&EPA
United States
Environmental Protection
Agency
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Toxicological Review of
Ammonia
FEBRUARY 2012
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EPA/635/R-11/013C-2
www.epa.gov/iris
Toxicological Review of Ammonia
(CAS No. 7664-41-7)
February 2012
NOTICE
This document is an Interagency Science Consultation draft. This information is distributed
solely for the purpose of pre-dissemination peer review under applicable information quality
guidelines. It has not been formally disseminated by EPA. It does not represent and should not
be construed to represent any Agency determination or policy. It is being circulated for review
of its technical accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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1 DISCLAIMER
2
3
4 This document is a preliminary draft for review purposes only. This information is
5 distributed solely for the purpose of pre-dissemination peer review under applicable
6 information quality guidelines. It has not been formally disseminated by EPA. It does not
7 represent and should not be construed to represent any Agency determination or policy.
8 Mention of trade names or commercial products does not constitute endorsement of
9 recommendation for use.
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CONTENTS
PREAMBLE vi
AUTHORS | CONTRIBUTORS | REVIEWERS xvi
PREFACE xviii
EXECUTIVE SUMMARY xxi
LITERATURE SEARCH STRATEGY & STUDY EVALUATION FOR HAZARD IDENTIFICATION xxv
1. HAZARD IDENTIFICATION 1
1.1. Synthesis of Major Toxicological Effects 1
1.1.1. Respiratory Effects 1
1.1.2. Gastrointestinal Effects 14
1.1.3. Reproductive and Developmental Effects 17
1.1.4. Immune System Effects 18
1.1.5. Other Systemic Effects 22
1.1.6. Cancer 31
1.1.7. Susceptible Populations and Life Stages 33
1.2. Weight of Evidence Evaluation for Toxicological Effects 33
2. DOSE-RESPONSE ANALYSIS 41
2.1. Oral Reference Dose for Effects other than Cancer 41
2.2. Inhalation Reference Concentration for Effects other than Cancer 43
2.2.1. Methods of Analysis 45
2.2.2. Derivation of Reference Concentration 46
2.2.3. Uncertainties in the Derivation of the RfC 47
2.2.4. Confidence Statement 48
2.2.5. Previous IRIS Assessment: Reference Concentration 49
2.3. Cancer Risk Estimates 49
3. REFERENCES 51
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LIST OF TABLES
Table 1-1. Respiratory effects in humans following inhalation exposure 6
Table 1-2. Respiratory effects in animals following inhalation exposure 9
Table 1-3. Gastrointestinal effects in animals following oral exposure 16
Table 1-4. Reproductive and developmental effects in animals following inhalation exposure 18
Table 1-5. Immune system effects in animals following inhalation exposure 20
Table 1-6. Systemic effects in humans following inhalation exposure 24
Table 1-7. Systemic effects in animals following oral exposure 24
Table 1-8. Systemic effects in animals following inhalation exposure 24
Table 1-9. Cancer bioassays following oral exposure 32
LIST OF FIGURES
Figure 1-1. Exposure-response array for respiratory effects following inhalation exposure 13
Figure 1-2. Exposure-response array for immune system effects following inhalation exposure 21
Figure 1-3. Exposure-response array for systemic effects following inhalation exposure 30
Figure 1-4. Exposure-response array for toxicological effects following inhalation exposure 38
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ABBREVIATIONS
ACGIH American Conference of Governmental 58 UFA
Industrial Hygienists 59 UFH
ALP alkaline phosphatase 60 UFL
ALT alanine aminotransferase 61 UFS
AST aspartate aminotransferase 62 UFD
ATSDR Agency for Toxic Substances and Disease 63
Registry 64
ATSG acid-treated silica gel
BAL bronchioalveolar lavage
BMD benchmark dose
BMI body mass index
BrDU 5-bromo-2-deoxyuridine
BUN blood urea nitrogen
CAC cumulative ammonia concentration
CASRN Chemical Abstracts Service Registry
Number
CI confidence interval
EPA Environmental Protection Agency
EU endotoxin unit
FEF forced expiratory flow
FEVi forced expiratory volume in 1 second
FVC forced vital capacity
GABA y-amino butyric acid
IgE immunoglobin E
IgG immunoglobin G
IRIS Integrated Risk Information System
LOAEL lowest-observed-adverse-effect level
MAO monoamine oxidase
MMEF mean midexpiratory flow
MNNG N-methy 1-N' -nitro -N-nitro soguanidine
MRM murine respiratory mycoplasmosis
NH3 ammonia
NH4+ ammonium ion
NIOSH National Institute for Occupational Safety
and Health
NOAEL no-observed-adverse-effect level
NOx nitrogen oxide
NRC National Research Council
OR odds ratio
PBPK physiologically based pharmacokinetic
PEF peak expiratory flow
PEFR peak expiratory flow rate
PHA phytohemagglutin
POD point of departure
PPD purified protein derivative
RfC inhalation reference concentration
RfD oral reference dose
RNA ribonucleic acid
SD standard deviation
SIFT-MS selected ion flow tube mass spectrometry
TLV threshold limit value
TWA time-weighted average
UF uncertainty factor
interspecies uncertainty factor
intraspecies uncertainty factor
LOAEL to NOAEL uncertainty factor
subchronic-to-chronic uncertainty factor
database deficiencies uncertainty factor
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PREAMBLE
51
1. Scope of the IRIS Program 52
Soon after EPA was established in 1970, it was at ^3
the forefront of developing risk assessment as a 54
science and applying it in decisions to protect human ^5
health and the enviromnent. The Clean Air Act, for ^6
example, mandates that EPA provide "an ample ^7
margin of safety to protect public health"; the Safe ^8
Drinking Water Act, that "no adverse effects on the ^9
health of persons may reasonably be anticipated to 60
occur, allowing an adequate margin of safety." ^ j
Accordingly, EPA relies on health assessments to 52
identify adverse effects and exposure levels below 53
which these effects are not anticipated to occur. 54
IRIS assessments critically review the publicly
available studies to identify adverse health effects of 65
chemicals and to characterize exposure-response 66
relationships. Exceptions are chemicals currently used 67
exclusively as pesticides, ionizing and non-ionizing 68
radiation and criteria air pollutants listed under 69
section 108 of the Clean Air Act (carbon monoxide, 70
lead, nitrogen oxides, ozone, particulate matter, and 71
sulfur oxides; EPA evaluates these in Integrated -jj
Science Assessments). An assessment may cover a 73
single chemical, a group of structurally or 74
toxicologically related chemicals, or a complex 75
mixture. 75
Once a year, the IRIS Program asks EPA 77
programs and regions, other federal agencies, state 73
governments, and the general public to nominate 79
chemicals and mixtures for future assessment or gQ
reassessment. These agents may be found in air, water, ^ |
soil, or sediment. Selection is based on program and ^2
regional office priorities and on availability of §3
adequate information to evaluate the potential for ^
adverse effects. IRIS can assess other agents as an ^
urgent public health need arises. IRIS also reassesses
agents as significant new data are published. 86
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2. Process for developing and peer- 88
reviewing IRIS assessments ^
The process for developing IRIS assessments 91
(revised in May 2009) involves systematic review of 92
the pertinent studies, opportunities for public input,
and multiple levels of scientific review. EPA revises ^
draft assessments after each review, and external drafts (j -
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and comments become part of the public record (U.S.
EPA, 2009).
Step 1. Development of a draft Toxicological
Review (usually about 11-1/2 months duration).
The draft assessment considers all pertinent
publicly available studies and applies consistent
criteria to evaluate the studies, identify health
effects, weigh the evidence of causation for each
effect, identify mechanistic events and pathways,
and derive toxicity values.
Step 2. Internal review by scientists in EPA
programs and regions (2 months). The draft
assessment is revised to address comments from
within EPA.
Step 3. Interagency science consultation with other
federal agencies and White House offices (1-1/2
months). The draft assessment is revised to
address the interagency comments. The science
consultation draft, interagency comments, and
EPA's response to major comments become part
of the public record.
Step 4. External peer review, after public review
and comment (3-1/2 months or more, depending
on the review process). EPA releases the draft
assessment for public review and comment,
followed by external peer review. The peer review
meeting is open to the public and includes time
for oral public comments. The peer reviewers also
receive the written public comments. The peer
reviewers assess whether the evidence has been
assembled and evaluated according to guidelines
and whether the conclusions are justified by the
evidence. The peer review draft, peer review
report, and written public comments become part
of the public record.
Step 5. Revision of draft Toxicological Review and
development of draft IRIS summary
(2 months). The draft assessment is revised to
reflect the peer review comments, public
comments, and newly available studies. The
disposition of peer review comments and public
comments becomes part of the public record.
Step 6. Final EPA review and interagency science
discussion with other federal agencies and
White House offices (1-1/2 months). The draft
assessment and summary are revised to address
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EPA and interagency comments. The science
discussion draft, written interagency comments,
and EPA's response to major comments become
part of the public record.
Step 7. Completion and posting (1 month). The
Toxicological Review and IRIS summary are
posted on the IRIS website (http://.epa.gov//).
The remainder of this Preamble addresses step 1,
the development of a draft Toxicological Review. IRIS
assessments follow standard practices of evidence
evaluation and peer review, many of which are
discussed in EPA guidelines (U.S. EPA, 1986a,
1986b, 1991, 1996, 1998, 2000a, 2005a, 2005b,
2006a) and other descriptions of "best practices" (U.S.
EPA, 1994, 2000b, 2002, 2006b, 2011a). Transparent
application of scientific judgment is of paramount
importance. To provide a harmonized approach across
IRIS assessments, this Preamble summarizes concepts
from these guidelines and emphasizes principles of
general applicability.
3. Identifying and selecting pertinent
studies
3.1 Identifying studies
Before beginning an assessment, EPA conducts a
comprehensive search of the primary scientific
literature. The literature search follows standard
practices and includes the PubMed and ToxNet
databases of the National Library of Medicine and
other databases listed in EPA's HERO system (Health
and Environmental Research Online, http://.epa.gov/).
Each assessment specifies the search strategies,
keywords, and cut-off dates of its literature searches.
EPA posts the results of the literature search on the
IRIS website and requests information from the public
on additional studies and ongoing research.
Each assessment also considers studies received
through the IRIS Submission Desk and studies
(typically unpublished) submitted to EPA under the
Toxic Substances Control Act. If a study that may be
critical to the conclusions of the assessment has not
been peer-reviewed, EPA will have it peer-reviewed.
EPA also examines the toxicokinetics of the agent
to identify other chemicals (for example, major
metabolites of the agent) to include in the assessment
if adequate information is available, in order to more
fully explain the toxicity of the agent and to suggest
dose metrics for subsequent modeling.
In assessments of chemical mixtures, mixture
studies are preferred for their ability to reflect
interactions among components (U.S. EPA, 1986a,
2000a). The literature search seeks, in decreasing
order of preference:
Studies of the mixture being assessed.
Studies of a sufficiently similar mixture. In
evaluating similarity, the assessment considers the
alteration of mixtures in the environment through
partitioning and transformation.
Studies of individual chemical components of the
mixture, if there are not adequate studies of
sufficiently similar mixtures.
3.2 Selecting pertinent epidemiologic studies
Study design is the key consideration for selecting
pertinent epidemiologic studies from the results of the
literature search.
Cohort studies and case-control studies provide
the strongest epidemiologic evidence, as they
collect information about individual exposures
and disease.
Cross-sectional studies provide useful evidence if
they relate exposures and disease at the individual
level and it is clear that exposure preceded the
onset of disease.
Ecologic studies (geographic correlation studies)
relate exposures and disease by geographic area.
They can provide strong evidence if there are
large exposure contrasts between geographic
areas, relatively little exposure variation within
study areas, and population migration is limited.
Case reports of high or accidental exposure lack
definition of the population at risk and the
expected number of cases. They can provide
information about a rare disease or about the
relevance of analogous results in animals.
The assessment briefly reviews ecologic studies
and case reports but includes details only if they
suggest effects not identified by other epidemiologic
studies.
3.3 Selecting pertinent experimental studies
Exposure route is a key design consideration for
selecting pertinent experimental studies from the
results of the literature search.
Studies of oral, inhalation, or dermal exposure
involve passage through an absorption barrier and
are considered most pertinent to human
environmental exposure.
Injection or implantation studies are often
considered less pertinent but may provide
valuable toxicokinetic or mechanistic information.
They also may be useful for identifying effects in
animals if deposition or absorption is problematic
(for example, for particles and fibers).
Exposure duration is also a key design
consideration for selecting pertinent experimental
studies.
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1 - Studies of effects from chronic exposure are most
2 pertinent to lifetime human exposure.
3 - Studies of effects from subchronic exposure are
4 pertinent but less preferred than studies of chronic
5 exposure.
6 - Short-term and acute studies are less pertinent but
7 are useful for obtaining toxicokinetic or
8 mechanistic information. The assessment reviews
9 short-term and acute studies if they suggest
10 distribution or effects at a site not identified by
11 longer-term studies.
12 - For developmental toxicity and reproductive
13 toxicity, irreversible effects may result from a
14 brief exposure during a critical period of
15 development. Accordingly, specialized study
16 designs are used for these effects (U.S. EPA,
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18 4. Evaluating the quality of individual
19 studies
20 4.1 Evaluating the quality of epidemiologic
21 studies
22 The assessment evaluates design and
23 methodologic aspects that can increase or decrease the
24 weight given to each epidemiologic study in the
25 overall evaluation (U.S. EPA, 1991, 1994, 1996a,
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Documentation of study design, methods,
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population characteristics, and results.
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Definition and selection of the study and
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comparison populations.
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Ascertainment of exposure and the potential for
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misclassification.
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Ascertainment of disease or effect and the
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potential for misclassification.
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Duration of exposure and follow-up and adequacy
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for assessing the occurrence of effects, including
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latent effects.
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Characterization of exposure during critical
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periods for the development of effects.
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Sample size and statistical power to detect
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anticipated effects.
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Participation rates and the resulting potential for
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selection bias.
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Potential confounding and other sources of bias
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are identified and addressed in the study design or
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in the analysis of results. The basis for
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consideration of confounding is a reasonable
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expectation that the confounder is prevalent in the
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population and is related to both exposure and
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outcome.
For developmental toxicity, reproductive toxicity,
neurotoxicity, and cancer there is further guidance on
the nuances of evaluating epidemiologic studies of
these effects (U.S. EPA, 1991, 1996, 1998, 2005a).
4.2 Evaluating the quality of experimental
studies
The assessment evaluates design and
methodologic aspects that can increase or decrease the
weight given to each experimental study in the overall
evaluation (U.S. EPA, 1991, 1994, 1996, 1998,
2005a):
Documentation of study design, animals or study
population, methods, basic data, and results.
Relevance of the animal model or study
population and the experimental methods.
Characterization of the nature and extent of
impurities and contaminants of the administered
chemical or mixture.
Characterization of dose and dosing regimen
(including age at exposure) and their adequacy to
elicit adverse effects, including latent effects.
Sample sizes and statistical power to detect dose-
related differences or trends.
Ascertainment of survival, vital signs, disease or
effects, and cause of death.
Control of other variables that could influence the
occurrence of effects.
The assessment uses statistical tests to evaluate
whether the observations may be due to chance. The
standard for determining statistical significance of a
response is a trend test or comparison of outcomes in
the exposed groups against those of concurrent
controls. In some situations, examination of historical
control data from the same laboratory within a few
years of the study may improve the analysis. For an
uncommon effect that is not statistically significant
compared with concurrent controls, historical controls
may show that the effect is unlikely to be due to
chance. For a response that appears significant against
a concurrent control response that is unusual, historical
controls may offer a different interpretation (U.S.
EPA, 2005a).
For developmental toxicity, reproductive toxicity,
neurotoxicity, and cancer there is further guidance on
the nuances of evaluating experimental studies of
these effects (U.S. EPA, 1991, 1996, 1998, 2005a). In
multi-generation studies, agents that produce
developmental effects at doses that are not toxic to the
maternal animal are of special concern. Effects that
occur at doses associated with mild maternal toxicity
are not assumed to result only from maternal toxicity.
Moreover, maternal effects may be reversible, while
effects on the offspring may be permanent (U.S. EPA,
1991, 1998).
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4.3 Reporting study results
The assessment uses evidence tables to report
details of the design and key results of pertinent
studies. There may be separate tables for each site of
toxicity or type of study.
If a large number of studies observe the same
effect, the assessment considers the study
characteristics in this section to identify the strongest
studies or types of study. The tables report details
from these studies, and the assessment explains the
reasons for not reporting details of other studies or
groups of studies that do not add new information.
Supplemental material provides references to all
studies considered, including those not summarized in
the tables.
The assessment discusses strengths and
limitations that affect the interpretation of each study.
If the interpretation of a study in the assessment differs
from that of the study authors, the assessment
discusses the basis for the difference.
As a check on the selection and evaluation of
pertinent studies, EPA asks peer reviewers to identify
studies that were not adequately considered.
5. Weighing the overall evidence of
each effect
5.1 Weighing epidemiologic evidence
For each effect, the assessment evaluates the
evidence from the epidemiologic studies as a whole to
determine the extent to which any observed
associations may be causal. Positive, negative, and
null results are given weight according to study
quality. This evaluation considers aspects of an
association that suggest causality, discussed by Hill
(1965) and elaborated by Rothman and Greenland
(1998) (U.S. EPA, 1994, 2002, 2005a; DHHS, 2004).
Strength of association: The finding of a large
relative risk with narrow confidence intervals
strongly suggests that an association is not due to
chance, bias, or other factors. Modest relative
risks, however, may reflect a small range of
exposures, an agent of low potency, an increase in
a disease that is common, exposure
misclassification, or other sources of bias.
Consistency of association: An inference of causality
is strengthened if elevated risks are observed in
independent studies of different populations and
exposure scenarios. Reproducibility of findings
constitutes one of the strongest arguments for
causality. Discordant results sometimes reflect
differences in exposure or in confounding factors.
Specificity of association: As originally intended, this
refers to one cause associated with one disease.
Current understanding that many agents cause
multiple diseases and many diseases have
multiple causes make this a less informative
aspect of causality, unless the effect is rare or
unlikely to have multiple causes.
Temporal relationship: A causal interpretation
requires that exposure precede development of the
disease.
Biologic gradient (exposure-response relationship):
Exposure-response relationships strongly suggest
causality. A monotonic increase is not the only
pattern consistent with causality. The presence of
an exposure-response gradient also weighs against
bias and confounding as the source of an
association.
Biologic plausibility: An inference of causality is
strengthened by data demonstrating plausible
biologic mechanisms, if available.
Coherence: An inference of causality is strengthened
by supportive results from animal experiments,
toxicokinetic studies, and short-term tests.
Coherence may also be found in other lines of
evidence, such as changing disease patterns in the
population.
"Natural experiments": A change in exposure that
brings about a change in disease frequency
provides strong evidence of causality.
Analogy: Information on structural analogues or on
chemicals that induce similar mechanistic events
can provide insight into causality.
These considerations are consistent with
contemporary guidelines that evaluate the quality and
weight of evidence. Confidence is increased if the
magnitude of effect is large, if there is evidence of an
exposure-response relationship, or if an association
was observed and the plausible biases would tend to
decrease the magnitude of the reported effect.
Confidence is decreased for study limitations,
inconsistency of results, indirectness of evidence,
imprecision, or reporting bias (Guyatt et al., 2008a,b).
To make clear how much the epidemiologic
evidence contributes to the overall weight of the
evidence, the assessment may choose a descriptor such
as sufficient evidence, suggestive evidence, inadequate
evidence, or evidence suggestive of no causal
relationship to characterize the epidemiologic
evidence of each effect (DHHS, 2004).
5.2 Weighing experimental evidence
For each effect, the assessment evaluates the
evidence from the animal experiments as a whole to
determine the extent to which they indicate a potential
for effects in humans. Consistent results across various
species and strains increase confidence that similar
results would occur in humans. Although causality is
not at issue in controlled experiments, several concepts
discussed by Hill (1965) affect the weight of
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experimental results: consistency of response, dose-
response relationships, strength of response, biologic
plausibility, and coherence (U.S. EPA, 1994, 2002,
2005a).
In weighing evidence from multiple experiments,
EPA (2005a) distinguishes
Conflicting evidence (that is, mixed positive and
negative results in the same sex and strain using a
similar study protocol) from
Differing results (that is, positive results and negative
results are in different sexes or strains or use
different study protocols).
Negative or null results do not invalidate positive
results in a different experimental system. EPA
regards all as valid observations and looks to
mechanistic information, if available, to reconcile
differing results.
It is well established that there are critical periods
for some developmental and reproductive effects.
Accordingly, the assessment determines whether
critical periods have been adequately investigated
(U.S. EPA, 1991, 1996, 1998, 2005a, 2005b).
Similarly, the assessment determines whether the
database is adequate to evaluate other critical sites and
effects.
5.3 Characterizing modes of action
For each effect, the assessment discusses the
available information on its modes of action and
associated key events (key events being empirically
observable, necessary precursor steps or biologic
markers of such steps; mode of action being a series of
key events involving interaction with cells, operational
and anatomic changes, and resulting in disease).
Pertinent information may also come from studies of
metabolites or of compounds that are structurally
similar or that act through similar mechanisms. The
assessment addresses several questions about each
hypothesized mode of action (U.S. EPA, 2005a).
(a) Is the hypothesized mode of action sufficiently
supported in test animals? Strong support for a
key event being necessary to a mode of action can
come from experimental challenge to the
hypothesized mode of action, where suppressing a
key event suppresses the disease. Support for a
mode of action is meaningfully strengthened by
consistent results in different experimental
models, but not by replicate experiments in the
same model. The assessment may consider
various aspects of causality in addressing this
question.
(b) Is the hypothesized mode of action relevant to
humans? The assessment reviews the key events
to identify critical similarities and differences
between the test animals and humans. Site
concordance is not assumed between animals and
humans, though it may hold for certain modes of
action. Information suggesting quantitative
differences is considered in dose-response
analyses but is not used to determine relevance.
Similarly, anticipated levels of human exposure
are not used to determine relevance.
(c) Which populations or life-stages can be
particularly susceptible to the hypothesized
mode of action? The assessment reviews the key
events to identify populations and life-stages that
might be susceptible to their occurrence.
Quantitative differences may result in separate
toxicity values for susceptible populations or life-
stages.
The assessment discusses the likelihood that an
agent operates through multiple modes of action. An
uneven level of support for different modes of action
can reflect disproportionate resources spent
investigating them (U.S. EPA, 2005a). It should be
noted that in clinical reviews, the quality of evidence
may be reduced if evidence is limited to studies
funded by one interested sector (Guyatt et al., 2008b).
Studies of genetic toxicity are often available, and
the assessment evaluates the evidence of a mutagenic
mode of action.
Demonstration of gene mutations, chromosome
aberrations, or aneuploidy in humans or
experimental mammals {in vivo) provides the
strongest evidence.
This is followed by positive results in lower
organisms or in cultured cells {in vitro) or for
other genetic events.
Negative results carry less weight, partly because
they cannot exclude the possibility of effects in
other tissues (IARC, 2006).
For germ-cell mutagenicity, EPA has defined
categories of evidence, ranging from positive results of
human germ-cell mutagenicity to negative results for
all effects of concern (U.S. EPA, 1986b).
5.4 Characterizing the overall weight of the
evidence
After weighing the epidemiologic and
experimental studies pertinent to each effect, the
assessment may select a standard descriptor to
characterize the overall weight of the evidence. For
example, the following standard descriptors combine
epidemiologic, experimental, and mechanistic
evidence of carcinogenicity (U.S. EPA, 2005a).
Carcinogenic to humans: There is convincing
epidemiologic evidence of a causal association
(that is, there is reasonable confidence that the
association cannot be fully explained by chance,
bias, or confounding); or there is strong human
evidence of cancer or its precursors, extensive
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animal evidence, identification of key precursor
events in animals, and strong evidence that they
are anticipated to occur in humans.
Likely to be carcinogenic to humans: The evidence
demonstrates a potential hazard to humans but
does not meet the criteria for carcinogenic. There
may be a plausible association in humans,
multiple positive results in animals, or a
combination of human, animal, or other
experimental data.
Suggestive evidence of carcinogenic potential: The
data raise concern for effects in humans but are
not sufficient for a stronger conclusion. This
descriptor covers a range of evidence, from a
positive result in the only available study to a
single positive result in an extensive database that
includes negative results in other species.
Inadequate information to assess carcinogenic
potential: No other descriptors apply. Conflicting
evidence can be classified as inadequate
information if all positive results are opposed by
negative studies of equal quality in the same sex
and strain. Differing results, however, can be
classified as suggestive evidence or as likely to be
carcinogenic.
Not likely to be carcinogenic to humans: There are
robust data for concluding that there is no basis
for concern. There may be no effects in both sexes
of at least two appropriate animal species; positive
animal results and strong, consistent evidence that
each mode of action in animals does not operate
in humans; or convincing evidence that effects are
not likely by a particular exposure route or below
a defined dose.
6. Selecting studies for derivation of
toxicity values
For each effect associated with an agent, the
assessment derives toxicity values if there are suitable
epidemiologic or experimental data. The derivation of
toxicity values may be linked to the weight-of-
evidence descriptor. For example, EPA typically
derives toxicity values for agents classified as
carcinogenic to humans or likely to be carcinogenic,
but not for agents with inadequate information or that
are not likely to be carcinogenic (U.S. EPA, 2005a).
Dose-response analysis requires quantitative
measures of dose and response. Then, other factors
being equal (U.S. EPA, 1994, 2005a):
Epidemiologic studies are preferred over animal
studies, if quantitative measures of exposure are
available and effects can be attributed to the
agent.
Among experimental animal models, those that
respond most like humans are preferred, if the
comparability of response can be determined.
Studies by a route of human environmental
exposure are preferred, although a validated
toxicokinetic model can be used to extrapolate
across exposure routes.
Studies of longer exposure duration and follow-up
are preferred, to minimize uncertainty about
whether effects are representative of lifetime
exposure.
Studies with multiple exposure levels are
preferred for their ability to provide information
about the shape of the exposure-response curve.
Studies that show an exposure-response gradient
are preferred, as long as lack of a monotonic
relationship at higher exposure levels can be
satisfactorily explained by factors such as
competing toxicity, saturation of absorption or
metabolism, misclassification bias, or selection
bias.
Among studies that show an exposure-response
gradient, those with adequate power to detect
effects at lower exposure levels are preferred, to
minimize the extent of extrapolation to levels
found in the environment.
If a large number of studies are suitable for dose-
response analysis, the assessment considers the study
characteristics in this section to focus on the most
informative data. The assessment explains the reasons
for not analyzing other groups of studies. As a check
on the selection of studies for dose-response analysis,
EPA asks peer reviewers to identify studies that were
not adequately considered.
7. Deriving toxicity values
7.1 General framework for dose-response
analysis
EPA uses a two-step approach that distinguishes
analysis of the observed dose-response data from
inferences about lower doses (U.S. EPA, 2005a).
Within the observed range, the preferred approach
is to use modeling to incorporate a wide range of data
into the analysis. The modeling yields a point of
departure (an exposure level near the lower end of the
observed range, without significant extrapolation to
lower doses) (sections 7.2-7.3).
Extrapolation to lower doses considers what is
known about the modes of action for each effect
(sections 7.4-7.5). An alternative to low-dose
extrapolation is derivation of reference values, which
are calculated by adjusting the point of departure by
factors that account for several sources of uncertainty
and variability (section 7.6).
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Increasingly, EPA is making use of multiple data
sets or combining multiple responses in deriving
toxicity values. EPA also considers multiple dose-
response approaches when they can be supported by
robust data.
7.2 Modeling dose
The preferred approach for analysis of dose is
toxicokinetic modeling because of its ability to
incorporate a wide range of data. The preferred dose
metric would refer to the active agent at the site of its
biologic effect or to a close, reliable surrogate
measure. The active agent may be the administered
chemical or a metabolite. Confidence in the use of a
toxicokinetic model depends on the robustness of its
validation process and on the results of sensitivity
analyses (U.S. EPA, 1994, 2005a, 2006b).
Because toxicokinetic modeling can require many
parameters and more data than are typically available,
EPA has developed standard approaches that can be
applied to typical data sets. These standard approaches
also facilitate comparison across exposure patterns and
species.
Intermittent study exposures are standardized to a
daily average over the duration of exposure. For
chronic effects, daily exposures are averaged over
the lifespan. Exposures during a critical period,
however, are not averaged over a longer duration
(U.S. EPA, 1991, 1996, 1998, 2005a).
Doses are standardized to equivalent human terms
to facilitate comparison of results from different
species.
Oral doses are scaled allometrically using
mg/kg3/4-d as the equivalent dose metric
across species. As allometric scaling is
typically based on adult body weight, it is not
used for early-life exposure or for
developmental effects (U.S. EPA, 2005a,
2011a).
Inhalation exposures are scaled using
dosimetry models that apply species-specific
physiologic and anatomic factors and
consider whether the effect occurs at the site
of first contact or after systemic circulation
(U.S. EPA, 1994).
It can be informative to convert doses across
exposure routes. If this is done, the assessment
describes the underlying data, algorithms, and
assumptions (U.S. EPA, 2005a).
7.3 Modeling response in the range of
observation
Toxicodynamic ("biologically based") modeling
can incorporate data on biologic processes leading to a
disease. Such models require sufficient data to
ascertain a mode of action and to quantitatively
support model parameters associated with its key
events. Because different models may provide
equivalent fits to the observed data but diverge
substantially at lower doses, critical biologic
parameters should be measured from laboratory
studies, not by model fitting. Confidence in the use of
a toxicodynamic model depends on the robustness of
its validation process and on the results of sensitivity
analyses. Peer review of the scientific basis and
performance of a model is essential (U.S. EPA,
2005a).
Because toxicodynamic modeling can require
many parameters and more knowledge and data than
are typically available, EPA has developed a standard
set of empirical ("curve-fitting") models (http://
www.epa.gov/ncea/bmds/) that can be applied to
typical data sets, including those that are nonlinear.
EPA has also developed guidance on modeling dose-
response data, assessing model fit, selecting suitable
models, and reporting modeling results (U.S. EPA,
2000b). Additional judgment or alternative analyses
are used when the procedure fails to yield reliable
results, for example, if the fit is poor, modeling may
be restricted to the lower doses, especially if there is
competing toxicity at higher doses (U.S. EPA, 2005a).
Modeling is used to derive a point of departure
(U.S. EPA, 2000b, 2005a). (See section 7.6 for
alternatives if a point of departure cannot be derived
by modeling.)
For dichotomous responses, the point of departure
is the 95% lower bound on the dose associated
with a small increase of a biologically significant
effect.
If linear extrapolation to lower doses will be
used, a standard value near the low end of the
observable range is used (10% response for
animal data, 1% for epidemiologic data,
depending on the observed response rates).
If nonlinear extrapolation will be used, both
statistical and biologic factors are considered
(10% response for minimally adverse effects,
5% or lower for more severe effects or for
developmental toxicity data on individual
offspring).
For continuous responses, the point of departure is
ideally a level where the effect is considered
minimally adverse. In the absence of such
definition, both statistical and biologic factors are
considered in selecting a response level.
7.4 Extrapolating to lower doses
The purpose of extrapolating to lower doses is to
estimate responses at exposures below the observed
data. Low-dose extrapolation is typically used for
known and likely carcinogens. Low-dose extrapolation
considers what is known about modes of action (U.S.
EPA, 2005a).
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1 (1) If a biologically based model has been developed
2 and validated for the agent, extrapolation may use
3 the fitted model beyond the observed range if
4 significant model uncertainty can be ruled out
5 with reasonable confidence. Below the range
6 where confidence bounds on the predictions are
7 reasonably precise, extrapolation may continue
8 using a linear model.
9 (2) Linear extrapolation is used if the dose-response
10 curve is expected to have a linear component
11 below the point of departure. This includes:
12 - Agents or their metabolites that are DNA-
13 reactive and have direct mutagenic activity.
14 - Agents or their metabolites for which human
15 exposures or body burdens are near doses
16 associated with key events leading to an
17 effect.
18 Linear extrapolation is also used if the evidence is
19 insufficient to establish a mode of action.
20 The result of linear extrapolation is described by
21 an oral slope factor or an inhalation unit risk,
22 which is the slope of the dose-response curve at
23 lower doses.
24 (3) Nonlinear extrapolation is used if there are
25 sufficient data to ascertain the mode of action and
26 to conclude that it is not linear at lower doses, and
27 the agent does not demonstrate mutagenic or other
28 activity consistent with linearity at lower doses. If
29 nonlinear extrapolation is appropriate but no
30 model is developed, a default is to calculate
31 reference values.
32 If linear extrapolation is used, the assessment
33 develops a candidate slope factor or unit risk for each
34 suitable data set. These results are arrayed, using
35 common dose metrics, to show the distribution of
36 relative potency across various effects and
37 experimental systems. The assessment then derives an
38 overall slope factor and an overall unit risk for the
39 agent, considering the various dose-response analyses,
40 the study preferences discussed in section 6, and the
41 possibility of basing a more robust result on multiple
42 data sets.
43 7.5 Considering susceptible populations and
44 life-stages
45 The assessment analyzes the available information
46 on populations and life-stages that may be particularly
47 susceptible to each effect. A tiered approach is used
48 (U.S. EPA, 2005a).
49 (1) If an epidemiologic or experimental study reports
50 quantitative results for a susceptible population or
51 life-stage, these data are analyzed to derive
52 separate toxicity values for susceptible
53 individuals.
(2) If data on risk-related parameters allow
comparison of the general population and
susceptible individuals, these data are used to
adjust the general-population toxicity values for
application to susceptible individuals.
(3) In the absence of chemical-specific data,
application of age-dependent adjustment factors is
recommended for early-life exposure to suspected
carcinogens. There is evidence of early-life
susceptibility to various carcinogenic agents, but
most epidemiologic studies and cancer bioassays
do not include early-life exposure. To address the
potential for early-life susceptibility, EPA
recommends:
10-fold adjustment for exposures before age 2
years.
3-fold adjustment for exposures between ages
2 and 16 years.
These adjustments are generally applied only for a
mutagenic mode of action, though early-life
susceptibility has been observed for several
carcinogens that are not mutagenic (U.S. EPA,
2005b).
7.6 Reference values and uncertainty factors
An oral reference dose or an inhalation reference
concentration is an estimate of an exposure (including
in susceptible subgroups) that is likely to be without
an appreciable risk of adverse health effects over a
lifetime (U.S. EPA, 2002). Reference values are
typically calculated for effects other than cancer and
for suspected carcinogens if a well characterized mode
of action indicates that a threshold can be based on
prevention of an early key event. Reference values
provide no information about risks at exposures above
the reference value.
The assessment characterizes effects that form the
basis for reference values as adverse, considered to be
adverse, or a precursor to an adverse effect. For
developmental, reproductive, and neurotoxicity there
is guidance on adverse effects and their biologic
markers (U.S. EPA, 1991, 1996, 1998).
To account for uncertainty and variability in the
derivation of a lifetime human exposure where effects
are not anticipated to occur, reference values are
calculated by adjusting the point of departure by a
series of uncertainty factors. If a point of departure
cannot be derived by modeling, a no-observed-
adverse-effect level or a lowest-observed-adverse-
effect level is substituted. The assessment discusses
scientific considerations involving several areas of
variability or uncertainty.
Human variation. A factor of 10 is applied to account
for variation in susceptibility across the human
population and the possibility that the available
data may not be representative of individuals who
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are most susceptible to the effect. This factor is
reduced only if the point of departure is derived
specifically for susceptible individuals (not for a
general population that includes both susceptible
and non-susceptible individuals) (U.S. EPA, 1991,
1994, 1996, 1998, 2002).
Animal-to-human extrapolation. A factor of 10 is
applied if animal results are used to make
inferences about humans. This factor is often
regarded as comprising toxicokinetics and
toxicodynamics in equal parts. Accordingly, if the
point of departure is based on toxicokinetic
modeling, dosimetry modeling, or allometric
scaling across species, a factor of 1012 (rounded
to 3) is applied to account for the remaining
uncertainty involving toxicodynamic differences.
An animal-to-human factor is not applied if a
biologically based model adjusts fully for
toxicokinetic and toxicodynamic differences and
residual uncertainty across species (U.S. EPA,
1991, 1994, 1996, 1998, 2002).
Adverse-effect level to no-observed-adverse-effect
level. If a point of departure is based on a lowest-
observed-adverse-effect level, the assessment
must infer a dose where such effects are not
expected. This can be a matter of great
uncertainty, especially if there is no evidence
available at lower doses. A factor of 10 is applied
to account for the uncertainty in making this
inference. A factor other than 10 may be used,
depending on the magnitude and nature of the
response and the shape of the dose-response curve
(U.S. EPA, 1991, 1994, 1996, 1998, 2002).
Subchronic-to-chronic exposure. If a point of
departure is based on subchronic studies, the
assessment considers whether lifetime exposure
would have effects at lower levels. A factor of 10
is applied to account for the uncertainty in using
subchronic studies to make inferences about
lifetime exposure. This factor may also be applied
for developmental or reproductive effects if
exposure covered less than the full critical period.
A factor other than 10 may be used, depending on
the duration of the studies and the nature of the
response (U.S. EPA, 1994, 1998, 2002).
Incomplete database. If an incomplete database
raises concern that further studies might identify a
more sensitive effect, organ system, or life-stage,
the assessment may apply a database uncertainty
factor (U.S. EPA, 1991, 1994, 1996, 1998, 2002).
EPA typically follows the suggestion that a factor
of 10 be applied if both a prenatal toxicity study
and a two-generation reproduction study are
missing, and a factor of 1012 if either is missing
(U.S. EPA, 2002).
In this way, the assessment derives candidate
reference values for each suitable data set and effect
that is plausibly associated with the agent. These
results are arrayed, using common dose metrics, to
show where effects occur across a range of exposures
(U.S. EPA, 1994). The assessment then selects an
overall reference dose and an overall reference
concentration for the agent to represent lifetime human
exposure levels where effects are not anticipated to
occur.
The assessment may also report reference values
for each effect. This would facilitate subsequent
cumulative risk assessments, where it may be
important to consider the combined effect of
chemicals acting at a common site or operating
through common mechanisms (U.S. EPA, 2002).
7.7 Confidence and uncertainty in the
reference values
The assessment selects a standard descriptor to
characterize the level of confidence in each reference
value, based on the likelihood that the value would
change with further testing. Confidence in reference
values is based on quality of the studies used and
completeness of the database, with more weight given
to the latter. The level of confidence is increased for
reference values based on human data supported by
animal data (U.S. EPA, 1994).
High confidence: The reference value is not likely to
change with further testing, except for
mechanistic studies that might affect the
interpretation of prior test results.
Medium confidence: This is a matter of judgment,
between high and low confidence.
Low confidence: The reference value is especially
vulnerable to change with further testing.
These criteria are consistent with contemporary
guidelines that evaluate the quality of evidence. These
also focus on whether further research would be likely
to change confidence in the estimate of effect (Guyatt
et al 2008a).
All assessments discuss the significant
uncertainties encountered in the analysis. EPA
provides guidance on characterization of uncertainty
(U.S. EPA, 2005a). For example, the discussion
distinguishes model uncertainty (lack of knowledge
about the most appropriate experimental or analytic
model), parameter uncertainty (lack of knowledge
about the parameters of a model), and human variation
(interpersonal differences in biologic susceptibility or
in exposures that modify the effects of the agent).
For other general information about this assessment or
other questions relating to IRIS, the reader is referred
to EPA's IRIS Hotline at (202) 566-1676 (phone),
(202) 566-1749 (fax), or hotline.iris@epa.gov (email
address).
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AUTHORS CONTRIBUTORS REVIEWERS
Authors
Audrey Galizia, Dr. PH (Chemical
Manager)
James Ball, Ph.D.
Christopher Brinkerhoff, PhD.
Keith Salazar, PhD.
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Edison, New Jersey
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Washington. DC
Contributors
Louis D'Amico, Ph.D.
Christopher Sheth, Ph.D.
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Washington, DC
Technical Support
Maureen Johnson
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Washington. DC
Contractor Support
SRC, Inc., Chemical and Biological Center, Syracuse, NY
Amber Bacon
Fernando Llados
Julie Stickney
Executive Direction
Vincent Cogliano, PhD.
Samantha Jones, PhD.
Susan Rieth, MPH
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Washington, DC
EPA Internal Reviewers
TedBerner, M.S
Marian Rutigliano, MD
John Whalan
Amanda S. Persad, PhD.
Paul Reinhart, PhD.
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Washington, DC
U.S. EPA
Office of Research and Development
National Center for Enviromnental Assessment
Research Triangle Park, NC
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Reviewers
This assessment was provided for review to scientists in EPA's Program and Regional Offices.
Comments were submitted by the Programs and Regions listed below.
This assessment was provided for review to other federal agencies and White House offices. The
federal agencies and White House offices that commented are listed below. Comments
submitted by the agencies listed below are available on the IRIS website.
Agency for Toxic Substances and Disease Registry, Public Health Service, Department of
Health & Human Services
Office of Public Health Service, Food Safety Inspection Service, U.S. Department of
Agriculture
A public listening session was held by EPA on [month] [date], [year]. Attendees external to the
EPA are listed below.
NAME Affiliation
NAME Affiliation
NAME Affiliation
NAME Affiliation
This assessment was released for public comment on [month] [date], 2012; the public comment
period ended on [month] [date], [year]. Comments were received from the following entities.
NAME Affiliation, Location
NAME Affiliation, Location
NAME Affiliation, Location
NAME Affiliation, Location
This assessment was peer-reviewed by independent expert scientists external to EPA (specify
SAB or NAS panel) and a peer review meeting was held on [month] [date], [year]. The external
peer review comments are available on the IRIS website. EPA's response to the external peer
review and public comments is included in Appendix C and is also available on the IRIS
website.
NAME Affiliation, Location
NAME Affiliation, Location
NAME Affiliation, Location
NAME Affiliation, Location
Rebecca Dzubow
Joyce Donohue
Marian Olsen
Office of Children's Health Protection, Washington DC
Office of Water, Washington, DC
Region 2, New York, New York
xvii
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PREFACE
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to exposure to ammonia. This
document presents background information and justification for the Integrated Risk Information
System (IRIS) Summary of the hazard and dose-response assessment of ammonia. The
appendices to this document include information addressing chemical and physical properties,
ammonium salts, toxicokinetics, toxicity study summaries, and external peer review, and are
included in a separate volume: the Supplemental Information for the Toxicological Review of
Ammonia.
The Toxicological Review of Ammonia is an update of a previous IRIS assessment for
ammonia posted to the IRIS database in 1991. The previous assessment included an inhalation
RfC only. A reassessment of ammonia was conducted because of concerns related to ammonia
emissions generated from its use in selective catalytic reduction-based diesel engine
aftertreatment technology to reduce nitrogen oxide (NOx) to N2 gas and the presence of
ammonia at hazardous waste National Priorities List (NPL) sites. Ammonia is found in over 8%
of the hazardous waste NPL sites (AT SDR, 2004).
Portions of this Toxicological Review were developed under a Memorandum of
Understanding with the Agency for Toxic Substances and Disease Registry (ATSDR) and were
adapted from the Toxicological Profile for Ammonia (ATSDR, 2004) as part of a collaborative
effort in the development of human health toxicological assessments for the purposes of making
more efficient use of available resources and to share scientific information.
Background
Ammonia is a corrosive gas with a very pungent odor (O'Neil et al., 2006). It is highly
soluble in water (4.82 x 105 mg/L) and is a weak base (Lide, 2008; Eggeman, 2001; Dean, 1985).
When ammonia (NH3) is present in water at environmental pH, a pKa of 9.25 indicates that the
equilibrium will favor the formation of the conjugate acid, the ammonium ion (NH4 ) (Lide,
2008). A solution of ammonia in water is sometimes referred to as ammonium hydroxide
because the ammonia and water both ionize to form ammonium cations and hydroxide anions
(Eggeman, 2001). Ammonium salts are easily dissolved in water and disassociate into the
ammonium ion and the anion. At physiological pH (7.4), the equilibrium between NH3 and
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NH4+ favors the formation of NH4+. Additional chemical and physical properties information for
ammonia is provided in Appendix A, Section A.l.
Low levels of ammonia occur naturally in the environment in air, soil, and water.
Ammonia is a major component of the geochemical nitrogen cycle and is essential for many
biological processes (Rosswall, 1981). Nitrogen-fixing bacteria convert atmospheric nitrogen
into ammonia available for plant uptake (Socolow, 1999; Rosswall, 1981). Organic nitrogen
released from biota is converted into ammonia through nitrogen mineralization (Rosswall, 1981).
Ammonia in water and soil is naturally converted into nitrite and nitrate through the process of
nitrification (Rosswall, 1981). Ammonia is endogenously produced in humans and animals, is
an essential mammalian metabolite used in nucleic acid and protein synthesis, is necessary for
maintaining acid-base balance, and is an integral part of nitrogen homeostasis (Nelson and Cox,
2008).
With regard to exogenous exposure, the largest and most significant use of ammonia is
the agricultural application of fertilizers, which represents about 80-85% of commercially-
produced ammonia in the form of urea, ammonium nitrate, ammonium sulfate, ammonium
phosphate, and other nitrogen compounds (Eggeman, 2001). Ammonia is also used as a
corrosion inhibitor, in the purification of water supplies, as a component of household cleaners,
as a refrigerant, as a chemical intermediate in pharmaceuticals, explosives and other chemicals,
as a stabilizer in the rubber industry, and as a hydrogen source for the hydrogenation of fats and
oils. Ammonia (generated from urea injected into the exhaust stream) is also used in the
reduction of NOx emissions from the exhaust of diesel vehicles and stationary combustion
sources such as industrial and municipal boilers and power generators (Eggeman, 2001; HSDB,
2009; Johnson et al., 2009).
Scope of the Assessment
This assessment presents a review of hazard and dose-response information for ammonia,
including gaseous ammonia (NH3) and ammonia dissolved in water (ammonium hydroxide,
NH4OH). Because ammonium salts (e.g., ammonium acetate, chloride, and sulfate) readily
dissolve in water through disassociation into the ammonium ion (NH4"1") and the anion, EPA
considered whether or not the literature on ammonium salts could inform the toxicity of
ammonia. The toxicology literature for ammonium salts includes several oral toxicity studies of
ammonium chloride and ammonium sulfate. No inhalation toxicity studies of ammonium salts
are available. The toxicity data for ammonium chloride and ammonium sulfate demonstrate that
these two salts present distinctly different toxicity profiles, suggesting that the anion can
influence the toxicity of the ammonium compound, and that the toxicity of the salts cannot
necessarily be attributed to the cation (i.e., NH4+) only (for detailed ammonium salts information
see Appendix A, Section A.2 and Table A-2). Accordingly, information on the toxicity of
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1 ammonium salts was not used to characterize the toxicity of ammonia or ammonium hydroxide
2 in this assessment.
3
4 Other Agency and International Assessments
5 Assessments and regulatory limits for ammonia developed by other health agencies,
6 including the Agency for Toxic Substances and Disease Registry (ATSDR), the National
7 Research Council (NRC), the American Conference of Governmental Industrial Hygienists
8 (ACGIH), the National Institute of Occupational Safety and Health (NIOSH), and the Food and
9 Drug Administration (FDA), are identified in Appendix B of the Supplemental Material.
10
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EXECUTIVE SUMMARY
Effects other than cancer observed following oral exposure
The oral toxicity database for ammonia is very limited. Gastric toxicity is identified as a
hazard for ammonia based on evidence from case reports in humans, two animal studies, and
mechanistic studies. Evidence in humans is limited to case reports of individuals suffering from
gastrointestinal effects from ingesting household cleaning solutions containing ammonia or
biting into capsules of ammonia smelling salts. In rats, gastrointestinal effects, characterized as
increased epithelial cell migration in the mucosa of the stomach and decreased thickness of the
gastric mucosa, were reported following subchronic and short-term exposure to ammonia. These
gastric mucosal effects observed in rats resemble mucosal changes in human atrophic gastritis;
indicating this effect is biological plausible and relevant to humans.
Given the limited number of studies available and the small number of toxicological
evaluations, there are uncertainties associated with the oral database for ammonia and a RfD for
ammonia was not derived.
Effects other than cancer observed following inhalation exposure
Respiratory effects have been identified as a hazard following inhalation exposure to
ammonia. Evidence for respiratory toxicity associated with exposure to ammonia comes from
studies in humans and animals. Cross-sectional occupational studies involving chronic exposure
to ammonia have consistently demonstrated an increased prevalence of respiratory effects and
decreased lung function. Cross-sectional studies of livestock farmers exposed to ammonia,
controlled human volunteer studies of ammonia inhalation, and case reports of injury in humans
with inhalation exposure to ammonia provide additional and consistent support for the
respiratory system as a target of ammonia toxicity. Additionally, respiratory effects were
observed in several animal species following subchronic and short-term exposures to ammonia.
The experimental toxicology literature for ammonia also provides evidence that inhaled
ammonia may be associated with toxicity to target organs other than the respiratory system,
including the liver, adrenal gland, kidney, spleen, heart, and immune system. The weight of
evidence for these effects is less robust than for respiratory effects.
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Inhalation reference concentration (RfC) for effects other than cancer
Table ES-1. Reference Concentration
Critical Effect
Point of Departure*
UF
Chronic RfC
Lack of decreased lung
function and increased
respiratory irritation
NOAELadj: 3.1 mg/m3
10
0.3 mg/m3
Occupational epidemiology study
Holness et al., 1989
*Because the POD (NOAEL = 8.8 mg/m3) involved workplace exposure conditions, the NOAEL was
adjusted for continuous exposure based on the ratio of VEho (human occupational default minute
volume of 10 m3 breathed during an 8-hour workday) to VEh (human ambient default minute volume
of 20 m3 breathed during the entire day) and an exposure of 5 days out of 7 days.
The occupational exposure study of ammonia exposure in workers in a soda ash plant by
Holness et al. (1989) was identified as the principal study for RfC derivation. Respiratory
effects, characterized as increased respiratory irritation and decreased lung function, observed in
workers exposed to ammonia were selected as the critical effect. In the evaluation of the
prevalence of increased respiratory irritation and decreased lung function in workers exposed to
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ammonia (Holness et al., 1989), aNOAELadj of 3.1 mg/m (adjusted for continuous exposure
"3
from 8.8 mg/m ; see calculation below) was identified based on the absence of statistically
significant increases in the prevalence of the respiratory effects. BMD modeling was not utilized
because ammonia concentrations in the Holness et al. (1989) study were not associated with
changes in respiratory effects in the study population (i.e., data from Holness et al. could not be
subjected to dose-response modeling). Thus, the Holness et al. (1989) data were analyzed using
-2
a NOAEL approach and the NOAELadj of 3.1 mg/m was used as the POD for RfC
derivation.
The RfC was calculated by dividing the POD (i.e., NOAELadj) by a composite
uncertainty factor (UF) of 10 to account for potentially susceptible individuals in the absence
of data evaluating variability of response to inhaled ammonia in the human population.
Confidence in the chronic inhalation RfC
Study - medium
Database - medium
RfC - medium
The overall confidence in the RfC is medium and reflects medium confidence in the
principal study (adequate design, conduct, and reporting of the principal study; limited by small
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sample size and identification of a NOAEL only) and medium confidence in the database, which
includes occupational and volunteer studies and studies in animals that are mostly of subchronic
duration. Although there are no studies of developmental toxicity and studies of reproductive
and other systemic endpoints are limited, reproductive, developmental, and other systemic
effects are not expected at the RfC because it is well documented that ammonia is endogenously
produced in humans and animals, ammonia concentrations in blood are homeostatically
regulated to remain at low levels, and ammonia concentrations in air at the POD are not expected
to alter homeostasis.
Evidence for human carcinogenicity
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is
"inadequate information to assess the carcinogenic potential" of ammonia based on the
absence of ammonia carcinogenicity studies in humans and a single lifetime drinking water study
of ammonia in mice that showed no evidence of carcinogenic potential. There is limited
evidence that ammonia may act as a cancer promoter based on the findings of H. pylori-induced
gastric cancer. The available studies of ammonia genotoxicity are inadequate to characterize the
genotoxic potential of this compound. A quantitative cancer assessment for ammonia was
not conducted.
Susceptible Populations and Life Stages
Hyperammonemia is a condition of elevated levels of circulating ammonia that can occur
in individuals with severe diseases of the liver or kidney or with hereditary urea cycle disorders.
These elevated ammonia levels can predispose an individual to encephalopathy due to the ability
of ammonia to cross the blood-brain barrier; these effects are especially marked in newborn
infants. Thus, individuals with disease conditions that lead to hyperammonemia may be more
susceptible to the effects of ammonia from external sources, but there are no studies that
specifically support this susceptibility. Studies of the toxicity of ammonia in children or young
animals compared to other life stages that would support an evaluation of childhood
susceptibility have not been conducted.
Key issues addressed in assessment
Endogenous ammonia
Ammonia, which is produced endogenously, has been detected in the expired air of
healthy volunteers. Higher and more variable ammonia concentrations are reported in breath
"3
exhaled from the mouth or oral cavity (0.09 to 2.1 mg/m ). These levels are largely attributed to
the production of ammonia via bacterial degradation of food protein in the oral cavity or
gastrointestinal tract, and can be influenced by factors such as diet, oral hygiene, age, and living
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conditions (i.e., urban vs. rural setting). In contrast, ammonia concentrations measured in breath
exhaled from the nose and trachea are lower (0.013-0.078 mg/m ) and more likely reflect levels
"3
of ammonia circulating in the blood. These levels are lower than the ammonia RfC of 0.3 mg/m
by a factor of approximately fourfold or more. Although the RfC falls within the range of breath
concentrations collected from the mouth or oral cavity, ammonia in exhaled breath is expected to
be rapidly diluted in the much larger volume of ambient air.
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LITERATURE SEARCH STRATEGY & STUDY
EVALUATION FOR HAZARD IDENTIFICATION
Literature Search Strategy and Study Selection
The literature search strategy employed for ammonia was conducted with the keywords
listed in Table LS-1. Primary, peer-reviewed literature was identified through a literature search
using the databases listed in Table LS-1. The literature search was last conducted on November
11, 2011. A data call-in was announced by EPA on December 21, 2007 (U.S. EPA, 2007); no
submissions in response to the data call-in were received. Other peer-reviewed information,
including health assessments developed by other health agencies, review articles, and
independent analyses of the health effects data were retrieved and may be included in the
assessment where appropriate.
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Table LS-1. Details of the search strategy employed for ammonia
Databases
Limits
Keywords
Pubmed
Search constraints: 2003-currentb
Chemical name and synonyms3:
Toxcenter
Pre-2003—ATSDR (2004) was
ammonia (7664-41-7); ammonium hydroxide
Toxline
used as the source of references
(1336-21-6); ammonium; spirit of hartshorn;
Current Contents (2008
published before 2003
aquammonia
& 2010 only)
Last search: November 11, 2011
Other keywords:
toxicity (including duration, effects to children
and occupational exposure); development;
reproduction; teratogenicity; exposure routes;
pharmacokinetics; toxicokinetics; metabolism;
body fluids; endocrinology; carcinogenicity;
genotoxicity; antagonists; inhibitors; respiration;
metabolism; breath tests; inhalation; air; breath;
exhalation; biological markers; analysis
TSCATS
2011
NA
ChemID
2011
NA
Chemfinder
2011
NA
CCRIS
2011
NA
HSDB
2011
NA
GENETOX
2008
NA
RTECS
2011
NA
aThe initial search conducted in 2008 included ammonia salts (i.e., ammonium nitrate [6484-52-2], ammonium fluoride
[12125-01-8], ammonium sulfate [7783-20-2], ammonium persulfate [7727-54-0], and ammonium chloride [12125-02-9]) as
keywords. Once the determination was made not to include data on ammonium salts in the assessment, updated searches
focused on ammonia and ammonium hydroxide only.
bThe search using search terms related to concentrations of ammonia in exhaled breath was conducted for the period
1/1/2002-11/11/2011.
Approximately 4,900 references were identified in the literature search for ammonia
using the literature search strategy identified in Table LS-1; the references captured in this search
can be found on the EPA's HERO website.1 From this list, approximately 250 references were
identified that provided information relevant to the human health effects of ammonia or
information on the physical and chemical properties of ammonia.
The references cited in this document, as well as those that were considered but not
included in the Toxicological Review of Ammonia, can be found on the HERO website
(http://hero.epa. gov/ i chemical!). This site contains HERO links to lists of references, including
bibliographic information and abstracts, which were considered for inclusion in the
Toxicological Review of Ammonia.
1 HERO (Health and Environmental Research On-line) is a database of scientific studies and other references used to
develop EPA's risk assessments aimed at understanding the health and environmental effects of pollutants and
chemicals. It is developed and managed in EPA's Office of Research and Development (ORD) by the National
Center for Environmental Assessment (NCEA). The database includes more than 300,000 scientific articles from
the peer-reviewed literature. New studies are added continuously to HERO.
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Study Evaluation for Hazard Identification
This document is not intended to be a comprehensive treatise on the chemical or
toxicological nature of ammonia. In general, the quality and relevance of health effects studies
were evaluated as outlined in the Preamble to this assessment. In addition, A Review of the
Reference Dose and Reference Concentration Processes (U.S. EPA, 2002) and Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry
(U.S. EPA, 1994) were consulted for guidance in evaluating the scientific quality of the available
studies.
The health effects literature for ammonia is not extensive; therefore, essentially all of the
available epidemiology and toxicity studies of ammonia and ammonium hydroxide were
considered in the characterization of the potential health hazards associated with ammonia
exposure. As discussed in the preface, literature on ammonium salts were not included in this
review because the available data suggest that the anion of the salt can influence the toxicity of
the ammonium compound. Approximately 100 case reports involving acute ammonia exposure
were identified; because case reports generally provide little information that would be useful for
characterizing chronic health hazard, these studies were only briefly reviewed and citations to
this literature are provided as supplemental materials in Appendix A. Human studies that
provided unreliable measures of exposure (e.g., self-reporting) or intentional dosing studies that
raised concerns of ethical conduct were excluded from consideration; two human studies fell into
this category.
The hazard identification analysis for each health endpoint in Chapter 1 includes a
synthesis of the relevant health effects literature and an analysis of the weight of the evidence for
an association between ammonia exposure and the health effects. The available studies
examining health effects of ammonia exposure in humans (four cross-sectional occupational
exposure studies, studies in livestock farmers and stable workers, and acute controlled-exposure
studies in volunteers) are discussed and evaluated, with specific limitations of individual studies
and of the collection of studies noted. The evaluation of the effects seen in experimental animal
studies focuses on the available subchronic toxicity studies and a single reproductive toxicity
study. Chronic toxicity studies were limited to oral exposure studies that did not adequately
evaluate the noncancer effects of ammonia.
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1. HAZARD IDENTIFICATION
1.1. Synthesis of Major Toxicological Effects
1.1.1. Respiratory Effects
Respiratory Irritation
The respiratory system is the primary target of toxicity of inhaled ammonia in humans
and experimental animals. Symptoms consistent with respiratory irritation were reported in two
cross-sectional studies of industrial worker populations exposed to ammonia (Rahman et al.,
2007; Ballal et al., 1998) (see Table 1-1). Rahman et al. (2007)2 found a higher prevalence, by
up to 20%, of respiratory irritation (cough, chest tightness, runny nose, stuffy nose, and
sneezing) in urea fertilizer factory workers exposed to a mean ammonia concentration of 18.5
mg/m (high-exposure group) for about 16 years compared to a control group (staff workers); the
prevalences of cough and chest tightness were statistically significantly elevated in the high-
exposure ammonia group compared to the control group. Respiratory irritation prevalence
between the low-exposure group exposed to a mean ammonia concentration of 4.9 mg/m was
not statistically significantly different from the control group. Significantly higher relative risks
for cough, phlegm, wheezing, dyspnea, and bronchial asthma were also observed in workers
from another cross-sectional study (Ballal et al., 1998) with ammonia exposure concentrations
higher than the American Conference of Governmental Industrial Hygienists [ACGIH] threshold
limit value [TLV] of 18 mg/m [25 ppm]) compared with workers exposed to levels below the
TLV. Distribution of respiratory irritation effects by cumulative ammonia concentration (CAC,
mg/m -years) also showed significantly higher relative risk for these respiratory irritation effect
among workers with higher CAC (>50 mg/m -years) compared to those with a lower CAC (< 50
mg/m -years) (Ballal et al., 1998). Only Ballal et al. (1998) evaluated respiratory endpoints in
terms of cumulative exposure.
In a third cross-sectional study of male ammonia-exposed workers, no differences were
observed in the prevalence of respiratory irritation, eye irritation, or odor detection threshold
between any of the ammonia-exposed workers and the control group (Holness et al., 1989),
either as one group or when stratified into three exposure categories: high = >8.8 mg/m ,
3 3
medium = 4.4-8.8 mg/m , or low = <4.4 mg/m . Although respiratory irritation prevalence was
similar across groups, the exposed workers reported that exposure in the plant aggravated some
2 Rahman et al. (2007) examined respiratory effects in workers from two plants in a urea fertilizer factory.
Workers in the urea plant were exposed to higher concentrations of ammonia (arithmetic mean ammonia
concentration of 18.5 mg/m3) than workers in the ammonia plant (arithmetic mean ammonia concentration of 4.9
mg/m3). Therefore, the urea plant workers represented the high-exposure group, and the ammonia plant workers
represented the low-exposure group.
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of their reported respiratory symptoms (cough, sputum, chronic bronchitis, wheeze, chest
tightness, dyspnea, chest pain, rhinitis) (no further information provided). Co-exposures to dust
and inorganic gases such as nitrogen dioxide and sulfur dioxide were possible in these cross-
sectional studies; however, except for the low levels of nitrogen dioxide identified in the Rahman
et al. (2007) study, these workplace exposures were not measured or reported.
Overall, these cross-sectional occupational epidemiology studies provide consistent
estimates of the effect level for respiratory irritation by ammonia. Rahman et al. (2007)
"3
observed that exposure to 18.5 mg/m ammonia increased the prevalence of respiratory effects.
This is consistent with the observation by Ballal et al. (1998) that workers in a factory with
-3
ammonia concentrations exceeding the TLV of 18 mg/m had significantly higher relative risks
for respiratory irritation effects. The prevalence of respiratory effects was not increased
"3
following occupational exposures at lower workplace concentrations (i.e., >8.8 mg/m ammonia
[Holness et al., 1989] and 4.9 mg/m3 [Rahman et al., 2007]).
Respiratory irritation, indicated by elevated prevalences of respiratory symptoms,
including cough, phlegm, wheezing, chest tightness, and eye, nasal and throat irritation, has been
reported in livestock farmers and stable workers compared to controls (Melbostad and Eduard,
2001; Preller et al., 1995; Choudat et al., 1994; Zejda et al., 1994; Crook et al., 1991; Heederik et
al., 1990). Additionally, bronchial hyperreactivity to methacholine or histamine challenge was
increased in farmers exposed to ammonia compared to control workers (Vogelzang et al., 2000,
1997; Choudat et al., 1994), indicating that exposure to ammonia and other air contaminants in
farm settings may contribute to chronic airway inflammation. In addition to ammonia, these
studies also documented exposures to airborne dust, bacteria, fungal spores, endotoxin, and
mold—agents that could also induce respiratory effects. The release of other volatiles on
livestock farms is likely, but measurements for other volatile chemicals were not conducted.
Therefore, while several studies have reported associations between ammonia exposure in
livestock farmers or stable workers and respiratory irritation, these findings are limited by
exposures to other constituents in air that likely confound the association between ammonia
exposure and the respiratory effects observed in the study populations.
Support for ammonia as a respiratory irritant is also provided by reports of irritation and
hyperventilation in volunteers acutely exposed to ammonia at concentrations ranging from
"3
11-354 mg/m ammonia for durations up to 4 hours under controlled exposure conditions
(Petrova et al., 2008; Smeets et al., 2007; Altmann et al., 2006; Ihrig et al., 2006; Verberk, 1977;
Silverman et al., 1949) (see Appendix A, Section A.4). Two controlled-exposure studies
reported habituation to eye, nose, and throat irritation in volunteers after several weeks of
ammonia exposure (Ihrig et al., 2006; Ferguson et al., 1977). Numerous case reports document
the acute respiratory effects of inhaled ammonia, ranging from mild symptoms (including nasal
and throat irritation and perceived tightness in the throat) to moderate effects (including
pharyngitis, tachycardia, dyspnea, rapid and shallow breathing, cyanosis, transient
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bronchospasm, and rhonchi in the lungs) to severe effects (including burns of the nasal passages,
soft palate, posterior pharyngeal wall, and larynx, upper airway obstruction, bronchospasm,
dyspnea, persistent, productive cough, bilateral diffuse rales and rhonchi, mucous production,
pulmonary edema, marked hypoxemia, and necrosis of the lung) (see Appendix A, Section A.4,
for more detailed information and references).
Experimental studies in laboratory animals also provide consistent evidence that
ammonia exposure for 35 days or more can produce respiratory irritation. Histopathological
changes in the nasal passages were observed in Sherman rats after 75 days of exposure to 106
3 3
mg/m ammonia or 35 days of exposure to 177 mg/m ammonia, with respiratory and olfactory
epithelium thickness increased three- to four times that of normal thickness (Broderson et al.,
1976). Thickening of nasal and tracheal epithelium (50 to 100%) was observed in pigs exposed
"3
to 71 mg/m ammonia continuously for 1-6 weeks (Doig and Willoughby, 1971). Nonspecific
inflammatory changes (not further described) were reported in the lungs of Sprague-Dawley and
"3
Longs-Evans rats continuously exposed to 127 mg/m ammonia for 90 days and rats and guinea
3 3
pigs intermittently exposed to 770 mg/m ammonia (or 183 mg/m , adjusted to continuous
exposure ) (Coon et al., 1970). Focal or diffuse interstitial pneumonitis was observed in all
Princeton-derived guinea pigs, New Zealand white rabbits, beagle dogs, and squirrel monkeys
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exposed to 470 mg/m ammonia that were examined (Coon et al., 1970). Additionally, under
these exposure conditions, dogs exhibited nasal discharge and other signs of irritation (marked
eye irritation, heavy lacrimation). Nasal discharge was observed in 25% of rats exposed to
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262 mg/m ammonia for 90 days (Coon et al., 1970).
At lower concentrations, approximately 50 mg/m and below, the majority of studies of
inhaled ammonia show that ammonia does not produce respiratory irritation effects in laboratory
animals. No increase in the incidence of respiratory or other diseases common to young pigs
were observed after continuous exposure to ammonia and inhalable dust at concentrations
"3
representative of those found in commercial pig farms (26 mg/m ammonia) for 5 weeks (Done
et al., 2005). No gross or histopathological changes in the turbinates, trachea, and lungs of pigs
"3
were observed after continuous exposure to 53 mg/m ammonia for up to 109 days (Curtis et al.,
"3
1975). No signs of toxicity in rats were observed after continuous exposure to 40 mg/m
3 3
ammonia for 114 days or after intermittent exposure to 155 mg/m ammonia (or 36.9 mg/m ,
adjusted to continuous exposure) for 6 weeks (Coon et al., 1970).
Lung Function
Decreased lung function in ammonia-exposed workers has been reported in two cross-
sectional studies of industrial worker populations (Rahman et al., 2007; Ali et al., 2001) of three
such studies that measured lung function (Rahman et al., 2007; Ali et al., 2001; Holness et al.,
(1989). Ammonia exposure was correlated with a significant decline in lung function over the
Adjusted = C x 8 hours/24 hours x 5 days/7 days, where C is the exposure concentration.
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course of a work shift (cross-shift) as measured by forced vital capacity (FVC) and forced
expiratory volume in one second (FEVi) in the high-exposure worker group (mean ammonia
concentration of 18.5 mg/m ) in a fertilizer factory (Rahman et al., 2007). In a second study (Ali
et al., 2001), the FVC% predicted was higher in fertilizer factory workers exposed to ammonia
than in controls (4.6% increase,/? < 0.002); FEVi was higher (1.5%) in the exposed workers but
the difference was not statistically significant. When Ali et al. (2001) based their analysis on
measures of cumulative exposure, workers with cumulative exposure >50 mg/m -years had
significantly lower FVC% predicted (5.4% decrease, p < 0.030) and FEVi% predicted (7.4%
"3
decrease,/? < 0.006) than workers with cumulative exposure <50 mg/m -years, but similar
FEVi/FVC%. The authors did not explain the inconsistent findings across the analyses of
noncumulative and cumulative exposures.
Lung function did not appear to be affected in worker populations chronically exposed to
ammonia at concentrations below approximately 18 mg/m . Baseline lung function, based on
spirometry conducted at the beginning and end of the work shift, differed very slightly relative to
3 3
control in workers exposed to ammonia concentrations ranging from <4.4 mg/m to >8.8 mg/m
in a cross-sectional study of male workers in a soda ash plant (Holness et al., 1989), but was not
statistically significant. Additionally, no changes in lung function were observed over either
work shift (days 1 or 2) or over the work week in the exposed group compared with controls.
Similarly, measures of lung function (FVC, FEVi, and PEFR [peak expiratory flow rate]) in
"3
workers exposed to a mean concentration of 4.9 mg/m ammonia in a urea fertilizer factory
showed no significant cross-shift changes (Rahman et al., 2007).
Decreased lung function (e.g., measured as decreased FEVi, FVC) was reported in
farmers with ammonia exposure (Cormier et al., 2000; Donham et al., 2000, 1995; Vogelzang et
al., 1998; Reynolds et al., 1996; Preller et al., 1995; Crook et al., 1991; Heederik et al., 1990).
These findings are limited by exposures to other constituents in air (including respirable dust,
bacteria, fungal spores, endotoxin, and mold) that can affect lung function, and likely confound
the association between exposure to ammonia and decreased lung function observed in the study
populations.
Changes in lung function following acute exposure to ammonia have been observed in
some but not all controlled exposure studies conducted in volunteers. Cole et al. (1977) reported
reduced lung function as measured by reduced expiratory minute volume and changes in exercise
tidal volume in volunteers exposed for a half-day in a chamber at ammonia concentrations
3 3
>106 mg/m but not at 71 mg/m . Bronchioconstriction was reported in volunteers exposed to
ammonia through a mouthpiece for 10 inhaled breaths of ammonia gas at a concentration of
"3
60 mg/m (Douglas and Coe, 1987); however, there were no bronchial symptoms reported in
"3
volunteers exposed to ammonia at concentrations of up to 35 mg/m for 10 minutes in an
exposure chamber (MacEwen et al., 1970). Similarly, no changes in bronchial responsiveness or
lung function (as measured by forced vital capacity and FEVi) were reported in healthy
4
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1
2
3
4
5
6
7
8
9
10
11
12
13
"3
volunteers exposed to ammonia at concentrations up to 18 mg/m for 1.5 hours during exercise
(Sundblad et al., 2004). There were no changes in lung function as measured by FEVi in 25
healthy volunteers and 15 mild/moderate persistent asthmatic volunteers exposed to ammonia
concentrations up to 354 mg/m ammonia for up to 2.5 hours (Petrova et al., 2008), or in six
-3
healthy volunteers and eight mildly asthmatic volunteers exposed to 11-18 mg/m ammonia for
30-minute sessions (Sigurdarson et al., 2004).
Lung function effects following ammonia exposure were not evaluated in the available
animal studies.
The evidence of respiratory effects in humans and experimental animals exposed to
ammonia is provided in Tables 1-1 and 1-2, respectively, and presented visually as an exposure-
response array in Figure 1-1.
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1
Table 1-1. Respiratory effects in humans following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEL/ LOAEL3
(mg/m3)
Respiratory
irritation
Cross-sectional occupational study
of soda ash plant workers in
Canada; 58 exposed workers and
31 controls (from stores and office
No statistically significant differences in
subjective symptomology relative to the
control.
NOAEL: 3.1
LOAEL: not
identified
areas of plant)b
Control
Exposed
p-value
Flu
3
7
0.6299
Low (<4.4 mg/m3), medium (4.4—
Cough
10
16
0.5289
8.8 mg/m3), high (>8.8 mg/m3);
Sputum
16
22
0.9770
adjusted0 concentration ranges
Bronchitis
19
22
0.6938
<1.6 mg/m3,1.6-3.1 mg/m3 and
Wheeze
10
10
0.9068
>3.1 mg/m3
Chest tightness
6
3
0.6221
Average exposure: 12 y
Dyspnea
13
7
0.0470
Chest pain
6
2
0.1563
Holness et al., 1989
Rhinitis
19
10
0.1185
Throat
3
7
0.5296
Cross-sectional occupational study
of urea fertilizer factory in
Bangladesh; 63 ammonia plant
workers, 77 urea plant workers,
and 25 controls (from
administration building)
Ammonia plant: 4.9 mg/m3d
(1.8 mg/m3 adjusted0)
Urea plant: 18.5 mg/m3d
(6.6 mg/m3 adjusted0)
Mean employment duration: 16 y
Rahman et al., 2007
Exposure-related increase in respiratory
symptoms.
Respiratory symptom prevalence (%):
NOAEL: 1.8
LOAEL: 6.6
Control
Ammonia
Urea
(admin)
plant
plant
Cough
8
17 (0.42)a
28 (0.05,
0.41)b
Chest
8
17 (0.42)a
33 (0.02,
tightness
0.19)b
Stuffy
4
12 (0.35)a
16 (0.17,
nose
1.0)b
Runny
4
4 (1.0)a
16 (0.17,
nose
0.28)b
Sneeze
8
0 (0.49)a
22 (0.22,
0.01)b
p-value for ammonia plant compared to
control
bp-value for urea plant compared to
control and urea plant compared to
ammonia plant
Cross-sectional study of two urea
fertilizer factories in Saudi Arabia;
161 exposed workers and 355
unexposed controls6
Exposures were stratified > or < the
ACGIH TLV of 18 mg/m3
Mean of employment duration:
51.8 mo (exposed workers) and
73.1 mo (controls)
Ballal et al., 1998
Higher relative risks for those exposed to
ammonia at concentrations >TLV as
compared to those exposed at levels
-------
Table 1-1. Respiratory effects in humans following inhalation exposure
NOAEL/ LOAEL3
Health Effect
Study Design and Reference
Results
(mg/m3)
Lung function
Cross-sectional occupational study
No statistically significant differences in
NOAEL: 3.1
of soda ash plant workers in
lung function relative to the control.
LOAEL: not
Canada; 58 exposed workers and
identified
31 controls (from stores and office
Exposed Control p value
areas of plant)b
Lung function (% predicted values):
FVC 96.8
98.6 0.0944
Low (<4.4 mg/m3), medium (4.4—
FEVi 94.1
95.1 0.3520
8.8 mg/m3), high (>8.8 mg/m3)
FEVi/FVC 97.1
96.5 0.4801
adjusted0 concentration ranges
Change in lung function over work shift:
<1.6 mg/m3,1.6-3.1 mg/m3 and
FVC dayl -0.8
-0.9 0.9940
>3.1 mg/m3
day 2 -0.0
+0.1 0.8378
Average exposure: 12 y
FEVi day 1 -0.2
-0.2 0.9363
day 2 +0.7
+0.5 0.8561
Holness et al., 1989
Cross-sectional occupational study
Dose-related decrease in lung function
NOAEL: 1.8
of urea fertilizer factory in
parameters.
LOAEL: 6.6
Bangladesh; 63 ammonia plant
workers, 77 urea plant workers,
Pre-shift
Post-shift p-value
and 25 controls (from
Ammonia plant
administration building)
FVC 3.308
3.332 0.67
Ammonia plant: 4.9 mg/m3d
FEVi 2.627
2.705 0.24
PEFR 8.081
8.313 0.22
(1.8 mg/m3 adjusted0)
Urea plant
Urea plant: 18.5 mg/m3d
FVC 3.362
3.258 0.01
(6.6 mg/m3 adjusted0)
FEVi 2.701
2.646 0.05
Mean employment duration: 16 y
PEFR 7.805
7.810 0.97
Rahman et al., 2007
Cross-sectional study of a urea
Lung function results based on exposure
NOAEL and
fertilizer factory in Saudi Arabia-
concentration and cumulative exposure:
LOAEL values
follow-up of Ballal et al. (1998); 73
were not
exposed workers and 343
Control
Exposed p-value
identified
unexposed controls
FVC]% 96.6
98.1 NS
because
predicted
exposures were
Exposures were stratified < or > the
FVC% 101.0
105.6 0.002
not adequately
ACGIH TLV of 18 mg/m3
predicted
characterized
Mean of employment duration: not
FEVi/FVCro 83.0
84.2 NS
reported
<50
>50
AN et al., 2001
mg/m3-
y mg/m3-y p-value
FVC]% 100.7
93.4 0.006
predicted
FVC% 105.6
100.2 0.03
predicted
FEVi/FVCro 84.7
83.4 NS
NS = not significant
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aThe NOAELand LOAEL values presented were identified by EPA.
bAt this plant, ammonia, carbon dioxide, and water were the reactants used to form ammonium bicarbonate,
which in turn was reacted with salt to produce sodium bicarbonate and subsequently processed to form sodium
carbonate. Ammonia and carbon dioxide were recovered in the process and reused.
cAdjusted to continuous exposure based on the ratio of VEho (human occupational default minute volume of
10 m3 breathed during an 8-hour workday) to VEh (human ambient default minute volume of 20 m3 breathed
during the entire day) and an exposure of 5 days out of 7 days (i.e., measured concentration x 10/20 x 5/7).
dExposure concentrations were determined by both the Drager tube and Drager PAC III methods. Using the
Drager tube method, concentrations of ammonia in the ammonia and urea plants were 17.7 and 88.1 mg/m3,
respectively; using the Drager PAC III method, ammonia concentrations were 4.9 and 18.5 mg/m3 (Rahman et al.,
2007). The study authors observed that their measurements indicated only relative differences in exposures
between workers and production areas, and that the validity of the exposure measures could not be evaluated
based on their results. Based on communication with technical support at Drager (telephone conversations and e-
mails dated June 22, 2010, from Michael Yanosky, Drager Safety Inc., Technical Support Detection Products to
Amber Bacom, SRC, Inc., contractor to NCEA, ORD, U.S. EPA), EPA considered the PAC III instrument to be a more
sensitive monitoring technology than the Drager tubes. Therefore, more confidence is attributed to the PAC III air
measurements of ammonia for the Rahman et al. (2007) study.
eThe process of fertilizer production involved synthesis of ammonia from natural gas, followed by reaction of the
ammonia and carbon dioxide to form ammonium carbamide, which was then converted to urea.
1
2
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1
Table 1-2. Respiratory effects in animals following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEL/ LOAEL3
(mg/m3)
0,155, or 770 mg/m3 8 hrs/d, 5 d/wk for
6 wks; (36.9,183 mg/m3 adjusted15)
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group and Sprague-Dawley and
Long-Evans rat; male and female; 15-
51/group
No visible signs of toxicity, gross
necropsies were normal, focal
pneumonitis in 1 of 3 monkeys at
36.9 mg/m3.
Nonspecific lung inflammation
observed in guinea pigs and rats
but not in other species at
183 mg/m3
NOAEL: 36.9
LOAEL: 183
Coon et al., 1970
Pulmonary
inflammation
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group
Coon et al., 1970
Focal or interstitial pneumonitis
in all animals, calcification of
bronchial epithelium was
observed in several animals.
Hemorrhagic lung lesion in 1 of 2
dogs. Moderate lung congestion
in 2 of 3 rabbits.
NOAEL: 40
LOAEL: 470
and congestion
0 or 40 mg/m3 for 114 d or 127, 262 or
470 mg/m3 for 90 d of 455 mg/m3 for 65 d
Sprague-Dawley or Long-Evans rat; male
and female; 15-51/group
Coon et al., 1970
Dyspnea (mild) at 455 mg/m3.
Focal or interstitial pneumonitis
in all animals, calcification of
bronchial epithelium observed in
several animals at 470 mg/m3.
(Exposure to 455 and 470 mg/m3
ammonia increased mortality in
rats.)
NOAEL: 262
LOAEL: 455
0 or 14 for 7-42 days or 35 mg/m3 for
42 days
Guinea pig (strain not specified); male and
female; 2/group
Pulmonary congestion, edema
and hemorrhage were observed
at 14 and 35 mg/m3 after 42 d.
NOAEL: NA
LOAEL: 14
Anderson et al., 1964
0 or 14 mg/m3 for 7-42 days
Swiss albino mouse; male and female;
4/group
Pulmonary congestion, edema
and hemorrhage were observed
at 14 mg/m3 after 42 d.
NOAEL: not
identified
LOAEL: 14
Anderson et al., 1964
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Table 1-2. Respiratory effects in animals following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEL/ LOAEL3
(mg/m3)
0, 0.4, 7,13.3, or 26 mg/m3 and 1.2, 2.7, 5.1,
or 9.9 mg/m3 inhalable dust for 5 wks
Exposure to ammonia and inhalable dust at
concentrations commonly found at pig
farms
No increase in the incidence of
respiratory or other disease.
NOAEL: 26
LOAEL: not
identified
Pig (several breeds); sex not specified;
24/group
Done et al., 2005
0, 35, or 53 mg/m3 for 109 d
Pig (crossbred); sex not specified; 4-8/group
Turbinates, trachea, and lungs of
all pigs were classified as normal.
NOAEL: 53
LOAEL: not
identified
Curtis et al., 1975
7 or 106 mg/m3 from bedding for 75 d
Sherman rat; 5/sex/group
Thickening of the nasal
epithelium (3-4 times) and nasal
lesions.
NOAEL: 7
LOAEL: 106
Broderson et al., 1976°
Thickening of
airway
epithelium
0 or 177 mg/m3 in an inhalation chamber
for 35 d
F344 rat; 6/sex/group
Broderson et al., 1976
Thickening of the nasal
epithelium (3-4 times) and nasal
lesions.
NOAEL: not
identified
LOAEL: 177
0 or 71 mg/m3 for 6 wks
Yorkshire-Landrace pig; sex not specified;
6/group
Thickening of nasal and tracheal
epithelium (50-100% increase).
NOAEL: not
identified
LOAEL:71
Doig and Willoughby, 1971
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Table 1-2. Respiratory effects in animals following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEL/ LOAEL3
(mg/m3)
Nasal
inflammation
and lesions
0,155, or 770 mg/m3 8 hrs/d, 5 d/wk for
6 wks; (36.9,183 mg/m3 adjusted15)
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group and Sprague-Dawley and
Long-Evans rat; male and female; 15-
51/group
Coon et al., 1970
No nasal irritation observed.
NOAEL: 183
LOAEL: not
identified
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Beagle dog; male; 2/group
Coon et al., 1970
Nasal discharge.
NOAEL: 40
LOAEL: 470
0 or 40 mg/m3 for 114 d or 127, 262 or
470 mg/m3 for 90 d or 455 mg/m3 for 65 d
Sprague-Dawley or Long-Evans rat; male
and female; 15-51/group
Coon et al., 1970
Nasal irritation in all animals at
455 mg/m3.
(Exposure to 455 and 470 mg/m3
ammonia increased mortality in
rats.)
NOAEL: 262
LOAEL: 455
7 or 106 mg/m3 from bedding for 75 d
Sherman rat; 5/sex/group
Broderson et al., 1976
Nasal lesions at 106 mg/m3.
NOAEL: 7
LOAEL: 106
0 or 177 mg/m3 in an inhalation chamber
for 35 d
F344 rat; 6/sex/group
Broderson et al., 1976
Nasal lesions at 177 mg/m3.
NOAEL: not
identified
LOAEL: 177
Ammonia vapor of 0 or 12% ammonia
solution for 15 min/d, 6 d/wk, for 8 wks
White albino mouse; male; 50
Gaafar et al., 1992
Histological changes in the nasal
mucosa.
NOAEL and
LOAEL values
were not
identified
because of
inadequate
reporting of
exposure
concentrations.
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Table 1-2. Respiratory effects in animals following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEL/ LOAEL3
(mg/m3)
8, 43, 73, or 103 mg/m3 for 5 wks
Duroc pig; both sexes; 9/group
Stombaugh et al., 1969
Excessive nasal, lacrimal, and
mouth secretions and increased
frequency of cough at 73 and
103 mg/m3.
NOAELand
LOAEL values
were not
identified
because of the
absence of a
control group.
aThe NOAELand LOAEL values presented were identified by EPA.
bAdjusted to continuous exposure based on the ratio of hours exposed per day and days exposed per week (i.e.,
measured concentration x 8/24 x 5/7).
cThe Broderson et al. (1976) paper includes a number of experiments in rats designed to examine whether
ammonia at concentrations commonly encountered in laboratory cage environments plays a role in the
pathogenesis of murine respiratory mycoplasmosis caused by the bacterium M. pulmonis. The experiments
conducted without co-exposure to M. pulmonis are summarized in this table; the results of experiments involving
co-exposure to M. pulmonis are discussed in Section 1.1.4, Immune System Effects.
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anoael
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XFEL
show exposures
• Intermediate dose
tested in study.
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EXPERIMENTAL ANIMAL STUDIES
| Thickening of airway epithelium |
Nasal inflammation and lesions
Figure 1-1. Exposure-response array for respiratory effects following inhalation exposure.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
> Mode of Action Analysis - Respiratory Effects
Data regarding the potential mode of action for respiratory effects associated with chronic
exposure to ammonia are limited. However, it is well established that injury to respiratory
tissues resulting from acute exposure to inhaled ammonia is primarily due to its alkaline
properties and its solubility. Given its high solubility, ammonia readily dissolves in the moisture
on the mucous membranes, forming ammonium hydroxide, which causes liquefaction necrosis of
the tissues. Ammonia directly denatures tissue proteins due to the production of alkaline
proteinates. Specifically, ammonium hydroxide causes saponification of cell membrane lipids
that leads to cell disruption and death (necrosis). As cell proteins break down, water is extracted,
resulting in an inflammatory response, which further damages the surrounding tissues (Amshel et
al, 2000; Mellea, 1989; Jarudi and Golden, 1973).
1.1.2. Gastrointestinal Effects
Reports of gastrointestinal effects of ammonia in humans are limited to case reports
involving intentional or accidental ingestion of household cleaning solutions or ammonia
inhalant capsules (Dworkin et al., 2004; Rosenbaum et al., 1998; Christesen, 1995; Wason et al.,
1990; Lopez et al., 1988; Klein et al., 1985; Klendshoj and Rejent, 1966). Clinical signs reported
in these case studies include stomachache, nausea, dizziness, diarrhea, drooling, erythematous
and edematous lips, reddened and blistered tongues, dysphagia, vomiting, oropharyngeal burns,
laryngeal and epiglottal edema, erythmatous esophagus with severe corrosive injury, and
hemorrhagic esophago-gastro-duodeno-enteritis.
In animals following oral exposure, statistically significant decreases of 40-60% in the
thickness of the gastric mucosa were reported in Sprague-Dawley rats administered 0.01%
ammonia in drinking water for durations of 2-8 weeks (Tsujii et al., 1993; Kawano et al., 1991);
estimated doses were 22 mg/kg-day (Kawano et al., 1991) and 33 mg/kg-day (Tsujii et al., 1993).
These studies were designed to investigate the hypothesis that the bacterium Helicobacter pylori,
which produces a potent urease that increases ammonia production, plays a significant role in the
etiology of chronic atrophic gastritis. Kawano et al. (1991) reported that the magnitude of the
decrease in gastric mucosal thickness increased with dose and duration of exposure and that the
effect was more prominent in the mucosa of the antrum region of the stomach than in the body
region of the stomach.4 As discussed further under Mode of Action - Gastrointestinal Effects
(see below), the difference in response to ammonia in drinking water in the two regions of the rat
stomach may be a function of differences in pH in these regions and resulting differences in the
extent of ionization of ammonia to NH4+. Parietal cell number per oxyntic gland also decreased
in a statistically significant dose- and time-dependent fashion, up to approximately 35% at 0.01%
4The body is the main, central region of the stomach. The antrum is located in the distal part of the stomach
adjacent to the body.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
ammonia in drinking water after 4 weeks. In a follow-up study (Tsujii et al., 1993), antral
mucosal thickness decreased significantly (by 56-59% of the tap water control) at 4 and 8 weeks
of exposure to 0.01% ammonia in drinking water, but there was no significant effect on the
thickness of the body mucosa. Increased mucosal cell proliferation and migration (as measured
by 5-bromo-2'-deoxyuridine [BrDU] labeling) were significantly increased. The authors
observed that it was not clear whether mucosal cell proliferation was primarily stimulated
directly by ammonia or indirectly by increased cell loss followed by compensatory cell
proliferation. They further observed that the ammonia-related changes in rat stomach resembled
mucosal changes in human atrophic gastritis (Tsujii et al., 1993; Kawano et al., 1991).
A relationship between ammonia ingestion and gastrointestinal effects is supported by
findings from two acute oral studies in rats following gavage administration of ammonium
hydroxide (Takeuchi et al., 1995; Nagy et al., 1996). Takeuchi et al. (1995) reported
hemorrhagic necrosis of the gastric mucosa in male Sprague-Dawley rats that received a single
gavage dose of ammonium hydroxide (concentration >1%). Nagy et al. (1996) observed severe
hemorrhagic mucosal lesions in female Sprague-Dawley rats 15 minutes after exposure to an
estimated dose of 48 mg/kg ammonium hydroxide via gavage.
The evidence of gastrointestinal effects in experimental animals following oral exposure
to ammonia is provided in Table 1-3.
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Table 1-3. Gastrointestinal effects in animals following oral exposures
Health Effect
Study Design and References
Results
NOAEL/LOAEL3
(mg/kg-day)
Histopathologic
changes of the
gastric mucosa
0, 22, or 220 mg/kg-day in
drinking water for 2 or 4 weeksb
Sprague-Dawley rat; male;
6/group
Kawano et al., 1991
Statistically significant decrease in the
thickness of the gastric mucosa that
was dose and duration related; effect
was more prominent in the mucosa of
the antrum region of the stomach than
the body region.
Thickness of mucosa relative to
control:
Antrum
Week 2: 96, 80d%
Week 4: 62d, 39d%
Body
Week 2: 99,103%
Week 4: 78, 71d%
NOAEL: not
identified
LOAEL: 22
0 or 33 mg/kg-day in drinking
water for 3 days or 1, 2, 4, or 8
weeks; tap water provided for
the balance of the 8-week
study0
Sprague-Dawley rat; male;
36/group
Tsujii et al., 1993
Antral mucosal thickness decreased
significantly at 4 and 8 weeks of
exposure; there was no significant
effect on the thickness of the body
mucosa. Cell migration was
significantly increased.
Thickness of mucosa relative to control
(d 3, wk 1, 2, 4, 8):
Antrum: 108, 96,106, 56d, 59d%
Body: 105, 101, 104, 99, 95%
(extracted from Figure 3 of Tsujii et al.,
1993)
NOAEL: not
identified
LOAEL: 33
aThe NOAELand LOAEL values presented were identified by EPA.
bAmmonia was provided in drinking water at concentrations of 0, 0.01 or 0.1%. Doses were estimated based on a
body weight of 230 g for male rats and estimated daily water intake of 50 mL/day.
cAmmonia was provided in drinking water at concentrations of 0 or 0.01%. Doses were estimated based on an
initial body weight of 150 g and estimated daily water intake of 50 mL.
Statistically significant from controls.
1
2 > Mode of Action Analysis - Gastrointestinal Effects
3 The mode of action for the gastric effects of ammonia has not been established; however,
4 relevant mechanistic information that informs ammonia mode of action comes largely from
5 investigation of the action of the bacterium Helicobacter pylori on the stomach. H. pylori
6 produces urease, which breaks down urea that is normally present in the stomach into ammonia
7 (Megraud et al. 1992; Tsujii et al. 1992a), and has been linked to chronic gastritis, gastric ulcers,
8 and stomach cancer in humans.
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This literature suggests that the alkalinity of the ammonia solution does not play a direct
role in the gastric effects associated with ammonia. An ammonia solution (pH 10.3) produced
dose-related acute macroscopic mucosal lesions, whereas a glycine-sodium hydroxide buffer (pH
10.3) or ammonium chloride (pH 4.5) did not (Tsujii et al., 1992b). Rather, the ability of
ammonia to damage the gastric mucosa may be related to its ionization state. Ammonia (NH3)
can easily penetrate cell membranes, subsequently reacting to form NH/ and OH" in the interior
of the membrane (Tsujii et al., 1992b). The finding that antral and body regions of the rat
stomach mucosa responded differently following administration of 33 mg/kg-day ammonia in
drinking water for 8 weeks (Tsujii et al., 1993) is consistent with the influence of ionization on
toxicity. The hydrogen chloride secreted by the mucosa in the body of the stomach resulted in a
decrease in pH and a corresponding decrease in the ratio of ammonia to ammonium ion. In
contrast, in the antral mucosa (a nonacid-secreting area), pH is higher, the ratio of ammonia to
ammonium ion is increased, and measures of gastric atrophy were increased compared to those
observed in the stomach body where there was relatively higher exposure to NH/.
Several specific events have been identified that may contribute to the induction of
gastric lesions by ammonia. Increased cell vacuolation and decreased viability of cells in vitro
were associated with increasing ammonia concentration in an in vitro system (Megraud et al.,
1992); the effect was not linked to pH change because of the high buffering properties of the
medium. Using an in situ rat stomach model, hemorrhagic mucosal lesions induced by ammonia
were associated with the rapid release and activation of cathepsins, mammalian cysteine
proteases that are released from lysosomes or activated in the cytosol and that can be damaging
to cells, tissues, or organs (Nagy et al., 1996). Ammonia also appears to inhibit cellular and
mitochondrial respiration, possibly by elevating intracellular or intraorganelle pH or by
impairing adenosine triphosphate (ATP) synthesis (Tsujii et al., 1992b). Mori et al. (1998)
proposed a role for increased release of endothelin-1 and thyrotropin releasing hormone from the
gastric mucosa in ammonia-induced gastric mucosal injury based on findings in rats given
ammonia intragastrically. Regardless of the specific mechanism(s) by which ammonia induces
cellular toxicity, gastric injury appears to accelerate mucosal cell desquamation and stimulate
cell proliferation via a compensatory mechanism (Tsujii et al., 1992a).
1.1.3. Reproductive and Developmental Effects
No statistically significant differences in reproductive or developmental endpoints were
found between two groups of female pigs (crossbred gilts) exposed to ammonia for 6 weeks at
-3
mean concentrations of 5 or 25 mg/m and then mated (Diekman et al., 1993) in the only study of
the reproductive and developmental toxicity potential of ammonia (see Table 1-4). Age at
"3
puberty did not differ significantly between the two groups. Gilts exposed to 25 mg/m ammonia
"3
weighed 7% less (p < 0.05) at puberty than those exposed to 5 mg/m ; however, body weights of
the two groups were similar at gestation day 30. Conception rates in the mated females were
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similar between the two groups (94.1 versus 100% in low- versus high-exposure groups). At
sacrifice on day 30 of gestation, there were no significant differences between the two exposed
groups in body weights of the pregnant gilts, number of corpora lutea, number of live fetuses, or
weight and length of the fetuses. The strength of the findings from this study are limited by the
absence of a control group and possible confounding by exposures to bacterial and mycoplasm
pathogens. The evidence of reproductive and developmental effects in experimental animals
exposed to ammonia is provided in Table 1-4.
Table 1-4. Reproductive and developmental effects in animals following inhalation
exposure
Health Effect
Study Design and Reference
Results
NOAEL/LOAEL3 (mg/m3)
Reproductive and
developmental
parameters
5 mg/m3 (range, 3-8.5
mg/m3) or 25 mg/m3 (range,
18-32 mg/m3) for 6 weeksb
Crossbred gilts (female pigs),
4.5 months old, 40/group
Diekman et al., 1993
No effect on any of the
reproductive or
developmental parameters
measured (age at puberty,
conception rates, body weight
of pregnant gilts, number of
corpora lutea, number of live
fetuses, and weight or length
of fetuses).
NOAEL: 5
LOAEL: not identified
aThe NOAELand LOAEL values presented were identified by EPA.
bA control group was not included. Prior to exposure to ammonia, pigs were also exposed naturally in
conventional grower units to Mycoplasma hypopneumoniae and Pasteurella multocida, which cause pneumonia
and atrophic rhinitis, respectively.
1.1.4. Immune System Effects
A limited number of studies have evaluated the immunotoxicity of ammonia in human
populations and in experimental animal models. Immunological function was evaluated in two
independent investigations of livestock farmers exposed to ammonia; immunoglobulin G (IgG)
and IgE-specific antibodies for pig skin and urine (Crook et al., 1991), elevated neutrophils from
nasal washes, and increased white blood cell counts (Cormier et al., 2000) were reported. These
data are suggestive of immunostimulatory effects; however, the test subjects were also exposed
to a number of other respirable agents in addition to ammonia such as endotoxin, bacteria, fungi,
and mold that are known to stimulate immune responses. Data in humans following exposure to
ammonia only are not available.
Animal studies that examined ammonia immunotoxicity were conducted using short-term
inhalation exposures and three general types of immune assays. Immunotoxicity studies of
ammonia using measures of host resistance provide the most relevant data for assessing immune
function since they directly measure the immune system's ability to control microorganism
growth. Other available studies of ammonia employed assays that evaluated immune function.
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Changes in immune cell populations without corresponding functional data are considered to be
the least predictive and were excluded from the hazard identification for ammonia (Neumann et
al, 1987; Gustin et al, 1994).
Evidence of immunosuppression was observed in several host resistance studies utilizing
lung pathogens to measure reduced bacterial clearance following ammonia exposure.
Inoculation with the respiratory pathogen Mycoplasma pulmonis causes murine respiratory
mycoplasmosis (MRM) characterized by lung lesions. Lung lesions, both gross and
microscopic, were positively correlated with ammonia concentration in F344 rats continuously
exposed to ammonia in an inhalation chamber for 7 days prior to inoculation withM pulmonis
o
(10 colony forming units [CFU]) followed by up to 42 days of ammonia exposure post
inoculation (Broderson et al., 1976). The incidence of lesions was significantly increased at
"3
ammonia concentrations >35 mg/m , and suggests that ammonia exposure decreased bacterial
clearance resulting in the development of M pulmonis-induced MRM. However, the increasing
ammonia concentration was not associated with increased CFU ofM pulmonis isolated from the
respiratory tract. The high number of inoculating CFU could have overwhelmed the immune
response and elicited a maximal response that could not be further magnified in
immunocompromised animals. Conversely, significantly increased CFU ofM pulmonis bacteria
isolated in the trachea, nasal passages, lungs, and larynx was observed in F344 rats continuously
exposed to 71 mg/m3 ammonia for 7 days prior toM pulmonis (104— 106 CFU) inoculation and
continued for 28 days post inoculation (Schoeb et al., 1982). This increase in bacterial
colonization indicates a reduction in bacterial clearance following exposure to ammonia.
Lesions were not assessed in this study. OF1 mice exposed to 354 mg/m ammonia for 7 days
prior to inoculation with a 50% lethal dose (LD50) of Pasteurella multocida significantly
increased mortality compared to controls (86% versus 50%, respectively); however, an 8-hour
exposure was insufficient to affect mortality (Richard et al., 1978a). The authors suggested that
the irritating action of ammonia destroyed the tracheobronchial mucosa and caused inflammatory
lesions thereby increasing sensitivity to respiratory infection with prolonged ammonia exposure.
Suppressed cell-mediated immunity and decreased T cell proliferation was also observed
following ammonia exposure. Using a delayed-type hypersensitivity (DTH) test to evaluate cell-
mediated immunity, Hartley guinea pigs were vaccinated with Mycobacterium bovis BCG and
exposed to ammonia followed by intradermal challenge with purified protein derivative (PPD).
Dermal lesion size was reduced in animals exposed to 64 mg/m indicating immunosuppression
(Targowski et al., 1984). Blood and bronchial lymphocytes harvested from naive guinea pigs
treated with the same 3 week ammonia exposure and stimulated with phytohaemagglutinin or
concanavalin A demonstrated reduced T cell proliferation (Targowski et al., 1984). Bactericidal
activity in alveolar macrophages isolated from ammonia-exposed guinea pigs was not affected.
Lymphocytes and macrophages isolated from unexposed guinea pigs and treated with ammonia
in vitro showed reduced proliferation and bactericidal capacity only at concentrations that
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1 reduced viability, indicating nonspecific effects of ammonia-induced immunosuppression
2 (Targowski et al., 1984). These data suggest that T cells may be the target of ammonia since
3 specific macrophage effects were not observed.
4 The evidence of immune system effects in experimental animals exposed to ammonia is
5 provided in Table 1-5, and presented visually in an exposure-response array in Figure 1-2.
6
Table 1-5. Immune system effects in animals following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEl7LOAELa
(mg/m3)
Host resistance
<3.5 (control), 18, 35, 71,177 mg/m3, 7
days (continuous exposure) pre
inoculation/28-42 days post inoculation
with M. pulmonis
F344 rat; male and female; 11-12/sex/
group
Broderson et al., 1976
Increased incidence of gross
lung lesions; no effect on CFU.
% of animals with gross
lesions: 16 (control), 46, 66b,
33, 83%
NOAEL: 18
LOAEL: 35°
<1.4 (control) or 71 mg/m3, 7 days
(continuous exposure) pre inoculation/ 28
days post inoculation with M. pulmonis
Increased bacterial
colonization (as a result of
reduced bacterial clearance).
NOAEL: not
identified
LOAEL: 71
F344 rat; 5-15/group (sex unknown)
No quantitative data available.
Schoeb et al., 1982
0 or 354 mg/m3, 8 hours or 7 days
(continuous exposure), prior to infection
with P. multocida
OF1 mouse; male; 99/group
Increased mortality.
Mouse mortality: 50% (control)
and 86%b
NOAEL: not
identified
LOAEL: 354
Richard et al., 1978
T cell
proliferation
<11 (control), 35 or 64 mg/m3, 3 weeks
(continuous exposure)
Hartley guinea pig; 8/group (sex unknown)
Targowski et al., 1984
Reduced proliferation in blood
and bronchial T cells.
No quantitative data available.
NOAEL: 35
LOAEL: 64
Delayed-type
hypersensitivity
<11 (control), 35 or 64 mg/m3, 3 weeks
(continuous exposure) followed by PPD
challenge in BCG immunized
Hartley guinea pig; 8/group (sex unknown)
Targowski et al., 1984
Reduced dermal lesion size.
Mean diameter (mm).12
(control), 12.6 and 8.7b
NOAEL: 35
LOAEL: 64
aThe NOAELand LOAEL values presented were identified by EPA.
Statistically significant from controls.
cStudy did not find statistical significance despite a large increase in the response at the lowest dose measured.
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1000
100 ¦
E
o>
E
a)
i_
3
(/>
O
Q.
X
111
I
I
10 -
¦LOAEL
ANOAEL
XFEL
• intermediate dose
Vertical lines show
range of exposures in
study. Closed circles
show exposures
tested in study.
Increased incidence of
gross lung lesions (rat);
Broderson et al. (1976)
Increased bacterial
colonization (rat);
Schoeb et al. (1982)
Increased mortality (mouse);
Richard et al. (1978)
Host resistance
Reduced proliferation
in blood and bronchial
lymphocytes (guinea pig);
Targowski et ai. (1984)
Mitogen stimulation
Reduced dermal
lesion size (guineapig);
Targowski et al. (1984)
Delayed-type hypersensitivity
Figure 1-2. Exposure-response array for immune system effects following inhalation exposure.
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1.1.5. Other Systemic Effects
Although the majority of information for ammonia suggests that ammonia induces effects
in and around the portal of entry, there is limited evidence that ammonia can produce effects on
organs distal from the portal of entry, including the liver, adrenal gland, kidney, spleen, and
heart. Alterations in liver function, based on elevated mean levels of aspartate aminotransferase
(AST), alanine aminotransferase (ALT), and blood urea, decreased hemoglobin, and inhibition of
catalase and monoamine oxidase (MAO) activities were observed in workers exposed to
ammonia over an average exposure duration of 12 years at an Egyptian urea production plant;
measurements of workplace exposure concentrations were not provided (Hamid and El-Gazzar,
1996). The authors suggested that inhibition of catalase can affect electrical stability,
permeability, and fluidity of membranes, which may lead to hepatotoxicity in occupationally
exposed workers (Hamid and El-Gazzar, 1996).
Evidence of hepatotoxicity in animals comes from observations of histopathological
alterations in the liver. Fatty changes in the liver were consistently reported at concentrations
"3
>470 mg/m ammonia in rats, guinea pigs, rabbits, dogs, and monkeys following identical
subchronic inhalation exposure regimens (Coon et al., 1970). Congestion of the liver was
observed in guinea pigs following subchronic and short-term inhalation exposure to 35 and
"3
120 mg/m (Anderson et al., 1964; Weatherby, 1952); no liver effects were observed in similarly
"3
exposed mice at 14 mg/m (Anderson et al., 1964; Weatherby, 1952). No histopathological or
hematological effects were observed in rats, guinea pigs, rabbits, dogs, or monkeys when these
animals were repeatedly, but not continuously, exposed to ammonia even at high concentrations
(e.g., 770 mg/m for 8 hours/day, 5 days/week), suggesting that mammals can recover from
short-term exposure to elevated ammonia levels (Coon et al., 1970). In addition, no effects were
"3
observed in mice exposed to 14 mg/m for up to 6 weeks (Anderson et al., 1964).
Adrenal effects were observed in animals following subchronic and short-term exposure
to ammonia; data in humans were not found. Increased mean adrenal weights and fat content of
the adrenal gland, as well as histological changes in the adrenal gland (enlarged cells of the zona
fasiculata of the adrenal cortex that were rich in lipid) were observed in rabbits exposed orally
via gavage to ammonium hydroxide for durations ranging from 5.5 days to 17 months (Fazekas,
1939). While the strength of these findings is limited by inadequate reporting and study design,
a separate study identified early degenerative changes in the adrenal glands of guinea pigs
-3
exposed to 120 mg/m ammonia by inhalation for 18 weeks (Weatherby, 1952), providing
additional limited evidence for effects on the adrenal gland.
Evidence that inhaled ammonia can affect the kidney and spleen is limited to studies in
experimental animals. Nonspecific degenerative changes in the kidneys (not further described)
"3
of rats exposed to 262 mg/m were reported (Coon et al., 1970). Histopathological evaluation of
-3
other animal species in the same study exposed to 470 mg/m , a concentration that induced a
high rate of mortality in rats, consistently showed alterations in the kidneys (calcification and
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proliferation of tubular epithelium; incidence not reported). Exposure of guinea pigs to inhaled
ammonia at a concentration of 120 mg/m for 18 weeks (but not 6 or 12 weeks) resulted in
histopathological alterations (congestion) of the kidneys and spleen, although incidence was not
reported (Weatherby, 1952). Enlarged and congested spleens were reported in guinea pigs
"3
exposed to 35 mg/m ammonia for 6 weeks in a separate study (Anderson et al., 1964).
Myocardial fibrosis was observed in monkeys, dogs, rabbits, guinea pigs, and rats
"3
following subchronic, inhalation exposure to 470 mg/m ammonia; no changes were observed at
lower concentrations (Coon et al., 1970). At the same concentration, ocular irritation
(characterized as heavy lacrimation, erythema, discharge and ocular opacity of the cornea) was
also reported by Coon et al. (1970) in dogs and rabbits, but not observed in similarly treated
monkeys and rats. Additionally, there is limited evidence of biochemical or metabolic effects of
acute or short-term ammonia exposure. Acidosis, as evidenced by a decrease in blood pH and an
increase in arterial blood carbon dioxide partial pressure (pC02), occurred in rats exposed to
-3
212 mg/m ammonia for 5-15 days (Manninen et al., 1988). Blood pH and pCC>2 did not change
in rats exposed to <818 mg/m for up to 24 hours, although statistically significant increases in
"3
oxygen partial pressure (PO2) were reported in rats exposed to 10.6 and 22.6 mg/m ammonia,
-3
but not at 219 and 818 mg/m over the same time period (Schaerdel et al., 1983).
Encephalopathy related to ammonia may occur following disruption of the body's normal
homeostatic regulation of the glutamine and urea cycles resulting in elevated ammonia levels in
blood, e.g., as a result of severe liver or kidney disease (Minana et al., 1995; Souba, 1987).
Acute inhalation exposure studies have identified alterations in amino acid levels and
neurotransmitter metabolism (including glutamine concentrations) in the brain of rats and mice
(Manninen and Savolainen, 1989; Manninen et al., 1988; Sadasivudu et al., 1979; Sadasivudu
and Murthy, 1978). It has been suggested that glutamate and y-amino butyric acid (GAB A) play
a role in ammonia-induced neurotoxicity (Jones, 2002). There is no evidence, however, that
ammonia is neurotoxic in humans or animals following chronic exposures.
The evidence of systemic toxicity in humans and experimental animals exposed to
ammonia is provided in Tables 1-6 to 1-8, and presented visually in an exposure-response array
in Figure 1-3.
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Table 1-6. Systemic effects in humans following inhalation exposure
Health Effect
Study Design and Reference
Results
NOAEL7LOAEL
Serum clinical
chemistry; liver
function
Occupational study workers in an
Egyptian urea plant; 30 exposed
and 30 control subjects
No measurement of exposure
concentrations
Average employment time: 12 y
Hamid and El-Gazzar, 1996
Elevated AST, ALT and blood
urea in exposed workers;
lower hemoglobin and
inhibition of catalase and
MAO.
Not identified because
the study did not report
measurements of
exposure.
1
2
Table 1-7. Systemic effects in animals following oral exposure
Health Effect
Study Design and Reference
Results
NOAEL7LOAEL
Adrenal effects
50-80 mL of a 0.5 or 1.0%
ammonium hydroxide solution by
gavage; initially every other day,
later daily; duration ranged from 5.5
days to 17 months; estimated dose:
61-110 mg/kg-day and 120-230
mg/kg-day, respectively3
Rabbits (strain and sex not
specified); 16-33/group
Fazekas, 1939
Increased mean adrenal
weights and fat content of
the adrenal gland.
Response relative to control
(adrenal weight): 95%
increase
Response relative to control
(fat): 4.5-fold increase
Not identified.
aAmmonia doses estimated using assumed average default body weight of 3.5-4.1 kg for adult rabbits (U.S. EPA,
1988).
3
Table 1-8. Systemic effects in animals following inhalation exposures
Health
Effect
Study Design and Reference
Results
NOAEL/LOAEL3
(mg/m3)
Liver
toxicity
0 or 120 mg/m3 6 h/day, 5 days/week for 6,
12 or 18 weeks; (24.1 mg/m3 adjusted15),
Guinea pig (strain not specified); male; 6-12/
group
Weatherby, 1952
Congestion of the liver at 18 weeks,
not observed at earlier times.
NOAEL: not
identified
LOAEL: 24.1
0 or 14 for 7-42 days or 35 mg/m3 for 42 days
Guinea pig (strain not specified); male and
female; 2/group
Anderson et al., 1964
Congestion of the liver at 35 mg/m3
for 42 days.
NOAEL: 14
LOAEL: 35
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Table 1-8. Systemic effects in animals following inhalation exposures
Health
Effect
Study Design and Reference
Results
NOAEL/LOAEL3
(mg/m3)
0 or 14 mg/m3 for 7-42 days
Swiss albino mouse; male and female;
4/group
No visible signs of liver toxicity.
NOAEL: 14
LOAEL: not
identified
Anderson et al., 1964
0,155, or 770 mg/m3 8 hrs/d, 5 d/wk for
6 wks; (36.9,183 mg/m3 adjusted15)
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group and Sprague-Dawley and
Long-Evans rat; male and female; 15-
51/group
No histopathologic changes
observed.
NOAEL: 183
LOAEL: not
identified
Coon et al., 1970
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Fatty liver changes in plate cells.
NOAEL: 40
LOAEL: 470
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group
Coon et al., 1970
0 or 40 mg/m3 for 114 d or 127, 262 or
470 mg/m3 for 90 d;
Sprague-Dawley or Long-Evans rat; male and
female; 15-51/group
Fatty liver changes in plate cells.
NOAEL: 262
LOAEL: 470
Coon et al., 1970
Adrenal
gland
toxicity
0 and 120 mg/m3 6 h/day, 5 days/week for 6,
12 or 18 weeks; (24.1 mg/m3 adjusted15)
Guinea pig (strain not specified); male; 6-12/
group
Weatherby, 1952
"Early" degenerative changes in the
adrenal gland (swelling of cells,
degeneration of the cytoplasm with
loss of normal granular structure) at
18 weeks, not observed at earlier
times.
NOAEL: not
identified
LOAEL: 24.1
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Table 1-8. Systemic effects in animals following inhalation exposures
Health
Effect
Study Design and Reference
Results
NOAEL/LOAEL3
(mg/m3)
Kidney and
spleen
toxicity
0,155, or 770 mg/m3 8 hrs/d, 5 d/wk for
6 wks; (36.9,183 mg/m3 adjusted15)
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group and Sprague-Dawley and
Long-Evans rat; male and female; 15-
51/group
Coon et al., 1970
No histopathologic changes
observed.
NOAEL: 183
LOAEL: not
identified
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Calcification and proliferation of
renal tubular epithelium.
NOAEL: 40
LOAEL: 470
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group
Coon et al., 1970
0 or 40 mg/m3 for 114 d or 127, 262 or
470 mg/m3 for 90 d;
Calcification and proliferation of
renal tubular epithelium.
NOAEL: 262
LOAEL: 470
Sprague-Dawley or Long-Evans rat; male and
female; 15-51/group
Coon et al., 1970
0 or 120 mg/m3 6 h/day, 5 days/week for 6,
12 or 18 weeks; (24.1 mg/m3 adjusted15)
Guinea pig (strain not specified); male; 6-12/
group
Congestion of the spleen and
kidneys.
NOAEL: not
identified
LOAEL: 24.1
Weatherby, 1952
0 or 14 for 7-42 days or 35 mg/m3 for 42 days
Guinea pig (strain not specified); male and
female; 2/group
Enlarged and congested spleens.
NOAEL: 14
LOAEL: 35
Anderson et al., 1964
0 or 14 mg/m3 for 7-42 days
Swiss albino mouse; male and female;
4/group
No visible signs of toxicity.
NOAEL: 14
LOAEL: not
identified
Anderson et al., 1964
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Table 1-8. Systemic effects in animals following inhalation exposures
Health
Effect
Study Design and Reference
Results
NOAEL/LOAEL3
(mg/m3)
Myocardial
toxicity
0,155, or 770 mg/m3 8 hrs/d, 5 d/wk for
6 wks; (36.9,183 mg/m3 adjusted15)
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group and Sprague-Dawley and
Long-Evans rat; male and female; 15-
51/group
Coon et al., 1970
No histopathologic changes
observed.
NOAEL: 183
LOAEL: not
identified
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Myocardial fibrosis.
NOAEL: 40
LOAEL: 470
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group
Coon et al., 1970
0 or 40 mg/m3 for 114 d or 127, 262 or
470 mg/m3 for 90 d;
Myocardial fibrosis.
NOAEL: 262
LOAEL: 470
Sprague-Dawley or Long-Evans rat; male and
female; 15-51/group
Coon et al., 1970
Ocular
Irritation
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Beagle dog; male; 2/group
Coon et al., 1970
Heavy lacrimation.
NOAEL: 40
LOAEL: 470
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Erythema, discharge and ocular
opacity over % to Yi of cornea.
NOAEL: 40
LOAEL: 470
New Zealand albino rabbit; male; 3/group
Coon et al., 1970
0 or 40 mg/m3 for 114 d or 470 mg/m3 for
90 d
Squirrel monkey (Saimiri sciureus); male;
3/group and 3/group and Princeton-derived
guinea pig; male and female; 15/group
No ocular irritation observed.
NOAEL: 470
LOAEL: not
identified
Coon et al., 1970
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Table 1-8. Systemic effects in animals following inhalation exposures
Health
Effect
Study Design and Reference
Results
NOAEL/LOAEL3
(mg/m3)
0 or 40 mg/m3 for 114 d or 127, 262 or
470 mg/m3 for 90 d;
Sprague-Dawley and Long-Evans rat; male
and female; 15-51/group
No ocular irritation observed.
NOAEL: 470
LOAEL: not
identified
Coon et al., 1970
0,155, or 770 mg/m3 8 hrs/d, 5 d/wk for
6 wks; (36.9,183 mg/m3 adjusted15)
Squirrel monkey (Saimiri sciureus); male;
3/group and beagle dog; male; 2/group and
New Zealand albino rabbit; male; 3/group
and Princeton-derived guinea pig; male and
female; 15/group and Sprague-Dawley and
Long-Evans rat; male and female; 15-
51/group
No ocular irritation observed.
NOAEL: 183
LOAEL: not
identified
Coon et al., 1970
Blood pH
changes
0,18, or 212 mg/m3 6 h/day for 5,10 or 15
days; (4.5, 53 mg/m3 adjusted15)
Wistar rat; female; 5/group
Manninen et al., 1988
Statistically significant decrease in
blood pH at 5 days. pH differences
"leveled off at later time points
(data not shown)".
Response difference from control:
0.09° and 0.07°
NOAEL: 53
LOAEL: not
identified
10.6-818 mg/m3 for 0, 8,12, 24 hours, 3 and
7 days
CrkCOBS CD(SD) rat; male; 32 and 70
Schaerdel et al., 1983
Statistically significant increase in
p02 at 10.6 and 22.6 mg/m3
exposure at 8,12 and 24 hours
(p<0.05). No change at higher
exposures. No change in blood pH or
pC02.
Response relative to control: 16, 6,
20% at 10.6 mg/m3 and at 8,12, 24
hrs; 18, 26,17% at 22.6 mg/m3 and
at 8,12, 24 hrs
NOAEL: 818
LOAEL: not
identified
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Table 1-8. Systemic effects in animals following inhalation exposures
Health
Effect
Study Design and Reference
Results
NOAEL/LOAEL3
(mg/m3)
Amino acid
levels and
neurotrans-
mitter
metabolism
in the brain
0,18, or 212 mg/m3 6 h/day for 5 days; (4.5,
53 mg/m3 adjusted15)
Wistar rat; female; 5/group
Manninen and Savolainen, 1989
Statistically significant increase in
brain glutamine (p< 0.05).
Response relative to control: 42°,
40c% for 18 and 212 mg/m3,
respectively
NOAEL: not
identified
LOAEL: 4.5e
0,18, or 212 mg/m3 6 h/day for 5,10 or 15
days; (4.5, 53 mg/m3 adjusted15)
Wistar rat; female; 5/group
Manninen et al., 1988
Brain and blood glutamine
statistically significantly increased
(p< 0.05 and 0.01, respectively) at
212 mg/m3 at 5 days, no statistically
significant difference from control at
10 and 15 days.
Response relative to control at
212 mg/m3: 44°, 13 and 14%
increase in blood glutamine at 5,10,
15 days; 40°, 4 and 2% increase in
brain glutamine at 5,10,15 days
NOAEL: 53
LOAEL: not
identified
aThe NOAELand LOAEL values presented were identified by EPA.
bAdjusted to continuous exposure based on the ratio of hours exposed per day and days exposed per week (i.e.,
measured concentration x 8/24 x 5/7).
Statistically significant difference from controls.
Measurements at time zero were used as a control. The study did not include an unexposed, control group.
1
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Figure 1-3. Exposure-response array for systemic effects following inhalation exposure.
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1.1.6. Cancer
No information is available regarding the carcinogenic effects of ammonia in humans
following oral or inhalation exposure. The carcinogenic potential of ammonia by the inhalation
route has not been assessed in animals, and animal carcinogenicity data by the oral route of
exposure are limited. Toth (1972) concluded that tumor incidence was not increased in Swiss
mice exposed for their lifetime (not further specified) to ammonium hydroxide in drinking water
at concentrations up to 0.3% (equivalent to 410 and 520 mg/kg-day in female and male mice,
respectively) or in C3H mice exposed to ammonium hydroxide in drinking water at a
concentration of 0.1% (equivalent to 214 and 191 mg/kg-day in female and male mice,
respectively). With the exception of mammary gland tumors in female C3H mice (a tumor with a
high background incidence), concurrent control tumor incidence data were not reported and
comparison of tumor incidence in exposed and control mice could not be performed. The
general lack of concurrent control data limits the ability to interpret the findings of this study.
The incidence of gastric cancer and the number of gastric tumors per tumor-bearing rat
were statistically significantly higher in rats exposed to 0.01% ammonia solution in drinking
water (equivalent to 10 mg/kg-day) for 24 weeks following pretreatment (for 24 weeks) with the
initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared with rats receiving only
MNNG and tap water (Tsujii et al., 1992a). In an almost identically designed study, reported by
Tsujii et al. (1995), similar increases in the incidence of gastric tumors were observed in rats
following exposure to MNNG and 10 mg/kg-day ammonia. Additionally, the size and
penetration to deeper tissue layers of the MNNG-initiated gastric tumors were enhanced in the
rats treated with ammonia (Tsujii et al., 1995). The investigators suggested that ammonia
administered in drinking water may act as a cancer promoter (Tsujii et al., 1995, 1992a);
however, in the absence of an ammonia-only exposure group in these studies, it is not possible to
distinguish between possible promotion and initiator activity.
The evidence of carcinogenicity in experimental animals exposed to ammonia is provided
in Table 1-9.
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Table 1-9. Cancer bioassays following oral exposure
Health Effect
Study Design and Reference
Results
Tumor incidence
250, 440, and 520 mg/kg-day (males);
240, 370, and 410 mg/kg-day (females)
[0.1, 0.2, and 0.3% ammonium hydroxide
in drinking water3] for their lifetime (not
further specified)
Swiss mouse, 50/sex/group
Toth, 1972
The authors reported that tumor incidence was
not increased in ammonia-exposed mice;
however, concurrent control tumor incidence
data were not reported and comparison of
tumor incidence in exposed and control mice
could not be performed.
191 (males) and 214 mg/kg-day (females)
[0.1% ammonium hydroxide in drinking
waterb] for their lifetime (not further
specified)
C3H mouse, 40/sex/group
Toth, 1972
The authors reported that tumor incidence was
not increased in ammonia-exposed mice;
however, with the exception of mammary gland
tumors in female mice, concurrent control tumor
incidence data were not reported and
comparison of tumor incidence in exposed and
control mice could not be performed.
Mammary gland adenocarcinoma: 76, 60%
0 or 10 mg/kg-day [0 or 0.01% ammonia
in drinking water0] for 24 weeks; both
groups pretreated for 24 weeks with the
tumor initiator MNNG
Sprague Dawley rat, male; 40/group
Tsujii et al.,1992a
Statistically significantly increased incidence of
gastric cancers and number of gastric tumors per
tumor-bearing rat in ammonia + MNNG group
compared to MNNG only group
Gastric tumor incidence: 31, 70d%
# of gastric tumors/tumor-bearing rat: 1.3, 2.1d
0 or 10 mg/kg-day [0 or 0.01% ammonia
in drinking water0] for 24 weeks; both
groups pretreated for 24 weeks with the
tumor initiator MNNG
Sprague Dawley rat; male; 43-44/group
Tsujii et al., 1995
Statistically significantly increased incidence of
gastric cancers, size, and penetration to deeper
tissue layers in ammonia + MNNG group
compared to MNNG only group
Gastric tumor incidence: 30, 66d%
Penetrated muscle layer or deeper: 12, 22d%
Size (mm): 4.4, 5.3d
aAmmonium hydroxide doses estimated based on reported average daily drinking water intakes of 9.2, 8.2, and 6.5
mL/day for males and 8.3, 6.5, and 4.8 mL/day for females in the 0.1, 0.2, and 0.3% groups, respectively, and
assumed average default body weights of 37.3 and 35.3 g for males and females, respectively (U.S. EPA, 1988).
bAmmonium hydroxide doses estimated based on reported average daily drinking water intakes of 7.9 and 8.4
mL/day for males and females, respectively, and assumed average default body weights of 37.3 and 35.3 g for
males and females, respectively (U.S. EPA, 1988).
cAmmonia doses estimated based on reported drinking water intake of 50 mL/day and assumed average default
body weight of 523 g for male Sprague-Dawley rats during chronic exposure (U.S. EPA, 1988).
Statistically significantly different from control.
1
2 A limited number of genotoxicity studies are available for ammonia vapor, including one
3 study in exposed fertilizer factory workers in India that reported chromosomal aberrations and
4 sister chromatid exchanges in lymphocytes (Yadav and Kaushik, 1997), mutation assays in S.
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typhimurium and E. coli (Shimizu et al., 1985; Demerec et al., 1951), a micronucleus assay in
mice (Yadav and Kaushik, 1997), studies in D. melanogaster (Auerbach and Robson, 1947;
Lobasov and Smirnov, 1934), and a chromosomal aberration test in chick fibroblast cells in vitro
(Rosenfeld, 1932) (see Appendix A, Section A. 5). Four of the six available studies were
published between 1932 and 1951, and the available genotoxicity database in general is
inadequate to characterize the genotoxic potential of ammonia.
1.1.7. Susceptible Populations and Life Stages
Studies of the toxicity of ammonia in children or young animals compared to other life
stages that would support an evaluation of childhood susceptibility have not been conducted.
Hyperammonemia is a condition of elevated levels of circulating ammonia that can occur
in individuals with severe diseases of the liver or kidney, organs that biotransform and excrete
ammonia, or with hereditary urea cycle disorders (Cordoba et al., 1998; Schubiger et al., 1991;
Gilbert, 1988; Jeffers et al., 1988; Souba, 1987). The elevated ammonia levels that accompany
human diseases such as acute liver or renal failure can predispose an individual to
encephalopathy due to the ability of ammonia to cross the blood-brain barrier; these effects are
especially marked in newborn infants (Minana et al., 1995; Souba, 1987). Thus, individuals with
disease conditions that lead to hyperammonemia may be more susceptible to the effects of
ammonia from external sources, but there are no studies that specifically support this
susceptibility.
Because the respiratory system is a target of ammonia toxicity, individuals with
respiratory disease (e.g., asthmatics) might be expected to be a susceptible population; however,
controlled human studies that examined both healthy volunteers and volunteers with asthma
exposed to ammonia as well as cross-sectional studies of livestock farmers exposed to ammonia
(Petrova et al., 2008; Sigurdarson et al., 2004; Vogelzang et al., 2000, 1998, 1997; Preller et al.,
1995) generally did not observe a greater sensitivity to respiratory effects in populations with
underlying respiratory disease.
1.2. Weight of Evidence Evaluation for Toxicological Effects
The available evidence for ammonia toxicity indicates that respiratory effects are
associated with inhalation exposure and gastrointestinal effects are associated with oral exposure
to ammonia. Ammonia exposure may not be associated with reproductive or developmental
toxicity, at least at levels in which respiratory and gastrointestinal effects are observed. Immune
system and other systemic effects (i.e., effects on the liver, kidney, heart, spleen, and adrenal
gland) may be associated with exposure to ammonia but are not sensitive targets of ammonia
toxicity. The evidence for these health effects are presented in more detail below. Figure 1-4 is
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an exposure-response array comparing effect levels for inhaled ammonia across a range of
toxicological effect categories.
Respiratory Effects
Evidence for respiratory toxicity associated with exposure to ammonia comes from
studies in humans and animals. Cross-sectional occupational studies involving chronic exposure
to ammonia have consistently demonstrated an increased prevalence of respiratory effects
(Rahman et al., 2007; Ballal et al., 1998) and decreased lung function (Rahman et al., 2007; Ali.,
2001). Cross-sectional studies of livestock farmers exposed to ammonia, controlled human
volunteer studies of ammonia inhalation, and case reports of injury in humans with inhalation
exposure to ammonia provide additional and consistent support for the respiratory system as a
target of ammonia toxicity.
Short-term and subchronic animals studies show respiratory effects in several animal
species (lung inflammation in guinea pigs and rats; focal or interstitial pneumonitis in monkeys,
dogs, rabbits and guinea pigs; pulmonary congestion in mice; thickening of nasal epithelium in
rats and pigs; nasal inflammation or lesions in rats and mice) across different dose regimens and
show respiratory effects across ranges of concentrations suggesting a dose-response (Coon et al.,
1970; Anderson et al., 1964; Broderson et al., 1976; Doig and Willoughby, 1971; Gaafar et al.,
1992). EPA considers the respiratory effects associated with ammonia exposure to be
biologically plausible and adverse. The evidence of observed respiratory effects seen across
multiple human and animal studies identifies the respiratory system as a hazard for ammonia.
Gastrointestinal Effects
Effects on gastric mucosa associated with oral exposure to ammonia are based on
evidence in animals and, to a more limited extent, in humans. Acute gastric toxicity observed in
case reports involving intentional or accidental ingestion of cleaning solutions or ammonia
inhalant capsules appears to reflect the corrosive properties of ammonia. Whether these acute
effects are relevant to toxicity following chronic low-level ammonia exposure is not known.
Indirect evidence is provided by the association between the stomach bacterium H. pylori, which
produces urease that catalyzes urea into ammonia, and human diseases of the upper
gastrointestinal tract (including chronic gastritis, gastric ulcers, and stomach cancer).
In vivo experimental evidence that ammonia is associated with gastric effects is provided
by two short-term studies in male Sprague-Dawley rats (Tsujii et al., 1993; Kawano et al., 1991).
These studies provide consistent findings of decreased gastric mucosal thickness that increased
with ammonia dose (Kawano et al., 1991) and duration of exposure (Tsujii et al., 1993; Kawano
et al., 1991); Tsujii et al. (1993) employed only one ammonia drinking water concentration and
therefore did not provide information on dose-response. Evidence for ammonia-related gastric
toxicity is limited to male rats of one strain and to investigations conducted by one research
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group (Kawano et al. and Tsujii et al. were both affiliated with Osaka University Medical
School).
Mechanistic studies in rodent models support the biological plausibility that ammonia
exposure may be associated with gastric effects. Conditions that favor the unionized form of
ammonia facilitate the penetration of the cell membrane and induce greater gastric toxicity.
Multiple specific mechanistic events have been proposed that may contribute to the induction of
gastric lesions, including ammonia-induced release of proteases, inhibition of mitochondrial
respiration, and increased release of endothelin-1 and thyrotropin-releasing hormone. EPA
considers the gastric effects associated with ammonia exposure to be biologically plausible and
adverse, and relevant to humans. Given the evidence from human, animal, and mechanistic
studies, gastric effects are identified as a hazard for ammonia.
Reproductive/Developmental Effects
No studies of the potential reproductive or developmental toxicity of ammonia in humans
are available, and only one animal study that examined the reproductive effects of ammonia in
the pig has been conducted. This study did not use a conventional test species and did not
include a control group with no ammonia exposure. Further, animals were exposed naturally to
bacterial and mycoplasm pathogens. Although the reproductive and developmental toxicity
database for ammonia is limited, evidence on the endogenous formation of ammonia can inform
the potential for ammonia to present a reproductive and developmental hazard.
Ammonia is endogenously produced in humans and animals during fetal and adult life
and concentrations in blood are homeostatically regulated to remain at low levels. Studies in
humans and animals demonstrate that ammonia is present in fetal circulation. In vivo studies in
several animal species and in vitro studies of human placenta suggest that ammonia is produced
within the uteroplacenta and released into the fetal and maternal circulations (Bell et al., 1989;
Johnson et al., 1986; Haugel et al., 1983; Meschia et al., 1980; Remesar et al., 1980; Holzman et
al., 1979, 1977; Rubaltelli and Formentin, 1968; Luschinsky, 1951). Jozwik et al. (2005)
reported that ammonia levels in human fetal blood (specifically, umbilical arterial and venous
blood) at birth were 1.0-1.4 |ig/mL, compared to 0.5 |ig/mL in the mothers' venous blood.
DeSanto et al. (1993) similarly collected human umbilical arterial and venous blood at delivery
(range of 25-43 weeks of gestation). Ammonia was present in blood samples, with umbilical
arterial ammonia concentrations significantly higher than venous concentrations; there was no
correlation between umbilical ammonia levels and gestational age. In sheep, uteroplacental
tissues are the main site of ammonia production, with outputs of ammonia into both the uterine
and umbilical circulations (Jozwik et al., 1999). In late-gestation pregnant sheep that were
catheterized to allow measurement of ammonia exposure to the fetus, concentrations of ammonia
in umbilical arterial and venous blood and uterine arterial and venous blood ranged from about
0.39 to 0.60 |ig/mL (Jozwik et al., 2005, 1999). Thus, the developing fetus and reproductive
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tissues are normally exposed to ammonia in blood, and external concentrations that do not alter
homeostasis would not be expected to pose a developmental or reproductive hazard.
Immune System Effects
The evidence for ammonia immunotoxicity is based on two epidemiological studies and
four animal studies. Available epidemiological studies that addressed immunological function
are confounded by exposures to a number of other respirable agents that have been demonstrated
to be immunostimulatory. Single-exposure human studies of ammonia evaluating immune
endpoints are not available. Therefore, human studies provide little support for ammonia
immunotoxicity.
Animal studies consistently provide evidence of elevated bacterial growth following
ammonia exposure. This is supported by observations of lung lesions (Broderson et al., 1976),
elevated CFU (Schoeb et al., 1982), and increased mortality (Richard et al., 1978a) in rats or
mice exposed to ammonia; however, the findings from the Broderson et al. (1976) study (% of
animals with gross lesions) were not dose-responsive, and the other studies used single
concentrations of ammonia and therefore did not provide information on dose-response. A
single study suggested that T cells are inhibited by ammonia, but the data were not dose
responsive (Targowski et al, 1984).
Mechanistic data are not available that would support a biologically plausible mechanism
for immunosuppression. Because ammonia damages the protective mucosal epithelium of the
respiratory tract, it is unclear if elevated bacterial colonization is the result of damage to this
barrier or the result of suppressed immunity. Overall, the evidence in humans and animals
indicates that ammonia exposure may be associated with these effects but does not support the
immune system as a sensitive target for ammonia toxicity.
Systemic Effects
Effects of ammonia exposure on organs distal from the portal of entry are based on
evidence in animals and, to a more limited extent, in humans. One occupational epidemiology
study of ammonia-exposed workers reported changes in serum enzymes indicative of altered
liver function (Hamid and El-Gazzar, 1996). Because the study population was small and
measurements of workplace ammonia concentrations were not provided, the evidence for liver
toxicity in humans associated with ammonia exposure is weak.
Effects on various organs, including liver, adrenal gland, kidney, spleen, and heart, were
observed in several studies that examined responses to ammonia exposure in a number of
laboratory species. While effects on many of these organs were observed in multiple species,
including monkey, dog, rabbit, guinea pig, and rat, effects were not consistent across exposure
protocols. For example, Coon et al. (1970) reported fatty liver and calcification and proliferation
of renal tubular epithelium in monkeys, dogs, rabbits, and guinea pigs exposed continuously to
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"3
ammonia for 90 days at a concentration of 470 mg/m , but no histopathological changes in these
organs were observed in the same species following intermittent exposure (8 hours/day, 5
"3
days/week for 6 weeks) to concentrations as high as 770 mg/m . It could be speculated that these
differences in response reflect recovery from short-term (i.e., 8-hour exposures), but the reason
for the inconsistent findings is not known.
Studies of ammonia toxicity that examined systemic effects were all published in the
older toxicological literature. The only oral study of ammonium hydroxide was published in
1939 (Fazekas, 1939), and three subchronic inhalation studies were published between 1952 and
1970 (Coon et al., 1970; Anderson et al., 1964; Weatherby, 1952). In general, the information
from these studies is limited by small group sizes, minimal characterization of some of the
reported responses (e.g., "congestion," "enlarged," "fatty liver"), insufficiently detailed reporting
of study results, and incomplete if any incidence data. In addition, Weatherby (1952), Anderson
et al. (1964), and some of the experiments reported by Coon et al. (1970) used only one ammonia
concentration in addition to the control, so no dose-response information is available the majority
of experimental studies to inform the evidence for systemic effects of ammonia.
As discussed above, ammonia is endogenously produced in all human and animal tissues,
and concentrations in all physiological fluids are homeostatically regulated to remain at low
levels (Souba, 1987). Thus, tissues are normally exposed to ammonia, and external
concentrations that do not alter homeostasis would not be expected to pose a hazard for systemic
effects. Overall, the evidence in humans and animals indicates that ammonia exposure may be
associated with these effects but does not support the liver, adrenal gland, kidney, spleen, or
heart as sensitive targets for ammonia toxicity.
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U> K> H-
Exposure (mg/m3)
o
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c=
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(D D
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c=
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Prevalence of respiratory sym |)tom s &
decreased lun|jfunction (male occupational);
HQlnessetal. (1989)
Prevalence of respiratory s^m ptoms &
decreased lung function (occupational);
Rahmanet al. (2007)
io
ro
in
to
O
*3
h>
Nonspecific lung inflam matidn dyspnea,
focaljor interstitial pneumonitis
& calcification of bronchial
epithelia (rat); Coon et all (1970)
thickining tracheal epithelium (pig);
Doig & Willoughby (1971)
Increased incidence of
gross lung lesions (rat);
Broderson dt al. (1976)
Congestion of the
liver(gpnea pig);
Weatherby (1952)
Fatty liverichanges in
plate cells (rat);
Coonetkl. (1970)
j calcification &
i of renal tubular e
Coonetal
prol
pithelii
iferation
ia (rat);
(1970)
Myocardial fibrosis (rat);
Coonetaj. (1970)
Ocular irritation (dog, rabbit);
Coonetal. (1970)
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Cancer
The available information on carcinogenicity following exposure to ammonia is limited
to oral animal studies. There was no evidence of carcinogenicity in Swiss or C3H mice
administered ammonium hydroxide in drinking water for a lifetime (Toth, 1972). There is
limited evidence that ammonia administered in drinking water may act as a cancer promoter
based on the findings of studies designed to examine//, pylori-induced gastric cancer (Tsujii et
al., 1995, 1992a). Additionally, the genotoxic potential cannot be characterized based on the
available genotoxicity information. Thus, under the Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 2005a), there is "inadequate information to assess the carcinogenic potential" of
ammonia.
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2. DOSE-RESPONSE ANALYSIS
2.1. Oral Reference Dose for Effects other than Cancer
The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty
spanning perhaps an order of magnitude) of a daily exposure to the human population (including
sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a
lifetime. It can be derived from a NOAEL, LOAEL, or benchmark dose (BMD), with
uncertainty factors (UFs) generally applied to reflect limitations of the data used.
The oral toxicity database for ammonia is very limited, although as noted in Section 1.2,
gastric toxicity is identified as a hazard for ammonia based on evidence from case reports in
humans, two animal studies, and mechanistic studies. Evidence in humans is limited to case
reports of individuals suffering from gastrointestinal (e.g., stomach ache, nausea, diarrhea,
distress, and burns along the digestive tract) effects from ingesting household cleaning solutions
containing ammonia or biting into capsules of ammonia smelling salts. The data in humans were
not considered for derivation of the RfD because although case reports can suggest the nature of
acute endpoints in humans they are inadequate for dose-response analysis and derivation of a
chronic reference value due to short duration of exposure and incomplete or missing quantitative
exposure information.
Two studies reported gastrointestinal effects, characterized as increased epithelial cell
migration in the mucosa of the stomach (in particular the antrum) leading to a statistically
significant decrease in the thickness of the antral mucosa, in rats following subchronic (Tsujii et
al., 1993) and short-term (Kawano et al., 1991) oral exposure to ammonia. These studies are
repeated dose studies that analyzed gastrointestinal effects of ammonia and did not evaluate a
comprehensive array of endpoints. Additionally, although both studies included a control group,
Tsujii et al. (1993) employed one dose group and Kawano et al. (1991) included two dose
groups. However, the decreased gastric antral mucosal thickness was consistently observed
across these two studies. Prevalence of this effect was observed to generally increase with dose
and duration, and the magnitude of decreases in thickness was 40-60%. Tsujii et al. (1993) and
Kawano et al. (1991) reported that the gastric mucosal effects observed in rats resemble mucosal
changes in human atrophic gastritis; indicating this effect is biological plausible and relevant to
humans. Therefore, decreased gastric antral mucosal thickness is an effect considered by EPA to
be adverse.
Given the limited number of studies available and the small number of toxicological
evaluations, there are uncertainties associated with the oral database for ammonia. Although the
oral database is limited, derivation of a RfD was considered due to the toxicological significance
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of the reported gastrointestinal effects. However, uncertainties with extrapolations from the
available data (described below) were too high to support derivation of a chronic RfD; thus, in
consideration of the limited oral database and associated uncertainties a RfD for ammonia was
not derived.
In considering the derivation of a RfD, the subchronic study by Tsujii et al. (1993) was
considered as a potential principal study due to the relatively longer duration of exposure
compared with the short-term study by Kawano et al. (1991). Decreased gastric antral mucosal
thickness was considered as a potential critical effect. This effect was characterized as a portal-
of-entry effect based on the following. Tsujii et al. (1993) postulated that the difference in
response of the mucosa in the stomach body versus the mucosa of the antrum relates to
differences in pH in the two stomach regions. Most ammonia is transformed to ammonium ion
in solution at physiological pH; the ratio of ammonia to ammonium ion increases 10-fold with
each unit rise in pH. In the mucosa of the stomach body—an acid-secreting mucosa—ammonia
is protonated to the ammonium ion, which reduces the cytotoxicity associated with nonionized
ammonia. In the antral mucosa—a nonacid secreting area of the stomach—the pH is higher,
resulting in a relatively higher concentration of ammonia and thus enhanced cytotoxicity.
EPA identified a potential point of departure (POD) based on the LOAEL of 33 mg/kg-
day, for decreased gastric antral mucosal thickness in rats, from this study. BMD modeling was
not utilized because the Tsujii et al. (1993) employed only one dose level and a control, a data set
that is not amenable to dose-response analysis.
In U.S. EPA's Recommended Use of Body Weight314 as the Default Method in Derivation
of the Oral Reference Dose (U.S. EPA, 201 la), the Agency endorses a hierarchy of approaches
to derive human equivalent oral exposures from data from laboratory animal species, with the
preferred approach being physiologically based pharmacokinetic modeling. Other approaches
may include using some chemical-specific information, without a complete physiologically
based pharmacokinetic model. In lieu of chemical-specific models or data to inform the
derivation of human equivalent oral exposures, EPA endorses body weight scaling to the %
power (i.e., BW3 4) as a default to extrapolate toxicologically equivalent doses of orally
administered agents from laboratory animals to humans for the purpose of deriving a RfD. More
specifically, the use of BW3 4 scaling for deriving a RfD is recommended when the observed
effects are associated with the parent compound or a stable metabolite, but not for portal-of-entry
effects.
No PBPK model or chemical-specific information exists to inform the generation of
human equivalent oral exposures for ammonia. Furthermore, because ammonia oral toxicity
appears to be a function of the physical/chemical environment at the mucosal surface (i.e., a
portal-of-entry effect) and it is not clear if regions of the stomach scale allometrically across
species, a surface area adjustment would be the most relevant for interspecies extrapolation;
however, a dose scaling approach involving mass per unit surface area has not been developed
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(U.S. EPA, 201 la). Therefore, because effects on the gastric antral mucosa are not expected to
scale allometrically, a BW3/4 scaling approach (in combination with a reduced default UF for
interspecies extrapolation) was not applied.
The composite UF for ammonia that would be applied to the POD (LOAEL of 33 mg/kg-
day) from the Tsujii et al. (1993) study would be 10,000, consisting of four areas of uncertainty.
These areas of uncertainty, and the UFs that address each, are based on EPA's A Review of the
Reference Dose and Reference Concentration Processes (U.S. EPA, 2002; Section 4.4.5) and
include the following: uncertainties associated with intraspecies extrapolation (i.e., to account for
human variability in susceptibility to ammonia; UFH = 10), uncertainties associated with
extrapolation of data from the rat to humans in the absence of information on species differences
in toxicokinetics and toxicodynamics (i.e., interspecies extrapolation; UFa = 10), uncertainties
associated with extrapolation of data from a subchronic study (i.e., 8-week study) to a reference
value for chronic exposure scenarios (UFS = 10), uncertainties associated with extrapolation
from a LOAEL to NOAEL (UFl = 10), and database deficiencies (UFd = 1; see Section 2.2.2 for
the justification for this UF).
In the report, A Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA, 2002), the RfD/RfC technical panel concluded that, in cases where
maximum uncertainty exists in four or more areas of uncertainty, or when the total UF is
>10,000, it is unlikely that the database is sufficient to derive a reference value. Therefore,
consistent with the recommendations in U.S. EPA (2002), the available oral data for ammonia
were considered insufficient to support reference value derivation and an RfD for ammonia was
not derived.
Route-to-route extrapolation from inhalation data was considered for deriving the oral
RfD; however, in the absence of a PBPK model and because the critical effect from the
inhalation literature is a portal-of-entry effect (respiratory irritation and decreased lung function),
route-to-route extrapolation is not supported (U.S. EPA, 1994).
Previous IRIS Assessment: Reference Dose
No RfD was derived in the previous IRIS assessment for ammonia
2.2. Inhalation Reference Concentration for Effects other than Cancer
"3
The RfC (expressed in units of mg/m ) is defined as an estimate (with uncertainty
spanning perhaps an order of magnitude) of a continuous inhalation exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or benchmark
concentration (BMC), with UFs generally applied to reflect limitations of the data used.
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As discussed in Section 1.2, respiratory effects have been identified as a hazard following
inhalation exposure to ammonia. The studies in humans and animals examining inhalation
exposure to ammonia provide evidence that inhaled ammonia is associated with toxicity to the
respiratory system. The experimental toxicology literature for ammonia also provides evidence
that inhaled ammonia may be associated with toxicity to target organs other than the respiratory
system, including the liver, adrenal gland, kidney, spleen, heart, and immune system. The
weight of evidence for these effects is less robust than for respiratory effects. Therefore, the
respiratory system is the primary and most sensitive target of inhaled ammonia toxicity in
humans and experimental animals.
Human data are preferred over animal data for deriving reference values when possible
because the use of human data is more relevant in the assessment of human health and avoids the
uncertainty associated with interspecies extrapolation introduced when animal data serve as the
basis for the RfC. Additionally, the respiratory effects in animals were observed at ammonia
concentrations higher than those associated with respiratory effects in humans and represent
much shorter durations (up to 114 days) of exposure, and thus were considered to carry less
weight than the available human data. Therefore, data in humans were considered for derivation
of the RfC and the respiratory effects in animals were not further considered.
Of the available human data, two occupational studies—Rahman et al. (2007) and
Holness et al. (1989)—provide information useful for examining the relationship between
chronic ammonia exposure and respiratory irritation and decreased lung function (quantitative
dose-response analysis of ammonia respiratory tract toxicity data). Both studies reported the
presence or absence of respiratory effects in workers exposed to ammonia over a range of
"3
concentrations (approximately 1 to 7 mg/m ). Both studies provide consistent estimates of the
"3
effect level for ammonia, with the NOAELadj of 3.1 mg/m identified from the Holness et al.
(1989) study falling between the NOAELadj and LOAELadj values (1.8 and 6.6 mg/m ,
respectively) from the Rahman et al. (2007) study. These studies are considered as candidate
principal studies for RfC derivation. Other occupational epidemiology studies (Ali et al., 2001;
Ballal et al., 1998) did not provide exposure information adequate for dose-response analysis and
thus were not useful for RfC derivation.
Consideration of analytical methods suggests that higher confidence is associated with
the exposure measures reported by Holness et al. (1989) than Rahman et al. (2007). Rahman et
al. (2007) used two analytical methods for measuring ammonia concentrations in workplace air
(Drager PAC III and Drager tube); concentrations measured by the two methods differed by
four- to fivefold, indicating some uncertainty in these measurements, although ammonia
concentrations measured by the two methods were strongly correlated. In contrast, the Holness
et al. (1989) study used an established analytical method for measuring exposure to ammonia
recommended by NIOSH that involved the collection of air samples on acid-treated silica gel
(ATSG) absorption tubes.
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Due to the greater confidence in the ammonia measurements in Holness et al. (1989) and
considering the range of NOAELs and LOAELs reported in both studies (in which a higher
NOAEL was reported by Holness et al. [1989]) the occupational exposure study of ammonia
exposure in workers in a soda ash plant by Holness et al. (1989) was identified as the principal
study for RfC derivation. Respiratory effects, characterized as increased respiratory irritation
and decreased lung function, observed in workers exposed to ammonia concentrations >6.6
"3
mg/m were selected as the critical effect. Respiratory effects, including changes in measures of
lung function and increased prevalence of wheezing, chest tightness, and cough/phlegm, have
been identified as adverse respiratory health effects by the American Thoracic Society (2000),
and are similarly noted as adverse in the EPA's Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994).
2.2.1. Methods of Analysis
In the evaluation of the prevalence of increased respiratory irritation and decreased lung
"3
function in workers exposed to ammonia (Holness et al., 1989), aNOAELADJ of 3.1 mg/m
(adjusted for continuous exposure from 8.8 mg/m ; see calculation below) was identified based
on the absence of statistically significant increases in the prevalence of the respiratory effects.
BMD modeling was not utilized because ammonia concentrations in the Holness et al. (1989)
study were not associated with changes in respiratory effects in the study population (i.e., data
from Holness et al. could not be subjected to dose-response modeling). Thus, the Holness et al.
•j
(1989) data were analyzed using a NOAEL approach and the NOAELadj of 3.1 mg/m was
used as the POD for RfC derivation.
Because the RfC is a measure that assumes continuous human exposure over a lifetime,
the POD derived from an occupational exposure was adjusted to account for the noncontinuous
exposure associated with occupational exposure (i.e., 8-hour workday and 5-day workweek).
The duration-adjusted POD was calculated as follows:
NOAELadj = NOAEL x VEho/VEh x 5 days/7 days
= 8.8 mg/m3 x 10 m3/20 m3 x 5 days/7 days
= 3.1 mg/m3
Where:
VEho = human occupational default minute volume (10 m3 breathed during the 8-hour workday,
corresponding to a light to moderate activity level [U.S. EPA, 201 lb])
VEh = human ambient default minute volume (20 m3 breathed during the entire day)
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2.2.2. Derivation of Reference Concentration
The UFs, selected based on EPA's A Review of the Reference Dose and Reference
Concentration Processes (U.S. EPA, 2002; Section 4.4.5) and described in the Preamble of this
document, address five areas of uncertainty resulting in a composite UF of 10. This composite
-3
UF was applied to the selected POD (3.1 mg/m ) to derive an RfC.
• An intraspecies uncertainty factor, UFH, of 10 was applied to account for potentially
susceptible individuals in the absence of data evaluating variability of response to inhaled
ammonia in the human population;
• An interspecies uncertainty factor, UFa, of 1 was applied to account for uncertainty in
extrapolating from laboratory animals to humans because the POD was based on human
data from an occupational study;
• A subchronic to chronic uncertainty factor, UFS, of 1 was applied because the
occupational exposure period in the principal study (Holness et al., 1989), i.e., mean
number of years at present job for exposed workers, of approximately 12 years was of
chronic duration;
• A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because a NOAEL value
was used as the POD; and
• A database uncertainty factor, UFD, of 1 was applied to account for deficiencies in the
database. The ammonia inhalation database consists of studies of occupational exposure
focused on effects of ammonia on respiratory irritation and lung function, studies in
livestock farmers, controlled exposure studies involving volunteers exposed to ammonia
vapors for short periods of time to evaluate irritation effects and changes in lung function,
and a large number of case reports of acute exposure to high ammonia concentrations
(e.g., accidental spills/releases). Studies of the toxicity of inhaled ammonia in
experimental animals include subchronic studies in rats, guinea pigs, and pigs that
examined respiratory and other systemic effects of ammonia and one limited,
reproductive toxicity study in young female pigs. The database lacks developmental and
multigeneration reproductive toxicity studies.
As noted in EPA's A Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA, 2002), "the size of the database factor to be applied will depend on
other information in the database and on how much impact the missing data may have on
determining the toxicity of a chemical and, consequently, the POD." Multigeneration
reproductive and developmental toxicity studies would not be expected to impact the
determination of ammonia toxicity at the POD, and therefore a database UF to account
for the lack of these studies is not necessary. This determination was based on the
observation that ammonia is endogenously produced and homeostatically regulated in
humans and animals during fetal and adult life. Baseline blood levels in healthy
individuals range from 0.1 to 1.0 |ig/mL (Monsen, 1987; Conn, 1972; Brown et al.,
1957). The fetoplacental unit produces ammonia, and concentrations in human umbilical
vein and artery blood (at term) have been shown to be higher than concentrations in
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maternal blood (Jozwik et al., 2005), providing some assurance that developmental
toxicity would not be associated with concentrations of ammonia at or below the POD.
DeSanto et al. (1993) reported that human fetal umbilical blood levels of ammonia at
birth were not influenced by gestational age based on deliveries ranging from gestation
week 25-43. Finally, evidence in animals (Manninen et al., 1988; Schaerdel et al., 1983)
suggests that exposure to ammonia at concentrations up to 18 mg/m3 does not alter blood
ammonia levels (see Appendix A, Section A.3, for a more detailed discussion of
ammonia distribution and elimination). Accordingly, exposure at the POD (3.1 mg/m3)
would not be expected to alter ammonia homeostasis or result in measureable increases in
blood ammonia concentrations. Thus, the concentration of ammonia at the POD for the
RfC would not be expected to result in systemic toxicity, including reproductive or
developmental toxicity.
The RfC for ammonia was calculated as follows:
RfC = NOAELadj - UF
= 3.1 mg/m3 ^ 10
3 3
= 0.31 mg/m or 0.3 mg/m (rounded to one significant figure)
2.2.3. Uncertainties in the Derivation of the RfC
As presented earlier in this section and in the Preamble, EPA standard practices and RfC
guidance (U.S. EPA, 2002, 1995, 1994a, b) were followed in applying a UF approach to a POD
to derive the RfC. Specific uncertainties were accounted for by the application of UFs (i.e., in
the case of the ammonia RfC, a factor to address the absence of data to evaluate the variability in
response to inhaled ammonia in the human population). The following discussion identifies
additional uncertainties associated with the quantification of the RfC for ammonia.
Use of a NOAEL as a POD
Data sets that support BMD modeling are generally preferred for reference value
derivation because the shape of the dose-response curve can be taken into account in establishing
the POD. For the ammonia RfC, no decreases in lung function or respiratory irritation were
observed in the worker population studied by Holness et al. (1989), i.e., the principal study used
to derive the RfC, and as such the data from this study did not support dose-response modeling.
Rather, a NOAEL from the Holness et al. (1989) study was used to estimate the POD. The
availability of dose-response data from a single study of ammonia would increase the confidence
in the estimation of the POD.
Endogenous ammonia
Ammonia, which is produced endogenously, has been detected in the expired air of
"3
healthy volunteers at levels generally ranging from 0.013 to 2.1 mg/m (Boshier et al., 2010;
Smith et al., 2008; Spanel et al., 2007a, b; Turner et al., 2006; Diskin et al., 2003; Kearney et al.,
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2002; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977). The higher and more
variable ammonia concentrations within this range are reported in breath exhaled from the mouth
or oral cavity, with the majority of ammonia concentrations from these sources ranging from
0.09 to 2.1 mg/m3 (Smith et al., 2008; Spanel et al., 2007a, b; Turner et al., 2006; Diskin et al.,
2003; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977). Ammonia in exhaled breath
from the mouth or oral cavity is largely attributed to the production of ammonia via bacterial
degradation of food protein in the oral cavity or gastrointestinal tract (Turner et al., 2006; Smith
et al., 1999; Vollmuth and Schlesinger, 1984), and can be influenced by factors such as diet, oral
hygiene, age, and living conditions (i.e., urban vs. rural setting). In contrast, ammonia
concentrations measured in breath exhaled from the nose and trachea are lower (range: 0.013-
0.078 mg/m ; Smith et al., 2008; Larson et al., 1977) and more likely reflect systemic levels of
ammonia (i.e., circulating levels in the blood) (Smith et al., 2008).
Ammonia concentrations measured in breath exhaled from the nose and trachea, i.e.,
concentrations expected to more closely correlate with circulating levels of ammonia in blood,
-3
are lower than the ammonia RfC of 0.3 mg/m by a factor of approximately fourfold or more;
however, the RfC does fall within the more variable range of breath concentrations collected
from the mouth or oral cavity. Although the contribution of ammonia generated endogenously
and expired through exhalation to ammonia present in ambient air is not known, this contribution
is expected to be minimal considering the ammonia in expired air should rapidly mix with and be
diluted in the much larger volume of ambient air.
2.2.4. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the
RfC, the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods
for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry
(U.S. EPA, 1994b). Confidence in the principal study (Holness et al., 1989) is medium. The
design, conduct, and reporting of this occupational exposure study were adequate, but the study
was limited by a small sample size and by the fact that workplace ammonia concentrations to
which the study population was exposed were below those associated with ammonia-related
effects (i.e., only a NOAEL was identified). However, this study is supported in the context of
the entire database, including the NOAEL and LOAEL values identified in the Rahman et al.
(2007) occupational exposure study, multiple studies of acute ammonia exposure in volunteers,
and the available inhalation data from animals. Confidence in the database is medium. The
inhalation ammonia database includes limited studies of reproductive toxicity and no studies of
developmental toxicity; however, reproductive, developmental, and other systemic effects are
not expected at the RfC because it is well documented that ammonia is endogenously produced
in humans and animals, ammonia concentrations in blood are homeostatically regulated to
remain at low levels, and ammonia concentrations in air at the POD are not expected to alter
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homeostasis. Reflecting medium confidence in the principal study and medium confidence in
the database, the overall confidence in the RfC is medium.
2.2.5. Previous IRIS Assessment: Reference Concentration
The previous IRIS assessment for ammonia (posted to the database in 1991) presented an
-3
RfC of 0.1 mg/m based on co-principal studies—the occupational exposure study of workers in
a soda ash plant by Holness et al. (1989) and the subchronic study by Broderson et al. (1976) that
examined the effects of ammonia exposure in F344 rats inoculated on day 7 of the study with the
-3
bacterium M pulmonis. The NOAEL of 6.4 mg/m (estimated as the mean concentration of the
entire exposed group) from the Holness et al. (1989) study (duration adjusted: NOAELadj =
2.3 mg/m3) was used as the POD.5
The previous RfC was derived by dividing the POD by a composite UF of 30: 10 to
account for the protection of sensitive individuals and 3 for database deficiencies to account for
the lack of chronic data, the proximity of the LOAEL from the subchronic inhalation study in the
rat (Broderson et al., 1976) to the NOAEL, and the lack of reproductive and developmental
toxicology studies. A UFd of 3 (rather than 10) was applied because studies in rats (Schaerdel et
al., 1983) showed no increase in blood ammonia levels at an inhalation exposure to 32 ppm (22.6
3 3
mg/m ) and only minimal increases at 300-1,000 ppm (212-707 mg/m ), suggesting that no
significant distribution is likely to occur at the human equivalent concentration. In this
document, a UFd of one was selected because a more thorough investigation of the literature on
ammonia homeostasis and literature published since 1991 on fetoplacental ammonia levels
provides further support that exposure to ammonia at the POD would not result in a measureable
increase in blood ammonia, including fetal blood levels.
2.3. Cancer Risk Estimates
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. Quantitative risk estimates may be derived from the application of a
low-dose extrapolation procedure. If derived, and unless otherwise stated, the oral slope factor is
a plausible upper bound on the estimate of risk per mg/kg-day of oral exposure. Similarly, an
"3
inhalation unit risk is a plausible upper bound on the estimate of risk per (J,g/m air breathed.
As discussed in Section 1.2, there is "inadequate information to assess the
carcinogenic potential" of ammonia. Therefore, a quantitative cancer assessment was not
conducted and cancer risk estimates were not derived for ammonia. The previous IRIS
assessment did not include a carcinogenicity assessment.
5In this document, the lower bound of the high exposure category from the Holness et al. (1989) study (8.8 mg/m3,
adjusted for continuous exposure to 3.1 mg/m3) was identified as the POD because workers in this high exposure
category, as well as those in the two lower exposure categories, showed no statistically significant increase in
respiratory irritation or decreases in pulmonary function.
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