v>EPA
EPA/635/R-16/163 Fa
www.epa.gov/iris
Toxicological Review of Ammonia
Noncancer Inhalation
[CASRN 7664-41-7]
September 2016
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of Ammonia
DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
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CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS vi
PREFACE ix
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xii
EXECUTIVE SUMMARY xx
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION xxvi
1. HAZARD IDENTIFICATION 1-1
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS 1-1
1.1.1. Chemical Properties 1-1
1.1.2. Toxicokinetics 1-2
1.2. SYNTHESIS OF EVIDENCE 1-3
1.2.1. Respiratory Effects 1-3
1.2.2. Immune System Effects 1-20
1.2.3. Other Systemic Effects 1-25
1.3. SUMMARY AND EVALUATION 1-33
1.3.1. Weight of Evidence for Effects Other than Cancer 1-33
1.3.2. Susceptible Populations and Lifestages 1-34
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis 2-1
2.1.2. Methods of Analysis 2-5
2.1.3. Derivation of the Reference Concentration 2-7
2.1.4. Uncertainties in the Derivation of the Reference Concentration 2-9
2.1.5. Confidence Statement 2-12
2.1.6. Previous IRIS Assessment 2-12
REFERENCES R-l
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TABLES
Table ES-1. Summary of reference concentration (RfC) derivation xxii
Table LS-1. Inclusion/exclusion criteria for inhalation health effect/toxicity studies (pre-peer review)
xxix
Table LS-2. Summary of epidemiology database xxxviii
Table LS-3. Considerations and relevant experimental information for evaluation of experimental
animal studies xxxix
Table LS-4. Summary of experimental animal database xlii
Table 1-1. Chemical and physical properties of ammonia 1-1
Table 1-2. Evidence pertaining to respiratory effects in humans following inhalation exposure in
industrial settings 1-8
Table 1-3. Evidence pertaining to respiratory effect in humans following inhalation exposure in
cleaning settings 1-12
Table 1-4. Evidence pertaining to respiratory effects in animals 1-16
Table 1-5. Evidence pertaining to immune system effects in animals 1-22
Table 1-6. Evidence pertaining to other systemic effects in animals 1-27
Table 2-1. Frequency distribution of ammonia exposure from Holness et al. (1989) 2-6
FIGURES
Figure LS-1. Summary of literature search and screening process for ammonia xxviii
Figure 1-1. Exposure-response array of respiratory effects following inhalation exposure to
ammonia 1-18
Figure 1-2. Exposure-response array of immune system effects following inhalation exposure to
ammonia 1-24
Figure 1-3. Exposure-response array of systemic effects following inhalation exposure to
ammonia 1-31
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ABBREVIATIONS
ADME absorption, distribution, metabolism, MRM
excretion NCEA
AEGL Acute Exposure Guideline Level
ACGIH American Conference of Governmental NH3
Industrial Hygienists NH4+
ALP alkaline phosphatase NIOSH
ATSDR Agency for Toxic Substances and
Disease Registry NOAEL
BCG bacillus Calmette-Guerin NRC
BMCL 95% lower bound on the benchmark ORD
concentration
BUN blood urea nitrogen PEF
CAAC Chemical Assessment Advisory PEFR
Committee PM
CAC cumulative ammonia concentration POD
CERCLA Comprehensive Environmental PPD
Response, Compensation, and Liability RfC
Act RfD
CFU colony forming unit RTECS
CI confidence interval
DAP diammonium phosphate SAB
EPA Environmental Protection Agency TLV
FEF forced expiratory flow TSCATS
FEVi forced expiratory volume in 1 second
FEVi% ratio of FEVi to FVC (FEVi/FVC) TWA
FVC forced vital capacity UF
HERO Health and Environmental Research UFa
Online UFh
HPV high production volume UFl
HPVIS high production volume information UFs
system
IgE immunoglobulin E UFd
IgG immunoglobulin G VEh
IOM Institute of Medicine
IRIS Integrated Risk Information System VEho
LC50 50% lethal concentration WOS
LD50 50% lethal dose
LOAEL lowest-observed-adverse-effect level
MLE maximum likelihood estimate
murine respiratory mycoplasmosis
National Center for Environmental
Assessment
ammonia
ammonium ion
National Institute for Occupational
Safely and Health
no-observed-adverse-effect level
National Research Council
EPA's Office of Research and
Development
peak expiratory flow
peak expiratory flow rate
particulate matter
point of departure
purified protein derivative
reference concentration
reference dose
Registry of Toxic Effects of Chemical
Substances
Science Advisory Board
Threshold Limit Value
Toxic Substance Control Act Test
Submission Database
time-weighted average
uncertainty factor
interspecies uncertainty factor
intraspecies uncertainty factor
LOAEL to NOAEL uncertainty factor
subchronic-to-chronic uncertainty
factor
database deficiencies uncertainty factor
human occupational default minute
volume
human ambient default minute volume
Web of Science
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AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Audrey Galizia, Dr. PH (Assessment U.S. EPA/ORD/NCEA
Manager)
James Ball, Ph.D. (no longer with EPA)
Glinda Cooper, Ph.D. (no longer with EPA)
Louis D'Amico, Ph.D.
Keith Salazar, Ph.D.
Christopher Sheth, Ph.D. (no longer with
EPA)
Christopher Brinkerhoff, Ph.D. ORISE Postdoctoral Fellow at the U.S. EPA
Washington, DC (currently with the Office of
Chemical Safety and Pollution Prevention)
Contributors
Ted Berner, MS U.S. EPA/ORD/NCEA
Vincent Cogliano, Ph.D.
Jason Fritz, Ph.D.
Martin Gehlhaus, MPH (currently with
EPA Region 3)
Samantha Jones, Ph.D.
John Stanek, Ph.D.
Jamie Strong, Ph.D. (currently with the
Office of Water)
Production Team
Maureen Johnson U.S. EPA/ORD/NCEA
Ellen F. Lorang, MA (Retired)
Vicki Soto
Dahnish Shams
Contractor Support
Amber Bacom, MS SRC, Inc., Syracuse, NY
Fernando Llados, Ph.D.
Julie Stickney, Ph.D.
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director - Retired) U.S. EPA/ORD/NCEA
Lynn Flowers, Ph.D., DABT (Associate Director for Health,
currently with the Office of Science Policy)
John Vandenberg, Ph.D. (HHRA National Program Director)
Vincent Cogliano, Ph.D. (IRIS Program Director)
Gina Perovich, M.S. (IRIS Program Deputy Director)
Samantha Jones, Ph.D. (IRIS Associate Director for Science)
Susan Rieth, MPH (Branch Chief)
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Internal Reviewers
Marian Rutigliano, DO, MSEV
Amanda S. Persad, Ph.D.
Paul Reinhart, Ph.D.
John Whalan, BS
U.S. EPA/ORD/NCEA
Reviewers
This assessment was provided for review to scientists in EPA's Program and Regional Offices.
Comments were submitted by:
Office of the Administrator/Office of Children's Health Protection
Office of Air and Radiation/Office of Air Quality and Planning Standards
Office of Air and Radiation/Office of Transportation and Air Quality
Office of Policy
Office of Water
Region 2, New York City
This assessment was provided for review to other federal agencies and the Executive Office of the
President (EOP). A summary and EPA's disposition of major comments from the other federal
agencies and EOP is available on the IRIS website. Comments were submitted by:
This assessment was released for public comment on June 8, 2012 and comments were due on
August 7, 2012. The public comments are available on Regulations.gov. A summary and EPA's
disposition of the comments from the public is included in the revised external review draft
assessment on the IRIS website. Comments were received from the following entities:
This assessment was peer reviewed by independent, expert scientists external to EPA convened by
EPA's Science Advisory Board (SAB), the Chemical Assessment Advisory Committee Augmented for
the IRIS Ammonia Assessment. A peer review meeting was held on July 14 to 16, 2014. The report
ofthe SAB's review of EPA's Draft Toxicological Review of Ammonia, dated August 6, 2015, is
available on the IRIS website. A summary and EPA's disposition of the comments received from the
SAB is included in Appendix D.
Dr. Michael Dourson (chair), Toxicology Excellence for Risk Assessment, Cincinnati, OH
Dr. Daniel Acosta, National Center for Toxicological Research, U. S. Food and Drug
Administration, Jefferson, AR
Dr. Henry Anderson, Wisconsin Division of Public Health, Madison, WI
Dr. Scott Bartell, Program in Public Health, University of California - Irvine, Irvine, CA
Dr. Arthur Cooper, Department of Biochemistry and Molecular Biology, New York Medical
College, Valhalla, NY
Dr. David Eastmond, Department of Cell Biology and Neuroscience and Environmental
Toxicology Graduate Program, University of California, Riverside, CA
Dr. William Michael Foster, Pulmonary and Critical Care Medicine, Duke University Medical
Center, Durham, NC
Department of Agriculture/Food Safety and Inspection Service
DHHS/Agency for Toxic Substances and Disease Registry
Executive Office of the President/Council on Environmental Quality
The American Chemistry Council
The Fertilizer Institute
Washington, DC
Washington, DC
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Dr. Russ Hauser, Department of Environmental Health, Harvard T. H. Chan School of Public
Health, Harvard University, Boston, MA
Dr. Abby A. Li, Health Science Practice, Exponent Incorporated, San Francisco, CA
Dr. Maria Morandi, Independent Consultant, Houston, TX
Dr. Victoria Persky, Epidemiology & Biostatistics Program, School of Public Health, University of
Illinois at Chicago, Chicago, IL
Dr. Kenneth Ramos, Precision Health Sciences and Professor of Medicine, Arizona Health
Sciences Center, Tucson, AZ
Dr. Alan H. Stern, New Jersey Department of Environmental Protection, Trenton, NJ, and
Department of Environmental and Occupational Health, Rutgers University School of Public
Health, NJ
Dr. I. David Weiner, Division of Nephrology, Hypertension & Renal Transplantation, University
of Florida, Gainesville, FL
Designated Federal Officer: Dr. Suhair Shallal
The post-external review draft of the assessment was provided for review to scientists in EPA's
Program and Regional Offices, and to other federal agencies and the Executive Office of the
President (EOP). A summary and EPA's disposition of major comments from the other federal
agencies and EOP is available on the IRIS website. Comments were submitted by:
Office of the Administrator/Office of Children's Health Protection
Office of Land and Emergency Management
Executive Office of the President/Office of Management and Budget
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PREFACE
This Toxicological Review critically reviews the publicly available studies on ammonia in
order to identify its adverse health effects and to characterize exposure-response relationships.
The assessment covers gaseous ammonia (NH3) and ammonia dissolved in water (ammonium
hydroxide, NH4OH). It was prepared under the auspices of the Environmental Protection Agency's
(EPA's) Integrated Risk Information System (IRIS) program.
Ammonia and ammonium hydroxide are listed as hazardous substances under the
Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA).
Ammonia is subject to reporting requirements for the Toxics Release Inventory under the
Emergency Planning and Community Right-to-Know Act of 1986 and to emergency planning
requirements under section 112(r) of the Clean Air Act
This assessment updates a previous IRIS assessment of ammonia that was developed in
1991. The previous assessment included only an inhalation reference concentration (RfC) for
effects other than cancer. This assessment provides an updated review of information on all
noncancer health effects by the inhalation route only.
This assessment was conducted in accordance with EPA guidance; relevant EPA guidance
documents can be found on the IRIS website (http://www.epa. gov/iris/l. The findings of this
assessment and related documents produced during its development are also available on the IRIS
website (http: / /www, ep a. gov/iris/l. Appendices for other health toxicity values, details of the
literature search strategy and study selection and evaluation, supporting information for hazard
identification and dose response, and other information are provided as Supplemental Information
to this assessment (see Appendices A to C).
Portions of this Toxicological Review were adapted from the Toxicological Profile for
Ammonia developed by the Agency for Toxic Substances and Disease Registiy (ATSDR. 20041 under
a Memorandum of Understanding that encourages interagency collaboration, sharing of scientific
information, and more efficient use of resources.
The IRIS program released this assessment for public comment and peer review in June
2012, as it was beginning to implement systematic review. The approach to implementation is to
use procedures and tools available at the time, without holding assessments until new methods
become available. Accordingly, the IRIS program edited this assessment to increase transparency
and clarity and to use more tables and figures. It conducted literature searches and evaluated
studies using tools and documentation standards then available. Problem formulation materials
and protocol development began with assessments started in 2015, after this assessment was well
into peer review. This assessment addresses peer review comments and retains the structure of
the peer review draft, to maintain fidelity with what the peer reviewers saw. Implementation of
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systematic review is a process of continuous improvement subject to periodic review by the
Chemical Assessment Advisory Committee of the EPA's Science Advisory Board. This assessment
represents a step in the evolution of the IRIS program.
Assessments by Other National and International Health Agencies
Toxicity information on ammonia has been evaluated by ATSDR, the National Research
Council (NRC), the National Institute for Occupational Safety and Health (NIOSH), and the Food and
Drug Administration. The results of these assessments are presented in Appendix A of the
Supplemental Information. It is important to recognize that these assessments may have been
prepared for different purposes and may utilize different methods, and that newer studies may be
included in the IRIS assessment
Overview of Uses, Sources, and Environmental Exposure
About 80% of commercially produced ammonia is used in agricultural fertilizers. Ammonia
is also used as a corrosion inhibitor, in water purification, as a household cleaner, as an
antimicrobial agent in food products, as a refrigerant, as a stabilizer in the rubber industry, in the
pulp and paper and metallurgy industries, as a source of hydrogen in the hydrogenation of fats and
oils, and as a chemical intermediate in the production of pharmaceuticals, explosives, and other
chemicals. Ammonia is also used to reduce nitrogen oxide emissions from combustion sources such
as industrial and municipal boilers, power generators, and diesel engines fHSDB. 2012: lohnson et
al.. 2009: Eggeman. 20011.
Major sources of ammonia gas include leaks and spills during commercial synthesis,
production, storage, processing, or transporting of ammonia; refrigeration equipment failure;
decaying manure from livestock; application of fertilizers; sewage or wastewater effluent; burning
of coal, wood, or other natural products; volcanic eruptions; forest fires; and the decomposition of
nitrogenous compounds. Ammonia from agricultural and other sources, along with sulfate and
nitrate salts, is an important contributor to fine inorganic particulate matter (PM2.5) mass (e.g., see
Paulotand lacob (2014)1. This literature on airborne particular matter is reviewed and evaluated
in EPA's Integrated Science Assessment for Particulate Matter (U.S. EPA. 2009bl.
Environmental exposures to ammonia in the air vary widely. Average ambient
concentrations of ammonia in the United States range from 0.28 to 15 |ig/m3, as measured in 2012
by the National Atmospheric Deposition Program's Ammonia Monitoring Network fAMoN. 20121.
Indoor residential ammonia concentrations can vary widely; one survey reported ammonia
concentrations in homes in Connecticut and southwest and central Virginia ranging from 0.09 to
166 |J.g/m3, depending on the season, use of air conditioning, type of heating, and other factors
(Leaderer etal.. 19991.
Ammonia is found naturally in the environment and is a component of the global nitrogen
cycle; it is essential to many biological processes. Nitrogen-fixing bacteria convert atmospheric
nitrogen to ammonia that is available for uptake into plants. Organic nitrogen released from biota
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can be converted to ammonia. Ammonia in water and soil can be converted to nitrite and nitrate
through the process of nitrification. Ammonia is also endogenously produced in humans and other
mammals, where it is an essential metabolite used in nucleic acid and protein synthesis, is
necessary for maintaining acid-base balance, and is an integral part of nitrogen homeostasis
fNelson and Cox. 2008: Socolow. 1999: Rosswall. 19811.
Scope of this Assessment
This assessment presents an evaluation of the noncancer health effects of ammonia by the
inhalation route of exposure. To address peer-review recommendations to expand the scope of the
oral toxicity literature to include ammonium salts and to allow expeditious completion of the
assessment of inhaled ammonia, ingested ammonia, including consideration of ammonium salts,
will be the focus of a separate assessment. Because carcinogenicity studies of ammonia have been
performed by the oral route of exposure only, the cancer assessment will be moved into the
separate oral assessment
For additional information about this assessment or for general questions regarding IRIS,
please contact EPA's IRIS Hotline at 202-566-1676 (phone), 202-566-1749 (fax), or
hotline.iris@epa.gov.
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The Preamble summarizes the objectives and scope of the IRIS program, general
principles and systematic review procedures used in developing IRIS assessments; and
the overall development process and document structure.
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
1. Objectives and Scope of the IRIS
Program
Soon after EPA was established in 1970, it
was at the forefront of developing risk
assessment as a science and applying it in
support of actions to protect human health and
the environment. EPA's IRIS program1
contributes to this endeavor by reviewing
epidemiologic and experimental studies of
chemicals in the environment to identify adverse
health effects and characterize exposure-
response relationships. Health agencies
worldwide use IRIS assessments, which are also
a scientific resource for researchers and the
public.
IRIS assessments cover the hazard
identification and dose-response steps of risk
assessment. Exposure assessment and risk
characterization are outside the scope of IRIS
assessments, as are political, economic, and
technical aspects of risk management. An IRIS
assessment may cover one chemical, a group of
structurally or toxicologically related chemicals,
or a chemical mixture. Exceptions outside the
scope of the IRIS program are radionuclides,
chemicals used only as pesticides, and the
"criteria air pollutants" (particulate matter,
ground-level ozone, carbon monoxide, sulfur
oxides, nitrogen oxides, and lead).
Enhancements to the IRIS program are
improving its science, transparency, and
productivity. To improve the science, the IRIS
program is adapting and implementing
principles of systematic review (i.e., using
explicit methods to identify, evaluate, and
synthesize study findings). To increase
transparency, the IRIS program discusses key
science issues with the scientific community and
the public as it begins an assessment External
peer review, independently managed and in
public, improves both science and transparency.
Increased productivity requires that
assessments be concise, focused on EPA's needs,
and completed without undue delay.
IRIS assessments follow EPA guidance2 and
standardized practices of systematic review.
This Preamble summarizes and does not change
IRIS operating procedures or EPA guidance.
Periodically, the IRIS program asks for
nomination of agents for future assessment or
reassessment. Selection depends on EPA's
priorities, relevance to public health, and
availability of pertinent studies. The IRIS
multiyear agenda3 lists upcoming assessments.
The IRIS program may also assess other agents
in anticipation of public health needs.
2. Planning an Assessment: Scoping,
Problem Formulation, and
Protocols
Early attention to planning ensures that IRIS
assessments meet their objectives and properly
frame science issues.
Scoping refers to the first step of planning,
where the IRIS program consults with EPA's
program and regional offices to ascertain their
needs. Scoping specifies the agents an
MRIS program website: http: / /www.epa.gov /iris/.
2EPA guidance documents: http://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem#guidance/.
3IRIS multiyear agenda: https: //www.epa.gov/iris/iris-agenda.
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assessment will address, routes and durations of
exposure, susceptible populations and lifestages,
and other topics of interest
Problem formulation refers to the science
issues an assessment will address and includes
input from the scientific community and the
public. A preliminary literature survey,
beginning with secondary sources (e.g.,
assessments by national and international health
agencies and comprehensive review articles),
identifies potential health outcomes and science
issues. It also identifies related chemicals (e.g.,
toxicologically active metabolites and
compounds that metabolize to the chemical of
interest).
Each IRIS assessment comprises multiple
systematic reviews for multiple health
outcomes. It also evaluates hypothesized
mechanistic pathways and characterizes
exposure-response relationships. An
assessment may focus on important health
outcomes and analyses rather than expand
beyond what is necessary to meet its objectives.
Protocols refer to the systematic review
procedures planned for use in an assessment.
They include strategies for literature searches,
criteria for study inclusion or exclusion,
considerations for evaluating study methods and
quality, and approaches to extracting data.
Protocols may evolve as an assessment
progresses and new agent-specific insights and
issues emerge.
3. Identifying and Selecting Pertinent
Studies
IRIS assessments conduct systematic
literature searches with criteria for inclusion and
exclusion. The objective is to retrieve the
pertinent primary studies (i.e., studies with
original data on health outcomes or their
mechanisms). PECO statements (Populations,
Exposures, Comparisons, Outcomes) govern the
literature searches and screening criteria.
"Populations" and animal species generally have
no restrictions. "Exposures" refers to the agent
and related chemicals identified during scoping
and problem formulation and may consider
route, duration, or timing of exposure.
"Comparisons" means studies that allow
comparison of effects across different levels of
exposure. "Outcomes" may become more specific
(e.g., from "toxicity" to "developmental toxicity"
to "hypospadias") as an assessment progresses.
For studies of absorption, distribution,
metabolism, and elimination, the first objective
is to create an inventory of pertinent studies.
Subsequent sorting and analysis facilitates
characterization and quantification of these
processes.
Studies on mechanistic events can be
numerous and diverse. Here, too, the objective is
to create an inventory of studies for later sorting
to support analyses of related data. The
inventory also facilitates generation and
evaluation of hypothesized mechanistic
pathways.
The IRIS program posts initial protocols for
literature searches on its website and adds
search results to EPA's HERO database.4 Then
the IRIS program takes extra steps to ensure
identification of pertinent studies: by
encouraging the scientific community and the
public to identify additional studies and ongoing
research; by searching for data submitted under
the Toxic Substances Control Act or the Federal
Insecticide, Fungicide, and Rodenticide Act; and
by considering late-breaking studies that would
impact the credibility of the conclusions, even
during the review process.5
4. Evaluating Study Methods and
Quality
IRIS assessments evaluate study methods
and quality, using uniform approaches for each
group of similar studies. The objective is that
subsequent syntheses can weigh study results on
their merits. Key concerns are potential bias
(factors that affect the magnitude or direction of
an effect) and insensitivity (factors that limit the
ability of a study to detect a true effect).
4Health and Environmental Research Online: https: //hero.epa.gov/hero/.
5IRIS "stopping rules": https: //www.epa.gov/sites/production/files/2014-06/documents/
iris stoppingrules.pdf.
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For human and animal studies, the
evaluation of study methods and quality
considers study design, exposure measures,
outcome measures, data analysis, selective
reporting, and study sensitivity. For human
studies, this evaluation also considers selection
of participant and referent groups and potential
confounding. Emphasis is on discerning bias that
could substantively change an effect estimate,
considering also the expected direction of the
bias. Low sensitivity is a bias towards the null.
Study-evaluation considerations are specific
to each study design, health effect, and agent
Subject-matter experts evaluate each group of
studies to identify characteristics that bear on
the informativeness of the results. For
carcinogenicity, neurotoxicity, reproductive
toxicity, and developmental toxicity, there is EPA
guidance for study evaluation (U.S. EPA. 2005a.
1998. 1996. 19911. As subject-matter experts
examine a group of studies, additional agent-
specific knowledge or methodologic concerns
may emerge and a second pass become
necessary.
Assessments use evidence tables to
summarize the design and results of pertinent
studies. If tables become too numerous or
unwieldy, they may focus on effects that are
more important or studies that are more
informative.
The IRIS program posts initial protocols for
study evaluation on its website, then considers
public input as it completes this step.
5. Integrating the Evidence of
Causation for Each Health Outcome
Synthesis within lines of evidence. For
each health outcome, IRIS assessments
synthesize the human evidence and the animal
evidence, augmenting each with informative
subsets of mechanistic data. Each synthesis
considers aspects of an association that may
suggest causation: consistency, exposure-
response relationship, strength of association,
temporal relationship, biological plausibility,
coherence, and "natural experiments" in humans
flJ.S. EPA. 1994. §2.1.3) flJ.S. EPA. 2005a. §2.5).
Each synthesis seeks to reconcile ostensible
inconsistencies between studies, taking into
account differences in study methods and
quality. This leads to a distinction between
conflicting evidence (unexplained positive and
negative results in similarly exposed human
populations or in similar animal models) and
differing results (mixed results attributable to
differences between human populations, animal
models, or exposure conditions) (U.S. EPA. 2005a.
§2.5).
Each synthesis of human evidence explores
alternative explanations (e.g., chance, bias, or
confounding) and determines whether they may
satisfactorily explain the results. Each synthesis
of animal evidence explores the potential for
analogous results in humans. Coherent results
across multiple species increase confidence that
the animal results are relevant to humans.
Mechanistic data are useful to augment the
human or animal evidence with information on
precursor events, to evaluate the human
relevance of animal results, or to identify
susceptible populations and lifestages. An agent
may operate through multiple mechanistic
pathways, even if one hypothesis dominates the
literature flJ.S. EPA. 2005a. §2.4.3.3).
Integration across lines of evidence. For
each health outcome, IRIS assessments integrate
the human, animal, and mechanistic evidence to
answer the question: What is the nature of the
association between exposure to the agent and the
health outcome?
For cancer, EPA includes a standardized
hazard descriptor in characterizing the strength
of the evidence of causation. The objective is to
promote clarity and consistency of conclusions
across assessments (U.S. EPA. 2005a. §2.5).
Carcinogenic to humans: convincing
epidemiologic evidence of a causal
association; or strong human evidence of
cancer or its key precursors, extensive
animal evidence, identification of mode-of-
action and its key precursors in animals, and
strong evidence that they are anticipated in
humans.
Likely to be carcinogenic to humans: evidence
that demonstrates a potential hazard to
humans. Examples include a plausible
association in humans with supporting
experimental evidence, multiple positive
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results in animals, a rare animal response, or
a positive study strengthened by other lines
of evidence.
Suggestive evidence of carcinogenic potential:
evidence that raises a concern for humans.
Examples include a positive result in the only
study, or a single positive result in an
extensive database.
Inadequate information to assess carcinogenic
potential: no other descriptors apply.
Examples include little or no pertinent
information, conflicting evidence, or negative
results not sufficiently robust for not likely.
Not likely to be carcinogenic to humans: robust
evidence to conclude that there is no basis
for concern. Examples include no effects in
well-conducted studies in both sexes of
multiple animal species, extensive evidence
showing that effects in animals arise through
modes-of-action that do not operate in
humans, or convincing evidence that effects
are not likely by a particular exposure route
or below a defined dose.
If there is credible evidence of
carcinogenicity, there is an evaluation of
mutagenicity, because this influences the
approach to dose-response assessment and
subsequent application of adjustment factors for
exposures early in life (U.S. EPA. 2005a. §3.3.1,
§3.5), flJ.S. EPA. 2005b. §5).
6. Selecting Studies for Derivation of
Toxicity Values
The purpose of toxicity values (slope factors,
unit risks, reference doses, reference
concentrations; see section 7) is to estimate
exposure levels likely to be without appreciable
risk of adverse health effects. EPA uses these
values to support its actions to protect human
health.
The health outcomes considered for
derivation of toxicity values may depend on the
hazard descriptors. For example, IRIS
assessments generally derive cancer values for
agents that are carcinogenic or likely to be
carcinogenic, and sometimes for agents with
suggestive evidence (U.S. EPA. 2005a. §3).
Derivation of toxicity values begins with a
new evaluation of studies, as some studies used
qualitatively for hazard identification may not be
useful quantitatively for exposure-response
assessment. Quantitative analyses require
quantitative measures of exposure and response.
An assessment weighs the merits of the human
and animal studies, of various animal models,
and of different routes and durations of exposure
(U.S. EPA. 1994. §2.1). Study selection is not
reducible to a formula, and each assessment
explains its approach.
Other biological determinants of study
quality include appropriate measures of
exposure and response, investigation of early
effects that precede overt toxicity, and
appropriate reporting of related effects (e.g.,
combining effects that comprise a syndrome, or
benign and malignant tumors in a specific
tissue).
Statistical determinants of study quality
include multiple levels of exposure (to
characterize the shape of the exposure-response
curve) and adequate exposure range and sample
sizes (to minimize extrapolation and maximize
precision) (U.S. EPA. 2012. §2.1).
Studies of low sensitivity may be less useful
if they fail to detect a true effect or yield toxicity
values with wide confidence limits.
7. Deriving Toxicity Values
General approach. EPA guidance describes
a two-step approach to dose-response
assessment: analysis in the range of observation,
then extrapolation to lower levels. Each toxicity
value pertains to a route (e.g., oral, inhalation,
dermal) and duration or timing of exposure (e.g.,
chronic, subchronic, gestational) fU.S. EPA. 2002.
§4).
IRIS assessments derive a candidate value
from each suitable data set Consideration of
candidate values yields a toxicity value for each
organ or system. Consideration of the organ/
system-specific values results in the selection of
an overall toxicity value to cover all health
outcomes. The organ/system-specific values are
useful for subsequent cumulative risk
assessments that consider the combined effect of
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multiple agents acting at a common anatomical
site.
Analysis in the range of observation.
Within the observed range, the preferred
approach is modeling to incorporate a wide
range of data. Toxicokinetic modeling has
become increasingly common for its ability to
support target-dose estimation, cross-species
adjustment, or exposure-route conversion. If
data are too limited to support toxicokinetic
modeling, there are standardized approaches to
estimate daily exposures and scale them from
animals to humans (U.S. EPA. 1994. §3), (U.S. EPA.
2005a. §3.1), flJ.S. EPA. 2011. 20061.
For human studies, an assessment may
develop exposure-response models that reflect
the structure of the available data (U.S. EPA.
2005a. §3.2.1). For animal studies, EPA has
developed a set of empirical ("curve-fitting")
models6 that can fit typical data sets fU.S. EPA.
2005a. §3.2.2). Such modeling yields a point of
departure, defined as a dose near the lower end
of the observed range, without significant
extrapolation to lower levels (e.g., the estimated
dose associated with an extra risk of 10% for
animal data or 1% for human data, or their 95%
lower confidence limits)(U.S. EPA. 2005a. §3.2.4),
CIJ.S. EPA. 2012. §2.2.1 "1.
When justified by the scope of the
assessment, toxicodynamic ("biologically
based") modeling is possible if data are sufficient
to ascertain the key events of a mode-of-action
and to estimate their parameters. Analysis of
model uncertainty can determine the range of
lower doses where data support further use of
the model fU.S. EPA. 2005a. §3.2.2, §3.3.2).
For a group of agents that act at a common
site or through common mechanisms, an
assessment may derive relative potency factors
based on relative toxicity, rates of absorption or
metabolism, quantitative structure-activity
relationships, or receptor-binding
characteristics (U.S. EPA. 2005a. §3.2.6).
Extrapolation: slope factors and unit
risks. An oral slope factor or an inhalation unit
risk facilitates subsequent estimation of human
cancer risks. Extrapolation proceeds linearly
(i.e., risk proportional to dose) from the point of
benchmark Dose Software: http: //www.epa.gov/bmds/.
departure to the levels of interest This is
appropriate for agents with direct mutagenic
activity. It is also the default if there is no
established mode-of-action fU.S. EPA. 2005a.
§3.3.1, §3.3.3).
Differences in susceptibility may warrant
derivation of multiple slope factors or unit risks.
For early-life exposure to carcinogens with a
mutagenic mode-of-action, EPA has developed
default age-dependent adjustment factors for
agents without chemical-specific susceptibility
data fU.S. EPA. 2005a. §3.5), CU.S. EPA. 2005b. §5).
If data are sufficient to ascertain the mode-
of-action and to conclude that it is not linear at
low levels, extrapolation may use the reference-
value approach fU.S. EPA. 2005a. §3.3.4).
Extrapolation: reference values. An oral
reference dose or an inhalation reference
concentration is an estimate of human exposure
(including in susceptible populations) likely to
be without appreciable risk of adverse health
effects over a lifetime (U.S. EPA. 2002. §4.2).
Reference values generally cover effects other
than cancer. They are also appropriate for
carcinogens with a nonlinear mode-of-action.
Calculation of reference values involves
dividing the point of departure by a set of
uncertainty factors (each typically 1, 3, or 10,
unless there are adequate chemical-specific
data) to account for different sources of
uncertainty and variability (U.S. EPA. 2002.
§4.4.5), flJ.S. EPA. 20141
Human variation: An uncertainty factor covers
susceptible populations and lifestages that
may respond at lower levels, unless the data
originate from a susceptible study
population.
Animal-to-human extrapolation: For reference
values based on animal results, an
uncertainty factor reflects cross-species
differences, which may cause humans to
respond at lower levels.
Subchronic-to-chronic exposure: For chronic
reference values based on subchronic
studies, an uncertainty factor reflects the
likelihood that a lower level over a longer
duration may induce a similar response. This
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factor may not be necessary for reference
values of shorter duration.
Adverse-effect level to no-observed-adverse-effect
level: For reference values based on a lowest-
observed-adverse-effect level, an
uncertainty factor reflects a level judged to
have no observable adverse effects.
Database deficiencies: If there is concern that
future studies may identify a more sensitive
effect, target organ, population, or lifestage, a
database uncertainty factor reflects the
nature of the database deficiency.
8. Process for Developing and Peer-
Reviewing IRIS Assessments
The IRIS process (revised in 2009 and
enhanced in 2013) involves extensive public
engagement and multiple levels of scientific
review and comment. IRIS program scientists
consider all comments. Materials released,
comments received from outside EPA, and
disposition of major comments (steps 3, 4, and 6
below) become part of the public record.
Step 1: Draft development. As outlined in
section 2 of this Preamble, IRIS program
scientists specify the scope of an assessment
and formulate science issues for discussion
with the scientific community and the public.
Next, they release initial protocols for the
systematic review procedures planned for
use in the assessment. IRIS program
scientists then develop a first draft, using
structured approaches to identify pertinent
studies, evaluate study methods and quality,
integrate the evidence of causation for each
health outcome, select studies for derivation
of toxicity values, and derive toxicity values,
as outlined in Preamble sections 3-7.
Step 2: Agency review. Health scientists across
EPA review the draft assessment
Step 3: Interagency science consultation.
Other federal agencies and the Executive
Office of the President review the draft
assessment
Step 4: Public comment, followed by external
peer review. The public reviews the draft
assessment IRIS program scientists release
a revised draft for independent external peer
review. The peer reviewers consider
whether the draft assessment assembled and
evaluated the evidence according to EPA
guidance and whether the evidence justifies
the conclusions.
Step 5: Revise assessment. IRIS program
scientists revise the assessment to address
the comments from the peer review.
Step 6: Final agency review and interagency
science discussion. The IRIS program
discusses the revised assessment with EPA's
program and regional offices and with other
federal agencies and the Executive Office of
the President
Step 7: Post final assessment. The IRIS
program posts the completed assessment
and a summary on its website.
9. General Structure of IRIS
Assessments
Main text. IRIS assessments generally
comprise two major sections: (1) Hazard
Identification and (2) Dose-Response
Assessment. Section 1.1 briefly reviews chemical
properties and toxicokinetics to describe the
disposition of the agent in the body. This section
identifies related chemicals and summarizes
their health outcomes, citing authoritative
reviews. If an assessment covers a chemical
mixture, this section discusses environmental
processes that alter the mixtures humans
encounter and compares them to mixtures
studied experimentally.
Section 1.2 includes a subsection for each
major health outcome. Each subsection
discusses the respective literature searches and
study considerations, as outlined in Preamble
sections 3 and 4, unless covered in the front
matter. Each subsection concludes with evidence
synthesis and integration, as outlined in
Preamble section 5.
Section 1.3 links health hazard information
to dose-response analyses for each health
outcome. One subsection identifies susceptible
populations and lifestages, as observed in human
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or animal studies or inferred from mechanistic
data. These may warrant further analysis to
quantify differences in susceptibility. Another
subsection identifies biological considerations
for selecting health outcomes, studies, or data
sets, as outlined in Preamble section 6.
Section 2 includes a subsection for each
toxicity value. Each subsection discusses study
selection, methods of analysis, and derivation of
a toxicity value, as outlined in Preamble sections
6 and 7.
Front matter. The Executive Summary
provides information historically included in
IRIS summaries on the IRIS program website. Its
structure reflects the needs and expectations of
EPA's program and regional offices.
A section on systematic review methods
summarizes key elements of the protocols,
including methods to identify and evaluate
pertinent studies. The final protocols appear as
an appendix.
The Preface specifies the scope of an
assessment and its relation to prior assessments.
It discusses issues that arose during assessment
development and emerging areas of concern.
This Preamble summarizes general
procedures for assessments begun after the date
below. The Preface identifies assessment-
specific approaches that differ from these
general procedures.
August 2016
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References
U.S. EPA. (1991). Guidelines for developmental toxicity risk assessment (pp. 1-83). (EPA/600/FR-
91/001). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum,
http: / /cfpub.epa.gov/ncea/cfm /recordisplav.cfm?deid=2 3162
U.S. EPA. (1994). Methods for derivation of inhalation reference concentrations and application of
inhalation dosimetry [EPA Report] (pp. 1-409). (EPA/600/8-90/066F). Research Triangle
Park, NC: U.S. Environmental Protection Agency, Office of Research and Development, Office
of Health and Environmental Assessment, Environmental Criteria and Assessment Office.
https://cfpub.epa. gov/ncea/risk/recordisplay.cfm?deid=71993&CFID=51174829&CFTOKE
N=25006317
U.S. EPA. (1996). Guidelines for reproductive toxicity risk assessment (pp. 1-143). (EPA/630/R-
96/009). Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
U.S. EPA. (1998). Guidelines for neurotoxicity risk assessment Fed Reg 63: 26926-26954.
U.S. EPA. (2002). A review of the reference dose and reference concentration processes (pp. 1-192).
(EPA/630/P-02/002F). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http://www.epa.gov/osa/review-reference-dose-and-reference-
concentration-processes
U.S. EPA. (2005a). Guidelines for carcinogen risk assessment [EPA Report] (pp. 1-166).
(EPA/630/P-03/001F). Washington, DC: U.S. Environmental Protection Agency, Risk
Assessment Forum, http://www2.epa.gov/osa/guidelines-carcinogen-risk-assessment
U.S. EPA. (2005b). Supplemental guidance for assessing susceptibility from early-life exposure to
carcinogens (pp. 1-125). (EPA/630/R-03/003F). Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum.
U.S. EPA. (2006). Approaches for the application of physiologically based pharmacokinetic (PBPK)
models and supporting data in risk assessment (Final Report) [EPA Report] (pp. 1-123).
(EPA/600/R-05/043F). Washington, DC: U.S. Environmental Protection Agency, Office of
Research and Development, National Center for Environmental Assessment.
http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=157668
U.S. EPA. (2011). Recommended use of body weight 3/4 as the default method in derivation of the
oral reference dose (pp. 1-50). (EPA/100/R11/0001). Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum, Office of the Science Advisor.
https://www.epa.gov/risk/recommended-use-bodv-weight-34-defaiilt-method-derivation-
oral-reference-dose
U.S. EPA. (2012). Benchmark dose technical guidance (pp. 1-99). (EPA/100/R-12/001).
Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum.
U.S. EPA. (2014). Guidance for applying quantitative data to develop data-derived extrapolation
factors for interspecies and intraspecies extrapolation. (EPA/100/R-14/002F). Washington,
DC: Risk Assessment Forum, Office of the Science Advisor.
http://www.epa.gov/raf/DDEF/pdf/ddef-final.pdf
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EXECUTIVE SUMMARY
Occurrence and Health Effects
Ammonia occurs naturally in air, soil, and water. Ammonia is also produced
by humans and other animals as part of normal biological processes.
Ammonia is used as an agricultural fertilizer and in many cleaning products.
Exposure to ammonia occurs primarily through breathing air containing ammonia
gas, and may also occur via diet, drinking water, or direct skin contact Measured
concentrations of ammonia range from 0.28 to 15 |ig/m:i in ambient outdoor air and
from 0.09 to 166 |ig/m3 in indoor air.
Health effects of inhaled ammonia observed at levels exceeding naturally-
occurring concentrations are generally limited to the respiratory tract, the site of
direct contact with ammonia. Short-term inhalation exposure to high levels of
ammonia in humans can cause irritation and serious burns in the mouth, lungs, and
eyes. Chronic exposure to airborne ammonia can increase the risk of respiratory
irritation, cough, wheezing tightness in the chest, and impaired lung function in
humans. Studies in experimental animals similarly indicate that breathing ammonia
at sufficiently high concentrations can result in effects on the respiratory system.
Animal studies also suggest that exposure to high levels of ammonia in air may
adversely affect other organs, such as the liver, kidney, and spleen.
This assessment presents an evaluation of the noncancer health effects of ammonia by the
inhalation route of exposure.
Chemical Properties
Ammonia (NH3) is a colorless alkaline gas with a pungent odor. In solution, ammonia exists
as ammonium hydroxide, a weak base that is only partially ionized in water according to the
following equilibrium fATSDR. 20041: NH3 + H2O *± NH4+ + OH". A decrease in pH results in an
increase in the concentration of ammonium ion (NH4+) and a decrease in the concentration of the
un-ionized form (NH3). At physiological pH (7.4), this equilibrium favors the formation of NH4+.
Toxicokinetics
Inhaled ammonia is almost completely retained in the upper respiratory tract Ammonia
produced endogenously in the intestines through the use of amino acids as an energy source and by
bacterial degradation of nitrogenous compounds from ingested food is largely absorbed. At
physiological pH, 98.3% of ammonia is present in the blood as the ammonium ion (NH4+). Given its
importance in amino acid metabolism, the urea cycle, and acid-base balance, ammonia is
homeostatically regulated to remain at low concentrations in the blood. Ammonia is present in
fetal circulation and in human breast milk as a source of nonprotein nitrogen. Ammonia production
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occurs endogenously by catabolism of amino acids by glutamate dehydrogenase or glutaminase
primarily in the liver, renal cortex, and intestines, but also in the brain and heart. Ammonia is
metabolized to glutamine via glutamine synthetase in the glutamine cycle or incorporated into urea
as part of the urea cycle. The principal means of excretion of ammonia is as urinary urea; lesser
amounts are eliminated in the feces, through sweat production, and in expired air.
Effects Other Than Cancer Observed Following Inhalation Exposure
Respiratory effects have been identified as a human health hazard following inhalation
exposure to ammonia. This hazard determination is based on findings from multiple epidemiology
studies in human populations exposed to ammonia in different settings (workers in industrial,
cleaning, and agricultural settings, volunteers exposed for up to 6 hours under controlled
conditions, and case reports) and animals (short-term and subchronic studies in several species
and across different exposure regimes).
Cross-sectional occupational studies involving chronic exposure to ammonia in industrial
settings provide evidence of an increased prevalence of respiratory symptoms (Rahman etal..
2007: Ballal etal.. 1998) and decreased lung function f Rah man etal.. 2007: Ali etal.. 2001: Ballal et
al.. 1998: Bhat and Ramaswamv. 1993). Other studies of exposure to ammonia when used as a
disinfectant or cleaning product provide evidence of asthma, asthma symptoms, and impaired
pulmonary function, using a variety of study designs fCasas etal.. 2013: Arif and Delclos. 2012:
Dumas etal.. 2012: Lemiere etal.. 2012: Vizcava etal.. 2011: Zock etal.. 2007: Medina-Ramon et al..
2006: Medina-Ramon etal.. 2005). Further evidence of respiratory effects of ammonia is seen in
studies of pulmonary function in an agricultural setting, specifically in studies that accounted for
effects of co-exposures to other agents such as endotoxin and dust (Donham etal.. 2000: Reynolds
etal.. 1996: Donham et al.. 1995: Preller etal.. 1995: Heederik et al.. 1990) and in one study that did
not control for co-exposures fLoftus etal.. 20151. Despite the variation in population
characteristics, level and pattern of exposure, and potential confounders across these three settings
of epidemiology studies, respiratory effects were consistently observed in these studies. Further,
but more limited, support for the respiratory system as a target of ammonia toxicity comes from
controlled human exposure studies of ammonia inhalation and case reports of injury in humans
with inhalation exposure to ammonia. Additionally, respiratory effects were observed in several
animal species following short-term and subchronic inhalation exposures to ammonia.
Overall, there are suggestions in experimental animals that ammonia exposure may be
associated with effects on organs distal from the portal of entry, but there is inadequate
information to draw conclusions about the liver, kidney, spleen, or heart as sensitive targets of
ammonia toxicity.
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Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
Table ES-1. Summary of reference concentration (RfC) derivation
Critical effect
Point of departure3
UF
Chronic RfC
Decreased lung function and respiratory symptoms
Occupational epidemiology studies
Holness et al. (1989), supported bv Rahman et al.
(2007), Ballal et al. (1998), and Ali et al. (2001)
NOAELadj: 4.9 mg/m3
10
0.5 mg/m3
aAn estimate of the 95% lower confidence bound of the mean exposure concentration in the high-exposure group
of the Holness et al. (1989) study was used as the NOAEL. Because the study involved workplace exposure
conditions, the NOAEL of 13.6 mg/m3 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.
NOAEL = no-observed-adverse-effect level; UF = uncertainty factor
The study of ammonia exposure in workers in a soda ash plant by Holness etal. (1989).
with support from three studies in urea fertilizer plants by Rahman etal. (2007). Ballal et al.
(1998). and Ali etal. (2001). was identified as the principal study for RfC derivation. Respiratory
effects, characterized as increased respiratory symptoms based on self-report (including cough,
wheezing, and other asthma-related symptoms) and decreased lung function in workers exposed to
ammonia, were selected as the critical effect. Rahman etal. f20071 observed an increased
prevalence of respiratory symptoms and decreased lung function in workers exposed in a plant
with a mean ammonia concentration of 18.5 mg/m3, but not in workers in a second plant exposed
to a mean concentration of 4.9 mg/m3. Ballal etal. (1998) observed an increased prevalence of
respiratory symptoms among workers in one factory with exposures ranging from 2 to 27.1
mg/m3,7 but no increase in another factory with exposures ranging from 0.02 to 7 mg/m3. A
companion study by Ali etal. (2001) also observed decreased lung function among workers
exposed to higher cumulative ammonia levels (>50 mg/m3-years), with an approximate 5-7%
decrease in FVC% predicted and FEVi% predicted (see definition of spirometry measures in Section
1.2.1). Holness etal. (1989). who investigated a plant with exposures generally lower than other
studies, found no differences in the prevalence of respiratory symptoms or lung function between
workers (mean exposure 6.5 mg/m3) and the control group, and no differences when stratified by
exposure level (highest exposure group, >8.8 mg/m3).
7This concentration range does not include exposures in the urea store (number of employees = 6; range of
ammonia concentrations = 90-130.4 mg/m3) because employees in this area were required to wear full
protective clothing, thus minimizing potential exposure.
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These four studies addressed smoking by a variety of methods (e.g., adjustment for
smoking, exclusion of smokers, stratification of the results by smoking status). Two of the
studies—Rahman et al. f20071 and Holness etal. f 19891—addressed other potential confounders
as appropriate. In particular, a high level of control of exposures in the facility studied by Holness
etal. f 19891 was reported, suggesting a low potential for co-exposures. As discussed in more detail
in the Literature Search Strategy | Study Selection and Evaluation section, confounding by other
workplace exposures, although a potential concern, was unlikely to be a major limitation of these
studies.
Considerations in selecting the principal study for RfC derivation include the higher
confidence placed in the measures of ammonia exposure in Holness et al. f 19891. evaluation of both
respiratory symptoms and lung function parameters in this study, and the fact that the estimate of
the no-observed-adverse-effect level (NOAEL) for respiratory effects of 13.6 mg/m3 from Holness et
al. (1989) was the highest of the studies with adequate exposure-response information. The
synthesis of findings from the full body of evidence demonstrates that there is a relationship
between ammonia exposure and respiratory effects. Although Holness etal. (19891 do not report
associations between ammonia exposure and respiratory effects, it is included in the body of
epidemiologic studies of industrial settings because it is informative of the levels above which
ammonia causes effects. Other epidemiology studies include those with higher workplace ammonia
concentrations associated with respiratory effects (i.e., higher concentrations relative to those
reported by Holness et al. (198911 and for which lowest-observed-adverse-effect levels (LOAELs)
could be identified. The Holness etal. (19891 study is identified as the principal study for RfC
derivation based on the quality of the exposure data and other factors, as stated above.
In summary, the study of ammonia exposure in workers in a soda ash plant by Holness et al.
Q9891 was identified as the principal study for RfC derivation, with support from Rahman et al.
(20071. Ballal etal. (19981. and Ali etal. (20011. and respiratory effects were identified as the
critical effect. The NOAEL, represented by an estimate of the 95% lower confidence bound of the
mean exposure concentration in the high-exposure group from the Holness etal. (19891 study, or
13.6 mg/m3, was used as the point of departure (POD) for RfC derivation. The NOAEL adjusted to
continuous exposure (NOAELadj) was 4.9 mg/m3.
An RfC of 0.5 (rounded) mg/m3 was calculated by dividing the POD (adjusted for
continuous exposure, 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.
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Confidence in the Chronic Inhalation RfC
Study - medium
Database - medium
RfC - medium
Consistent with Environmental Protection Agency (EPA) Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA. 19941. 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 sample size and
identification of a NOAEL only) and medium confidence in the database, which includes
occupational, cleaner, agricultural, and human exposure studies and studies in animals that are
mostly of subchronic duration. There are no studies of developmental toxicity, and studies of
reproductive and other systemic endpoints are limited; however, the likelihood of reproductive,
developmental, and other systemic effects at the RfC is considered small because it is well
documented that ammonia is endogenously produced in humans and animals, and any changes in
blood ammonia levels at the POD would be small relative to normal blood ammonia levels. Further,
EPA is not aware of any mechanisms by which ammonia can exert effects at the point of contact
(i.e., respiratory system) that could directly or indirectly impact tissues or organs distal to the point
of contact
Susceptible Populations and Lifestages
Studies of the toxicity of ammonia in children that would support an evaluation of
childhood susceptibility are limited. Casas etal. (2013) and Loftus etal. (2015) reported evidence
of an association between ammonia exposure and decrements in lung function in children;
however, these studies did not report information that would allow a comparison of children and
adults.
A limited number of studies provides inconsistent evidence of greater respiratory
sensitivity to ammonia exposure in asthmatics (Loftus etal.. 2015: Petrova etal.. 2008: Sigurdarson
etal.. 2004: Preller etal.. 19951. Loftus etal. T2 0151 reported no increase in asthma symptoms and
medication use in asthmatic children living near animal feeding operations; however, ammonia
exposure was associated with lower FEVi.
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 [CO(NH2)2] 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
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more susceptible to the effects of ammonia from external sources, but there are no studies that
specifically support this susceptibility.
Key Issues Addressed in This Assessment
Comparison of Exhaled Ammonia to the RfC
Ammonia is generated endogenously in multiple organs and plays central roles in nitrogen
balance and acid-base homeostasis (Weiner etal.. 2014: Weiner and Verlander. 20131. Given its
important metabolic role, free ammonia is homeostatically regulated to remain at low
concentrations in blood fSouba. 19871. Elimination of ammonia occurs primarily in urine and
exhaled breath. Consideration was given to the presence of ammonia in exhaled air because the
range of ammonia concentrations in exhaled breath (0.009-2 mg/m3) overlaps the ammonia RfC
(0.5 mg/m3).
In general, higher and more variable ammonia concentrations (0.03-2 mg/m3) are reported
in human breath exhaled from the mouth or oral cavity (Schmidt etal.. 2013: Smith etal.. 2008:
Spanel etal.. 2007a. b; Turner etal.. 2006: Diskin etal.. 2003: Smith etal.. 1999: Norwood etal..
1992: Larson etal.. 19771. Ammonia concentrations measured in breath derived from oral
breathing largely reflect the production of ammonia via bacterial degradation of food protein in the
oral cavity or gastrointestinal tract, and can be influenced by diet, oral hygiene, age, and saliva pH.
In contrast, concentrations of ammonia in breath exhaled from the nose and trachea of humans
(0.0092-0.1 mg/m3) are lower than those in air exhaled from the mouth f Schmidt etal.. 2013:
Smith etal.. 2008: Larson etal.. 19771. and are generally lower than the RfC by a factor of five or
more. Concentrations in breath exhaled from the nose appear to better represent levels at the
alveolar interface of the lung and are more relevant to understanding systemic levels of ammonia
than breath exhaled from the mouth f Schmidt etal.. 2013: Smith etal.. 20081: however,
concentrations in breath from neither the mouth nor the nose can be used to predict blood
ammonia concentration or previous exposure to environmental (ambient) concentrations of
ammonia (see Appendix C, Section C.1.4).
Regardless of the source of expired ammonia (mouth or nose), the level of ammonia in
breath, even at concentrations that exceed the RfC, does not necessarily raise questions about the
appropriateness of the RfC. The exhalation of ammonia is a clearance mechanism for a product of
metabolism that is otherwise toxic in the body at sufficiently high concentrations. Thus, ammonia
concentrations in exhaled breath may be higher than inhaled concentrations. However, the
presence of ammonia in exhaled breath is not considered an uncertainty in the RfC.
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LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
Literature Search and Screening Strategy
The literature search for ammonia was conducted in six online scientific databases,
including PubMed, Toxline, the Toxic Substances Control Act Test Submissions (TSCATS) database,
Web of Science (WOS), Health and Environmental Research Online (HERO)8, and Toxcenter. The
initial search was performed in March 2012 (PubMed, Toxline, TSCATS, HERO, and Toxcenter) and
literature search updates were conducted in March 2013 (PubMed, Toxline, TSCATS, HERO, and
WOS) and September 2015 (PubMed, Toxline, TSCATS, and WOS). Toxcenter is a database in which
titles may be viewed for free after a fee-based search, but full citations and abstracts are purchased.
The use of Toxcenter was discontinued in 2013. No unique relevant hits were returned in the 2013
update search of HERO; therefore, this search was notrepeated in 2015. The detailed search
approach, including the query strings, is presented in Appendix B, Table B-l. This search of online
databases identified approximately ~28,000 unique citations (after electronically eliminating
duplicates).
The core computerized database searches were supplemented by a review of citations in
other national and international health agency documents (see Table B-2). The ATSDR (2004)
Toxicological Profile of Ammonia9 was used to identify toxicokinetic studies for ammonia. A search
of online chemical assessment-related websites was performed in 2012 and 2015; links to the
websites that were searched are provided in Table B-2. An additional focused search strategy was
also employed to obtain studies of cleaning and hospital workers to address a new area of research
identified during the 2013 literature search update. This strategy involved a manual reference list
review of several seminal studies published in 2012 (see Appendix B, Table B-2). In addition,
8HER0 is a database of scientific studies and other references used to develop Environmental Protection
Agency (EPA) 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 1.6 million scientific
references, including articles from the peer-reviewed literature. New studies are added continuously to
HERO. For each IRIS assessment, a HERO project page is created that stores all citations identified from that
chemical-specific literature search. These citations may be organized using various tags to indicate if the
citations are used in the assessment and how they are categorized.
9Portions 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 ATSDR (2004)
Toxicological Profile for Ammonia and the references cited in that document, 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 sharing scientific information.
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electronic forward searches were conducted in WOS in 2013 and 2015, using a methods paper
describing the development of a job exposure matrix focusing on asthma as a health outcome
fKennedv et al.. 20001. The disposition of studies obtained from the manual backward and
electronic forward searches is presented in Table B-3.
In Federal Register notices announcing annual Integrated Risk Information System (IRIS)
agendas and on the IRIS website, EPA encouraged the public to submit information on IRIS
chemicals throughout the assessment development process, and specifically requested that the
public submit additional data to support development of the ammonia assessment on December 21,
2007 and November 2, 2009 (U.S. EPA. 2009a. 20071. No public submissions were received in
response to these calls for data.
Figure LS-1 depicts a summary of the literature search and screening process and the
number of references included or excluded at each step. In 2012, the initial literature search was
conducted in core computerized databases. These citations were electronically screened in an
EndNote database using a set of terms intended to prioritize "on-topic" references for title and
abstract review. The electronic screening process created two broad categories: one of all citations
that contain (in title, abstract, or keywords) at least one inclusion term related to health outcomes,
epidemiological or toxicological study design, absorption/distribution/metabolism/excretion
(ADME) or toxicokinetics, or mechanistic information (see Appendix B, Table B-4), and one that did
not contain any of the terms. Some of the electronic inclusion terms listed in Table B-4 are generic
(i.e., not chemical specific) and are intended to capture health effect studies of any type. Other
terms are specific to ammonia and are based on previous knowledge of health effects and possible
mechanisms of toxicity summarized in other health agency review documents (see Appendix A).
Citations that did not contain at least one inclusion term in Table B-4 (i.e., excluded by the
electronic screening) were subjected to a quality control check to verify that relevant references
were not missed. Specifically, a random sample (approximately 10%) of the electronically excluded
citations were subjected to title and abstract review by a toxicologist to confirm that the electronic
screening process produced acceptable results (i.e., no relevant citations were inadvertently
missed). Relevant items were added to the HERO project page for ammonia and retrieved for full-
text review. The results from the updated literature searches performed in March 2013 and
September 2015 were not screened electronically in EndNote. All titles and abstracts obtained
from these search updates were reviewed manually by a toxicologist.
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PubMed
n=16,644
Toxline
n=2,550
Database Searches
(see Table B-l for query strings and date limits)
TSCATS
n=58
WOS
n=3,816
Toxcenter
n=2,591
HERO
n=5,410
n=~28,000 (After duplicates removed electronically and manually)
Additional Search Strategies
(see Table B-2 for methods and results)
—>
Excluded by electronic screen
n=~13,000
Combined Dataset
n=~15,000
—>
Supplementary Studies
Excluded (n=~14,750)
See exclusion criteria in Table LS-1
Secondary Literature
9 Reviews and regulatory documents
Title, Abstract, and Full Text Screening
Health Effect/Toxicity Studies
(see inclusions criteria in Table LS-1)
26 Human studies (includingstudies in
occupational, agricultural, and cleaner
settings)
17 Animal studies
Supplementary Health Effect/
Toxicity Studies
(see inclusion criteria in Table LS-1)
35 Case reports
13 Controlled human exposure studies
27 Inhalation acute and short-term studies
Physical-Chemical/Mechanistic/
Toxicokinetic Studies
13 Physical-chemical property studies
2 Mode of action studies
76 Toxicokinetic studies
Figure LS-1. Summary of literature search and screening process for
ammonia.
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Manual screening of titles/abstracts and full text was accomplished using a set of inclusion
and exclusion criteria to identify sources of primary human health effects data and sources of
primary data that supplement the assessment of ammonia health effects (i.e., bottom boxes in
Figure LS-1). The inclusion/exclusion criteria that were used prior to peer review are presented in
Table LS-1. Manual screening of the post-peer review literature search update (i.e., September
2015) was performed using more stringent inclusion and exclusion criteria to capture studies that
would impact the credibility of the assessment's conclusions consistent with EPA's IRIS Stopping
Rules (http://www.epa.gov/sites/production/files/2014-06/documents/iris stoppingrules.pdf).
For ammonia, those references identified in the post-peer review literature search that were
considered for inclusion in hazard identification were in vivo animal toxicity and epidemiology
studies. No additional in vivo animal toxicity studies of ammonia were identified in the post-peer
review search. The disposition of epidemiology studies obtained from the post-peer review
literature search update (i.e., September 2015) is provided in Table B-5.
Specific inclusion/exclusion criteria were not applied in identifying sources of mechanistic
and toxicokinetic data. Because ammonia is produced endogenously and serum ammonia levels are
measured in certain disease states, the toxicokinetics literature is large and complex; relevant
toxicokinetic studies for ammonia were initially identified using the ATSDR f20041 Toxicological
Profile of Ammonia and supplemented by more recent studies identified in literature search
updates. The number of mechanistic studies identified for ammonia was not large, and therefore,
all mechanistic studies were included.
Table LS-1. Inclusion/exclusion criteria for inhalation health effect/toxicity
studies (pre-peer review)3
Inclusion criteria
Exclusion criteria
Population
• Humans, including occupational workers,
livestock workers and those in close
proximity to agricultural operations, hospital
workers/cleaners and volunteers
• Standard mammalian animal models,
including rat, mouse, hamster, rabbit, guinea
pig, monkey, dog
• Pigs
• Ecological species/ecosystem effects
• Nonmammalian species
• Agricultural species/livestock (except pigs)
Exposure
• Exposure is to ammonia by the inhalation
route (any duration)
• Exposure is measured as a concentration in
air
• Exposure is in vivo
• Not chemical specific (i.e., not ammonia-
specific)
• Animal studies: exposure is to a mixture only
• Human studies: exposure is inferred but not
measured (e.g., some cleaning and hospital
worker studies)
• Exposure by oral, dermal, injection or
instillation routes
• Studies of quaternary ammonia
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Inclusion criteria
Exclusion criteria
Outcome
• One or more of the following health effect
endpoints is evaluated: effects on the
cardiovascular, dermal/integumentary,
endocrine, gastrointestinal, immune,
musculoskeletal, nervous, reproductive,
respiratory, hepatic, or renal (urinary)
systems; effects on the eyes, survival,
growth, or development
• No health outcome evaluated
• Pathogenic effects of Helicobacter pylori
infection
Other
• Review article or abstract only (i.e., no
primary data)
• Environmental fate and transport of
ammonia
• Analytical methods for measuring ammonia
in environmental media, and use in sample
preparations and assays
• Study of physical-chemical properties
• Study of in vitro or in vivo toxicokinetics
• Study of in vitro or in vivo mechanistic
endpoints
• Other studies not on topic and not captured
by other exclusion criteria
aReviews and regulatory documents were retained as Secondary Literature. Studies that provided primary
information on the physical-chemical properties, mode of action, or toxicokinetics of ammonia were also
retained, but were not screened as sources of health effect/toxicity information for ammonia.
The results of the pre- and post-peer review literature screening are described below and
graphically in Figure LS-1:
• 43 references (including 26 human studies and 17 animal studies) were identified as
studies with health effects data and were considered for data extraction to evidence tables
and exposure-response arrays.
• Supplementary health effect/toxicity studies included 35 case reports, 13 acute-duration
controlled human exposure studies, and 27 acute or short-term animal studies. Information
from these studies was not extracted into evidence tables; however, these studies were
considered as supplementary studies for assessing ammonia health effects.
• 91 studies were identified as physical-chemical, mode-of-action, or toxicokinetic studies,
including 13 studies of physical-chemical properties, 2 studies providing mode-of-action
information, and 76 toxicokinetic studies. Information from these studies was not extracted
into evidence tables; however, these studies were considered as supplementary studies for
assessing ammonia health effects (e.g., consideration of toxicokinetic information in
assessing the health effects literature).
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• Nine reviews or regulatory/health assessment documents were identified as secondary
literature. These references were retained as additional resources in developing the
Toxicological Review.
• More than 27,000 references were identified as not pertinent to an evaluation of the
inhalation health effects of ammonia. Approximately 13,000 were excluded by electronic
screening (see Table B-4) and approximately 14,750 were excluded by manual screening
(see Table LS-1 for exclusion criteria).
Study Selection and Evaluation
Selection of studies for inclusion in the Toxicological Review was based on consideration of
the extent to which the study was informative and relevant to the assessment as well as on general
study quality considerations. In general, the relevance and scientific quality of the available studies
were evaluated as outlined in the Preamble and in EPA guidance (i.e., A Review of the Reference Dose
and Reference Concentration Processes (U.S. EPA. 20021 and Methods for Derivation of Inhalation
Reference Concentrations and Application of Inhaled Dosimetry (U.S. EPA. 199411. The scientific
considerations used to evaluate and select studies and the relevance of these studies to the
assessment are described in the section below.
Considerations for Evaluation of Epidemiology Studies
Case reports are often anecdotal and describe unusual or extreme exposure situations,
providing little information that would be useful for characterizing chronic health hazards.
Ammonia case studies were only briefly reviewed; representative citations from the collection of
case reports are provided as supplemental information in Appendix C, Section C.2.4. Similarly,
acute controlled human exposure studies would not be useful for characterizing chronic health
effects; these studies were therefore briefly reviewed and are provided as supplemental
information in Appendix C, Section C.2.3.
Epidemiology studies of chronic exposure to ammonia have primarily focused on industrial
worker populations, workers exposed to ammonia as a cleaning or disinfectant product, and those
exposed in an agricultural setting. There is considerable variation in population characteristics,
level and pattern of exposure, and potential confounders across the three categories of studies.
Evaluations of the observational epidemiology studies of industrial worker populations and
workers exposed to ammonia as a cleaning or disinfectant product identified in Figure LS-1 (i.e., the
studies considered most informative for evaluating ammonia toxicity from chronic exposure) are
provided in Appendix B (Tables B-6 to B-8). The process used to evaluate these studies addressed
aspects relating to the selection of study participants, exposure parameters, outcome measurement,
confounding, and statistical analysis. As discussed below, studies of populations exposed in
agricultural settings were considered to be supporting material because of the variety of potential
co-exposures in these studies (including dust, endotoxin, mold, and disinfectant products). The
process for evaluating studies in an agricultural setting considered the same five aspects (selection
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of study participants, exposure parameters, outcome measurement, confounding, and statistical
analysis); however, specific study evaluation tables were not provided in Appendix B for this set of
studies.
For study evaluation purposes, EPA differentiated between "major" limitations, defined as
biases or deficiencies that could materially affect the interpretation of the study, and "minor"
limitations, defined as limitations that are not likely to be severe or to have a substantive impact on
the results. These categories are similar to the "serious risk of bias" and "moderate risk of bias"
categories, respectively, described by Stearne etal. (2014) in the Cochrane Collaborative
Assessment Tool for non-randomized studies of clinical interventions. Identification of major
limitations in the epidemiology studies of populations exposed in industrial, cleaning, and
agricultural settings is included in the broader evaluation of study quality below. Uninformative
studies are also noted.
Studies of Industrial Settings
Selection of study participants
All of the studies were cross-sectional analyses in occupational settings. The workers were
healthy enough to remain in the work area for a considerable time; with one exception, mean
duration ranged from 52 months to 16 years. One study fBhatand Ramaswamv. 19931 grouped
workers into those exposed for up to 10 years and those with more than 10 years of exposure; a
minimum exposure duration was not provided. As in inherent property of occupational studies,
these designs may result in a "healthy worker" bias. In addition, the workers in these studies are
not representative of the general population, as they do not include children and only one study of
ammonia exposure in hair salons included women (Nemer etal.. 2015). These aspects of the study
design may result in an underestimate of the risk of health effects of ammonia exposure, as the
worker population may not exhibit health effects (such as decreased lung function or increased
prevalence of respiratory symptoms) to the same degree that would be seen in the general
population under the same conditions. In addition to the "healthy worker" effect, the Nemer etal.
(2015) study exhibited a potential selection bias in the controls due to differences in recruitment
(self-selected based on interest) or workload.
Exposure parameters
Exposure methods differ across these occupational studies, which makes comparison of
ammonia measurements among the studies difficult Spectrophotometric absorption measures of
areas samples (Ali etal.. 2001: Ballal etal.. 1998) are not directly comparable to direct-reading
diffusion methods f Rah man etal.. 2007) or electrochemical sensor methods (Nemer etal.. 2015)
used to analyze personal samples. Nor are they comparable to the National Institute for
Occupational Safety and Health (NIOSH)-recommended protocol for personal sampling and analysis
of airborne contaminants fHolness etal.. 19891. In the study by Rahman etal. f20071. exposure
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concentrations were determined by both the Drager tube and Drager PAC III methods. The Drager
tube method yielded concentrations of ammonia in the two plants studied that were approximately
4-fold higher than the concentrations obtained by the Drager PAC III method; a strong correlation
between measurements by the two methods was reported. Rahman etal. (2007) stated that their
measurements indicated only relative differences in exposures between workers and production
areas, and did not identify one analytical measure as the more valid of the two. Based on
communication with technical support at Drager Safety Inc. (Bacom and Yanoskv. 20101. EPA
considered the PAC III instrument to be a more sensitive monitoring technology than the Drager
tubes. Ammonia concentrations based on the PAC III method were also in line with concentrations
reported in other studies. Therefore, exposure levels based on PAC III air measurements of
ammonia were used in the current health assessment to characterize the exposure-response
relationship in the Rahman etal. f20071 study.
In the Abdel Hamid and El-Gazzar (1996) study, no direct measurement of ammonia
exposure was made; blood urea was used as a surrogate measure of ammonia exposure. The
correlation of blood urea with ammonia is not reported by the authors. EPA considered this a
major limitation of this study, based on other data indicating no correlation between ammonia
levels in air and serum urea levels in a study of six groups of workers with varying types of
exposure fGiroux and Ferrieres. 1998). No exposure measurements of ammonia were used in the
study by Bhat and Ramaswamv Q993I EPA considered the lack of exposure measure in this study
to be a major limitation. In the Nemer etal. (2015) study, the measurement device had limited
specificity for measuring ammonia relative to other gases and could therefore have produced false
positive results in the presence of other gases. In addition, few exposure measurements were made
in the Nemer etal. (2015) study. EPA considered the limited specificity for measuring ammonia,
the limited number of exposure measurements, and the possible misclassification of exposure in
the Nemer etal. (2015) study to be major limitations.
Outcome measurement
Assessment of respiratory symptoms in Rahman etal. (2007). Ballal etal. (1998). Holness et
al. (1989). and Nemer etal. (2015) was based on four different questionnaires; each of these,
however, is a standardized, validated questionnaire. Self-reporting of types and severity of
respiratory symptoms could be biased by the knowledge of exposure, for example, in studies
comparing factory workers to office workers. EPA evaluated this non-blinded outcome assessment
as a potential bias. In each of these studies, comparisons were made across exposure categories
among the exposed; EPA concluded that the non-blinded outcome assessment as a potential bias is
unlikely in these types of comparisons. One study also compared exposed to nonexposed, and
observed little differences in symptom prevalence between these groups (Holness etal.. 1989).
Thus, EPA concluded that the non-blinded outcome assessment was not a major bias in this analysis
either. Assessment of lung function was performed by standard spirometry protocols in five
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studies (Nemer etal.. 2015: Rahman etal.. 2007: Ali etal.. 2001: Bhatand Ramaswamv. 1993:
Holness et al.. 19891. EPA did not consider any of these procedures for assessing lung function to
be a source of bias.
Confounding
Co-exposures to other ambient chemicals in urea fertilizer factories included inorganic
gases (nitrogen dioxide and sulfur dioxide) and dust In one of these studies (Rahman etal.. 20071.
nitrogen dioxide was measured concurrently with ammonia and found to be below detection limits
for all areas (urea plant, ammonia plant, and administration area). The other urea fertilizer studies
(Ali etal.. 2001: Ballal etal.. 1998: Abdel Hamid and El-Gazzar. 19961 did not describe potential co-
exposures. [It appears from the exposure measurements that the plant in Ali etal. f 2 0 011 is
"Factory A" in Ballal etal. Q9981.] In the fertilizer plant in Bhat and Ramaswamv f 19931. co-
exposures were not discussed, but the workers were grouped based on different parts of the plant
(ammonia, urea, and diammonium phosphate); effects observed with respect to lung function tests
were similar in magnitude, albeit slightly stronger, in the ammonia plant workers compared with
the urea plant workers. One study was conducted in a soda ash production plant (Holness etal..
19891. No measurements of co-exposures were described in this study, but the authors noted the
high level of control of exposures (resulting in low ammonia levels) in this facility. Because of the
lack of demonstration of co-exposures correlated with ammonia levels in these studies, and lack of
demonstration of stronger associations between potential co-exposures and respiratory outcomes,
EPA concluded that confounding by other workplace exposures, although a potential concern, was
unlikely to be a major limitation for the urea plant and soda ash plant studies. However, in a study
of ammonia exposure among hairdressers (Nemer etal.. 20151. co-exposures to other workplace
contaminants (such as persulfates and paraphenylenediamine) were not measured or controlled
for in the analysis; therefore, possible confounding is considered to be a limitation in this study.
The analyses of respiratory symptoms and lung function may also be confounded by
smoking. In six studies, analyses accounted for smoking as follows: the analysis included either an
adjustment for smoking (Rahman et al.. 2 0 0 7: Holness etal.. 19891. the exclusion of smokers
(Nemer etal.. 2015: Bhatand Ramaswamv. 19931. or stratification of the results by smoking status
(Ali etal.. 2001: Ballal etal.. 19981. Thus, EPA did not consider potential confounding by smoking
to be a major limitation of these studies.
Ammonia is present in both tobacco and cigarette smoke fCallicuttetal.. 20061. Typical
concentrations of ammonia in commercial U.S. tobacco blends range from 0.02 to 0.4% fSeeman
and Carchman. 20081. Thus, there is some potential for additional exposure to ammonia associated
with use of ammonia-containing tobacco products and/or inhalation of tobacco smoke. This finding
reinforces the importance of controlling for smoking in the analyses of the respiratory symptoms
and lung function. EPA did not consider potential confounding by smoking of ammonia-containing
tobacco or by inhaling tobacco smoke to be a major limitation of these studies because smoking as a
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Toxicological Review of Ammonia
potential confounder was adequately addressed in the studies that examined effects on the
respiratory system.
Information on smoking habits and use of alcohol (an exposure potentially affecting liver
function tests) was not documented in the study of liver function by Abdel Hamid and El-Gazzar
Q996I The lack of information and potential failure to control for these confounders is considered
a major limitation.
Statistical analysis
EPA considered the statistical analysis in the epidemiological studies (Nemer etal.. 2015:
Rahman etal.. 2007: Ali etal.. 2001: Ballal etal.. 1998: Abdel Hamid and El-Gazzar. 1996: Bhatand
Ramaswamv. 1993: Holness etal.. 19891 to be adequate and appropriate. Although the type of
statistical testing was not specified in Abdel Hamid and El-Gazzar f!996I the results were
presented in sufficient detail to allow interpretation of the data and analysis. Sample size, an
important consideration with respect to statistical power, was also considered. EPA noted the
small number of exposed workers and low levels of exposure in the study by Holness etal. (1989)
as limitations that could result in "false negative" results (i.e., a test result indicating a lack of
association, whereas a positive association between exposure and a health effect exists).
Identification of uninformative studies
The study by Abdel Hamid and El-Gazzar T19961 was determined to have major limitations.
Air concentrations of ammonia were not directly measured, and the use of blood urea has not been
established as a reliable surrogate of ammonia exposure. Further, the lack of information on
smoking and alcohol use, factors that could affect liver function, in a study intended to examine the
association between liver function and ammonia exposure, was considered a significant flaw.
Therefore, Abdel Hamid and El-Gazzar T19961 was not further considered in this assessment
Major limitations were also identified in the Nemer etal. (2015) study: potential selection
bias in the control group due to differences in recruitment (self-selected based on interest in the
study) or workload; limited specificity of the analytical method used to measure ammonia (i.e.,
potential for false positives from other gases); and failure to control for confounders. In addition,
the study used small sample sizes and only a single measurement of ammonia for each location
(which may not have been representative of workplace exposures). Therefore, the Nemer etal.
(2015) study was deemed to be uninformative and was not further considered in this assessment.
Studies of Health Care and Cleaning Settings
Selection of study participants
EPA evaluated the studies that examined exposure to ammonia when used as a cleaning or
disinfectant product EPA noted the potential for the "healthy worker" bias arising from movement
out of jobs by affected individuals in most of these studies (Le Moual et al.. 2008). This issue was
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Toxicological Review of Ammonia
less of a concern in the study by Zock etal. (2007). which was conducted in a general (non-
occupational) population sample, focusing on cleaning activities in the home. In a birth cohort that
evaluated the association between exposure to cleaning products and children's respiratory health
(Casas etal.. 2013). 35% of the recruited population was excluded because information on the use
of cleaning products and/or respiratory tests was not available, representing a potential study
limitation. However, the authors of this study noted that the children included were not different
from those excluded regarding most study characteristics (sex, atopy, asthma, parental asthma, and
parental smoking).
Exposure parameters
None of these studies used a direct measure of ammonia exposure in the analysis,
precluding interpretation of the results in relation to an absolute level of exposure. The limited
data available concerning exposure levels in cleaning scenarios found median exposures of
0.6-5.4 ppm (0.4-3.8 mg/m3), with peaks exceeding 50 ppm (35 mg/m3), in a small study (n = 9)
using personal samples during a domestic cleaning session (Medina-Ramon etal.. 2005). Although
an absolute level of exposure is not available, the relative ranking of exposure used in these studies
does allow examination of risk by relative levels of exposure.
Key considerations regarding the validity of the exposure measures are the specificity of the
classification and the extent to which classification could be influenced by knowledge of the disease
or symptoms under study. Methodological research has reported underestimation of self-reported
exposure to specific products by health care workers, and differential reporting by disease status
(i.e., asthma) for self-reported use of cleaning products in patient care, but not in instrument
cleaning or building materials (Donnav etal.. 2011: Delclos etal.. 2009: Kennedy et al.. 2000). Two
of these studies used an exposure assessment protocol that incorporated an independent, expert
review, blinded to disease status fDumas etal.. 2012: Lemiere etal.. 20121: one study collected
exposure information using a 2-week daily diary (Medina-Ramon et al.. 20061 and one study
developed a composite exposure score based on an interviewer-led questionnaire concerned with
the frequency of use and number of products used (Casas etal.. 2013). EPA considered these two
studies to be the strongest of the exposure protocols used within this set of studies.
Outcome measures
Six of the studies in this set used standard protocols for the assessment of respiratory
symptoms in epidemiological studies (Casas etal.. 2013: Arif and Delclos. 2012: Dumas etal.. 2012:
Vizcava etal.. 2011: Zock etal.. 2007: Medina-Ramon etal.. 2005). and one study included a clinical
assessment protocol designed specifically for the assessment of occupational asthma (Lemiere et
al.. 20121. Details of the specific questions were provided, and EPA did not consider any of these
methods to be a limitation in terms of specificity of the outcome. The study by Medina-Ramon et al.
f20061 collected information on daily respiratory symptoms in a 2-week diaiy, and also trained the
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participants to measure peak expiratory flow 3 times daily. A potential limitation in the Casas et al.
(2013) study was the lack of information about the reliability of the pulmonary function measures.
Confounding
All of these studies addressed the potential for smoking to act as a confounder in the
analysis. Two of the studies reported relatively weak correlations between ammonia and other
products assessed (Zock etal.. 2007: Medina-Ramon etal.. 20051 and one study reported stronger
associations with ammonia than with bleach fDumas etal.. 20121. Based on this information, EPA
did not consider potential confounding to be a major limitation of this set of studies.
Statistical analysis
EPA considered the statistical analysis in this set of studies to be appropriate. One study,
however, was limited in terms of the level of detail provided pertaining to the results for ammonia
from multivariate models fMedina-Ramon etal.. 20051.
Studies of Agricultural Settings
Selection of study participants
EPA evaluated a set of studies conducted among livestock farmers and one study of
asthmatic children in close proximity to animal feeding operations fLoftus etal.. 20151. As with the
other occupational studies discussed above, the selection of sensitive individuals out of the
workforce ("healthy worker bias") would be a potential bias in cross-sectional studies of livestock
farmers.
Exposure parameters
Among the studies examining pulmonary function, one study collected 24-hour air sampling
from 14 ammonia monitoring devices located outside the home of a subset of the participants every
6 days for at least 3 months during the air monitoring period fLoftus etal.. 2015). two studies used
area-based exposure sampling in animal confinement buildings (Monso etal.. 2004: Zeida etal..
1994). one study used area samples taken in conjunction with specific tasks and calculated a
personal exposure measure taking into account duration spent in specific locations and tasks
fHeederik etal.. 19901. four studies collected personal samples over a work shift fDonham etal..
2000: Reynolds etal.. 1996: Preller etal.. 19951 or an unspecified time period fDonham etal..
1995). and two studies used colorimetric tubes, which are generally less precise, to measure
ammonia exposure (Monso etal.. 2004: Zeida etal.. 1994). EPA considered the use of the area-
based samples without consideration of exposure duration to be limitations of the studies by Zeida
etal. (1994) and Monso etal. (2004).
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Outcome measures
All of the studies reported using a standard spirometric technique; one study fLoftus etal..
20151 used twice daily home lung function measurements taken by the test subject; four studies
compared two measures per individual (i.e., pre- and post-shift) fMonso etal.. 2004: Donham etal..
2000: Reynolds etal.. 1996: Heederik etal.. 19901: and two studies used a single pulmonary
function measure, adjusted for height, age, and smoking variables fPreller etal.. 1995: Zeida etal..
19941. EPA did not consider any of these outcome measures to be limitations in these studies,
although the self-administered spirometry testing in the Loftus etal. (20151 study is a potential
limitation.
Confounding
Six of these studies addressed confounding in some way. Four studies controlled for co-
exposures (e.g., endotoxin, dust, disinfectants) (Melbostad and Eduard. 2001: Reynolds etal.. 1996:
Donham etal.. 1995: Preller etal.. 19951. one study noted only weak correlations (i.e., Spearman
r < 0.20) between ammonia and dust or endotoxin f Don ham et al.. 20001. and one study observed
associations with ammonia but not with endotoxin or dust measures fHeederik etal.. 19901. Three
studies did not address confounding fLoftus etal.. 2015: Monso etal.. 2004: Zeida etal.. 19941.
Based on these considerations, EPA considered the studies by Reynolds etal. Q9961. Preller
etal. f 19951. Donham etal. f20001. Donham etal. Q9951. and Heederik etal. Q9901 to be the
methodologically strongest studies of this set
Based on the evaluation of the epidemiology studies of ammonia in terms of selection of
study participants, exposure parameters, outcome measurement, confounding, and statistical
analysis, the studies listed in Table LS-2 were selected for data extraction into evidence tables in
Chapter 1.
Table LS-2. Summary of epidemiology database
Study setting
Reference
Industrial
Rahman et al. (2007)
AN et al. (2001)
Ballal etal. (1998)
Bhat and Ramaswamy (1993)
Holness et al. (1989)
Cleaning
Casas et al. (2013)
Arif and Delclos (2012)
Dumas et al. (2012)
Lemiere et al. (2012)
Vizcava et al. (2011)
Zock et al. (2007)
Medina-Ramon et al. (2006)
Medina-Ramon et al. (2005)
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Study setting
Reference
Agricultural
Loft us et al. (2015)
Monso et al. (2004)
Melbostad and Eduard (2001)
Donham et al. (2000)
Reynolds et al. (1996)
Donham et al. (1995)
Preller et al. (1995)
Choudat et al. (1994)
Zeida et al. (1994)
Crook et al. (1991)
Heederik et al. (1990)
Considerations for Evaluation of Animal Studies
Repeat-exposure toxicity studies of ammonia in experimental animals were evaluated using
the study quality considerations outlined in the Preamble and discussed in various EPA guidance
documents (U.S. EPA. 2005a. 2002.19941. including consideration of aspects of design, conduct, or
reporting that could affect the interpretation of results, overall contribution to the synthesis of
evidence, and determination of hazard potential. The objective was to identify the stronger, more
informative studies based on a uniform evaluation of quality characteristics across studies of
similar design.
Additionally, a number of general questions, presented in Table LS-3, were considered in
evaluating the animal studies. Much of the key information for conducting this evaluation can be
determined based on study methods and how the study results were reported.
Table LS-3. Considerations and relevant experimental information for
evaluation of experimental animal studies
Methodological feature
Considerations (relevant information extracted into evidence tables)
Test animal
Suitability of the species, strain, sex, and source of the test animals
Experimental design
Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and
duration of exposure (e.g., hrs/d, d/wk); timing of endpoint evaluations; sample size
and experimental unit (e.g., animals, dams, litters)
Exposure
Characterization of test article source, composition, purity, and stability; suitability
of the control (e.g., vehicle control); documentation of exposure techniques (e.g.,
chamber type); verification of exposure levels (e.g., consideration of homogeneity,
stability, analytical methods)
Endpoint evaluation
Suitability of specific methods for assessing the endpoint(s) of interest
Results presentation
Data presentation for endpoint(s) of interest (including measures of variability) and
for other relevant endpoints needed for results interpretation (e.g., decrements in
body weight in relation to organ weight)
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Information relevant to study evaluation is reported in evidence tables and was considered
in the synthesis of evidence. Discussions of study strengths and limitations (that ultimately
supported preferences for the studies and data relied upon) were included in the text where
relevant The general findings of this evaluation are presented in the remainder of this section.
Study evaluation considerations that are outcome specific are discussed in the relevant hazard
section in Section 1.2.
Test animal
The ammonia database consists of toxicology studies conducted in rats (F344, Sprague-
Dawley, Long-Evans, Sherman, Wistar), mice (0F1, Swiss albino), New Zealand White rabbits,
guinea pigs (Princeton-derived, Hartley), beagle dogs, squirrel monkeys, and pigs (several strains).
The species and strains of animals used are consistent with those typically used in laboratory
studies, and all were considered relevant to assessing the potential human health effects of
ammonia. The species, strain, and sex of the animals used in the experimental studies were
recorded in the evidence tables. The Anderson et al. (1964) and Weatherbv (1952) guinea pig
studies provided no information on the strain of the test animal; this is considered a minor
limitation of these studies.
Experimental design
General aspects of study design and experimental design were evaluated to determine if
they were appropriate for evaluation of specific endpoints. Key features of the experimental
design, including the periodicity and duration of exposure and sample sizes, were summarized in
the evidence tables in Chapter 1.
A single exposure group was used in a number of the general toxicity studies fGaafar etal..
1992: Broderson et al.. 1976: Doigand Willoughbv. 1971: Anderson et al.. 1964: Weatherbv. 19521.
and in about half of the studies that examined immune endpoints (Hamilton etal.. 1999: Hamilton
etal.. 1998: Schoeb etal.. 1982: Richard etal.. 1978a). Use of a single exposure group limits the
extent to which conclusions about a dose-response relationship can be drawn.
Sample size was not a basis for excluding a study from consideration, as studies with small
numbers of animals can still inform the consistency of effects observed for a specific endpoint
Nevertheless, the following studies with small sample sizes were considered relatively less
informative: Anderson etal. T19641 studies in the mouse (four animals/exposure interval) and
guinea pigs (two animals/exposure interval); the Weatherbv (1952) study in guinea pigs
(two control and four exposed animals/exposure interval); and the Coon etal. (1970) studies in the
rabbit (three animals/group), monkey (three animals/group), and dog (two animals/group).
Exposure
Because inhalation toxicity studies can be technically difficult to perform, particular
attention was paid to each study's exposure methods and documentation for assurance that the
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animals were properly exposed to gaseous ammonia. Exposure evaluation focused on those studies
that reported effects on the respiratory system. Of the studies evaluated for exposure quality, six
provided information on generation method, analytical method used to measure ammonia
concentrations, analytical chamber concentrations, and chamber type; exposure characterization
for these studies was considered robust fDone etal.. 2005: Diekman etal.. 1993: Broderson etal..
1976: Doig and Willoughbv. 1971: Coon etal.. 1970: Stombaugh etal.. 19691. Studies by Anderson
etal. (1964) and Curtis etal. (19751 failed to report analytical chamber concentrations, but
exposures were otherwise considered to be adequately characterized. Exposure characterization in
two studies (Gaafar etal.. 1992: Weatherbv. 19521 was considered poor because the studies failed
to report analytical chamber concentrations, analytical method, and type of inhalation chamber
used. One of these two studies (Gaafar etal.. 19921 also failed to describe how gaseous ammonia
was generated from a 12% "ammonia solution."
Endpoint evaluation
Respiratory system and other noncancer effects were largely evaluated based on clinical
signs (in the case of respiratory system effects) and histopathologic examination. All studies
identified the tissues taken for histopathologic examination; however, the extent to which
histopathologic methods were described varied across studies. Because histopathology is
considered a relatively routine measure, limited reporting of methodologic details was not
considered a significant study deficiency.
Essentially all studies examined tissues from the lung and approximately half of the studies
examined upper respiratory tissues. This is a concern because the highest exposure would have
been to the upper respiratory tract due to the fact that ammonia is both water soluble and highly
reactive. Gaafar etal. (19921 examined only the nasal mucosa. Tissues from other organs remote
from the point of entry were inconsistently examined. Coon etal. f 19701 examined sections from
the heart, lung, liver, kidney, and spleen from all surviving monkeys, dogs, and rabbits, but from
approximately half of the surviving guinea pigs and rats only; this incomplete histopathological
investigation of guinea pigs and rats is considered a limitation. Anderson etal. (19641 examined
only the liver and spleen from exposed mice and guinea pigs. Broderson etal. (19761 examined
sections from the liver, kidney, adrenal gland, pancreas, testicle, spleen, mediastinal nodes, and
thymus. Curtis et al. f 19751 noted that "visceral organs" were taken at necropsy for subsequent
histopathologic examination, but provided no further details. Weatherbv Q9521 examined the
heart, liver, stomach, small intestines, spleen, kidney, and suprarenal gland, but only reported
limited incidence and severity information for the exposed and control guinea pigs. The extent of
histopathological examination of the tissues was taken into consideration in evaluating animal
findings.
Methodological considerations related to immune-specific endpoints are discussed in
Section 1.2.2.
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Results presentation
The majority of studies reported only limited qualitative results. With the exception of
Broderson etal. f 19761. none provided information on the incidence of histopathologic lesions.
In summary, relatively few repeat-dose toxicity studies of inhaled ammonia in experimental
animals are available. The majority of these studies come from the older toxicological literature
and were generally limited in terms of study design (e.g., small group sizes), documentation of
methods, and reporting of results. Nevertheless, no study was considered sufficiently flawed as to
be uninformative. Therefore, all in vivo animal toxicity studies, as listed in Table LS-4, were
considered in hazard identification and data extraction to evidence tables.
Table LS-4. Summary of experimental animal database
Reference and study description (duration, route, species/strain)
Done et al. (2005): 5-wk inhalation study in pigs (several breeds)
Andreasen et al. (2000): 63-d inhalation study in Landrace X large white pigs
Hamilton et al. (1999): 4-wk inhalation study in large white pigs
Hamilton et al. (1998): 14-d inhalation study in large white pigs
Diekman et al. (1993): 6-wk inhalation study in crossbred gilts (female pigs)
Gaafar et al. (1992): 8-wk inhalation study in white albino mice
Gustin et al. (1994): 6-d inhalation study in pigs
Manninen and Savolainen (1989): 5-d inhalation study in Wistar rats3
Manninen et al. (1988): 15-d inhalation study in Wistar rats3
Neumann et al. (1987a): 35-d inhalation study in unweaned piglets
Targowski et al. (1984): 3-wk inhalation study in Hartley guinea pigs
Schaerdel et al. (1983): 24-hr inhalation study in CrkCOBS CD(SD) rats3
Schoeb et al. (1982): 35-d study in F344 rats
Richard et al. (1978a): 7-d study in OF1 mice
Broderson et al. (1976): 35-75-d inhalation studies in Sherman rats and F344 rats
Curtis et al. (1975): 109-d inhalation study in crossbred pigs
Doig and Willoughbv (1971): 6-wk inhalation study in Yorkshire-Landrace pigs
Coon et al. (1970): 42-90-d inhalation studies in Sprague-Dawley and Long-Evans rats, New Zealand albino
rabbits, Princeton-derived guinea pigs, squirrel monkeys, and beagle dogs
Stombaugh et al. (1969): 5-wk inhalation study in Duroc pigs
Anderson et al. (1964): 7-42-d inhalation studies in Swiss albino mice and guinea pigs (strain not specified)
Weatherbv (1952): 6-18-wk inhalation study in guinea pigs (strain not provided)
3These studies were not identified as health effect/toxicity studies in Figure LS-1, but were included in Table 1-6
(evidence pertaining to other system effects in animals) as studies that provided useful quantitative information
on the biochemical/metabolic effects of ammonia.
The references considered and cited in this document, including bibliographic information
and abstracts, can be found on the HERO website (http: //hero.epa.gov/ammonial.
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1. HAZARD IDENTIFICATION
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS
1.1.1. Chemical Properties
Ammonia (NH3) is a colorless alkaline gas with a pungent odor. Ammonia is very soluble in
water fNRC. 20081: in solution, it exists as ammonium hydroxide. Ammonium hydroxide is a weak
base that is partially ionized in water according to the following equilibrium fATSDR. 20041:
NH3 + H20 NH4+ + OH"
Ammonium hydroxide ionizes with a dissociation constant of 1.77 x 10"5 at 25°C that
increases slightly with increasing temperature fRead. 19821. A decrease in pH results in an
increase in the concentration of ammonium ion (NH4+ or protonated form), a decrease in the
concentration of the un-ionized form (NH3), and an increase in solubility of ammonia in water. At
pH 9.25, half of the ammonia will be ionized (NH4+) and half will be un-ionized (NH3). AtpH values
of 8.25 and 7.25, 90% and 99%, respectively, of ammonia will be ionized (NH4+) fATSDR. 20041.
Thus, at physiological pH (7.4), the equilibrium between NH3 and NH4+ favors the formation of
NH4+. Chemical and physical properties of ammonia are listed in Table 1-1.
Table 1-1. Chemical and physical properties of ammonia
Parameter
Value
Reference
Chemical name
Ammonia3
Synonym(s)
AM-Fol; anhydrous ammonia; ammonia
gas; Nitro-sil; R 717; Spirit of hartshorn
NLM (2012)
Structure
H
1
.Nk
H H
NLM (2012)
Chemical formula
NHs
NLM (2012)
CASRN
7664-41-7a
NLM (2012)
Molecular weight
17.031
Lide (2008), pp. 4.46-4.48, 8.40
Form
Colorless gas; corrosive
O'Neil etal. (2006)
Melting point
-77.73°C
Lide (2008), pp. 4.46-4.48, 8.40
Boiling point
-33.33°C
Lide (2008), pp. 4.46-4.48, 8.40
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Parameter
Value
Reference
Odor threshold
53 ppm (37 mg/m3)
2.6 ppm (2 mg/m3)
O'Neil etal. (2006)
Smeets et al. (2007)
Density
0.7714 g/Lat 25°C
O'Neil etal. (2006)
Vapor density
0.5967 (air = 1)
O'Neil etal. (2006)
pKa (ammonium ion)
9.25
Lide (2008), dd. 4.46-4.48, 8.40
Solubility:
Water
Organic solvents
4.82 x 105 mg/L at 24°C
Soluble in ethanol, chloroform, and
ether
Lange and Dean (1985), dd. 10-3,10-23;
Lide (2008), dd. 4.46-4.48, 8.40; O'Neil et al.
(2006)
Vapor pressure
7.51 x 103 mm Hg at 25°C
AlChE (1999)
Henry's law constant
1.61 x 10-5 atm-m3/mol at 25°C
Betterton (1992)
Conversion factors
ppm to mg/m3
mg/m3 to ppm
1 ppm = 0.707 mg/m3
1 mg/m3 = 1.414 ppm
Verschueren (2001)
aAmmonia dissolved in water is sometimes referred to as ammonium hydroxide (CASRN 1336-21-6). Ammonium
hydroxide does not exist outside of solution.
1.1.2. Toxicokinetics
Ammonia is absorbed by the inhalation route of exposure. Most inhaled ammonia is
retained in the upper respiratory tract and is subsequently eliminated in expired air. Ammonia (as
NH4+) is produced endogenously in the human intestines through the use of amino acids as an
energy source (glutamine deamination) and by bacterial degradation of nitrogenous compounds
from ingested food. At physiological pH, 98.3% of ammonia is present in the blood as the
ammonium ion (NH4+). Given its importance in amino acid metabolism, the urea cycle, and acid-
base balance, ammonia is homeostatically regulated to remain at low concentrations in the blood.
Ammonia is normally present in circulation at all lifestages (with fetal blood ammonia
concentrations higher than maternal concentrations) and in human breast milk as one of the
sources of nonprotein nitrogen. Ammonia is produced endogenously by catabolism of amino acids
by glutamate dehydrogenase or glutaminase primarily in the liver, renal cortex, and intestines, but
also in the brain and heart. Ammonia is metabolized to glutamine via glutamine synthetase in the
glutamine cycle or incorporated into urea as part of the urea cycle. The liver removes an amount of
ammonia from circulation equal to the amount added by the intestines at metabolic steady state,
such that the gut does not contribute significantly to systemic ammonia release under normal
conditions. Renal elimination via the kidney is a major contributor to ammonia homeostasis;
however, the kidneys are themselves a source of systemic ammonia. The principal means of
excretion of ammonia is as urinary urea; lesser amounts are eliminated in the feces, through sweat
production, and in expired air. A more detailed summary of ammonia toxicokinetics is provided in
Appendix C, Section C.l.
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1.2. SYNTHESIS OF EVIDENCE
Section 1.2 provides a synthesis and evaluation of the literature on the health effects of
inhaled ammonia in humans and experimental animals organized by organ/system. Evidence for
ammonia health effects is also summarized both in organ/system-specific evidence tables, which
present key study design information and results, and in exposure-response arrays. More detailed
study design information and results are provided in individual study summaries in Appendix C in
the Supplemental Information.
1.2.1. Respiratory Effects
The respiratory system is the primary target of toxicity of inhaled ammonia in humans and
experimental animals. Five cross-sectional occupational epidemiology studies in industrial settings
(Rahman etal.. 2007: Ali etal.. 2001: Ballal etal.. 1998: Bhatand Ramaswamv. 1993: Holness etal..
19891 examined the association between inhaled ammonia and prevalence of respiratory
symptoms or changes in lung function (Table 1-2). Another set of studies examined pulmonary
function or asthma symptoms in relation to ammonia exposure in health care workers and
domestic cleaners fArif and Delclos. 2012: Dumas etal.. 2012: Lemiere etal.. 2012: Vizcava etal..
2011: Zock etal.. 2007: Medina-Ramon etal.. 2006: Medina-Ramon etal.. 20051 (Table 1-3). The
association between ammonia exposure and respiratory effects indicated by these studies is also
informed by studies of pulmonary function in individuals in agricultural settings and subchronic
inhalation toxicity studies in various experimental animal species (Table 1-4). The evidence of
respiratory effects in humans and experimental animals exposed to ammonia is summarized in an
exposure-response array in Figure 1-1 at the end of this section.
Respiratory Symptoms
Respiratory symptoms (including cough, wheezing, and other asthma-related symptoms)
were reported in two cross-sectional studies of industrial worker populations exposed to ammonia
at levels greater than or equal to approximately 18 mg/m3 fRahman et al.. 2 0 0 7: Ballal etal.. 19981
(Table 1-2). One of these studies also examined frequency of respiratory symptoms by cumulative
ammonia concentration (CAC, mg/m3-years) and observed significantly higher relative risks
(2.4-5.3) with a higher CAC (>50 mg/m3-years) compared to those with a lower CAC
(<50 mg/m3-years) f Ballal etal.. 19981. In three studies examining lower exposure settings
fRahman etal.. 2007: Ballal etal.. 1998: Holness etal.. 19891 (Table 1-2), no differences were
observed in the prevalence of respiratory symptoms between ammonia-exposed workers and
controls. Ammonia concentrations reported in these lower exposure settings included a mean
ammonia concentration of <6.5 mg/m3 and a high-exposure group defined as >8.8 mg/m3 in
Holness et al. (19891. an exposure range of 0.2—7 mg/m3 in "Factory B" of Ballal etal. (19981. and a
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mean concentration of 4.9 mg/m3 in Rahman etal. (2007). The primary limitation noted in all of
these studies was the potential under-ascertainment of effects inherent in the study of a long-term
worker population (i.e., "healthy worker" effect) (see Literature Search Strategy | Study Selection
and Evaluation section and Table B-6 in the Supplemental Information). Confounding by other
workplace exposures, although a potential concern, was unlikely to be a major limitation affecting
the interpretation of the pattern of results seen in these studies, given the lack of nitrogen dioxide
measurements above the detection limit in one study (Rahman etal.. 2007) and the high level of
control of exposures in another study fHolness etal.. 1989).
Studies of health care workers or hospital workers fArif and Delclos. 2012: Dumas etal..
20121 (Table 1-3) provide evidence that exposure to ammonia as a cleaning or disinfectant product
is associated with increased risk of asthma or asthma symptoms. Use of ammonia as a cleaning
product in other settings has also been associated with asthma and respiratory symptoms fCasas et
al.. 2013: Vizcava etal.. 2011: Zock etal.. 2007: Medina-Ramon et al.. 2005) (Table 1-3).
Occupational exposure to ammonia was associated with work-exacerbated asthma (compared to
nonwork-related asthma) in a study at two occupational asthma specialty clinics by Lemiere et al.
(2012) (Table 1-3). Six studies, from Europe, Canada, and the United States, observed elevated
odds ratios, generally between 1.5 and 2.0, with varying degrees of precision. These studies were
conducted using a variety of designs, including a prospective study (Zock etal.. 2007) and two
nested case-control studies fMedina- Ramon et al.. 2006: Medina-Ramon etal.. 20051. Criteria used
to define current asthma or asthma symptoms were generally well defined and based on validated
methods. A major limitation of this collection of studies is the lack of direct measures of ammonia
exposure. Two of the studies included expert assessment of exposure (blinded to case status);
expert assessment improves reliance on self-reported exposure (Dumas etal.. 2012: Lemiere etal..
20121. Confounding by other cleaning products is an unlikely explanation for these results, as two
of the studies noted only weak correlations between ammonia and other product use (Zock etal..
2007: Medina-Ramon etal.. 20051. and another study observed stronger associations with
ammonia than with bleach (Dumas etal.. 2012). All of the studies addressed smoking as a potential
confounder.
Studies in populations exposed in agricultural settings, including swine and dairy farmers,
that analyzed for prevalence of respiratory symptoms (including cough, phlegm, wheezing, chest
tightness, and eye, nasal, and throat irritation) in relation to ammonia exposure provided generally
negative results fLoftus etal.. 2015: Melbostad andEduard. 2001: Preller etal.. 1995: Zeidaetal..
19941 (Appendix C, Table C-7). Two other studies that measured ammonia, but did not present an
analysis in relation to variability in ammonia levels, reported an increased prevalence of
respiratory symptoms in pig farmers exposed to ammonia from animal waste (Choudatetal.. 1994:
Crook etal.. 1991) (Appendix C, Table C-8). With the exception of the Loftus etal. (2015) study, all
studies involving exposure in agricultural settings documented exposures to compounds in
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addition to ammonia, such as airborne dust, endotoxin, mold, and disinfectants; Loftus etal. (2015)
did not analyze for other contaminants.
Reports of irritation and hyperventilation in volunteers acutely exposed to ammonia at
concentrations ranging from 11 to 354 mg/m3 ammonia for durations up to 4 hours under
controlled exposure conditions fPetrova et al.. 2008: Smeets etal.. 2007: Ihrigetal.. 2006: Verberk.
1977: Silverman etal.. 19491 provide support for ammonia as a respiratory irritant (Appendix C,
Section C.2.3 and Table C-9). Two controlled-exposure studies provide some evidence of
habituation to eye, nose, and throat irritation in volunteers after repeated ammonia exposure.
Following exposure to ammonia at concentrations ranging from 7 to 35 mg/m3 for 4 hours/day on
5 consecutive days, Ihrigetal. (20061 reported higher mean intensities for irritative, olfactory, and
respiratory symptoms in male volunteers unfamiliar with ammonia when compared to male
chemical company workers exposed to ammonia vapor for several years in a urea department
Differences were statistically significant only for olfactory symptoms; however, the sample size was
small. In a more limited study with only four male volunteers each exposed to 18, 35, or 71 mg/m3
ammonia (exposure to each concentration was for 1 week, 2-6 hours/day, 5 days/week), fewer
occurrences of irritation occurred upon the second weekly exposure to the same concentration
fFerguson etal.. 19771.
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 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; persistent, productive cough; bilateral diffuse rales and rhonchi; mucous
production; pulmonary edema; marked hypoxemia; and necrosis of the lung) (Appendix C,
Section C.2.3).
Experimental studies in laboratory animals also provide consistent evidence that repeated
exposure to ammonia can affect the respiratory system (Table 1-4 and Appendix C, Section C.3).
The majority of available animal studies did not look at measures of respiratory irritation, in
contrast to the majority of human studies, but rather examined histopathological changes of
respiratory tract tissues. Histopathological changes in the nasal passages were observed in
Sherman rats after 75 days of exposure to 106 mg/m3 ammonia and in F344 rats after 35 days of
exposure to 177 mg/m3 ammonia, with respiratory and nasal epithelium thicknesses increased
3-4 times that of normal fBroderson etal.. 19761. Thickening of nasal and tracheal epithelium
(50-100%) was also observed in pigs exposed to 71 mg/m3 ammonia continuously for 1-6 weeks
(Doig and Willoughby. 19711. Nonspecific inflammatory changes (not further described) were
reported in the lungs of Sprague-Dawley and Long-Evans rats and guinea pigs intermittently
exposed to 770 mg/m3 ammonia for 6 weeks; continuous exposure to 455 and 470 mg/m3
ammonia increased mortality in rats fCoon etal.. 19701. Focal or diffuse interstitial pneumonitis
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was observed in all Princeton-derived guinea pigs, New Zealand White rabbits, beagle dogs, and
squirrel monkeys exposed to 470 mg/m3 ammonia (Coon etal.. 19701. 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 262 mg/m3
ammonia for 90 days fCoon etal.. 19701.
At lower concentrations, approximately 50 mg/m3 and below, the majority of studies of
inhaled ammonia did not identify respiratory effects in laboratory animals exposed to ammonia.
No increase in the incidence of respiratory or other diseases common to young pigs was observed
after continuous exposure to ammonia and inhalable dust at concentrations representative of those
found in commercial pig farms (<26 mg/m3 ammonia) for 5 weeks fDone etal.. 20051. No gross or
histopathological changes in the turbinates, trachea, or lungs of pigs were observed after
continuous exposure to 35 or 53 mg/m3 ammonia for up to 109 days fCurtis etal.. 19751. No signs
of toxicity in rats or dogs were observed after continuous exposure to 40 mg/m3 ammonia for
114 days or after intermittent exposure (8 hours/day) to 155 mg/m3 ammonia for 6 weeks (Coon
etal.. 19701. Only one study reported respiratory effects at concentrations <50 mg/m3 (i.e., lung
congestion, edema, and hemorrhage in guinea pigs and mice exposed to 14 mg/m3 ammonia for up
to 42 days; Anderson et al. f196411. but confidence in the findings from this study is limited by
inadequate reporting and the small numbers of animals tested.
Lung Function
Decreased lung function in ammonia-exposed workers has been reported in three of the
four studies examining this outcome measure (Rahman etal.. 2007: Ali etal.. 2001: Bhat and
Ramaswamv. 19931: the exception is the study by Holness etal. (19891 in which no significant
changes in lung function were observed in workers exposed to ammonia in an industrial setting
with relatively low ammonia exposure levels (Table 1-2). These effects were observed in short-
term scenarios (i.e., cross-work shift changes in lung function) in fertilizer factory workers (mean
ammonia concentration of 18.5 mg/m3) compared with administrative staff controls f Rah man etal..
20071. and in longer-term scenarios, in workers with a cumulative exposure of >50 mg/m3-years
when compared with workers with a lower cumulative exposure of <50 mg/m3-years (with an
approximate 5-7% decrease in forced vital capacity [FVC]% predicted and forced expiratory
volume in 1 second [FEVi]% predicted)10 (Ali etal.. 20011. There were no decrements in the
percent of predicted lung function values when comparing the total exposed group to a control
group of office workers in the latter study, in the relatively low exposure scenario examined in
Holness etal. (19891 (mean ammonia concentration of 6.5 mg/m3 and high-exposure group defined
10 FVC = total amount of air exhaled during a maximal forced expiratory effort; FEVi = volume of air exhaled
in the first second of forced expiration; FEVi% = ratio of FEVi to FVC; when expressed as % predicted,
indicates that value for the subject is divided by the average value in the population for any person of similar
age, sex, and body composition.
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as >8.8 mg/m3), or in the low-exposure group (mean ammonia concentration of 4.9 mg/m3) in
Rahman etal. (2007). Another study of ammonia plant fertilizer workers reported statistically
significant decreases in FEVi and peak expiratory flow rate (PEFR/minute) in workers compared to
controls (Bhatand Ramaswamv. 19931: however, measurements of ammonia levels were not
included in this study. As discussed previously in the summary of respiratory symptoms studies,
the primary limitation within this set of studies is the potential under-ascertainment of effects in
these studies of long-term worker populations.
One of the studies of domestic cleaning workers described in Table 1-3 included a measure
of pulmonary function (Medina-Ramon et al.. 20061. Ammonia use was associated with a decrease
in peak expiratory flow (PEF) (-9.4; 95% confidence interval [CI] -17, -2.3]). A limitation of this
study was the use of lung function measurements conducted by the participant; the reliability of
this procedure has not been established. In a study by Casas etal. f 2 0131 on the effects of cleaning
product use on the respiratory health of children, ammonia exposure was associated with
decreased lung function (FEVi: -28 [95% CI -131, 76]) (Table 1-3).
Impaired respiratory function (e.g., decreased FEVi and/or FVC) in an agricultural setting
was associated with ammonia exposure in six of the eight studies that included pulmonary function
measures fLoftus etal.. 2015: Monso etal.. 2004: Donham etal.. 2000: Reynolds etal.. 1996:
Donhametal.. 1995: Preller etal.. 1995: Zeidaetal.. 1994: Heederiketal.. 1990) (AppendixC,
Table C-7). In general, the U.S. Environmental Protection Agency (EPA) considered these eight
studies to be the strongest with respect to methodology, based on considerations of exposure
assessment as well as assessment of potential confounding (see Literature Search Strategy | Study
Selection and Evaluation section).
Changes in lung function following acute exposure to ammonia have been observed in some,
but not all, controlled human exposure studies conducted in volunteers (Appendix C, Section C.2.3
and Table C-9). Cole etal. (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 >106 mg/m3, but not at 71 mg/m3. Bronchoconstriction
was reported in volunteers exposed to ammonia through a mouthpiece for 10 inhaled breaths of
ammonia gas at a concentration of 60 mg/m3 fDouglas and Coe. 19871: however, there were no
bronchial symptoms reported in volunteers exposed to ammonia in an exposure chamber at
concentrations of up to 35 mg/m3 for 10 minutes fMacEwen etal.. 19701. Similarly, no changes in
bronchial responsiveness or lung function (as measured by FVC and FEVi) were reported in healthy
volunteers exposed to ammonia at concentrations up to 18 mg/m3 for 1.5 hours during exercise
(Sundblad etal.. 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/m3 ammonia for up to 2.5 hours (Petrova etal.. 2008). or in 6 healthy
volunteers and 8 mildly asthmatic volunteers exposed to 11-18 mg/m3 ammonia for 30-minute
sessions fSigurdarson etal.. 20041.
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Toxicological Review of Ammonia
Lung function effects following ammonia exposure were not evaluated in the available
animal studies.
Table 1-2. Evidence pertaining to respiratory effects in humans following
inhalation exposure in industrial settings
Study reference and design
Results
Respiratory symptoms
Rahman et al. (2007) (Bangladesh)
Urea fertilizer factory worker (all men);
24 ammonia plant workers, 64 urea plant workers,
and 25 controls (staff from administration
building). Mean employment duration: 16 yrs
Exposure: Personal samples (two methods3;
correlation = 0.80)
Low-exposure group (ammonia plant), mean:
6.9 ppm (4.9 mg/m3); range: 2.8-11.1 ppm
(2-8 mg/m3)
High-exposure group (urea plant), mean: 26.1 ppm
(18.5 mg/m3); range: 13.4-43.5 ppm (9-31 mg/m3)
Outcome: Respiratory symptoms (five-point scale
for severity over last shift), based on Optimal
Symptom Score Questionnaire
Percentage of workers reporting symptoms (p-value):
Low
exposed High exposed
Controls (n = 24) (n = 64)
(n = 25) (p-value)* (p-value)* (p-value)f
Cough 8 17 (0.42) 28 (0.05) (0.41)
Chest 8 17(0.42) 33(0.02) (0.19)
tightness
Stuffy nose 4 12(0.35) 16(0.17) (1.0)
Runny nose 4 4(1.0) 16(0.17) (0.28)
Sneeze 8 0 (0.49) 22 (0.22) (0.01)
*p-value for ammonia plant compared to control
*p-value for urea plant compared to control
fp-value for urea plant compared to ammonia plant
Ballal et al. (1998) (Saudi Arabia)
Urea fertilizer factory workers (two factories) (all
men); 161 exposed workers and 355 unexposed
controls'5. Mean employment duration: 51.8 mo
(exposed workers) and 73.1 mo (controls)
Exposure: Area monitors (3 sets in each work
section taken at least 3 mo apart, mean
16 measures per set).
Factory A (high-exposure factory): 2-130° mg/m3
(mid-point = 66 mg/m3); geometric mean
<18 mg/m3, except for urea packaging and store
areas (geometric means = 18.6 and 115 mg/m3,
respectively)
Factory B (low-exposure factory): 0.02-7 mg/m3;
geometric mean <18 mg/m3
Cumulative exposure calculated based on exposure
and duration; dichotomized to high and low at
50 mg/m3-yrs
Outcome: Respiratory symptoms based on British
Medical Research Council questionnaire
Relative risk (95% CI), compared with controls
Factory B* Factory A*
(0.02-7 mg/m3; (2-27.1 mg/m3;
n = 77) n = 78)c
Cough No cases 2.0 (0.38,10.4)
Phlegm No cases 2.0 (0.38,10.4)
Wheezing 0.97(0.21,4.5) 3.4(1.2,9.5)
Dyspnea 0.45 (0.11,1.9) 1.8 (0.81,4.2)
* Factory-specific analyses stratified by smoking status;
results presented here are for nonsmokers. Similar patterns
seen in other smoking categories.
Relative risk (95% CI), compared with lower exposure
setting (<18 mg/m3 [n = 138] or <50 mg/m3-years [n = 130])
Cumulative
>18 mg/m3 >50 mg/m3-years
(n = 17) (n = 30)
Cough 3.5 (1.8,6.6) 2.8 (1.6,5.0)
Phlegm 3.8(2.0.7.1) 3.0(1.7,5.5)
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Study reference and design
Results
Wheezing
Dyspnea
Asthma
Chronic bronchitis
5.0 (2.4,10.6)
4.6 (2.4, 8.8)
4.3 (2.1, 9.0)
2.3 (0.31, 17)
5.2 (2.9, 9.5)
2.6 (1.3, 5.4)
2.4 (1.1, 5.4)
5.3 (1.7, 16)
Approximate 1.3-1.5 relative risk (p < 0.05) per unit increase
in ammonia concentration for cough, phlegm, wheezing,
and asthma, adjusting for duration of work, cumulative
exposure, smoking, and age.
Holness et al. (1989) (Canada)
Soda ash plant workers (all men); 58 exposed
workers and 31 controls (from stores and office
areas of plant)0. Average exposure: 12.2 yrs
Exposure: Personal samples, one work-shift per
person, mean 8.4 hrs
Low: <6.25 ppm (<4.4 mg/m3); n = 34
Medium: 6.25-12.5 ppm (4.4-8.8 mg/m3); n = 12
High: >12.5 ppm (>8.8 mg/m3); n = 12
All exposed workers (mean): 6.5 mg/m3
Outcome: Respiratory symptoms based on
American Thoracic Society questionnaire
Percentage of workers reporting symptoms (%):
Control
(n = 31)
Exposed
(n = 58)
p-value
Cough
Sputum
Bronchitis
Wheeze
Chest
tightness
Dyspnea
(shortness of
breath)
Chest pain
Rhinitis (nasal
complaints)
Throat
irritation
10
16
19
10
6
13
6
19
16
22
22
10
3
2
10
0.53
0.98
0.69
0.91
0.62
0.05
0.16
0.12
0.53
No increased risk seen in analyses stratified by exposure
group.
Lung function
Rahman et al. (2007) (Bangladesh)
Urea fertilizer factory workers (all men);
24 ammonia plant workers, 64 urea plant workers,
and 25 controls (staff from administration
building). Mean employment duration: 16 yrs
Exposure: Personal samples (2 methods3;
correlation = 0.80)
Low-exposure group (ammonia plant), mean:
6.9 ppm (4.9 mg/m3); range: 2.8-11.1 ppm
(2-8 mg/m3)
Pre-shift
Post-shift
p-value
Ammonia plant (low-exposure group, 4.9 mg/m3);
n = 24 ammonia plant workers
FVC
FEVi
PEFR
3.308
2.627
8.081
3.332
2.705
8.313
0.67
0.24
0.22
Urea plant (high-exposure group, 18.5 mg/m3); n = 64 urea
plant workers
FVC
3.362
3.258
0.01
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Toxicological Review of Ammonia
Study reference and design
Results
High-exposure group (urea plant), mean:
26.1 ppm (18.5 mg/m3); range: 13.4-43.5 ppm
(9-31 mg/m3)
Outcome: Lung function (standard spirometry)
FEVi 2.701 2.646 0.05
PEFR 7.805 7.810 0.97
p-value reflects the comparison of pre- and post-shift
values.
Multiple regression model (data from 23 ammonia and urea
plant workers with concurrent measurements of ammonia
exposure and lung function):
Concentration of ammonia and exposure duration (yrs of
employment as proxy for duration) were significantly
correlated with percentage cross-shirt decrease in FEVi%
(AFEVi%).
Each year of work in a production section was associated
with a decrease in AFEVi% of 0.6%. [Limitation of analysis:
failure to explore the age parameter; age and years of
work were highly correlated (Pearson correlation
coefficient 0.97).]
Ali et al. (2001) (Saudi Arabia)
Urea fertilizer factory workers (all men)—
(additional studv of "Factorv A" in Ballal et al.
(1998)); 73 exposed workers and 348 unexposed
controls. Mean employment duration: not
reported
Exposure: 4-hr measurements. Cumulative
exposure calculated based on exposure and
duration; dichotomized to high and low at
50 mg/m3-yrs
Outcome: Lung function (standard spirometry;
morning measurement)
<50 mg/m3-yr >50 mg/m3-yr
(n = 45) (n = 28) p-value
FVCi% 100.7 93.4 0.006
predicted
FVC% 105.6 100.2 0.03
predicted
FEVi/FVC% 84.7 83.4 NS
NS = not significant (p-values not provided by study authors)
Bhat and Ramaswamv (1993) (India)
Fertilizer chemical plant workers; 30 diammonium
phospate (DAP) plant workers, 30 urea plant
workers, 31 ammonia plant workers, and
68 controls (people with comparable body surface
area chosen from the same socio-economic status
and sex as exposed workers)
Exposure: Measurements not reported; duration
dichotomized as <10 and >10 yrs
Outcome: Lung function (standard spirometry)
Ammonia
Controls DAP plant Urea plant plant
(n = 68) (n = 30) (n = 30) (n = 31)
FVC 3.4 ±0.21 2.5 ±0.06* 3.3 ±0.11 3.2 ±0.07
FEVi 2.8 ±0.10 2.1 ±0.08* 2.7 ±0.10 2.5 ±0.1*
PEFR 383 ±7.6 228 ± 18* 307 ± 19* 314 ± 20*
*p < 0.05
Holness et al. (1989) (Canada)
Soda ash plant workers (all men); 58 exposed
workers and 31 controls (from stores and office
areas of plant)d. Average exposure: 12.2 yrs
Exposure: Personal samples, one work-shift per
person, mean 8.4 hrs
Control Exposed
(n = 31) (n = 58) p-value*
Lung function (% predicted values)*:
FVC 98.6 ± 11.3 96.8 ±11.0 0.094
FEVi 95.1 ± 12.5 94.1 ±12.9 0.35
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Study reference and design
Results
Low: <6.25 ppm (<4.4 mg/m3); n = 34
FEVi/FVC 96.5 ±6.1 97.1 ±7.1
0.48
Medium: 6.25-12.5 ppm (4.4-8.8 mg/m3); n = 12
High: >12.5 ppm (>8.8 mg/m3); n = 12
Change in lung function over work shift:
All exposed workers (mean): 6.5 mg/m3
FVC day 1 -0.9 -0.8
0.99
Outcome: Lung function (standard spirometry;
beginning and end of shift, at least 2 test days per
day 2 +0.1 -0.0
0.84
worker)
FEVi day 1 -0.2 -0.2
0.94
day 2 +0.5 +0.7
0.86
*p-value for difference between exposed and control
workers calculated by using actual baseline values and
correcting for age, height, and pack-years smoked
determined by multiple regression analysis.
Percentage of the subject's predicted value (% predicted)
has been widely adopted as follows: % predicted =
recorded
value x 100/predicted value); this value is now calculated on
automated spirometers based on sex, race, age, and height.
aExposure 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, respectively
(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 Safety Inc.
(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 the National Center for Environmental
Assessment [NCEA], Office of Research and Development [ORD], EPA), EPA considered the PAC III instrument to
be a more sensitive monitoring technology than the Drager tubes. Therefore, higher confidence is attributed to
the PAC III air measurements of ammonia for the Rahman et al. (2007) study.
bThe 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.
cThe ammonia concentration range in Factory A is better represented as 2-27.1 mg/m3. This range excludes the
employees in the urea store (n = 6; range of ammonia concentrations = 90-130.4 mg/m3) who were required to
wear full protective clothing, thus minimizing potential exposure. Number of workers in Factory A excluding urea
store workers = 78.
dAt 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.
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Table 1-3. Evidence pertaining to respiratory effect in humans following
inhalation exposure in cleaning settings
Study reference and design
Results
Asthma or asthma symptoms
Dumas et al. (2012) (France)
Hybrid design, hospital workers, drawn from
population-based case-control study; 179 hospital
workers (136 women), 545 other workers
(333 women).
Exposure: Asthma-specific job exposure matrix plus
expert review (blinded), ever exposed, 18 specific
products, based on all jobs held at least 3 mo;
ammonia prevalence 23% in female hospital workers
Outcome: Current asthma: Asthma attack, respiratory
symptoms or asthma treatment in the last 12 mo
(based on standardized questionnaire)
Odds ratio (95% CI), current asthma
Women: 3.05 (1.19, 7.82)
Men: no associations with any specific products
(prevalence low)
Adjusted for age and smoking, and accounting for
familial dependence (due to sampling of cases and first-
degree relatives)
Arif and Delclos (2012) (United States. Texas)
Population survey of 3,650 health care workers
(physicians, nurses, respiratory therapists,
occupational therapists), (total n = 5,600, response
rate 66%)
Exposure: Structured questionnaire—frequency of use
of products for longest job held; ever contact with list
of 28 products; ammonia prevalence 23%
Outcome: Structured questionnaire
• Work-related asthma symptoms:
wheezing/whistling at work or shortness of breath
at works that gets better away from work or worse
at work
• Work-exacerbated asthma: onset before began
work
• Occupational asthma: onset after began work
Odds ratio (95% CI) [n cases]
Work-related asthma symptoms [n = 132]
2.45 (1.28, 4.69)
Work-exacerbated asthma [n = 41]
1.58 (0.56, 4.43)
Occupational asthma [n = 33]
1.86 (0.49, 7.13)
Adjusted for age, sex, race/ethnicity, body mass index,
seniority, atopy, and smoking status
Lemiere et al. (2012) (Quebec. Canada)
Case-control study, workers seen at two tertiary care
centers specializing in occupational asthma. Asthma
(defined below) based on reversible airflow limitation
or airway hyper-responsiveness tests; referent
group = non-work-related asthma seen at same clinics
but symptoms did not worsen at work (n = 33).
Exposure: Structured interview focusing on
last/current job, combined with expert review
(blinded); ammonia prevalence 19/153 = 12%
Outcome: Diagnoses made based on reference tests
• Occupational asthma if specific inhalation
challenge test was positive
• Work-exacerbated asthma if specific inhalation test
was negative but symptoms worsened at work
Odds ratio (95% CI) [n cases]
Work exacerbation [n = 53]
8.4(1.1, 371.7)
Occupational asthma [n = 67]
3.7 (0.4,173.4)
Age, smoking, and occupational exposure to heat, cold,
humidity, dryness, and physical strain assessed as
confounders.
[Wide CIs reflect sparseness in referent group, with only
1 of the 33 classified as exposed to ammonia.]
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Toxicological Review of Ammonia
Study reference and design
Results
Vizcava et al. (2011) (Spain)
Survey of cleaning service workers (n = 917) from
37 businesses (19% response rate to questionnaire
distributed through the employers); 761 current
cleaners, 86 former cleaners, 70 never cleaners;
referent group = never cleaners and current cleaners
who had not used any of the specified cleaning
products in the past year (n = 161)
Exposure: Structured questionnaire, use of cleaning
tasks and 12 products; ammonia prevalence 66%
Outcome: Structured questionnaire
• Current asthma: in past 12 mo, woken by an attack
of shortness of breath, had an attack of asthma, or
currently taking any asthma medications (including
inhalers, aerosols, or tablets)
• Asthma score: Sum of "yes" answers to five
symptoms in last 12 mo (wheeze with
breathlessness, woken up with chest tightness,
attack of shortness of breath at rest, attack of
shortness of breath after exercise, woken by attack
of shortness of breath)
Odds ratio (95% CI) (among current cleaners) [n]
Current asthma 1.4 (0.6, 3.2) [81]
Wheeze without having a cold 2.1 (0.9, 4.7) [83]
Chronic cough 1.6 (0.8, 3.3) [95]
Asthma score 1.6 (1.0, 2.5)
[mean 0.59, SD 1.12]
Adjusted for age, country of birth (Spanish versus non-
Spanish), sex, and smoking status
Zock et al. (2007) (Europe. 22 sites)
Longitudinal study, n = 3,503, 9-yr follow-up of
European Community Respiratory Health Survey,
population-based sample, ages 20-44 yrs. Excluded
764 individuals with asthma at baseline; limited to
individuals reporting doing the cleaning or washing in
their home.
Exposure: Structured interview at follow-up; frequency
of use of 15 products
Outcome: Structured interview at follow-up
• New onset (since baseline survey) current asthma,
defined by asthma attack or nocturnal shortness of
breath in the past 12 mo or current use of
medication for asthma
• Current wheeze defined as wheezing or whistling in
the chest in last 12 mo when not having a cold
• New onset physician-diagnosed asthma, asthma
defined as above with confirmation by a physician
and information on age or date of first attack
Odds ratio (95% CI) [n]
Current asthma 1.4 (0.87, 2.23) [199]
Current wheeze 1.3 (0.81, 2.13) [226]
Physician-diagnosed asthma 0.92 (0.33, 2.59) [71]
Adjusted for sex, age, smoking, employment in a
cleaning job during follow-up, and study center;
heterogeneity by center also assessed. Correlations
among products were generally weak (Spearman
rho < 0.3).
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Study reference and design
Results
Medina-Ramon et al. (2005) (Spain)
Nested case-control, cleaning workers; case (n = 40;
74% participation rate) based on asthma and/or
bronchitis at both assessments. Controls (n = 155, 69%
participation rate)—no history of respiratory symptoms
in preceding year and no asthma at either assessment.
Exposure: Structured interview; frequency of use of
22 products; ammonia prevalence 16% undiluted, 56%
diluted
Outcome: Asthma: asthma attack or being woken by
attack or shortness of breath in past 12 mo;
Chronic bronchitis: regular cough or regular bringing
up phlegm for at least 3 mo each year
Odds ratio (95% CI) (unadjusted), >12 compared with
<12 times per year
Undiluted 3.1(1.2,8.0)
Diluted 0.8 (0.4,1.7)
Fraction of exhaled nitric oxide (FeNO) and pulmonary function
Casas et al. (2013) (Spain)
Adjusted* associations of FeNO, FVC and FEVi!
Population based cross sectional birth cohort study;
n = 432 infants enrolled; n = 295 total number of
individuals recruited that completed the 10-yr follow
up visit and the cleaning products questionnaire and
performed the FeNO and/or lung function test; 35% of
recruited population were excluded because
information on use of cleaning products and/or
respiratory tests was not available; only 46 individuals
reported use of ammonia Exposure: Interviewer-led
questionnaire; frequency of use of 10 different
cleaning products (bleach, ammonia, polishes or
waxes, acids, solvents, furniture sprays, glass cleaning
sprays, degreasing sprays, air freshening sprays, and air
freshening plug- ins); exposure score developed based
on frequency of use and number of products used
Outcome: Questionnaires on wheezing asthma,
treatment and allergies were administered by mother
from birth to age 10; at age 10-13 yrs, FeNO and lung
function tests were carried out
with weekly use of ammonia (n = 46; 16%)
FeNOfppb FVC mL FEVi mL
GM ratio (95% CI) P (95% CI) P (95% CI)
0.86 (0.66,1.12) 3 (-127, 133) -28 (-131, 76)
* Adjusted for sex, age, asthma medication, season of
respiratory measurement, maternal education, and
parental smoking; FVC and FEVi models were
additionally adjusted for height and weight.
bChange in FeNO, FVC and FEVi per interquartile range
increase of the score (interquartile range = 6.5 d of
product use per wk).
cFeNO (fraction of exhaled nitric oxide) is used to
characterize asthma or other conditions associated with
airway inflammation; it is measured in a breath test.
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Study reference and design
Results
Medina-Ramon et al. (2006) (Spain)
Panel study, sample selected from participants in
nested case-control studv bv Medina-Ramon et al.
(2005). Current asthma symptoms or chronic
bronchitis in 2000-2001 survey; n = 51 of 80 (64%);
8 excluded for possible recording errors, outliers,
learning effects
Exposure: Daily diary of use of products
Outcome: Respiratory symptoms based on 2-wk daily
diary (seven symptoms, five-point intensity scale);
summed score for upper respiratory symptoms
(blocked nose, throat irritation, watery eyes) and lower
respiratory symptoms (chest tightness, wheezing,
shortness of breath, and cough); PEF measured with
mini-Wright peak flow meter (with training and written
instructions); measured morning, lunchtime, night
(three measurements each; highest recorded)
Diluted and Diluted
undiluted only
Odds ratio (95% CI)
Upper respiratory 1.8 (0.7, 4.9) 1.3 (0.3, 5.0)
symptoms
Lower respiratory 1.6(0.6,4.4) 3.0(1.0,9.1)
symptoms
Beta (95% CI)
PEF at night -9.4 (-17,-2.3) -10.3 (-18,-2.7)
PEF, following -1.2 (-8.5, 6.2) -2.9 (-11, 6.2)
morning
Adjusted for respiratory infection, use of maintenance
medication, and age; daily number of cigarettes smoked,
years of employment in domestic cleaning, and/or
weekly working hours in domestic cleaning also assessed
as potential confounders
GM= geometric mean; SD = standard deviation.
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Toxicological Review of Ammonia
Table 1-4. Evidence pertaining to respiratory effects in animals
Study reference and design
Results
Effects on the lungs
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15/group
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female;
15/group
Squirrel monkey (Saimiri sciureus); male; 3/group
Beagle dog; male; 2/group
0,155, or 770 mg/m3 for 8 hrs/d, 5 d/wk for 6 wks
Gross necropsies were normal; focal pneumonitis in one
of three monkeys at 155 mg/m3.
Nonspecific lung inflammation observed in guinea pigs
and rats, but not in other species, at 770 mg/m3.a
Coon et al. (1970)
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female;
15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
At 470 mg/m3, focal or diffuse interstitial pneumonitis in
all animals. Calcification of bronchial epithelium
observed in several animals. Hemorrhagic lung lesion in
one of two dogs; moderate lung congestion in two of
three rabbits.3 (This exposure was lethal to ~25% of the
guinea pigs.)
Coon et al. (1970)
Sprague-Dawley or Long-Evans rat; male and female;
15-51/group
0 or 40 mg/m3 for 114 d, 127, 262, or 470 mg/m3 for
90 d, or 455 mg/m3 for 65 d
Focal or diffuse interstitial pneumonitis in all animals, and
calcification of bronchial epithelium observed in several
animals at 470 mg/m3, an exposure that was lethal to
most of the rats.3
Anderson et al. (1964)
Swiss albino mouse; male and female; 4/exposure
interval
0 or 20 ppm (0 or 14 mg/m3) for 7,14, 21, 28, or 42 d
Lung congestion, edema, and hemorrhage observed at
14 mg/m3 after 42 d.3
Anderson et al. (1964)
Guinea pig (strain not specified); male and female;
2/exposure interval at 20 ppm, 6/exposure interval at
50 ppm
0 or 20 ppm (0 or 14 mg/m3) for 7,14, 21, 28, or 42 d
or 50 ppm (35 mg/m3) for 42 d
Lung congestion, edema, and hemorrhage observed at
14 and 35 mg/m3 after 42 d.3
Done et al. (2005)
Pig (several breeds); sex not specified; 24/group
0, 0.6, 10, 18.8, or 37 ppm (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
diseases.
Curtis et al. (1975)
Pig (crossbred); sex not specified; 4-8/group
0, 50, or 75 ppm (0, 35, or 53 mg/m3 for 109 d)
Turbinates, trachea, and lungs of all pigs were classified
as normal.
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Study reference and design
Results
Effects on the upper respiratory tract
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15/group
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female;
15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0,155, or 770 mg/m3 for 8 hrs/d, 5 d/wk for 6 wks
Dyspnea in rabbits and dogs exposed to 770 mg/m3
during wk 1 only; no indication of irritation after wk 1;
nasal tissues not examined for gross or histopathologic
changes.
Broderson et al. (1976)b
Sherman rat; 5/sex/group
10 or 150 ppm (7 or 106 mg/m3) from bedding for
75 d
1" thickness of the nasal epithelium (3-4 times) and nasal
lesions at 106 mg/m3.a
Broderson et al. (1976)b
F344 rat; 6/sex/group
0 or 250 ppm (0 or 177 mg/m3) in an inhalation
chamber for 35 d
1" thickness of the nasal epithelium (3-4 times) and nasal
lesions at 177 mg/m3.a
Coon et al. (1970)
Sprague-Dawley or Long-Evans rat; male and female;
15-51/group
0 or 40 mg/m3 for 114 d, 127, 262, or 470 mg/m3 for
90 d, or 455 mg/m3 for 65 d
Nasal discharge at 262 mg/m3 (25% of rats). Dyspnea and
nasal irritation/discharge in all animals at 455 and
470 mg/m3, an exposure that was lethal to the majority
of the rats.3
Gaafar et al. (1992)
White albino mouse; male; 50
Ammonia vapor of 0 or 12% ammonia solution for
15 min/d, 6 d/wk, for 8 wks
Histological changes in the nasal mucosa.3
Doig and Willoughbv (1971)
Yorkshire-Landrace pig; sex not specified; 6/group
0 or 100 ppm (0 or 71 mg/m3) for 6 wks
1" thickness of nasal and tracheal epithelium (50-100%
increase).3
Stombaugh et al. (1969)
Duroc pig; both sexes; 9/group
12, 61,103,145 ppm (8, 43, 73, or 103 mg/m3) for
5 wks
Excessive nasal, lacrimal, and mouth secretions and T*
frequency of cough at 73 and 103 mg/m3.3
Coon et al. (1970)
Beagle dog; male; 2/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
Nasal discharge at 470 mg/m3.3
incidence data not provided.
bThe 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, Mycoplasma 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 1.2.2, Immune System Effects.
1-17
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Toxicological Review of Ammonia
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HUMAN STUDIES
Effects on the lung
EXPERIMENTAL ANIMAL STUDIES
Effects on the upper respiratory tract
Figure 1-1. Exposure-response array of respiratory effects following inhalation exposure to ammonia.
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Toxicological Review of Ammonia
Mode-of-Action Analysis—Respiratory Effects
Data on the potential mode of action for respiratory effects associated with chronic
exposure to ammonia are limited. However, acute exposure data demonstrate that injury to
respiratory tissues is primarily due to ammonia's alkaline (i.e., caustic) properties from the
formation of hydroxide ion when it comes in contact with water and is solubilized. Ammonia
readily dissolves in the moisture on the mucous membranes, forming ammonium hydroxide, which
causes liquefactive necrosis of the tissues. Specifically, ammonia directly denatures tissue proteins
and causes saponification of cell membrane lipids, which leads to cell disruption and death
(necrosis). In addition, the cellular breakdown of proteins results in an inflammatory response,
which further damages the surrounding tissues fAmshel etal.. 2000: Millea etal.. 1989: larudi and
Golden. 19731
Summary of Respiratory Effects
Evidence for respiratory toxicity associated with exposure to ammonia comes from studies
in humans and animals. Multiple occupational studies involving chronic exposure to ammonia in
industrial settings provide evidence of an increased prevalence of respiratory symptoms (Rahman
etal.. 2007: Ballal etal.. 19981 and decreased lung function f Rah man et al.. 2 0 0 7: Ali etal.. 2001:
Bhatand Ramaswamv. 1993) (Table 1-2 and Appendix C, Section C.2.1). An increase in respiratory
effects was reported both with higher workplace ammonia concentrations (Rahman et al.. 2007:
Ballal etal.. 1998) and with greater cumulative ammonia concentration (expressed in
mg/m3-years) (Ali etal.. 2001: Ballal etal.. 1998). Evidence of respiratory effects is provided by
studies of asthma, asthma symptoms, and pulmonary function in workers and others exposed to
cleaning agents containing ammonia, in a variety of study designs and populations fCasas etal..
2013: Arif and Delclos. 2012: Dumas etal.. 2012: Lemiere etal.. 2012: Vizcava etal.. 2011: Zock et
al.. 2007: Medina-Ramon etal.. 2006: Medina-Ramon et al.. 2005) (Table 1-3). Additional evidence
of respiratory effects of ammonia is seen in studies of pulmonary function in an agricultural setting,
specifically in livestock farmer studies that accounted for effects of co-exposures to other agents
such as endotoxin and dust (Don ham et al.. 2000: Reynolds etal.. 1996: Donham etal.. 1995: Preller
etal.. 1995: Heederik etal.. 1990). and in one study of asthmatic children who lived near animal
feeding operations that did not control for co-exposures fLoftus etal.. 20151 (Appendix C,
Table C-7). The livestock farmer studies, however, do notprovide evidence of associations between
ammonia and respiratory symptoms. Controlled human exposure studies of ammonia inhalation
and case reports of injury in humans with inhalation exposure to ammonia provide additional
support for the respiratory system as a target of ammonia toxicity when inhaled (Appendix C,
Section C.2.3). Overall, the consistency of findings across three categories of epidemiological
studies (industrial, cleaner, and agricultural settings) that differed in population characteristics,
level and pattern of exposure, and potential confounders, and support from studies of acute
1-19
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Toxicological Review of Ammonia
exposures, adds strength to the evidence for an association between respiratory effects and
ammonia exposure.
Evidence from animal studies supports an association between inhaled ammonia and
respiratory effects. Short-term and subchronic animal studies show histopathological changes of
respiratory tissues 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 dosing regimens (Gaafar etal.. 1992: Broderson etal.. 1976: Doig and Willoughbv.
1971: Coon etal.. 1970: Anderson etal.. 19641 (Table 1-4 and Appendix C, Section C.3). In general,
responses in respiratory tissues increased with increasing ammonia exposure concentration.
Based on evidence of respiratory effects in multiple human and animal studies (including
epidemiological studies in different settings and populations), respiratory system effects are
identified as a hazard associated with inhalation exposure to ammonia.
1.2.2. 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 via inhalation.
Immunoglobulin G- (IgG) and E-specific (IgE) antibodies for pig skin and urine fCrook etal.. 19911.
elevated neutrophils from nasal washes, and increased white blood cell counts fCormier etal..
20001 were reported. These data on immunological function 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 were measured by three general types of immune assays: host resistance,
T cell proliferation, and delayed-type hypersensitivity. Immunotoxicity studies of ammonia using
measures of host resistance provide the most relevant data for assessing immune function since
they directly measure the ability of the immune system to control microorganism growth. Other
available studies of ammonia employed assays that evaluated immune function. Changes in
immune cell populations without corresponding functional data are considered to be the least
predictive, and studies that looked only at these endpoints fGustin etal.. 1994: Neumann etal..
1987b) were considered less informative and were not further considered in evaluating the
immune system effects of ammonia.
Several host resistance studies utilized lung pathogens to assess bacterial clearance
following ammonia exposure; however, these studies were not designed to discriminate between
direct immunosuppression associated with ammonia exposure or immune effects secondary to
damage to the protective mucosal epithelium of the respiratory tract. The available studies also do
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Toxicological Review of Ammonia
not correlate increased bacterial colonization with reduced immune function. 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 with
108 colony forming units [CFU] of Mycoplasma pulmonis followed by up to 42 days of ammonia
exposure post inoculation fBroderson etal.. 19761. (Inoculation with the respiratory pathogen
M. pulmonis causes murine respiratory mycoplasmosis [MRM] characterized by lung lesions.) The
incidence of lung lesions was significantly increased at ammonia concentrations >35 mg/m3,
suggesting that ammonia exposure decreased bacterial clearance resulting in the development of
M. pulmonis-induced MRM. However, increasing ammonia concentration was not associated with
increased CFU of M. pulmonis isolated from the respiratory tract The high number of inoculating
CFU could have overwhelmed the innate immune response and elicited a maximal response that
could not be further increased in immunocompromised animals.
Conversely, significantly increased CFU of M. pulmonis bacteria isolated in the trachea, nasal
passages, lungs, and larynx were observed in F344 rats continuously exposed to 71 mg/m3
ammonia for 7 days prior to M. 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.
0F1 mice exposed to 354 mg/m3 ammonia for 7 days prior to inoculation with a 50% lethal
dose (LD50) of Pasteurella multocida exhibited significantly increased mortality compared to
controls (86% versus 50%, respectively); however, an 8-hour exposure was insufficient to affect
mortality (Richard etal.. 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.
Pig studies support the findings observed in the rodent studies that ammonia exposure
increases the colonization of respiratory pathogens. Andreasen et al. (2000) demonstrated that
63 days of ammonia exposure increased the number of bacterial-positive nasal swabs following
inoculation with P. multocida and Mycoplasma hyopneumoniae; however, the effect was not dose
responsive and did not result in an increase in lung lesions. Additional data obtained from pigs
suggest that ammonia exposure eliminates the commensal flora of the nasal cavities, which allows
for increased colonization of P. multocida; however, this effect abates following cessation of
ammonia exposure (Hamilton et al.. 1999: Hamilton et al.. 1998).
Suppressed cell-mediated immunity and decreased T cell proliferation was observed
following ammonia exposure. Using a delayed-type hypersensitivity test to evaluate cell-mediated
immunity, Hartley guinea pigs were vaccinated with Mycobacterium bovis bacillus Calmette-Guerin
(BCG) and exposed to ammonia followed by intradermal challenge with a purified protein
derivative (PPD). Dermal lesion size was reduced in animals exposed to 64 mg/m3 ammonia,
indicating immunosuppression (Targowski etal.. 19841. Blood and bronchial lymphocytes
harvested from naive guinea pigs treated with the same 3-week ammonia exposure and stimulated
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Toxicological Review of Ammonia
with phytohaemagglutinin or concanavalin A demonstrated reduced T cell proliferation (Targowski
et al.. 19841. 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 reduced viability, indicating nonspecific effects of ammonia-induced
immunosuppression fTargowski et al.. 19841. These data suggest that T cells may be the target of
ammonia exposure since specific macrophage effects were not observed.
The evidence of immune system effects in experimental animals exposed to ammonia is
summarized in Table 1-5 and is presented graphically as an exposure-response array in Figure 1-2.
Table 1-5. Evidence pertaining to immune system effects in animals
Study reference and design
Results
Host resistance
Broderson et al. (1976)
F344 rat; male and female; 11-12/sex/group
<5 (control), 25, 50,100, or 250 ppm (<3.5 [control], 18, 35,
71, or 177 mg/m3), 7 d (continuous exposure) pre-
inoculation/28-42 d post-inoculation with M. pulmonis
% of animals with gross lung lesions: 16, 46, 66*, 33,
and 83%
No effect on CFU
Schoeb et al. (1982)
F344 rat; 5-15/group (sex unknown)
<2 or 100 ppm (<1.4 [control] or 71 mg/m3), 7 d
(continuous exposure) pre-inoculation/28 d post-
inoculation with M. pulmonis
1" bacterial colonization (as a result of reduced
bacterial clearance).
Richard et al. (1978a)
OF1 mouse; male; 99/group
0 or 500 ppm (0 or 354 mg/m3), 8 hrs or 7 d (continuous
exposure), prior to infection with P. multocida
% Mortality: 50 and 86%*
Andreasen et al. (2000)
Landrace X large white pigs; 10/group (sex unknown)
<5 (control), 50, or 100 ppm (3.5, 35, or 71 mg/m3), 63 d
(continuous exposure) inoculated with M. hyopneumoniae
on day 9 and P. multocida on d 28, 42, and 56
% of animals with positive day 49 nasal swab:
24, 100*, and 90%*
Hamilton et al. (1998)
Large white pigs; 4-7/group (sex unknown)
0 or 20 ppm (0 or 14 mg/m3), 14 d (continuous exposure),
inoculated with P. multocida on d 0
1" bacterial colonization
Hamilton et al. (1999)
Large white pigs; 5/group (sex unknown)
0 or 50 ppm (0 or 35 mg/m3), 1 wk pre-inoculation with P.
multocida, 3 wks post-inoculation
1" bacterial colonization
Bacteria isolated from nasal cavities: 3.18 and 4.30*
CFU
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Toxicological Review of Ammonia
Study reference and design
Results
T cell proliferation
Tareowski et al. (1984)
Hartley guinea pig; 8/group (sex unknown)
<15, 50, or 90 ppm (<11 [control], 35, or 64 mg/m3), 3 wks
(continuous exposure)
4/ proliferation in blood and bronchial T cells
Delayed-type hypersensitivity
Tareowski et al. (1984)
Hartley guinea pig, BCG immunized; 8/group (sex unknown)
<15, 50, or 90 ppm (<11 [control], 35, or 64 mg/m3), 3 wks
(continuous exposure) followed by PPD challenge
Mean diameter of dermal lesion (mm): 12,12.6, and
8.7*
^Statistically significantly different from the control (p < 0.05).
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Toxicological Review of Ammonia
1000
E
*S| 100
i
w
1
8
Ł
I
o
fr
10
1
A
¦ LOAEL
Vertical lines show range of
ANOAEL concentrations in study.
# Additional concentrations
"T1 incidence of f1 bacterial
gross iurg lesions colonization (rat};
(rat); Broderson et Schoeb et al.
a!. (1976) (1982)
mortality 1" bacterial bacterial bacterial
(mouse); Richard colonization (pig); colonization (pig); colonization (pig);
etaS, (1978a) Andreasen etal. Hamilton etal. Hamilton et al.
(2000) (1998) (1999)
Host resistance
4/ proliferation in
blood and
bronchial
lymphocytes
(guinea pig);
Targowski et al.
(1984)
T cell proliferation
4- dermal lesion
size (guinea pig);
Targowski et ai.
(1984)
Delayed-type
hypersensitivity
Figure 1-2. Exposure-response array of immune system effects following inhalation exposure to ammonia.
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Toxicological Review of Ammonia
Summary of Immune System Effects
The evidence for ammonia immunotoxicity is based on epidemiological and 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 are not particularly informative for evaluating whether
ammonia has immunotoxic properties.
Animal studies provide consistent evidence of elevated bacterial growth following ammonia
exposure. This is supported by observations of lung lesions fBroderson etal.. 19761. elevated CFU
fSchoeb et al.. 19821. and increased mortality (Richard etal.. 1978al in rats or mice exposed to
ammonia; however, the findings from the Broderson et al. Q9761 study (which described the
percent 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 thatT cells are inhibited by ammonia (Targowski et al.. 19841. but the data were
not dose responsive.
Overall, there are suggestions that ammonia exposure may be associated with
immunotoxicity, but it is unclear if elevated bacterial colonization is the result of damage to the
protective mucosal epithelium of the respiratory tract or the result of suppressed immunity.
Therefore, there is inadequate information to draw a conclusions about the immune system as a
potential hazard of ammonia exposure.
1.2.3. Other Systemic Effects
The majority of information suggests that ammonia induces effects in and around the portal
of entry. As discussed below, there is limited evidence from experimental animals that ammonia
can produce effects on organs distal from the portal of entry, including the liver, kidney, spleen, and
heart.
Evidence of liver toxicity in animals comes from observations of histopathological
alterations in the liver. Histopathologic changes described as "fatty changes of the liver plate cells"
were reported at an exposure concentration of 470 mg/m3 ammonia in rats, guinea pigs, rabbits,
dogs, and monkeys following the same subchronic inhalation exposure regimens fCoon et al..
19701: this concentration was lethal to approximately 25% of exposed guinea pigs and the majority
of exposed rats. Congestion of the liver was reported in guinea pigs following inhalation exposure
to 35 mg/m3 for 42 days and 120 mg/m318 weeks (Anderson et al.. 1964: Weatherbv. 19521: no
liver effects were observed in similarly exposed mice at 14 mg/m3 (Anderson etal.. 19641.
Experimental animal studies provide some evidence that inhaled ammonia can affect the
kidney and spleen. Alterations in the kidneys (calcification and proliferation of tubular epithelium)
were reported in rats, rabbits, guinea pigs, monkeys, and dogs exposed to 470 mg/m3, an ammonia
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Toxicological Review of Ammonia
concentration that was lethal to rats and guinea pigs (Coon etal.. 19701. "Congestion" of the
kidneys and spleen was reported in four guinea pigs exposed to 120 mg/m3 ammonia for 18 weeks
(but not 6 or 12 weeks) fWeatherbv. 19521. Enlarged and "congested" spleens were reported in
guinea pigs exposed to 35 mg/m3 ammonia for 6 weeks (Anderson et al.. 19641. None of these
studies provided incidence of histopathologic lesions.
Myocardial fibrosis was observed in monkeys, dogs, rabbits, guinea pigs, and rats following
subchronic inhalation exposure to 470 mg/m3 ammonia, a concentration lethal to exposed guinea
pigs and rats; no changes were observed at lower concentrations (Coon etal.. 19701. 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. Q9701 in small numbers of dogs and
rabbits, but was not observed in similarly exposed monkeys or rats.
"Early degenerative changes" in the adrenal gland were reported in four guinea pigs
exposed to 120 mg/m3 ammonia by inhalation for 18 weeks, but not in guinea pigs exposed for 6 or
12 weeks (Weatherbv. 1952). With the exception ofBroderson etal. (1976). no other investigators
examined effects on the adrenal gland following exposure to inhaled ammonia, and Broderson etal.
(1976) did not describe effects on nonrespiratory tissues. These limited findings are insufficient to
draw conclusions about possible effects of ammonia on the adrenal gland.
As discussed above, Coon etal. (1970) reported effects on the liver, kidney, and heart
following continuous exposure to 470 mg/m3; however, no histopathological changes 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/m3 for
8 hours/day, 5 days/week; Table 1-6). These findings suggest that animals can recover from
intermittent exposure to elevated ammonia levels (Coon etal.. 1970). although the evidence to
support this observation is limited.
Additionally, there is limited evidence of biochemical or metabolic effects of acute or short-
term ammonia exposure. Evidence of slight acidosis, as indicated by a decrease in blood pH, was
reported in rats exposed to 18 or 212 mg/m3 ammonia for 5 days; the study authors stated that
differences in pH leveled off at 10 and 15 days (Manninen etal.. 19881. In another study, blood pH
in rats was not affected by exposure to ammonia at concentrations up to 818 mg/m3 for up to
24 hours (Schaerdel etal.. 1983).
Encephalopathy related to ammonia may occur in humans following disruption of the
body's normal homeostatic regulation of the glutamine and urea cycles (e.g., due to severe liver
disease resulting in elevated ammonia levels in blood) fMinana etal.. 1995: Souba. 19871. 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 etal.. 1979: Sadasivudu and Radha Krishna
Murthv. 19781. It has been suggested that glutamate and y-amino butyric acid play a role in
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Toxicological Review of Ammonia
ammonia-induced neurotoxicity (Tones. 20021. There is no evidence, however, that ammonia is
neurotoxic in humans or animals following chronic inhalation exposure.
In the only study of the reproductive and developmental toxicity of ammonia, no changes in
reproductive or developmental endpoints were found between two groups of female pigs
(crossbred gilts) exposed to ammonia via inhalation for 6 weeks at mean concentrations of 5 or
25 mg/m3 and then mated fDiekman et al.. 19931. A control group without ammonia exposure was
not evaluated. Age at puberty did not differ significantly between the two groups. Gilts exposed to
25 mg/m3 ammonia weighed 7% less (p < 0.05) at puberty than those exposed to 5 mg/m3;
however, body weights of the two groups were similar at gestation day 30. Conception rates in the
mated females were similar between the two groups (94.1 versus 100% in 5- versus 25-mg/m3
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 with no ammonia exposure and possible confounding by
exposures to bacterial and mycoplasm pathogens.
The evidence of systemic toxicity in experimental animals exposed to ammonia is
summarized in Table 1-6 and displayed graphically as an exposure-response array in Figure 1-3.
Table 1-6. Evidence pertaining to other systemic effects in animals
Study reference and design
Results
Liver effects
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15/group
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0,155, or 770 mg/m3 for 8 hrs/d, 5 d/wk for 6 wks
No histopathologic changes observed.
Coon et al. (1970)
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
"Fatty changes of the liver plate cells" in several
animals of each species at 470 mg/m3.a
Coon et al. (1970)
Sprague-Dawley or Long-Evans rat; male and female;
15-51/group
0 or 40 mg/m3 for 114 d, 127, 262, or 470 mg/m3 for 90 d, or
455 mg/m3 for 65 d
"Fatty changes of the liver plate cells" in several
rats at 470 mg/m3, an exposure that was lethal
to the majority of the rats.3
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Toxicological Review of Ammonia
Study reference and design
Results
Anderson et al. (1964)
Swiss albino mouse; male and female; 4/exposure interval
0 or 20 ppm (0 or 14 mg/m3) for 7,14, 21, 28, or 42 d
No visible signs of liver toxicity.
Weatherbv (1952)
Guinea pig (strain not specified); male; 2 control and
4 exposed/exposure interval
0 or 170 ppm (0 or 120 mg/m3) for 6 hrs/d, 5 d/wk for 6, 12, or
18 wks
Congestion of the liver at 18 wks, not reported
at earlier times.3
Anderson et al. (1964)
Guinea pig (strain not specified); male and female; 2/exposure
interval at 20 ppm, 6/exposure interval at 50 ppm
0 or 20 ppm (0 or 14 mg/m3) for 7,14, 21, 28, or 42 d or 50 ppm
(35 mg/m3) for 42 d
Congestion of the liver at 35 mg/m3 for 42 d.a
Adrenal gland effects
Weatherbv (1952)
Guinea pig (strain not specified); male; 2 control and
4 exposed/exposure interval
0 and 170 ppm (0 and 120 mg/m3) 6 hrs/d, 5 d/wk for 6,12, or
18 wks
"Early" degenerative changes in the adrenal
gland (swelling of cells, degeneration of the
cytoplasm with loss of normal granular
structure) at 18 wks, not observed at earlier
times.3
Kidney and spleen effects
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15/group
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0,155, or 770 mg/m3 for 8 hrs/d, 5 d/wk for 6 wks
No histopathologic changes reported.
Coon et al. (1970)
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
Calcification and proliferation of renal tubular
epithelium at 470 mg/m3.a (This exposure was
lethal to ~25% of guinea pigs.)
Coon et al. (1970)
Sprague-Dawley or Long-Evans rat; male and female;
15-51/group
0 or 40 mg/m3 for 114 d, 127, 262, or 470 mg/m3 for 90 d, or
455 mg/m3 for 65 d
Calcification and proliferation of renal tubular
epithelium at 470 mg/m3, an exposure that was
lethal to the majority of the rats.3
Anderson et al. (1964)
Swiss albino mouse; male and female; 4/exposure interval
0 or 20 ppm (0 or 14 mg/m3) for 7,14, 21, 28, or 42 d
No visible signs of toxicity.
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Toxicological Review of Ammonia
Study reference and design
Results
Weatherbv (1952)
Guinea pig (strain not specified); male; 2 control and
4 exposed/exposure interval
0 or 170 ppm (0 or 120 mg/m3) 6 hrs/d, 5 d/wk for 6,12, or
18 wks
Congestion of the spleen and kidneys.3
Anderson et al. (1964)
Guinea pig (strain not specified); male and female; 2/exposure
interval at 20 ppm, 6/exposure interval at 50 ppm
0 or 20 ppm (0 or 14 mg/m3) for 7,14, 21, 28, or 42 d or 50 ppm
(35 mg/m3) for 42 d
Enlarged and congested spleens at 35 mg/m3.a
Myocardial effects
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15/group
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0,155, or 770 mg/m3 for 8 hrs/d, 5 d/wk for 6 wks
No histopathologic changes reported.
Coon et al. (1970)
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
Myocardial fibrosis at 470 mg/m3.a (This
exposure was lethal to ~25% of guinea pigs.)
Coon et al. (1970)
Sprague-Dawley or Long-Evans rat; male and female;
15-51/group
0 or 40 mg/m3 for 114 d, 127, 262, or 470 mg/m3 for 90 d, or
455 mg/m3 for 65 d
Myocardial fibrosis at 470 mg/m3, an exposure
that was lethal to the majority of the rats.3
Ocular effects
Coon et al. (1970)
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
No ocular irritation reported.
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15/group
New Zealand albino rabbit; male; 3/group
Princeton-derived guinea pig; male and female; 15/group
Squirrel monkey (S. sciureus); male; 3/group
Beagle dog; male; 2/group
0,155, or 770 mg/m3 for 8 hrs/d, 5 d/wk for 6 wks
No ocular irritation reported.
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Toxicological Review of Ammonia
Study reference and design
Results
Coon et al. (1970)
Sprague-Dawley and Long-Evans rat; male and female;
15-51/group
0 or 40 mg/m3 for 114 d, 127, 262, or 470 mg/m3 for 90 d, or
455 mg/m3 for 65 d
No ocular irritation reported.
Coon et al. (1970)
New Zealand albino rabbit; male; 3/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
Erythema, discharge, and ocular opacity over
Zi-Zi of cornea at 470 mg/m3.a
Coon et al. (1970)
Beagle dog; male; 2/group
0 or 40 mg/m3 for 114 d or 470 mg/m3 for 90 d
Heavy lacrimation at 470 mg/m3.a
Blood pH changes
Manninen et al. (1988)
Wistar rat; female; 5/group
0, 25 or 300 ppm (0,18, or 212 mg/m3) 6 hrs/d for 5,10 or 15 d
4/ blood pH at 5 days; pH differences "leveled
off at later time points (data not shown)".
Blood pH (day 5): 7.43, 7.34*, 7.36*
Schaerdel et al. (1983)
CrkCOBS CD(SD) rat; male; 8/group (blood oxygen partial
pressure [pCh] based on n = 5)
15, 32, 310, or 1,157 ppm (11, 23, 219, or 818 mg/m3) for
0 (control), 8,12, or 24 hrs
1" blood pC>2 at 11 and 23 mg/m3 at 8-, 12-, and
24-hr time points; no change at higher
concentrations; no change in blood pH.
Percent change in pC>2 from time 0 (at 24 hours
of exposure0-. 20*, 17*, 1, -2%
Amino acid levels and neurotransmitter metabolism in the brain
Manninen and Savolainen (1989)
Wistar rat; female; 5/group
0, 25, or 300 ppm (0,18, or 212 mg/m3) 6 hrs/d for 5 d
% change compared to control:c
Brain glutamine: 42*, 40*%
Manninen et al. (1988)
Wistar rat; female; 5/group
0, 25, or 300 ppm (0,18, or 212 mg/m3) 6 hrs/d for 5,10, or
15 d
% change compared to control at 212 mg/m3:c
Blood glutamine (5,10,15 d): 44*, 13,14%
Brain glutamine (5,10,15 d): 40*, 4, 2%
Reproductive and developmental effects
Diekman et al. (1993)
Crossbred gilt (female pig); 4.5 mo old; 40/group
7 ppm (5 mg/m3), range 4-12 ppm (3-8.5 mg/m3) or 35 ppm
(25 mg/m3), range 26-45 (18-32 mg/m3) for 6 wksd
No change in 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).
incidence data not provided.
bMeasurements at time zero were used as a control; the study did not include an unexposed control group.
cPercent change compared to control calculated as: (treated value - control value)/control value x 100.
dA control group was not included. Prior to exposure to ammonia, pigs were also exposed naturally in
conventional grower units to M. hyopneumoniae and P. multocida, which cause pneumonia and atrophic rhinitis,
respectively.
^Statistically significantly different from the control (p < 0.05).
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guinea pig, monkey, dog); Coon et al. (1970) *:
Fatty liver changes in plate cells (rat, rabbit,;
guinea pig, monkey, dog); Coon et al. (1970);
Fatty liver changes in plate cells (rat);:
Coon et al. (1970)
No liver effects (mouse);:
Anderson et al. (1964) :
Liver congestion (guinea pig);;
Weatherby (1952) *
Liver congestion (guinea pig);:
Anderson et al. (1964)
Early degenerative changes in the adrenal:
gland (guinea pig); Weatherby (1952) * :
No histopathological changes (rat, rabbit,
guinea pig, monkey, dog); Coon et al. (1970)!
Calcification & proliferation renal tubular epithelium
S (rabbit, guinea pig, monkey, dog); Coonetal. (1970):
=s
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tubularepithelium (rat); Coon etal. (1970):
"E.
{g No kidney or spleen effects (mouse);;
^ Anderson et al. (1964)
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Ł Congestion of the spleen & kidneys (guinea pig);:
Weatherby (1952) *
Enlarged & congested spleen (guinea pig);:
Anderson et al. (1964)
No histopathological changes (rat, rabbit,
guinea pig, monkey, dog); Coon et al. (1970) *
Myocardial fibrosis (rabbit, guinea pig,
monkey, dog); Coon et al. (1970)
Myocardial fibrosis (rat);
Coon et al. (1970)
No irritationobserved (guinea pig, monkey);
Coon et al. (1970)
No irritationobserved (rat, rabbit, guinea pig,
monkey, dog); Coon et al. (1970) *
No irritationobserved (rat);
Coon et al. (1970)
Erythema, discharge & corneal opacity (rabbit);
Coon et al. (1970)
Heavy lacrimation (dog);
Coon et al. (1970)
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-------
Toxicological Review of Ammonia
Summary of Other Systemic Effects
Effects of ammonia exposure on organs distal from the portal of entry (systemic effects) are
based on evidence in animals. Effects on various organs, including liver, kidney, spleen, and heart,
were observed in several studies that examined responses to ammonia exposure in a number of
laboratory animal 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. Evidence of ocular irritation in experimental animals was inconsistently
observed, and then only at high ammonia concentrations (470 mg/m3).
Studies of ammonia toxicity that examined other systemic effects were all published in the
older toxicological literature. Three subchronic inhalation studies were published between 1952
and 1970 fCoon etal.. 1970: Anderson et al.. 1964: Weatherbv. 19521. In general, the information
from these studies is limited by small group sizes, minimal characterization of reported
histopathological changes (e.g., "congestion," "enlarged," "fatty liver"), insufficiently detailed
reporting of study results, and incomplete, if any, incidence data. In addition, Weatherbv (1952).
Anderson et al. (1964). and some of the experiments reported by Coon etal. (1970) used only one
ammonia concentration in addition to the control, so no dose-response information is available
from the majority of experimental studies to inform the evidence for systemic effects of ammonia.
Finally, exposure characterization in Weatherbv f!9521 was considered poor.
Overall, there are suggestions in experimental animals that ammonia exposure may be
associated with effects on organs distal from the portal of entry, but there is inadequate
information to draw conclusions about the liver, kidney, spleen, or heart as sensitive targets of
ammonia toxicity.
Given the inadequacies of the available toxicology literature for other systemic effects, the
potential toxicity of inhaled ammonia at sites distal from the respiratory system was evaluated by
considering ammonia levels normally present in blood. As discussed in more detail in Appendix C,
Section C.1.2, ammonia is produced endogenously in all human and animal tissues during all
lifestages, including prenatal. In adults, the normal range of ammonia in venous blood is 0.1-
0.8 [ig/mL. Concentrations in fetal circulation are higher than maternal blood concentrations; two
studies reported that mean umbilical concentrations of ammonia in venous blood at delivery were
50% to 3-fold higher than mean concentrations in maternal blood, with umbilical concentrations
ranging from approximately 0.5 to 5 |J.g/mL flozwik etal.. 2005: DeSanto etal.. 19931. Human fetal
umbilical blood levels of ammonia at birth were not influenced by gestational age based on
deliveries ranging from gestation week 25 to 43 (DeSanto etal.. 1993).
At external concentrations that do not measurably change normal (baseline) levels of
ammonia, the likelihood is low that exposures would pose a hazard for systemic effects. In rats,
exposure to ammonia concentrations <18 mg/m3 did not produce a statistically significant change
in blood or brain ammonia concentrations fManninen et al.. 1988: Schaerdel etal.. 1983). Higher
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Toxicological Review of Ammonia
external ammonia concentrations (>212 mg/m3) were associated with elevated blood ammonia
levels, but even at these relatively high concentrations, experimental findings in rats indicate that
compensation readily occurs fManninen etal.. 19881. In a 24-hour exposure duration study, blood
ammonia concentrations at 12 hours of exposure to >219 mg/m3 ammonia in air were lower than
at 8 hours; in a second 15-day exposure duration study, blood ammonia concentrations that were
elevated on day 5 of exposure to 212 mg/m3 ammonia in air were not significantly different from
control values on days 10 and 15 of exposure (Schaerdel etal.. 19831. See Appendix C,
Section C.1.3, Metabolism/Endogenous Production of Ammonia, for a more detailed summary of the
available literature that describes the relationship between environmental ammonia
concentrations and blood ammonia levels. Therefore, the available experimental data suggest that
any changes in blood ammonia at external concentrations <18 mg/m3 would be small relative to
levels normally present in blood. The potential for systemic effects (i.e., on tissues/organs distal
from the respiratory system), including reproductive and developmental effects, at these
concentrations cannot be ruled out, but the likelihood of such effects is considered small.
Because the health effects literature identified the respiratory system as the primary target
of ammonia toxicity, EPA also considered the possibility that point-of-contact effects could translate
into effects on tissues or organs distal from the respiratory system. EPA is not aware of any
mechanisms by which point-of-contact effects could directly or indirectly impact distal tissues or
organs.
1.3. SUMMARY AND EVALUATION
1.3.1. Weight of Evidence for Effects Other than Cancer
The respiratory system is the primary and most sensitive target of inhaled ammonia toxicity
in humans and experimental animals. Evidence for respiratory system toxicity in humans comes
from cross-sectional occupational studies in industrial settings that reported changes in lung
function and an increased prevalence of respiratory symptoms. The findings of respiratory effects
in workers exposed to ammonia as a disinfectant or cleaning product (primarily studies of asthma
or asthma symptoms), studies in agricultural settings (primarily lung function studies), controlled
human exposure studies, and case reports of injury following acute exposure provide additional
evidence that the respiratory system is a target of inhaled ammonia. Short-term and subchronic
animal studies show respiratory effects in several animal species across different dose regimens.
Thus, the weight of evidence of observed respiratory effects observed across multiple human and
animal studies identifies respiratory system effects as a hazard from ammonia exposure.
Evidence for an association between inhaled ammonia exposure and effects on other organ
systems distal from the portal of entry is less compelling than for the respiratory system. Overall,
there are suggestions in experimental animals that ammonia exposure may be associated with
effects on the liver, kidney, spleen, or heart, but the available information is inadequate to draw
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Toxicological Review of Ammonia
conclusions. The two epidemiological studies that addressed immunological function are
confounded by exposures to a number of other respirable agents that have been demonstrated to
be immunostimulatory and provide little support for ammonia immunotoxicity. Animal studies
provide consistent evidence of elevated bacterial growth following ammonia exposure. It is
unclear, however, whether elevated bacterial colonization is the result of suppressed immunity or
damage to the barrier provided by the mucosal epithelium of the respiratory tract. Overall, the
weight of evidence does not support the immune system as a target of ammonia toxicity.
Studies of the potential reproductive or developmental toxicity of ammonia in humans are
not available. Reproductive effects were not associated with inhaled ammonia in the only animal
study that examined the reproductive effects of ammonia (i.e., a limited-design inhalation study in
the pig). As discussed in Section 1.1.2, ammonia is produced endogenously in human and animal
tissues during all lifestages, including prenatal. Although the potential for effects on reproduction
and the developing fetus cannot be ruled out at external concentrations that do not alter normal
blood or tissue ammonia levels, there is no evidence that raises concerns for the developing fetus or
reproduction or to other distal tissues/organs.
1.3.2. Susceptible Populations and Lifestages
Studies of the toxicity of ammonia in children or young animals that would support an
evaluation of childhood susceptibility are limited. Casas etal. f20131 found evidence of airway
inflammation (as indicated by increased exhaled nitric oxide) and decreased lung function in
school-age children exposed to cleaning products. No studies were identified that would support
the comparison of response to ammonia at different lifestages.
Infants are exposed to ammonia in human breast milk. Ammonia in human breast milk is
present as one of the sources of nonprotein nitrogen, with approximately 1% of the nitrogen
derived from nonprotein nitrogen sources coming from ammonia f Atkinson etal.. 19801. No
evidence was identified that inhaled ammonia would influence the ammonia nitrogen content of
breast milk.
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. Loftus etal. (2015)
reported no increase in asthma symptoms and medication use in asthmatic children living near
animal feeding operations; however, ammonia exposure was associated with lower FEVi.
Controlled human exposure studies that examined both healthy adult volunteers and volunteers
with asthma fPetrova etal.. 2008: Sigurdarson et al.. 20041 did not demonstrate greater respiratory
sensitivity in asthmatics than healthy volunteers after acute exposure to ammonia. Under longer-
term exposure conditions, however, as seen among livestock farmers, one study observed
associations between ammonia exposure and decreased lung function among workers with chronic
respiratory symptoms, but not among the asymptomatic workers (Preller etal.. 1995). Additional
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Toxicological Review of Ammonia
research focusing on the question of susceptibility and variability in response to ammonia exposure
in these populations is needed.
Individuals with disease conditions that lead to hyperammonemia, a condition of elevated
levels of circulating ammonia, may be more susceptible to the effects of ammonia from external
sources. Hyperammonemia can occur in individuals with severe diseases of the liver (e.g.,
cirrhosis) or kidney, organs that biotransform and excrete ammonia, urea cycle disorders, and
other conditions such as fatty acid oxidation defects and Reye syndrome (Biirki etal.. 2015: Auron
and Brophv. 2012: Romero-Gomez etal.. 2004: Cordoba etal.. 1998: Davies etal.. 1997: Schubiger
etal.. 1991: Gilbert. 1988: leffers etal.. 1988: Souba. 19871. Elevated ammonia levels can
predispose an individual to encephalopathy as a result of the ability of ammonia to cross the blood-
brain barrier and subsequent disturbances in amino acid synthesis and alterations in
neurotransmission systems. Neonates and infants are particularly susceptible to the neurological
effects of elevated levels of ammonia; hyperammonemia can cause irreparable damage to the
developing brain (Auron and Brophv. 2012: Mi nana etal.. 1995: Souba. 19871. While patients with
hyperammonemia could plausibly be considered a susceptible population, there are no studies that
specifically support this hypothesized susceptibility.
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Toxicological Review of Ammonia
2. DOSE-RESPONSE ANALYSIS
2.1. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The reference concentration (RfC; expressed in units of mg/m3) 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 no-observed-adverse-effect
level (NOAEL), lowest-observed-adverse-effect level (LOAEL), or the 95% lower bound on the
benchmark concentration (BMCL), with uncertainty factors (UFs) generally applied to these points
of departure (PODs) to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis
As discussed in Section 1.2, the respiratory system is the primary and most sensitive target
of inhaled ammonia in humans and experimental animals, and respiratory effects have been
identified as a hazard following inhalation exposure to ammonia. The experimental toxicology
literature for ammonia provides evidence that inhaled ammonia may be associated with toxicity to
target organs other than the respiratory system, including the liver, kidney, spleen, heart, and
immune system. Effects in these other (nonrespiratory) target organs were not considered as the
basis for RfC derivation because the evidence for these associations is weak relative to that for
respiratory effects.
Respiratory effects, characterized as increased prevalence of respiratory symptoms or
decreased lung function, have been observed in worker populations exposed to ammonia
concentrations >18.5 mg/m3 fRahman etal.. 2007: Ali etal.. 2001: Ballal etal.. 19981. Decrements
in lung function parameters and increased prevalence of respiratory symptoms, such as wheezing
chest tightness, and cough/phlegm, have been identified as adverse respiratory health effects by
the American Thoracic Society (ATS. 2000) and are similarly noted as adverse in the Environmental
Protection Agency (EPA) Methods for Derivation of Inhalation Reference Concentrations and
Application of Inhalation Dosimetry (U.S. EPA. 1994). At the population level, ATS (2000) stated that
"any detectable level of permanent pulmonary function loss attributable to air pollution exposure
should be considered as adverse" and that:
"It should be emphasized that a small but significant reduction in a population
mean FEVi or FEV0.75 is probably medically significant, as such a difference may
indicate an increase in the number of persons with respiratory impairment in the
population. In other words, a small part of the population may manifest a marked
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Toxicological Review of Ammonia
change that is medically significant to them, but when diluted with the rest of the
population the change appears to be small fATS. 20001."
Thus, even a small change in the average (mean) of a distribution of pulmonary function
parameters is considered adverse for purposes of deriving an RfC.
In general, human data are preferred over animal data for deriving reference values
because these data are more relevant for assessing human health effects than animal studies and
avoid the uncertainty associated with interspecies extrapolation when animal data serve as the
basis for the RfC. In the case of ammonia, the available occupational studies provide adequate data
for the quantitative analysis of health outcomes considered relevant to potential general population
exposures. Respiratory effects have also been observed in animals, but at ammonia concentrations
higher than those associated with respiratory effects in humans and in studies involving exposure
durations (up to 114 days) shorter than those in occupational studies (Section 1.2.1). Therefore,
data on respiratory effects in humans were used for the derivation of the RfC and respiratory
effects in animals were not further considered.
Of the available human data, associations between ammonia exposure and respiratory
effects have been examined in epidemiology studies of industrial worker populations (Table 1-2), in
studies of ammonia exposure in a cleaning setting (Table 1-3), and in studies of populations in
agricultural settings. Studies using ammonia as a cleaning product provide evidence of an
association between ammonia exposure and an increased risk of asthma; however, these studies
did not measure ammonia concentrations and are thus not useful for dose-response analysis.
Studies in agricultural settings also support an association between ammonia exposure and
decreased pulmonary function; however, because of co-exposures to other agents (including dust,
endotoxin, mold, and disinfectant products) and the availability of studies with fewer co-exposures,
studies in agricultural settings were considered to be supportive of the association between
ammonia exposure and respiratory effects, but were not carried forward for dose-response
analysis. In addition, several controlled-exposure studies in volunteers evaluated the effects of
ammonia on irritation and lung function following acute exposures. These human exposure studies
have several methodological strengths compared to epidemiological studies of worker populations,
including well-characterized exposures and resistance to confounding; however, the short exposure
durations used in these studies (i.e., 15 seconds to 6 hours) make them inappropriate for evaluating
the effects of chronic exposure to ammonia.
Of the available studies of ammonia exposure in industrial settings, four cross-sectional
epidemiology studies of industrial worker populations—three studies in urea fertilizer plants by
Rahman etal. (2007). Ballal etal. (1998). and Ali etal. (2001). and a study in a soda ash plant by
Holness etal. f 19891—provide information useful for examining the relationship between chronic
ammonia exposure and increased prevalence of respiratory symptoms and/or decreased lung
function. Bhat and Ramaswamv (1993) evaluated lung function in ammonia plant workers, but did
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Toxicological Review of Ammonia
not measure ammonia concentrations in workplace air. Therefore, this study was not considered
useful for RfC derivation.
In general, these four cross-sectional occupational studies provide a coherent set of
estimated NOAELs and effect levels, and are considered candidate principal studies for RfC
derivation. A brief description of these studies and the contribution of each to the understanding of
the dose-response relationship between ammonia exposure and respiratory effects follow. More
study details are provided in the Supplemental Information, Section C.2.1 and in Table 1-2, and
evaluation of the strengths and limitations are more fully considered in the Literature Search
Strategy | Study Selection and Evaluation section.
• Rahman etal. (2007) observed an increased prevalence of respiratory symptoms (coughing,
chest tightness) in urea fertilizer plant workers (mean employment duration: 16 years)
exposed to a mean ammonia concentration of 18.5 mg/m3 (range: 9-31 mg/m3), but not in
workers in a second plant exposed to a mean ammonia concentration of 4.9 mg/m3 (range:
2-8 mg/m3). Decrements in lung function (forced vital capacity [FVC] and forced expiratory
volume in 1 second [FEVi]) between pre- and post-shift in the high-exposure group (2-3%)
were statistically significant Exposure was measured by personal samples using two
different analytical methods.
• Ballal etal. (1998) observed an increased prevalence of respiratory symptoms (cough,
phlegm, wheezing, and dyspnea) among urea fertilizer factory workers (mean employment
duration: 4.3 years) in one factory (Factory A) with ammonia exposures ranging from 2 to
27.1 mg/m3,11 but no increase in symptoms in another factory (Factory B) with exposures
ranging from 0.02 to 7 mg/m3. Lung function was not measured.
• A companion study by Ali etal. (2001) examined lung function among workers in Factory A
from Ballal etal. (1998): respiratory symptoms were not evaluated. Workers with
cumulative exposure >50 mg/m3-years had significantly lower lung function values
(declines of 5-7% in FVC% predicted and FEVi% predicted) than workers with cumulative
exposure <50 mg/m3-years. In this and the Ballal etal. f 199 81 study, exposure was
measured by air monitors.
• Holness etal. (1989) found no differences in the prevalence of respiratory symptoms or
lung function between soda ash plant workers (mean exposure: 6.5 mg/m3; mean exposure
duration: 12.2 years) and the control group, and also no differences in respiratory
symptoms or lung function when workers were stratified by ammonia exposure level
(lowest exposure group, <4.4 mg/m3; middle exposure group, 4.4-8.8 mg/m3; highest
exposure group, >8.8 mg/m3). Exposure was measured by personal samples. EPA
identified the concentration range for the high-exposure group (i.e., >8.8 mg/m3) as the
NOAEL from this study. The authors stated that 3 of the 12 workers in the high-exposure
group were exposed to concentrations >17.7 mg/m3; therefore, the majority of workers in
"This concentration range does not include exposures in the urea store (number of employees = 6; range of
ammonia concentrations = 90-130.4 mg/m3) because employees in this area were required to wear full
protective clothing, thus minimizing potential exposure.
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Toxicological Review of Ammonia
the high-exposure group (9 of 12) would have been exposed to ammonia concentrations in
the range of 8.8-17.7 mg/m3.
In selecting the principal study for RfC derivation, consideration was given to exposure
measures, assessment of outcomes, potential for co-exposures, and the value of the NOAEL. Of the
four candidate principal studies, higher confidence was associated with the exposure measures
from Holness etal. (1989). Both Holness etal. (1989) and Rahman etal. (2007) collected personal
air samples, but confidence in the analytical method used by Holness etal. (1989) is higher than
that used by Rahman etal. (2007). Rahman etal. (2007) used two analytical methods for
measuring ammonia concentrations in workplace air (i.e., Drager PAC III and Drager tube);
concentrations measured by the two methods differed by 4-5-fold, indicating some uncertainty
across the two measurement methods, although ammonia concentrations measured by the two
methods were strongly correlated (correlation coefficient of 0.8). In contrast, the Holness et al.
(1989) study used an established analytical method for measuring exposure to ammonia
recommended by the National Institute for Occupational Safety and Health (NIOSH) that involved
the collection of air samples on acid-treated silica gel absorption tubes. Ballal etal. (1998) used
area monitors rather than personal air sampling methods; the latter method provides a better
estimate of an individual's exposure.
As discussed in the Literature Search Strategy | Study Selection and Evaluation section,
assessment of respiratory symptoms in all studies that measured this outcome was based on self-
reporting by questionnaire, and assessment of lung function was performed using standard
spirometry protocols. While considered unlikely, nonblinded outcome assessments of respiratory
symptoms could introduce bias. Therefore, both Holness etal. (1989) and Rahman et al. (2007). the
two studies of industrial populations that examined both respiratory symptoms and lung function,
provide stronger evidence of respiratory effects than studies that evaluated symptom data only
(notably Ballal etal. (1998)).
Also as discussed in the Literature Search Strategy | Study Selection and Evaluation section,
confounding by other workplace exposures is a potential concern, although not likely to be a major
limitation of the studies considered for dose-response analysis. Only Rahman etal. (2007)
measured another workplace chemical (nitrogen dioxide; below detection limits); other studies did
not describe potential co-exposures. Therefore, a more rigorous examination of the potential for
confounding by co-exposure to other workplace chemicals could not be performed. Holness et al.
(1989) noted the high level of control of exposures in the facility used in their study, resulting in
low ammonia levels.
Three of the four occupational studies supported the identification of a NOAEL (or, more
correctly, an exposure range not associated with an increase in respiratory effects). Rahman et al.
(2007) did not observe a change in respiratory effects in workers exposed to a mean ammonia
concentration of 4.9 mg/m3 (range: 2-8 mg/m3). Holness etal. T19891 found no differences in
respiratory effects in soda ash plant workers when compared to a control group or when workers
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Toxicological Review of Ammonia
were stratified by exposure level (low, medium, and high); the concentration range for the high-
exposure group (i.e., >8.8 mg/m3) was identified as the NOAEL. Ballal etal. (1998) reported no
increase in respiratory symptoms in a factory with exposures ranging from 0.02 to 7 mg/m3.
Because Ali etal. (2001). the companion study to Ballal etal. (1998). evaluated only workers in a
second factory with higher exposures, study findings did not support the identification of an
estimated NOAEL.
In light of the above considerations, overall confidence in the Holness etal. (1989) study as
the principal study for RfC derivation was higher than other candidate studies in terms of:
measurement of ammonia exposure, evaluation of both respiratory symptoms and lung function
parameters, smaller potential for co-exposures to other workplace chemicals, and the fact that the
estimated NOAEL for respiratory effects of >8.8 mg/m3 was the highest of the NOAELs estimated
from the candidate principal studies. The Holness etal. T19891 study does not demonstrate a
relationship between ammonia exposure and respiratory effects. The relationship between
ammonia exposure and respiratory effects is based on the body of evidence, and the Holness etal.
(1989) study is identified as the principal study for derivation of the RfC for the reasons given
above.
In summary, the occupational study of ammonia exposure in workers in a soda ash plant by
Holness etal. (1989) was identified as the principal study for RfC derivation, with support
from Rahman etal. (2007). Ballal etal. (1998). and Ali etal. (2001). and respiratory effects
were identified as the critical effect.
2.1.2. Methods of Analysis
A NOAEL of 13.6 mg/m3, or an estimate of the lower confidence bound of the mean
exposure concentration in the high-exposure group of the Holness etal. fl9891 study, was
used as the POD for RfC derivation. The POD for respiratory effects was based on the NOAEL
representing the high-exposure group in Holness etal. Q989I The individual subject data from
this study were no longer available (call from S. Rieth, EPA, to C. Clayton, administrative assistant to
Dr. Holness, St. Michael's Hospital, Center for Research Expertise in Occupational Health, Toronto,
Canada, February 11, 2015), so that the mean exposure in the high-exposure group could not be
calculated precisely based on the data. Therefore, the mean was estimated assuming that the data
in the study followed a skewed probability distribution, specifically the lognormal distribution. The
frequency distribution provided in Holness etal. T19891 fsee Table 2-1) was used to estimate the
parameters (log-scale mean and standard deviation) of the lognormal distribution that best fit the
data.
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Table 2-1. Frequency distribution of ammonia exposure from Holness et al.
fl989)
Exposure group
Interval of exposures
(mg/m3)
Interval of exposures
(ppm)
Number of exposed
workers
Low
0-4.4
0-6.25
34
Medium
4.4-8.8
6.25-12.5
12
High3
8.8-17.7
12.5-25
9
>17.7
>25
3
aEPA divided the high-exposure group into two subgroups based on the statement in Holness et al. (1989): "Three
workers were exposed to TWA concentrations of ammonia in excess of 25 ppm, the current exposure guideline."
TWA = time-weighted average
Lognormal parameter estimates were obtained by applying the maximum likelihood
method to this frequency distribution. Using the estimated distribution defined by these parameter
estimates, the estimated mean exposure in the high-exposure group and 95% lower confidence
bound on this mean were calculated as follows (see Appendix C, Section C.4 for detailed
documentation of this calculation):
mean exposure estimate (high-exposure group) = 17.9 mg/m3
95% lower confidence bound on this mean (high-exposure group) = 13.6 mg/m3
The lower confidence bound of 13.6 mg/m3 was used as the POD for respiratory effects.
Because the RfC assumes continuous human exposure over a lifetime, the POD was adjusted
to account for the noncontinuous exposure associated with occupational exposure (i.e., 8-hour
workday and 5-day workweek). Cross-shift data for FVC and FEVi from the Rahman et al. f20071
study provide some evidence of an immediate effect of ammonia exposure on lung function12, which
could argue against adjustment from noncontinuous to continuous exposure; however, Rahman et
al. (2007) also reported that duration of exposure (using years of employment as a proxy for
exposure duration) was significantly associated with percentage cross-shift decrease in FEVi%. In
addition, Ballal etal. (1998) found a significant correlation between respiratory symptoms (cough,
phlegm, and wheezing) and duration of service (a proxy for exposure duration). In the absence of
clear evidence that respiratory effects in occupationally-exposed populations are an acute
response, and given evidence for contributions of exposure duration (cumulative exposure) to the
12Rahman et al. (2007) reported that mean pre-shift FVC and FEVi values in ammonia and urea plants
workers were similar, suggesting similar lung function in low- and high-exposure workers upon arrival at
work. Cross-shift changes in FVC and FEVi were statistically significant decreased in the urea plant (more
highly-exposed) workers only.
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respiratory effects of ammonia, the standard adjustment to continuous exposure was applied. The
duration-adjusted POD was calculated as follows:
NOAELadj = NOAEL x VEho/VEh x 5 days/7 days
= 13.6 mg/m3 x 10 m3/20 m3 x 5 days/7 days
= 4.9 mg/m3 or 5 mg/m3 (rounded)
Where:
VEho = human occupational default minute volume (10 m3 breathed during an 8-hour
workday) (U.S. EPA. 1994). This inhalation rate corresponds to more current
inhalation rates for light to moderate activity levels from U.S. EPA (2009c). as cited
in U.S. EPA (2011a). An occupational inhalation rate of 10.8 m3 for an 8-hour
workday, similar to the default value from U.S. EPA (1994). can be derived as an
average of activity-specific inhalation rates for males, in age groups of 21-60 years,
for combined light and moderate activity from Table 6-17 of U.S. EPA (2011a). The
average inhalation rate of 1.3 m3/hour (0.022 m3/minute) can be multiplied by
8 hours to obtain an inhalation rate of 10.8 m3/8-hour workday.
VEh = human ambient default minute volume (20 m3 breathed during the entire day)
fU.S. EPA. 19941. This value is consistent with the average of the daily average
inhalation rates for males, in age groups of 21-60 years, of 20.2 m3/day, from U.S.
EPA (2009c). as summarized in Table 6-14 of U.S. EPA (2011a).
2.1.3. Derivation of the Reference Concentration
Consistent with EPA's A Review of the Reference Dose and Reference Concentration Processes
(U.S. EPA. 2002: Section 4.4.5). also described in the Preamble, five possible areas of uncertainty
and variability were considered when deriving the RfC. A composite UF of 10 was applied to the
selected duration-adjusted POD of 4.9 mg/m3 to derive the RfC of 0.5 mg/m3. An explanation of the
five possible areas of uncertainty and variability follows:
• 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 fHolness etal.. 1989). defined as the mean number of
years at the present job for exposed workers, of approximately 12 years was considered to
be of chronic duration;
• An uncertainty factor for extrapolation from a LOAEL to a NOAEL, UFl, of 1 was applied
because a NOAEL was used as the POD; and
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Toxicological Review of Ammonia
• A database uncertainty factor, UFd, of 1 was applied to account for deficiencies in the
database. As discussed in Section 1.2, available epidemiological studies include studies of
workers exposed in industrial settings, in agriculture, or through use of cleaning products.
There are also controlled human exposure studies involving short-duration exposure to
ammonia vapors and many case reports of acute exposures to high concentrations.
Available animal studies include subchronic studies that investigated respiratory and
systemic effects in rats, guinea pigs, and pigs. There are also several immunotoxicity
studies and one limited reproductive toxicity study in young female pigs. The database
lacks developmental and multigenerational reproductive toxicity studies. The EPA's review
of RfD and RfC processes (U.S. EPA. 20021 states that "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." U.S. EPA ("20021 also states,
"If data from the available toxicology studies raise suspicions of
developmental toxicity and signal the need for developmental data on
specific organ systems (e.g., detailed nervous system, immune system,
carcinogenesis, or endocrine system), then the database factor should take
into account whether or not these data are available and used in the
assessment and their potential to affect the POD ..."
Although the database lacks developmental and multigene ration reproductive toxicity
studies, these studies are not expected to impact the determination of ammonia toxicity at
the POD. Accordingly, a database UF to account for the lack of these studies is not
considered necessary. This determination is based on the observation that ammonia is
produced endogenously in all human and animal tissues during all lifestages, including the
prenatal period. Ammonia produced within the uteroplacenta is released into the fetal and
maternal circulations (see Appendix C, Section C.1.2). As discussed in Section 1.2.3,
concentrations in fetal circulation are higher than maternal blood concentrations, by as
much as 50% to 3-fold (lozwik etal.. 2005: DeSanto etal.. 19931. Further, human fetal
umbilical blood levels of ammonia at birth were not influenced by gestational age based on
deliveries ranging from gestation week 25 to 43 (DeSanto etal.. 19931. This evidence
provides some assurance that endogenous ammonia concentrations in the fetus are similar
to other lifestages, and that baseline ammonia concentrations would not be associated with
developmental toxicity. Additionally, evidence in animals fManninen et al.. 1988: Schaerdel
etal.. 19831 suggests that exposures to ammonia at concentrations up to 18 mg/m3 do not
produce a statistically significant change in blood or brain ammonia concentrations (see
Section 1.2.3 and Appendix C, Section C.1.3). Accordingly, any changes in blood ammonia
concentrations at the duration-adjusted POD (3.1 mg/m3) would be small relative to levels
normally present in blood. The likelihood of developmental or reproductive effects at these
concentrations, while they cannot be ruled out, is considered small.
The RfC for ammonia was calculated as follows:
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Toxicological Review of Ammonia
RfC = NOAELadj - UF
= 4.9 mg/m3 4- 10
= 0.49 mg/m3 or 0.5 mg/m3 (rounded to one significant figure)
2.1.4. Uncertainties in the Derivation of the Reference Concentration
As presented earlier in this section and in the Preamble, EPA standard practices and RfC
guidance (U.S. EPA. 2002.1995.19941 were followed in applying a UF approach to a POD (from a
NOAEL) 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 benchmark dose 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 increases in the
prevalence of respiratory symptoms 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 etal. (1989) study was
used to estimate the POD. The availability of dose-response data from a study of ammonia,
especially in humans, would increase the confidence in the estimation of the POD.
Comparison of Exhaled Ammonia to the RfC
Ammonia is generated endogenously in multiple organs, including the liver, kidneys,
intestines, brain, and skeletal muscle, as a product of amino acid catabolism. Ammonia plays
central roles in nitrogen balance and acid-base homeostasis fWeiner etal.. 2014: Weiner and
Verlander. 2013). Given its important metabolic role, free ammonia is homeostatically regulated to
remain at low concentrations in blood (Souba. 1987). Elimination of ammonia occurs primarily in
urine and exhaled breath. (See Appendix C, Section C.1.3 for additional information on production
and regulation of endogenous ammonia.)
Further consideration was given to the presence of ammonia in exhaled air because the
range of ammonia concentrations in exhaled breath overlaps the ammonia RfC. Specifically,
ammonia has been measured in exhaled breath at concentrations ranging from 0.009 to 2 mg/m3
(see Appendix C, Table C-l), a range that exceeds the RfC of 0.5 mg/m3. This section reviews
information related to the exhalation of ammonia that provides context for this comparison.
In general, higher and more variable ammonia concentrations are reported in human breath
exhaled from the mouth or oral cavity. Investigators reported concentrations ranging from 0.03 to
2 mg/m3, with the majority of concentrations >0.2 mg/m3 fSchmidt et al.. 2 013: Smith etal.. 2008:
Spanel etal.. 2007a. b; Turner etal.. 2006: Diskin etal.. 2003: Smith etal.. 1999: Norwood etal..
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Toxicological Review of Ammonia
1992: Larson etal.. 19771. Ammonia concentrations measured in breath derived from oral
breathing largely reflect the production of ammonia via bacterial degradation of food protein in the
oral cavity or gastrointestinal tract fTurner etal.. 2006: Smith etal.. 1999: Vollmuth and
Schlesinger. 19841. Ammonia concentrations from exhaled breath can be influenced by factors such
as diet, oral hygiene, and age fSolga etal.. 2013: Spanel etal.. 2007a. b; Turner etal.. 2006: Diskin et
al.. 2003: Norwood et al.. 19921. Schmidt etal. T20131 reported that ammonia concentrations in
breath from the mouth strongly depended on saliva pH.
Concentrations of ammonia in breath exhaled from the nose and trachea of humans
(0.0092-0.1 mg/m3) are lower than those in air exhaled from the mouth f Schmidt etal.. 2013:
Smith etal.. 2008: Larson etal.. 19771. Whereas the upper end of the range of ammonia
concentrations in mouth breath exceeds the RfC of 0.5 mg/m3, concentrations from the nose and
trachea are generally lower than the ammonia RfC by a factor of five or more. Ammonia
concentrations in breath exhaled from the nose appear to better represent levels at the alveolar
interface of the lung and are thought to be more relevant to understanding systemic levels of
ammonia than breath exhaled from the mouth (Schmidt etal.. 2013: Smith etal.. 20081.
Nevertheless, the relationship between nose ammonia concentrations and systemic levels is
complicated by the possibility that nose ammonia concentrations are still influenced by the oral
cavity (e.g., in individuals with the soft palate incompletely closed) and tracheobronchial fluids that,
like saliva, can influence the airway concentration of ammonia. Further, measurements of exhaled
ammonia reported in the literature were generally not conducted in ammonia-free environments,
and the ammonia in inhaled air may thus account for some of the ammonia measured in exhaled air
(e.g., see Spanel etal. (201311.
Thus, ammonia concentrations in exhaled breath, and particularly those exhaled through
the mouth, are not correlated with blood ammonia; factors identified as influencing exhaled
ammonia concentrations include bacterial populations in the oral cavity, salivary pH, diet, oral
hygiene, and age (see Appendix C, Section C.1.4). Concentration in breath cannot be used to predict
blood ammonia concentration or previous exposure to environmental (ambient) concentrations of
ammonia.
Regardless, the level of ammonia in breath, even at concentrations that exceed the RfC, does
not necessarily raise questions about the appropriateness of the RfC. The exhalation of ammonia is
a clearance mechanism for a product of metabolism that is otherwise toxic in the body at
sufficiently high concentrations. Ammonia concentrations in exhaled breath may be higher than
inhaled concentrations, particularly when compared to exhaled air from the mouth or oral cavity.
However, the fact that humans may exhale ammonia at concentrations higher than 0.5 mg/m3
(i.e., the RfC) is not considered an uncertainty in the RfC.
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Consideration of Tolerance and the Healthy Worker Effect on Selection of the POD
As discussed in Section 1.2.1, two controlled-exposure studies provide some evidence of
habituation to eye, nose, and throat irritation in volunteers after repeated ammonia exposure.
Following exposure to ammonia at concentrations ranging from 7 to 35 mg/m3 for 4 hours/day on
5 consecutive days, Ihrigetal. (20061 reported higher mean intensities for irritative, olfactory, and
respiratory symptoms in male volunteers unfamiliar with ammonia when compared to male
chemical company workers exposed to ammonia vapor for several years in a urea department;
differences were statistically significant only for olfactory symptoms. In a more limited study with
only four male volunteers each exposed to 18, 35, or 71 mg/m3 ammonia (exposure to each
concentration was for 1 week, 2-6 hours/day, 5 days/week; individuals were exposed to each
concentration twice), fewer occurrences of irritation were reported during week 2 than during
week 1 at the same exposure concentration (Ferguson etal.. 19771. However, in the same Ferguson
etal. (1977) study, the occurrences of irritation in two individuals exposed to 50 ppm for
6 hours/day, 5 days/week for 6 weeks were variable from week to week and did not show any clear
trend. The study by Ihrigetal. (20061. and to a lesser extent the study by Ferguson etal. (19771.
provide some evidence of decreased irritation following repeated exposure; the results of Ihriget
al. f 2 0 0 61 may also be influenced by attrition out of the workforce of those most affected by the
irritation symptoms. These studies raise the possibility that repeated exposure could lead to the
development of tolerance to ammonia (i.e., to decreased sensory responsiveness). It is possible,
therefore, that industrially-exposed populations considered in deriving the RfC for ammonia (i.e.,
Holness et al. (19891. Rahman etal. (20071. Ballal etal. (19981. and Ali etal. (200111 may have
developed some degree of tolerance to ammonia, and may underpredict responses to ammonia that
would be observed in the general population. The magnitude of tolerance, if any, cannot be
estimated from the available studies.
In addition, as discussed in the Literature Search Strategy | Study Selection and Evaluation
section, the workers in the cross-sectional occupational studies used to derive the RfC were healthy
enough to remain in the plant for a considerable time; mean employment duration ranged from
52 months to 18 years. In general, studies in these populations may result in a "healthy worker
survivor" bias and in an underestimate of the risk of health effects of ammonia exposure, as a
healthy worker population may not exhibit health effects (such as decreased lung function or
increased prevalence of respiratory symptoms) to the same degree that would be seen in the
general population under the same conditions.
Therefore, there is potential for tolerance development in populations exposed
occupationally to ammonia and "healthy worker" bias, both of which may result in underestimation
of the general population response. However, the evidence is limited and not conclusive, and thus,
does not warrant increasing the intraspecies uncertainty factor.
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2.1.5. 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.
19941. Confidence in the principal study fHolness etal.. 19891 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, the results from the principal study are supported by the results from other
cross-sectional studies of workers in industrial settings, studies of ammonia exposure in a cleaning
setting, studies in agricultural settings, multiple studies of acute ammonia exposure in volunteers,
and available inhalation data from animals.
Confidence in the database is medium. The ammonia database includes a number of
epidemiological studies of workers exposed in different occupational settings, and multiple repeat-
exposure experimental animal studies, the majority from the older toxicological literature. The
inhalation ammonia database includes one limited study of reproductive and developmental
toxicity in pigs that did not examine a complete set of reproductive or developmental endpoints.
Normally, confidence in a database lacking reproductive and developmental toxicity studies is
considered to be lower due to the uncertainty surrounding the use of any one or several studies to
adequately address all potential endpoints following chemical exposure at critical lifestages. Unless
a comprehensive array of endpoints is addressed by the database, there is uncertainty as to
whether the critical effect chosen for RfC derivation is the most sensitive or appropriate. However,
the likelihood of reproductive, developmental, and other systemic effects at the RfC is considered
small because it is well documented that ammonia is endogenously produced in humans and
animals, and any changes in blood ammonia levels at the POD would be small relative to normal
blood ammonia levels. Further, EPA is not aware of any mechanisms by which effects at the point
of contact (i.e., respiratory system) could directly or indirectly impact tissues or organs distal to the
point of contact Thus, confidence in the database, in the absence of these types of studies, is
medium.
Reflecting medium confidence in the principal study and medium confidence in the
database, the overall confidence in the RfC is medium.
2.1.6. Previous IRIS Assessment
The previous Integrated Risk Information System (IRIS) assessment for ammonia (posted to
the database in 1991) presented an RfC of 0.1 mg/m3 based on co-principal studies—the
occupational exposure study of workers in a soda ash plant by Holness etal. (1989) and the
subchronic study by Broderson etal. Q9761 that examined the effects of ammonia exposure in
F344 rats inoculated on day 7 of the study with the bacterium, Mycoplasma pulmonis. The NOAEL
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of 6.4 mg/m3 (estimated as the mean concentration of the entire exposed group) from the Holness
etal. (1989) study (duration adjusted: NOAELadj = 2.3 mg/m3) was used as the POD.13
The previous RfC was derived by dividing the exposure-adjusted POD of 2.3 mg/m3 (from a
NOAEL of 6.4 mg/m3) 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 fBroderson etal.. 19761 to the NOAEL,
and the lack of reproductive and developmental toxicity studies. A UFd of 3 (rather than 10) was
applied because studies in rats (Schaerdel etal.. 19831 showed no increase in blood ammonia levels
at an inhalation exposure up to 32 ppm (22.6 mg/m3) and only minimal increases at
300-1,000 ppm (212-707 mg/m3), suggesting that no significant distribution is likely to occur at
the human equivalent concentration.
13In this document, the lower confidence bound of the estimated mean exposure concentration in the high-
exposure group from the Holness etal. (19891 study (13.6 mg/m3, adjusted for continuous exposure to
4.9 mg/m3) was identified as the POD because workers in this high-exposure group, as well as those in the
two lower-exposure groups, showed no statistically significant increase in the prevalence of respiratory
symptoms or decreases in lung function.
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