
from ammonia or organic nitrogen compounds. As material passes through the
intestine, some nitrite is converted to nitrate. Absorbed nitrate is re-
cycled into saliva via the salivary glands, the stomach via the parietal
glands, and the bladder via the urine. Absorbed nitrite is rapidly de-
stroyed in the blood.
The amount of nitrosamine formed at any site is affected by many factors
such as nucleophilicity of the amine, substrate concentration, and pH. A
detailed discussion is provided by Mirvish (1975). Conditions in the stom-
ach of monogastric animals following a meal (pH range 1 to 5) particularly
favor nitrosation. Tannenbaum, et al. (1978a) suggest that nitrite origi-
nating in the intestine may react to form N-nitroso compounds in the cecum
C-3
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and colon, which are relatively more acidic than the small intestine. Ni-
trosamine formation has also been shown to be possible in saliva even at
neutral pH, although the amount formed is small (Tannenbaum, et al. 1978b).
Some substances, such as thiocyanate, increase the rate of nitrosamine for-
mation (Boyland, et al. 1971). Thiocyanate occurs in saliva, especially
that of smokers, and in gastric juice. Others, such as ascorbic acid, in-
hibit the reaction (Mirvish, et al. 1972).
The situation with regard to inhaled potential nitrosamine precursors is
considerably more speculative. Nitrous acid is rapidly formed when a mix-
ture of nitric oxide (NO), nitrogen dioxide (NO^), and water interact in
systems of high surface-to-volume ratio (Wayne and Yost, 1951; Graham and
Tyler, 1972). It therefore seems reasonable to expect that if these gases
are inhaled as pollutants of ambient air, they will rapidly equilibrate in
the lung to form nitrous acid. The neutral, buffered pH of the lung is not
normally regarded as favorable to formation of N-nitroso compounds (al-
though, as indicated above, nitrosamine formation in saliva has been ob-
served at neutral pH). However, it has been suggested (U.S. EPA, 1976) that
if nitric acid, sulfuric acid, or other common atmospheric acidic pollutants
were inhaled in sufficient amount to produce a local acidity within the re-
spiratory tract, nitrosation could occur by interaction between inhaled ni-
trogen oxides and tissue amines and amides. It is also said (U.S. EPA,
1976) to be theoretically possible for all the precursors necessary for ni-
trosamine formation to be generated in acid aerosol droplets in an atmo-
sphere containing significant amounts of nitrogen oxides, sulfur oxides, and
ammonium ion.
It is evident that the human population is exposed to both preformed N-
nitroso compounds in the environment and to similar compounds formed endo-
C-4
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genously from precursors in the environment. Assessment of the relative
significance of various exposure pathways is clearly invalid unless both
"nitrosamines" and their precursors are considered.
Ingestion from Water
Precursor chemicals of nitrosamines are ubiquitous in soils and water.
The concentration of simple aliphatic amines is normally low (nanogram-to-
miHi gram per kilogram amounts) since they are rapidly metabolized by micro-
organisms [National Academy of Sciences (NAS), 1978]. Many pesticides have
been shown to be nitrosatable, and some, such as atrazine, are only slowly
degraded and persist in soil and water. Nitrite concentrations in soil and
water are normally low (<1 mg/kg nitrite N). However, the concentrations of
nitrite {and its precursors, ammonia and nitrate) and nitrosatable compounds
can be much greater in soils heavily fertilized with organic waste matter or
in waters receiving runoff from agricultural areas or discharges of indus-
trial or municipal wastewater containing substantial amounts of amines.
Levels of nitrate in municipal drinking waters in the United States seldom
exceed 10 mg/1 nitrate N, although some smaller water supplies and private
wells contain much more nitrate. Concentrations as high as 100 to 500 mg/1
of nitrate N have been reported in polluted wells (NAS, 1977).
It has been amply demonstrated that nitrosamines are formed in soils,
water, and sewage after addition of relatively large amounts of secondary or
tertiary amines and nitrite or nitrate (Ayanaba, et al. 1973; Ayanaba and
Alexander, 1974). N-nitrosodimethylamine has been found in a number of soil
samples (Fine, et al. 1977c) at the 1 to 8 vg/kg (dry basis) level. Fine,
et al. (1977c) speculate that this may have arisen from absorption of pre-
formed nitrosodimethylamine from the air or absorption of dimethyl amine with
subsequent nitrosation. Another possible source is pesticide application.
C-5
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Several pesticides (carbamates and N,N-disubstituted amides) have been shown
to yield nitrosodimethylamine upon nitrosation (Mirvish, 1975). Others,
such as the phenoxyacetic acid derivatives, are formulated as amine salts;
some commercial preparations have been found to contain as much as 0.06 per-
cent nitrosodimethylamine as a contaminant (Fine, et al. I977a). Nitros-
amines are readily leached through the soil profile by percolating water and
thus may eventually contaminate surface and ground waters if formed in the
soil (Dean-Raymond and Alexander, 1976). These authors have also found
N-nitrosodimethylamine to be taken up from soil by spinach and lettuce; the
percentage taken up from the soil varied from 0,02 to 5=1 with the experi-
mental conditions. However, under natural conditions, nitrosamines are not
commonly found in plants.
Significant concentrations of nitrosamines have been reported for a lim-
ited number of samples of ocean water, river water, and waste treatment
plant effluent adjacent to or receiving wastewater from industries using
nitrosamines or secondary amines in production operations. Nitrosodimethyl-
amine has been reported at the 3 to 4 ug/1 level in waste water samples
(Fine, et al. 1977c). To what extent the m'trosamine arose from impurities
in the amine process or from nitrosation in the waste treatment plant is not
known. In water samples from wells characterized by both high nitrate lev-
els and coliform counts, the concentration of volatile and nonvolatile non-
ionic nitrosamines was less than 0.015 ug/1 (U.S. EPA, 1977). Volatile
nitrosamines have not been detected in drinking water (Fine, et al. 1975).
However, there is a unconfirmed report indicating existence of nonvolatile
nitrosamines (including N-nitrosoatrazine) in New Orleans water at levels of
0.1 to 0.5 ug/1 (Fine, et al. 1976).
C-6
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Nitrosamines are rapidly decomposed by photolysis and do not persist for
a significant time in water illuminated in sunlight. Thus, it is unlikely
that they will be present in high (greater than 1 mg/1) concentrations in
surface waters. However, in the absence of light they can be expected to
persist (Tate and Alexander, 1976). No degradation of N-nitrosodimethyl-
amine, N-nitrosodiethyl amine, or N-nitrosodipropylamine was observed in lake
water during a 3.5 month period (Tate and Alexander, 1975). Fine, et al.
(1977a) have shown that nitrosodimethylamines can exist for extended periods
of time in the aquatic environment.
Ingestion from Food
Many food constituents are either directly capable of conversion to N-
nitroso compounds or give rise through chemical action or metabolic process-
es to nitrosatable products. Walters (1977) has listed some of these com-
pounds. Amino acids such as proline, hydroxyproline, tryptophan, arginine,
etc., are nitrosatable. The action of heat on other amino acids can give
rise to degradation products, such as pipecolic acid, containing secondary
amino groups. There is no evidence that proteins are nitrosated directly,
but they release nitrosatable amino acids during food processing or diges-
tion. Walters (1977) suggests that prolyl peptides may be more readily
nitrosated than proline itself. A number of other tissue components, such
as choline and phospholipids, contain tertiary amines and quaternary ammoni-
um groups which can be dealkylated to secondary amines. Many of the purine
and pyrimidine bases of the nucleic acids contain amino groups capable of
forming N-nitroso derivatives, as do some vitamins, for example folic acid.
Other nitrosatable compounds include caffeine in coffee, amines in tea, and
orotic acid in milk. Some pesticides (for example, atrazine, carbaryl, fer-
C-7
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bam, simazin) are m'trosatable, and hence their release in or on food repre-
sents another source of precursors of N-nitroso compounds (Elsperu and
Lijinsky, 1973).
Nitrate and nitrite are also well supplied in the diet. The mean intake
in food of nitrate plus nitrite in the United States has been calculated to
be approximately 120 mg per day (White, 1975), although there must be con-
siderable individual variability. According to these estimates, 86 percent
of the nitrate comes from vegetables such as celery, potatoes, lettuce,
melons, cabbage, spinach, and root vegetables; some, such as spinach and
beets, contain 2,000 to 3,000 ppm of nitrate. Cured meat supplies nine per-
cent of the nitrate. Only 0.2 percent of the nitrite is supplied by vege-
tables; 21 percent comes from cured meat {White, 1975).
Nitrate is secreted in the saliva, the mean amount being approximately
40 mg per day. Of this, about 10 mg per day is reduced to nitrite in the
mouth by the oral flora (Tannenbaum, et al. 1974). These quantities, al-
though internally derived, also represent inputs to the gastrointestinal
tract. Ingestion of vegetables containing high levels of nitrate has been
shown to lead to extremely high concentrations of nitrite in saliva, and
these levels may persist for several hours (Tannenbaum, et al. 1976).
Preformed nitrosamines have been found in food, particularly in meats
such as sausages, ham, and bacon which have been cured with nitrite. To
date, analyses have been confined largely to the volatile N-nitroso com-
pounds. N-nitrosodimethylamine has been found to be present in a variety of
foods (including smoked, dried or salted fish, cheese, salami, frankfurters,
and cured meats) in the 1 to 100 yg/kg range, but more usually in the 1 to
10 yg/kg range (Montesano and Bartsch, 1976). Other nitrosamines tentative-
ly identified in meat products are N-nitrosodiethylamine, N-nitrosopiperi-
C-8
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dine, and N-nitro .upyrrolidine (Montesano and Bartsch, 1976). N-nitrosopyr-
rolidine has been consistently found to be present in cooked bacon at the 10
to 50 wg/kg concentration level, but not in raw bacon (Fine, et al. 1977a).
It apparently arises from N-nitrosoproline by decarboxylation during the
cooking process (Lijinsky, et al. 1970). The source of nitrosamines in meat
products is undoubtedly nitrosation; a report from a USDA Expert Panel on
Nitrites and Nitrosamines (U.S. Dep. Agric., 1978), therefore recommends
substantial reductions in the amounts of nitrate and nitrite used in cured
meats.
Recently data have become available on human exposure to nitrosamines in
beverages (Soff and Fine, 1979). Eighteen brands of domestic and imported
beer contained N-nitrosodimethylamine at levels ranging from 0.4 to 7.0
wg/1, and six out of seven brands of Scotch whiskey were also shown to con-
tain N-nitrosodimethylamine, at levels between 0.3 and 2.0 pg/1. Analysis
was performed using gas chromatograph interfaced to a Thermal Energy Analy-
ser (TEA).
It is necessary to note that studies prior to 1970 reporting the pres-
ence of nitrosamines in foods are open to question since the analytical
methodology employed has been shown to be non-specific.
N-nitroso compounds are difficult to analyze for two reasons. First,
they are usually present at ppb levels which require specialized instrumen-
tation to confirm their positive identity. For example, high resolution
mass spectrometry with peak matching or Thermal Energy Analyzer (TEA) is
generally regarded as acceptable. Second, if the identity of the nitros-
amine is established, proof must be provided that it was present in the
environment and was not formed artificially during analysis. This is a dif-
ficult question to answer, since nitrosamines are generally found in the
C-9
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presence of much larger concentrations of their precursors [International
Agency for Research on Cancer (IARC), 1972, 1974, 1976, 1978].
A bioconcentration factor (BCF) relates the concentration of a chemical
in aquatic animals to the concentration in the water in which they live.
The steady-state BCFs for a lipid-soluble compound in the tissues of various
aquatic animals seem to be proportional to the percent lipid in the tissue.
Thus the per capita ingestion of a lipid-soluble chemical car be estimated
from the per capita consumption of fish and shellfish, the weighted average
percent lipids of consumed fish and shellfish, and a steady-state BCF for
the chemical.
Data from a recent survey on fish and shellfish consumption in the
United States were analyzed by SRI International (U.S. EPA, 1980). These
data were used to estimate that the per capita consumption of freshwater and
estuarine fish and shellfish in the United States is 6.5 g/day (Stephan,
1980). In addition, these data were used with data on the fat content of
the edible portion of the same species to estimate that the weighted average
percent lipids for consumed freshwater and estuarine fish and shellfish is
3.0 percent.
No measured steady-state BCF is available for any of the following com-
pounds, but the equation "Log BCF - (0.85 Log P) - 0.70" can be used (Veith,
et al. 1979) to estimate the steady-state BCF for aquatic organisms that
contain about 7.6 percent lipids (Veith, 1980) from the octanol/water parti-
tion coefficient (P). the measured log P values were obtained from Hansch
and Leo (1979). The adjustment factor of 3.0/7.6 = 0.395 is used to adjust
the estimated BCF from the 7.6 percent lipids on which the equation is based
to the 3.0 percent lipids that is the weighted average for consumed fish and
C-10
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shellfish in order to obtain the weighted average bioconcentration factor
for the edible portion of all freshwater and estuarine aquatic organisms
consumed by Americans.
Chemical
N-ni trosodimethyl amine
N-n i trosod i ethyl ami ne
N-ni trosodi butyl amine
N-n i trosopyrrol i dine
Meas. Log P
-0.575
0.48
1.92
-0.19
Estimated Steady
State BCF
0.065
0.51
8.55
0.138
Weighted
Average BCF
0.026
0.20
3.38
0.055
A measured steady-state bioconcentration factor of 217 was obtained for
N-nitrosodiphenylamine using bluegills (U.S. EPA, 1978). Similar bluegills
contained an average of 4.8 percent lipids (Johnson, 1980). An adjustment
factor of 3.0/4.8 » 0.625 can be used to adjust the measured BCF from the
4.8 percent lipids of the bluegill to the 3.0 percent lipids that is the
weighted average for consumed fish and shellfish. Thus, the weighted aver-
age bioconcentration factor for N-nitrosodiphenylamine and the edible por-
tion of all freshwater and estuarine aquatic organisms consumed by Americans
is calculated to be 217 x 0.625 . 136.
Inhalation
In theory there are several possible routes to the formation of nitros-
amines in the atmosphere. These have been discussed in some detail (U.S.
EPA, 1976, 1977). Due to the photolabile nature of nitrosamines, it seems
unlikely that concentrations in ambient air would exceed a few ppb except
very near sources of direct emissions of nitrosamines. This has since been
confirmed by recent observations of Fine, et al. (1977a). N-nitrosodi-
C-ll
-------
methylamine was identified as an air pollutant near two chemical plants, one
using the amine as a raw material and the other discharging it as an unwant-
ed by-product. Typical levels at the first factory were 6 to 36 yg/nr on
site, 1 ug/m in the residential neighborhood adjacent to the factory, and
0.1 ug/nr two miles away. Typical daily human exposures were calculated
to be 39 u9 on site, 10 u9 in the adjacent residential neighborhood, and 0.3
ug two miles away. Typical levels adjacent to the second site were 0.001 to
0.04 ug/m . However, nitrosamines were detected only twice at 40 collec-
tion points in New Jersey and New York City, and then only below the 0.01
ug/nr level. Fine, et al. (1977a) conclude that airborne N-nitroso com-
pounds may not represent a daily widespread air pollution problem, but rath-
er a localized problem associated with a particular segment of a specialized
industry or with a particularly severe pollution level.
Many drugs and medicines contain secondary or tertiary amine groups.
Model and animal experiments have demonstrated that these compounds can be
readily nitrosated and thus suggest that they are precursors of N-nitroso
compounds in vivo (Lijinsky and Taylor, 1977).
Tobacco and tobacco smoke contain both secondary amines and nitros-
amines. Nitrosamines are not present in fresh tobacco, but are found during
curing (Hoffman, et al. 1974). In relatively high concentrations (in the
order of 100 mg/nr), secondary amines and nitrogen dioxide can react rap-
idly to form nitrosamines; this reaction apparently occurs in tobacco smoke
(U.S. EPA, 1977). The mainstream smoke from an 85 mm U.S. blended cigarette
without a filter tip has been found to contain 0.084 yg N-nitrosodimethyl-
amine, 0.030 yg N-nitrosomethylethylamine, 0.137 ug N-nitrosonornicotine,
and traces of N-nitrosodiethylamine (Hoffman, et al. 1974). It can be esti-
mated that the intake from smoking 20 cigarettes per day would therefore be
C-12
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approximately 2 u9 N-nitrosodimethylamine, 1 pg N-nitrosomethylethyl amine,
and 3 ug N-m'trosonornicotine. Walker, et al. (1976) have attempted to
evaluate the exposure to nitrosamines of a non-smoker exposed to tobacco
smoke. Assuming exposure to the smoke of five simultaneously burning ciga-
rettes under crowded conditions with no ventilation, levels in air were cal-
culated to be approximately 0.015 ug/nr N-nitrosodimethylamine, 0.004
ug/nr N-nitrosomethylethylamine, and 0.015 yg/nr N-nitrosonornicotine
with traces of N-nitrosodiethylamine.
Dermal
N-nitroso-bis(2-hydroxyethyl)amine (N-nitrosodiethanolamine) has been
reported to occur in cosmetic preparations, including facial creams, hand
lotions, and hair shampoos, in concentrations ranging from 20 to 48,000
ug/kg (Fan, et al. 1977). The extent to which this compound is absorbed
from the skin is unknown.
Commercial pesticide formulations available for home use have been found
to contain as much as 0.06 percent N-nitrosodimethylamine as a contaminant
(Fine, et al. 1977a). The contamination could have arisen during the manu-
facturing process or from nitrosation of dimethylamine by nitrate rust in-
hibitors added to prevent corrosion of the can. The main routes of exposure
from home use of pesticides can be expected to be inhalation and absorption
through the skin during spraying operations. Severn (1977), using data from
three studies on inhalation and dermal exposure to pesticides during spray-
ing of orchards, estimated that the intake from skin deposition, assuming 50
percent absorption, averaged about 325 times more than the intake via inha-
lation and concluded that the same ratio would hold for individuals perform-
ing hand spot-spraying. Inhalation, dermal, and/or oral exposure could also
occur from careless use of these pesticides.
C-13
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Fine, et al. (1977a) have calculated the daily exposure to preformed N-
nitrosamines under worst-case conditions (Table 1). The intake from nitrite
preserved foods assumes 100 g cooked bacon to be consumed daily. Air expo-
sure is based on the highest concentrations measured on a factory site. For
the general population, exposure information is very limited. It has been
estimated that air, diet, and smoking all play a roughly equivalent role in
direct human exposure, contributing a few micrograms per day, with direct
intake from drinking water probably much less than 1 uy/ciay (U.S. EPA, 1976).
There is even greater uncertainty with regard to the significance of
exposure to precursors. The chief source of nitrate exposure, except in the
newborn, is ingested vegetables, unless rural well water high in nitrate is
consumed. Food and water normally contribute approximately 100 ug/day.
Inhalation may also contribute several hundred ug/day (U.S. EPA, 1977). On
a daily basis, the major source of nitrite is saliva (Table 2). However,
salivary nitrite is presented to the body as a continuous, low-level input,
in comparison with the relatively high concentrations over short periods
resulting from ingestion of cured meats. This may be significant since the
rate of nitrosation is a function of the square of the nitrite concentration
(U.S. EPA, 1977). Estimates of the contribution to the daily intake of
N-nitroso compounds (as nitrosodimethylamine) have been attempted (MAS,
1978). Using blood levels of nitrosamines measured in one human subject
before and after consuming a lunch consisting of spinach, cooked bacon,
tomato, bread, and beer (Fine, 1977b), it was calculated that in vivo forma-
tion contributed 2.8 yg/day nitrosodimethylamine. For various reasons it is
believed that the total amount of nitrosamine formed may have been consider-
ably more than this. A second approach assumed that the rate of formation
of nitrosamines is equal to 5 percent of the amount of nitrite present.
C-14
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TABLE 1
Calculated Daily Human Exposure to N-nitroso Compounds**
Nitrite preserved foods, 100 g.
Tobacco smoke, 20 cigarettes
Drinking water, New Orleans
Air, factory site
Herbicide formulation, 1 ml spill
0)
c
•r™
E
p2
>^
Si
l +
T3
O
I/I
O
i-
4J
Z
1
2
40
640
C
•r;
§
r—
^)
-C
• 4->
•F"
•o
o
I/)
o
S-
z
Dai
0)
c
•o
•*—
r^
0
i.
^)
o.
o
CO
o
2
5
ly intake
01
c
"^
0
u
c
t-
o
c
o
I/I
o
t-
•p—
z
3
t-
OJ
o
8*
10*
*Tentative, unconfirmed identification as N-nitroso compound.
**Source: Fine, et al. 1977a
C-15
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TABLE 2
Calculated Average Daily Exposure to Nitrate and Nitrite**
Nitrate
Source
Vegetables
Fruits, juices
Milk and products
Bread
Water
Cured meats
Saliva
Total
mg
86.1
1.4
0.2
2.0
0.7
9.4
(30.0)*
99.8
%
86.3
1.4
0.2
2.0
0.7
9.4
100
Nitrite
mg
0.20
0.00
0.00
0.02
0.00
2.38
8.62
11.22
%
1.8
0.0
0.0
0.2
0.0
21.2
76.8
100
*Not included in total
**Source: White, 1975
C-16
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This yielded an estimated daily production from precursors of 962 u9 nitros-
odimethylamine. However, this approach is likely to give a substantial
overestimate. The conclusion appears inescapable that _i£ vivo nitrosation
provides a major contribution to the total body burden of N-nitroso com-
pounds.
It must also be concluded that water supplies are a relatively minor
source when compared with other potential sources of either preformed N-
nitroso compounds or their precursors.
PHARMACOKINETICS
Distribution
Following intravenous injection into rats, nitrosamides (e.g., N-nitros-
omethylurea, N-nitrosoethylurea) and nitrosamines (e.g., N-nitrosodimethyl-
amine, N-nitrosomorpholine) are rapidly, and apparently uniformly, distrib-
uted in the body (Magee, 1972; Stewart, et al. 1974). Orally administered
nitrosodiethylamine is found in the milk of lactating rats (Schoental, et
al. 1974). Both nitrosamines (e.g., nitrosodiethylamine) and nitrosamides
(e.g., N-nitrosoethylurea) can presumably cross the placenta since they are
capable of inducing neoplasms in the offspring if administered to rats in
late pregnancy (Magee, et al. 1976).
Metabolism
The nitrosamides are rapidly metabolized in the animal body. The half-
lives of intravenously administered N-nitrosomethylurea and N-nitrosoethyl-
urea in rats are about two minutes and five to six minutes, respectively.
The metabolism of * C-labeled N-methyl-N'-nitro-N-nitroso-guanidine has
been studied in some detail. Following an oral dose, most of the radio-
activity was excreted in the urine within 24 hours and less than 3 percent
in the feces. Less than 3 percent of the radioactivity remained in the body
as acid-insoluble materials at 24 to 48 hours (Magee, et al. 1976).
C-17
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The nitrosamines are metabolized less rapidly and persist in the body
unchanged for a longer period than nitrosamides. The rate of metabolism
depends upon the chemical structure. In the rat or mouse, administration of
^C-labeled nitrosodimethylamine leads to about 60 percent of the isotope
appearing as ^CO^ within 12 hours. Corresponding figures for labeled
nitrosodiethylamine and nitrosomorpholine are about 45 percent and 3 per-
cent, respectively. For the three compounds, corresponding urinary excre-
tions are 4, 14, and 80 percent, respectively. Metabolic products of dial-
kylnitrosamines found in the urine which contains the nitroso group are
formed by u-oxielation of the alkyl groups to give the corresponding alcohols
and carboxylic acids (Magee, et al. 1976).
ln_ vitro studies have demonstrated that the organs in the rat with the
greatest capacity for metabolism of nitrosodimethylamine are the liver and
kidney and that this compound is metabolized to a DNA-methylating agent by
human liver slices at a rate slightly slower than, but comparable with, that
of rat liver slices (Montesano and Magee, 1974).
The product(s) of metabolism of N-nitrosamines are thought to be respon-
sible for the mutagenicity and/or carcinogenicity of many of these com-
pounds. One hypothesis is that these active intermediates alkylate DNA at
specific sites. Although the liver appears to be the major site of decompo-
sition, other organs, such as kidney and lung, possess varying capacity to
metabolize nitrosamines. The relative metabolic activity of different or-
gans toward the same compound varies among species (Magee, et al. 1976).
Evidence to support the various proposed metabolic pathways of N-nitroso
compounds is inconclusive. However, the rn_ vitro studies of Montesano and
Magee (1974) indicate that nitrosamines are metabolized similarly by human,
guinea pig, and rat tissue.
C-18
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EFFECTS
Acute, Subacute, and Chronic Toxicity
N-nitroso compounds are acutely toxic to every animal species and are
also poisonous to humans.
The dialkyl and cyclic N-nitrosamines are characteristically hepatotox-
ins, producing hemorrhagic centrilobular necrosis. In experimental animals
acute exposure to nitrosodimethylamine or nitrosodiethylamine produces liver
lesions in 24 to 48 hours; deaths occur in three to four days, or the ani-
mals survive and apparently recover completely in about three weeks. Other
organs than the liver are less severely affected; the main features are
peritoneal and sometimes pleura! exudates, which may contain a high propor-
tion of blood, and a tendency to hemorrhage into the lungs and other organs.
Kidney lesions, limited to the convoluted renal tubules, and testicular
necrosis have been described in protein-deficient rats following treatment
with nitrosodimethylamine (Magee, et al. 1976).
The livers of rats and other species chronically exposed to nitrosamines
exhibit various pathological changes, including biliary hyperplasia, fibro-
sis, nodular parenchymal hyperplasia, and the formation of enlarged hepatic
parenchymal cells with large nuclei (Magee, et al. 1976). Chronic adminis-
tration of many nitrosamines induces tumors of the liver and other organs
(see Carcinogenicity section).
The N-nitrosamides also induce a liver necrosis, but it is not as pro-
nounced as that seen with the N-nitrosamines and is localized in the peri-
portal areas. Unlike the nitrosamines, the nitrosamides cause severe tissue
injury at the site of contact. The degree of local damage may be related to
the rate at which the compound decomposes at the site since the damage is
probably caused by a breakdown product rather than by the compound itself.
C-19
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The systemic targets of the nitrosamides are mainly the organs of rapid cell
turnover, including the bone marrow, crypt cells of the small intestine, and
lymphoid tissues (Magee, et al. 1976).
The effects of human exposure to nitrosodimethylamine were first report-
ed by Freund in 1937. The following description is from weisburger and
Raineri (1975).
"Freund recorded the case of a young chemist engaged in the synthe-
sis of dimethylnitrosamine, who presented with a number of syn-
dromes eventually traced to occupational exposure. The patient had
ill-defined pains in the abdomen, exhaustion, headaches, and dis-
tended abdomen. A second case, which involved an accidental single
severe exposure due to a spill of nitrosamine, again led to abdomi-
nal fluid accumulation. During an exploratory laparotomy, ascitic
fluid was found and the liver was enlarged. This patient failed to
survive. Microscopic findings at autopsy revealed liver necrosis
and areas of intense regenerative proliferation of the liver cells."
Further cases are now on record. Of two men accidentally exposed to
nitrosodimethylamine used as a solvent in an automobile factory, one recov-
ered after exhibiting signs of liver damage; the other died in a clinical
accident, and a necroposy revealed a cirrhotic liver with regenerating nod-
ules. Two of three men in an industrial research laboratory, working with
nitrosodimethylamine over a period of ten months, showed signs of liver in-
jury. One died of bronchopneumonia, and a necropsy found liver cirrhosis.
The other developed a hard liver with an irregular surface, but recovered
after exposure was terminated (Shank, 1975). The two individuals surviving
this 1953 episode were still alive in 1976 (Weisburger and Raineri, 1975).
The acute toxicity of the N-nitroso compounds varies considerably. Sin-
gle dose oral LD^Q values in adult rats range from 18 mg/kg for N-nitroso-
methylbenzyl amine to more than 7,500 mg/kg for N-nitrosoethyl-2-hydroxy-
ethylamine (Table 3). The acute oral LD5Q in the rat for nitrosodiphenyl-
amine, the only nitrosamine now produced in the U.S. in amounts greater than
450 kg/year, is given as 1,650 mg/kg [National Institute for Occupational
C-20
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TABLE 3
Acute Oral LDso Values (Druckrey, et al. 1967) and Relative
Carcinogenic Potency Expressed as Log (LDso) (Wishnok and Archer, 1976)
in BD Rats. Classification of N-nitroso compounds follows that of
Druckrey, et al. (1967)
_
Compound (mg/kg) Log(l/D5o)**
Symmetrical dialkyl(aryl)nitrosamines:
N-nitrosodimethylamine 40 2.27
N-nitrosodiethylamine 280 3.20
N-nitrosodi-n-propylamine 480 2.05
N-nitrosodi-iso-propylamine 850 0.97
N-nitrosodiallylamine* 800
N-nitrosodi-n-butylamine 1,200 1.61
N-nitrosodi-n-amylamine 3,000 0.59
N-nitrosodicyclohexylamine* 5,000
N-nitrosodiphenylamine* 3,000
N-nitrosodibenzyl amine* 900
Asymmetrical alkyl(aryl)m'trosamines:
N-nitrosomethyl ethyl ami r>e 90 2.32
N-nitrosomethylvinyl amine 24 2.89
N-nitrosomethylallylamine 340 2.10
N-nitrosomethyl-n-amylamine 120 2.60
N-nitrosomethylcyclohexylamine 30 2.98
N-nitrosomethyl-n-heptylamine - 1.53
N-nitrosomethylphenylamine 280 1.60
N-nitrosomethylbenzylamine 18 3.10
N-nitrosomethyl-(2-phenylethyl)amine 48 3.01
N,N'-dimethyl-N,N'-dinitrosoethylenediamine 150 2.40
N-nitrosoethylvinylamine 88 2.64
N-nitrosoethyl-iso-propylamine 1,100 1.49
N-nitrosoethyl-n-butylamine 380 2.11
N-nitrosoethyl-tert-butylamine* 1,600
N-nitroso-n-butyl-n-amylamine 2,500 1.00
Cyclic nitrosamines:
N-nitrosopyrrolidine 900 1.41
N-nitrosoproline (ethyl ester)* 5,000
N-nitrosopiperidine 200 1.91
N,N'-dinitrosopiperazine 160 1.95
N-nitroso-N'-methylpiperazine 1,000 0.95
N-nitroso-N'-carbethoxypiperazine 400 1.91
N-nitrosoindoline 320 0.88
N-nitrosomorpholine 320 1.95
N-nitrosohexamethyleneimine 340
N-nitrosoheptamethyleneimine 280
N-nitrosoctamethyleneimine 570
C-21
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TABLE 3 (Continued)
Compound
N — nitroso compounds with functional
3_( N-n i troso-N-methyl ami no )-su 1 f ol ane
N-n i troso-N-phenyl hydroxyl ami ne
N-nitrosotrimethylhydrazine
N-nitrosoethyl-2-hydroxyethylamine
N-n i troso-b i s ( 2-hydr oxyethy 1 ) ami ne
N-n i troso-b i s ( 2-acetoxyethy 1 ) ami ne
N-n i tr oso-n-butyl - ( 4-hydroxy-n-buty 1 ) ami ne
N-nitrosomethyl-2-chloroethylamine
N-n i tr osomethy 1 cyanomethy 1 ami ne
N-n i troso-b i s ( cyanomethy 1 ) ami ne
N-nitrososarcosine
N-n i trosoethyl sarcos i nate
2 -methyl -2 ( N-n i troso-N-methyl ami ne )-
pentan-4-one
Nitrosamides:
N , n ' -d i n i troso-N , N ' -d i methyl oxami de
N-methyl -N-n itrosoacetamide
N-methyl -N-n i trosourethane
N-ethyl-N-nitrosourethane
N-methyl -N-n itrosourea
N,N '-dimethyl -N-n itrosourea
N-n i trosotri methyl urea
N-ethyl-N-nitrosourea
N-n-butyl -N-n itrosourea
Hydrazodicarboxylic acid bis (methyl-
nitrosamide)
N-methyl -N ' -n i tro-N-n i trosoguan i di ne
N-nitrosoimidazolidone
LD50
(mg/kg)
substituent
750
2,000
95
7,500
7,500
5,000
1,800
22
45
163
5,000
4,000
2,100
96
20
240
-
110
280
240
240
1,200
200
420
250
Log(l/D50)**
groups:
1.82
1.15
2.24
0.18
-
0.74
1.51
3.21
2.18
1.95
0.60
1.18
1.04
2.40
2.31
2.01
1.96
2.18
1.95
2.00
2.67
2.10
2.38
2.51
2.26
*Non-carcinogenic in BD rats (Druckrey, et al. 1967)
**1/D50 mean total carcinogenic dose
C-22
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Safety and Health (NIOSH), 1976] or 3,000 mg/kg (Druckrey, et al. 1967).
The relationship between structure and acute toxicity is not fully under-
stood; however, for the dialkylnitrosamines acute toxicity appears to de-
crease with chain length (Shank, 1975). The predominantly hepatotoxic ef-
fects of these compounds are consistent with the hypothesis that the biolog-
ically active species is a metabolite and not the parent compound since the
liver is generally the most active organ for metabolism. It is unlikely
that under environmental conditions N-nitroso compounds would be present in
su, icient quantity to provide an acutely toxic dose.
Teratogenicity
N-nitroso compounds can also be teratogens. The effects of experimental
administration to pregnant animals have been studied systematically by
Druckrey (1973a). In summary, whereas the N-nitrosamides were found to be
teratogenic over an extended period of gestation, the N-nitrosamines were
active only when administered late in pregnancy. Thus, near-ID™ levels
of N-nitrosoalkylureas and N-nitrosoalkylanilines given to pregnant rats on
day 9 or 13 of gestation produced malformations of the eye and brain in the
offspring; similar levels of N-nitrosodimethylamine or N-nitrosodiethylamine
did not (Napalkov and Alexandrov, 1968). Given at other periods of develop-
ment, both N-nitrosamines and N-nitrosamides have been shown to be embryo-
toxic or carcinogenic (Druckrey, 1973b).
The two principal factors determining the response appear to be the
state of differentiation of the various embryonic tissues and the metabolic
competence of these tissues. Magee (1973) has adduced evidence suggesting
that the lack of teratogenic and carcinogenic response to N-nitrosamines in
early and mid-pregnancy is because the embryonic tissues have not yet ac-
quired the competence for metabolic activation.
C-23
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In Druckrey's studies (1973a), it was observed that some malformations,
mainly those of the central and peripheral nervous systems, were associated
with good survival times and that no tumors appeared at the sites of mal-
formation. This led Druckrey to suggest that teratogenesis and carcinogene-
sis are two independent processes and that the molecular mechanisms of in-
duction may be different.
Mutagenicity
The N-nitroso compounds include some of the most powerful chemical muta-
gens known. Montesano and Bartsch (1976) reported on the mutagenicity of 90
N-nitroso compounds, observed in direct mutagenicity assays and dominant
lethal tests. Data on chromosome observations and tests in Drosophila mela-
nogaster were also listed. As with other biological effects, there is a
clear distinction between the mutagenic actions of N-nitrosamides and
N-nitrosamines. N-nitrosamides are mutagenic in almost all test systems,
due to nonenzymic formation of degradation products. N-nitrosamines, on the
other hand, are not mutagenic in microbial test systems without metabolic
activation.
Liver microsomal preparations from mouse, rat, hamster, and man are cap-
able of activating nitrosamines. Czygan, et al. (1973), using human liver
microsomes, found considerable variations in the capacity of the microsomes
to activate N-nitrosodimethylamine to a mutagenic product. The cytochrome
P-450 content showed proportional variations. (Cytochrome P-450 is the ter-
minal enzyme in the microsomal system responsible for metabolism of foreign
compounds). Czygan, et al. (1973) attributed the variations in cytochrome
P-450 content to "diseases, therapy, or environmental pollutants." Czygan,
et al. (1974) later demonstrated a positive correlation between the protein
and choline content of the diet and the microsomal P-450 content, and con-
C-24
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eluded that activation of nitrosamines can be influenced by nutritional fac-
tors. Extracts from organs other than liver are either ineffective or much
less effective in activating nitrosamines to bacterial mutagens. Yet these
organs may be the target for tumor induction jn_ vivo by the same compounds.
Thus, both nitrosodimethylamine and nitrosodiethylamine induce tumors in
mouse lung and rat kidney; yet rat, mouse, and hamster lung microsomal pre-
parations and mouse kidney preparations are ineffective in activating those
compounds to mutagens in Salmonella typhimurium and Escherichia coli, re-
spectively (Montesano and Bartsch, 1976).
Nitrosodimethylamine and nitrosodiethylamine have been reported to in-
duce forward and reverse mutations in several bacterial species including _S_.
typhimurium, _E_. coli, Neurospora crassa, gene recombination and conversion
in Saccharomyces cerevisiae, "recessive lethal mutation" in Drosophila mela-
nogaster, and chromosome aberrations in mammalian cells (Montesano and
Bartsch, 1976). These compounds gave a negative response in the mouse domi-
nant lethal test, probably due to the inability of the germ cells in the
male to metabolize these compounds.
Not all N-nitroso compounds have been found to be mutagenic, although
many have been tested only in microbial systems. Of the 23 N-nitrosamines
listed by Montesano and Bartsch as having been tested in systems that in-
cluded metabolic activation, six show no mutagenic activity. These include
N-nitrosodiphenylamine, which is reported to give a negative response in
both ^. typhimurium and _E. coli after activation with a rat liver microsomal
preparation (Bartsch, et al. 1976; Nakajima, et al. 1974).
Carcinogenicity
Magee, et al. (1976) summarized data from studies through about 1975 on
the carcinogenic activity of N-nitroso and related compounds. Of the 107
C-25
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N-nitroso compounds (including 83 N-nitrosamines) listed, 87 (including 67
N-nitrosamines) are reported as having carcinogenic activity. Since that
time more compounds have been tested and, to date, approximately 100 N-ni-
troso compounds are known to be carcinogenic in one or more species of ex-
perimental animals (Lijinsky and Taylor, 1977).
All animal species tested are susceptible, including the following:
mice; rats; Chinese, Syrian, and European hamsters; gerbils; guinea pigs;
rabbits; mink; dogs; pigs; and monkeys. Sensitivity varies with species.
The African white-tailed rat, Mystromys albicaudatus, apparently remarkably
free from spontaneous tumors, developed liver tumors after treatment with
nitrosodiethylamine, although only after about 40 weeks of exposure to 50 to
100 mg/1 in the drinking water; by comparison rats showed extensive hepato-
cellular carcinomas ten weeks after a ten-week exposure to 40 mg/1 (Yama-
moto, et al. 1972). Not all carcinogenic N-nitroso compounds have induced
tumors in all species. The cyclic nitrosamine N-nitrosoazetidine (N-nitros-
otrimethyleneimine) is reported to induce lung, liver, and kidney tumors in
the rat and lung and liver tumors in the mouse, but induced no tumors under
the test conditions used in the Syrian golden hamster. Toluene-p-sulfonyl-
methylnitrosamide is reported to have failed to induce tumors in the rat but
produced lung tumors in the mouse (Magee, et al. 1976). The most recent
addition to the list is N-nitrosodiphenylamine, previously thought to be a
non-carcinogen. Cardy, et al (1979) have noted induction of neoplastic and
non-neoplastic urinary bladder lesions in rats after two years of feeding
N-nitrosodiphenylamine mixed in food at an average daily intake of 50 or 200
mg/kg body weight.
Not all N-nitrosamines have been found to induce tumors, although in
most cases only one test species has been used, usually the rat. Those com-
C-26
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pounds observed by Druckrey, et al. (1967) to give a negative response in
rats are indicated in Table 3. Others include N-nitrosoethyl-(3-hydroxypro-
pyl)-amine, N-nitroso-n-butylcarboxymethylamine, N-nitroso-£-butyl-(3-hy-
droxypropyl)amine, N-nitroso-n_-butyl-(3-hydroxybutyl)amine, N-nitroso-t-bu-
tyl-(4-hydroxybutyl)amine (Okada, et al. 1976), and guvacoline (Lijinsky and
Taylor, 1977). It is interesting to note that apparently all N,N-dialkylni-
trosamines containing a tert-butyl group are noncarcinogenic (Heath and
Magee, 1962). The list includes N-nitroso-1-proline, found in cured meats,
particularly bacon. Although noncarcinogenic itself, nitrosoproline gives
rise to the carcinogenic N-nitrosopyrrolidine during cooking (Lijinsky, et
al. 1970). Aromatic nitrosamines are capable of transnitrosation, i.e.,
under suitable conditions, their nitroso group can be transferred to appro-
priate amine-type compounds. It is thus possible that noncarcinogenic
transnitrosating agents could form new carcinogenic N-nitroso compounds in
the stomach (Singer, et al. 1977).
The carcinogenic N-nitroso compounds are capable of inducing tumors in a
wide variety of tissues, many compounds exhibiting a remarkable target organ
specificity (organotropism) sometimes modified by the route of administra-
tion. Druckrey, et al. (1967) studied the effects of a large number of
N-nitroso compounds following prolonged administration to adult rats of the
BD strain (said to exhibit a spontaneous malignant tumor rate of one percent
at 500 days). In general, daily doses were approximately 2.5 percent of the
LDgQ values listed in Table 3 and were administered in drinking water over
the life span. Pilot experiments used higher dose rates (five percent or
more of the LD^), and some animals received the N-nitroso compounds by
subcutaneous or intravenous injection or inhalation. The mean time to tumor
C-27
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varied, with dose rate and compound, between 160 and 840 days. Druckrey, et
al. (1967) made the following general observations (paraphrased from the
English summary to their paper).
All symmetrically substituted dialkylnitrosamines produced carcinomas of
the liver. The only exception was N-nitrosodi-n-butylamine, which produces
carcinomas of the urinary bladder. Subcutaneous injection of this compound
produced only bladder tumors. N-nitrosodiamylamine given subcutaneously
selectively produced lung cancer.
Asymmetrical dialkylnitrosamines, especially those possessing a methyl
group and with the second substituent group amyl, cyclohexyl, phenyl, benzyl
or phenylethyl, and also N,N'-dimethyl-N,N'-dinitrosoethylene diamine, N-ni-
trosethylvinylamine, and N-nitrosoethyl-jr-butylamine, selectively produced
carcinomas of the esophagus following both oral and parenteral administra-
tion. N-nitrosomethylalkylamines induced malignant tumors of the kidney,
particularly after intravenous injection.
The cyclic nitrosamines, N-nitrosopyrrolidine, N-nitrosomorpholine, and
N-nitroso-N'-carbethoxypiperazine induced cancer of the liver. N-nitrosopi-
peridine and N,N'-dinitrosopiperazine produced carcinomas of the esophagus
after both oral and intravenous administration but tumors of the nasal cav-
ity, mostly esthesioneuroepitheliomas, after subcutaneous injection.
Nitrosamines with functional substituent groups also produced malignant
tumors in different organs. 3-(N-nitroso-N-methylamino)-sulfolane and N-ni-
trososarcosine and its ethyl ester induced esophageal cancer. N-nitroso-n-
butyl-(4-hydroxy-n-buty1)amine selectively induced carcinomas of the urinary
bladder. N-nitrosoethyl-2-hydroxyethylamine and N-nitroso-bis-(2-hydroxy-
ethyl)amine regularly produced liver tumors following chronic exposure but
exhibited minimal toxicity in acute experiments (LDcg, 7,500 mg/kg).
C-28
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Several N-nitrosamides produced carcinomas of the forestomach after oral
administration or local sarcomas at the site of injection. Intravenous N-
methyl-N-nitrosourethane selectively produced lung cancer. Methylnitrosour-
eas induced malignant tumors in the brain, spinal cord, and/or peripheral
nervous system.
Lijinsky and his co-workers (1977) have systematically studied the ef-
fects of modification of chemical structure on the biological activity and
organ specificity of the nitrosamines. They have found that minor changes
can have a profound effect on which organ becomes the target organ. For
example, chronic administration in the drinking water of N-nitrosohexa-
methyleneimine induces liver tumors in rats; N-nitrosoheptamethyleneimine
produces lung tumors. Lijinsky (1977) has discussed his findings in rela-
tion to what is known of the mechanism of action of nitrosamines. His con-
clusion is that the major factor responsible for variations in biological
activity is the reactivity of hydrogen atoms on carbon atoms adjacent to the
nitroso group (alpha hydrogen atoms).
The response to a particular compound also varies among species. The
following attempt to illustrate the diversity of responses is derived from
Magee, et al. (1976). In most species, as in the rat, the predominant
tumors following prolonged oral administration of dialkyl cyclic and many
other N-nitrosamines are in the liver. Tumors in rats have been described
as hepatomas and hepatocellular carcinomas, cholangiomas and cholangiocarci-
nomas, fibrosarcomas, and angiosarcomas. The tumor type(s) observed in mice
depend upon both the strain and the compound. Nitrosodimethylamine produced
mainly hemangiomatous tumors, with few parenchyma! cell tumors. Nitrosodi-
ethylamine induced mainly parenchyma! tumors in seven strains of mice but
predominantly hemangiosarcomas and hemangioendotheliomas in two strains.
C-29
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Nitrosodiethylamine given to Syrian golden hamsters by the intragastric,
intraperitoneal, or intradertnal routes produced hepatocellular carcinomas
that metastasized and were transplantable; continuous oral administration
induced cholangiocarcinomas. However, following single or multiple subcuta-
neous injections, both adult and newborn hamsters developed mainly respira-
tory tumors and very few liver tumors. In the Syrian golden hamster, respi-
ratory tract tumors induced by nitrosodiethylamine are confined mainly to
the nasal cavities, larynx, and trachea irrespective of the route of admin-
istration. In the mouse, guinea pig, and rabbit, liver tumors following
prolonged oral administration of nitrosodiethylamine are accompanied by
adenocarcinomas of the lung.
In their first studies demonstrating the carcinogenicity of nitrosodi-
methylamine, Magee and Barnes (1956) reported that 19 of 20 rats continuous-
ly fed 50 mg/kg in the diet developed primary hepatic tumors within 40
weeks. However, they later (Magee and Barnes, 1959) found that in rats ex-
posed for one week at 100 or 200 mg/kg in the diet, kidney tumors predomi-
nated over liver tumors. A single, near-ID-- dose {30 mg/kg body weight)
of nitrosodimethyl amine produced no progressive liver lesions nor liver
tumors but a 20 percent incidence of kidney tumors. A single intraperitone-
al injection given to newborn mice induced hepatocellular carcinomas (Toth,
et al. 1964). A single dose to partially hepatectomized adult rats (Crad-
dock, 1973) or to rats previously treated with a single dose of carbon
tetrachloride (Pound, et al. 1973) induced liver tumors. Both treatments
induce liver cells to divide, and these observations prompted Craddock
(1973) to speculate that both injury to the genetic material and the occur-
rence of cell replication before the damage has been repaired are required
C-30
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for carcinogenesis. However, the incidence of liver tumors following chron-
ic administration of either nitrosodimethylamine or nitrosodiethylamine was
the same in both intact and partially hepatectomized rats (Rajewski, et al.
1966). There seems to be no simple explanation as to why a single oral dose
of nitrosodimethylamine, while ineffective in the adult mouse or rat, is
capable of inducing liver tumors in the adult Syrian golden hamster (Tomatis
and Cefis, 1967).
Some N-nitroso compounds administered during pregnancy induce cancer not
only in the mother but also in the offspring. A single administration of
N-nitrosoethylurea to pregnant rats resulted in malignant tumors of the
vagina, uterus, or ovaries. Given on days 15 through 18 of gestation (but
not before day 11), the compound produced brain and spinal cord tumors in
the offspring. Ethylurea and nitrite given orally to pregnant rats also
produced nervous system tumors. The sensitivity of the nervous system dur-
ing prenatal development was estimated to be about 50 times that of adults
(Druckrey, et al. 1969). Exposure during days 10 through 21 of gestation
led to renal tumors in the offspring several months after treatment (Shank,
1975). The N-nitrosoamines, including nitrosodimethylamine, nitrosodiethyl-
amine, nitrosomethylbutylamine, nitrosoethylvinylamine, and nitrosopiperi-
dine, have induced tumors in the offspring of mice, rats, and Syrian golden
hamsters only when administered during the last days of pregnancy. Subcuta-
neous, intraperitoneal, intravenous, and oral administration and inhalation
exposure were equally effective (Tomatis, 1973). In rats the tumors ob-
served were mainly neurogenic. However, Mohr, et al. (1966) observed tra-
cheal papillomas in almost half the offspring of pregnant Syrian golden ham-
sters within 25 weeks of subcutaneous administration of N-nitrosodiethyl-
amine on days 9 through 15 of gestation. In mice, treatment with nitrosodi-
C-31
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ethylamine on day 16, 17, or 18 of gestation induced mainly lung tumors. It
has been suggested that the inefficacy of the nitrosamines in early pregnan-
cy is due to the lack in the fetus of enzyme systems necessary for metabolic
activation (Druckrey, 1973b). Presumably, although active products are pro-
duced in the maternal tissues, they are generally too unstable to survive
crossing the placenta and hence do not affect the fetus.
Exposure to N-nitrosamides during pregnancy may result in a risk not
only to the immediate offspring but for at least two more generations of
animals. An increased incidence of tumors has been reported in the F,,
f^. and ^3 descendants of rats treated with N-nitrosomethylurethane or
N-nitrosomethylurea during pregnancy (Montesano and Bartsch, 1976). There
is not experimental evidence to indicate that N-nitrosamines pose a similar
threat.
Nitrosodiethylamine has been found in the stomach contents of suckling
rats following oral administration to the dam. The young rats subsequently
developed multiple tumors (Schoental and Appleby, 1973).
The carcinogenic action of the N-nitroso compounds can be modified by
appropriate treatment. The effect of partial hepatectomy or prior adminis-
tration of carbon tetrachloride has already been mentioned. Other interac-
tions have also been demonstrated. The intragastric administration of
methylcholanthrene to mice (which would be expected to increase the activity
of liver nitrosamine-metabolizing enzymes) together with intraperitoneal in-
jection of nitrosodimethylamine resulted in increased incidence and de-
creased latency period to tumors as compared with mice treated with either
compound alone (Cardesa, et al. 1973). Intratracheal instillation of ferric
oxide and subcutaneous injection of nitrosodimethylamine in Syrian golden
hamsters induced esthesioneuroepitheliomas of the nasal cavity, a type of
C-32
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tumor not induced in hamsters by nitrosodimethylamine alone (Stenback, et
al. 1973). Ferric oxide is frequently used as a carrier for introducing
carcinogenic chemicals into the lung by intratracheal instillation. It is
believed to facilitate the penetration and retention of the carcinogen in
the lung tissue. However, in the present instance, ferric oxide can be con-
sidered a cocarcinogen. Other studies have shown enhanced bronchial meta-
plasia and trachea! papilloma formation in hamsters treated with nitrosodi-
ethylamine by subseqeuent exposure to cigarette smoke, volatile acids, alde-
hydes, and methyl nitrite and increased incidence of lung tumors by subse-
quent intratracheal instillation of benzo(a)pyrene and/or ferric oxide par-
ticles (Magee, et al. 1976). The toxicity and carcinogenicty of various
alkylnitrosoureas are said to be increased when administered with copper,
nickel, or cobalt ions (Magee, et al. 1976). Magee, et al. (1976) cite ex-
amples of agents known to depress the activity of drug metabolizing enzymes
and which have been reported to modify the action of N-nitrosamines. A pro-
tein-deficient diet protected against acute liver damage in rats and result-
ed in an almost twofold increase in the ID™; however, the incidence of
kidney tumors in survivors was 100 percent. Aminoacetonitrile, which inhib-
its the metabolism of nitrosodimethylamine both in_ vivo and J_n_ vitro, pre-
vented its toxic and carcinogenic effect in rat liver. At the present time,
these interactions appear to be of academic rather than practical interest.
Although there is a wealth of reported studies on the carcinogenicity of
N-nitroso compounds, these tend to address structure-activity relationships
or mechanisms of action; information on dose-response characteristics is
sparse. Table 4 includes experimental data culled from studies in the pub-
lished literature in which nitrosamines were administered over the lifetime
C-33
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TABLE 4
Dose-response Data from Studies Involving Lifetime
Exposure to Four N-nitrosamines
Animals with Malignant Benign
Tumors of Listed Organs
Compound: N-Nitrosodimethylamine
Vehicle: Diet
Species: Rat
Target Organ: Liver
(Terracini, et al. 1967)
ICompound: N-Nitrosodiethylamine
Vehicle: Drinking Water
Species: Rat
Target orqan: Liver
(Druckrey, et al. 1963)
Compound: N-Nitrosodi-n-butylamine
Vehicle: Drinking Water
Species: Mouse
Target organ: Urinary Bladder
and/or Esophagus
(Bertram and Craig, 1970)
^Compound: N-Nitrosopyrrolidine
Vehicle: Drinking Water
Species: Rat
Target organ: Liver
(Preussmann, et al. 1977)
Daily Dose
(mg/kg
Body wt. )
0
0.67
0.12
0.17
0.30
0.60
1.2
6.0
0.075
0.15
0.30
0.60
1.2
7.6
8.2
29.1
30.9
0
0.30
1.0
3.0
10.0
Animals
(Animal
Male
0(12)
1(19)
1(6)
5(60)
22(45)
63 80
51(60)
36(40)
46(47)
45(45)
0(61)
3(60)
17 62
31(38)
14(24)
with Tumors
s Exposed)
Female
0(29)
0(18)
4(62)
2(5)
15(23)
10(12)
40(42)
45(45)
C-34
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TABLE 4 (cont.)
Animals with Malignant Benign Daily Dose Animals with Tumors
Tumors of Listed Organs (mg/kg (Animals Exposed)
Bodywt.) HaleFemale
Compound: N-Nitrosod i phenylami ne
Vehicle: Diet 0 0(19) 0(18)
Species: Rat 50 0(46) 0(48)
Target organ: Urinary Bladder 200 16(45) 40(49)
(Cardy, et al. 1979)
It is assumed that these are male BOI rats.
sex difference.
C-35
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of experimental animals at two or more daily dose levels which induced tum-
ors in some but not all animals exposed. Although only tumors (benign and
malignant) occurring in the principal target organ are listed, in all cases
other organs were also affected. Some comments are necessary. The data of
Druckrey, et al. (1963) are difficult to interpret since many animals were
lost through intercurrent infections. Thus, of the 60 animals originally
exposed to nitrosodiethylamine at the 0.075 mg/kg body weight level, 40 suc-
cumbed to a "pneumonia infection" during the first 600 days of the experi-
ment and, by the time the first (and only) hepatic carcinoma had been iden-
tified in this group, there were only three survivors. The sex of the ani-
mals used in this study is not specified. However, it is probable that they
were male BO II (albino) rats. In addition to tumors of the urinary blad-
der, Bertram and Craig (1970) report a very high incidence of esophageal
tumors following administration of N-nitrosodi-n-butylamine. The incidence
of bladder tumors in females was relatively low, but these tumors developed
significantly later than in males. The authors speculate that, had not
death from esophageal tumors intervened, both sexes would have had a uni-
formly high bladder tumor incidence. Preussman, et al. (1977) report that
other dose response studies (initially with N-nitrosopiperidine) are under
way or planned. In other studies with nitrosodimethylamine, mink, apparent-
ly the most sensitive species, developed tumors when fed 0.05 mg/kg body
weight two days per week (MAS, 1978). An increase in the incidence of
malignant liver and kidney tumors was found in male but not in female rats,
and not in mice of either sex when the animals continuously inhaled air con-
taining 200 ug/m of dinitrosomethylamine for 17 months (mice) or 25
months (rats). A concentration of 5 ug/m produced no increase in tumors
(MAS, 1978). Preussman, et al. (1977) have attempted to derive "no-effect
C-36
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levels" for rats for nitrosodimethylamine and other carcinogenic nitros-
amines (although they themselves Question the validity of such levels). Ex-
pressed as dietary levels, the estimates are: nitrosodimethylamine, 1 to 2
mg/kg; nitrosodiethylamine, <1 mg/kg; N-nitrosopyrrolidine, 3 to 5 mg/kg
(corresponding to a daily intake of approximately 0.1, <0.1, and 0.3 mg/kg
body weight, respectively).
Attempts have been made to derive some measure of the relative carcino-
genic potency of N-nitroso compounds in the absence of complete dose-re-
sponse information. The favored data base is the review of Druckrey, et al.
(1967) of studies in which adult BD rats received small daily doses (usually
orally) of 51 N-nitrosamines and 13 N-nitrosamides. Druckrey, et al. calcu-
lated the mean total carcinogenic dose reouired for production of tumors in
50 percent of the animals (D^g). Wishnok and Archer (1976) have used only
those D^Q values corresponding to a daily dose that was an approximately
constant fraction (one to three percent) of the acute oral ID™ (a dose
which gave a mean induction time for appearance of tumors of about 300 to
600 days), and, in order to have increasing carcinogenicity represented by
increasing (and manageably small) numbers, have expressed carcinogenic po-
tency as log I/DCQ. Table 3 lists the values given by Wishnok and Archer
(1976) for most of the carcinogenic N-nitroso compounds examined by Druck-
rey, et al. (1967). For the four N-nitrosamines for which dose-response
data are available, the order of increasing potency as measured by log
1/D50 is: N-nitrosopyrrolidine (1.41); N-nitroso-n-butylamine (1.61);
N-nitrosodimethylamine (2.27}; N-nitrosodiethylamine (3.20). Analysis of
experimental dose-response data places these compounds in the same order
(Table 5). Despite this possibly fortuitous agreement, log (1/D50) values
can be regarded only as providing general guidance.
C-37
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TABLE 5
Concentrations in Water Estimated to Induce no more than one
Excess Cancer per 100,000 Individuals Exposed over a Lifetime
Compound
Estimated
Concentration
(ng/1)
Data Base
N-Ni trosod imethylami ne
N-Ni trosodi ethyl ami ne
N-Nitrosodi-n-butylamine
N-Nitrosopyrrolidine
14
8
64
160
Rats (female)
(Druckrey, et al. 1967)
Rats (male?)
(Druckrey, et al. 1963)
Mice (male)
(Bertram and Craig, 1970)
Rats (mixed sexes)
(Preussman, et al. 1977)
C-38
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Wishnok and Archer (1976) and Wishnok, et al. (1978), using the log
) value as a measure of carcinogenicity, have attempted to relate
carcinogenicity to the chemical and physical properties of N-nitroso com-
pounds. Wishnok, et al. (1978) have derived an equation that takes into
account not only chemical structure, but also the partition coefficient of
the N-nitroso compounds and their electronic factors as expressed by
Taft G * values of substituents on the a-carbon atoms. With certain ex-
plainable exceptions, which are discussed by Wishnok, et al., the equation
appears to serve as a reasonably reliable method for assessing carcinogenic-
ity.
There is no instance known of occupational exposure to specific nitros-
amines having resulted in a cancer in man. The epidemiologic evidence for
the association of N-nitroso compounds with human cancer is also very limit-
ed. These data have been reviewed by a panel convened by the National Acad-
emy of Sciences (1978), and the following is taken in its entirety from this
report.
A few epidemiological studies have attempted to associate
environmental nitrates, nitrites, and nitroso compounds with human
cancer. A problem common to all the early studies was the inabili-
ty to measure with high specificity N-nitroso compounds in biologi-
cal samples. For example, African studies associating esophageal
cancer with a nitrosamine in a local alcoholic beverage (McGlashan,
1969) and a study relating carcinoma of the cervix with nitrosamine
formation in the vagina of South African women (Harington, et al.
1973) were done without the advantage of mass spectroscopic confir-
mation that is needed to identify the nitrosamines.
The International Agency for Research on Cancer has investi-
gated the possible association between N-nitroso compounds in the
diet and esophageal cancer in specific areas of Iran and France,
where these tumors occur at a high rate, and in nearby areas where
the tumor rates are not elevated (Bogovski, 1974). Complete stud-
ies of possible sources of exposure to the carcinogens have not
been made, but 15 of 29 samples of cider contained 1 to 10 yq/kg
OWN (nitrosodimethylamine) and two samples also contained DEN (ni-
trosodiethylamine) (less than 1 yg/kg). Correlations between diet-
ary intake of N-nitroso compounds and incidence of esophageal can-
cer have not yet been made.
C-39
-------
The Chinese conducted a similar study in the Anyang region,
where it is claimed that approximately 20 percent or all deaths
(not just cancer deaths) result from esophageal cancer (Coordina-
tion Group, 1975). Twenty-three percent of the food samples from
areas with the highest cancer rates were reported to contain DMN,
DEN, and methylbenzyl nitrosamine. However, confirmation of this
analysis by gas chromatography and mass spectroscopy is required
before the finding can be accepted. Dietary nitrite levels were
higher in areas of high cancer incidence than in low incidence
areas. Chickens in areas where there were high rates of esophageal
cancer in humans also had a high incidence of similar tumors, sug-
gesting an environmental etiology for the disease.
Zaldivar and Wetterstand (1975) demonstrated a linear regres-
sion between death rates from stomach cancer and the use of NaNOs
as fertilizer in various Chilean provinces. Fertilizer use was
presumed equitable to human exposure to nitrates and nitrosamines,
but no actual exposure data were reported. Armijo and Coulson
(1975) have shown similar correlations. These reports suggest that
nitrate from fertilizer enters the diet in meat, vegetables, and
drinking water, is reduced to nitrite by microbial action, and thus
is available for j£ vivo nitrosation of secondary amines in the
diet, to form carcinogenic nitrosamines, which induce stomach can-
cer. As yet, no scientific data have been gathered that support
this hypothesized etiology, and the suggested causal relationship
remains highly speculative.
Hill, et al. (1973) correlated differences in rates of stomach
cancer with the nitrate content of drinking water in two English
towns; but again, the evidence required to demonstrate a causative
role for nitrate is not available. Gelperin, et al. (1975) com-
pared death rates ascribed to cancer of the gastrointestinal tract
and liver with nitrate levels of drinking water in three unmatched
population groups in Illinois used in an infant mortality study.
No significant differences in cancer rates were found among the
three groups (the level of significance was not stated). It is
doubtful, however, whether the available mortality data permitted
an analysis that could have detected an effect in the high nitrate
population.
Increased rates of stomach cancer have been observed in Japan
in occupational groups and other populations characterized by an
unusually high consumption of salt-preserved foods (Sato, et al.
1959); presumably, these foods are high in nitrate and perhaps in
nitrite.
A statistical correlation is presented of the incidence of cancer mor-
tality with estimated exposures of urban populations in the United States to
various environmental and dietary factors. Again to quote the report (NAS,
1978): "Strong positive correlations were shown between the aggregate rate
C-40
-------
of cancer mortality and components of the diet, particularly nitrite and
protein; however, insufficient biological evidence is available to confirm
the hypothesized causal pathway (involving formations of N-nitroso compounds
from nitrite and amines, reacting in the stomach)." There is, in fact, di-
rect evidence for formation of nitrosamines from precursors in the human
stomach (see, for example, Fine, et al. 1977b); still in contention is the
extent to which m'trosation occurs.
Although N-nitrosamines such as N-nitrosodimethylamine and N-nitroso-
morpholine are rapidly and fairly evenly distributed throughout the bodies
of rats after injection (Magee, 1972; Stewart, et al. 1974), the acute toxic
damage they produce is more severe in the liver than elsewhere, and tumors
following chronic exposure are confined mainly to the liver and kidney
(Druckrey, et al. 1967). _In_ vitro studies have shown that the liver and
kidney possess the greatest capacity for metabolism of N-nitrosodimethyl-
amine (Montesano and Magee, 1974). These observations are most readily ex-
plained on the assumption that carcinogenesis and other biological actions
of nitrosamines are mediated by metabolic products. The lack of mutagenic
activity exhibited by nitrosamines in bacterial test systems in the absence
of a metabolic activating system (Montesano and Bartsch, 1976) supports this
hypothesis, which is now generally accepted.
The N-nitrosamides differ from the N-nitrosamines in that they are chem-
ically unstable at physiological pH and decompose nonenzymically, again into
active metabolic products, upon contact with the tissues. They therefore
tend to produce damage or tumors at the site of administration.
The nature of the metabolite(s) responsible for the carcinogenic activi-
ty of N-nitroso compounds is still in debate. Magee (1977) has adduced con-
siderable evidence in support of the commonly accepted hypothesis that the
C-41
-------
active agents are electrophilic alkylating agents which bind to DNA. The
major product formed in rat liver after administration of nitrosodimethyl-
amine or nitrosodiethylamine is the corresponding 7-alkylguanine. However,
little correlation has been found between the occurence of 7-alkylguanine in
DNA and the tumor-producing activity of the nitrosamines. A much better
correlation has been demonstrated between the formation and persistence of
06-alkylguanines and tumor incidence (Pegg and Nicoll, 1976). These auth-
ors postulate that the formation and persistence until cell division of cer-
tain promutagenic products such as 0-methylguanine might be responsible:
for the initiation of tumors and that the differing abilities of various
tissues to catalyze DNA repair might account for part of the differing sus-
ceptibilities of these tissues to the carcinogenic action of the N-nitroso
compounds (Pegg and Nicoll, 1976). However, Lijinsky and coworkers found
that a series of cyclic nitrosamines, while as carcinogenic as the aliphatic
nitrosamines, gave rise to much smaller amounts of alkylated guanines; in
some cases none could be detected (Lijinsky, 1977). For this and other rea-
sons, Lijinsky concludes that the initial step cannot be a simple alkylation
of DNA.
Many N-nitrosamines (and N-nitrosamides) are teratogenic, mutagenic, or
carcinogenic. Evidence from experimental animals suggests that, as carcino-
gens, they are most effective by the oral route and when given as multiple
small doses. However, some are capable of inducing tumors after a single
dose, and they are also capable of inducing tumors in certain organs and
tissues regardless of the route of administration, i.e., they are systemic
carcinogens. In the rat, at least, every organ is probably susceptible to
tumor induction by some nitrosamine. There is a strong relationship between
C-42
-------
chemical structure and type of tumor induced. There are large differences
in tumor response among species, both in type of tumor produced and in sus-
ceptibility.
The late fetus and neonate appear to be highly susceptible to the carci-
nogenic action of both N-nitrosamines and N-nitrosamides. The sensitivity
of the nervous system to some N-nitrosamides during prenatal development is
about 50 times that in the adult. A single exposure to some nitrosamides
during pregnancy may result in development of tumors not only in the immedi-
ate descendants but in at least two succeeding generations. Although pro-
longed exposure to some nitrosamides is needed to elicit tumors in adult
animals, a single dose of the compound will induce tumors in the newborn.
The epidemiological studies to date have been inadequate to establish
any correlation between exposure to N-nitroso compounds or their precursors
and human cancer as valid causal relationships. Nevertheless, the ability
of N-nitrosamines to induce tumors in a wide range of species other than
man, together with the fact that human liver tissue is capable of forming
alkylating and mutagenic metabolites, suggest strongly that it is improbable
that humans are refractory to the carcinogenic action of these compounds.
C-43
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CRITERION FORMULATION
Existing Guidelines and Standards
Current Levels of Exposure
For the general population, exposure information is very limited. It
has been estimated that air, diet, and smoking all play roughly equivalent
roles in direct human exposure, contributing a few micrograms per day, with
direct intake from ingested water probably much less than 1 yg/day (U.S.
EPA, 1976).
There is even greater uncertainty with regard to the significance of
exposure to precursors. The chief source of nitrate exposure, except in the
newborn, is ingested vegetables, unless rural well water high in nitrate is
consumed. Food and water normally contribute approximately a few hundred
milligrams per day. Inhalation may also contribute several hundred micro-
grams per day (U.S. EPA, 1977). On a daily basis, the major source of ni-
trite is saliva. However, salivary nitrate is presented to the body as a
continuous, low-level input, as contrasted with the relatively high concen-
trations over short periods resulting from ingestion of cured meats. This
may be significant since the rate of nitrosation is a function of the square
of the nitrite concentraton (U.S. EPA, 1977).
The concentrations of nitrite (and its precursors, ammonia and nitrate)
and nitrosatable compounds can be much greater in soils heavily fertilized
with organic waste matter or in waters receiving runoff from agricultural
areas or discharges of industrial or municipal waste waters containing sub-
stantial amounts of amines. Levels of nitrate in municipal drinking water
in the U.S. seldom exceed 10 mg/1 nitrate N, although some private supplies
contain much more.
C-44
-------
Significant concentrations of nitrosamines have been reported for a lim-
ited number of samples of ocean water, river water, and waste treatment
plant effluent adjacent to or receiving wastewater from industries using
nitrosamines or secondary amines in production operations. Nitrosodimethyl-
amine has been reported at the 3-4 ng/1 level in these samples. Nitros-
amines, however, are rapidly decomposed by photolysis and do not persist for
a significant time in water exposed to sunlight.
Although it is difficult to analyze this wide spectrum of exposure po-
tential, it must be concluded that ingested water is a relatively minor
source of exposure when compared with other potential sources of either pre-
formed N-nitroso compounds or their precursors.
Special Groups at Risk
Because of the ubiquitous nature of nitrosatable compounds and nitrosat-
ing agents in the environment (food, air, drugs, tobacco, water, soil) spe-
cial risk groups would have to include those individuals who are exposed to
multiple exposures. To quantify this, however, is almost impossible at this
point because of the need to create exposure scenarios for which the bound-
ing factors are unknown or relatively wide ranging.
Basis and Derivation of Criterion
Both N-nitrosamines and N-nitrosamides exhibit acute toxicity, teratoge-
nicity, mutagenicity, and/or carcinogenicity. For most, it is the latter
capability which demands consideration in the context of human exposure
since the toxicological evidence is such that they must be treated as poten-
tial human carcinogens. Thus, nitrosamines are included in a list from the
American Conference of Governmental Industrial Hygienists (ACGIH, 1977) "In-
C-45
-------
dustrial Substances Suspect of Carcinogenic Potential for Man." No Thresh-
old Limit Value (TLV) is given. The guidelines which follow are based upon
the assumption that N-nitrosamines are human carcinogens.
Adequate dose-response data to permit an assessment of the carcinogenic
risk to man are available from studies involving lifetime exposure of rats
or mice to five nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine,
N-nitrosodi-n-butylamine, N-nitrosopyrrolidine, and N-nitrosodiphenylamine)
in their drinking water or food (see Table 4). These data have been used to
derive estimates of the concentrations in water which, if used as the source
for man of drinking water and edible fish and shellfish, would increase the
risk of a tumor by not more than one in 100,000 individuals exposed for the
duration of their life span. The methods of extrapolation are discussed in
the Human health Methodology Appendices to the October 1980 Federal Register
notice which announced the availability of this document. The water crite-
ria shown in Table 5 are based on parameters listed in the Appendix.
Table 3 lists one measure of the relative carcinogenic potential of a
number of N-nitrosamines. The value of N-nitrosodiethylamine (3.20) is ex-
ceeded only by that for N-nitrosomethyl-2-chloroethylamine (3.21) and ap-
proached only by the values for N-nitroso-methylbenzylamine (3.10) and N-ni-
trosomethyl-(2-phenylethyl) amine (3.01). Hence, N-nitrosodiethylamine can
reasonably be considered to be one of the most carcinogenic nitrosamines.
It is, therefore, appropriate to recommend a water criterion value for the
nitrosamine class based on the value obtained for N-nitrosodiethylamine. If
sufficient evidence exists to indicate that some nitrosamines may be less
potent carcinogens than N-nitrosodiethylamine, then a separate criterion
should be derived. This has been done in four cases. In addition, if there
C-46
-------
is sufficient experimental evidence that a particular nitrosamine is not
carcinogenic to mammals, then a noncarcinogenic-based criterion should be
allowed.
Criteria have been derived by considering only the excess cancer risk
imposed by exposure to contaminated drinking water, fish, and shellfish.
However, the average daily intake of preformed nitrosamines from other
sources (air, diet, and smoking) is estimated to be on the order of a few
micrograms per day (U.S. EPA, 1976). There is an additional and, at the
present time, ill-defined contribution to the body burden from the _i_n_ vivo
nitrosation of precursors. This contribution has been variously estimated
to range from a few micrograms to several hundred micrograms daily (NAS,
1978). Thus, present evidence suggests that control of exposure to N-ni-
trosamines should take into account both preformed nitrosamines and their
precursors in the environment.
Under the Consent Decree in NRDC v. Train, criteria are to state "recom-
mended maximum permissible concentrations (including, where appropriate,
zero) consistent with the protection of aquatic organisms, human health, and
recreational activities." Nitrosamines are suspected of being human carcin-
ogens. Because there is no recognized safe concentration for a human car-
cinogen, the recommended concentration of nitrosamines in water for maximum
protection of human health is zero.
Because attaining a zero concentration level may be infeasible in some
cases and in order to assist the Agency and states in the possible future
development of water quality regulations, the concentrations of nitrosamines
corresponding to several incremental lifetime cancer risk levels have been
estimated. A cancer risk level provides an estimate of the additional inci-
dence of cancer that may be expected in an exposed population. A risk of
C-47
-------
10~^» for example, indicates a probability of one additional case of can-
cer for every 100,000 people exposed, a risk of 10 indicates one addi-
tional case of cancer for every million people exposed, and so forth.
In the Federal Register notice of availability of draft ambient water
Quality criteria, EPA stated that it is considering setting criteria at an
interim target risk level of 10~5, 10~6, or 10~7 as shown in the table
below.
Exposure Assumptions Risk Levels and Corresponding Criteria (1)
(per day)ng/1
2 liters of drinking water £ 10~7 10~6 10~
and consumption of 6.5
grams fish and shellfish (2)
N-nitrosodimethylamine 0 0.14 1.4 14.0
N-nitrosodiethylamine 0 0.08 0.8 8.0
N-nitrosodi-n-butylamine 0 0.64 6.4 64
N-nitrosopyrrolidine 0 1.60 16.0 160
N-nitrosodiphenylamine 0 490 4,900 49,000
Consumption of fish and
shellfish only. —
N-nitrosodimethylamine 0 1,600 16,000 160,000
N-nitrosodiethylamine 0 124 1,240 12,400
N-nitrosodi-n-butylamine 0 58.7 587 5,868
N-nitrosopyrrolidine 0 9,190 91,900 919,000
N-nitrosodiphenylamine 0 1,610 16,100 161,000
(1) Calculated by applying either a linearized multistage model (N-nitroso-
di-n-butylamine, N-nitrosopyrrolidine, and N-nitrosodiphenylamine) or a
time to tumor model (N-nitrosodimethylamine and N-nitrosodiethylamine),
as discussed in the Human Health Methodology Appendices to the October
1980 Federal Register notice which announced the availability of this
document, to the animal bioassay data presented in the Appendix and in
Table 4. Since the extrapolation models are linear at low doses, the
additional lifetime risk is directly proportional to the water concen-
C-48
-------
traton. Therefore, water concentrations corresponding to other risk
levels can be derived by multiplying or dividing one of the risk levels
and corresponding water concentrations shown in the table by factors
such as 10, 100, 1,000, and so forth.
(2) Approximately zero percent of the exposure of these first four nitros-
amines results from the consumption of aquatic organisms which exhibit
an average bioconcentration potential near zero. The remaining 100 per-
cent of these nitrosamines' exposure results from drinking water. In
the case of N-m'trosodiphenylamine 31 percent of the exposure results
from the consumption of aquatic organisms which exhibit an average bio-
concentration potential of 136 I/kg. The remaining 69 percent reflects
exposure from drinking water.
Concentration levels were derived assuming a lifetime exposure to vari-
ous amounts of nitrosamines, (1) occurring from the consumption of both
drinking water and aquatic life grown in waters containing the corresponding
nitrosamines concentrations, and (2) occurring solely from consumption of
aquatic life grown in the waters containing the corresponding nitrosamines
concentrations. Because data indicating other sources of nitrosamines expo-
sure and their contributions to total body burden are inadequate for quanti-
tative use, the figures reflect incremental risks associated with the indi-
cated routes only.
C-49
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APPENDIX
Derivation of Criterion for Dimethyl nitrosamine
Druckrey et al. (1967) summarized a series of experiments in which a
large series of nitrosamine compounds were given to BD rats for a lifetime.
He found that the incidence of liver tumors increased with daily dose, d,
and that the median time when tumors were observed, ten, was less at high-
2 3
er doses and the relationship between d and t was d(tr) * * k,
4
where k is a constant eaual to 0.81 x 10 mM/kg/day when teg is ex-
pressed in units of days.
The water Quality extrapolation model uses dose units of mg/kg/day and
time units of fractions of a lifetime. Converting k to these units by using
728 days (two years) as the lifetime and a molecular weight of 74 mg/mM
gives the following:
0.81 x 104mM/kg/day x 74 mg/mM
0.15661
(728)2.3
Therefore the parameters of the dose-response model are:
nt/Nt = 0.5 dtn = 0.15661
n /N = 0 R = 0.026 I/kg
c c
w = 0.35 kg
With these parameters, the carcinogenic potency factor for humans, Bu,
n
is 25.88 (mg/kg/day)~ . The result is that the water concentration should
be less than 14 ng/1 in order to keep the individual lifetime risk below
io-5.
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Derivation of Criterion for Diethylnitrosamine
Druckrey, et aT. (1963) administered diethylnitrosamine to BD rats via
drinking water in nine dose groups ranging from 0.075 to 14.2 mg/kg/day.
They found that the incidence of liver tumors increased with daily dose,
d, and that the median time when tumors were observed, t^Q, was less at
2 3
higher doses and the relationship between d and t was dft) * = k,
where k is a constant. The value of the constant was not given in the 1963
publication, but a later paper by Drucker, et al. (1967) stated that k =
0.35 x 104 mM/kg/day.
When this is converted to the units of mg/kg/day for dose and fractions
of a lifetime (which is 728 days) for time, the value of k becomes:
0.35 x 104 mM/kg/day x 102 mg/mM
= 0.09328.
(728)2-3
Therefore, the parameters of the dose -response model are:
nt/Nt = 0.5 dtn = 0.09328
nc/Nc =0 R = 0.20 I/kg
w = 0.35 kg
With these parameters the carcinogenic potency factor for humans, B^,
is 43.46 (mg/kg/day) . The result is that the water concentration should
be less than 8.0 ng/1 in order to keep the individual lifetime risk below
10 -5.
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Derivation of Criterion for Dibutylnitrosamine
Bertram and Grain (1970) administered dibutylnitrosamine via drinking
water to C57BL/6 mice at dose levels of about 8 and 30 mg/kg/day until the
animals became moribund or died. They found that dibutylnitrosamine induced
tumors of the bladder and esophagus in both sexes. Using the bladder and/or
esophageal tumor induction in males, the parameters of the extrapolation are:
Dose Incidence
(mg/kg/day) (No. responding/No, tested)
0 a
7.6 46/47
29.1 45/45
le (low dose) = 630 days w = 0.028 kg
le (high dose) = 414 days R = 3.38 I/kg
Le = 630 days
L = 630 days
With these parameters the carcinogenic potency factor for humans, q,*,
is 5.43 (mg/kg/day) . The result is that the water concentration should
be less than 64 ng/1 in order to keep the individual lifetime risk below
10~5.
aSpecific incidence was not reported. Very low spontaneous incidence in
controls was stated.
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Derivation of Criterion of N-Nitrosopyrrolidine
Preussnian, et al. (1977) found a dose-related incidence of hepatocellu-
lar carcinomas in Spragus-Dawley rats in a lifetime feeding study of N-ni-
trosopyrrolidine at levels of 0,3, 1,0, 3,0, and 10 mg/kg/day. The parame-
ters of the extrapolation are:
Dose Incidence
(mg/kg/day) (No. responding/No, tested)
0.0 0/61
0.3 3/60
1.0 17/62
3.0 31/38
10 14/24
le » 728 days w = 0.350 kg
Le = 630 days R = 0.055 I/kg
L - 728 days
With these parameters the carcinogenic potency factor for humans, c^*,
is 2.13 (mg/kg/day)~ . The result is that the water concentration should
be less than 160 ng/1 in order to keep the individual lifetime risk below
io-5.
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Derivation of Criterion of N-Nitrosodiphenylamine
Cardy, et al. (1979) found in a lifetime feeding study of F344 rats that
n-nitrosodiphenylamine induced transitional-cell carcinomas of the urinary
bladder in both sexes at significant incidences over matched controls. Us-
ing the female data the parameters of the extrapolation are:
Dose Incidence
(mg/kg/day) (No. responding/No, tested)
0 0/18
50 0/48
200 40/49
le * 700 days w = 0.250 kg
Le - 700 days R = 136 I/kg
L - 700 days
With these parameters the carcinogenic potency factor for humans, a,*,
is 4.92 x 10" (mg/kg/day)~ . The result is that the water concentra-
tion should not exceed 49 yg/1 in order to keep the lifetime human cancer
risk below 10~5.
» V. S. GOVERMMENT PRINTING OFFICE : ?9«0 723-C16/5957
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