United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711 •-, k
Research and Development
EPA/600/S1 -87/008 May 1988
&EPA Project Summary
Formation and
Distribution of Organic
N-Chloramines from the
Ingestion of Chlorinated
Drinking Water
Frank E. Scully, Jr., and Daniel E. Sonenshine
The chemical reactions that hypo-
chlorite undergoes in the body when
chlorinated water is ingested have
received very little attention. Because
amino nitrogen compounds are impor-
tant components of the average diet,
the reactions of hypochlorite with
amino compounds in the stomach were
investigated.
Stomach fluid was recovered from
Sprague-Dawley rats that had been
fasted for 48 hr and administered 4 ml
deionized water. The chlorine demand
of the stomach fluid was determined.
An average volume-independent de-
mand of 2.7 mg chlorine was meas-
ured. At doses below 40 mg/L chlorine
reducing reactions appeared to account
for reduction of all oxidizing species
within 15 min as measured by the FAS-
DPD titrimetric method.
At least part of the chlorine demand
is associated with amino acids present
in the stomach fluid. Amino acids were
identified and quantified in the stomach
fluid by pre-column derivatization with
ortho-phthalaldehyde and high-pres-
sure liquid chromatography (HPLC).
When stomach fluid is chlorinated to
concentrations of chlorine between
200 and 1000 mg/L, organic N-
chloramines are formed. After deriva-
tization of chlorinated stomach fluid
with dansyl sulfinic acid, fluorescent
derivatives of chloramines were sepa-
rated by HPLC. Three chloramino acid
derivatives, N-chloroalanine, N-
chloroglycine, and N-chlorophenyl-
alanine, were identified by co-chroma-
tography with known standards using
two chromatographic methods.
The yield of a chloramine that would
form in stomach fluid on administration
of hypochlorite to animals was deter-
mined using tritiated piperidine and
doses of 200 and 1000 mg/L chlorine.
Yields of tritiated N-chloropiperidine
(NCP) in recovered stomach fluid were
70% and 42%, respectively, of the
theoretical amount expected.
The stability of 36CI-N-chloropiper-
idine was examined at typical pHs
found in stomach fluid (pH 2-7). N-
Chloropiperidine was found to transfer
its chlorine atom slowly to unchlori-
nated amines at pHs below 3 with a
half-life at 37°C (pH 2.35) of 292 min.
36CI-N-chloropiperidine slowly under-
goes isotope exchange in a phosphate
buffer (0.01 M at pH 2.5) with 0.1 M
chloride without equally fast decompo-
sition of the chloramine. However, both
chlorine transfer and isotope exchange
are too slow to be of significance in
the toxicological studies reported here.
When 36CI-N-chloropiperidine is
incubated with rat stomach fluid at
37°C for 30 min, 34% is reduced to
36CI-chloride, and 66% reacts with
organic components in the fluid to form
a mixture of 3*CI-chlorinated organic
compounds of unknown identity.
A series of pharmacokinetic studies
was conducted in male and female
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Sprague-Dawley rats employing 3H-N-
chloropiperidine and 36CI-chloro-
piperidine as test compounds and 3H-
piperidine and 36CI-chloride as control
compounds. Studies showed that tri-
tiated compounds were absorbed into
blood, excreted, and distributed in
tissues in a similar manner. However,
36CI-activity was retained in the tissues
of animals administered 36CI-N-
chloropiperidine to a much greater
extent and eliminated at a much slower
rate than from animals given 36CI-
chloride. A relationship between the
retention of the 36CI-activity in the
pharmacokinetic studies and formation
of 36CI-chloroorganic compounds in
vitro is discussed.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering
information at back).
Introduction
Over the past 10 years it has been
recognized that chlorine, used to disin-
fect drinking water, reacts with trace
organic compounds dissolved in natural
waters to produce by-products which
may have adverse health effects in
humans. Consequently, evaluation of the
potential health effects of water disin-
fection of these by-products and deter-
mination of the quantities of these
compounds typically ingested by the
population at large. Water treatment
policies have been primarily concerned
with minimizing the concentrations of
these trace contaminants, particularly
the trihalomethanes.
In the United States the average
person drinks between 2 and 3 liters of
water each day which may typically
contain between 1 and 2 mg/L of the
residual chlorine oxidant such as aque-
ous chlorine (CI2). By comparison with
the U.S. Environmental Protection Agen-
cy's (EPAs) Maximum Contaminant Level
for chloroform, the molar concentration
of chlorine in a drinking water containing
2 mg/L is 30 times higher. Furthermore,
when drinking water is ingested, it enters
a medium with a total organic carbon
(TOC) content that is several orders of
magnitude higher than that found in
natural waters. However, little attention
has been given to the possible reactions
of hypochlorous acid which may take
place in the organic-rich medium of the
stomach on ingestion of chlorinated
drinking water.
The average person's daily diet
includes a minimum of 30 to 45 grams
of protein. Through the action of digestive
enzymes in the stomach, proteins are
broken down into peptones, large poly-
peptides, and about 15% amino acids.
Since stomach fluid contains high con-
centrations of organic amino nitrogen
compounds, and since hypochlorite
reacts rapidly with these types of com-
pounds to form chloramines, it was
hypothesized that, upon ingestion, chlo-
rinated water would react rapidly with
amino nitrogen compounds in the stom-
ach to produce N-chloramino by-
products which may be distributed
throughout the body. Therefore, two
main objectives were identified at the
beginning of the project:
1. To determine if organic N-
chloramines can form in the stom-
ach upon ingestion of hypochlorous
acid and inorganic chloramines.
2. To determine what chemical reac-
tions chloramines, both organic and
inorganic, may undergo in the stom-
ach and whether they can be ab-
sorbed into the bloodstream for
circulation to other parts of the body.
N-Chloropiperidine (NCR) was used
extensively in this project as a model to
study the reactions of chloramines in the
stomach. Initially, this compound was
chosen for four reasons. First, the parent
amino, piperidine, had been identified in
drinking water and had been shown to
be excreted in urine at the rate of 5 mg/
L/day by normal human males. It was,
therefore, recognized as an endogenous
amine in humans. Secondly, the oxidiz-
ing power of NCR was recognized to be
similar to that of other organic mono-
chloramines, but it is a relatively stable
chloramine. From an experimental
sta ndpoint this property makes it possible
to track the fate of this chloramine in the
body. The mono-N-chlorinated deriva-
tives of primary amines and ammonia are
not as stable below pH 8 and are
converted to their dichloramino ana-
logues by chlorine exchange. NCR does
not undergo this reaction because it has
no remaining exchangeable hydrogens
that can be replaced with chlorines. The
third reason NCR was chosen was that
conditions for chromatographing NCR
directly without extensive work-up or
pre-column derivatization have been
developed. The analysis of the compoum
in aqueous solution can be carried ou
simply and rapidly.
The fourth reason NCR was selectee
was because both a tritium-labeled N-
chloropiperidine (3H-NCP) and a 36C|-
labeled N-chloropiperidine (36CI-NCP)
could be synthesized. Chloramines may
act as either chlorinating agents or as
aminating agents. Therefore, if the
pharmacokinetics of absorption and
excretion of 3H-NCP differed from those
of this control compound, tritiated pip-
eridine, it might be inferred that chlor-
amines acted as aminating species in
biological systems. On the other hand,
if the pharmacokinetics of 36CI-NCP
differed from its control compound, 36Cr,
it -might b® inferred that chloramines
acted as chlorinating agents in the body.
Recently, a series of studies on the
toxicity and pharmacokinetics of 36CI-
labeled hypochlorous acid and 36CI-
labeled monochloramine were reported.
These studies showed that the 38CI used
in these studies is retained in non-fasted
animals much longer than 36CI-enriched
chloride. Therefore, it is important to
determine what chemistry that can take
place in the stomach can account for this
greater degree of retention.
Part of the impetus for this research
is the previous observation that at least
one organic N-chloramine, NCR, is
mutagenic by Ames' assay, is cytotoxic,
and induces chromosomal aberrations in
mammalian cells the frequency of which
is proportional to the concentration of
NCR.
In addition to NCR, N-chloroglycine
was used to probe the reactions of
hypochlorite and the stabilities of chlor-
amines in the stomach. N-Chloroglycine
is a relatively stable chloramino acid
formed from glycine. The chloroglycine
is not present in the stomach because
of ingestion or as a product of proteolytic
activity, but glycine would be. It can then
serve as a precursor for formation of
chloroglycine by reacting with ingested
hypochlorus acid or another chloramine.
Recently, a method for the derivatiza-
tion and analysis of organic N-
chloramines in dilute aqueous solution
was described. In the method, solutions
containing N-chloramines were reacted
with 5-dimethylaminonaphthalene-1 -
sulfinic acid (DANSOaH) to produce
highly fluorescent sulfonamide deriva-
tives (dansyl derivatives) which could be
analyzed by HPLC. In the present study,
this derivatization method is used to
detect the formation of N-chloroglycine 4
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in stomach fluid, but results are corrob-
orated by chromatography of an unde-
rivatized chloramine (NCP) and its radio-
labeled counterpart.
Discussion
When rats were treated with either the
tritium-labeled organic chloramine, NCP,
or its tritium-labeled parent amine,
piperidine, the radioactivity was rapidly
eliminated from the body. Except in the
case of the male animals administered
3H-NCP (where plasma decay and excre-
tion rates were faster than in the other
studies), plasma decay rates were similar
for both compounds and in all cases less
than 5% of the label remained in the
animal at the end of the 120-hr study
period. The study of the pharmacoktnet-
ics of 3H-NCP in male rats is being
repeated to determine if the apparent
deviafTqn^from"*the' kinetic§.,observed in
the Sfljgr studi'es'is real. These data
suggeigkJhat the 3H-1ab^led,c^mpounfl
absorbatUfrto blgp^Tand^tBratgd in bot,
Jjtudjes 4O» boiaa frgatad- *n«*he sam
piperidine in urine and tissues from
animals administered 3H-NCP suggests
that the chloramine, 3H-NCP, is rapidly
being dechlorinated to its parent amine,
3-H-piperidine (the control compound),
which is then absorbed into blood and
excreted by the body with the same
kinetics as the control compound.
By contrast, the kinetics of 36CI-NCP
plasma decay and excretion differed
dramatically from those of the control
compound, 36CI-chloride. The half-life of
elimination of 36CI" (53 hr in males and
50 hr in females), was comparable to that
found in other studies (51.9 hr). However,
the rate of elimination of 36CI-chloride
from plasma was approximately twice as
fast as elimination of 36CI-activity after
administration of 36CI-NCP (half-lives of
173 hr and 116 hr, respectively, in male
and female,jcats). By the end of the test
periodTess,Ahan 50% of the,radioactivity
had b§en excreted in "Cl-NCP-treated
. 36CJUactivitfrm admjfllgiftrtiorvof the 36CI-
At first glance the results of the
pharmacokinetic studies involving 3H-
NCP might seem to contradict the results
using 36CI-NCP. However, taken to-
gether, these data suggest that the 36CI-
label originally associated with the
administered chloramine is rapidly
becoming associated with another mole-
cule in the stomach and this new
chlorinated compound is exhibiting
different pharmacokinetics than chlo-
ride.
To support this hypothesis, we have
examined the in vitro reactions of 36CI-
NCP. As described in the full report it
was observed that at concentrations of
576 ppm (CI2) 36CI-NCP reacts with
organic constituents in stomach fluid to
form a new 36CI-chloroorganic fraction
of compounds that is chemically distinct
from either 36CI-NCP or 36CI-chloride. It
would appear that this 36CI-chloroorganic
fraction is likely to account for the
distribution and retention of the 36CI-
activity in the pharmacokinetic study of
36CI-NCP.
In .a comparative study of the pharma-
cokinetics of HO36CI and NH236CI, inves-
tigators found that 36CI-activity was
>^Timirt£te£ from plasma, distributed, and
^^xcreted* Vom fasted Sprague-Dawley
V, rkfs at raj\s similar to that of 36CI-
chloride. - «J
i*ftowSver/a pharmacokinetic study of
hyB^^isxjom plica ted by the fact that the
labwhsn hypochlorous acid undergoes
extremely rapid isotope exchange with
unlabeled chloride. Other studies found
that the rate of isotope exchange is
dependent on the concentrations of
HOCI, chloride, and hydrogen ion and
measured a third order rate constant at
27°C of 3.16 x 1013 /W"1min"1. In the
stomach, which can contain as much as
0.1 M chloride, this reaction would be
greater than 99% complete in 8.7 x 10~4
sec, assuming an initial pH of 7.0y%
temperature of 27°C and no compejffng
reactions. Consequently, the similarity
between the pharmacokinetics of Hu^CI
and 36CI-chloride is likely to be duetto
the rapid formation of 36CI-chloride frottn
HO36CI by isotope exchange with unla-
beled chloride in the stomach.
On the other hand, it was observed that
m^tfe for elimination of 36CI-
vity from^on-fasted male rats admin-
H0369\ was twice (88.5 hr) that
if the half-life* or elimination from fasted
»ratH(44«Hhr).£jnfortunately, the half-life
of ?4f Ig^hloride in non-fasted rats is
unknown. However, in light of the
observed formation of a 36CI-chloro-
organic fraction when 36CI-NCP is mixed
with stomach fluid, it is possible that HO-
36CI reacts rapidly with the higher
concentration of food-based organic
compounds in the stomachs of non-
fasted rats before it has the opportunity
to undergo isotope exchange.
Other investigators also studied the
pharmacokinetics of 370 mg/L (as Ck)
NH236CI in male Sprague-Dawley rats.
They note that the 36CI-activity in the
plasma reached a peak 8 hr following
adminstration. The 36CI-activity re-
mained at a plateau from 8 to 48 hr after
administration before it was eliminated
with a rate constant similar to that of
chloride. Consequently, over 70% of the
amount of radioactivity administered was
retained in the animals at the end of the
5-day test-period. The rate of isotope
exchange between NH236CI and unla-
beled chloride has not been measured.
Therefore, it is possible to determine how
much of the label was lost by this
reaction. However, because of the dif-
ferences between the kinetrcs'of N Hsase I
and H036CI and 36CI-chloride, the greater
degree of retention of the label in the
study involving NH236CI is likely to be due
to retention of the chloramine with
organics in the stomach to form a 36CI-
chloroorganic fraction similar to the one
found in the reaction of 36CI-NCP with
rat stomach fluid.
Studies showed that hypochlorite
reacts with fetal calf serum to produce
products that inhibit division of porcine
aortic vascular endothelial cells, but do
not kill them. A reduction in cell growth
of at least 20% compared with controls
was noted when any combination of fetal
bovine serum and NaOCI was preincu-
bated in the growth medium.
In the present study radiolabeled NCP
\Q fetal calf serum was incubated with
cells or 3T3 NIH cells., At approx-
the safn^~8ionpeift(gtrens of
ilorine us§cTJ«2.Q. nta2£
ijum actiuity,*f?oraJfcl-J^CP was""
by cells, jn 15 min, -ajthougir"""
the activity., was tost over th'e^
taming 4 hr. ^Gl-aetivitywas accum- „
ulated within 30 min iri 3T3 NIH celts
to a much greater extent after incubation
of the cells with 36CI-NCP than after
incubation with seci-chloride. Evidence
was obtained that there was a time-
dependent accumulation of a very small
amount of cell nuclei.
The 36CI-chloroorganic fraction formed
when 36CI-NCP js incubated with rat
stomach fluid is believed to be due to
a reaction of the chloramine with pro-
teinaceous components of the stomach
fluid. Since both hypochlorite and chlor-
amines are chlorinating agents, and
since the chloramine is incubated with
MEM containing 2% fetal calf serum in
the cell culture studies, it is pdssible that
products similar to the 36CI-chloroorganic
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fraction are being formed which are
being taken up into the cell in the present
study and which account for the
inhibition of growth in the studies with
porcine aortic endothelium cells.
Conclusions
Based on results of this study, it may
be concluded that the rates of reaction
of hypochlorite with amines are suffi-
ciently fast that organic chloramines can
be formed on ingestion of aqueous
hypochlorite. In low concentration they
are short-lived and appear to be reduced
to non-oxidizing species. However,
organic chloramines appear to undergo
subsequent reaction with other organics
in stomach fluid to form new covalently
bonded chlorine compounds, probably
chlorocarbon compounds. It is possible
that these compounds are intermediates
in the detoxification and/or elimination
of active chlorine compounds in the body.
On the other hand, the pharmacokinetic
data suggests that much of seci-labeled
organic fraction is retained in the body
after five days. Since these compounds
have not yet been characterized, their
health effects are unknown. However,
they appear to be the end-products for
active chlorine compounds in the body
and, as such, related to the health effects
of both hypochlorite and inorganic
chloramine disinfectants. On the other
hand, the fact that a chloramine can be
absorbed into blood is remarkable in. it-
self and suggests that direct toxicological
effects of chloramines cannot be ignored.
Frank E. Scully, Jr. and Daniel E. Sonenshine are with Old Dominion University,
Norfolk, VA 23508.
Frederick C. Kopfler is the EPA Project Officer (see below).
The complete report, entitled "Formation and Distribution of Organic /V-
Chloramines from the Ingestion of Chlorinated Drinking Water," (Order No.
PB 88-103 742/AS; Cost: $14.95, subject to change) will be available only
from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park. NC 27711
United States
Environmental Protection
Agency
Official Business
Penalty for Private Use $300
EPA/600/S1-87/008
Center for Environmental Research
Information
Cincinnati OH 45268
1 -&U.S GOVERNMENT PRINTING OFFICE 1988—548-013/8
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