FINAL DRAFT
ECAO-CIN-D002
March, 1994
DRINKING WATER CRITERIA DOCUMENT FOR
CHLORAMINES
HEALTH AND ECOLOGICAL CRITERIA DIVISION
OFFICE OF SCIENCE AND TECHNOLOGY
OFFICE OF WATER
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DISCLAIMER
This report is an external draft for review purposes only and does not constitute
Agency policy. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended in 1986, requires
the Administrator of the Environmental Protection Agency to publish maximum
contaminant level goals (MCLGs) and promulgate National Primary Drinking Water
Regulations for each contaminant, which, in the judgment of the Administrator, may have
an adverse effect on public health and which is known or anticipated to occur in public
water systems. The MCLG is nonenforceable and is set at a level at which no known or
anticipated adverse health effects in humans occur and which allows for an adequate
margin of safety. Factors considered in setting the MCLG include health effects data and
sources of exposure other than drinking water.
This document provides the health effects basis to be considered in establishing the
MCLG. To achieve this objective, data on pharmacokinetics, human exposure, acute and
chronic toxicity to animals and humans, epidemiology and mechanisms of toxicity are
evaluated. Specific emphasis is placed on literature data providing dose-response
information. Thus, while the literature search and evaluation performed in support of this
document has been comprehensive, only the reports considered most pertinent in the
derivation of the MCLG are cited in the document. The comprehensive literature data
base in support of this document includes information published up to 1986; however,
more recent data have been added during the review process, and final revisions updating
this document were made.
When adequate health effects data exist, Health Advisory values for less than lifetime
exposures (1-day, 10-day and longer-term, 10% of an individual's lifetime) are included
in this document. These values are not used in setting the MCLG, but serve as informal
guidance to municipalities and other organizations when emergency spills or
contamination situations occur.
Tudor Davies, Director
Office of Science and
Technology
James Elder, Director
Office of Ground Water
and Drinking Water
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DOCUMENT DEVELOPMENT
Carolyn L. Smallwood, A.B., Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Authors
Carolyn L. Smallwood
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Patricia A. Murphy
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Annette M. Gatchett
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Rita Schoeny
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Frank E. Scully, Jr.
Dept. of Chemical Sciences
Old Dominion University
Norfolk, VA 23508
Scientific Reviewers
Randall F. Bruins
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
W.B. Peirano
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
David J. Reisman
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Cynthia Sonich-Mullin
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Joseph R. Bianchine
Medical Research and Development
American Critical Care
McGaw Park, IL
Joseph P. Gould
Environmental Chemistry
Georgia Institute of Technology
Atlanta, GA
Richard J. Bull
College of Pharmacy
Washington State University
Pullman, WA
Herbert H. Cornish
Professor Emeritus
University of Michigan
Ypsilanti, Ml
Curtis D. Klaassen
University of Kansas Medical Center
Dept. of Pharmacology and Toxicology
Kansas City, KS
Loren K. Koller
College of Veterinary Medicine
Oregan State University
Corvallis, Oregan
IV
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DOCUMENT DEVELOPMENT (cont.)
David Jollow
Medical University of South Carolina
Charleston, SC
Editorial Reviewers
Erma Durden, B.S.
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Judith Olsen, B.A.
Environmental Criteria and Assessment
Office, Cincinnati
U.S. Environmental Protection Agency
Document Preparation
Technical Support Services: Bette Zwayer, Environmental Criteria and Assessment
Office, Cincinnati
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TABLE OF CONTENTS
I. SUMMARY 1-1
II. PHYSICAL AND CHEMICAL PROPERTIES 11-1
INTRODUCTION 11-1
PHYSICAL AND CHEMICAL PROPERTIES OF CHLORAMINES II-2
USES OF CHLORAMINES 11-11
ANALYTICAL METHODS 11-12
ENVIRONMENTAL FATE AND TRANSPORT 11-16
SUMMARY 11-17
III. TOXICOKINETICS 111-1
INTRODUCTION 111-1
ABSORPTION 111-1
DISTRIBUTION III-2
METABOLISM III-6
EXCRETION 111-11
DISCUSSION 111-14
SUMMARY 111-16
IV. HUMAN EXPOSURE IV-1
[To be provided by the Office of Water]
V. HEALTH EFFECTS IN ANIMALS V-1
INTRODUCTION V-1
SHORT-TERM EXPOSURE V-1
OTHER SHORT-TERM EFFECTS V-15
LONG-TERM EXPOSURE V-16
DEVELOPMENTAL TOXICITY V-20
REPRODUCTIVE TOXICITY V-21
MUTAGENECITY V-23
CARCINOGENICITY V-26
SUMMARY AND DISCUSSION V-31
VI
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TABLE OF CONTENTS (cont.)
VI. HEALTH EFFECTS IN HUMANS VI-1
INTRODUCTION VI-1
CLINICAL REPORTS AND EXPERIMENTS VI-1
EPIDEMIOLOGY STUDIES VI-4
HIGH-RISK SUBPOPULATIONS VI-6
SUMMARY AND DISCUSSION VI-7
VII. MECHANISMS OF TOXICITY VII-1
GENERAL THEORIES VII-1
SUMMARY VII-3
VIII. QUANTIFICATION OF TOXICOLOGIC EFFECTS VIII-1
INTRODUCTION VIII-1
CURRENT LEVELS OF EXPOSURE VIII-7
NONCARCINOGENIC EFFECTS VIII-7
QUANTIFICATION OF NONCARCINOGENIC EFFECTS VIII-14
Derivation of 1-Day Health Advisory VIII-14
Derivation of 10-Day Health Advisory VIII-15
Derivation of Longer-term HA VIII-16
Assessment of Lifetime Exposure and Derivation of a DWEL .... VIII-17
CARCINOGENIC EFFECTS VIII-19
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS . . VIII-21
SPECIAL GROUPS AT RISK VIII-21
RISK CHARACTERIZATION VIII-22
REFERENCES IX-1
VII
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LIST OF TABLES
No. Title
11-1 Summry of Chemical and Physical Properties of Mono-
and Trichloramines II-3
II-2 Proportions of Monochloramine (NH2CI) and Dichloramine(NHCI2)
Formed in Water Chlorination with Equimolar Concentrations of
Ammonia and Chlorine II-5
111-1 Metabolism of NH236CI in Rat Plasma III-7
III-2 Excretion of 36CI in Rat 120 Hours After Single Oral Administration
ofNH236CI 111-11
III-3 Excretion of 36CI in Rat 120 Hours After Single Oral Administration
ofNa36CI 111-12
V-1 Summary of Studies on Acute and Subchronic Oral Exposure to
Monochloramine V-2
V-2 Summary of Studies on Chronic Oral Exposure to Monochloramine
in Drinking Water V-5
VI11-1 Summary of HAs and Dwel for Noncarcinogenic Effects VIII-20
VIM
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LIST OF FIGURES
No. Title
11-1 Graphic Description of Breakdown Phenomenon II-7
111-1 NH236CI Radioactivity Distribution in the Rat 120 Hours After the
Administration of NH236CI (1.1 mg) Orally III-4
III-2 36CI Distribution in the Rat III-5
IX
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LIST OF ABBREVIATIONS
Alk-P Alkaline phosphatase
AST Aspartate aminotransferase
ATP Adenosine triphosphate
BUN Blood urea nitrogen
bw Body weight
CHO Chinese hamster ovary
CNS Central nervous system
CSF Cerebrospinal fluid
DNA Deoxyribonucleic acid
DPD Diethyl-p-phenylenediamine
DWEL Drinking water equivalent level
EAA Essential amino acid
EKG Electrocardiogram
FEV Forced expiratory volume
FVC Forced vital capacity
GGTP q-Glutamyltranspeptidase
Gl Gastrointestinal
GSH Glutathione
HA Health advisory
HCT Hematocrit
HGB Hemoglobin percent
LIST OF ABBREVIATIONS (cont.)
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i.p. Intraperitoneal
i.v. Intravenous
LOAEL Lowest-observed-adverse-effect level
MCH Mean corpuscular hemoglobin
MCHC Mean corpuscular hemoglobin concentration
MCV Mean corpuscular volume
NADH Nicotinamide-adenine dinucleotide dehydrogenase
NCR N-chloropiperidine
NOAEL No-observed-adverse-effect level
NOEL No-observed-effect level
PHA Phytohemagglutinin
RBC Red blood cell
RfD Reference dose
s.c. Subcutaneous
SCE Sister chromatid exchange
T4 Plasma thyroxine
TCA Trichloroacetic acid
TPA 12-o-Tetradecanoylphorbol-13-acetate
TrpC Tryptophan C
TWA Time-weighted average
XI
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XII
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I. SUMMARY
Inorganic chloramines are alternate disinfectants that are rapidly formed when free
chlorine is added to water containing ammonia. To achieve the desired chloramine
concentration, chlorine may also be intentionally added to the already naturally occurring
ammonia in the water source. Under the usual conditions of water and wastewater
chlorination, monochloramine is the principal chloramine encountered. In the event of
excess hypochlorite or at lower pH, ammonia can be di- and trichlorinated and organic
amines can be dichlorinated. Comparatively little is known about the physical properties
of pure dichloramine because it is not stable and is difficult to synthesize. Trichloramine
is formed in acidic solutions where chlorine concentrations are much greater than those
of ammonia. This document will deal primarily with inorganic chloramines; however,
where data are limited or not available various chlorinated amino compounds (including
organic chloramines) will be provided as supplemental information.
Inorganic dichloramine is unstable and decomposes to nitrogen, hypochlorous acid
and other products. This reaction accounts to a large extent for the breakpoint reaction
seen in water chlorination. Inorganic monochloramine is a much poorer disinfectant than
hypochlorous acid, and it reacts slowly with organic amino nitrogen compounds to
produce organic N-chloramines, which are even poorer disinfectants.
Conventional methods of monochloramine analysis have led to confusion in the
use of the term "chloramines." The term has been used to describe many compounds in
CLORAMIN.1 1-1 03/08/94
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complex mixtures that oxidize iodide to iodine, the amount of which is subsequently
determined by colorimetric or amperiometric analysis. Organic N-chloramines and
N-chloramides respond similarly to monochloramine; however, they are poor disinfectants
compared with inorganic chloramine. From a public health viewpoint, these ambiguities
can present serious problems since the disinfecting capabilities of waters that contain
organic N-chloramines could be overestimated. Improved analytical methods are needed
for the determination of organic and inorganic N-chloramines in chlorinated waters. For
the purpose of this document, the term "chloramines" will refer to a combination of
inorganic mono-, di- or trichloramines unless otherwise stated.
Human exposure to chloramines is through ingestion of chlorinated water
containing ammonia or chloraminated water. Chloramination is a technique that is being
adopted by many communities to avoid formation of trihalomethanes on water
disinfection. Organic chloramines have been shown to form upon chlorination of stomach
fluid in vitro. However, the significance of their formation on ingestion of chlorinated water
is not clear.
Information on the absorption of inorganic chloramines is extremely limited. In one
study an absorption rate constant was calculated for monochloramine at 0.278 mg/hour
(after 8 hours) with an absorption half-life of 2.5 hours after a single oral dose of ~4.6
mg/kg/day was administered to Sprague-Dawley rats. After 48 hours, the rate constant
was 0.018 mg/hour. Absorption rates with respect to various dosage media and different
routes of exposure were not available.
CLORAMIN.1 I-2 03/08/94
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The distribution of radiolabeled chlorine in the subtractions of rat liver
homogenates in organs, tissues, and fluids was similar (120 hours) after oral
administration of either 36Cr (200 mg/L as Na36CI) or NH236CI (370 mg/L NH236CI). Plasma
contained the highest concentrations of 36CI radioactivity for NH236CI followed by whole
blood, skin, testes, packed cells, bone marrow, kidney, lung, stomach, thyroid, thymus,
duodenum, spleen, carcass, liver, ileum and fat. The major metabolite of NH236CI was
36C|-
Information on the metabolism of chloramines is also extremely limited. One
experiment indicated that chloramines are transformed to the chloride moiety and
eliminated primarily in this form.
Chloramines are eliminated primarily through the urine. During the first 24 hours
after a single dose of NH236CI (1.1 mg/animal) to Sprague-Dawley rats, only 0.40 and
0.08% of the total dose was eliminated in the urine and feces, respectively. At the end
of 120 hours, 25.15 and 1.98% of the dose was eliminated through the urine and feces,
respectively. By comparison 16.1 and 0.92% of 36Cr radiolabel (200 mg/L as Na36CI) was
eliminated in the urine and feces, respectively, in the first 24 hours. Over twice as much
of the 36CI" radiolabel was eliminated over a 120-hour period. The major difference
between the NH236CI and 36CI" studies was in the total amount of label excreted over the
test period.
CLORAMIN.1 I-3 03/08/94
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Several short-term studies showed no observed adverse hematologic effects in
mice, rats and monkeys. In A/J mice administered chloramine solutions between 2.5 and
200 mg/L (pH 8.9) for 30 days, the only observable effect was a slight increase in
hematocrit. In another study of similar duration (45 days) rats treated with 10, 50 or 100
mg/L monochloramine experienced a decrease in the amount of methemoglobin present
in the blood, the opposite of what was expected. Monochloramine in drinking water for
6 weeks at 100 mg/L had no detectable effects on 18 hematologic tests of 12 African
Green monkeys.
In a 12-month study using Sprague-Dawley rats administered 1,10 and 100 mg/L
monochloramine, glutathione levels, red blood cell count and hematocrit were found to
be decreased at sporadic intervals. However, there was a lack of dose- and
time-dependent response in the results. Plasma thyroxine levels were significantly
decreased and cholesterol elevated in pigeons administered 15 mg/L monochloramine
for 3 months.
Rats and mice were administered monochloramine in drinking water at
concentrations of 0, 25, 50, 100, 200 or 400 ppm (0, 25, 50, 100, 200 or 400 mg/L) for 91
days. Decreased body weight gain and liver damage were observed at 200 and 400 mg/L
in rats and 100, 200 and 400 mg/L in mice. Histopathologic observation revealed mild
to moderate cytologic alteration in the liver of male mice administered 200 and 400 mg/L
chloramines. Chronic liver inflammatory changes occurred at 100, 200 and 400 mg/L in
female mice and to a lesser extent in male mice at the 100 ppm level. Microscopic
CLORAMIN.1 I-4 03/08/94
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examination of rat tissues at the 400 mg/L level did not reveal any treatment-related
lesions. The investigators suggested a NOEL of 50 mg/L or ~8.3 mg/kg/day
monochloramine based on chronic liver inflammatory changes in mice.
In a 90-day study, male and female Sprague-Dawley rats were administered
monochloramine in drinking water at concentrations of 0, 25, 50, 100 and 200 mg/L. At
200 mg/L the average weight gain was 51 % of controls. There were also reductions in
organ weights (absolute, relative or both) at the high dose level. The authors identified
the 100 mg/L dose as the NOAEL.
B6C3F1 mice were administered monochloramine in their drinking water for 90
days at 0, 12.5, 25, 50, 100 and 200 mg/L. There were weight gain reductions, and
reductions in absolute and relative organ weights at the 100 and 200 mg/L dose levels.
Based on these reductions the authors identified a NOAEL of 50 mg/L.
In a 2-year study, F344/N rats and B6C3F1 mice were administered 0, 50, 100 and
200 ppm monochloramine in their drinking water. There was a decrease in mean body
weight in high-dose rats. The high-dose group had decreases in organ weights and
increases in organ-to-body weight ratios at 14- or 66-week evaluations. There was also
a dose-related decrease in mean body weights of dosed male and female mice
throughout the study. There were decreases in organ weights and increases in
organ-to-body weight ratios observed in high-dose mice at 15- or 66-week evaluations.
CLORAMIN.1 I-5 03/08/94
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Monochloramine was not teratogenic in mature female Sprague-Dawley rats
exposed to 1, 10 or 100 mg/L in drinking water, nor did 40, 100 and 200 mg/L solutions
induce sperm-head anomalies in B6C3F1 mice. In addition, no significant differences in
fertility, viability, litter size, day of eye opening or day of vaginal patency were observed
between control and exposed Long-Evans rats given >10 mg/kg chloramines. There were
no alterations in sperm count, direct progressive sperm movement, percent mobility or
sperm morphologic characteristics.
Results on the mutagenicity of chloramines are inconclusive. Monochloramine has
been found to be marginally mutagenic in Bacillus subtilis and in Vicia faba plant seeds.
In Salmonella typhimurium (TA97, TA100 and TA102), chloramines (40 um) marginally
increased the number of revertant colonies over untreated controls. It was responsible
for cellular hypertrophy, increased mitotic figures and bizarre chromatin patterns in
B6C3F1 mice exposed to 200 and 400 mg/L in drinking water. In another study,
monochloramine at 40, 100 and 200 mg/L did not induce chromosomal aberrations or
micronuclei in bone marrow of CD-1 mice.
The organic chloramine, N-chloropiperidine, was found to be marginally mutagenic
in the reverse mutation plate incorporation assay (Ames test). It was cytotoxic and
cytostatic in CHO cells and produced chromosomal aberrations, the frequency of which
was proportional to the concentration of the compound. It produced SCEs in CHO cells,
but not in baby hamster kidney cells. The analogous chloramine, N-chlorodiethylamine,
was more toxic but nonmutagenic. When the synthetic N-CI compound chloramine T
CLORAMIN.1 I-6 03/08/94
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(sodium p-toluene-sulfonyl chloramide) was tested, SCEs were significantly increased in
a dose-dependent manner in CHO cells.
Organic concentrates of water treated with monochloramine produced papillomas,
squamous cell carcinomas and lung adenomas in SENCAR mice. These data are
inadequate, however, to assess the carcinogenic potential of monochloramine. In a
2-year study using male and female F344 rats and B6C3F1 mice, monochloramine was
administered in drinking water at 0, 50, 100 and 200 ppm. Equivocal evidence of
carcinogenic activity was found in female rats because of the slightly increased incidence
of mononuclear cell leukemia. There was no evidence of carcinogenic activity in male
rats or female or male mice, which was attributed to chloraminated drinking water.
Information concerning human exposure to chloramines is extremely limited. In
humans, acute exposure by inhalation of chloramine fumes has been observed after
mixing 4-5% solutions of ammonia and sodium hypochlorite in a small room. Pneumonitis
resulted, but no permanent pulmonary damage occurred. Very few experimental studies
have been conducted. Individuals ingesting levels of chloramines between 0.01 and 24.0
mg/L for 1 day or 5 mg/L for 12 weeks showed no hematologic or detrimental physiologic
responses resulting from chloramine ingestion.
There are no epidemiologic studies that have been designed to address
specifically the potential adverse effects of exposure to chloramines on human health.
One study was conducted to see if there was a difference in cancer mortality among
CLORAMIN.1 I-7 03/08/94
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communities using chlorine compared with communities using chloramine for disinfection.
This study was not designed to assess adverse effects from exposure to chloramine but
rather to consider the chloramine-exposed participants as controls.
Chloramines appear to be produced by normal human neutrophils as part of their
bactericidal action. Chloramines are believed to exert their effects by interfering with
enzymatic reactions. Monochloramine oxidizes and denatures hemoglobin and inhibits
the hexose monophosphate shunt. It also causes strand-breaks in DMA.
Lack of sufficient data preclude the derivation of a 1 -day HA for Chloramines. It is
recommended that the 10-day HA of 1 mg/L be adopted as the 1 -day HA. The 10-day HA
was derived from a drinking water study using African Green monkeys. The 10-day HA
for a 10 kg child is 1 mg/L based on the absence of hematologic effects. The longer-term
HAs for a 10 kg child and 70 kg adult are 1 and 4 mg/L, respectively. These HAs are
based on a NOAEL for body and organ weight changes in rats exposed to Chloramines
in drinking water. The DWEL of 4 mg/L is derived from a proposed RfD of 0.1 mg/kg/day
from a NOAEL in a chronic drinking water study using rats, based on absence of
decreased organ weight changes.
There was one 2-year bioassay with equivocal evidence of carcinogenic activity
in female rats. The CRAVE Work Group verified (12/02/92) a classification for
monochloramine of group D, not classifiable as to human carcinogenicity, meaning that
there is inadequate human and animal evidence of carcinogenicity.
CLORAMIN.1 I-8 03/08/94
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CLORAMIN.1 I-9 03/08/94
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II. PHYSICAL AND CHEMICAL PROPERTIES
Introduction
Inorganic chloramines are alternate disinfectants that are rapidly formed when free
chlorine is added to water containing ammonia. This reaction is represented by the
following equation with its respective reaction rate constant (at 25°C = 298°K) (Morris,
1967):
NH3 + HOCI = NH2CI + H20
= 6.1x106M-1sec-1)
Since ammonia has more than one hydrogen that can be replaced by a chlorine
atom, it reacts with an excess of hypochlorous acid to form dichloramine (Morris, 1967;
Grayetal., 1979):
NH2CI + HOCI = NHCI2 + H20
= 3.4x102M'1 sec'1)
ortrichloramine (Morris and Isaac, 1983):
NHCI2 + HOCI = NCI3 + H20
= 2.1 M-1sec-1)
CLORAMIN.2 11-1 03/08/94
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The distribution of mono-, di- and trichloramine is dependent on pH, temperature and
relative concentrations of ammonia and hypochlorite as described in the following section
on physical and chemical properties.
Physical and Chemical Properties of Chloramines
Under the usual conditions of water and wastewater chlorination, monochloramine
is the principal chloramine encountered. Anhydrous monochloramine is a colorless,
water-soluble liquid (Kirk-Othmer, 1979) that freezes at -66°C. The pure liquid
decomposes above -50°C with formation of nitrogen, chlorine and nitrogen trichloride
(Colton and Jones, 1955; Kovacic et al., 1970). The known chemical and physical
properties of pure monochloramine are summarized in Table 11-1. The environmental
significance of monochloramine, however, is generally restricted to its aqueous solutions,
where it is useful in water treatment for destruction of pathogenic bacteria (Butterfield,
1948; Wolfe et al., 1984). The rate of its formation is so rapid that determination of rates
between pH 6.5 and 10 were impossible because of the rapidity of the reaction in this
range (Weil and Morris, 1949). At pH 8.5 the rate of its formation reaction reaches a
maximum (Weil and Morris, 1949). Monochloramine is the only chloramine formed when
the pH of ammonia containing water is >8 and the molar ratio of hypochlorite to ammonia
is<1 (Grayetal., 1979).
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CLORAMIN.2 11-3 03/08/94
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At hypochlorite to ammonia ratios >1 or at lower pH values, dichloramine and
trichloramine are formed. At pH values <5.5 monochloramine slowly converts to form
dichloramine (Gray et al., 1979):
2 NH2CI + H+ = NHCI2 + NH4+
The relative proportions of monochloramine and dichloramine formed as a function of pH
and temperature are listed in Table II-2.
Comparatively little is known about the physical properties of pure dichloramine
because of its instability and difficulty of preparation. Its odor, volatility from aqueous
solution, and relative solubility in various solvents are intermediate between those of
monochloramine and trichloramine (NRC, 1980). Under equilibrium conditions at pH 4 it
is the only product of the reaction of equimolar concentrations of chlorine and ammonia.
Under normal conditions, however, dichloramine solutions are unstable (Corbett et al.,
1953) and decompose by several mechanisms, not all of which have been elucidated
(Chapin, 1931; Wei and Morris, 1974; Hand and Margerum, 1983).
Trichloramine (nitrogen trichloride) is formed in acid solutions where chlorine
concentrations are much greater than those of ammonia. At these high chlorine
concentrations and at pH values <3, trichloramine is the only chloramine present. Nitrogen
CLORAMIN.2 II-4 03/08/94
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CLORAMIN.2 11-5 03/08/94
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trichloride occurs in diminishing proportions at chlorine-to-ammonia mole ratios >2 and pH
values of <7.5. At pH >7.5, no trichloramine is found, regardless of the ratio of chlorine
to ammonia. Pure trichloramine is a bright yellow liquid that, because of its limited
solubility, can be isolated from aqueous solution by solvent extraction (Dowell and Bray,
1917). Its chemical and physical properties are summarized in Table 11-1. It can be
explosive in concentrated solutions and is an effective chlorinating agent, particularly in
nonaqueous media (Dowell and Bray, 1917; Jander, 1955; Kovacic et al., 1970). In
aqueous solutions at neutral pH it decomposes slowly to ammonia and hypochlorous acid
(Ryan et al., 1980) by an autocatalytic pathway (Hand and Margerum, 1983). Aqueous
solutions of trichloramine are stabilized by small amounts of acid (Corbett et al., 1953).
When chlorine is added to waters containing ammonia, the breakpoint phenomenon
becomes significant in the pH range of 6-9. The breakpoint is that dosage of chlorine that
produces the first detectable amount of free chlorine residual. At chlorine-to-ammonia
weight ratios of <5:1 ~pH 7, monochloramine is formed and the combined residual
increases to a maximum. This is the case up to the hump in the curve as displayed in
Figure 11-1. At chlorine-to-ammonia weight ratios 5:1, dichloramine is formed. The
residuals formed in this reaction occur between the top and the dip of the curve. With the
addition of chlorine the previously formed dichloramine is oxidized to nitrous oxide (N20),
nitrogen trichloride (NCI3) and nitrogen (N2). The formation of these nitrogen compounds
oxidize chlorine which in turn results in a decrease of ammonia nitrogen. As the
chlorine-to-ammonia nitrogen weight ratio reaches 10:1 at ~pH 7, the breakpoint
CLORAMIN.2 II-6 03/08/94
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COMBINED RESIDUAL
NITROGEN (110
DESTRUCTION
j[ / CHLORINE
" *.*/ DEMAND
ORGANIC CHLORAM1NES
NOT DESTROYED 8Y
FREE CHLORINE
FREE RESIDUAL
COMDINED RESIDUAL
APPLIED CHLORINE DOSAGE
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FIGURE II-l
Graphic Description of Breakdown Phenomenon
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phenomenon occurs (NRC, 1980; White, 1972). At the breakpoint dosage, some resistant
chloramines are present (primarily di- and trichloramine); however, they are not of any
importance (NRC, 1980).
Chlorine that is added after the breakpoint exists as free chlorine [that is, elemental
chlorine, hypochlorous acid (HOCI) and the hypochlorite ion (OCT)] (Pressley et al., 1973;
Wei and Morris, 1974). When natural waters and wastewater are chlorinated, there is a
residual oxidant formed, which remains stable at the breakpoint and in the presence of
hypochlorite beyond the breakpoint. This residual oxidant responds to conventional
methods of analysis in the same way monochloramine does. It can be shown that some
organic amino nitrogen compounds form very stable organic N-chloramines that, unlike
inorganic chloramines, do not decompose in the presence of excess hypochlorite. This
residual oxidant is, therefore, believed to be due to organic N-chloramines.
Monochloramine is less effective as a chlorinating agent than hypochlorous acid by
a factor of ~104 (Morris, 1967). However, when chloramines (mostly monochloramine)
were used to treat raw water, Stevens et al. (1978) determined that trihalomethane (THM)
formation was minimized. Thus, during the chlorination of water, when the ammonia
breakpoint is not achieved, THM production may be significantly reduced. Rickabaugh
and Kinman (1978) determined that chloramination of Ohio River water with
monochloramine at 10 mg/L, pH 7-9 and 25°C resulted in 90.7-99.9% less THM formation,
as compared with THM production from chlorination with 10 mg/L chlorine as hypochlorous
CLORAMIN.2 II-8 03/08/94
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acid or hypochlorite. Presumably many of the reaction products of water treated with free
chlorine (Cl, OCT and HOCI) are produced by the reaction of combined chlorine residual
(chlorinated waters that contain chloramines) with free chlorine. This is a result of the slow
hydrolysis of chloramines to hypochlorous acid. The products should occur in low
concentrations because of the low equilibrium concentration of the hypochlorous acid
formed (Margerum et al., 1979).
Margerum et al. (1979) indicated that the formation of hydroxylamine (NH2OH)
at pH 8 has a reaction half-time of 350 years. Therefore, it probably does not occur in
water treatment.
When low concentrations (mg/L range) of monochloramine and phenol are mixed,
chlorophenols appear after a reaction time of several days (Burttschell et al., 1 959). This
probably results from the hydrolysis of NH2CI and subsequent reaction of HOCI with
phenols.
Organic amines and amino acids in natural waters also react rapidly with hypochlorite
to form organic N-chloramines (Calvert, 1940; Wright, 1926, 1936; Taras, 1950; Crane et
al., 1946; Ellis and Soper, 1954; Mauger and Soper, 1946; Sandford et al., 1971; Edmond
CLORAMIN.2 II-9 03/08/94
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and Soper, 1949; Ingols et al., 1953; Wajon and Morris, 1980). Morris (1967) concluded
that the reaction rates of free chlorine with amino nitrogen compounds increases with the
basicity of the compound. As with ammonia, organic amines can also form dichloramines,
but the reaction rates are considerably slower for the addition of the second chlorine atom
than the addition of the first chlorine atom. Formation of N,N-dichloromethylamine from
N-chloromethylamine occurs with a second order reaction rate constant at 25°C of 1.1x103
M"1 sec"1 which is faster for organic than inorganic dichloramine (Morris, 1967):
CH3NHCI + HOCI = CH3NCI2 + H20
Both inorganic chloramines and organic N-chloramines are formed when wastewater
effluents are chlorinated. The relative amounts depend on the concentration ratios of
ammonia to organic amino-N, the temperature, pH and the relative reaction rates (Isaac
and Morris, 1980).
Organic N-chloramines can also form slowly by the reaction of inorganic chloramine
with organic amines (Snyderand Margerum, 1982; Isaac and Morris, 1983, 1985):
NH2CI + RNH2 = NH3 + RNHCI
The transfer of chlorine from monochloramine to organic amines has been shown to
involve two mechanisms: hydrolysis of the monochloramine to ammonia and hypochlorous
CLORAMIN.2 11-10 03/08/94
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acid and direct chlorine transfer from a protonated monochloramine NH3CI+ to the organic
amine (Snyderand Margerum, 1982; Isaac and Morris, 1983, 1985). Because the chlorine
transfer reaction is slow, its significance may be limited to large water distribution systems
using inorganic chloramine as the disinfectant where retention time in the system becomes
considerable.
From a water treatment standpoint organic N-chloramines are undesirable, since they
are not effective disinfectants (Feng, 1966; Marks and Styandskov, 1950; Wolfe et al.,
1984; Wolfe and Olson, 1985).
Uses of Chloramines
The combining of ammonia with chlorine for the purpose of forming chloramines to
treat drinking water has been called combined residual chlorination, chloramination or the
chloramine process (NRC, 1979). This process has been employed to provide a more
persistent disinfecting residual than free chlorine. It also generally reduces the unpleasant
taste and odors resulting from the formation of chlorophenolic compounds (Symons et al.,
1978).
Inorganic chloramines have been considered poorer disinfectants than hypochlorous
acid, since nearly 25 times as much chloramine as free chlorine was required to obtain a
100% kill with equivalent contact times (Butterfield, 1948). Brodtmann and Russo (1979)
refuted the idea that chloramine was a poor biocide for use in the treatment of drinking
CLORAMIN.2 11-11 03/08/94
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water. They found that chloramine treatment of drinking water was effective in destroying
~60% of the total bacterial population remaining after clarification, with a contact time of
<10 minutes. Furthermore, the chloramine treatment was effective in destroying ~88% of
the remaining coliform bacteria before sand filtration. Thus, the authors concluded that
chloramine properly applied at effective dosages (1.5-1.8 mg/L) produced 100% kills of
pathogenic bacterial species and reduced the total population of bacteria to an acceptable
range.
Analytical Methods
Methods deemed acceptable for the determination of chlorine residuals in natural and
treated waters include the following: colorometric methods, amperometric titration,
stabilized neutral orthotolidine (SNORT) method, ferrous diethyl-p-phenylenediamine
(DPD) method, and the leucocrystal violet (LCV) method.
The most common methods of analysis of monochloramine and most monochlorinated
organic N-chloramines use the ability of these compounds to oxidize iodide to iodine
followed by determination of the amount of iodine formed (APHA, 1980). The iodine
formed can be titrated with a standardized solution of sodium thiosulfate and the endpoint
detected visually with the aid of a starch solution. It can also be detected colorimetrically
by addition of DPD (diethyl-p-phenylenediamine), which is converted to a red oxidized
form. The intensity of the red color is measured with a spectrophotometer at 515 nm or
with a filter photometer equipped with a filter having maximum transmission in the
CLORAMIN.2 11-12 03/08/94
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wavelength range 490-530 nm. The oxidized DPD can also be titrated with standardized
ferrous ammonium sulfate. This method is known as the FAS-DPD titrimetric method.
Another method of measuring the iodine formed involves amperometric titration using a
standardized solution of phenylarsine oxide. Leucocrystal violet is a colorless form of a
dye that is converted by iodine to its colored form and measured with a spectrophotometer
(592 nm), using a filter photometer or Messier tubes.
Organic amines and albumenoid nitrogen compounds interfere with the analysis of
monochloramine in chlorinated natural waters because they respond in a similar manner.
In addition, as described below, the use of iodometric methods for analysis of chloramines
has contributed a considerable amount of confusion to the meaning of the term
"chloramines" (Johnson, 1978; Jolley and Carpenter, 1983; Wajon and Morris, 1980;
Cooper et al., 1982). There is a need for new methods for distinguishing the various
chemical compounds that respond to conventional analyses as "free residual chlorine" and
"combined residual chlorine."
Most kinetic measurements of the formation, reactions and decomposition of inorganic
mono- di- and trichloramine have employed a direct spectrophotometric determination of
their concentrations. Each has characteristic absorption spectra (Hand and Margerum,
1983). However, because of their low molar extinction coefficients the method is only good
for measurements of solutions with concentrations >10"4 M.
CLORAMIN.2 11-13 03/08/94
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Evans (1982) reported a voltammetric method for analysis of inorganic chloramines
in aqueous solution from pH 4-12. However, the method has approximately the same
sensitivity as the spectrophotometric method.
Scully et al. (1984a) studied solutions of organic and inorganic N-chloramines in acid
solution (pH 2) by cyclic voltammetry. Monochloramines can be distinguished from
dichloramines and hypochlorous acid, but the method also lacks the sensitivity needed to
measure concentrations <10"4 M.
Scully et al. (1984b) developed a method for the analysis of organic and inorganic
chloramines in dilute aqueous solution. The method involves derivatization of the
chloramines with the sodium salt of 5-dimethylaminonaphthylene-1-sulfinic acid in
bicarbonate buffer to form highly fluorescent sulfonamide derivatives. These derivatives
can be separated by high pressure liquid chromatography so that organic chloramines can
be measured in the presence of inorganic chloramine. The method has not yet been used
successfully to identify organic N-chloramines in chlorinated natural waters. This will be
necessary before the importance of organic N-chloramines in drinking water can be
determined.
From a strictly chemical standpoint only ammonia and organic amines (R = alkyl or
aryl) can form chloramines. Considerable confusion, however, has been introduced into
the nomenclature of chloramines because of the methods used in the analysis of these
CLORAMIN.2 11-14 03/08/94
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compounds in chlorinated natural waters and wastewaters (Johnson, 1978; Jolley and
Carpenter, 1983; Wajon and Morris, 1980; Cooper et al., 1982). The term "combined
residual chlorine" has been used to describe compounds in chlorinated waters that can be
analyzed by amperometric and colorimetric methods only after addition of iodide, and the
term "free residual chlorine" has been used to describe compounds that are generally
analyzed by the same methods before the addition of iodide (APHA, 1980). Because
solutions of organic and inorganic chloramines respond to these analyses as "combined
residual chlorine" and because natural waters that contain high concentrations of ammonia
form correspondingly high concentrations of combined residual chlorine, the terms
combined residual chlorine and chloramines have become almost synonymous.
Consequently, chlorinated waters that contain combined residual chlorine are said to
contain chloramines. While the major fraction of combined residual chlorine is probably
inorganic chloramines, other compounds including organic amines that are likely to
contaminate natural waters and wastewaters react with chlorine to form compounds that
respond to analyses as combined residual chlorine. For instance, proteinaceous or
albumenoid nitrogen compounds contain amide linkages that react slowly with hypochlorite
to form chloramides, R(C=0)NCIR, which respond to analyses as combined residual
chlorine.
In addition, because solutions of hypochlorite are most commonly associated with free
residual chlorine, hypochlorite and free residual chlorine also have become almost
synonymous. Nevertheless, N-chlorosuccinimide, which is a chlorimide, and
CLORAMIN.2 11-15 03/08/94
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trichloroisocyanuric acid respond at least partially to conventional analyses as free
residual chlorine (Morris et al., 1980). The term free residual chlorine most accurately
refers to elemental chlorine, hypochlorus acid (HOCI) and hypochlorite ion (OCT). From
a public health viewpoint these ambiguities can present serious problems; for instance,
organic chloramines are poor disinfectants compared with inorganic chloramine (NH2CI)
(Johnson, 1978). Jolley and Carpenter (1983) suggested the terms "combined oxidant"
and "free oxidant" to avoid the ambiguities that have become associated with the residual
chlorine term.
Further ambiguity exists in the term chloramine because it implies simply that a
compound, organic or inorganic, contains both a chlorine atom and an amino nitrogen
functional group. This would then include the highly carcinogenic nitrogen mustards that
are not formed when water containing ammonia or organic amines is chlorinated. For the
purposes of this document, only the toxicology and health effects data of organic and
inorganic N-chlorinated amino nitrogen compounds will be reviewed.
Environmental Fate and Transport
The major source of chloramines in waters is the reaction of ammonia compounds with
added chlorine. This occurs as a by-product of chlorination (i.e., in drinking water
processing and sewage effluents) and from use of the chlorine-ammonia process
(chloramination). An inventory of municipal water supplies undertaken in 1963 indicated
that 308/11,590 surveyed used an ammonia-chlorine process (chloramination) (Moore and
CLORAMIN.2 11-16 03/08/94
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Calabrese, 1980). For a discussion of rates of formation and breakdown of chloramines
see the Physical and Chemical Properties Section and the Uses of Chloramines Section
of this chapter. A 1984 survey of some U.S. utilities using chloramines (Trussell and Kreft,
1984) showed a possible distribution of chloramine residuals. A typical range of
chloramine concentrations in drinking water supplies where it is used as a primary
disinfectant or to provide a residual in the distribution system is 1.5-2.5 mg/L.
Summary
The alternate disinfectant inorganic chloramines are formed when water containing
ammonia is chlorinated. The type and extent of each is dependent on pH, temperature
and relative concentrations of ammonia and hypochlorite. Under the usual concentrations
and conditions of water and wastewater treatment, monochloramine is the principal
chloramine formed. Ammonia reacts with an excess of hypochlorous acid to form
dichloramine ortrichloramine. Dichloramine solutions, however, are unstable and readily
decompose. When chlorine concentrations are much greater than ammonia and the
solutions are acidic (pH<3), trichloramine is formed. Monochloramine at standard
temperature and pressure is a liquid, whereas trichloramine at standard temperature and
pressure is a solid.
Organic amines or amino acids react rapidly with hypochlorite in natural waters to form
organic N-chloramines. Organic N-chloramines are also formed by the slow reaction of
CLORAMIN.2 11-17 03/08/94
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inorganic chloramines with organic amines. Information on organic chloramines and their
reactions is very limited.
Chloramines are used primarily as disinfectants since they are a more persistent
disinfecting residual than free chlorine. They also reduce the unpleasant taste and odors
in drinking water resulting from chlorophenolic compound formation.
There are several acceptable methods for determining chlorine residuals in natural
and treated waters. They are as follows: colorimetric methods, amperometric titration,
stabilized neutral orthotolidine (SNORT) method, ferrous diethyl-p-phenylenediamine
(DPD) method, and the leucocrystal violet (LCV) method. New methods with greater
sensitivity and the ability to distinguish various chemical compounds are needed.
The reaction of ammonia compounds with added chlorine is the major source of
chloramine release to the environment. This is due to processes and by-products of
chlorination.
CLORAMIN.2 11-18 03/08/94
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TABLE 11-1
Summary of Chemical and Physical Properties of Mono- and Trichloramines
Properties
Chemical structure
Molecular weight
Chemical Abstracts
Registry Number
Registry of Toxic Effects of Chemical
Substances (RTECS) Number
Synonyms
Color
Physical state
(25°C, 1 atm)
Melting point, °C
Boiling point, °C
Density
Solubility in water
Other solubilities
Monochloramine3
NH,CI
51.48
10599-90-3
FN0275000
-
Yellow (liquid)
Colorless crystals
(solid)
Liquid
-66°
-
_
Soluble
ETOH, ether,
CCI4, benzene
Trichloromineb'c
NCI,
120.38
10025-85-1
-
Nitrogen trichloride
Nitrogen chloride
Chlorine nitride
Trichlorine nitride
Yellow oil (liquid)
Rhombic crystals
(solid)
Thick oil or rhombic
crystals
<-40°
<71°
Explodes at 93°
1.653
Insoluble in cold,
decomposes in hot
CS2, PCI3,
benzene, CCI4,
CHCI3
aRTECS, 1984
bMerck Index, 1983
cWeast, 1983
-------
TABLE 11-2
Proportions of Monochloramine (NH2CI) and Dichloramine (NHCI2)
Formed in Water Chlorination with Equimolar Concentrations
of Ammonia and Chlorinea'b
PH
4
5
6
7
8
9
Proportion (%) at 0°C
NH2CI
0
34
77
94
99
100
NHCI2
100
66
23
6
1
0
Proportion (%) at 10°C
NH2CI
0
20
67
81
98
100
NHCI2
100
80
33
9
2
0
Proportion (%) at 25°C
NH2CI
0
13
57
88
97
100
NHCI2
100
87
43
12
3
0
aSource: NRC, 1980
Consideration of kinetic effects leads to calculated values with lower proportions of NHCI2
-------
III. TOXICOKINETICS
Introduction
Studies relevant to the toxicokinetics of inorganic chloramines are severely
limited. However, studies done with various chlorinated amino compounds (including
organic chloramines) give information on the phaimacokinetics of chloramines.
Absorption
Scully et al. (1985) have shown that solutions of hypochlorite can react with
amines and amino acids in stomach fluid to form the corresponding N-chloramines. In
separate experiments piperidine (0.2 M solution) and glycine (0.5 M solution) were
administered to Sprague-Dawley rats followed by a solution (either ~200 or 1000 mg/L)
of aqueous hypochlorite. Stomach fluid was recovered from these animals and shown
to contain the corresponding N-chloramino derivatives. The "chlorine demand" of
stomach fluid was determined by chlorinating the stomach fluid to various levels,
incubating the chlorinated fluid in the dark for 1 hour and measuring the residual chlorine
- in essence developing a breakpoint curve for the fluid. The chlorine dosage at which
a breakpoint was found was taken as the chlorine demand. The shape of the breakpoint
curve was very similar to that of wastewater, exhibiting a significant irreducible minimum
at the breakpoint and a high chlorine demand (400-800 mg/L) (Scully et al., 1986).
Scully et al. (1985) also found that when N-chloropiperidine (1.3 ml of 1700 mg/L as
chlorine containing 100 //Ci tritium labeled N-chloropiperidine) was administered to
CLORAMIN.3 111-1 03/04/94
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Sprague-Dawley rats, small amounts (1-3 //Ci) of the added chloramine appeared in the
plasma at 30, 60 and 120 minutes after the exposure.
Abdel-Rahman et al. (1983) administered single oral doses of 3 ml NH236CI (370
mg/L) to four male Sprague-Dawley rats that had been fasted overnight. Each rat
received 1.1 mg of test material (~4.6 mg/kg/day). A peak 36CI plasma level (10.3 //g/L)
was reached 8 hours after administration, and the absorption rate constant was 0.278
mg/hour with an absorption half-life of 2.5 hours. The 36CI plasma level remained at a
plateau from 8 to 48 hours after administration. After 48 hours the radiolabel was
eliminated from the plasma with a half-life of 38.8 hours and a rate constant of 0.018
mg/hour.
Toxicokinetics data on other absorption rates with respect to various dosage
media and different routes of exposure were not available.
Distribution
At 24 hours following the single oral treatment to rats of NH236CI as described
above, Abdel-Rahman et al. (1983) found the level of 36CI radioactivity in the plasma to
be 0.87%/ml_ of the administered dose. During the first 24 hours the portion of the
administered NH236CI dose eliminated through the urine was 0.40% and 0.08% by the
feces. After the addition of TCA, 0.14%/ml_ was precipitated from 1 ml of plasma,
possibly indicating binding to the protein fraction. Packed cells had an activity of
0.20%/ml_ that decreased to 0.06%/ml_ after washing twice with saline, indicating that
CLORAMIN.3 III-2 03/04/94
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most of the 36CI radioactivity in the packed cells was loosely bound to the erythrocyte
membrane or was exchangeable with the chloride in saline.
Abdel-Rahman et al. (1983) also characterized the subcellular distribution of 36CI
radioactivity in rat liver preparations 24 hours following NH236CI administration. The
major portion of the activity (75%) in the whole liver homogenate was recovered in the
cytosol, 2.5% was recovered in the microsomal, 1.5% in the nuclear and <0.1% in the
mitochondrial fractions. Only 4.0% of the total 36CI radioactivity of the whole homogenate
was precipitated by TCA. In the control experiment using 36CI" Suh and Abdel-Rahman
(1983) found a similar liver subcellular distribution of 36CI radioactivity: 0.82 //g/g in the
whole homogenate, 0.66 //g/g in the cytosol, 0.03 //g/g in the microsomes, 0.01 //g/g in
nuclei, and 0.001 //g/g in mitochondria. 36CI was administered as 200 mg/L Na36CI (0.6
mg/animal) solution orally.
The distribution of 36CI in various tissues was determined 120 hours following
NH236CI administration (Abdel-Rahman et al., 1983). Plasma contained the highest
concentration of 36CI radioactivity (3.15 //g/g), followed by whole blood (2.66 //g/g), skin
(2.13 //g/g), testes (2.09 //g/g), packed cells (1.90 //g/g), bone marrow (1.82 //g/g),
kidney (1.62 //g/g), lung (1.58 //g/g), stomach (1.53 //g/g), thyroid (1.36 //g/g), thymus
(1.36//g/g), duodenum (1.20//g/g), spleen (1.11 //g/g), carcass (0.77//g/g), liver (0.74
//g/g), ileum (0.59//g/g) and fat (0.18//g/g) (Figure 111-1). In the control experiment with
36CI-labeled chloride Suh and Abdel-Rahman (1983) using 47% as much labeled
CLORAMIN.3 III-3 03/04/94
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material as in the NH236CI study found an approximately proportional amount of 36CI
distributed similarly
CLORAMIN.3 111-4 03/04/94
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J.5
3.0
H
1
o>
^ 2.5
A
n 2.0
<4-
o
1 u
L.
4->
§ '.o
c
o
o
0.5
0
—
_
—
—
-
—
a
Q.
1
o
0
o
_J
CD
O
i
ll
z
in
-------
throughout the same organs, fluid and tissues (Figure 111-2). The 36CI radioactivity was
highest in plasma (1.4 //g/g) followed by whole blood (1.3 //g/g), testes (1.2 //g/g),
packed cells (1.1 //g/g), skin (0.9 //g/g), kidney (0.8 //g/g), lung (0.8 //g), bone marrow
(0.8 //g/g), stomach (0.7 //g/g), thymus (0.7 //g/g), spleen (0.6 //g/g), duodenum (0.5
//g/g), carcass (0.3 //g/g), liver (0.3 //g/g), ileum (0.4 //g/g) and fat (0.2 //g/g).
Metabolism
Abdel-Rahman et al. (1983) measured chloride, chlorite and chlorate in plasma
120 hours after administration of NH236CI in rats for determination of its metabolites.
Neither 36CI-labeled chlorite nor chlorate was detected in rat plasma. Most of the total
36CI was identified as36CI-chloride, which (according to the investigators) indicated that
the chlorine moiety was eliminated primarily in this form (Table 111-1).
Scully et al. (1986) identified three organic N-chloramines (N-chloroglycine,
N-chloroalanine and N-chlorophenylalanine) formed in vitro when stomach fluid from
Sprague-Dawley rats was chlorinated with a concentration of 400 mg/L hypochlorite.
After in vitro chlorination of stomach fluid from rats fasted for 8, 24 and 48 hours, Scully
et al. (1990) identified and quantified N-chloroleucine or N-chloroisoleucine,
N-chloroglycine and N-chlorophenylalanine by GC/MS. Scully et al. (1986) also found
that because stomach fluid already contains high concentrations of amino nitrogen, the
percent conversion of a single exogenous amine to its chloramino compound is low.
They determined the actual yield of chloramine by administration of tritium-labeled
piperidine followed by aqueous hypochlorite. The quantity of N-chloropiperidine formed
CLORAMIN.3 III-6 03/04/94
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FIGURE 111-2
36CT distribution in the rat. Four fasted rats were each administered 200 mg/L
Na36CI in a 3 ml solution orally. The rats were sacrificed 120 hours after administration,
and 36CT was determined in different organs. Values represent the mean ± SE as //g
36Cr/ml_ (or per gram tissue) from four rats.
Source: Suh and Abdel-Rahman, 1983
CLORAMIN.3 III-7 03/04/94
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15
u
X
05
l
1
l
Al
FIGURE III-2
3*C1- distribution 1n the rat. Four fasted rats were each
administered 200 mg/l Na36Cl 1n a 3 mi solution orally. The rats were
sacrificed 120 hours after administration, and 3«C1~ was determined 1n
different organs. Values represent the mean _f SE as yg "ClVma {or
per gram tissue) from four rats.
Source: Sun and Abdel-Rahman, 1983
03960
III-5
07/11/89
-------
CLORAMIN.3 111-8 03/04/94
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was determined by direct liquid chromatography of the compound. This quantity
combined with the average chlorine demand determined for stomach fluid was used to
calculate the yield of chloramine formed. For concentrations of hypochlorite ranging
from 200-1000 mg/L, yields of organic chloramines varied from 50-75% of the theoretical
amount expected for these compounds. However, Scully et al. (1986) showed that
administration of hypochlorite to animals at low (<40 mg/L), medium (40-800 mg/L) and
high (>800 mg/L) dosages can produce very different results. This may be due to
different chemical reactions taking place at varying hypochlorite concentrations below
the minimum chlorine demand. They cautioned that toxicologic studies conducted at
high oxidant concentrations may not reflect chloramine reactions that would account for
any toxicologic effects observed at lower oxidant concentrations or at actual drinking
water concentrations.
Under conditions designed to simulate the Gl tract, Bercz and Bawa (1986) found
that monochloramine caused covalent binding of radioiodide to nutrient biochemicals.
Saliva and gastric juice were obtained from rhesus monkeys under mild anesthesia.
Depending on their solubilities, nutrient substrates were dissolved in solvents and added
to a mixture of 600 ppm monochloramine, 0.02 N HCI and 0.1 M Kl. Monochloramine
was believed to oxidize iodide to iodine, which subsequently reacted with nutrient
chemicals to form iodinated organic compounds. Tyrosine, 4-aminobenzoic acid,
arachidonic acid, and folic acid were among the compounds that became iodinated
under the conditions of the experiment. Some of the reactions occurred under basic pH
conditions. The observed percent binding was generally lower for reactions occurring
CLORAMIN.3 III-9 03/04/94
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at lower pHs. While complex mixtures of nutrients such as gastric juice and saliva
appeared to bind iodine in dilute aqueous solution, it is important that these results be
correctly extrapolated to physiologic pH before their significance is fully understood.
The biologic effects of chloramine, its persistence in biologic fluids and its
possible conversion to more toxic products were examined by the U.S. EPA (1990). The
persistence of monochloramine in saliva and gastric fluid was examined to determine the
extent of formation of the products dichloramine, trichloramine and molecular chlorine.
Pooled human saliva and gastric fluid samples were treated with monochloramine to
produce samples with initial concentrations of monochloramine between 1.0 and 20 ppm.
These samples were continuously analyzed by membrane introduction mass
spectrometry and tandem mass spectrometry for monochloramine, dichloramine,
trichloramine and chlorine. The continuous monitoring experiments for the saliva
monochloramine mixtures were done using the technique of selected ion monitoring.
Because of constituents present in the gastric fluid samples, selected ion monitoring
experiments were shown not to give reliable results and the multiple reaction monitoring
procedure was used.
Monochloramine was completely depleted in saliva in ~5 minutes at the 1 ppm
level, the 5 ppm solution was incompletely depleted in 2 hours and the monochloramine
in the higher concentration solutions was largely unaffected. Dichloramine, trichloramine
and molecular chlorine were not produced in detectable levels from the
chloramine-treated saliva samples.
CLORAMIN.3 111-10 03/04/94
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Gastric fluid monochloramine disappeared completely in <30 seconds at all
concentrations, and dichloramine, trichloramine and molecular chlorine were not
observed.
Excretion
Abdel-Rahman et al. (1983) collected the urine, feces and expired air of
Sprague-Dawley rats over a 5-day period after the administration of 370 mg/L (1.1
mg/animal or ~4.6 mg/kg/day) NH236CI to four Sprague-Dawley rats. The amount of
36CI-radiolabeled material excreted by urinary and intestinal routes are summarized in
Table III-2. During the first 24 hours after administration of NH236CI, only 0.40 and 0.08%
of the total dose administered was eliminated in the urine and feces, respectively. The
proportion of the dose eliminated through the urine and feces at the end of the 120-hour
study period was 25.15 and 1.98%, respectively. By comparison, Suh and
Abdel-Rahman (1983) found that over twice as much of the 36CI-radiolabel was
eliminated over the 120-hour study period when 36CI-radiolabeled chloride ion (200 mg/L
as Na36CI) was administered (Table III-3). They found that 57.2% of the administered
36CI-radiolabel was eliminated in urine and 3.0% was eliminated in feces. Unlike
NH236CI, more of the 36CI-label was eliminated in the first 24 hours, 16.1% in urine and
0.92% in feces. After 48 hours 36CI was eliminated with a half-life similar to that found
after 24 hours in the NH236CI study (t1/2 = 24 hours).
CLORAMIN.3 111-11 03/04/94
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CLORAMIN.3 111-12 03/04/94
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CLORAMIN.3 111-13 03/04/94
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Discussion
In order to interpret the results of the toxicokinetics correctly, it is necessary to
understand the complications of the synthesis of NH236CI and possible interferences.
36CI-radiolabeled chloride ion is available commercially. H036CI is synthesized
by acidic permanganate oxidation of the 36CI" to 36CI2:
2KMn04 + 16H36CI = 2KCI + 2MnCI2 + 536CI2 + 8H20.
The 36CI-labeled CI2 gas is passed from the generation flask to a receiving flask where
it is dissolved in deionized water:
36CI2 + H20 = H036CI + H36CI
The resulting hydrolysis converts half the labeled chlorine to H036CI, the desired
compound, and half to 36CI~.
Chlorine-36 radiolabeled inorganic chloramine is synthesized by reaction of the
solution of H036CI (containing an equivalent amount of 36CI~) with aqueous ammonia in
bicarbonate buffer:
NH, + 36CIO- = NH 36CI + HCr
CLORAMIN.3 111-14 03/04/94
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However, the observed kinetics of NH236CI absorption and elimination are necessarily
affected by the presence of an equivalent amount of 36CI~ formed during the preparation
oftheH036CI.
In addition, the stomach contains high concentrations of Cl~. There is a lack of
information on the rate of chloramine-chloride exchange
NH236CI + Cr = NH2CI + 36Cr
in solutions of high chloride concentration. If this exchange rate is significant, the
toxicokinetics of NH236CI could appear to resemble chloride when, in fact, the compound
has simply lost its radiolabel through exchange. This is the case for H036CI (Anbar et
al., 1959) where the exchange reaction is so fast that at 25°C in the presence of 0.1 M
chloride the exchange is 99% complete in <0.1 sec at any pH <10.8. In the absence of
specific information on the isotopic exchange rates for NH236CI, the rate of hydrolysis of
NH2CI as reported by Margerum et al. (1979) (k = 1.9x10"5 sec"1) and by Anbar and Yagil
(1962) (k = 6.3x10"5 M"1 sec"1) would suggest that the rate of exchange is slow and that
it would require between 4 and 10 hours for half the radiolabel to be lost at 25°C.
Nevertheless, monochloramine exchanges its chlorine with organic amino nitrogen
compounds at a rate faster than hydrolysis (Snyder and Margerum, 1982; Isaac and
Morris, 1983, 1985). Since the stomach contains high concentrations of organic amino
nitrogen, the chlorine from the NH236CI can be transferred to these organic amino
nitrogen compounds. How this affects the rate of isotope exchange is unknown.
CLORAMIN.3 111-15 03/04/94
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The control reagent used in the above toxicokinetic study (Anbar et al., 1959) of
NH236CI was 36CT. It is not likely that an oxidant as strong as NH2CI will survive intact
absorption, distribution, and excretion from an animal. If the chloramine is rapidly
detoxified in the stomach to 36CI-radiolabeled chloride and ammonia, the observed
toxicokinetics will be identical to the control kinetics. However, if the chloramine acts as
a chlorinating agent, other more stable chlorinated organic compounds may form. The
observed kinetics may then be due to exposure to these chlorinated compounds.
Summary
Information on the absorption of inorganic chloramines is extremely limited. In one
study Abdel-Rahman et al. (1983) calculated an absorption rate constant for 36CI at 0.278
mg/hour (after 8 hours) with an absorption half-life of 2.5 hours after male
Sprague-Dawley rats were administered a single oral dose of ~4.6 mg/kg/day. After 48
hours the radiolabel was eliminated from the plasma with a half-life of 38.8 hours and a
rate constant of 0.018 mg/hour. Absorption rates with respect to various dosage media
and different routes of exposure were not available. However, studies done with various
chlorinated amino compounds (including organic chloramines) give information on the
pharmacokinetics of chloramines. Scully et al. (1985) found that when the organic
chloramine, N-chloropiperidine, was administered to Sprague-Dawley rats, 1-3 //Ci of the
chloramine appeared in the plasma at 30, 60 and 120 minutes after the exposure.
The distribution of radiolabeled chlorine in the subfractions of rat liver homogenates,
in organs, tissues and fluids was similar (120 hours) after oral administration of either
CLORAMIN.3 111-16 03/04/94
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36CI (200 mg/L as Na36CI) or NHfCI (370 mg/L NHf Cl) (Abdel-Rahman et al. 1983; Suh
and Abdel-Rahman (1983). Plasma contained the highest concentrations of 36CI
radioactivity for NH36CI followed by whole blood skin, testes, packed cells, bone marrow,
kidney, lung, stomach, thyroid, thymus, duodenum, spleen, carcass, liver, ileum and fat
(Abdel-Rahman et al., 1983).
Information on the metabolism of chloramines is also extremely limited.
Abdel-Rahman et al. (1983) indicated that chloramine (NH236CI) administered to rats was
metabolized primarily to 36CI-chloride, which indicated that the chlorine moiety was
eliminated in this form.
When pooled saliva and gastric fluid were treated with monochloramine (U.S. EPA,
1990), saliva monochloramine was depleted in 5 minutes at 1 ppm, and in higher
concentrations was not affected, while gastric monochloramine disappeared in <30
seconds at all concentrations tested. Dichloramine, trichloramine and molecular chlorine
were not produced in detectable levels from the chloramine-treated saliva or gastric
samples.
Chloramines or their metabolites (principally chloride) are eliminated primarily
through the urine. Abdel-Rahman et al. (1983) found that during the first 24 hours after
a single administration of NH236CI (1.1 mg/animal) to Sprague-Dawley rats, only
0.40-0.08% of the total dose was eliminated in the urine and feces, respectively. At the
end of 120 hours 25.15 and 1.98% of the dose was eliminated in the urine and feces,
CLORAMIN.3 111-17 03/04/94
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respectively. By comparison, Suh and Abdel-Rahman (1983) found that over twice as
much of the 36CI-radiolabel was eliminated over a 120-hour period when 200 mg/L of
Na36CI was administered orally to rats. Unlike NH236CI, more of the 36CI-label was
eliminated in the first 24 hours, 16.1% in urine and 0.92% in feces. The major difference
between the NH236CI and Na36CI studies (Abdel-Rahman et al., 1983; Suh and
Abdel-Rahman 1983) was in the total amount of label excreted over the test period. Only
half as much label was eliminated in the chloramine study as in the chloride study.
Interpretation of the toxicokinetics of inorganic chloramines is difficult since
synthesis of NH236CI is complicated by possible interferences. There is a lack of
information on the rate of chloramine/chloride exchange in solutions of high chloride
concentrations.
36m-
NH236CI + Cr = NH2CI + 36CI
If this exchange rate is significant the toxicokinetics of NH236Cr could appear to
resemble chloride when, in fact, the compound has simply lost its radiolabel through
exchange. Since the stomach contains high dose concentrations of organic amino
nitrogen, the chlorine from the NH236CI can be transferred to these organic amino
nitrogen compounds (Snyderand Margerum, 1982; Issac and Morris, 1983, 1985). How
this affects the rate of exchange is unknown.
CLORAMIN.3 111-18 03/04/94
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TABLE
Metabolism of NH236
Treatment
NH236CI(1.1 mg)
MM
Cl in Rat Plasma3
Analyte (%/mL)b'c
Total 36
0.41 ±0
Cl 36CI
.08 0.35 ±0.08
aSource: Abdel-Rahman et al., 1983
bNeither 36CI02 nor 36CI03 was detected in rat plasma at the time period studied.
cValues represent mean ± SE from seven (fasted) rats after 120 hours following
NH236CI treatment expressed as percentage of total administered dose per ml of
plasma.
-------
TABLE II 1-2
Excretion of 36CI in Rat 120 Hours After
Single Oral Administration of NH236Cla
Collection
Period
(hours)
0-8
8-16
16-24
0-24
24-48
48-72
72-96
96-120
0-120
Proportion of NH236CI Excreted (%)b
Urine
0.07 ±0.07
0.13 ±0.02
0.21 ±0.08
0.40 ± 0.02
6.28 ±2.87
6.30 ±5.52
5.03 ±3.03
7.16±1.93
25.15 ±13.32
Feces
0.08 ±0.05
0.48 ± 0.24
0.31 ±0.29
0.26 ±0.21
0.87 ±0.49
1.98 ±0.29
Total
0.48 ± 0.02
6.75 ±3.10
6.60 ±5.80
5.29 ±3.24
8.02 ±1.44
27.13 ±13.61
aSource: Abdel-Rahman et al., 1983
Values represent the mean ± SE from four treated (fasted) rats expressed as the
proportion of administered dose. 36CI was not detected in expired air throughout the
120 hours studied.
-------
TABLE II 1-3
Excretion of 36CI~ in Rat 120 Hours After
Single Oral Administration of Na36Cla
Collection
Period
(hours)
0-12
12-24
0-24
24-48
48-72
72-96
96-120
0-120
Proportion of Na36CI Excreted (%)b
Urine
8.2 ±1.9
9.7 ±1.9
16.1 ±3.8
8.2 ±1.8
14.8 ±3.2
9.5 ±1.9
8.6 ±1.6
57. 2 ±10.6
Feces
0.92 ± 0.43
0.43 ±0.12
0.55 ±0.50
0.93 ±0.17
0.17 ±0.07
3.0 ±0.91
Total
17.0 ±3.4
8.6 ±1.8
15.4 ±2.8
10.4 ±2.0
8.8 ±1.6
60.2 ±9.7
aSource: Suh and Abdel-Rahman, 1983
Values represent the mean ± SE from four treated rats, expressed as percentage of
administered dose. 36CI~was not detected in expired air through the 120-hour study.
-------
V. HEALTH EFFECTS IN ANIMALS
Introduction
Animal studies on chloramines are limited. Few health-related studies have been
conducted on chloramines in drinking water. Since chloramines may be used as an
alternate means of disinfection, more studies are being conducted. Summaries of the
acute, subchronic and chronic oral studies are presented in Tables V-1 and V-2.
Short-Term Exposure
Because individuals undergoing long-term hemodialysis have been shown to
develop hemolytic anemia when water containing monochloramine (NH2CI) has been
inadvertently used in the dialysis treatment, hematologic parameters have been examined
in several studies. Moore et al. (1980) exposed 12 male A/J mice per group for 30 days
to a bicarbonate buffered solution (pH 8.9) of monochloramine in drinking water at
concentrations of 2.5-200 mg/L. They found no statistically significant (two-way analysis
and Kruskal-Wallis one-way analysis of variance) changes in nine measured blood
parameters; however, hematocrit did rise slightly with increasing levels of monochlor-
amine. The authors concluded that monochloramine ingested by A/J strain mice did not
appear to produce hemolysis even at very high dose levels.
Abdel-Rahman et al. (1984) investigated the toxicity of NH2CI in male
Sprague-Dawley rats. Acute exposure to a single dose at 10, 20 or 40 mg/L NH2CI
induced a statistically significant (p<0.05) increase in blood glutathione levels within 30
CLORAMIN.5 V-1 03/08/94
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TABLE V-1
Summary of Studies on Acute and Suijctirorwc Oral Exposure to MonoditorBmine
Mod* of
Administration
Cavage
Oral/drinking
water
Oral/drinking
water
Oral/drinking
water
Species
Sprague-Dawley
rats
African Green
monkeys
CD-1 mice
rat
Sex, Weight
4 males/group,
150-1 70 g
5 males,
7 females, 3.0-57
kg
10 males,
10 females
NS
Duration
1 dose
6 weeks
30 days
45 days
Dose
10mg/Lx 3 ml ({].!&-
0 ,2 mg/kg bw)
20mg/lx3mL(0.35-
0.4 mg,'kg bw)
40mg/Lx3mL(07l-
0.8 mgrttg bw)
100 mg,'L
100x"
400x'
10, 50 or lOOmgfl.
Effect
Significant increase in blood glutathione at 30 and
60 minutes (7 9 and 26%. respectively), but not at
15 w 120 minutes, No significant effect on blood
osmotic fragility.
Significant increase in blood glytathiocie at 15, 30
and 60 minutes (17, 15 and 25%, respectively) but
not at 120 minutes. No significant effect on Slood
osmotic fragility.
Significant increase in blood glutalhiorie at 16. 30
and 80 minutes (13, 1? and 33%, respectively) but
not at 1 20 minutes. No significant effect on blood
osmotic fragility.
No detects bte effect in 1 8 hemaiologic iesis .
No akjnificarst differences in body wetg'hl; signiftcant
increase in liver weights in females; stgnifcant
decrease in lung weights of both sexes, significant
decrease in brain and kidney weights in males.
No significant difference in body weight:
significantly decreased kidney and liver weights in
mates ancl significantly increased ovary weigWS in
females.
No effect on weight gain or h-ernatologtc parameters
Reference
Abetet-Rahman
etal., 19B4
Berczetal., 19S2
Milter eial., 1986
Bull, 1980
CLORAMIN.5
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3/08/94
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TABLE V-1 fcont.)
Mode of
Administration
Oral/drinking water
OralttJrmking wate-r
Species
36C3F1 -niw
Fischer 344 rats
Sprague-
Dawlfly rats
Sex, Weight
10,'sex/graup
1Q]1 sax/group
Duration
91 days
90 days
Dose
2 5 and 50 mg.'t
100, 200 and 400 mg/L
25, 55 and 100 mg/L
200 and 400 mg/L
25 mglL
SO me/L
lOOrwyL
200 mg/L
Effect
No adverse effects
Chronic liver inflammatory changes ; increased
frequency of miSotic figures, hypertrophy and
bizarre chromatin panems; decreased liver
weights at 400 mg/l_ in males and >1
-------
TABLE V-1 (conl.)
Mode of
Administration
Oral/dn diking water
Oral/drinking water
Species
B6C3F1 mice
Sprague-Dawtoy
rats
Sex, Weight
Wwx/group
12 males/dose,
1Q09
Duration
90 days
9 we&ks
Dose
12.5 and 25 mglL
SO mg/L
100 and 200 mg/L
9, 19 and 38 mg/L
Effect
Decrease in MCV in females, decrease in Alk-P (25 mg/L}
(neither effect considered treatment related).
Decrease in MCV in fe-mates, significant decreases in Aid-
P in males and significant mcr in AST in females
(none considered treatment related).
Significant differences in weight gam and water
consumption. Increase in lymphocytes in mates aid
decrease in kidney and tung weights in both sexes. In
males decreased absolute testes and spleen weights (200
rnp/l} Also at 200 mg/L signficant increases in relative
brain, kidney, lung and tesles in males and brain and
kidney in females.
Significant reduction in spleen weight (38 mg/L); decreased
antibody syntnesis (9 and 19 mg/L); augmented PGE2
production (19 and 38 mgrt.)
Reference
Daniel etaL 1991
Exon etal., 1987
*Samples were oofioenlrat&d equal to ihe original concentrate (400x) dr one-fourth of the ortginaJ concentrate (iQQx). The onojirtal concentrate or monochloramine residual was 2.1 mg/L. The
cjriginal oonoentrate of chtorarmnes was not S
MS =• Not specified
CLORAMIN.5
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3/08/94
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TABLE V-2
Summary erf Studies on Chronic Oral Exposure to Monochloramine m Drinking Water
Species
Sex. Weight
Duration
Dose
Effect
Reference
rats
4 males/group, 150-
570 q
12 months
1 mg/L
(-0.1 rng/kg bwMay)
Significant decrease m blood giufathiorte at 4, 6 and 12 months (27,24
and 25%. respectively). No effect on Wood osmotic fragility. No effect
on Mood cell compartment Increase in [•"H]-thymicSir»e incorporation in
nuclei, significant in kidney (190% f and spleen (230%) but not liver,
testes or intestinal mucosa, at 3 months. No effect on body weight.
Abet-Rahman
etal., 1964
10mg,'L
(- 1.0 mg/kg bw/day)
Significant decrease in blood gimathione at 6 and 12 months (20 and
24%, respectively); significant increase at 10 months (27%)
Significant increase in blood osmotic fragility at 2 and 10 rnontna (44
and 39%, respectively). Significant decrease in RBC count (8.8%) arid
HCT (11%) at 3 months, no effect on HGB MCV, MCH or MCHC.
Significant increase m {JH]-ttiymtdtns incorporation in nuclei of kidney
(290%) and spleen, (160%) but not qther organs, at 3 months. No
effect on body weight,
lOOrngrL
(-10 mgAg bw/day>
Significant decrease in blood giutathione at 4, 6 ant) 12 months (20, 23
and 22%, respectively). Significant increase in blood osmotic fragility
at 2 and 6 months (27 ano) 67%, respectively). Significant decf»as« in
RBC count (6.8%) and HCT (12%} at 3 months, and decrease in HGB
(22%) and MCH (23%) at 10 months. Significant increase in []H]-
ine incorporation m nuctei of ieates (200%) at 3 months. Body
signiiciyijjy rgjiucgd at
F344/N rats
10/sex/group
2 years
50 pprn
(2.1-2.8 mg/kg/dayj.
No irealment-relaied eftecls
NTP, 1990
100ppm
[4.8-5,
200 ppm
(8,7-9,5mg/kg/day)
Lower mean body weights and decreases in liver and kidney weights in
males and increases in brain and kidney-le-body weight ratios at 14- or
66-week evaluations.
CLORAMIN.5
V-5
3/08/94
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TABLE V-2 (cgnt.)
Specie*
B6C3F1 mice
Sex, Weight
10/sex/group
Duration
2 years
Oesa
SO ppm
(5.0-44 9 mgAg/dayl
100 ppm
(8.9-9.0 mg/kg/day)
200 ppm
{15.9-17.2mg,'kgKlay)
Effect
No treatment-related effects
Mean body weights of mica were 10-22% lower alter waek 37 and
body weights of female mioa ware 10-35% iowsr after waefi. fl. At 15 or
66 wee-ks decreases in liver weights and increases in brain or ksdney-lo-
txxiy- weigh! ratios occurred.
Reference
WP. 1990
CLORAMIN.5
V-6
3/08/94
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minutes after administration of a 3 ml aqueous solution by gavage. On the other hand,
chronic administration at 1, 10 or 100 mg/L doses in drinking water induced a statistically
significant (p<0.05) decrease in glutathione after 4 months of exposure at the 1 and 100
mg/L dose levels. The difference in glutathione blood levels following subchronic versus
chronic exposures may be due to different physiologic responses such as 1) an increase
in reduced glutathione levels resulting in increased RBC glutathione reductase activity
in subchronically exposed animals, and 2) a decrease in reduced glutathione levels in
chronically exposed animals, which is due to depletion of glutathione body stores.
Bercz et al. (1982) studied the toxicity of monochloramine administered in drinking
water to 5 adult males and 7 adult female African Green monkeys with body weights
ranging from 3.0-5.7 kg. Monochloramine was administered for 6 weeks at 100 mg/L.
The authors estimated the mean daily dose to be ~10 mg/kg/day. Such ingestion of
monochloramine had no detectable effect in 18 hematologic tests on the 12 monkeys,
including red cell GSH levels. No evidence of thyroid suppression was detected in
serum. Maziarka et al. (1976) also found that rats exposed for 9 months to 9.0 mg/L
chloramines showed no observable hematologic effects. However, the study was not a
dose-response type and many of the blood parameters reflecting oxidant stress were not
measured.
In a review of alternate disinfectants Bull (1980) reported the results of a 45-day
study in which 10, 50 or 100 mg/L (1.4, 7.0 and 14.0 mg/kg assuming rats consume 0.14
L/day) monochloramine in drinking water was administered to laboratory rats. Body
CLORAMIN.5 V-7 03/08/94
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weight gain and hematologic parameters in exposed animals did not differ significantly
from control animals. The only significant finding was a decrease in the amount of
methemoglobin present in the blood at 100 mg/L monochloramine. This is the opposite
of what was expected. There were no signs of overt toxicity (Bull, 1980). Details of this
study were not reported.
A subchronic 30-day toxicity test was performed on groups of 10 male and 10
female CD-1 mice using concentrated drinking water samples collected at a pilot scale
drinking water treatment plant using monochloramine for disinfection purposes (Miller et
al., 1986). The residual monochloramine level was 2.1 mg/L before concentration of
organics by reverse osmosis. Samples were concentrated (100x or 400x) before
toxicologic testing. Post exposure, the mice were examined for gross pathologic changes.
Body weights were not significantly different from controls. There was a statistically
significant increase (p<0.05) in liver-to-body weight ratios of females and a statistically
significant decrease (p<0.05) in the lung weights of males and females at 100x. The
brain and kidney weights were significantly decreased (p<0.05) in males at 10Ox. At 400x
the kidney and liver weights of males were significantly decreased (p<0.05) and the ovary
weights of females were significantly increased (p<0.05). The authors conclude that
these data did not reveal any overt toxicity; therefore, histopathologic examinations of
major organs was not performed.
A draft report by GSRI (1981) investigated the effects following exposure to
monochloramine in Fischer 344 rats and B6C3F1 mice. Rats (10/sex/group) were
CLORAMIN.5 V-8 03/08/94
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administered concentrations of 0, 25, 50, 100, 200 and 400 ppm (25, 50, 100, 200 and
400 mg/L) monochloramine in drinking water ad libitum for 91 days. Using water and food
consumption data provided in the report, corresponding doses were calculated to be 2.5,
4.9, 10.2, 18.8 and 40.7 mg/kg/day for males and 3.8, 6.5, 13.8, 26.6 and 53.9 mg/kg/day
for females. No animals died during the course of the study. After 25 days the buffer
system for monochloramines was changed because of a palatability problem at the
high-dose levels. Terminal body weight gains in male rats decreased at the 200 and 400
ppm levels by 13 and 24%, respectively, when compared with their controls. Female rats
gained 8 and 14% less weight than their controls at the 200 and 400 ppm levels,
respectively. The decrease in the percentage of body weight gain was most marked at
0-4 weeks and appeared to be related to the palatability of the 200 and 400 ppm
solutions. Absolute liver weights showed a trend toward reduced liver size; however,
when liver weights were expressed as a percentage of total body weight no significant
differences were observed. Protein excretion increased in male rats when 200 and 400
ppm monochloramine was administered. Microscopic examination of tissues at the 400
ppm level did not reveal any treatment-related lesions. This study identified a NOAEL for
rats of 100 ppm (10.2 mg/kg/day). (Confidence in this study is low because of questions
regarding the conduct of this study, the histopathologic evaluations and lack of
corroboration of its findings.)
Similarly, mice (10/sex/group) were administered concentrations of 0, 25, 50, 100,
200 and 400 ppm (25, 50, 100, 200 and 400 mg/L) monochloramines in drinking water ad
libitum for 91 days. Using water and food consumption data provided in the report
CLORAMIN.5 V-9 03/08/94
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corresponding doses were 4.9, 8.3, 14.5, 31.3 and 50.7 mg/kg/day for males and 7.7,
12.1, 21.9, 34.6 and 88.5 mg/kg/day for females. During the course of the study no
animals died. The buffer system for monochloramines was changed after 25 days since
palatability was a problem at the high dose levels. Male and female mice gained less
weight than the controls at the 200 and 400 ppm levels. There was a reduction in
absolute liver weights and liver-to-body weight ratios in male mice at the 400 ppm levels
and in female mice at >100 ppm. Histopathologic observations revealed necrotic
changes at the low doses and mild to moderate cytologic alteration in the livers of male
mice administered 200 and 400 ppm. Chronic liver inflammatory changes occurred at
100, 200 and 400 ppm in female mice and to a lesser extent in male mice at the 100 ppm
level. At concentrations of 100, 200 and 400 ppm increased frequency of mitotic figures,
hypertrophy and bizarre chromatin patterns occurred in males and in one female at 200
ppm. This study identified a NOAEL for mice of 50 ppm (8.4 mg/kg/day).
In a more recent study, Daniel et al. (1990) administered 0, 25, 50, 100 and 200
mg/L monochloramine to male and female Sprague-Dawley rats (10/sex/dose) in their
drinking water for 90 consecutive days. Using food and water consumption data provided
in the report, corresponding doses were calculated to be 0, 1.8, 3.4, 5.8 and 9.0
mg/kg/day for males and 0, 2.6, 4.3, 7.7 and 12.1 mg/kg/day for females. A control group
received distilled water buffered with sodium bicarbonate to a pH of 8.0-8.5. Mortality,
clinical signs, body weight, organ weights, food consumption, hematology, clinical
chemistry, gross pathology, and histopathology were examined. Water consumption was
significantly decreased at all monochloramine doses. At 200 mg in males water
CLORAMIN.5 V-10 03/08/94
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consumption was ~31% of controls and in females at 200 mg water consumption was
-34% of controls.
Food consumption was significantly reduced in males at the highest dose tested.
In males there was a significant reduction in body weight gain at doses of 50 mg/L and
higher. The reduced body weight gain in females was significant only at 200 mg/L. For
males and females the average weight gain at 200 mg/L was ~51 % of controls. At the
200 mg/L dose, reductions in the absolute weight of liver and spleen were observed in
both sexes. In females, the absolute weight of the thymus was also decreased as was
the weight of the lung and heart of the males. Also at 200 mg/L, females had increased
relative kidney and decreased relative liver weights while males had increased relative
brain, testes and kidney weights.
At 100 mg/L males had significantly decreased absolute liver weights and at 50
mg/L males had decreased absolute lung weights. At the 25 mg/L dose, there was no
significant effect on absolute organ weights in either sex. The authors concluded that the
100 mg/L dose is considered the NOAEL. Although reductions in organ weights
appeared to be dose-related in males, subsequent histopathologic examination did not
reveal any target organ or treatment-related changes. Although reduced RBC count (200
mg/L) and decreased calcium levels in males were significant, the authors did not
consider them biologically significant, dosage-related or within the normal range for rats
of this age and strain.
CLORAMIN.5 V-11 03/08/94
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Daniel etal. (1991) administered monochloramine in drinking water for 90 days to
male and female B6C3F1 mice (10/sex/dose) at 0, 12.5, 25, 50, 100 and 200 mg/L. Using
food and water consumption data provided in the report, corresponding doses were
calculated to be 0, 2.5, 5.0, 8.6, 11.1 and 15.6 mg/kg/day for males and 0, 2.8, 5.3, 9.2,
12.9 and 15.8 mg/kg/day for females. Mortality, clinical signs, body weight, organ
weights, food consumption, hematology, clinical chemistry, gross pathology and
histopathology were examined. Food consumption was decreased in males and females
at the two highest dose levels with a statistically significant decrease in females. Water
consumption was decreased in all treated groups with statistically significant decreases
in female mice at all treatment levels and male mice at the two highest levels.
In males there was an increase in lymphocytes at 100 and 200 mg/L and in
females there was a decrease in neutrophils at 100 mg/L and a decrease in MCV in all
dose levels. None of the hematology results were considered to be dose related. Alk-P
was decreased in males at all concentrations, statistically significant at 25, 50 and 100
mg/L, while AST was increased in females at all dose levels, and statistically significant
at 50 and 200 mg/L.
There were significant reductions in body weight gain in males at the three highest
dose levels, and at the two highest dose levels in females. Significant decreases in water
consumption occurred in all doses in female mice and at the two highest dose levels in
males. Significant reductions in absolute organ weights were evident in males and
females drinking 200 mg/L monochloramine. In male and female mice, liver and heart
CLORAMIN.5 V-12 03/08/94
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weights were decreased at the two highest dose levels, and relative and absolute spleen
weights were decresed in the two highest doses for females. At the highest dose, the
kidney and lung weights were decreased in males and females; also at the highest dose,
absolute testes and spleen weights were decreased in males. When analyzed relative
to body weight, brain, kidney, lung and testes in males and brain and kidney in females
were significantly increased at the highest dose compared with the control value. Based
on relatively minor changes at 100 mg/L of monochloramine, including <10% depression
of body weight and the greater changes at 200 mg/L (19-25% decreased body weight),
the 100 mg/L dose (12.9 mg/kg/day) can be identified as a NOAEL in this study.
No compound-related gross or microscopic lesions were observed, and no target
tissues were identified in the monochloramine-treated animals.
Exon et al. (1987) administered monochloramine at 0, 9, 19 and 38 ppm (mg/L) in
the drinking water of male Sprague-Dawley rats (12/sex/dose) from weaning to 12 weeks
of age (9 weeks of exposure). Based on reference body weight and water consumption
values for subchronic exposure (U.S. EPA, 1986), the corresponding intake of
monochloramine was 0, 1.3, 2.6 and 5.3 mg/kg/day. Following treatment, the test animals
were assessed for immune competence. Parameters of immunity measured were spleen
and thymus weights, antibody production, delayed-type hypersensitivity (DTH) reactions,
natural killer cell (NKC) cytotoxicity, oxidative metabolism response and phagocytosis by
macrophages and production of two immunoregulatory cytokines, interleukin 2 (112) and
prostaglandin E2 (PGE2). Significant (p<0.05) reduction of spleen weights (38 ppm),
CLORAMIN.5 V-13 03/08/94
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decreased antibody synthesis (9 and 19 ppm) and augmented PGE2 production (19 and
38 ppm) were reported. The authors state that these results indicate that B lymphocyte,
as well as macrophage function may be adversely affected. While monochloramine
appears to exert immunotoxic effects, the biologic importance of effects on the immune
system as a factor in the toxicity of monochloramines is not clear. In addition, there is a
lack of correlation among various endpoints examined, as well as deficiencies in some
of the methodologies (i.e., the use of the ELISA test for antibody analysis). Based on
these considerations, a NOAEL or LOAEL cannot be determined from this study.
Since pigeons may be susceptible to cardiovascular disease, Revis et al. (1986)
investigated the relationship of monochloramine to plasma cholesterol and thyroid levels.
Groups of 12 male white carneau pigeons (age 3-4 months) were fed altered diets and
drinking water containing either 0 (deionized water), 2 or 15 ppm monochloramine ad
libitum for 3 months. The treatment diets consisted of either (A) a diet reduced to 0.35%
calcium (80% of the minimal daily requirement for a pigeon) i.e., a normal diet, or (B) a
diet reduced to 0.35% calcium with the addition of 10% lard and 0.5% cholesterol, i.e.,
a high cholesterol diet. The controls were given either diet and deionized water ad
libitum. Treated drinking water was prepared and changed daily. At 1-month intervals
blood samples were collected and plasma levels of cholesterol and T4 were determined.
The study reported that plasma T4 levels were significantly decreased in pigeons fed a
normal diet (A) or high cholesterol diet (B) and drinking water containing 15 ppm
monochloramine. Following a 3-month exposure to diet B and receiving either deionized
water or water containing 15 ppm monochloramine, plasma cholesterol levels were
CLORAMIN.5 V-14 03/08/94
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1266±172 and 2049±212 mg/dL, respectively, a difference of 783 mg/dL. Significant
increases in plasma cholesterol were also observed in pigeons at the 2 ppm level
compared with deionized water and when both groups were fed only the calcium-deficient
diet (A). No significant changes were observed in the comparative 15 ppm diet (A) group.
T4 levels were not significantly altered when 2 ppm monochloramine and diet (A) were
administered. There was no clear dose-response effect for plasma cholesterol observed
in any of the treatment groups. Thus, factors associated with the effects of
monochloramine on plasma cholesterol are not known. The authors suggested that the
changes in plasma cholesterol may be mediated by products formed when
monochloramine reacts with organic matter in the upper Gl tract. Pigeons may or may
not mimic humans in thyroid function, so the significance of effects in the pigeons
relationship to humans is unknown. Also, pigeons were maintained on a calcium deficient
diet in order to depict an average human diet. It is not known if pigeons metabolize
calcium in the same manner as humans.
Other Short-term Effects
Robinson et al. (1986) treated female Sencar mice (5/dose) with aqueous solutions
of monochloramine by whole-body exposure (except head) for a 10-minute period for 4
days to assess hyperplastic effects. The backs of the animals were shaved 3 days before
treatment of 1, 10, 100 and 1000 mg/L of monochloramine. Animals were sacrificed the
day following the last treatment and skin thickness measured. TPA
(12-o-tetradecanoylphorbol13-acetate) was applied at a dose of 1.0 //g in 0.2 ml
acetone/mouse to the positive control mice. Epidermal thickness on the fifth experimental
CLORAMIN.5 V-15 03/08/94
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day measured ~32.8 //m in the positive controls. The epidermal layer in control animals
given water measured ~15.4//m. Exposure to 1, 10, 100 and 1000 mg/Lmonochloramine
decreased epidermal thickness (-14.0, 14.4, 13.1 and 13.6, respectively); however, this
was not significant when compared with controls. In addition, cell counts were not
increased at any of the doses when compared with controls.
Flour bleached with trichloramine administered in the diet has been shown to
produce "canine hysteria" or "running fits" in dogs. However, one study suggests that this
is a species-specific phenomenon for dogs (Mellanby, 1946; Silver et al., 1947a,b,c;
Newell et al., 1947) and does not affect humans (Pollock, 1949). Trichloramine is formed
in waters at high chlorine-to-ammonia ratio concentrations and at lower pHs than normally
found in drinking water.
Long-Term Exposure
Abdel-Rahman et al. (1984) conducted a study to investigate the toxicity of NH2CI
in drinking water (see Table V-2). Groups of four male Sprague-Dawley rats drank either
0, 1, 10 or 100 mg/L NH2CI in deionized water daily for <12 months. The levels of
chlorine, dichloramine and trichloramine in the NH2CI solution were <1%, <1% and 0%,
respectively, of the total NH2CI added. Food was available ad libitum and body weight
was measured during the treatment. Cardiac puncture and collection of heparinized
blood was performed at 2, 4, 6, 8, 10 and 12 months after treatment. Hematologic
parameters, blood glutathione (GSH) levels and osmotic fragility were monitored
throughout the treatment period. 3H-Thymidine incorporation was studied in liver, kidney,
CLORAMIN.5 V-16 03/08/94
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testes, intestinal mucosa and spleen after 3 months of treatment. Increased incorporation
of 3H-thymidine was observed in the kidney and spleen at 1 and 10 mg/L and in testes at
100 mg/L at 3 months; other time periods were not examined. Body weights were
significatnly reduced after 3 months of treatment at 100 mg/L and remained lower than
controls throughout the experiment. Blood levels of chloroform monitored during the
study were no different than in untreated rats. Results varied over the 12-month study
period, but at 6 and 12 months after initiation of the study statistically lower GSH levels
were observed at all doses. After 3 months of treatment significant decreases in RBC
count and hematocrit were observed at the higher dose levels. A reduction in hemoglobin
concentration and mean corpuscular hemoglobin occurred at 100 mg/L after 10 months
of treatment. The decrease in blood GSH could be due to a protective role by GSH
against damage caused by oxidants. The health significance of these types of changes
is uncertain. Furthermore, results were analyzed by inappropriate statistical methods and
a number of "significant" changes in hematologic parameters relative to control values
were identified that had no consistent relationship with dose and were not observed
consistently throughout the period of exposure.
The NTP (1990) conducted two studies to determine the potential chronic toxicity
or carcinogenic activity of chloraminated drinking water. In the first study F344/N
(70/sex/dose) rats were administered chloramine for 2 years at doses of 0, 50, 100 and
200 ppm in pH 9 buffered charcoal filtered deionized drinking water. These doses were
calculated on the basis of a time-weighted average to be 0, 2.1, 4.8 and 8.7 mg/kg/day
for male rats and 0, 2.8, 5.3 and 9.5 mg/kg/day for female rats. There was a dose-related
CLORAMIN.5 V-17 03/08/94
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decrease in the amount of water consumed by both sexes; this decrease was noted
during the first week and continued throughout the study. Food consumption of treated
rats was the same as the controls with males consuming more. Mean body weights of
200 ppm dosed rats were lower than their control groups. However, mean body weights
of rats receiving chloraminated drinking water were within 10% of controls until week 97
for females and week 101 for males. At the end of the study all animals were given a
complete histopathologic examination. The authors determined that no clinical changes
were attributable to chloraminated drinking water. Survival of rats receiving
chloraminated drinking water was not significantly different than controls except that, for
the 50 ppm dose groups, survival was greater than that of controls.
Groups of rats (10/sex/group) were predestined for incremental sacrifice and
evaluation at 14 and 66 weeks. At these times a complete hematologic examination and
necropsy were performed on all animals and histopathologic examinations were
performed on all control and high-dose rats. In F344/N rats at the 14-week evaluation the
mean body weight at necropsy of 200 ppm dosed males was significantly lower (p<0.01)
than that of controls while mean body weights of low- and mid-dose males was similar to
that of controls. At the 66-week evaluation, there was a dose-related decrease in body
weights of chloraminated treated male rats. The mean body weights of high-dose male
and female rats were 94 and 92% of controls, respectively. Slight decreases (p<0.05) in
the liver and kidney weight in high-dose males and kidney-to-body weight ratios in male
and female high-dose rats were related to the lower body weights in these groups.
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The second NTP (1990) study was a 2-year study to determine the potential
chronic toxicity or carcinogenic activity of chloraminated drinking water. B6C3F1 mice
(70/sex/dose) were administered chloramine at doses of 0, 50, 100 and 200 ppm in pH
9 buffered charcoal filtered deionized drinking water. These doses were calculated based
on a time-weighted average to be 0, 5.0, 8.9 and 15.9 mg/kg/day for male mice and 0,
4.9, 9.0 and 17.2 mg/kg/day for female mice. There was a dose-related decrease in the
amount of water consumed by both sexes; this decrease was noted during the first week
and continued throughout the study. Male and female mice had similar food
consumptions as controls except the 200 ppm dose females that were slightly lower than
controls. There was a dose-related decrease in mean body weights of dosed male and
female mice throughout the study. Mean body weights of high-dose male mice were
10-22% lower than their control group after week 37 and the body weights of high-dose
female mice were 10-35% lower after week 8. Survival of mice receiving chloraminated
drinking water was not significantly different than controls. All animals were given a
complete histopathologic examination at the end of the study. The authors did not
attribute any clinical findings to the consumption of chloraminated drinking water.
Groups of mice (10/sex/group) were predestined for incremental sacrifice and
evaluation at 15 and 66 weeks. A complete hematologic examination and necropsy were
performed on all animals and a histologic examination was performed on all controls and
high-dose rats at the incremental sacrifices. At 15 weeks the group mean body weights
of high-dose male and female mice were 91 and 84% of controls, respectively. At the
66-week evaluation, the differences in body weight between the high-dose mice and
CLORAMIN.5 V-19 03/08/94
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controls was 87% for females but 91% of controls for mid- and high-dose males.
Decreases in liver weights and increases in brain- or kidney-to-body weight ratios
observed in high-dose mice at 15 or 66 weeks were attributable, according to NTP, to the
lower body weights in these groups.
Because specific organic N-chloramines have not yet been identified in chlorinated
water or in vivo on ingestion of chlorinated water, Bempong and Scully (1980a) studied
N-chloropiperidine (NCR), a model for the health effects of organic chloramines as a class
of compounds. They found it was toxic to C57BI/J6 mice after i.p. injection. Groups of
10 female and 10 male mice were injected with either 50, 100, 200, 300 or 400 mg/kg bw
of NCR. The animals were observed daily for toxic effects, food intake and body weight.
The LD50 for NCR was slightly greater than 300 mg/kg as presented by the available data.
However, the toxicity of NCR was increased when allowed to stand at room temperature
in an aqueous solution, indicating that the degradation products are more toxic. The
toxicity of the chloramine was less than that of the parent amine, but the toxicity of
aqueous solutions of the chloramine increased on standing at room temperature for 24
hours or more.
Developmental Toxicitv
Abdel-Rahman et al. (1982) investigated the effects of monochloramine
administered in drinking water to adult female Sprague-Dawley rats. Six animals/group
were administered 0, 1, 10 or 100 mg/L NH2CI daily in the drinking water both 2.5 months
before and throughout gestation. Sacrifice of rats on the 20th day of gestation was
CLORAMIN.5 V-20 03/08/94
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performed for soft-tissue and skeletal examination of the progeny. Monochloramine did
not produce any significant changes in rat fetuses at any dose level; in fact, there was a
slight increase in fetal weight in all NH2CI groups compared with controls.
Reproductive Toxicitv
Eight- to 10-week-old hybrid male mice (C57BI/J6 x DBA2) and 10-week-old inbred
male golden Syrian hamsters (numbers not specified) were given daily exposures (ad
libitum) to 400 mg/kg/day NCR in 0.001 % ethanol for 100 days. Significant increases of
abnormal sperm (either tail or head anomalies) were observed in both hamsters and mice
after 5 weeks of exposure (Bempong and Scully, 1983). Another significant result among
NCR-treated hamsters was reduced sperm count after 70 days of daily exposure. Greater
than 80% of the hamster population was classified as sterile based on their reproductive
performance. A few (number not provided) were reported as having testicular atrophy
after 16 weeks of daily exposure. Gradual reductions in percent abnormal sperm were
observed after the 75th day of treatment in hamsters; however, sperm counts did not
increase. In mice the level of sperm abnormalities remained the same for more than 5
weeks except there was a reduction in sperm tail damage with concomitant increased
anomalous sperm heads. Sperm count was also reduced in mice but did not affect
percent anomalous sperm. The data support the concept that species and strain
differences in sperm abnormalities are suggestive of genotoxic control of sperm
development. In addition, the investigators propose that since increased levels of sperm
abnormalities were observed >5 weeks after treatment, the most sensitive cells were early
CLORAMIN.5 V-21 03/08/94
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spermatocytes > differentiating spermatogonia > premeiotic cells (Bempong and Scully,
1983).
Fertility studies were also undertaken wherein male mice and male hamsters were
caged with two respective females after 1 week of continuous exposure as described
previously (Bempong and Scully, 1983). The duration of each mating period was 7 days.
Six treatment groups of 15-25 females were constituted in the following manner:
1) untreated (UT) female x UT male; 2) UT female x ethylmethane sulfonate (EMS)-
treated male; 3) NCR-treated female x NCR-treated male; 4) UT female x NCR male;
5) NCR female x UT male; and 6) piperidine-treated female x piperidine-treated male.
EMS served as a positive control and piperidine was included to ascertain the extent to
which effects could be ascribed to the monochloramine group. Reduced fertility indices
were observed on week 5 and subsequently for all matings of NCR-treated animals,
except when females only were exposed (group 5). To obtain data on potential
embryotoxic effects of NCR, five pregnant animals/group were allowed to reach term and
the offspring were counted. Numbers of offspring/pregnancy were variable for the
NCR-treated animals, but was lowest for the group (3) wherein both males and females
received NCR. Treatment with NCR increased the number of uterine moles, particularly
when the female was exposed. Implant frequency was reduced in all NCR treatment
groups; exposure of both males and females resulted in a significantly greater reduction
than treatment of either single parent (Bempong and Scully, 1983).
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In a more recent study, Carlton et al. (1986) administered monochloramine by
gavage at doses of 0, 2.5, 5.0 and 10 mg/kg bw/day to male (12/dose) and female
(24/dose) Long-Evans rats for 66-76 days. Males were treated for 56 days and females
for 14 days before mating. The administration was continued during the 10-day mating
trial period, and thereafter females were gavaged with monochloramine daily throughout
gestation and lactation. Males were necropsied at the end of the mating period and bled
for complete blood counts and thyroid hormone levels. Their sperm was examined for
normalcy and histologic examinations were performed on the reproductive tract including
the testis, epididymus, prostate and seminal vesicles. Dams and some offspring were
necropsied at weaning 21 days after birth of the pups. At necropsy dams were bled for
complete blood counts and thyroid hormone levels. In addition, the reproductive tract was
removed for histopathologic examination. Other offspring were administered chloramines
after weaning until they were 28-40 days old; these pups were evaluated for vaginal
patency and thyroid hormone levels. The authors reported no significant (statistical
analysis not provided) differences between control and exposed rats in fertility, viability,
litter size, day of eye opening, or average day of vaginal patency (day 31.8-32.6). There
were no alterations in sperm count, direct progressive sperm movement, percent mobility,
or sperm morphologic characteristics in adult males. Weights of male and female
reproductive organs were not significantly different among test and control groups, and
there were no significant morbid anatomic changes evident on tissue examination. There
were no signs of toxicity, changes in hematologic parameters, or body weight suppression
in adult rats of either sex at any dose level. The mean weight of the pups was unchanged
from that of control litters. Based on these data, a NOAEL of 10 mg/kg/day is identified.
CLORAMIN.5 V-23 03/08/94
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Mutaqenicitv
Lu Shih and Lederberg (1976) showed chloramine to be weakly mutagenic at the
trpC locus of Bacillus subtilis. They further investigated the biologic and physical effects
of chloramine on B. subtilis after treatment of the bacterial cells or the bacterial DMA.
Both resulted in single-strand breaks and a few double-strand scissions at higher
chloramine doses, with loss of DMA-transforming activity. Since some DMA
repair-defective mutants seem to be more sensitive to chloramine, it would appear that
chloramine's bacteriotoxic effect may be due in part to its ability to damage DMA.
Thomas et al. (1987) reported that monochloramine (40 //m) slightly increased the
number of revertant colonies over untreated control levels in assays employing
Salmonella typhimurium (TA97, TA100 and TA102). Positive controls were run
concurrently. Lipophilic dichloramines were the most active mutagens in this study.
Fetner (1962) found that distilled water containing monochloramine produced
chromosome breakage when used for soaking Vicia faba seeds. A 1-hour exposure to
10"4 M monochloramine produced 24% abnormal anaphases (including chromosomal
bridges and fragments) in the embryonic Vicia roots. As there was no visible browning
of the plant embryos, Fetner concluded that monochloramine produced chromosome
breakage at a concentration that exhibited little evidence of tissue damage.
Meier et al. (1985) evaluated the ability of monochloramine and other oxidants to
induce chromosomal aberrations (5/sex/dose) and micronuclei in the bone marrow of
CLORAMIN.5 V-24 03/08/94
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CD-1 mice (4/sex/dose), and sperm-head abnormalities in male B6C3F1 mice (10/dose).
Monochloramine administered by gavage at concentrations of 40, 100 and 200 mg/L
showed no evidence of any significant effects in any of the tests.
Bempong and Scully (1980a) found that the organic chloramine, NCR, was weakly
mutagenic in a preincubation Salmonella reverse mutation assay (Ames test). Mutagenic
activity was detected in the absence of exogenous S9 activation in strains TA1535 and
TA100. The toxicity of the chloramine appeared to overshadow its mutagenic property
at doses >64 //g/plate with a concomitant reduction in the number of revertants/plate.
N-Chlorodiethylamine, on the other hand, was highly toxic but nonmutagenic (Scully et
al., 1983).
The effect of increasing concentrations of NCR on the toxicity, mitotic indices and
chromosomal aberrations were studied in Chinese hamster cells (CHO). CHO cells
exposed to NCR (0.4-2.4 //g/L) for 3 hours and analyzed during a 96-hour period
proliferate less rapidly than control cells and as the concentration of NCR increased,
mitotic indices were reduced and the peaks of mitotic activity shifted resulting in
chromosome separation and nuclear distribution. CHO cells were exposed to increasing
concentrations of NCR (0, 0.4, 0.8 and 1.2 //g/ml_) for a 3-hour period, harvested 48 hours
after treatment and analyzed for chromatid aberrations (breaks, exchanges, fragments,
ring and centric errors). The increases in the frequency of aberrations were proportional
to the increase in NCR concentration with breaks accounting for the major proportion of
aberrations. Chromosomal aberrations were further investigated after a 3-hour incubation
CLORAMIN.5 V-25 03/08/94
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and a 60-hour recovery period to allow for delayed mitotic activities, and increased
populations of metaphases. A nonlinear dose-dependent increase in bridges, dicentrics,
exchanges, fragments and ring configurations occurred (Bempong and Scully, 1980b;
Bempong etal., 1981, 1986; Scully and Bempong, 1982).
Chloramine T (sodium p-toluenesulfonylchloramide) tested negative in a
Salmonella reverse mutation assay (Ames test) in the presence of S-9 in strains TA98,
TA100, TA1535 and TA1538 (Anderson and Styles, 1978), and negative in strains TA98,
TA100, TA1535, TA1537 and TA1538 with and without S-9 fractions (Gocke et al., 1981).
Weitberg (1987) tested the ability of chloramine T (sodium p-toluene-
sulfonylchloramide) to produce SCEs in cultured CHO cells. A significant
dose-dependent increase was observed at 10"5 to 10"7 M and at 10"8 M when compared
with controls. This effect was significantly diminished when cells were treated with
methionine, a thioether, which reduces N-CI back to the parent amine.
Sussmuth (1982) examined the mutagenicity of chlorinated solutions of several
amino acids using several strains of bacteria including the recombination-deficient strain
of B. M45 (red), Es. coll P3478 (polA1), and three histidine auxotrophic strains of S.
himurium. Chlorinated solutions of methionine, tyrosine, phenylalanine and glycine were
mutagenic in more than one strain of bacteria. The active mutagens in Sussmuth's
solutions were not defined clearly. Since the amino acids react rapidly in chlorinated
water to form N-chloramino acids, the first group of compounds to form would be the
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chloramine acids. These may be the active mutagens; however, because some of the
chloramine acids are labile, their degradation by-products may be the active mutagens.
An Ames Salmonella assay was performed using concentrated drinking water
samples collected at a pilot scale drinking water treatment plant disinfected with
monochloramine (Miller et al., 1986). The reverse osmosis concentrates ranged from
0.025-1.0 ml. The mutagenic response was negative for strains TA98 or TA100 when
tested with or without the metabolic activation system S9.
Carcinoqenicitv
Several initiation-promotion studies have been conducted. Although these studies
are not useful for quantitative risk assessment, they may support findings in bioassay
studies.
Settled, coagulated and sand-filtered Ohio River water was treated with
monochloramine at 3 mg/L (Bull, 1980; Bull et al., 1982). The residual disinfectant was
dissipated within 48 hours. The water was then concentrated by reverse osmosis and the
concentrate subjected to a mouse skin initiation-promotion assay in SENCAR mice. A
total of 1.5 ml of the reverse osmosis concentrate was applied to the backs of the mice
in six doses (0.25 ml each) over a 2-week period. This was followed by thrice weekly
applications of 2.5 ug of the promoting agent phorbol myristate acetate in acetone for 18
weeks. Nondisinfected water concentrates produced no tumors, while concentrated
monochloramine-treated water samples induced neoplasms in 5/25 animals. Lesions
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included papillomas (1/25), squamous cell carcinomas (2/25) and lung adenomas (5/25).
Overall chi-square analysis comparing numbers of animals with systemic tumors indicated
that the increase was not significant. Subsequent to the results published by Bull (1980),
five additional experiments were undertaken using new samples of disinfected waters.
Results of two of these were published by Bull et al. (1982). In initiation-promotion
assays of concentrated monochloramine-treated water samples at a dose of 1.0 //g for
20 weeks, 23% and 15% of animals developed papillomas respective to the two studies.
For these assays, however, papillomas were also observed in 15 and 13% of mice treated
with saline and in 20% of mice exposed to nondisinfected water concentrates. The
authors pointed out that the average tumor incidence of all the monochloramine
concentrated water-exposed animals is almost twice that of the simultaneous controls and
is more than double the historical control response. It is likely that the initiating activity
of the water concentrates is not due to the monochloramine per se, but rather to organic
chloramines or other materials formed as by-products of the disinfection process. The
authors noted that as the reverse osmosis process used to produce the water
concentrates does not concentrate low molecular weight organics, trihalomethanes were
not included in these test samples.
Herren-Freund and Pereira (1986) tested chloramines for initiation-promotion
activity in rat liver by using the rat liver foci bioassay. The endpoint of the assay is the
occurrence of altered foci of hepatocytes. This assay uses an increased incidence of
Y-glutamyltranspeptidase positive foci (GGT foci) as an indicator of carcinogenicity since
initiation-promotion bioassays using GGT foci have detected both hepatic and nonhepatic
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carcinogens. Male rats (authors do not clarify if Fischer 344 or Sprague-Dawley or both
were used) were administered chloramine (route not specified) 14.75 mg/kg bw 24 hours
following a 2/3 partial hepatectomy. Seven days after initiation, promotion by 500 ppm
phenobarbital in drinking water was begun. After 10 weeks of exposure to phenobarbital,
the rats were removed from the exposure to the promoter for 1 week and then sacrificed.
Positive and negative controls were run concurrently. The authors reported that under
conditions of this study chloramine did not initiate GGTfoci.
Similar results were reported by Miller et al. (1986) when rats (10/group) were
administered concentrated drinking water samples collected at a pilot scale drinking water
treatment plant using monochloramine for disinfection purposes, which were concentrated
(4000x or 2000x the residual concentration of 2.1 mg/L) by the macroreticular resin
process. Rats were partially (2/3) hepatectomized on day 0 and treated 24 hours later
with chloramines for 1 week. On day 7 the rats received 500 ppm sodium phenobarbital
in their drinking water for 56 days. Chloramine-treated water did not initiate the incidence
of GGT foci above that of the vehicle control group.
One of the toxicologic tests used for determining tumor-initiating potential of
concentrated drinking water samples is the mouse lung adenoma assay. Concentrated
drinking water samples from a pilot treatment plant using monochloramine for disinfectant
purposes was administered to 6-week-old strain A mice in drinking water samples
concentrated (4000x and 2000x) by the macroreticular resin process (Miller et al., 1986).
The residual chloramine level before concentration was 2.1 mg/L. Both positive and
CLORAMIN.5 V-29 03/08/94
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negative controls were run concurrently. The vehicle controls had 0.1 adenoma/animal
and the treated animals had <0.20 adenoma/animal, indicating there was no
treatment-related effect.
The National Toxicology Program (NTP, 1990) conducted a 2-year study of
chloraminated drinking water in F344/N rats at doses of 0, 50, 100 and 200 ppm. This
dose was calculated to beO, 2.1, 4.8 and 8.7 mg/kg/day for males and 0, 2.8, 5.3 and 9.5
mg/kg/day for females. The purpose of this study was to determine the potential chronic
toxicity or carcinogenic activity of chloraminated water. The water from all treatment
groups was charcoal filtered and deionized to remove organic substances and other
residues.
According to the authors there were no neoplasms or nonneoplastic lesions in rats
of the 2-year study that were attributable to the consumption of chloraminated drinking
water. The incidence of mononuclear cell leukemia was slightly increased relative to that
of controls in the mid- and high-dose range. Although female rats receiving
chloraminated water had significantly greater leukemia incidence in the high-dose group
than the controls, there was no clear dose response and the incidence of leukemia in the
female control group (16%) was less than the untreated historical controls (25%). There
was also no evidence of a reduced latency in the occurrence of leukemia in female rats
consuming chloraminated water. However, because of the marginal statistical
significance the increased incidence of leukemia was considered equivocal evidence of
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carcinogenic activity in female rats. There was no evidence of carcinogenic activity of
chloraminated drinking water for male rats.
The National Toxicology Program (NTP, 1990) conducted another 2-year study of
chloraminated drinking water in B6C3F1 mice at doses of 0, 50, 100 and 200 ppm. These
doses were calculated to be 0, 5.0, 8.9 and 15.9 mg/kg/day for males and 0, 4.9, 9.0 and
17.2 mg/kg/day for females. The purpose of this study, as with the rat study, was to
determine the potential chronic toxicity or carcinogenic activity of chloraminated water.
The water from all treatment groups was charcoal filtered and deionized to remove
organic substances and other residues.
Renal tubular cell adenomas occurred in two high-dose and one low-dose male
mice; focal tubule hyperplasia was seen in one low-dose, three mid-dose and one
high-dose male. Hyperplasia was also observed in male control animals. There were no
other lesions in the kidneys of male or female mice receiving chloraminated water.
Histopathology evaluation of the kidneys failed to identify any difference in the incidence
or severity of renal tubular atrophy, dilation, regeneration, focal mineralization, or protein
casts. Step sections of the kidneys of male mice did not provide supportive evidence of
a chemical effect. There were no neoplasms or nonneoplastic lesions in mice in the
2-year study that were attributable to the consumption of chloraminated drinking water,
according to the authors. There was no evidence of carcinogenic activity of
chloraminated drinking water for male or female mice.
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Summary and Discussion
Several short-term studies showed no observed adverse hematologic effects in
mice, rats and monkeys (Moore et al., 1980; Bercz et al., 1982; Bull, 1980). In A/J mice
administered chloramine solutions between 2.5 and 200 mg/L (pH 8.9) for 30 days, the
only observable effect was a slight increase in hematocrit (Moore et al., 1980). In another
study of similar duration (45 days) rats treated with 10, 50 or 100 mg/L monochloramines
experienced a decrease in the amount of methemoglobin present in the blood, the
opposite of what was expected (Bull, 1980). Monochloramine in drinking water for 6
weeks at 100 mg/L resulted in no detectable effects on 18 hematologic tests of 12 African
Green monkeys (Bercz et al., 1982). The results from the three studies described above
are somewhat conflicting in that similar dose levels and duration produced different
changes in hematologic parameters (i.e., increased hematocrit, decreased
methemoglobin in blood) while others produced no detectable effects. In one longer-term
study in which blood glutathione levels were decreased after 12 months of exposure to
monochloramine, there was a lack of dose- and time-dependency in the observed effects
(Abdel-Rahman et al., 1984).
One possible reason for the varying observations between laboratories may be the
conditions under which the solutions of monochloramine are generated. It is understood
that, as the pH of a solution of monochloramine is lowered, some of the monochloramine
is converted to dichloramine. To avoid this, solutions are frequently buffered to pH 8 to
avoid dichloramine formation. However, in doing this, buffer concentrations may be
exerting an unusually strong effect on the reactions of disinfectants in the stomach where
CLORAMIN.5 V-32 03/08/94
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enzyme activity is usually greatest (lower pH). The pH and buffer concentrations of the
monochloramine solutions used in toxicity studies may vary widely from laboratory to
laboratory.
A draft report by GSRI (1981) investigated the effects in rats and mice
administered drinking water containing 0, 25, 50, 100, 200 or 400 mg/L monochloramine
for 91 days. Using water and food consumption data provided in the report,
corresponding respective doses were 2.5, 4.9, 10.2, 18.8 and 40.7 mg/kg/day for male
rats; 3.8, 6.5, 13.8, 26.6 and 53.9 mg/kg/day for female rats; 4.9, 8.3, 14.5, 31.3 and 50.7
mg/kg/day for male mice; and 7.7, 12.1, 21.9, 34.6 and 88.5 mg/kg/day for female mice.
Animals experienced decreased body weight gain and liver damage at 200 and 400 mg/L
monochloramine in rats and 100, 200 and 400 mg/L monochloramine in mice. Based on
these observations, the investigators suggested a NOEL of 50 mg/L or ~8.3 mg/kg/day
monochloramine.
In addition monochloramine at 9, 19 and 38 ppm in drinking water may exert
immunotoxic effects to male Sprague-Dawley rats. Significant (p<0.05) reduction of
spleen weight (38 ppm), decreased antibody synthesis (9 and 19 ppm) and augmented
PGE2 production (19 and 38 ppm) were reported (Exon et al., 1987). The biologic
importance of effects on the immune system as a factor in the toxicity of monochloramines
is not clear.
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Daniel et al. (1990) investigated the effects of monochloramine administered for
90 days in the drinking water of Sprague-Dawley rats at 0, 25, 50, 100 and 200 mg/L.
Based on food and water consumption data, doses were calculated to be 0, 1.8, 3.4, 5.8
and 9.0 mg/kg/day for males and 0, 2.6, 4.3, 7.7 and 12.1 mg/kg/day for females. For
males and females the average weight gain at 200 mg/L dose was 51% of controls.
Reductions in absolute and relative organ weights occurred at 200 mg/L. The authors
concluded that 100 mg/L is considered the NOAEL.
Daniel et al. (1991) also investigated the effects of monochloramine administered
for 90 days in the drinking water of B6C3F1 mice at 0, 12.5, 25, 50, 100 and 200 mg/L.
Based on food and water consumption data, doses were calculated to be 0, 2.5, 5.0, 8.6,
11.1 and 15.6 mg/kg/day for males and 0, 2.8, 5.3, 9.2, 12.9 and 15.8 mg/kg/day for
females. Based on decreases in liver, heart and lung weights in males and liver, heart
and spleen weights in females at 100 mg/L and decreased weight gain and food
consumption in both sexes and reduced water consumption in females, the 50 mg/L
concentration is considered the NOAEL.
NTP (1990) conducted two studies for 2 years (F344/N rats and B6C3F1 mice) to
determine the potential chronic toxicity for chloraminated drinking water. Chloramine was
administered at doses of 0, 50, 100 and 200 ppm. These doses were calculated to be 0,
2.1, 4.8 and 8.7 mg/kg/day for male rats and 0, 2.8, 5.3 and 9.5 mg/kg/day for female rats,
and 0, 5.0, 8.9 and 15.9 mg/kg/day for male mice and 0, 4.9, 9.0 and 17.2 mg/kg/day for
female mice. Mean body weights of 200 ppm dosed rats were lower than controls. Slight
CLORAMIN.5 V-34 03/08/94
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decreases in organ-to-body weight ratios in high-dose rats were related to the lower body
weights. There was a dose-related decrease in mean body weights of dosed male and
female mice throughout the study. No clinical changes were attributable to chloraminated
drinking water in the rat or mouse study.
Studies suggest that flour treated with trichloramine may cause "canine hysteria"
but one study suggests that this is a species-specific phenomenon in dogs (Mellanby,
1946; Silver et al., 1947a,b,c; Newell etal., 1947) and does not affect humans (Pollock,
1949).
Few studies have been conducted on organic N-chloramines. Studies conducted
with N-chloropiperidine suggest that organic chloramines may be toxic, but determination
of their environmental significance will rely on identification of specific organic
N-chloramines actually formed on chlorination of natural waters (Bempong and Scully,
1980a).
Monochloramine was not teratogenic in mature female Sprague-Dawley rats
exposed to 1, 10 or 100 mg/L in drinking water nor did 40, 100 and 200 mg/L solutions
induce sperm-head anomalies in B6C3F1 mice (Abdel-Rahman et al., 1982).
In a more recent study there were no significant differences in fertility, viability,
litter size, day of eye opening or day of vaginal potency between control and exposed
Long-Evans rats given >10 mg/kg chloramines. There were no alterations in sperm
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count, direct progressive sperm movement, percent mobility or sperm morphologic
characteristics in adult males. Weights of male and female reproductive organs were not
significantly different among test and control groups.
Data on the mutagenicity of chloramines are inconclusive. A number of studies
suggest that monochloramine and some organic N-chloramines and N-chloramino acids
may be weakly mutagenic. Monochloramine has been found to be marginally mutagenic
in Bacillus subtilis bacteria (Lu Shih and Lederberg, 1976) and in Vicia faba plant seeds
(Fetner, 1962). It was responsible for cellular hypertrophy, increased mitotic figures and
bizarre chromatin patterns in B6C3F1 mice exposed to 200 and 400 mg/L in drinking
water (Wolfe et al., 1984). Thomas et al. (1987) reported monochloramine (40 um)
slightly increased the number of revertant colonies over untreated control levels in assays
employing Salmonella typhimurium (TA97, TA100 and TA102). In addition,
monochloramine showed no evidence of producing spermhead abnormalities in B6C3F1
mice or inducing chromosomal aberrations and micronuclei in bone marrow of CD-1 mice
(Meier etal., 1985).
The organic chloramine, N-chloropiperidine, was found to be marginally mutagenic
in the reverse mutation plate incorporation assay (Ames test). It was cytotoxic and
cytostatic in CHO cells and produced chromosomal aberrations, the frequency of which
was proportional to the concentration of the compound (Bempong and Scully, 1980b;
Bempong et al., 1981, 1986; Scully and Bempong, 1982). It produced SCEs in CHO
cells, but not in baby hamster kidney cells (Scully et al., 1983). The analogous
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chloramine, N-chlorodiethylamine, was more toxic but nonmutagenic (Scully et al., 1983).
When the synthetic N-CI compound chloramine T (sodium p-toluenesulfonylchloramide)
was tested, SCEs were significantly increased in a dose-dependent manner in CHO cells
(Weitberg, 1987).
Data indicate that chloramine perse is not an initiator (Bull et al., 1982; Bull, 1980;
Herren-Freund and Pereira, 1986). Two-year studies by the National Toxicology Program
(NTP, 1990) have shown equivocal evidence of carcinogenic activity in female rats that
is due to the slightly increased incidence of mononuclear cell leukemia compared with
that of concurrent controls, although incidence in historical controls was higher. There
was no evidence of carcinogenic activity that isdue to chloraminated drinking water in
male rats or in male or female mice. Until the carcinogenicity of chloramines is further
delineated, it is categorized in group D, not classifiable as to human carcinogenicity,
meaning that there is inadequate human and animal evidence of carcinogenicity (U.S.
EPA, 1986).
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VI. HEALTH EFFECTS IN HUMANS
Introduction
Data on the human health effects observed following exposure to chloramines are
limited to a few clinical reports and epidemiologic studies. Several cases have been
reported where chloramine-T has caused allergic contact dermatitis. Clinical reports
indicate that acute chloramine exposure either by inhalation or ingestion results in
burning eyes and throat, dyspnea, coughing, nausea, reversible pulmonary damage and
allergic responses. One epidemiologic study looked at a population exposed to
chloramine in its drinking water and used the disease risk as a baseline for comparing
the risk in a population exposed to chlorine in its drinking water. These findings will be
addressed only briefly as they are not directly relevant to this document. Damage to red
blood cells has been observed in high risk subpopulations such as hemodialyzed
individuals.
Clinical Reports and Experiments
Lombardi et al. (1989) reported the work related case of a 38-year-old nurse who
developed subacute eczema that appeared to be caused by the use of chloramine-T as
an antiseptic disinfectant in the cleansing of burns. Only a few cases of sensitization to
chloramine-T have been reported and usually from nonoccupational contact even though
it has widespread use in Italy as an antiseptic, disinfectant and chemical reagent.
CLORAMIN.6 VI-1 03/08/94
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Laakso et al. (1982) reported the case of a 27-year-old woman who mixed ~500
ml of 4-5% household ammonia with the same amount of 5% sodium hypochlorite
bleach in a small, poorly ventilated bathroom. The vapors from the mixture caused
burning in the eyes and throat, dyspnea, coughing, nausea and vomiting. Inhalation of
the chloramine fumes resulted in pneumonitis, which did not result in permanent
pulmonary damage.
Beck (1983) reported the case of a 28-year-old nurse who suffered from rhinitis
when she was in contact with a chloramine solution. Chloramine is often used as an
antiseptic in the treatment of infected ulcers, and when the nurse was treated with a 2%
chloramine solution for a dental abscess, a severe angioneurotic edema developed.
Since this Type I reaction to chloramine was reported only once before, the authors
recommended that a prick test with chloramine be used before treatment of patients with
previous exposures.
A clinical study was conducted by Lubbers et al. (1981) to assess the safety of
chronically administered chlorine water disinfectants in humans. This study was
conducted in three parts over a 12-week period. Phase I and Phase II subjects were
male college students between the ages of 21 and 35 years of age, of normal body
weight and free of any history of disease or any medical or surgical condition that might
interfere with the absorption, excretion or metabolism of substances by the body. Phase
III subjects were glucose-6-phosphate dehydrogenase deficient, but were normal in all
other respects.
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Phase I consisted of an increasing dose tolerance analysis in which progressive
doses of chlorine were administered in water as chlorate, chlorine dioxide, chlorite,
chlorine and chloramine to six groups, 10 subjects/compound with 10 subjects in the
control group that received untreated water. Chloramine was given every 3 days for a
total of 15 days, at concentrations of 0.01, 1.0, 8.0, 18.0 and 24.0 mg/L (corresponding
doses of 0.14, 14, 110, 260 and 340 ug/kg/day assuming a body weight of 70 kg) in a
total volume of 1000 ml.
Phase II consisted of 60 subjects randomly assigned into six treatment groups of
10 subjects/group with one group receiving untreated water. A daily concentration of 5
mg/L chloramines in a volume of 500 ml of water was administered for 12 consecutive
weeks. Physicals and collection of blood and urine were conducted on a weekly basis
during the treatment period and for 8 weeks following.
Phase III was conducted on male students who were deficient in
glucose-6-phosphate dehydrogenase and were considered to be more susceptible to
oxidative stress. These students were given 5 mg/L of sodium chlorite daily in a volume
of 500 ml for 12 consecutive weeks.
Blood and urine samples were collected and physical exams were given, including
blood pressure measurements and taste tests. During all three phases of this study a
massive volume of raw data was acquired. No definitive finding of detrimental
physiologic impact was made in any of the three phases of this human investigation of
CLORAMIN.6 VI-3 03/08/94
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the relative safety and tolerance of oral chlorine disinfectant ingestion. Other possible
confounders such as diet and other sources of drinking water were not addressed. The
fact that there were no overt adverse health effects within the limitations of this study
suggests that ingestion of chloramine at these levels over a relatively short period of time
produces no toxicity in healthy adult males, but does not rule out the possibility that
longer treatment periods would result in any detectable adverse outcomes of biologic
significance.
Epidemiology Studies
There are no epidemiologic studies that have been designed to address
specifically the potential adverse effects of exposure to chloramines on human health.
The study of Zierler et al. (1986, 1988) was designed in response to earlier ecologic
studies that indicated that areas using chlorinated surface waters for drinking water were
associated with higher cancer mortality than areas using other sources of drinking water
and disinfectant practices. The addition of chlorine to surface water is known to form
organic micropollutants (Murphy and Craun, 1990). Specifically, the reaction of free
chlorine with naturally occurring precursor substances, primarily humic and fulvic acids,
produces a group of halogen-substituted single-carbon compounds known as
trihalomethanes (THMs) (Craun, 1988). The predominant THMs formed are chloroform
and bromodichloromethane. The process of chloramination produces only small
amounts of THMs. It was originally thought that the early findings of increased cancer
mortality associated with chlorinated drinking water might be due to exposure to the
THMs themselves. Zierler's study in Massachusetts was conducted to see if there was
CLORAMIN.6 VI-4 03/08/94
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a difference in cancer mortality among communities using chlorine compared with
communities using chloramine for disinfection. In this sense, the persons who were
exposed to chloraminated drinking water were used as controls with the assumption
being they would be much less exposed to chlorination by-products.
The first phase of this study (Zierler et al., 1986) looked at the patterns of cancer
mortality among 43 communities using either chlorine or chloramine since 1938. All
resident Massachusetts deaths among those 45 years and older and occurring during
1969-1983 were eligible for the study. Deaths were selected for inclusion if the last
residence listed on the death certificate was in a community using chlorine or chloramine
for disinfection. Cancers of the bladder, colon, kidney, pancreas, rectum, stomach, lung
and female breast were thought to be related to chlorinated by-products of disinfection
and were therefore treated as cases for a mortality odds ratio (MOR) analysis. Deaths
from cardiovascular and cerebravascular disease, chronic obstructive lung disease and
lymphatic cancer (N=214,988), considered to be unrelated to chlorinated by-products,
were used for comparison. In general, cancer mortality was not associated with type of
disinfectant in the MOR analysis. There was a slight association (MOR=1.05) for
chlorine use noted only with bladder cancer that increased slightly (MOR=1.15, 95%
confidence interval = 1.06-1.26) when lung cancer deaths were used for controls.
Standardized mortality ratio analysis of the data set were generally unremarkable. There
was a small increase in mortality (SMR=118, 95% confidence interval = 116-120) from
influenza and pneumonia in the chloraminated communities.
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The second phase of this mortality study (Zierler et al., 1988) was designed to
further pursue the bladder cancer findings in a more refined case-control analysis, which
included decedent next-of-kin interviews. Information on cigarette smoking, occupation
and residential history was obtained. The relationship with bladder cancer and
residence in communities using chlorine for disinfection again persisted. The crude
association was highest for lifetime residents of chlorinated drinking water communities
relative to lifetime residents of chloraminated drinking water communities (MOR=1.5,
95% confidence interval = 1.1-2.2) when lymphatic cancers were used for controls. The
MOR increased to 2.7 after controlling for the joint effects of age, gender, occupation and
pack-years of cigarette smoking. Although the exposure estimates in these analyses are
relatively crude, there is a consistent relationship, albeit small, with bladder cancer
mortality and years of exposure to chlorinated drinking water when compared with years
of exposure to chloraminated drinking water. The study was not designed to assess
adverse effects from exposure to chloramine, but rather considers the
chloramine-exposed participants as controls. At this time there are no epidemiologic
studies that have evaluated chloraminated drinking water as the exposure of interest and
not the control exposure.
Hiqh-Risk Subpopulations
Eaton et al. (1973) studied hemodialyzed patients (number of subjects not
reported) from three University of Minnesota hospitals. They observed significant
methemoglobinemia (>5% statistical analysis not provided) and Heinz body inclusions
in the red cells of the patients in two hospitals, which used unpurified tap water and
CLORAMIN.6 VI-6 03/08/94
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water purified with the reverse osmosis (RO) technique during dialysis. The RO
technique removes particulate matter and trace metals from the dialysis water.
Charcoal-filtered RO water that did not contain chloramines was used in the third
hospital, and did not result in significant methemoglobinemia. Serial observation of
several patients exposed to chloramines through dialysis suggested that the RBC
oxidant damage was cumulative. In addition to oxidant damage to the RBC, chloramines
inhibit the metabolic pathway used by these cells to prevent and repair such damage.
Thus, chloramine-containing dialysis water presented a severe threat of acute hemolytic
anemia to uremic patients undergoing dialysis.
Kjellstrand et al. (1974) also observed similar effects in patients undergoing
hemodialysis. The authors concluded that chloramines induced their deleterious effects
through the formation of methemoglobin from the direct oxidation of hemoglobin, and
through damage of the hexosemonophosphate shunt (HMPS), with which red cells
defend themselves against oxidant damage. The authors suggested that
chloramine-induced hemolysis may be reduced by the addition of ascorbic acid to the
treatment water.
Summary and Discussion
Few acute exposures to chloramines have been reported in the literature. Laakso
et al. (1982) reported pneumonitis as a result of inhalation of chloramine fumes from a
mixture of household ammonia and sodium hypochlorite. Permanent pulmonary damage
CLORAMIN.6 VI-7 03/08/94
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was not sustained. Beck (1983) reported a Type I allergic response after treatment with
a 2% chloramine solution for a dental abscess.
One experimental study on chloramines was located in the available literature.
Lubbers et al. (1981) reported no hematologic or abnormal effects following routine
clinical tests in individuals ingesting 0.01, 1.0, 8.0, 18.0 and 24.0 mg/L (0.14, 14, 110,
260 and 340 mg/kg/day, respectively) chloramines for 1 day in drinking water or 5 mg/L
chloramines for 12 weeks in drinking water. It is unknown, however, whether exposure
beyond this time would have any impact.
Although mentioned here for the sake of completeness, the work of Zierler et al.
(1986, 1988) should not be used as definitive evidence that chloramine exposure is not
associated with adverse health effects in humans. Although statistically stable because
they are based on large numbers of deaths, the community based SMR analyses
represent a relatively crude means of assessing the relationship of a specific cause of
death with a specific drinking water disinfection practice. If there is a positive
relationship between these variables, it may go undetected because of the attenuating
effects of random misclassification of the observed deaths into exposure categories. The
case-control study of bladder cancer was designed to use the chloramine exposure as
the comparison group and does not address the potential adverse effects from
chloramine, although often misinterpreted in this way.
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Hemodialyzed patients are a high-risk subpopulation for chloramine exposure
through chloraminated dialysis water. Chloramines cause oxidant damage to red blood
cells and inhibition of the hexose-monophosphate shunt with which red blood cells
defend themselves against oxidant damage (Eaton et al., 1973; Kjellstrand et al., 1974).
Thus, this high-risk group must also be considered when using monochloramine for
disinfection of water used in dialysis.
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VII. MECHANISMS OF TOXICITY
General Theories
The literature on the mechanism of inactivation of microorganisms by chloramines
is limited. Since low levels of inorganic chloramines are effective in inactivating bacteria,
Nusbaum (1952) proposed that the mechanism of action must essentially be the same as
that of hypochlorous acid on enzymes; that is, the chloramine molecules enter the
cytoplasm and interfere with enzymatic reations. Ingols et al. (1953) studied bactericidal
mechanisms of chlorine compounds on unspecified bacterial suspensions. They noted
that monochloroamine bacteriotoxicity was not completely reversed by the addition of
sulfhydryl radicals; and thus, conclude that sulfhydryl radical oxidation was not the
exclusive mechanism of toxicity. They proposed that the sulfhydryl group of critical
enzymes may be the point of vulnerability to a strong oxidant (for example, chlorine
dioxide) but that changes in other functional groups may also be involved in cell death.
Thus, a weak oxidant like monochloramine may lead to microbial inactivation by changes
in groups other than sulfhydryls.
Lu Shih and Lederberg (1976) showed that chloramine applied to both intact
Bacillus subtilis cells or to the extracted bacterial DMA resulted in double and single strand
breaks. Some microbial toxicity may, therefore, be attributable to DMA damage.
Monochloramine produced chromosome breaks in Vicia faba (Fetner, 1962), and organic
chloramines have been shown to produce chromosomal abnormalities in rodent cells
(NIEHS, 1982).
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Through observation of patients undergoing long-term hemodialysis and
concomitant analysis of the water used for this process, Eaton et al. (1973) ascertained
that chloramines were probably the oxidants responsible for hemolytic anemia in the
patients. In vitro studies using human RBCs indicated that chloramine produced
denaturation of hemoglobin through direct oxidant damage to erythrocytes. It also
inhibited the hexose monophosphate shunt, which generates NADPH that in turn protects
RBCs from oxidant damage.
A study by Grisham et al. (1984) demonstrated that organic N-chloramines formed
in vivo play an important role in ameliorating oxidant effects but can also contribute to
monochloramine formation. These workers stimulated isolated human neutrophilic
leukocytes to produce hydrogen peroxide (H202) and secrete cytoplasmic granule
components such as myeloperoxidase into the medium. Myeloperoxidase catalyzed the
oxidation of chloride (Cl") by H202 to yield hypochlorous acid (HOCI). The HOCI, in turn,
reacted with endogenous nitrogen compounds to yield derivatives containing
nitrogen-chlorine (N-CI) bonds, such as hydrophilic, low molecular weight,
mono-N-chloramine (RNHCI) derivatives. The RNHCI derivatives were of low toxicity, but
reacted with ammonium ion (NH4+) to yield monochloramine (NH2CI). The bactericidal,
cytotoxic and cytolytic activities of the organic N-chloramines (RNHCI) result from reaction
of RNHCI derivatives and the ammonium ion. This indicates that neutrophil amines act as
a trap for HOCI, and by competing with endogenous NH4+ for reaction with HOCI, protect
neutrophils and other cells from oxidative attack. The RNHCI derivatives remain as a
CLORAMIN.7 VII-2 07/27/93
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reserve of oxidizing equivalents that convert to a toxic form when an increase in NH4+
concentration favors formation of monochloramine.
Nusbaum (1952) proposed that the mechanism of action of dichloramine was similar
to monochloramine, but there are insufficient data to support this contention. Silver et al.
(1947c) demonstrated that trichloramine reacted with cystine and cysteine residues to
produce a reagent that was responsible for causing canine hysteria.
Summary
Bactericidal, cytotoxic and cytolytic activities of organic N-chloramines (RNHCI)
result from the reaction of RNHCI derivatives and the ammonium ion to yield
monochloramine (NH2CI). The RNHCI derivatives remain as a reserve of oxidizing
equivalents, which convert to a toxic form when the ammonium ion concentration increases
favoring monochloramine formation (Grisham et al., 1984).
It has been suggested that the chloramine molecules enter the cytoplasm and
interfere with enzymatic reactions (Nusbaum, 1952). Ingols et al. (1953) found
monochloramine required higher concentrations and longer contact times to destroy
bacteria than hypochlorous acid, suggesting that monochloramine led to microbial
inactivation through enzyme changes that may not have been involved in bacterial
inactivation by hypochlorous acid. Chloramines are also capable of causing DNA damage
in bacterial plant and mammalian cells, which may contribute to cytotoxicity. Eaton et al.
(1973) found that chloramines produced denaturation of hemoglobin through both direct
oxidant damage to RBCs and inhibition of the hexose monophosphate shunt.
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VIM. QUANTIFICATION OF TOXICOLOGIC EFFECTS
Introduction
The quantification of toxicologic effects of a chemical consists of separate
assessments of noncarcinogenic and carcinogenic health effects. Chemicals that do not
produce carcinogenic effects are believed to have a threshold dose below which no
adverse, noncarcinogenic health effects occur, while carcinogens are assumed to act
without a threshold.
In the quantification of noncarcinogenic effects, a Reference Dose (RfD), [formerly
termed the Acceptable Daily Intake (ADI)] is calculated. The RfD is an estimate (with
uncertainty spanning perhaps an order magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk
of deleterious health effects during a lifetime. The RfD is derived from a no-observed-
adverse-effect level (NOAEL), or lowest-observed-adverse-effect level (LOAEL), identified
from a subchronic or chronic study, and divided by an uncertainty factor(s) times a
modifying factor. The RfD is calculated as follows:
RfD - (NOAEL or LOAEL) = _ mg/kg/day
[Uncertainty Factor (s) x Modifying Factor ]
Selection of the uncertainty factor to be employed in the calculation of the RfD is
based upon professional judgment, while considering the entire data base of toxicologic
effects for the chemical. In order to ensure that uncertainty factors are selected and
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applied in a consistent manner, the U.S. EPA (1994) employs a modification to the
guidelines proposed by the National Academy of Sciences (MAS, 1977, 1980) as follows:
Standard Uncertainty Factors (UFs)
• Use a 10-fold factor when extrapolating from valid
experimental results from studies using prolonged exposure to
average healthy humans. This factor is intended to account
for the variation in sensitivity among the members of the
human population. [10H]
• Use an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
inadequate. This factor is intended to account for the uncer-
tainty in extrapolating animal data to the case of humans.
[10A]
• Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there is no
useful long-term human data. This factor is intended to
account for the uncertainty in extrapolating from less than
chronic NOAELs to chronic NOAELs. [10S]
• Use an additional 10-fold factor when deriving an RfD from a
LOAEL instead of a NOAEL. This factor is intended to account
for the uncertainty in extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (MF)
• Use professional judgment to determine another uncertainty
factor (MF) that is greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of the
overall data base and the number of species tested. The
default value for the MF is 1.
The uncertainty factor used for a specific risk assessment is based principally upon
scientific judgment rather than scientific fact and accounts for possible intra- and
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interspecies differences. Additional considerations not incorporated in the NAS/ODW
guidelines for selection of an uncertainty factor include the use of a less than lifetime study
for deriving an RfD, the significance of the adverse health effects and the counterbalancing
of beneficial effects.
From the RfD, a Drinking Water Equivalent Level (DWEL) can be calculated. The
DWEL represents a medium specific (i.e., drinking water) lifetime exposure at which
adverse, noncarcinogenic health effects are not anticipated to occur. The DWEL assumes
100% exposure from drinking water. The DWEL provides the noncarcinogenic health
effects basis for establishing a drinking water standard. For ingestion data, the DWEL is
derived as follows:
DWEL = (RfD) x (Body weight in kg) = /L
Drinking Water Volume in Llday
where:
Body weight = assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 L/day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for exposures of shorter
duration (1-day, 10-day and longer-term) are determined. The HA values are used as
informal guidance to municipalities and other organizations when emergency spills or
contamination situations occur. The HAs are calculated using an equation similar to the RfD
and DWEL; however, the NOAELs or LOAELs are identified from acute or subchronic
studies. The HAs are derived as follows:
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HA _ (NOAEL or LOAEL) x (bw) _
(UF)x( L/cfay)
Using the above equation, the following drinking water HAs are developed for
noncarcinogenic effects:
1. 1 -day HA for a 10 kg child ingesting 1 L water per day.
2. 10-day HA for a 10 kg child ingesting 1 L water per day.
3. Longer-term HA for a 10 kg child ingesting 1 L water per day.
4. Longer-term HA for a 70 kg adult ingesting 2 L water per day.
The 1-day HA calculated for a 10 kg child assumes a single acute exposure to the
chemical and is generally derived from a study of <7 days duration. The 10-day HA
assumes a limited exposure period of 1-2 weeks and is generally derived from a study of
<30 days duration. The longer-term HA is derived for both the 10 kg child and a 70 kg
adult and assumes an exposure period of ~7 years (or 10% of an individual's lifetime). The
longer-term HA is generally derived from a study of subchronic duration (exposure for 10%
of animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chemical, based on the
overall weight-of-evidence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from epidemiology
studies to support a causal association between exposure to the chemical and
human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of carcinogenicity in
animals with limited (Group B1) or inadequate (Group B2) evidence in humans.
Group C: Possible Human Carcinogen. Limited evidence of carcinogenicity in
animals in the absence of human data.
CLORAMIN.VIII VIII-4 03/08/94
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Group D: Not Classified as to Human Carcinogenicity. Inadequate human and
animal evidence of carcinogenicity or for which no data are available.
Group E: Evidence of Noncarcinogenicity for Humans. No evidence of
carcinogenicity in at least two adequate animal tests in different species or in both
adequate epidemiologic and animal studies.
If toxicologic evidence leads to the classification of the contaminant as a known,
probable or possible human carcinogen, mathematical models are used to calculate the
estimated excess cancer risk associated with the ingestion of the contaminant in drinking
water. The data used in these estimates usually come from lifetime exposure studies
using animals. In order to predict the risk for humans from animal data, animal doses must
be converted to equivalent human doses. This conversion includes correction for
noncontinuous exposure, less than lifetime studies and for differences in size. The factor
that compensates for the size difference is the cube root of the ratio of the animal and
human body weights. It is assumed that the average adult human body weight is 70 kg
and that the average water consumption of an adult human is 2 L of water per day.
For contaminants with a carcinogenic potential, chemical levels are correlated with a
carcinogenic risk estimate by employing a cancer potency (unit risk) value together with
the assumption for lifetime exposure from ingestion of water. The cancer unit risk is
usually derived from a linearized multistage model with a 95% upper confidence limit
providing a low dose estimate; that is, the true risk to humans, while not identifiable, is not
likely to exceed the upper limit estimate and, in fact, may be lower. Excess cancer risk
estimates may also be calculated using other models such as the one-hit, Weibull, logit
and probit. There is little basis in the current understanding of the biologic mechanisms
CLORAMIN.VIII VIII-5 03/08/94
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involved in cancer to suggest that any one of these models is able to predict risk more
accurately than any other. Because each model is based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of cancer risk rate
levels has an inherent uncertainty that is due to the systematic and random errors in
scientific measurement. In most cases, only studies using experimental animals have
been performed. Thus, there is uncertainty when the data are extrapolated to humans.
When developing cancer risk rate levels, several other areas of uncertainty exist, such as
the incomplete knowledge concerning the health effects of contaminants in drinking water,
the impact of the experimental animal's age, sex and species, the nature of the target
organ system(s) examined and the actual rate of exposure of the internal targets in
experimental animals or humans. Dose-response data usually are available only for high
levels of exposure and not for the lower levels of exposure closer to where a standard may
be set. When there is exposure to more than one contaminant, additional uncertainty
results from a lack of information about possible synergistic or antagonistic effects.
Current Levels of Exposure
Monochloramine has been used as a disinfectant of drinking water. An inventory of
municipal water supplies done in the early 1960s revealed that 308 of 11,590 supplies
surveyed used an ammonia chlorine process (Moore and Calabrese, 1980).
CLORAMIN.VIII VIII-6 03/08/94
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According to Morris et al. (1980), the extent of knowledge of natural water
contamination by N-containing aromatic compounds or the degree to which these
compounds react with aqueous chlorine is very limited. It is known that both inorganic
chloramines and organic N-chloramines are found upon chlorination of wastewater
effluents (Isaac and Morris, 1980). One survey reported the presence of monochloramine
ranging from 0.0321-0.9979 mg/L and dichloramine ranging from 0.0020-0.6950 mg/L in
secondary sewage effluents and cooling water samples (Jolley et al., 1978). Because
inorganic and organic chloramines cannot be identified separately, the levels are based
on the mixture.
Noncarcinogenic Effects
The chlorination of water containing ammonia or organic amines may result in the
formation of monochloramine, dichloramine, and trichloramine (nitrogen trichloride) through
the reaction between ammonia and hypochlorous acid. The amount of chloramines
produced depends upon the levels of chlorine and ammonia, and the pH of the water being
treated. Some studies on the oral effects of chloramines were presented in Tables V-1
and V-2.
Results on the mutagenicity of chloramines are inconclusive. Chloramine is weakly
mutagenic for Bacillis subtilis and causes DMA breaks in this bacterial species (Lu Shih
and Lederberg, 1976). Thomas et al. (1987) reported that monochloramine (40 um)
marginally increased the numbers of revertant colonies over untreated control levels in
assays employing Salmonella typhimurium (TA97, TA100 and TA102). Monochloramines
CLORAMIN.VIII VIII-7 03/08/94
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produce chromosome abnormalities in both Vicia faba and rodent cells (Fetner, 1962;
NIEHS, 1982). It was responsible for cellular hypertrophy, increased mitotic figures and
bizarre chromatin patterns in B6C3F1 mice exposed to 200 and 400 mg/L in drinking water
(Wolfe et al., 1984), while Meier et al. (1985) found that monochloramine at 0, 40, 100 and
200 mg/L did not induce chromosomal aberrations or micronuclei in bone marrow of CD-1
mice or spermhead abnormalities in B6C3F1 mice. Organic chloramines are mutagenic
or bacteriotoxic in S. typhimurium and have been reported to produce SCEs and other
chromosomal changes in mammalian cells (Scully et al., 1983; Bempong and Scully,
1980b; Bempong et al., 1981, 1986; Scully and Bempong, 1982). Tests for teratogenic,
reproductive and carcinogenic effects have been negative or inconclusive.
Abdel-Rahman et al. (1984) investigated the toxicity of monochloramine (NH2CI) in
groups of four male Sprague-Dawley rats/treatment weighing -160 g. Acute exposure to
a single dose at 10 (0.19 mg/kg/day), 20 (0.38 mg/kg/day) or 40 (0.75 mg/kg/day) mg/L
NH2CI induced a significant increase in blood glutathione levels within 30 minutes after
administration of 3 mL aqueous solution by gavage. As part of the same study, the long-
term toxicity of NH2CI in drinking water was also investigated. Groups of four male
Sprague-Dawley rats drank either 0, 1 (0.067 mg/kg/day), 10 (0.67 mg/kg/day) or 100 (6.7
mg/kg/day) mg/L NH2CI in deionized water daily for <12 months. The levels of chlorine,
dichloramine and trichloramine in the NH2CI solution were <1%, <1% and 0% respectively
of the total NH2CI added. Food was available ad libitum and body weight was measured
during the treatment. Heparinized blood was collected by cardiac puncture at 2, 4, 6, 8,
10 and 12 months after treatment, and blood GSH and osmotic fragility were determined
CLORAMIN.VIII VIII-8 03/08/94
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at each of these intervals. Results varied over the 12-month study period, but at 6 and 12
months after initiation of the study, statistically lower GSH levels were observed in all
treated rats. After 3 months of treatment significant decreases in RBC count and
hematocrit were observed at the higher dosage levels. Hemoglobin concentration and
MCH decreased significantly in the 100 mg/L group after 10 months of treatment. The
health significance of these types of changes is uncertain. Furthermore, results of
treatment effects were analyzed by inappropriate statistical methods (ANOVA). The
multiple comparison test used for paired comparisons (Duncan's) is a nonconservative
appproach. A more conservative approach would have taken into account correlations or
nonindependences. In addition, the control variability was greater than variability between
dose groups. A number of "significant" changes in hematologic parameters relative to
control values were identified that had no consistent relationship with dose and were not
observed consistently throughout the period of exposure. This could be due to the use of
a multiple comparison procedure, which is liberal such as the Duncan's.
Several short-term studies showed no observed adverse hematologic effects in mice,
rats and monkeys (Moore et al., 1980; Bercz et al., 1982; Bull, 1980). In A/J mice
administered chloramine solutions between 2.5 and 200 mg/L (pH 8.9) for 30 days, the
only observable effect was a slight increase in hematocrit (Moore et al., 1980). In another
study of similar duration (45 days) rats treated with 10, 50 or 100 mg/L monochloramine
experienced a decrease in the amount of methemoglobin present in the blood, the
opposite of what was expected (Bull, 1980). Bercz et al. (1982) studied the toxicity of
monochloramine administered in drinking water to 5 adult male and 7 adult female African
CLORAMIN.VIII VIII-9 03/08/94
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Green monkeys (3.0-5.7 kg). Monochloramine was administered for 6 weeks at 0 and 100
mg/L. The authors estimated the mean daily dose at ~10 mg/kg/day. Treatment with
monochloramine had no detectable effect in 18 hematologic tests on the 12 monkeys,
including red cell GSH levels. No evidence of thyroid suppression was detected from
measurements of serum T4. The results of these data are somewhat conflicting in that
similar dose levels and duration produced different changes, if any, in hematologic
parameters. One explanation for the varying observations between laboratories may be
the conditions under which the solutions of monochloroamine were generated.
In 1981 a draft report investigated the effects of monochloramine in Fischer 344 rats
and B6C3F1 mice (GSRI, 1981). Rats and mice (10 animals/sex/group) were administered
concentrations of 0, 25, 50, 100, 200 and 400 ppm monochloramine in drinking water for
91 days. Using food and water consumption data provided in the report, corresponding
dose levels are as follows: 2.5, 4.9, 10.2, 18.8, 40.7 mg/kg/day for male rats; 3.8, 6.5,
13.8, 26.6, 53.9 mg/kg/day for female rats; 4.9, 8.3, 14.5, 31.3, 50.7 mg/kg/day for male
mice; 7.7, 12.1, 21.9, 34.6, 88.5 mg/kg/day for female mice. After 25 days the buffer
system for monochloramine was changed because of a palatibility problem at the higher
dose levels. Decreased body weight gain and decreased relative liver weight were
observed in male and female rats at concentrations of 200 and 400 mg/L. A statistical
analysis was not provided. Protein excretion increased in male rats only when 200 and
400 mg/L monochloramine was administered. Microscopic examination of rat tissues at
the 400 ppm level did not reveal any treatment-related lesions.
CLORAMIN.VIII VIII-10 03/08/94
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As with rats, male and female mice gained less weight than the controls at the 200
and 400 ppm levels. There was a reduction in absolute liver weights and liver-to-body
weight ratios in male mice at the 400 ppm level and in female mice at >100 ppm.
Histopathologic observations revealed mild to moderate cytologic alteration in male mice
administered 200 and 400 ppm levels. Chronic liver inflammatory changes occurred at
100, 200 and 400 ppm in female mice and to a lesser extent in male mice at the 100 ppm
level. At concentrations of 100, 200 and 400 ppm increased frequency of mitotic figures,
hypertrophy and unusual chromatin patterns occurred in males and in a female at >200
ppm. The results suggest a LOAEL for liver toxicity of 100 ppm and a NOEL of 50 ppm
based on chronic liver inflammatory changes in mice.
Daniel et al. (1990) administered 0, 25, 50, 100 and 200 mg/L monochloramine to
male and female Sprague-Dawley rats (10/sex/dose) in their drinking water for 90
consecutive days. These doses correspond to 0, 1.8, 3.4, 5.8 and 9.0 mg/kg/day for males
and 0, 2.6, 4.3, 7.7 and 12.1 mg/kg/day for females. At >50 mg/L in males there were
reductions in body weight gain with significant reductions only at the highest dose (200
mg/L). For males and females in the 200 mg/L dose group the average weight gain was
51% of that of the controls; however, water consumption for the 200 mg/L dose was 31
and 33% of controls for males and females, respectively. At the 200 mg/L dose level there
were also reductions in organ weights (absolute, relative or both) and liver and spleen
weight reductions in both sexes. Although authors concluded that 100 mg/L dose is
considered the NOAEL, they suggest that a matched watering and feeding study would be
CLORAMIN.VIII VIII-11 03/08/94
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useful for distinguishing between systemic toxic effects and weight loss from taste aversion
to more clearly identify the NOAEL.
Daniel etal. (1991) administered 0, 12.5, 25, 50, 100 and 200 mg/L monochloramine
to male and female B6C3F1 mice (10/sex/dose). These doses correspond to 0, 2.5, 5.0,
8.6, 11.1 and 15.6 mg/kg/day for males and 0, 2.8, 5.3, 9.2, 12.9 and 15.8 mg/kg/dayfor
females. Water consumption was decreased in all treated groups. There were significant
water and food consumption decreases and weight gain reductions at the two highest dose
groups. There were reductions in absolute and relative organ weights in male and female
mice in the two highest dose groups. Based on relatively minor changes at 100 mg/L)
including <10% decrease in body weight gain at 100 mg/L, and 19-25% decrease in body
weight gain at 200 mg/L, a NOAEL of 100 mg/L (12.9 mg/kg/day) was identified based on
a decrease in body weight gain and decreased organ weights in B6C3F1 mice consuming
200 mg/L monochloramine in the drinking water for 90 days. The authors state that the
lower levels of serum enzyme and reduced organ weights were considered consistent with
decreased water and nutrient consumption and altered electrolyte balance rather than
disinfectant-induced toxicity. The authors conclude that the absence of histopathology or
observable clinical signs of toxicity suggest that these monochloramine exposures induce
a relatively mild, nonspecific toxicity by an indirect mechanism (nutritional and electrolyte
deficiencies) rather than a direct toxicologic effect on specific organs or tissues.
NTP (1990) conducted 2-year studies using monochloramine administered in the
drinking water. In the first study monochloramine was administered to male and female
CLORAMIN.VIII VMM 2 03/08/94
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F344/N rats at 0, 50, 100 and 200 ppm. These doses were calculated on a time-weighted
average to be 0,2.1,4.8 and 8.7 mg/kg/day for males and 0, 2.8, 5.3 and 9.5 mg/kg/day
for females. Mean body weights of high-dose rats were lower than their respective
controls. However, mean body weights of rats receiving chloraminated drinking water were
within 10% of controls until week 97 for females and week 101 for males. Interim
evaluations made at 14 weeks revealed that body weights of high-dose males were lower
than controls. At the 66-week evaluation, there was a dose-related decrease in body
weight in male chloraminated-treated rats and the mean body weights of high-dose rats
were 94% and 92% of controls for males and females, respectively. Decreases in liver and
kidney weight in the high-dose males and increases in brain and kidney-to-body weight
ratios in high-dose male and female rats were observed in the 14- and 66-week
evaluations.
The NTP (1990) second study was a 2-year study of momochloramine administered
in drinking water to male and female B6C3F1 mice at 0, 50, 100 and 200 ppm. These
doses were calculated on a time-weighted average to be 0, 5.0, 8.9 and 15.9 mg/kg/day
for males and 0, 4.9, 9.0 and 17.2 mg/kg/day for females. Mean body weights of
high-dose male mice were 10-22% lower than controls after week 37 and the body weights
of high-dose female mice were 10-35% lower after week 8. At the 15-week interim
sacrifice the mean body weights of high-dose mice were 91% and 84% of controls for
males and females, respectively. At the 66-week evaluation the differences in body weight
between the high-dose and controls was 87% for females but 91 % of controls for the mid-
and high-dose males. Decreases in liver weights and increases in brain and
CLORAMIN.VIII VMM 3 03/08/94
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kidney-to-body weight ratios were observed in high-dose male and female mice at 15 and
66 weeks.
Quantification of Noncarcinogenic Effects
Derivation of 1-Day Health Advisory. The only report of an acute exposure to
monochloramine in the literature is the study by Abdel-Rahman et al. (1984) wherein the
authors observed a drop in blood GSH levels at the lowest dose administered by gavage
(3 ml of 10 mg/L solution). As the health implications of these effects are uncertain, they
were transitory and were not corroborated by other investigations, it would seem
inappropriate to use this study as the basis for a 1-day HA. Therefore, no suitable study
is available for the derivation of a 1 -day HA. It is recommended that the 10-day HA of 1.0
mg/L be adopted as the 1-day HA.
Derivation of 10-Day Health Advisory. Similar NOAELs or NOELs for
monochloramine of 200, 100 and 100 ug/L for hematologic parameters were identified by
Bercz et al. (1982), Moore et al. (1980) and Bull (1980), respectively. Both Moore et al.
(1980) and Bull (1980) did observe some changes in hematologic parameters. Moore et
al. (1980) found a slight increase in hematocrit in A/J mice after 30 days of exposure
whereas after 45 days of exposure to rats there was a decrease in the amount of
methemoglobin present in the blood, the opposite of what was expected (Bull, 1980).
Bercz et al. (1982) reported a NOEL in monkeys after treatment with 100 mg/L
chloramines in drinking water. There were no detectable effects in 18 hematologic tests.
The authors calculated that by the termination of the study the test animals were
CLORAMIN.VIII VIII-14 03/08/94
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consuming 10 mg chloramine/kg/day. One explanation for the varying observations
between laboratories may be the conditions under which the solutions of monochloramine
were generated. It is recommended that the NOEL of 100 mg/L from the Bercz et al.
(1982) study be used to derive the 10-day HA for two reasons: 1) the Bull (1980) study
provides an incomplete description of methods concerning study design and clinical
analysis, and 2) monkeys may be a better animal model since the A/J strain of mice used
in the Moore et al. (1980) study maintain an erythrocyte glucose-6-phosphate
dehydrogenase activity 3 times that of human cells and may be more resistant to oxidant
stress (Kiese, 1974). A NOEL is, therefore, set at 10 mg/kg/day, which the authors
determined to be the rate of consumption of chloramine at the 6-week termination of the
study.
A provisional 10-day HA is calculated as follows:
Fora 10 kg child:
10-day HA - 1° ^kg/day x 10 kg __ 1
100 x 1 L/day
where:
10 mg/kg/day = NOAEL based on the absence of hematologic effects in
monkeys (Bercz et al., 1982)
10 kg = assumed body weight of a child
10 = uncertainty factor for use of an animal NOAEL and to protect
sensitive members of the human population
1 L/day = assumed water consumption of a child
CLORAMIN.VIII VIII-15 03/08/94
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Thus, the proposed 10-day HA is 1 mg/L for a 10 kg child. The small number of animals
used by Bercz et al. (1982) and the lack of other shorter- term studies for supporting data
should be considered when evaluating this HA.
Derivation of Longer-Term HA. Several longer-term studies were considered for
serving as the basis for the longer-term HA. The 1981 draft report by GSRI had
methodology and data inconsistencies that make it unsuitable for a longer-term HA. In the
Daniel et al. (1990) study, male and female rats in the 200 mg/L dose group (highest dose
tested) had an average weight gain of 51% of controls and drinking water consumption
was decreased 31-34%, which may suggest that the effect of weight loss may be due to
a taste aversion rather than a systemic toxic effect. Although the authors identified a
NOAEL of 100 mg/L (5.8 and 7.7 mg/kg/day) they also suggested that the NOAEL could
be more clearly identified by a matched feeding and water study to more clearly distinguish
between systemic toxic effects and weight loss from taste aversion. In the Daniel et al.
(1991) study a NOAEL of 50 mg/L (8.6-9.2 mg/kg/day) was identified for B6C3F1 mice.
At the two higher dose levels tested, the lower levels of serum enzymes and reduced
organ weights were considered by the authors to be consistent with decreased water and
nutrient consumption and altered electrolyte balance rather than chemical-induced toxicity.
The studies by Daniel et al. are supportive of the NTP (1990) study, which is used as the
basis for the DWEL. Using the data from either of the two studies by Daniel et al. would
result in a longer-term HA of 3 mg/L, slightly less than the DWEL of 4 mg/L. The DWEL
is based on a well conducted chronic NTP (1990) study with a NOAEL of 9.5 mg/kg/day.
Because of the problems in interpreting the subchronic studies, it is recommended that the
CLORAMIN.VIII VIII-16 03/08/94
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DWEL of 4 mg/L be adopted for the longer-term HA for adults. For the 10 kg child drinking
1 L/day, the DWEL is modified resulting in a longer-term HA of 1 mg/L.
Assessment of Lifetime Exposure and Derivation of a DWEL. It is recommended
that the study by NTP (1990) be used as a basis for a DWEL because it is a chronic 2-year
study using rats and mice with chloramine in the drinking water. When monochloramine
was administered to rats and mice in their drinking water there were statistically significant
changes in body and several organ weights (as previously described) at the high-dose
level. However, the significance of these changes is unclear because test animals
consumed a reduced amount of water, which was perhaps due to palatability, and NTP
does not consider these changes in body and organ weight biologically significant. It is
recommended that the highest dose tested be used as the NOAEL. The NOAEL identified
in the rat study was chosen over the NOAEL in the mouse study to calculate the proposed
RfD because data from acute and shorter term studies indicate that the rat is the more
sensitive species. The calculation of the RfD is as follows:
RfD = 9'5 m9! k9lday = QQQ5 mglkglday (rouncjed to 0.1 mglkg/day)
100
where:
9.5 mg/kg/day = NOAEL based on an absence of biologically significant
adverse effects in rats (NTP, 1990)
100 = uncertainty factor for protection of sensitive members of the
human population and for use of an animal NOAEL
CLORAMIN.VIII VIII-17 03/08/94
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DWEL = 0.1 mg I kg/day x 70 kg = 3 g mg/L (munded fo 4
2 L/cfay
where:
0.1 mg/kg/day = RfD
2 L/day = assumed water consumption by an adult
70 kg = assumed body weight of an adult
The RfD of 0.1 mg/kg/day was verified (06/23/92) by the RfD/RfC Work Group of the
U.S. EPA (1994). The Work Group expressed a high degree of confidence in the critical
study. Although ideally higher doses should have been tested, this was not possible due
to the taste aversion. The study by NTP (1990) examined relevant endpoints in two animal
species exposed to chloramine by a relevant route of exposure for a prolonged period of
time. Several dosage levels were included, the number of animals per dose group was
adequate, and the statistical analyses were used. The Work Group expressed a medium
level of confidence in the data base. Information is available on mice, rat and monkeys for
the noncarcinogenic toxicity of oral exposure to monochloramine for subchronic periods.
The developmental toxicity and reproductive toxicity of monochloramine have been
examined in rats but a developmental toxicity study in a second species and a
2-generation reproductive study are not available. (Information available for chlorine can
be used to satisfy data gaps for monochloramine.) Confidence in the data base is limited
CLORAMIN.VIII VMM 8 03/08/94
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by the lack of information on health effects in humans. Overall confidence in the RfD is
considered to be medium. A summary of the HAs and DWEL is presented in Table VIII-1.
Carcinogenic Effects
In a 2-year study by NTP (1990), there was equivocal evidence of carcinogenic activity
of chloraminated drinking water in female rats, which was due to the slightly increased
incidence of mononuclear cell leukemia compared with that of controls. There was no
CLORAMIN.VIII VIII-19 03/08/94
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TABLE VMM
Summary of HAs and DWEL for Noncareinogenic Effects
HAs and DWEL
1 -Day HA
10-Day HA
Longer-term HA
DWEL
Dose Level
(mg/kcj/day)
10
10
9.5
9,5
10 kg Child
(mg/L)
r
1
1
--
70 kg Adult
(mg/L)
—
—
4*
4
Reference
Bercz et al.,
1982
Bercz et al.,
1982
NTP, 1990
NTP, 1990
aAdopted from the 10-day HA
bAdopted from the DWEL
CLORAMINL5
VIII-20
3/08/94
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evidence of carcinogenic activity because of chloraminated drinking water in male rats or
in male or female mice. Monochloramine was verified by the CRAVE Work Group
(12/02/92) and is classified in group D, not classifiable as to human carcinogenicity,
meaning that there is inadequate human and animal evidence of carcinogenicity (U.S.
EPA, 1986).
Existing Guidelines. Recommendations and Standards
The MAS (1987) calculated a suggested no-adverse-response level (SNARL) of 0.581
mg/L. This was based on a NOEL of 50 ppm (estimated by NAS to be 8.3 mg/kg bw/day)
from a 90-day study in mice, which showed reduced body weight and liver toxicity (GSRI,
1981). It was assumed that a 70 kg human consumes 2 L of water daily, which contributes
20% of total intake. This value should be viewed cautiously since the data in this study
were not verified.
Special Groups at Risk
Long-term hemodialysis patients have displayed higher risks of hemolytic anemia
caused by chloramines present in dialysis baths. These chloramines denature hemoglobin
through oxidation and inhibit the hexose monophosphate shunt (Eaton et al., 1973). This
problem can be eliminated by using charcoal-filtered water in the dialysis bath; therefore,
this group should not be given special consideration in the development of water quality
criteria (U.S. EPA, 1981).
Risk Characterization
CLORAMIN.VIII VIII-21 03/08/94
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In characterizing the risk that chloramines pose in drinking water, the health effects
must be considered in light of the widespread use of the chloramine-ammonia process
(chloramination) for disinfection purposes. As stated earlier in this document, the range of
residual in the distribution system (1.5-2.5 mg/L) is well below the DWEL of 4 mg/L. In
humans, health effects do not appear to be associated with levels of residual chloramine
typically found in drinking water.
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IX. REFERENCES
Abdel-Rahman, M.S., M.R. Berardi and R.J. Bull. 1982. Effect of chlorine and
monochloramine in drinking water on the developing rat fetus. J. Appl. Toxicol. 2(3):
156-159.
Abdel-Rahman, M.S., D.M. Waldron and R.J. Bull. 1983. A comparative kinetics study
of monochloramine and hypochlorous acid in rat. J. Appl. Toxicol. 3(4): 175-179.
Abdel-Rahman, M.S., D.H. Suh and R.J. Bull. 1984. Toxicity of monochloramine in rat:
An alternative drinking water disinfectant. J. Toxicol. Environ. Health. 13: 825-834.
Anbar, M. and G. Yagil. 1962. The hydrolysis of chloramine in alkaline solution. J. Am.
Chem. Soc. 84: 1790-1796.
Anbar, M., S. Guttmann and R. Rein. 1959. The isotopic exchange between
hypochlorite and halide ions. II. The exchange between hypochlorous acid and chloride
ions. J. Am. Chem. Soc. 81: 1816-1821.
CLORAMIN.9 IX-1 03/08/94
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Anderson, D. and J.A. Styles. 1978. An evaluation of 6 short-term tests for detecting
organic chemical carcinogens. Appendix 2. The bacterial mutation test. Br. J. Cancer.
37: 924-930.
APHA (American Public Health Association). 1980. Standard Methods for the
Examination of Water and Wastewater, 15th ed. p. 286-288.
Beck, H.I. 1983. Type I reaction to chloramine. Contact Dermatitis. 9(2): 155-156.
Bempong, M.A. and F.E. Scully, Jr. 1980a. Mutagenic activity of N-chloropiperidine. J.
Environ. Path. Toxicol. 4(2,3): 345-354.
Bempong, M.A. and F.E. Scully, Jr. 1980b. In vitro cytological effect of
N-chloropiperidine: Induction of mitotic anomalies in Chinese hamster ovary cells. ]n:
Water Chlorination: Environmental Impact and Health Effects, Vol. 3, R.L. Jolley et al.,
Ed. Ann Arbor Science, Ann Arbor, Ml. p. 817-825.
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