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TOXICOLOGICAL REVIEW
OF
AMMONIA
(CAS No. 7664-41-7)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
October 2011
NOTICE
This document is an Interagency Science Consultation draft. This information is distributed
solely for the purpose of pre-dissemination peer review under applicable information quality
guidelines. It has not been formally disseminated by EPA. It does not represent and should not
be construed to represent any Agency determination or policy. It is being circulated for review
of its technical accuracy and science policy implications.
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and
should not be construed to represent any Agency determination or policy. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
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CONTENTS—TOXICOLOGICAL REVIEW OF AMMONIA (CAS No. 7664-41-7)
LIST OF TABLES v
LIST OF FIGURES vi
LIST OF ABBREVIATIONS AND ACRONYMS vii
FOREWORD ix
AUTHORS, CONTRIBUTORS, AND REVIEWERS x
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 5
3.1. ABSORPTION 5
3.1.1. Inhalation Exposure 5
3.1.2. Oral Exposure 6
3.1.3. Dermal Exposure 6
3.2. DISTRIBUTION 7
3.2.1. Inhalation Exposure 8
3.2.2. Oral Exposure 8
3.2.3. Dermal Exposure 8
3.3. METABOLISM 8
3.4. ELIMINATION 11
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS 13
4. HAZARD IDENTIFICATION 14
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 15
4.1.1. Case Reports - Oral and Inhalation Exposures 15
4.1.2. Controlled Human Inhalation Exposure Studies 15
4.1.3. Cross Sectional Studies in Farmers Exposed to Inhaled Ammonia 18
4.1.4. Occupational Studies in Industrial Worker Populations 19
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN
ANIMALS—ORAL AND INHALATION 27
4.2.1. Oral Exposure 27
4.2.2. Inhalation Exposure 31
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND
INHALATION 38
4.4. OTHER DURATION-OR ENDPOINT-SPECIFIC STUDIES 39
4.4.1. Acute Oral Studies 39
4.4.2. Acute and Short-Term Inhalation Studies 39
4.4.3. Immunotoxicity 40
4.5. MECHANISTIC STUDIES 46
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 46
4.6.1. Oral 46
4.6.2. Inhalation 47
4.6.3. Mode-of-Action Information 51
4.7. EVALUATION OF CARCINOGENICITY 51
4.7.1. Summary of Overall Weight-of-Evidence 51
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 51
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4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 51
4.8.1. Possible Childhood Susceptibility 51
4.8.2. Possible Gender Differences 52
4.8.3. Other Susceptible Populations 52
5. DOSE-RESPONSE ASSESSMENTS 53
5.1. ORAL REFERENCE DOSE (RfD) 53
5.1.1. Previous RfD Assessment 55
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 55
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 55
5.2.2. Methods of Analysis 60
5.2.3. RfC Derivation—Including Application of Uncertainty Factors (UFs) 60
5.2.4. Previous RfC Assessment 62
5.3. CANCER ASSESSMENT 63
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE 64
6.1. HUMAN HAZARD POTENTIAL 64
6.2. DOSE RESPONSE 65
6.2.1. Noncancer/Oral 65
6.2.2. Noncancer/Inhalation 65
6.2.3. Cancer 66
7. REFERENCES 67
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION A-l
APPENDIX B. SUMMARY OF REPEAT DOSE TOXICITY INFORMATION FOR
SELECTED AMMONIUM SALTS B-l
APPENDIX C. SUPPLEMENTAL INFORMATION ON AMMONIA C-l
C.I. AMMONIA LEVELS MEASURED IN EXPIRED AIR IN HUMANS C-l
C.2. HUMAN CASE STUDIES AND REPORTS OF HUMAN EXPOSURE TO
AMMONIA C-8
C.3. CONTROLLED HUMAN EXPOSURE STUDIES OF AMMONIA
INHALATION C-33
C.4. CROSS SECTIONAL STUDIES OF LIVESTOCK FARMERS EXPOSED TO
AMMONIA C-36
C.5. ACUTE AND SHORT-TERM INHALATION TOXICITY STUDIES OF
AMMONIA IN EXPERIMENTAL ANIMALS C-40
C.6. MECHANISTIC STUDIES C-48
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LIST OF TABLES
Table 2-1. Chemical and physical properties of ammonia
Table 4-1. Symptoms and lung function results of workers exposed to different levels of TWA
ammonia concentrations 21
Table 4-2. The prevalence of respiratory symptoms and disease in urea fertilizer workers
exposed to ammonia 22
Table 4-3. Logistic regression analysis of the relationship between ammonia concentration and
respiratory symptoms or disease in exposed urea fertilizer workers 22
Table 4-4. Prevalence of acute respiratory symptoms and cross-shift changes in lung function
among workers exposed to ammonia in a urea fertilizer factory 25
Table 4-5. Summary of significant changes in serum from workers occupationally exposed to
ammonia at a fertilizer plant 26
Table 4-6. Effect of ammonia in drinking water on the thickness of the gastric antral and body
mucosaoftherat stomach 27
Table 4-7. Effect of ammonia in drinking water on gastric antral and body mucosa in the
stomach of Sprague-Dawley rats administered 0.01% ammonia in drinking water 28
Table 4-8. Summary of histological changes observed in rats exposed to ammonia for 6 weeks34
Table 4-9. Incidence of pulmonary lesions in rats inoculated withM pulmonis and exposed to
ammonia (7 days later for 28-42 days) 41
Table 4-10. Mortality in P. multocida-infected mice exposed to ammonia for 8 or 168 hours .. 42
Table 4-11. Dermal response to the injection of tuberculin in animals exposed to ammonia for 3
weeks (mean diameter of redness in mm) 44
Table 4-12. Summary of noncancer results of human occupational studies and repeat-dose
studies in experimental animals involving inhalation exposure to ammonia 49
Table B-l. Summary of noncancer results of repeat dose studies of oral exposure of
experimental animals to selected ammonium salts B-l
Table C-l. Ammonia levels in exhaled breath of volunteers C-2
Table C-2. Human case studies and reports of human exposure to ammonia C-10
Table C-3. Controlled human exposure studies of ammonia inhalation C-33
Table C-4. Cross sectional studies of livestock farmers exposed to ammonia C-36
Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals C-41
Table C-6. In vivo genotoxicity studies of ammonia C-51
Table C-7. In vitro genotoxicity studies of ammonia C-52
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LIST OF FIGURES
Figure 3-1. Glutamine cycle in the liver 9
Figure 3-2. The urea cycle showing the compartmentalization of its steps within liver cells 10
Figure 5-1. Exposure-response array comparing workers occupationally exposed and human
volunteers acutely exposed to ammonia 57
Figure 5-2. Exposure-response array comparing noncancer effects in occupationally exposed
workers and experimental animals exposed to ammonia by inhalation 59
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LIST OF ABBREVIATIONS AND ACRONYMS
ACGIH
ALP
ALT
AMP
AST
ATSDR
ATSG
BMD
BrDU
BUN
C3
CAC
CASRN
CI
FEF
FEV
FVC
GABA
IgE
IgG
IRIS
LOAEL
MAO
MMEF
MNNG
MRM
NAD+
NADH
NH4+
NIOSH
NMDA
NOAEL
NOx
NRC
OR
PARP
PEF
PEFR
PHA
POD
PPD
PRRSV
RfC
RfD
SIFT-MS
American Conference of Governmental Industrial Hygienists
alkaline phosphatase
alanine aminotransferase
adenosine monophosphate
aspartate aminotransferase
Agency for Toxic Substances and Disease Registry
acid-treated silica gel
benchmark dose
5-bromo-2-deoxyuridine
blood urea nitrogen
complement 3
cumulative ammonia concentration
Chemical Abstracts Service Registry Number
confidence interval
forced expiratory flow
forced expiratory volume
forced vital capacity
y-amino butyric acid
immunoglobin E
immunoglobin G
Integrated Risk Information System
lowest-observed-adverse-effect level
monoamine oxidase
mean midexpiratory flow
N-m ethyl -N'-nitro-N-nitrosoguani dine
murine respiratory mycoplasmosis
nicotineamide adenine dinucleotide, oxidized
nicotineamide adenine dinucleotide, reduced
ammonia
ammonium ion
National Institute for Occupational Safety and Health
N-methyl D-aspartate
no-observed-adverse-effect level
nitrogen oxides
National Research Council
odds ratio
poly(ADP-ribose) polymerase
peak expiratory flow
peak expiratory flow rate
phytohemagglutin
point of departure
purified protein derivative
porcine reproductive and respiratory syndrome virus
reference concentration
reference dose
selected ion flow tube mass spectrometry
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TLV threshold limit value
TWA time-weighted average
UF uncertainty factor
U.S. EPA U.S. Environmental Protection Agency
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to ammonia.
It is not intended to be a comprehensive treatise on the chemical or toxicological nature of
ammonia.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration, and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of data and related uncertainties. The discussion is intended to convey the limitations
of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk
assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/ AUTHOR
Audrey Galizia, Dr. PH
Office of Research and Development
U.S. Environmental Protection Agency
Edison, NJ
CONTRIBUTORS
James Ball, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Washington, DC
Christopher Brinkerhoff, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Washington, DC
Christopher Sheth, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Washington, DC
John Whalan
National Center for Environmental Assessment
Office of Research and Development
Washington, DC
CONTRACTOR SUPPORT
Amber B acorn
Fernando Llados
Julie Stickney
Chemical, Biological and Environmental Center
SRC, Inc.
Portions of this Toxicological Review were developed under a Memorandum of
Understanding with the Agency for Toxic Substances and Disease Registry (ATSDR) and were
adapted from the Toxicological Profile for Ammonia (ATSDR, 2004) as part of a collaborative
effort in the development of human health toxicological assessments for the purposes of making
more efficient use of available resources and to share scientific information.
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REVIEWERS
INTERNAL EPA REVIEWERS
TedBerner, M.S.
National Center for Environmental Assessment
Office of Research and Development
Research Triangle Park, NC
Amanda S. Persad, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Research Triangle Park, NC
Paul Reinhart, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Research Triangle Park, NC
Marian Rutigliano, MD
National Center for Environmental Assessment
Office of Research and Development
Washington, DC
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1 1. INTRODUCTION
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4 This document presents background information and justification for the Integrated Risk
5 Information System (IRIS) Summary of the hazard and dose-response assessment of ammonia.
6 IRIS Summaries may include oral reference dose (RfD) and inhalation reference concentration
7 (RfC) values for chronic and other exposure durations, and a carcinogenicity assessment.
8 The RfD and RfC, if derived, provide quantitative information for use in risk assessments
9 for health effects known or assumed to be produced through a nonlinear (presumed threshold)
10 mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
11 uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
12 population (including sensitive subgroups) that is likely to be without an appreciable risk of
13 deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
14 analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
15 inhalation RfC considers toxic effects for both the respiratory system (portal-of-entry) and for
16 effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference
17 values are generally derived for chronic exposures (up to a lifetime), but may also be derived for
18 acute (<24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of
19 lifetime) exposure durations, all of which are derived based on an assumption of continuous
20 exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are
21 derived for chronic exposure duration.
22 The carcinogenicity assessment provides information on the carcinogenic hazard
23 potential of the substance in question and quantitative estimates of risk from oral and inhalation
24 exposure may be derived. The information includes a weight-of-evidence judgment of the
25 likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
26 effects may be expressed. Quantitative risk estimates may be derived from the application of a
27 low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on
28 the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a
29 plausible upper bound on the estimate of risk per ug/m3 air breathed.
30 Development of these hazard identification and dose-response assessments for ammonia
31 has followed the general guidelines for risk assessment as set forth by the National Research
32 Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. EPA) Guidelines and Risk
33 Assessment Forum technical panel reports that may have been used in the development of this
34 assessment include the following: Guidelines for the Health Risk Assessment of Chemical
35 Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b),
36 Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S.
37 EPA, 1988), Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Interim
38 Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA,
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1 1994a), Methods for Derivation of Inhalation Reference Concentrations and Application of
2 Inhalation Dosimetry (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk
3 Assessment (U.S. EPA, 1995), Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA,
4 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council
5 Handbook: Risk Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance
6 Document (U.S. EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment
7 of Chemical Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference
8 Concentration Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S.
9 EPA, 2005a), Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
10 Carcinogens (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA,
11 2006a), A Framework for Assessing Health Risks of Environmental Exposures to Children (U. S.
12 EPA, 2006b), and Recommended Use of Body Weight3'4 as the Default Method in Derivation of
13 the Oral Reference Dose (U.S. EPA, 201 la).
14 The literature search strategy employed for ammonia was based on the Chemical
15 Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
16 scientific information submitted by the public to the IRIS Submission Desk was also considered
17 in the development of this document. Primary, peer-reviewed literature identified through June
18 2011 was included where that literature was determined to be critical to the assessment. The
19 relevant literature included publications on ammonia which were identified through Toxicology
20 Literature Online (TOXLINE), PubMed, the Toxic Substance Control Act Test Submission
21 Database (TSCATS), the Registry of Toxic Effects of Chemical Substances (RTECS), the
22 Chemical Carcinogenesis Research Information System (CCRIS), the Developmental and
23 Reproductive Toxicology/Environmental Teratology Information Center (DART/ETIC), the
24 Hazardous Substances Data Bank (HSDB), the Genetic Toxicology Data Bank (GENE-TOX),
25 Chemical abstracts, and Current Contents. Other peer-reviewed information, including health
26 assessments developed by other organizations, review articles, and independent analyses of the
27 health effects data were retrieved and may be included in the assessment where appropriate.
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2. CHEMICAL AND PHYSICAL INFORMATION
Ammonia is a corrosive gas with a very pungent odor (O'Neil et al., 2006). It is highly
soluble in water (4.82 x io5 mg/L) and is a weak base (Lide, 2008; Eggeman, 2001; Dean, 1985).
When ammonia (NH3+) is present in water at environmental pH, a pKa of 9.25 indicates that the
equilibrium will favor the formation of the conjugate acid, the ammonium ion (NH4+) (Lide,
2008). A solution of ammonia in water is sometimes referred to as ammonium hydroxide
because the ammonia and water both ionize to form ammonium cations and hydroxide anions
(Eggeman, 2001). Ammonium salts are easily dissolved in water and disassociate into the
ammonium ion and the anion.
Many physical and chemical properties of ammonia are related to the pH of ammonia in
solution (ammonium hydroxide). Ammonium hydroxide is a weak base that is partially ionized
in water with a dissociation constant of 1.77 x IO"5 at 25 °C that increases slightly with
increasing temperature (Reed, 1982). At a pH of 8.25, 90% of ammonia will be protonated. At a
pH of 7.25, 99% of ammonia will be protonated. Thus, a decrease in pH would result in an
increase in the ammonium ion concentration and an increase in solubility of ammonia in water.
At physiological pH (7.4), the equilibrium between NHa and NH4+ favors the formation of NH4+.
Chemical and physical properties of ammonia are listed in Table 2-1.
Table 2-1. Chemical and physical properties of ammonia
Chemical name
Synonym(s)
Structure
Chemical formula
CASRN
Molecular weight
Form
Melting point
Boiling point
Odor threshold
Density
Vapor density
pKa (ammonium ion)
Solubility:
Water
Organic solvents
Ammonia3
AM-Fol; anhydrous ammonia; ammonia gas;
Nitro-sil; R 717; Spirit of hartshorn
H
HAH
NH3
7664-4 l-7a
17.031
Colorless gas; corrosive
-77.73°C
-33.33°C
53 ppm
0.7714g/Lat25°C
0.5967 (air = 1)
9.25
4.82 x io5 mg/L at 24°C
Soluble in ethanol, chloroform, and ether
ChemID Plus, 2009
ChemID Plus, 2009
ChemID Plus, 2009
ChemID Plus, 2009
Lide, 2008
O'Neil et al., 2006
Lide, 2008
Lide, 2008
O'Neil et al., 2006
O'Neil et al., 2006
O'Neil et al., 2006
Lide, 2008
Dean, 1985
Lide, 2008; O'Neil et al., 2006
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Table 2-1. Chemical and physical properties of ammonia
Vapor pressure
Henry's law constant
Conversion factors
ppm to mg/m3
mg/m3 to ppm
7.51 x 103mmHgat25°C
1.61 x ID'5 atm-nrVmol at 25°C
1 ppm = 0.707 mg/m3
1 mg/m3 = 1.414 ppm
AIChE, 1999
Betterton, 1992
Verschueren, 2001
Verschueren, 2001
"Ammonia dissolved in water is sometimes referred to as ammonium hydroxide (CASRN 1336-21-6). Ammonium
hydroxide does not exist outside of solution.
1
2 Ammonia is a major component of the geochemical nitrogen cycle and is essential for
3 many biological processes (Rosswall, 1981). Nitrogen-fixing bacteria convert atmospheric
4 nitrogen into ammonia available for plant uptake (Socolow, 1999; Rosswall, 1981). Organic
5 nitrogen released from biota is converted into ammonia through nitrogen mineralization
6 (Rosswall, 1981). Ammonia in water and soil is naturally converted into nitrite and nitrate
7 through the process of nitrification (Rosswall, 1981).
8 Commercially produced ammonia is obtained through the Haber-Bosch process, which
9 involves mixing nitrogen from the atmosphere with hydrogen obtained from natural gas in a 1 to
10 3 ratio and passing the mixture over a catalyst at high temperature and pressure (Eggeman,
11 2001). Over the past century, world-wide anthropogenic nitrogen fixation (conversion to
12 ammonia) has risen to approximately 140 million metric tons, exceeding the amount of nitrogen
13 fixed through natural processes (NSF, 1999; Socolow, 1999).
14 Large amounts (thousands of tons) of ammonia are transported and stored using large
15 pipelines and refrigerated, low pressure tanks (Eggeman, 2001). Approximately 80-85% of
16 commercially produced ammonia is used in the production of agricultural fertilizers in the form
17 of urea, ammonium nitrate, ammonium sulfate, ammonium phosphate, and other nitrogen
18 compounds (Eggeman, 2001). The remaining uses of ammonia mostly involve formation of
19 chemical intermediates (Eggeman, 2001). Ammonia is used in metal treating operations, in
20 water treatment operations, in catalytic reactors, as a convenient source of hydrogen for the
21 hydrogenation of fats and oils, as a neutralizer in the petroleum industry, and as a stabilizer in the
22 rubber industry (HSDB, 2009). Ammonia may be emitted during these processes and may also
23 be emitted from light duty vehicles, heavy-duty diesel trucks and some non-road engines.
24 Ammonia has been used to reduce nitrogen oxides (NOx) emissions from the exhaust of
25 stationary combustion sources such as industrial and municipal boilers and power generators
26 since the 1980's (Johnson et al., 2009), but more recently, ammonia (generated from urea
27 injected into the exhaust stream) is being used in a selective catalytic reduction-based diesel
28 engine aftertreatment technology to reduce NOx emissions.
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3. TOXICOKINETICS1
4 Ammonia can be absorbed by the inhalation and oral routes of exposure. There is less
5 certainty regarding absorption through the skin, although absorption through the eye has been
6 documented. Most of the inhaled ammonia is retained in the upper respiratory tract and is
7 subsequently eliminated in expired air. Ammonia that reaches systemic circulation is widely
8 distributed to all body compartments, although substantial first-pass metabolism occurs in the
9 liver, where biotransformation into urea and glutamine occur. Ammonia exists in the blood as
10 ammonium ion (NH4+). Ammonia is transported in the circulatory system primarily via
11 glutamine and alanine, amino acids that are used to transport ammonia to and from tissues.
12 When transported to the liver and kidney, the amide moiety is hydrolyzed via glutaminase
13 forming glutamatic acid (glutamate) and ammonium ion, which is synthesized into urea and
14 excreted in the urine. Ammonia or ammonium ion reaching the tissues is utilized for glutamate
15 production, which participates in transamination and other reactions. The principal means of
16 excretion of absorbed ammonia in mammals is as urinary urea; minimal amounts are excreted in
17 the feces and in expired air.
18 Ammonia is endogenously produced in humans and animals. It is an essential
19 mammalian metabolite used in nucleic acid and protein synthesis, is necessary for maintaining
20 acid-base balance, and an integral part of nitrogen homeostasis. Given its important metabolic
21 role, ammonia exists in a homeostatically regulated equilibrium in the body.
22
23 3.1. ABSORPTION
24 3.1.1. Inhalation Exposure
25 Experiments with volunteers2 show that ammonia, regardless of its tested concentration
26 in air (range, 57-500 ppm or 40-354 mg/m3), is almost completely retained in the nasal mucosa
27 (83-92%) during short-term acute exposure, i.e., up to 120 seconds (Landahl and Hermann,
28 1950). However, longer-term acute exposure (10-27 minutes) to a concentration of 500 ppm
29 (354 mg/m3) resulted in lower retention (4-30%), with expired breath concentrations of 350-400
30 ppm (247-283 mg/m3) observed by the end of the exposure period (Silverman et al., 1949),
31 suggesting saturation of absorption into the nasal mucosa. Nasal and pharyngeal irritation, but
32 not tracheal irritation, suggests that ammonia is retained in the upper respiratory tract.
Portions of this section were adapted from the Toxicokinetics Section (Section 3.4) of the Toxicological Profile for
Ammonia (ATSDR, 2004) under a Memorandum of Understanding (MOU) with ATSDR.
2The human toxicokinetic studies cited in this section did not provide information on the human subjects research
ethics procedures undertaken in the studies; however, there is no evidence that the conduct of the research was
fundamentally unethical or significantly deficient relative to the ethical standards prevailing at the time the research
was conducted.
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1 Unchanged levels of blood urea nitrogen (BUN), nonprotein nitrogen, urinary urea, and urinary
2 ammonia following these acute exposures are evidence of low absorption into the blood.
3 Exposure to a common occupational limit of ammonia in air (25 ppm or 18 mg/m3), assuming
4 30% uptake into blood, would yield an increase in blood ammonia concentration of 0.09 |ig/mL
5 (calculated by WHO, 1986). This calculated rise would likely be indistinguishable from the
6 observed baseline levels of 0.1-1.0 |ig/mL (Monsen, 1987; Conn, 1972; Brown et al., 1957) for
7 healthy controls.
8 Data in rabbits and dogs provide supporting evidence for high-percentage nasal retention,
9 resulting in a lower fraction of the inhaled dose reaching the lower respiratory tract (Egle, 1973;
10 Dalhamn, 1963; Boyd et al., 1944). Continuous exposure of rats to up to 32 ppm (23 mg/m3) for
11 24 hours did not result in a statistically significant increase in blood ammonia levels (0.1 |ig/mL
12 above preexposure levels), whereas exposures to 310-1,157 ppm (219-818 mg/m3) led to
13 significantly increased blood concentrations of ammonia within 8 hours of exposure initiation;
14 blood ammonia returned to preexposure values within 12 hours of continuous exposure
15 (Schaerdeletal., 1983).
16
17 3.1.2. Oral Exposure
18 Case reports of human ingestion of household ammonia (ammonium hydroxide) provide
19 evidence of oral absorption, but few quantitative data are available. For example, in a fatal case
20 of a man who drank an unknown amount of a 2.4% solution of ammonium hydroxide, analysis of
21 the contents of the stomach and blood showed ammonium ion levels of 15.3 mg in the stomach
22 and 33 |ig/mL in the blood (Klendshoj and Rejent, 1966). This blood concentration is about 30-
23 fold higher than the concentration of 1 |ig/mL in fasting volunteers, as reported by Conn (1972).
24 Ammonium ion is endogenously produced in the human digestive tract, much of it arising
25 from the bacterial degradation of nitrogenous compounds from ingested food. About
26 4,200 mg/day are produced, >70% of which is synthesized or liberated within the colon and its
27 fecal contents (Summerskill and Wolpert, 1970). About 99% of the total amount produced
28 (4,150 mg) is systemically absorbed. Evidence suggests that fractional absorption of ammonia
29 increases as the lumen pH increases, and that active transport occurs at the lower pH levels
30 (absorption has been detected at a pH as low as 5) (Castell and Moore, 1971; Mossberg and
31 Ross, 1967). Ammonium ion absorbed from the gastrointestinal tract travels via the hepatic
32 portal vein directly to the liver, where in healthy individuals, most of it is converted to urea and
33 glutamine.
34
35 3.1.3. Dermal Exposure
36 Quantitative data on absorption from exposure by the dermal route are not available. One
37 report of five case histories of workers exposed to anhydrous ammonia via a burst gas pipe
38 indicated that there was systemic toxicity (vomiting, renal congestion, and delirium), suggesting
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1 dermal absorption; however, the fractional dose from dermal exposure could not be determined
2 (Slot, 1938). WHO (1986) concluded that systemic effects from skin and eye exposure are not
3 quantitatively important. Ammonia is readily absorbed into the eye; and it was found to diffuse
4 within seconds into the cornea, lens, drainage system, and retina (Beare et al., 1988; Jarudi and
5 Golden, 1973). However, amounts absorbed were not quantified, and absorption into systemic
6 circulation was not investigated.
7
8 3.2. DISTRIBUTION
9 The range of mean ammonia concentrations in humans as a result of endogenous
10 production was reported as 0.1 to 0.6 |ig/mL in arterial blood and 0.2 to 1.7 |ig/mL in venous
11 blood (Huizenga et al., 1994). Other baseline levels observed in experimental volunteers range
12 from 1 to 5.5 |ig/mL (Conn, 1972; Brown et al., 1957). Ammonia is homeostatically regulated to
13 remain at low concentrations, with 95-98% existing in the blood (at physiological pH) as NH4+
14 ion (da Fonseca-Wolhheim, 1995; Souba, 1987).
15 Ammonia is present in fetal circulation. In vivo studies in several animal species and in
16 vitro studies of human placenta suggest that ammonia is produced within the uteroplacenta and
17 released into the fetal and maternal circulations (Bell et al., 1989; Johnson et al., 1986; Haugel et
18 al., 1983; Meschia et al., 1980; Remesar et al., 1980; Holzman et al., 1979, 1977; Rubaltelli et
19 al., 1968; Luschinsky, 1951). Jozwik et al. (2005) reported that ammonia levels in human fetal
20 blood (specifically, umbilical arterial and venous blood) at birth were 1.0-1.4 |ig/mL, compared
21 to 0.5 |ig/mL in the mothers' venous blood. DeSanto et al. (1993) similarly collected human
22 umbilical arterial and venous blood at delivery, and found umbilical arterial ammonia
23 concentrations were significantly higher than venous concentrations; there was no correlation
24 between umbilical ammonia levels and gestational age (range of 25-43 weeks of gestation). In
25 sheep, uteroplacental tissues are the main site of ammonia production, with outputs of ammonia
26 into both the uterine and umbilical circulations (Jozwik et al., 1999). In late-gestation pregnant
27 sheep that were catheterized to allow measurement of ammonia exposure to the fetus,
28 concentrations of ammonia in umbilical arterial and venous blood and uterine arterial and venous
29 blood ranged from about 0.39-0.60 |ig/mL (Jozwik et al., 2005, 1999).
30 Ammonia is present in human breast milk as one of the sources of nonprotein nitrogen
31 (Atkinson et al., 1980).
32
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1 3.2.1. Inhalation Exposure
2 Little information was found in the available literature for distribution of inhaled
3 ammonia. Information on the distribution of endogenously-produced ammonia suggests that any
4 ammonia absorbed through inhalation would be distributed to all body compartments via the
5 blood, where it would be used in protein synthesis, as a buffer, reduced to normal concentrations
6 by urinary excretion, or converted by the liver to glutamine and urea (Takagaki et al., 1961).
7 Rats inhaling 300 ppm (212 mg/m3) ammonia for 6 hours/day for 15 days exhibited
8 increased blood ammonia (200%) and brain (28%) glutamine levels at 5 days of exposure, but
9 not at 10 or 15 days (Manninen et al., 1988), demonstrating transient distribution of ammonia to
10 the brain (metabolic adaptation).
11
12 3.2.2. Oral Exposure
13 Human oral exposure data indicate that ammonia readily enters the portal circulation and
14 is delivered to the liver, as has been shown to be the case for endogenously produced ammonia
15 (Pitts, 1971; Summerskill and Wolpert, 1970). Unionized ammonia is freely diffusible, whereas
16 the ammonium ion is less so, and is relatively confined to the extracellular compartment
17 (Stabenau et al., 1958).
18 3.2.3. Dermal Exposure
19 No quantitative data on distribution of ammonia from dermal exposure were located in
20 the available literature.
21
22 3.3. METABOLISM
23 Endogenously, ammonia is produced by catabolism of amino acids by glutamate
24 dehydrogenase primarily in the liver and renal cortex, but also in the brain and heart (Souba,
25 1987). In skeletal muscle, ammonia may be produced by metabolism of adenosine
26 monophosphate (AMP) via adenylate deaminase. Information on the metabolism of
27 exogenously-introduced ammonia was not found in the available literature. Ammonia and
28 ammonium ion are metabolized to glutamine mainly in the liver via glutamine synthetase in the
29 glutamine cycle (Figure 3-1), or incorporated into urea as part of the urea cycle as observed in
30 the hepatic mitochondria and cytosol (Figure 3-2) (Souba, 1987). Ammonia can be rapidly
31 converted to glutamine in the brain as well (Takagaki et al., 1961). Van de Poll (2008) reported
32 that the liver removes an amount of ammonia from circulation equal to the amount added by the
33 intestines at metabolic steady state, indicating that the gut does not contribute significantly to
34 systemic ammonia release.
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NH
H,O
Glutamate
ATP
lutaminase
(in liver
mitochondria)
Glutamine
ADP
glutamine
synthetase
2
o
J
4
5
6
7
y-Glutamyl
phosphate
P; NH4
Adapted from: Nelson and Cox (2008).
Figure 3-1. Glutamine cycle.
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C02+NH4+
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
H2O
2 ATP
2ADP
Pi4
3H+
MITOCHONDRIA!/
MATRIX
Carbamoyl
phosphate
synthase I
Carbamoyl
phosphate
Ornithine Citrulline
Omithine
transcarbamoylase
Ornithine
Urea
H90
Citrulline
ATP
Arginase
Arginmosuccmate ^Aspartate
synthase A
» ^-^
Arginine Argininosuccinate
Argininosuccinate
lyase
CYTOSOL
Fumarate
Adapted from: Nelson and Cox (2008).
Figure 3-2. The urea cycle showing the compartmentalization of its steps
within liver cells.
Given its important metabolic role, ammonia exists in a homeostatically regulated
equilibrium in the body. In particular, free ammonia has been shown to be homeostatically
regulated to remain at low concentrations, with 95-98% of body burden existing in the blood (at
physiological pH) as NH4+ ion (da Fonseca-Wolhheim, 1995; Souba, 1987). Two studies in rats
(Manninen et al., 1988; Schaerdel et al., 1983) provided evidence that ammonia concentrations
in air below 25 ppm (18 mg/m3) do not alter blood ammonia concentrations. Schaerdel et al.
(1983) exposed rats to ammonia for 24 hours at concentrations ranging from 15-1,157 ppm (11-
818 mg/m3). Exposure to 15 ppm (11 mg/m3) ammonia did not increase blood ammonia
concentrations after 24 hours; concentrations of >32 ppm caused an exposure-released increase
in blood ammonia, but concentrations at 12- and 24-hour sampling periods were lower than at 8
hours, suggesting compensation by increasing ammonia metabolism through conversion to urea,
pyrimidine and polyamine synthesis, incorporation into amino acid substrates, and metabolism in
nervous system tissue. Rats inhaling 25 ppm (18 mg/m3) ammonia for 6 hours/day for 15 days
10
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1 did not exhibit blood or brain ammonia or glutamine levels that were different from controls;
2 however, rats inhaling 300 ppm (212 mg/m3) for the same duration exhibited statistically
3 significantly increased levels of blood ammonia (threefold) and brain glutamine (approximately
4 40%) at 5 days of exposure, but not at 10 or 15 days (Manninen et al., 1988). The return of
5 blood and brain ammonia and glutamine levels to control levels with time is consistent with
6 metabolic adaptation, and these data suggest that animals have a large capacity to handle high
7 concentrations of inhaled ammonia.
8 Various disease states can affect the rate of glutamine uptake and catabolism and,
9 thereby, affect the blood and tissue levels of ammonia. Abnormally elevated levels of ammonia
10 are indicative of end-stage renal failure (Davies et al., 1997). Acute renal failure can result in
11 increased renal glutamine consumption and ammonia production with a decreased capability of
12 eliminating urea in the urine (Souba, 1987). End-stage liver failure due to fulminant hepatitis or
13 hepatic cirrhosis may result in decreased ureagenesis and increased levels of ammonia in blood
14 (hyperammonemia), leading to increased uptake into the brain and the onset of hepatic
15 encephalopathy. The increased metabolic alkalosis associated with hepatic encephalopathy may
16 result in a shift in the NH4+/NH3 ratio in the direction of ammonia, which could pass through the
17 blood-brain barrier (Katayama, 2004). In patients with liver cirrhosis and acute clinical hepatic
18 encephalopathy, the observed trapping of [13N]-ammonia in the brain appeared to be related to a
19 fivefold increase, relative to healthy controls, of ammonia permeability across the blood-brain
20 barrier (Keiding et al., 2010, 2006). Furthermore, Sorensen et al. (2009) demonstrated greater
21 unidirectional clearance of ammonia from the blood to brain cells than metabolic clearance of
22 ammonia from the blood in both healthy controls and in cirrhotic patients with and without
23 hepatic encephalopathy.
24
25 3.4. ELIMINATION
26 Absorbed ammonia, as well as endogenously produced ammonia, is excreted by the
27 kidneys as urea (Summerskill and Wolpert, 1970; Gay et al., 1969; Muntwyler et al., 1956;
28 Davies and Yudkin, 1952; Van Slyke et al., 1943) and is a component of sweat (Guyton, 1981;
29 Wands, 1981). Lee and colleagues observed that acidosis-stimulated renal excretion of ammonia
30 is mediated by intercalated cell-specific Rh B glycoprotein expression in mice (Bishop et al.,
31 2010; Lee et al., 2010, 2009). In rat kidney, ammonium ion is secreted into the lumen of the
32 outer medullary collecting duct via H+ secretion and parallels ammonia diffusion (Flessner et al.,
33 1992). The inner medullary collecting duct exhibits a Na+- and K+-independent NH4+/H+
34 exchange activity that may be mediated by an Rh C glycoprotein (Handlogten et al., 2005),
35 which is also expressed in human kidneys (Han et al., 2006).
36 Additionally, ammonia is known to be excreted through expired air and is present in the
37 expired air of all humans (Manolis, 1983). Two investigators specifically measured ammonia in
38 breath exhaled from the nose (Smith et al., 2008; Larson et al., 1977). Smith et al. (2008)
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1 reported median ammonia concentrations in exhaled breath from the nose of three healthy
2 volunteers (with samples collected daily over a 4-week period) of 0.059-0.078 mg/m3; these
3 concentrations were similar to or slightly higher than the mean laboratory air level of ammonia
4 of 0.056 mg/m3 reported in this study. Larson et al. (1977) reported that the median
5 concentration of ammonia collected from air samples exhaled from the nose ranged from 0.013
6 to 0.046 mg/m3. One sample collected from the trachea (via a tube inserted through the nose of
7 one subject) was 0.029 mg/mg3—a concentration within the range of concentrations in breath
8 exhaled through the nose (Larson et al., 1977).
9 Higher and more variable ammonia concentrations are reported in breath exhaled from
10 the mouth or oral cavity than from air exhaled from the nose. In studies that reported ammonia
11 in breath samples from the mouth or oral cavity, the majority of ammonia concentrations ranged
12 from 0.085 to 2.1 mg/m3 (Smith et al., 2008; Spanel et al., 2007a; Spanel et al., 2007b; Turner et
13 al., 2006; Diskin et al., 2003; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977).
14 These higher concentrations are largely attributed to the production of ammonia by bacterial
15 degradation of food protein in the oral cavity or GI tract (Turner et al., 2006; Smith et al., 1999;
16 Vollmuth and Schlesinger, 1984). This source of ammonia in breath was demonstrated by Smith
17 et al. (1999), who observed elevated ammonia concentrations in the expired air of 6 healthy
18 volunteers following the ingestion of a protein-rich meal.
19 Other factors that can affect ammonia levels in breath exhaled from the mouth or oral
20 cavity include diet, oral hygiene, age, living conditions, and disease state. Norwood et al. (1992)
21 reported decreases in baseline ammonia levels (0.085-0.905 mg/m3) in exhaled breath following
22 tooth brushing (<50% depletion), a distilled water oral rinse (<50% depletion), and an acid oral
23 rinse (80-90% depletion). These findings are consistent with ammonia generation in the oral
24 cavity by bacterial and/or enzymatic activity. Several investigators have reported that ammonia
25 in breath from the mouth and oral cavity increases with age (Spanel et al., 2007a; Spanel et al.,
26 2007b; Turner et al., 2006), with ammonia concentrations increasing, on average, by about 0.1
27 mg/m3 for each 10 years of life (Spanel et al., 2007b). Turner et al. (2006) reported that the age
28 of the individual accounts for about 25% of the variation in observed mean breath ammonia
29 levels and the remaining 75% is due to factors other than age. Certain disease states can also
30 influence ammonia levels in exhaled breath. Ammonia is greatly elevated in the breath of
31 patients in renal failure (Spanel et al., 2007b; Davies, 1997). Increases in breath ammonia are
32 associated with decreasing kidney function with increasing age (Diskin et al., 2003; Epstein,
33 1996). These studies are further described in Appendix C.I in Table C-l.
34 Because ammonia measured in samples of breath exhaled from the mouth or oral cavity
35 can be generated in the oral cavity and thus may be substantially influenced by diet and other
36 factors, ammonia levels measured in mouth or oral cavity breath samples do not likely reflect
37 systemic (blood) levels of ammonia. Ammonia concentrations in breath exhaled from the nose
38 appear to better represent systemic or background levels (Smith et al., 2008).
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1 Ammonia has also been detected in the expired air of animals. Whittaker et al. (2009)
2 observed a significant association between ambient ammonia concentrations and increases in
3 exhaled ammonia in stabled horses. Analysis of endogenous ammonia levels in the expired air
4 of rats showed concentrations ranging from 0.01 to 0.353 ppm (0.007-0.250 mg/m3)
5 (mean = 0.08 ppm or 0.06 mg/m3) in nose-breathing animals (Barrow and Steinhagen, 1980).
6 Larson et al. (1980) reported ammonia concentrations measured in the larynx of dogs exposed to
7 sulfuric acid ranging between 0.03 and 0.225 ppm (0.02 and 0.16 mg/m3) following mouth
8 breathing, and between 0.05 and 0.220 ppm (0.04 and 0.16 mg/m3) following nose breathing.
9
10 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS
11 No physiologically based pharmacokinetic (PBPK) models have been developed for
12 ammonia. An expanded one-compartment toxicokinetic model in rats was developed by Diack
13 and Bois (2005), which used physiological values to represent first-order uptake and elimination
14 of inhaled ammonia (and other chemicals). The model is not useful for dose-response
15 assessment of ammonia because: (1) it cannot specify time-dependent amounts or concentrations
16 of ammonia in specific target tissues, (2) it has not been verified against experimental data for
17 ammonia, glutamate, or urea levels in tissues, and (3) it cannot extrapolate internal doses of
18 ammonia between animals and humans.
19
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4. HAZARD IDENTIFICATION
4 As noted in Section 2, ammonium salts (e.g., ammonium acetate, chloride, and sulfate)
5 readily dissolve in water through disassociation into the ammonium ion (NH4+) and the anion.
6 At physiological pH (7.4), the equilibrium favors the formation of NH4+. Because of this
7 equilibrium at physiological pH, the literature on the toxicity of ammonium salts was reviewed
8 to determine whether this literature could inform the toxicity of ammonia. In rats following
9 exposure to ammonium chloride in the diet for subchronic and chronic exposure durations at
10 doses ranging from approximately 500 to 1,800 mg/kg-day, the primary effect of this salt was
11 related to the acid-base balance in the body (Lina and Kuijpers, 2004; Barzel et al., 1969).
12 Ammonium chloride treatment induced a dose-related hyperchloremic metabolic acidosis in rats
13 as evidenced by decreases in blood pH, base excess, and bicarbonate concentration, and
14 increased plasma chloride levels. Lina and Kuijpers (2004) also observed a significant dose-
15 related depression in body weight gain at exposure to ammonium chloride doses >1,590 mg/kg-
16 day for 13 weeks. The administration of ammonium chloride was also associated with zona
17 glomerulosa hypertrophy of the adrenal gland in both the 13-week and 18- and 30-month studies
18 by Lina and Kuijpers (2004).
19 In contrast, metabolic acidosis was not induced in rats exposed to ammonium sulfate in
20 the diet at doses up to 1,527 mg/kg-day for 52 weeks or histopathologic changes in the adrenal
21 gland at doses up to 1,371 mg/kg-day for 104 weeks (Ota et al., 2006). The only dose-related
22 effects associated with the 52-week exposure to ammonium sulfate were increased liver and
23 kidney weights (~7 and 10%, respectively) at a dose of 1,490 mg/kg-day (female rat) or 1,527
24 mg/kg-day (male rat) (Ota et al., 2006). In the 104-week study, the incidence of chronic
25 nephropathy was statistically significantly increased in low-dose (564 mg/kg-day), but not in
26 high-dose (1,288 mg/kg-day) male rats (Ota et al., 2006).
27 Appendix B, Table B-l, presents a summary of repeat-dose oral toxicity studies for
28 selected ammonium salts. Studies of ammonium sulfamate, a broad spectrum herbicide, were
29 not included in this summary table because the pesticidal properties of this ammonium salt were
30 not considered relevant to ammonia toxicity. No studies of the toxicity of ammonium salts by
31 the inhalation pathway are available.
32 Because the toxicity information for the ammonium salts that have been adequately
33 studied (i.e., the chloride and sulfate) indicates that their toxicity profiles differ, it appears that
34 the anion can influence the toxicity of the ammonium salt. Therefore, it is uncertain whether
35 toxicity data for ammonium salts can be used to inform the toxicity of ammonia. Accordingly,
36 the toxicity of ammonium salts as the basis for characterizing the toxicity of ammonia was not
37 further evaluated in this assessment.
38
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1 4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
2 CONTROLS
3 4.1.1. Case Reports - Oral and Inhalation Exposures
4 Ammonia exposure has occurred frequently in occupational settings (both industrial and
5 agricultural) when equipment failure or operator error resulted in the sudden release of
6 pressurized ammonia. Transportation accidents and catastrophic releases have also resulted in
7 exposure to high concentrations of ammonia. Oral exposure to ammonia has resulted from
8 intentional or accidental ingestion of household products containing ammonia. Numerous case
9 reports of injury in adults and children due to exposure to ammonia via inhalation of vapors,
10 dermal contact, or ingestion of household cleaning solutions or ammonia inhalant capsules exist.
11 These case reports indicate that the clinical signs of acute oral exposure to ammonia
12 were headache, stomachache, nausea, dizziness, diarrhea, drooling, erythematous and edematous
13 lips, reddened and blistered tongues, dysphagia, vomiting, oropharyngeal burns, laryngeal and
14 epiglottal edema, erythmatous esophagus with severe corrosive injury, and hemorrhagic
15 esophago-gastro-duodeno-enteritis. Acute inhalation or dermal exposure to ammonia resulted in
16 first-, second-, and third-degree burns to body surfaces, mild to severe erythema and edema
17 throughout the mouth, nasal passages, pharynx, larynx, trachea and esophagus, mild to severe
18 respiratory distress with diffuse or scattered rales and rhonchi, hypoxemia, wheezing, and upper
19 airway obstruction, eye irritation, blepharospasm, lacrimation, ocular erythema, edema, and
20 death. In addition, delayed effects and complications involving the eyes and respiratory system
21 were often encountered even after initial improvement in acute conditions. Delayed effects
22 included complete or partial loss of vision, persistent and/or progressive shortness of breath
23 (especially on exertion), persistent, productive cough, and progressive obstructive and restrictive
24 pulmonary disease with development of cylindrical and/or saccular bronchiectasis with or
25 without obliterative fibrous adhesions in the pleural space, and death. The details of these case
26 reports are given in Appendix C.2.
27
28 4.1.2. Controlled Human Inhalation Exposure Studies
29 Several controlled exposure studies were conducted in volunteers to evaluate irritation
30 effects and changes in pulmonary function following acute inhalation exposure to ammonia;
31 some of these studies describe the occurrence of eye, nose, and throat irritation. These studies
32 are presented in more detail in Appendix C.3. Some of the studies included in Appendix C.3 did
33 not provide information on the human subjects research ethics procedures undertaken in the
34 study (Altmann et al., 2006 (abstract only); Douglas and Coe, 1987; Kalandarov et al., 1984;
35 Ferguson et al., 1977; Verberk, 1977; Silverman et al., 1949), but there is no evidence that the
36 conduct of the research was fundamentally unethical or significantly deficient relative to the
37 ethical standards prevailing at the time the research was conducted. Other studies reported that
38 informed consent by volunteers and/or study approval by local boards/officials regarding ethical
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1 conduct was obtained (Petrova et al., 2008; Smeets et al., 2007; Ihrig et al., 2006; Sigurdarson et
2 al., 2004; Sundblad et al., 2004; Cole et al., 1977; MacEwan et al., 1970).
3 Altmann et al. (2006) showed a dose-dependent increase in the intensity of odor
4 annoyance and irritation in healthy male and female volunteers during inhalation exposure to
5 ammonia; strong olfactory and moderate to strong irritation sensations occurred at concentrations
6 >15 ppm (11 mg/m3), with odor detection thresholds at <20 ppm (14 mg/m3). In another study,
7 12 healthy volunteers exposed to 5 and 25 ppm (4 and 18 mg/m3) ammonia on three different
8 occasions for 1.5 hours in an exposure chamber while exercising on a stationary bike reported
9 discomfort in the eyes and odor detection at 5 ppm (4 mg/m3) (Sundblad et al., 2004). Eye
10 irritation was also shown to increase in a concentration-dependent manner in 15 volunteers
11 exposed to ammonia for 2 hours in an exposure chamber at concentrations of 50, 80, 110, and
12 140 ppm (35, 57, 78, and 99 mg/m3); ammonia concentrations of 140 ppm (99 mg/m3) caused
13 severe and intolerable irritation (Verberk, 1977). The lachrymatory threshold was determined to
14 be 55 ppm (39 mg/m3) in volunteers exposed to ammonia gas inside tight-fitting goggles for an
15 acute duration of up to 15 seconds (Douglas and Coe, 1987). In contrast, exposures to up to 90
16 ppm (64 mg/m3) ammonia gas did not produce severe lacrimation in seven volunteers after 10
17 minutes in an exposure chamber, although increased eye erythema was reported (MacEwen et
18 al., 1970). Exposure to 500 ppm (354 mg/m3) of ammonia gas for 30 minutes through a masked
19 nose and throat inhalation apparatus resulted in 2/7 volunteers reporting lacrimation, and 2/7
20 reporting nose and throat irritation that lasted up to 24 hours after exposure (Silverman et al.,
21 1949).
22 Petrova et al. (2008) investigated irritation threshold differences between 25 healthy
23 volunteers and 15 mild-to-moderate persistent asthmatic volunteers exposed to ammonia via the
24 eyes and nose at concentrations ranging from 2 to 500 ppm (1-354 mg/m3) for durations lasting
25 up to 2.5 hours. Irritation threshold, odor intensity, and annoyance were not significantly
26 different between the two groups. The nasal and eye irritation thresholds were reported to be
27 129 ppm (91 mg/m3) and 175 ppm (124 mg/m3), respectively. Smeets et al. (2007) investigated
28 odor and irritation thresholds for ammonia vapor in 24 healthy female volunteers at
29 concentrations ranging from 0.03 to 615 ppm (0.02 to 435 mg/m3). This study observed a mean
30 odor detection threshold of 2.6 ppm (2 mg/m3) and a mean irritation threshold of 31.7 or 60.9
31 ppm (22 or 43 mg/m3), depending on the olfactometry methodology followed (static versus
32 dynamic, respectively). Irritation thresholds may be higher in people who have had prior
33 experience with ammonia exposure (Ihrig et al., 2006). Thirty male volunteers who had not
34 experienced the smell of ammonia and 10 male volunteers who had regular workplace exposure
35 to ammonia were exposed to ammonia vapors at concentrations of 0, 10, 20, and 50 ppm (0, 7,
36 14, and 35 mg/m3) on 5 consecutive days (4 hours/day) in an exposure chamber; volunteers in
37 the group familiar to the smell of ammonia reported fewer symptoms than the nonhabituated
38 group, but at a concentration of 20 ppm (14 mg/m3), there were no differences in perceived
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1 symptoms between the groups. However, the perceived intensity of symptoms was
2 concentration-dependent in both groups, but was only significant in the group of volunteers not
3 familiar with ammonia exposure (Ihrig et al., 2006). Ferguson et al. (1977) reported habituation
4 to eye, nose, and throat irritation in six male and female volunteers after 2-3 weeks of exposure
5 to ammonia concentrations of 25, 50, and 100 ppm (18, 35, and 71 mg/m3) during a 6-week
6 study (6 hours/day, 1 time/week). Continuous exposure to even the highest concentration tested
7 became easily tolerated with no general health effects occurring after acclimation occurred.
8 Several studies evaluated pulmonary functions following acute inhalation exposure to
9 ammonia. Volunteers exposed to ammonia (lung only) through a mouthpiece for 10 inhaled
10 breaths of gas experienced bronchioconstriction at a concentration of 85 ppm (60 mg/m3)
11 (Douglas and Coe, 1987); however, there were no bronchial symptoms reported in seven
12 volunteers exposed to ammonia at concentrations of 30, 50, or 90 ppm (21, 35, and 64 mg/m3)
13 for 10 minutes in an exposure chamber (MacEwen et al., 1970). Similarly, 12 healthy volunteers
14 exposed to ammonia on three separate occasions to 5 and 25 ppm (4 and 18 mg/m3) for 1.5 hours
15 in an exposure chamber while exercising on a stationary bike did not have changes in bronchial
16 responsiveness, upper airway inflammation, exhaled nitric oxide levels, or lung function as
17 measured by vital capacity and FEVi (Sundblad et al., 2004). In another study, 18 healthy
18 servicemen volunteers were placed in an exposure chamber for 3 consecutive half-day sessions.
19 Exposure to ammonia at concentrations of 50-344 mg/m3 (70-486 ppm) occurred on the 2nd
20 session, with sessions 1 and 3 acting as controls (Cole et al., 1977). The no-effect concentration
21 was determined to be 71 mg/m3 (100 ppm). Exercise tidal volume was increased at 106 mg/m3
22 (150 ppm), but then decreased at higher concentrations in a concentration-dependent manner
23 (Cole et al., 1977). Decreased FEVi and forced vital capacity (FVC) were reported in eight
24 healthy male volunteers exposed to a mean airborne ammonia concentration of 20.7 ppm (15
25 mg/m3) in swine confinement buildings for 4 hours at one-week intervals; however, swine
26 confinement buildings also include confounding exposures to dust, bacteria, endotoxin, and
27 molds, thereby making measurement of effects due to ammonia uncertain in this study (Cormier
28 et al., 2000).
29 Differences in pulmonary function between healthy and asthmatic volunteers exposed to
30 ammonia were evaluated in several studies. There were no changes in lung function as measured
31 by FEVi in 25 healthy volunteers and 15 mild/moderate persistent asthmatic volunteers after
32 ocular and nasal exposure to 2-500 ppm (1-354 mg/m3) ammonia at durations lasting up to
33 2.5 hours (Petrova et al., 2008). In another study, six healthy volunteers and eight mildly
34 asthmatic volunteers were exposed to 16-25 ppm (11-18 mg/m3) ammonia, ammonia and dust,
35 and dust alone for 30-minute sessions, with 1 week between sessions (Sigurdarson et al., 2004).
36 There were no significant changes in pulmonary function as measured by FEVi in the healthy
37 volunteers for any exposure. A decrease in FEVi was reported in asthmatics exposed to dust and
38 ammonia, but not ammonia alone; similarly, increased bronchial hyperreactivity was reported in
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1 asthmatics after exposure to dust and ammonia, but not to ammonia alone. Exposure to dust
2 alone caused similar effects, suggesting that dust was responsible for decreased pulmonary
3 function (Sigurdarson et al., 2004).
4 Kalandarov et al. (1984) investigated the effect of ammonia exposure on the function of
5 the sympathico-adrenal system and the adrenal cortex in male volunteers and found that
6 exposure to 2 mg/m3 (3.0 ppm) ammonia in combination with increased temperature and
7 humidity did not result in any significant changes to the function of the sympathico-adrenal
8 system, although the adrenal cortex function was affected exhibiting increased 11-
9 oxycorticosteroids in plasma. Ammonia concentrations of 5 mg/m3 (7.2 ppm), in combination
10 with increased temperature and humidity, resulted in increased levels of adrenaline, 17-
11 oxycorticosteroids, and free 11-oxycorticosteroids fraction in plasma. These results suggest that
12 both sympathico-adrenal system and adrenal cortex function is altered at an ammonia
13 concentration of 5 mg/m3 (7.2 ppm) (Kalandarov et al., 1984).
14 In summary, volunteer studies demonstrate that eye irritation can occur following acute
15 exposure to ammonia at concentrations as low as 5 ppm (4 mg/m3). Irritation thresholds may be
16 higher in people who have had prior experience with ammonia exposure and habituation to eye,
17 nose, and throat irritation occurs over time. Pulmonary function was not affected in workers
18 acutely exposed to ammonia concentrations as high as 71 mg/m3 (100 ppm). Studies comparing
19 the pulmonary function of asthmatics and healthy volunteers exposed to ammonia do not suggest
20 that asthmatics are more sensitive to the pulmonary effects of ammonia.
21
22 4.1.3. Cross Sectional Studies in Farmers Exposed to Inhaled Ammonia
23 Several studies have evaluated respiratory symptoms and changes in pulmonary function
24 in livestock farmers and stable workers exposed to ammonia (see Appendix C.4 for detailed
25 descriptions). In addition to ammonia, these studies also documented exposures to airborne dust,
26 bacteria, fungal spores, endotoxin, and mold. The release of other volatiles on livestock farms is
27 likely, but measurements for other volatile chemicals were not conducted. Although studies of
28 farm workers summarized here focused on exposure to ammonia, these and other studies have
29 also demonstrated respiratory effects associated with exposure to other constituents in farm
30 worker air (e.g., respirable dust, endotoxin).
31 Swine and dairy farmers had a higher prevalence of respiratory symptoms including
32 cough, phlegm, wheezing, chest tightness, and eye, nasal and throat irritation compared to
33 controls (Melbostad and Eduard, 2001; Preller et al., 1995; Choudat et al., 1994; Zejda et al.,
34 1994; Crook et al., 1991; Heederik et al., 1990). Impaired respiratory function in farmers was
35 associated with ammonia exposure in several studies (e.g., decreased FEVi, FVC) (Cormier et
36 al., 2000; Donham et al., 2000, 1995; Vogelzang et al., 1998; Reynolds et al., 1996; Preller et al.,
37 1995; Crook et al., 1991; Heederik et al., 1990). Bronchial hyperreactivity to methacholine or
38 histamine challenge was increased in farmers exposed to ammonia compared to control workers
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1 (Vogelzang et al., 2000, 1997; Choudat et al., 1994). Stable workers showed signs of bronchial
2 obstruction with increased peak expiratory flow (PEF) variability as well as increased pulmonary
3 inflammation related to allergies (Elfman et al., 2009). Other findings that suggest an allergic or
4 inflammatory response in livestock farmers exposed to ammonia include the presence of
5 immunoglobin E (IgE) and immunoglobin G (IgG) antibodies to pig squames and urine in blood
6 (Crook et al., 1991), increased neutrophils in the nasal wash (Cormier et al., 2000) and increased
7 white blood cell count (Cormier et al., 2000). In summary, several studies have demonstrated an
8 association between ammonia exposure in livestock farmers and respiratory symptoms and
9 impaired respiratory function; however, farmers are additionally exposed to several constituents
10 that likely contribute to these effects, including respirable dust, endotoxin, bacteria, fungi, and
11 mold.
12
13 4.1.4. Occupational Studies in Industrial Worker Populations
14 Holness et al. (1989) conducted a cross-sectional study of workers in a soda ash (sodium
15 carbonate) plant3 who had chronic low-level exposure to ammonia. The cohort consisted of
16 58 workers and 31 controls from stores and office areas of the plant. All workers were males
17 (average age 40.5 years) and the average exposure duration for the exposed workers at the plant
18 was 12.2 years. The mean time-weighted average (TWA) ammonia exposure of the exposed
19 group based on personal sampling over one work shift (mean sample collection time, 8.4 hours)
20 was 9.2 ppm (6.5 mg/m3), compared to 0.3 ppm (0.2 mg/m3) for the control group. The average
21 concentrations of ammonia to which workers were exposed were determined using the procedure
22 recommended by the National Institute for Occupational Safety and Health (NIOSH) which
23 involves the collection of air samples on sulfuric acid-treated silica gel (ATSG) adsorption tubes
24 (NIOSH, 1979).
25 No statistically significant differences were observed in age, height, years worked,
26 percentage of smokers, or pack-years smoked for exposed versus control workers. Exposed
27 workers weighed approximately 8% (p < 0.05) more than control workers. Information
28 regarding past occupational exposures, working conditions, and medical and smoking history, as
29 well as respiratory symptoms and eye and skin complaints was obtained by means of a
30 questionnaire that was based on an American Thoracic Society questionnaire (Ferris, 1978).
31 Each participant's sense of smell was evaluated at the beginning and end of the work week using
32 several concentrations of pyridine (0.4, 0.66, or 10 ppm). Lung function tests were conducted at
33 the beginning and end of the work shift on the first and last days of their work week (four tests
34 administered). Differences in reported symptoms and lung function between groups were
35 evaluated using the actual exposure values with age, height, and pack-years smoked as covariates
3At 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 recoved in the process and reused.
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1 in linear regression analysis. Exposed workers were grouped into three exposure categories
2 (high = >12.5 ppm [>8.8 mg/m3], medium = 6.25-12.5 ppm [4.4-8.8 mg/m3], and low =
3 <6.25 ppm [<4.4 mg/m3]) for analysis of symptom reporting and pulmonary function data.
4 Endpoints evaluated in the study included sense of smell, prevalence of respiratory
5 symptoms (cough, bronchitis, wheeze, dyspnea, and others), eye and throat irritation, skin
6 problems and lung function parameters (FVC, FEVi, FEVi/FVC, forced expiratory flow [FEFso],
7 and FEFys). No statistical differences in the prevalence of respiratory symptoms or eye irritation
8 were evident between the exposed and control groups (Table 4-1). There was a statistically
9 significant increase (p < 0.05) in the prevalence of skin problems in workers in the lowest
10 exposure category (<4.4 mg/m3) compared to controls; however, the prevalence was not
11 increased among workers in the two higher exposure groups. Workers also reported that
12 exposure at the plant had aggravated specific symptoms including coughing, wheezing, nasal
13 complaints, eye irritation, throat discomfort, and skin problems. Odor detection threshold and
14 baseline lung functions were similar in the exposed and control groups. No changes in lung
15 function were demonstrated over either work shift (days 1 or 2) or over the work week in the
16 exposed group compared with controls. No relationship was demonstrated between chronic
17 ammonia exposure and baseline lung function changes either in terms of the level or duration of
18 exposure. Study investigators noted that this finding was limited by the lack of adequate
19 exposure data collected over time, precluding development of a meaningful index accounting for
20 both level and length of exposure. Based on the lack of exposure-related differences in
21 subjective symptomatology, sense of smell, and measures of lung function, EPA identified 8.8
22 mg/m3 (12.5 ppm) as the no-observed-adverse-effect level (NOAEL). A lowest-observed-
23 adverse-effect level (LOAEL) was not identified for this study.
24
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Table 4-1. Symptoms and lung function results of workers exposed to
different levels of TWA ammonia concentrations
Parameter
Ammonia concentration
Control
0.2 mg/m3
(0.3 ppm)
Exposed
<4.4 mg/m3
(<6.25 ppm)
Exposed
4.4-8.8 mg/m3
(6.25-12.5 ppm)
Exposed
>8.8 mg/m3
(>12.5 ppm)
Symptom
Cough
Sputum
Wheeze
Chest tightness
Shortness of breath
Nasal complaints
Eye irritation
Throat irritation
Skin problems
3/31(10)a
5/31(16)
3/31(10)
2/31(6)
4/31(13)
6/31(19)
6/31(19)
1/31(3)
2/31(6)
6/34 (18)
9/34 (26)
5/34 (15)
2/34 (6)
3/34 (9)
4/34 (12)
2/34 (6)
2/34 (6)
10/34b(29)
1/12 (8)
3/12 (25)
1/12 (8)
0/12 (0)
1/12 (8)
2/12 (17)
2/12 (17)
1/12 (8)
1/12 (8)
2/12 (17)
1/12 (8)
0/12 (0)
0/12 (0)
0/12 (0)
0/12 (0)
1/12 (8)
1/12 (8)
1/12 (8)
Lung function (% predicted)
FVC
FEVj
FEF50
FEF75
98.6
95.1
108.4
65.2
96.7
93.7
106.9
71.0
96.9
93.9
106.2
67.8
96.8
95.3
111.2
78.8
FVC = forced vital capacity; FEV^forced expiratory volume in 1 second; FEF50=forced expiratory flow rates at
50%; FEF75=forced expiratory flow rates at 75%
aNumber affected/number examined. The percentage of workers reporting symptoms is indicated in parentheses.
bSignificantly different from controls, p < 0.05, by Fisher's exact test performed for this review.
Source: Holnessetal. (1989).
1
2 Ballal et al. (1998) conducted a cross-sectional study of male workers at two urea
3 fertilizer factories in Saudi Arabia4. The cohort consisted of 161 exposed subjects (84 from
4 factory A and 77 from factory B) and 355 unexposed controls. Workers in factory A were
5 exposed to air ammonia levels of 2-130 mg/m3 (2.8-184.4 ppm), and workers in factory B were
6 exposed to 0.02-7 mg/m3 (0.03-9.9 ppm). Mean duration of employment was 51.8 months for
7 exposed workers and 73.1 months for controls. Exposure levels were estimated by analyzing a
8 total of 97 air samples collected over 8-hour shifts close to the employee's work site. The
9 prevalence of respiratory symptoms and diseases was determined by administration of a
10 questionnaire. The authors stated that there were no other chemical pollutants in the workplace
11 that might have affected the respiratory system. Smoking habits were similar for exposed
12 workers and controls. Stratifying the workers by ammonia exposure levels (above or below the
13 American Conference of Governmental Industrial Hygienists [ACGIH] threshold limit value
4The 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.
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[TLV] of 18 mg/m3 [25 ppm]) showed that those exposed to ammonia concentrations higher than
the TLV had significantly higher relative risks for cough, phlegm, wheezing, dyspnea, and
asthma than workers exposed to levels below the TLV (Table 4-2). The relative risk for
wheezing was also elevated among those exposed to ammonia levels at or below the TLV.
Distribution of symptoms by cumulative ammonia concentration (CAC, mg/m3-years) also
showed significantly higher relative risk for all the above symptoms among those with higher
CAC (Table 4-2). Results of the logistic regression analysis showed that ammonia concentration
was significantly related to cough, phlegm, wheezing with and without shortness of breath, and
asthma (Table 4-3).
Table 4-2. The prevalence of respiratory symptoms and disease in urea
fertilizer workers exposed to ammonia
Respiratory
symptom/disease
Cough
Wheezing
Phlegm
Dyspnea
Chronic bronchitis
Bronchial asthma
Chronic bronchitis and
bronchial asthma
Relative risk (95% CI)
Exposure category
ACGIH TLV
(18 mg/m3)
(n = 17)
3.48 (1.84-6.57)
5.01 (2.38-10.57)
3.75(1.97-7.11)
4.57 (2.37-8.81)
2.32(0.31-17.28)
4.32 (2.08-8.98)
6.96 (0.76-63.47)
Cumulative concentration
(mg/m3 of air-yrs)
<50
(n = 130)
0.72 (0.38-1.35)
1.86 (1.04-3.32)
0.63 (0.31-1.26)
1.19(0.66-2.17)
0.61(0.13-2.77)
1.22 (0.66-2.28)
1.82(0.31-10.77)
>50
(n = 30)
2.82 (1.58-5.03)
5.24 (2.85-9.52)
3.03 (1.69-5.45)
2.59 (1.25-5.36)
5.32 (1.72-16.08)
2.44(1.10-5.43)
8.38 (1.37-45.4)
11
Source: Ballaletal. (1998).
Table 4-3. Logistic regression analysis of the relationship between ammonia
concentration and respiratory symptoms or disease in exposed urea fertilizer
workers
Respiratory symptom/disease
Cough
Phlegm
Shortness of breath with wheezing
Wheezing alone
Dyspnea on effort
Diagnosis of asthma
OR (95% CI)
1.32 (1.08-1.62)3
1.36(1.10-1.67)a
1.26 (1.04-1.54)3
1.55(1.17-2.06)a
0.83 (0.68-1.02)
1.33 (1.07-1.65)3
Bp < 0.05.
Source: Ballaletal. (1998).
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1 Results from limited spirometry testing of workers from factory A were reported in a
2 followup study (Ali, 2001). The pulmonary function indices measured in 73 ammonia workers
3 and 343 control workers included FEVi and FVC. Prediction equations for these indices were
4 developed for several nationalities (Saudis, Arabs, Indians, and other Asians) and corrected
5 values were expressed as the percentage of the predicted value for age and height. The FVC%
6 predicted was higher in exposed workers than in controls (4.6% increase, p < 0.002); however,
7 workers with cumulative exposure >50 mg/m3-years had significantly lower FEVi% predicted
8 (7.4% decrease,p < 0.006) and FVC% predicted (5.4% decrease,p < 0.030) than workers with
9 cumulative exposure <50 mg/m3-years. A comparison between symptomatic and asymptomatic
10 exposed workers showed that FEVi% predicted and FEVi/FVC% were significantly lower
11 among symptomatic workers (9.2% decrease in FEVi% predicted, p< 0.001 and 4.6% decrease
12 in FEVi/FVC%, p < 0.02). Although Ballal et al. (1998) and Ali (2001) suggest that exposure to
13 ammonia concentrations above 18 mg/m3 (50 mg/m3-years) is associated with respiratory
14 symptoms and altered pulmonary function, NOAEL and LOAEL values could not be identified
15 by EPA from these studies due to inadequate reporting of exposure concentrations.
16 Rahman et al. (2007) conducted a cross-sectional study of workers at a urea fertilizer
17 factory in Bangladesh that consisted of an ammonia plant and a urea plant. The exposed group
18 studied consisted of 63 operators in the ammonia plant and 77 in the urea plant; 25 individuals
19 from the administration building served as a control group. Mean duration of employment
20 exceeded 16 years in all groups. Personal ammonia exposures were measured by two different
21 methods (Drager PAC III and Drager tube) in five to nine exposed workers per day for 10
22 morning shifts in the urea plant (for a total of 64 workers) and in five to nine exposed workers
23 per day for 4 morning shifts from the ammonia plant (for a total of 24 workers). Four to seven
24 volunteer workers per day were selected from the administration building as controls for a total
25 of 25 workers over a five-day period. Questionnaires were administered to inquire about
26 demographics, past chronic respiratory disease, past and present occupational history, smoking
27 status, acute respiratory symptoms (cough, chest tightness, runny nose, stuffy nose, and
28 sneezing), and use of protective devices. Lung function tests (FVC, FEVi, and peak expiratory
29 flow rate [PEFR]) were administered preshift and postshift (8-hour shifts) to the 88 exposed
30 workers after exclusion of workers who planned to have less than a four-hour working day; lung
31 function was not tested in the control group. Personal ammonia exposure and pulmonary
32 function were measured on the same shift for 28 exposed workers. Linear multiple regression
33 was used to analyze the relationship between workplace and the percentage cross-shift change in
34 FEVi (AFEVi%) while adjusting for current smoking.
35 Mean exposure levels at the ammonia plant determined by the Drager tube and Drager
36 PAC III methods were 25.0 and 6.9 ppm (17.7 and 4.9 mg/m3), respectively; the corresponding
37 means in the urea plant were 124.6 and 26.1 ppm (88.1 and 18.5 mg/m3) (Rahman et al., 2007).
38 Although the Drager tube measurements indicated ammonia exposure about 4-5 times higher
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1 than those obtained with the PAC III instrument, there was a significant correlation between the
2 ammonia concentrations measured by the two methods (p = 0.001). No ammonia was detected
3 in the control area using the Drager tube (concentrations less than the measuring range of 2.5 to
4 200 ppm [1.8 to 141 mg/m3]). Based on an evaluation of the two monitoring methods and
5 communication with technical support at Drager5, EPA considered the PAC III instrument to be
6 a more sensitive monitoring technology than the Drager tubes. Therefore, the PAC III air
7 measurements were considered the more reliable measurement of exposure to ammonia for the
8 Rahman et al. (2007) study. The study authors, however, observed that their measurements
9 indicated only relative differences in exposures between workers and production areas, and that
10 the validity of the exposure measures could not be evaluated based on their results.
11 The prevalence of acute respiratory symptoms was higher in the urea plant than in the
12 ammonia plant or in the administration building. Comparison between the urea plant and the
13 administration building showed that cough and chest tightness were statistically higher in the
14 former; a similar comparison of the ammonia plant and the administration building showed no
15 statistical difference in symptom prevalence between the two groups (Table 4-4). Preshift
16 measurement of FVC, FEVi, and PEFR did not differ between urea plant and ammonia plant
17 workers. Significant cross-shift reductions in FVC and FEVi were reported in the urea plant
18 (2 and 3%, respectively,/* < 0.05), but not in the ammonia plant. When controlled for current
19 smoking, a significant decrease in AFEVi% was observed in the urea plant (p < 0.05). Among
20 23 workers with concurrent measurements of ammonia and lung function on the same shift,
21 ammonia exposure was correlated with a cross-shift decline in FEVi of 3.9% per unit of log-
22 transformed ammonia concentration in ppm. EPA identified a NOAEL of 6.9 ppm (4.9 mg/m3)
23 and a LOAEL of 26.1 ppm (18.5 mg/m3) in the Rahman et al. (2007) study based on increased
24 prevalence of respiratory symptoms and an acute decrease in lung function.
25
telephone conversations and e-mails dated June 22, 2010, from Michael Yanosky, Drager Safety Inc., Technical
Support Detection Products to Amber Bacom, Syracuse Research Corporation [contractor to National Center for
Environmental Assessment (NCEA), ORD, U.S. EPA].
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Table 4-4. Prevalence of acute respiratory symptoms and cross-shift
changes in lung function among workers exposed to ammonia in a urea
fertilizer factory
Parameter
Ammonia plant
(4.9 mg/m3)a
Urea plant
(18.5 mg/m3)3
Administration building
(concentration not
determined)1"
Respiratory symptoms
Cough
Chest tightness
Stuffy nose
Runny nose
Sneeze
4/24 (17%)c
4/24 (17%)
3/24 (12%)
1/24 (4%)
0/24 (0%)
18/64 (28%)d
21/64 (33%)d
10/64 (16%)
10/64 (16%)
14/64 (22%)
2/25 (8%)
2/25 (8%)
1/25 (4%)
1/25 (4%)
2/25 (8%)
Lung function parameters (cross-shift percentage change)6' f
FVC
FEVj
PEFR
0.2 ±9.3
3.4 ±13.3
2.9±11.1
-2.3 ±8.8
-1.4 ±8.9
-1.0 ±16.2
ND
ND
ND
ND: no data
"Mean ammonia concentrations measured by the Drager PAC III method.
bConcentrations in the administration building were rejected by study authors due to relatively large drift in the zero
levels.
0Values are presented as incidence (prevalence expressed as a percentage).
dp < 0.05 by Fisher's exact test, comparing exposed workers to administrators.
"Calculated as ((Post shift-preshift)/preshift) x 100.
Values are presented as mean ± SD.
Source: Rahman et al. (2007).
1
2 Tepper et al. (1991) evaluated pulmonary function (FEVi) changes among firefighters
3 from the city of Baltimore 6-10 years after a baseline examination. The eligible study
4 population consisted of 963 firefighters of which 695 participated in the follow-up study.
5 Pulmonary function tests were performed for 628 firefighters. Information about exposures was
6 obtained by questionnaire and by combining data from fire department records regarding the
7 number of fires fought by fire fighting units with individual work histories. To determine the
8 effects of occupational exposures while accounting for confounding by or interaction with other
9 risk factors, multiple linear regression techniques with dichotomous indicator variables were
10 used. Reported exposure to specific chemicals was rare, except for ammonia and chlorine;
11 160 men reported exposure to ammonia and 128 to chlorine. Men with self-reported ammonia
12 exposure experienced a rate of decline in FEVi 1-7 times greater than men without ammonia
13 exposure, but the difference was not statistically significant. NOAEL and LOAEL values were
14 not identified in this study.
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Hamid and El-Gazzar (1996) evaluated changes in serum clinical chemistry as measures
of neurochemical alterations and liver function among workers at a urea production plant in
Alexandria, Egypt. The study group consisted of 60 male workers from the fertilizer plant,
including 30 workers with known exposures to ammonia and 30 workers from the administrative
departments with no known history of exposure to ammonia. The authors indicated that the
exposed population had worked at the fertilizer plant on average for 12 years. The exposed and
reference populations were matched on demographic characteristics including age, educational
status, and socioeconomic status. No information is reported on exposure levels. Blood samples
were collected from each subject and analyzed for aspartate aminotransferase (AST), alanine
aminotransferase (ALT), hemoglobin, blood urea, and monoamine oxidase (MAO) and catalase
activity. Table 4-5 shows statistically significant changes in hemoglobin and serum chemistry.
Mean levels of AST, ALT, and blood urea were significantly elevated among exposed workers
over controls. Mean levels of hemoglobin were significantly lower, and MAO and catalase
enzyme activities were significantly depressed among exposed workers compared to controls. A
correlation analysis showed a positive correlation between catalase activity and levels of
hemoglobin, AST and ALT, and MAO activities. Hamid and El-Gazaar (1996) noted that
inhibition of catalase can affect electrical stability, permeability, and fluidity of membranes,
which may lead to hepatotoxic and neurotoxic alterations in occupationally exposed workers.
NOAEL and LOAEL values were not identified in this study due to the absence of information
on exposures at this fertilizer plant.
Table 4-5. Summary of significant changes in serum from workers
occupationally exposed to ammonia at a fertilizer plant
Parameter
ALT (U/mL)
AST (U/mL)
Hb (%)
Blood urea (mg/mL)
MAO (units)
Catalase (lU/mL)
Controls3
16.0 ±5.59
14.5 ±4.67
14.8 ±2.62
0.203 ±0.0512
31.9±10.1
119.3 ±4.76
Exposed3
19.4±5.69b
17.9±4.14b
12.2±2.29C
0.319 ±0.0755C
20.8±4.30C
80.9±9.31C
"Mean ± standard deviation.
bSignificantly different from controls (p < 0.05).
Significantly different from controls (p < 0.01).
Source: Hamid and El-Gazzar (1996).
22
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1 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
2 ANIMALS—ORAL AND INHALATION
3 4.2.1. Oral Exposure
4 Kawano et al. (1991) investigated the hypothesis that the bacterium Helicobacterpylori
5 (H. pylori), which produces a potent urease that increases ammonia production, plays a
6 significant role in the etiology of chronic atropic gastritis. Male Sprague Dawley rats (6/group)
7 were given tap water, 0.01%, or 0.1% ammonia ad libitum for two or four weeks. The daily dose
8 of 0.01% and 0.1% ammonia in drinking water, based on a weight of 230 g for male rats and a
9 water consumption of 50 mL/day, was estimated to be 22 and 220 mg/kg-day, respectively. The
10 effect of ammonia on the antral mucosa was estimated by three measurements of the thickness of
11 the mucosa about 175 microns from the pyloric ring in the antral mucosa. The parietal cell
12 number per gland was determined at three locations in the oxyntic glandular area. Mucosal
13 lesions were not observed macro- or microscopically. There was a statistically significant
14 decrease in mean antral mucosal thickness with increasing dose and duration of exposure (Table
15 4-6). Parietal cell number per oxyntic gland decreased in a statistically significant dose- and
16 time-dependent fashion. The index of periodic acid-Schiff Alician blue positive intracellular
17 mucin was significantly lower in the antral and body mucosa with 0.1% ammonia; the index was
18 significantly lower only for the antral mucosa with 0.01% ammonia. The authors suggested that
19 administration of ammonia in drinking water causes gastric mucosal atrophy. Based on the
20 reduction in antral mucosal thickness, EPA identified a LOAEL of 22 mg/kg-day; a NOAEL was
21 not identified.
22
Table 4-6. Effect of ammonia in drinking water on the thickness of the
gastric antral and body mucosa of the rat stomach
Length of treatment
Thickness of mucosa (uM); mean ± sem
Control (tap water)
Percent ammonia in drinking water
0.01%
0.1%
Antral mucosa
2 weeks
4 weeks
270 ± 18
276 ± 39
258 ± 22
171±22a
217±40a
109 ± 12b'c
Body mucosa
2 weeks
4 weeks
574 ±116
618 ±154
568 ± 159
484 ± 123
591 ±183
440 ± SO3-0
p < 0.05 vs. control group
p < 0.01 vs. control group
'p < 0.01 versus 2 week treatment group
Source: Kawano etal. (1991).
23
24 In a follow-up study of the effect of ammonia produced from H. pylori, Tsujii et al.
25 (1993) studied the subchronic effect of ammonia in drinking water on the cell kinetics of the
26 gastric mucosa of the stomach. Six groups of Sprague Dawley male rats (36 rats/group) were
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1 given 0.01% ammonia in drinking water for 3 days, or 1, 2, 4 or 8 weeks; ammonia solutions
2 were changed daily. Tap water was provided for the balance of the 8-week study. A control
3 group was given tap water for eight weeks. Based on the initial body weight (150 g) and
4 estimated daily water intake (50 mL), the daily dose at a drinking water concentration of 0.01%
5 ammonia was estimated to be 33 mg/kg-day. Cellular migration was measured by labeling cells
6 with 5-bromo-2-deoxyuridine (BrDU) at different time periods and measuring the incorporation
7 of this modified nucleoside with a histochemical technique using anti BrDU monoclonal
8 antibodies. Antral and body mucosa thickness was measured as described in Kawano et al.
9 (1991). The measurement of cell proliferation in the gastric mucosa was estimated using the
10 labeling index in gastric pits (ratio of labeled nuclei to total nuclei in the proliferation zone). The
11 antral mucosal thickness decreased significantly at 4 and 8 weeks of treatment (Table 4-7) but
12 there was no effect on the body mucosa. Cell migration preceded the decrease in thickness of the
13 antral mucosa. The rate of cell migration (cells/day) toward the mucosal surface was
14 significantly greater for 0.01% ammonia-treated rats compared to the control at 4 and 8 weeks of
15 treatment. Cell proliferation, as estimated from the labeling index, was significantly increased
16 after one week for the antral and body mucosa. The authors concluded that 0.01% ammonia
17 increased epithelial cell migration in the antrum leading to mucosal atrophy. The EPA identified
18 a LOAEL of 33 mg/kg-day based on decreased thickness of the gastric antrum; a NOAEL was
19 not identified.
20
Table 4-7. Effect of ammonia in drinking water on gastric antral and body
mucosa in the stomach of Sprague-Dawley rats administered 0.01%
ammonia in drinking water
Length of treatment
Control (tap water only)
3 Days
1 Week
2 Weeks
4 Weeks
8 Weeks
Thickness of mucosa (jiM)
Antral mucosa
283 ± 26
305 ± 45
272 ±31
299 ± 26
159±29b
168 ± 26b
Body mucosa
534 ±27
559 ±50
542 ± 28
555 ±37
531 ±32
508 ± 29
'Extracted from Figure 3 of Tsujii et al. (1993); mean± SD
'p < 0.05 vs. control (tap w;
Source: Tsujii et al. (1993)
bp < 0.05 vs. control (tap water only) group
21
22 Fazekas (1939) administered ammonium hydroxide to 51 rabbits (strain and sex not
23 specified) via gavage every other day initially and later daily in increasing amounts of 50-80 mL
24 as either a 0.5 or 1.0% solution over a long period of time. The daily dose (mg/kg-day) was
25 estimated using the weight of adult rabbits from standard growth curve for rabbits (3.5-4.1 kg)
26 (U.S. EPA, 1988). Based on daily water consumption of 50-80 mL, a daily dose for the rabbits
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1 receiving 0.5 and 1.0% ammonia in drinking water was approximately 61-110 mg/kg-day and
2 120-230 mg/kg-day, respectively. The exact duration of the study is not reported, but it is clear
3 from the data that by the end of the experiment some rabbits received only three or four doses
4 before dying as a result of intoxication in 5.5 days and other rabbits received over 80 doses and
5 survived for up to 17 months. Toxicological endpoints evaluated included fluctuations in body
6 weights, changes in blood pressure measured at the central artery of the ear in 10 rabbits after
7 lengthy treatment, and changes in the weight, fat and cholesterol content of adrenals. For
8 comparison purposes, the weight of the adrenals from 41 healthy rabbits of similar age and body
9 weight were also determined. The average weight of adrenals from these 41 control rabbits was
10 400.0 ± 13.4 mg.
11 Fazekas (1939) reported that differences in mean adrenal weight in ammonium
12 hydroxide-treated animals were significant, although there was no description of the statistical
13 analysis performed in this study. Chemical evaluation of the adrenals from treated rabbits
14 revealed fat content 4.5 times greater and cholesterol content 6.5 times greater than controls. At
15 the beginning of the experiment, a greater weight loss was observed among those rabbits
16 receiving ammonium hydroxide more frequently (daily) at higher doses. Body weights
17 fluctuated among treated rabbits and generally decreased initially and gradually increased in the
18 later months only to drop again a few weeks before death. Body weights for controls were not
19 reported. Thirteen rabbits exhibited weight increases after the initial loss that persisted until the
20 end of the experiment. Dissection of these rabbits revealed enlarged adrenals (800-1,340 mg),
21 and fatty tissue surrounding the kidneys, mesentery, and the pericardium. This fat accumulation
22 was not observed in untreated controls. Histology revealed enlarged cells of the zona fasciculata
23 of the adrenal cortex that were rich in lipid. The blood pressure of rabbits before dosing ranged
24 from 60 to 74 mm Hg and dropped with initial exposure (during the first 5-10 minutes that lasted
25 up to 7 hours) to 20-30 Hg/mm. Following several months of ammonium hydroxide treatment, a
26 moderate elevation in blood pressure of 10-30 mm Hg was found in 8/10 rabbits. In the other
27 two rabbits, the blood pressure increased from the initial values of 62 and 65 to 90 Hg/mm
28 during the first 7 months of treatment and remained almost unchanged at this level until sacrifice.
29 In summary, Fazekas (1939) concluded that initial decreases in blood pressure and effects
30 of emaciation in rabbits following gavage treatment with ammonium hydroxide is associated
31 with the hypofunction of the cortical or medullary substance of the adrenal gland. The authors
32 also concluded that the subsequent increases in blood pressure and body weight could be
33 attributed to hypertrophy of the adrenal cortex. This study is limited by lack of reporting detail
34 and inadequate study design. The EPA did not identify a NOAEL or LOAEL from this study.
35 Toth (1972) evaluated whether hydrazine, methylhydrazines, and ammonium hydroxide
36 play a role in tumorigenesis in mice. Solutions of hydrazine (0.001%), methyl hydrazine
37 (0.01%), methyl hydrazine sulfate (0.001%), and ammonium hydroxide (0.1, 0.2, and 0.3%)
38 were administered continuously in the drinking water of 5- and 6-week-old randomly bred Swiss
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1 mice (50/sex) for their entire lifetime. For ammonium hydroxide, the study authors reported the
2 average daily drinking water intakes as 9.2, 8.2, and 6.5 mL/day for males for the 0.1, 0.2, and
3 0.3% groups, respectively, and as 8.3, 6.5, and 4.8 mL/day for females, respectively. Given
4 these rates and assuming average default body weights of 37.3 and 35.3 g for males and females,
5 respectively (U.S. EPA, 1988), the approximate continuous doses for ammonium hydroxide are
6 250, 440, and 520 mg/kg-day for males and 240, 370, and 410 mg/kg-day for females.
7 Additionally, groups of CsH mice (40/sex) were exposed to ammonium hydroxide in the
8 drinking water at a concentration of 0.1% for their lifetime. Average daily water consumption
9 for these mice was reported as 7.9 and 8.4 mL/day for males and females, respectively. The
10 approximate equivalent doses for these mice assuming the same default body weights as above
11 (U.S. EPA, 1988) are 191 and 214 mg/kg-day for males and females, respectively. Data were
12 not reported for a concurrent control group. Mice were monitored weekly for changes in body
13 weights and gross pathological changes were recorded. The animals were either allowed to die
14 or were killed when found in poor condition. Complete necropsies were performed on all mice
15 and the liver, kidney, spleen, lung, and organs with gross lesions were processed for
16 histopathological examination. Data on body weights were not reported.
17 For Swiss mice, tumor incidence at the 0.3% ammonium hydroxide concentration was as
18 follows: malignant lymphomas: 3/50 (males), 9/50 (females); and lung adenoma or
19 adenocarcinoma: 7/50 (male), 4/50 (female). Tumor incidence at the 0.2% ammonium
20 hydroxide concentration was: malignant lymphomas: 7/50 (males), 10/50 (females); lung
21 adenoma or adenocarcinoma: 5/50 (male), 8/50 (female); and breast tumors: 4/50 (females).
22 Tumor incidence at the 0.1% ammonium hydroxide concentration was: malignant lymphomas:
23 4/50 (males), 10/50 (females); lung adenoma or adenocarcinoma: 5/50 (male), 12/50 (female);
24 and breast tumors: 1/50 (females). The denominators were not adjusted for survival, and
25 concurrent control data were not provided. For a second strain of mice (CjH) that received 0.1%
26 ammonium hydroxide in drinking water, the incidence of adenocarcinomas of the mammary
27 gland in female mice was 60%. The incidence of breast tumors in the corresponding untreated
28 control mice was 76%. Other tumors were identified in treated rats, but were of low incidence.
29 Toth (1972) concluded that ammonium hydroxide was not carcinogenic in either strain of mouse.
30 Because concurrent control tumor incidence was not provided other than the incidence of breast
31 tumors in CsH female mice, the incidence of tumors in treated mice cannot be independently
32 compared to control tumor incidence.
33 Tsujii et al. (1995, 1992a) evaluated the role of ammonia in H. pylori-related gastric
34 carcinogenesis. H. pylori is a bacterium that produces a potent urease, which generates ammonia
35 from urea in the stomach, and has been implicated in the development of gastric cancer. Tsujii et
36 al. (1995, 1992a) pretreated groups of 40-44 male Sprague-Dawley rats with the initiator N-
37 methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the drinking water for 24 weeks before
38 administering 0.01% ammonium solution as a drinking fluid for 24 weeks. Based on an average
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1 body weight of 523 g for male Sprague-Dawley rats during chronic exposure (U.S. EPA, 1988)
2 and a reported water consumption rate of 0.05 L/day, the approximate continuous dose
3 administered to these rats is 10 mg/kg-day. In each study, an additional group of 40-43 rats
4 given tap water for 24 weeks following pretreatment with MNNG served as controls. The study
5 protocol did not include a dose group that received ammonia only in drinking water. Stomachs
6 from rats surviving beyond 45 weeks were examined histologically for evidence of ulcers,
7 lesions, and tumors. Tsujii et al. (1995) also evaluated serum gastrin levels from blood collected
8 at 30 and 46 weeks and mucosal cell proliferation in animals surviving to 48 weeks by
9 calculating the labeling index (percentage ratio of labeled nuclei to total number of nuclei in the
10 proliferation zone) and the proliferation zone index (fraction of the gastric pit occupied by the
11 proliferation zone).
12 Tsujii et al. (1995, 1992a) observed a significantly greater incidence of gastric cancers
13 among rats receiving ammonia after pretreatment with MNNG compared to rats receiving only
14 MNNG and tap water (p < 0.01, %2 test). Seventy percent of MNNG+ammonia-treated rats
15 versus 31% of control rats developed gastric tumors in the first study (Tsujii et al., 1992a). The
16 number of gastric cancers per tumor-bearing rat in this study was 2.1 ± 1.4 among treated rats
17 and 1.3 ± 0.6 among control rats (p < 0.01, ^ test).
18 In the second study, 66% of rats dosed with ammonia and pretreated with MNNG
19 developed gastric cancers compared to 30% of the control rats (Tsujii et al., 1995). The numbers
20 of gastric tumors per rat in this study were also significantly higher among MNNG+ammonia-
21 exposed rats than controls (p < 0.001, Mann-Whitney test) suggesting that ammonia was a
22 promoter. In the absence of an ammonia-only treatment group, however, it is not possible to
23 distinguish with certainty between possible promotion and initiator activity. The degree of
24 differentiation of adenocarcinomas in control and ammonia-treated rats was significantly
25 different. Ammonia-treated rats also demonstrated a significantly higher incidence of larger
26 tumors (5.3 mm compared to 4.4 mm for controls) and of gastric cancers penetrating the
27 muscularis propria or deeper (p < 0.01, 22% compared to controls 12%). In this study, the
28 labeling index and the proliferation zone index were statistically significantly elevated in
29 ammonia exposed rats compared to controls in the fundic mucosa and antral mucosa.
30 Tsujii et al. (1995) explored the hypothesis that ammonia might increase intragastric pH,
31 leading to an increase in serum gastrin, a trophic hormone in the gastric fundus mucosa, and a
32 possible proliferating factor in gastric epithelial cells. The investigators found no significant
33 effects on serum gastrin levels and concluded that serum gastrin does not appear to play a
34 significant role in ammonia-induced promotion.
35
36 4.2.2. Inhalation Exposure
37 Anderson et al. (1964) exposed a group of 10 guinea pigs (strain not given) and 10 Swiss
38 albino mice of both sexes continuously to 20 ppm (14 mg/m3) ammonia vapors for up to 6 weeks
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1 (anhydrous ammonia, purity not reported). Controls (number not specified) were maintained
2 under identical conditions except for the exposure to ammonia. An additional group of six
3 guinea pigs was exposed to 50 ppm (35 mg/m3) for 6 weeks. The animals were observed daily
4 for abnormal signs or lesions. At termination, the mice and guinea pigs were sacrificed (two per
5 group at 1, 2, 3, 4, and 6 weeks of exposure) and selected tissues (lungs, trachea, turbinates,
6 liver, and spleen) were examined for gross and microscopic pathological changes. No significant
7 effects were observed in animals exposed for up to 4 weeks, but exposure to 20 ppm (14 mg/m3)
8 for 6 weeks caused darkening, edema, congestion, and hemorrhage in the lung. Exposure of
9 guinea pigs to 50 ppm (35 mg/m3) ammonia for 6 weeks caused grossly enlarged and congested
10 spleens, congested livers and lungs, and pulmonary edema. NOAEL and LOAEL values were
11 not identified in this study because only two animals per group were examined at a single time
12 period for the 20 ppm (14 mg/m3) exposure groups (mice and guinea pigs).
13 Coon et al. (1970) exposed groups of male and female Sprague-Dawley and Long-Evans
14 rats, male and female Princeton-derived guinea pigs, male New Zealand rabbits, male squirrel
15 monkeys, and purebred male beagle dogs to 0, 155, or 770 mg/m3 ammonia (0, 219, or
16 1,089 ppm) 8 hours/day, 5 days/week for 6 weeks (anhydrous ammonia, >99% pure). The
17 investigators stated that a typical loaded chamber contained 15 rats, 15 guinea pigs, 3 rabbits,
18 3 monkeys, and 2 dogs. Blood samples were taken before and after the exposures for
19 determination of hemoglobin concentration, packed erythrocyte volume, and total leukocyte
20 counts. Animals were routinely checked for clinical signs of toxicity. At termination, sections
21 of the heart, lung, liver, kidney, and spleen were processed for microscopic examination in
22 approximately half of the surviving rats and guinea pigs and all of the surviving dogs and
23 monkeys. Sections of the brain, spinal cord, and adrenals from dogs and monkeys were also
24 retained, as were sections of the thyroid from the dogs. The nasal passages were not examined in
25 this study.
26 Exposure to 155 mg/m3 ammonia did not result in any deaths or adverse clinical signs of
27 toxicity in any of the animals. Hematological values were within normal limits for the laboratory
28 and there were no significant gross alterations in the organs examined. Microscopic examination
29 showed evidence of focal pneumonitis in the lung of one of three monkeys. Exposure to 770
30 mg/m3 caused initial mild to moderate lacrimation and dyspnea in rabbits and dogs. However,
31 these clinical signs disappeared by the second week of exposure. No significant alterations were
32 observed in hematology tests or upon gross or microscopic examinations at the highest dose.
33 However, consistent nonspecific inflammatory changes (not further described) were observed in
34 the lungs from rats and guinea pigs in the high-dose group that were more extensive than in
35 control animals (incidence not reported).
36 Coon et al. (1970) also exposed rats (15-51/group) continuously to ammonia (anhydrous
37 ammonia, >99% pure) at 0, 40, 127, 262, 455, or 470 mg/m3 (0, 57, 180, 371, 644, or 665 ppm)
38 for 90-114 days. Fifteen guinea pigs, three rabbits, two dogs, and three monkeys were also
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1 exposed continuously under similar conditions to ammonia at 40 mg/m3 (57 ppm) or 470 mg/m3
2 (665 ppm). No significant effects were reported in any animals exposed to 40 mg/m3 and
3 nonspecific inflammatory changes in the lungs and kidneys (not further described) were seen in
4 rats exposed to 127 mg/m3 (180 ppm) ammonia. Exposure to 262 mg/m3 (371 ppm) caused
5 nasal discharge in 25% of the rats and nonspecific circulatory and degenerative changes in the
6 lungs and kidneys (not further described, incidence not reported). A frank effect level at
7 455 mg/m3 (644 ppm) was observed due to high mortality in the rats (50/51). Thirty-two of
8 51 rats died by day 25 of exposure; no histopathological examinations were conducted in these
9 rats. Exposure to 470 mg/m3 (665 ppm) caused death in 13/15 rats and 4/15 guinea pigs and
10 marked eye irritation in dogs and rabbits. Dogs experienced heavy lacrimation and nasal
11 discharge, and corneal opacity was noted in rabbits. Hematological values did not differ
12 significantly from controls in animals exposed to 470 mg/m3 (665 ppm) ammonia.
13 Histopathological evaluation of animals exposed to 470 mg/m3 (665 ppm) consistently showed
14 focal or diffuse interstitial pneumonitis in all animals and also alterations in the kidneys
15 (calcification and proliferation of tubular epithelium), heart (myocardial fibrosis), and liver (fatty
16 change) in several animals of each species (incidence not reported). The study authors did not
17 determine a NOAEL or LOAEL concentration from this study. EPA identified a NOAEL of
18 40 mg/m3 (57 ppm) and a LOAEL of 127 mg/m3 (180 ppm) based on nonspecific inflammatory
19 changes in the lungs and kidneys in rats exposed to ammonia for 90 days.
20 Stombaugh et al. (1969) exposed groups of Duroc pigs (9/group) to measured
21 concentrations of 12, 61, 103, or 145 ppm ammonia (8, 43, 73, or 103 mg/m3) continuously for
22 5 weeks (anhydrous ammonia, purity not reported). Endpoints evaluated included clinical signs,
23 food consumption (measured 3 times/week), weight gain (measured weekly), and gross and
24 microscopic examination of the respiratory tract at termination. A control group was not
25 included. In general, exposure to ammonia reduced food consumption and body weight gain, but
26 since a control group was not used, it is impossible to determine whether this reduction was
27 statistically significant. Food efficiency (food consumed/kg body weight gain) was not affected.
28 Exposure to >103 ppm (>73 mg/m3) ammonia appeared to cause excessive nasal, lacrimal, and
29 mouth secretions and increased the frequency of cough (incidence data for these effects were not
30 reported). Examination of the respiratory tract did not reveal any significant exposure-related
31 alterations. The study authors did not identify a NOAEL or LOAEL concentration from this
32 study. EPA did not identify a NOAEL or LOAEL value for this study due to the absence of a
33 control group.
34 Doig and Willoughby (1971) exposed groups of six specific-pathogen-free derived
35 Yorkshire Landrace pigs to 0 or 100 ppm ammonia (0 or 71 mg/m3) continuously for up to
36 6 weeks. The mean concentration of ammonia in the control chamber was 8 ppm (6 mg/m3).
37 Additional groups of pigs were exposed to similar levels of ammonia as well as to 0.3 mg/ft3 of
38 ground corn dust to simulate conditions on commercial farms. Pigs were monitored daily for
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1
2
o
J
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
clinical signs and changes in behavior. Initial and terminal body weights were measured to
determine body weight gain during the exposure period. Blood samples were collected prior to
the start of each experiment and at study termination for hematology (packed cell volume, white
blood cell, differential leukocyte percentage, and total serum lactate dehydrogenase). Two pigs
(one exposed and one control) were necropsied at weekly intervals and tracheal swabs for
bacterial and fungal culture were taken. Histological examination was conducted on tissue
samples from the lung, trachea, and bronchial lymph nodes.
During the first week of exposure, exposed pigs exhibited slight signs of conjunctival
irritation including photophobia and excessive lacrimation. These irritation effects were not
apparent beyond the first week. Measured air concentrations in the exposure chambers increased
to more than 150 ppm (106 mg/m3) on two occasions. Doig and Willoughby (1971) reported
that at this concentration, the signs of conjunctival irritation were more pronounced in all pigs.
No adverse effects on body weight gain were apparent. Hematological parameters and gross
pathology were comparable between exposed and control pigs. Histopathology revealed
epithelial thickening in the trachea of exposed pigs and a corresponding decrease in the numbers
of goblet cells as shown in Table 4-8. Tracheal thickening was characterized by thinning and
irregularity of the ciliated brush border and an increased number of cell layers. Changes in
bronchi and bronchioles characterized as lymphocytic cuffing were comparable between exposed
and control pigs. Similarly, intraalveolar hemorrhage and lobular atelectasis were common
findings in both exposed and control pigs. Pigs exposed to both ammonia and dust exhibited
similar reactions as those pigs exposed only to ammonia although initial signs of conjunctival
irritation were more severe in these pigs and these pigs demonstrated lesions in the nasal
epithelium similar to those observed in the tracheal epithelium of pigs exposed only to ammonia.
Table 4-8. Summary of histological changes observed in rats exposed to
ammonia for 6 weeks
Duration of exposure
(wks)
1
2
3
4
5
6
Mean ± standard deviation
Thickness of tracheal epithelium (um)
Control
15.7
20.4
20.4
21.8
19.3
18.9
19.4 ±2.1
71 mg/m3 NH3
21.0
29.3
36.6
36.2
33.2
41.6
32.9 ±7.2
Number of tracheal goblet cells (per
500 um)
Control
13.6
22.7
18.9
18.3
20.2
20.0
18.9 ±3.0
71 mg/m3 NH3
24.0
10.3
7.3
10.7
10.0
1.3
10.6 ±7.5
Source: Doig and Willoughby (1971).
25
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1 Doig and Willoughby (1971) concluded that ammonia exposure at 71 mg/m3 may be
2 detrimental to young pigs. The authors suggested that although the structural damage to the
3 upper respiratory epithelium was slight, such changes may cause severe functional impairment.
4 The study authors did not identify a NOAEL or LOAEL concentration from this study. EPA
5 identified a LOAEL of 71 mg/m3 (100 ppm), based on damage to the upper respiratory
6 epithelium. A NOAEL could not be identified from this single-concentration study.
7 Broderson et al. (1976) exposed groups of Sherman rats (5/sex/dose) continuously to
8 10 or 150 ppm ammonia (7 or 106 mg/m3, respectively) for 75 days (anhydrous ammonia, purity
9 not reported). The 10-ppm exposure level represented the background ammonia concentration
10 resulting from cage bedding that was changed 3 times/week. The 150-ppm concentration
11 resulted from cage bedding that was replaced occasionally, but never completely changed. F344
12 rats (6/sex/group) were exposed to ammonia in an inhalation chamber at concentrations of 0 or
13 250 ppm (177 mg/m3) continuously for 35 days. Rats were sacrificed at the end of the exposure
14 period and tissues were prepared for histopathological examination of nasal passages, middle ear,
15 trachea, lungs, liver, kidneys, adrenal, pancreas, testicle, mediastinal lymph nodes, and spleen.
16 Histopathological changes were observed in the nasal passage of rats exposed to 150 ppm
17 (106 mg/m3) for 75 days (from bedding) or 250 ppm (177 mg/m3) for 35 days (inhalation
18 chamber). Nasal lesions were most extensive in the anterior portions of the nose compared with
19 posterior sections of the nasal cavity. The respiratory and olfactory mucosa was similarly
20 affected with a three- to fourfold increase in the thickness of the epithelium. Pyknotic nuclei and
21 eosinophilic cytoplasm were observed in epithelial cells located along the basement membrane.
22 Epithelial cell hyperplasia and formation of glandular crypts were observed and neutrophils were
23 located in the epithelial layer, the lumina of submucosal glands and the nasal passages. Dilation
24 of small blood vessels and edema were observed in the submucosa of affected areas. Collagen
25 replacement of submucosal glands and the presence of lymphocytes and neutrophils were also
26 observed. No histopathological alterations were seen in control rats (10 ppm from bedding or
27 0 ppm from the inhalation chamber). Broderson et al. (1976) did not identify a NOAEL or
28 LOAEL from this study. EPA identified a NOAEL of 10 ppm (7 mg/m3) and a LOAEL of 150
29 ppm (106 mg/m3) based on nasal lesions in rats exposed to ammonia (from bedding) for 75 days.
30 Broderson et al. (1976) also studied the effect of ammonia inhalation on the incidence and
31 severity of murine respiratory plasmosis in mice inoculated with Mycoplasmapulmonis. These
32 results are presented in Section 4.4.3.
33 Gaafar et al. (1992) exposed 50 adult male white albino mice under unspecified
34 conditions to ammonia vapor derived from a 12% ammonia solution (air concentrations were not
35 reported) 15 minutes/day, 6 days/week for up to 8 weeks. Twenty-five additional mice served as
36 controls. Starting the fourth week, 10 exposed and five control mice were sacrificed weekly.
37 Following sacrifice, the nasal mucosa was removed and examined histologically. Frozen
38 sections of the nasal mucosa were subjected to histochemical analysis (succinic dehydrogenase,
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1 nonspecific estrase, acid phosphatase, and alkaline phosphatase [ALP]). Histological
2 examination revealed a progression of changes in the nasal mucosa from exposed rats from the
3 formation of crypts and irregular cell arrangements at 4 and 5 weeks, epithelial hyperplasia,
4 patches of squamous metaplasia, and loss of cilia at 6 weeks, and dysplasia in the nasal
5 epithelium at 7 weeks. Similar changes were exaggerated in the nasal mucosa from rats
6 sacrificed at 8 weeks. Neoplastic changes included a carcinoma in situ in the nostril of one rat
7 sacrificed at 7 weeks that presented with loss of polarity of the epithelium, hyperchromatism and
8 mitotic figures with an intact basement membrane, and an invasive adenocarcinoma in one rat
9 sacrificed at 8 weeks. Histochemical results revealed changes in succinic dehydrogenase, acid
10 phosphatase, and ALP in exposed mice compared to controls (magnitude of change not
11 reported), especially in areas of the epithelium characterized by dysplasia. Succinic
12 dehydrogenase and acid phosphatase changes were largest in the superficial layer of the
13 epithelium, although the acid phosphatase reaction was stronger in the basal and intermediate
14 layers in areas of squamous metaplasia. ALP was strongest in the goblet cells from the basal part
15 of the epithelium and basement membrane.
16 In summary, Gaafar et al. (1992) observed that ammonia exposure induces histological
17 changes in the nasal mucosa of male mice that increase in severity over longer exposure periods.
18 Corresponding abnormalities in histochemistry suggest altered cell metabolism and energy
19 production, cell injury, cell proliferation, and possible chronic inflammation and neoplastic
20 transformation. The study authors did not determine a NOAEL or LOAEL concentration from
21 this study. EPA did not identify a NOAEL or LOAEL because air concentrations were not
22 reported in the study.
23 Done et al. (2005) continuously exposed groups of 24 weaned pigs of several breeds in an
24 experimental facility to atmospheric ammonia at 0, 0.6, 10, 18.8, or 37 ppm (0, 0.4, 7, 13.3, or 26
25 mg/m3) and 1.2, 2.7, 5.1, or 9.9 mg/m3 inhalable dust for 5 weeks (16 treatment combinations).
26 The concentrations of ammonia and dust used were representative of those found commercially.
27 A split-plot design was used in which one dust concentration was allocated to a "batch" (which
28 involved five lots of 24 pigs each) and the four ammonia concentrations were allocated to the
29 four lots within that batch. The fifth lot served as a control. Each batch was replicated. In other
30 words, there were four dust concentrations x four ammonia concentrations plus four controls
31 each replicated once giving 40 lots in total. In total, 960 pigs (460 males and 500 females) were
32 used in the study; 560 pigs were given postmortem examinations. Blood was collected from 15
33 sows before the start of the experiment and tested for porcine reproductive and respiratory
34 syndrome virus (PRRSV) and swine influenza. Five sentinel pigs were sacrificed at the start of
35 each batch and lung, nasal cavity, and trachea, together with material from any lesions, were
36 examined postmortem and subjected to bacteriological examination. Postmortem examination
37 involved examining the pigs' external surfaces for condition and abnormalities, examination of
38 the abdomen for peritonitis and lymph node size, internal gross examination of the stomach for
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1 abnormalities, and gross examination of the nasal turbinates, thorax, larynx, trachea,
2 tracheobronchial lymph nodes, and lung. Pigs were monitored for clinical signs (daily), growth
3 rate, feed consumption, and feed conversion efficiency (frequency of observations not specified).
4 After 37 days of exposure, eight pigs from each lot were sacrificed. Swabs of the nasal cavity
5 and trachea were taken immediately after death for microbiological analysis and the pigs were
6 grossly examined postmortem. On day 42, the remaining pigs were removed from the exposure
7 facility and transferred to a naturally ventilated building for a recovery period of 2 weeks. Six
8 pigs from each lot were assessed for evidence of recovery and the remaining 10 pigs were
9 sacrificed and examined postmortem.
10 The pigs in this study demonstrated signs of respiratory infection and disease common to
11 young pigs raised on a commercial farm (Done et al., 2005). The different concentrations of
12 ammonia and dust did not have a significant effect on the pathological findings in pigs or on the
13 incidence of pathogens. In summary, exposure to ammonia and inhalable dust at concentrations
14 commonly found at pig farms was not associated with increase in the incidence of respiratory or
15 other disease. The study authors did not identify a NOAEL or LOAEL concentration from this
16 study. EPA identified a NOAEL of 26 mg/m3 (37 ppm), based on the lack of respiratory or other
17 disease following exposure to ammonia in the presence of respirable dust.
18 Weatherby (1952) exposed a group of 12 guinea pigs (strain not reported) to a target
19 concentration of 170 ppm (120 mg/m3) 6 hours/day, 5 days/week for up to 18 weeks (anhydrous
20 ammonia, purity not reported). The actual concentration measured in the exposure chamber
21 varied between 140 (99 mg/m3) and 200 ppm (141 mg/m3). A control group of six guinea pigs
22 was exposed to room air. All animals were weighed weekly. Interim sacrifices were conducted
23 at intervals of 6 weeks (four exposed and two control guinea pigs) and the heart, lungs, liver,
24 stomach and small intestine, spleen, kidneys, and adrenal glands were removed for microscopic
25 examination; the upper respiratory tract was not examined. No exposure-related effects were
26 observed in guinea pigs sacrificed after 6 or 12 weeks of exposure. However, guinea pigs
27 exposed to ammonia for 18 weeks showed considerable congestion of the spleen, liver, and
28 kidneys, and early degenerative changes in the adrenal gland. The most severe changes occurred
29 in the spleen and the least severe changes occurred in the liver. The spleen of exposed guinea
30 pigs contained a large amount of hemosiderin and kidney tubules showed cloudy swelling with
31 precipitated albumin in the lumens and some urinary casts (cylindrical structures indicative of
32 disease). The incidence of histopathological lesions was not reported. EPA identified the
33 ammonia concentration of 170 ppm (120 mg/m3) to be a LOAEL based on congestion of the
34 spleen, liver, and kidneys and early degenerative changes in the adrenal gland. A NOAEL could
35 not be identified in this single-concentration study.
36 Curtis et al. (1975) exposed groups of crossbred pigs (4-8/group) to 0, 50, or 75 ppm
37 ammonia (0, 35, or 53 mg/m3) continuously for up to 109 days (anhydrous ammonia, >99.9%
38 pure). Endpoints evaluated included clinical signs and body weight gain. At termination, all
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1 pigs were subjected to a complete gross examination and representative tissues from the
2 respiratory tract, the eye and its associated structures, and the visceral organs (not specified)
3 were taken for subsequent microscopic examination. Weight gain was not significantly affected
4 by exposure to ammonia and the results of the evaluations of tissues and organs were
5 unremarkable. The turbinates, trachea, and lungs of all pigs were classified as normal. The
6 study authors did not identify a NOAEL or LOAEL from this study. EPA identified a NOAEL
7 of 75 ppm (53 mg/m3) based on the absence of effects occurring in pigs exposed to ammonia; a
8 LOAEL was not identified from this study.
9
10 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
11 Diekman et al. (1993) reared 80 crossbred gilts (young female pigs) in a conventional
12 grower from 2 to 4.5 months of age; pigs were exposed naturally during that time to Mycoplasma
13 hypopneumoniae and Pasteurella multocida, which causes pneumonia and atrophic rhinitis,
14 respectively. At 4.5 months of age, the pigs were transferred to environmentally regulated rooms
15 where they were exposed continuously to a mean concentration of ammonia of 7 ppm (range, 4-
16 12 ppm) (5 mg/m3; range, 3-8.5 mg/m3) or 35 ppm (range, 26-45 ppm) (25 mg/m3; range, 18-32
17 mg/m3) for 6 weeks (Diekman et al., 1993). A control group was not included in this study. The
18 low concentration of ammonia was obtained by the flushing of manure pits weekly and the
19 higher concentration of ammonia was maintained by adding anhydrous ammonia (purity not
20 reported) to manure pits that were not flushed. After 6 weeks of exposure, 20 gilts from each
21 group were sacrificed and sections of the lungs and snout were examined for gross lesions. In
22 addition, the ovaries, uterus, and adrenal glands were weighed. The remaining 20 gilts/group
23 were mated with mature boars and continued being exposed to ammonia until gestation day 30,
24 at which time they were sacrificed. Fetuses were examined for viability, weight, and length and
25 the number of copora lutea were counted.
26 Gilts exposed to 35 ppm (25 mg/m3) ammonia gained less weight than gilts exposed to
27 7 ppm (5 mg/m3) during the first 2 weeks of exposure (7% decrease, p < 0.01), but growth rate
28 recovered thereafter. Mean scores for lesions in the lungs and snout were not statistically
29 different between the two exposure groups and there were no differences in the weight of the
30 ovaries, uterus, and adrenals. Age at puberty did not differ significantly between the two groups,
31 but gilts exposed to 35 ppm (25 mg/m3) ammonia weighed 7% less (p < 0.05) at puberty than
32 those exposed to 7 ppm (5 mg/m3). In gilts that were mated, conception rates were similar
33 between the two groups (94.1 vs. 100% in low vs. high exposure). At sacrifice on day 30 of
34 gestation, body weights were not significantly different between the two groups. In addition,
35 there were no significant differences between the two groups regarding percentage lung tissue
36 with lesions and mean snout grade. Number of corpora lutea, number of live fetuses, and weight
37 and length of the fetuses on day 30 of gestation were not significantly different between
38 treatment groups. Diekman et al. (1993) did not identify NOAEL or LOAEL concentrations for
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1 maternal or fetal effects in this study. The EPA did not identify NOAEL or LOAEL values from
2 this study due to the absence of a control group and due to confounding by exposure to bacterial
3 and mycoplasm pathogens.
4
5 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES
6 4.4.1. Acute Oral Studies
7 Two acute oral studies have been conducted using ammonia. Gastrointestinal effects
8 were observed in rats following gavage with ammonium hydroxide (Nagy et al., 1996; Takeuchi
9 et al., 1995). Takeuchi et al. (1995) reported hemorrhagic necrosis of the gastric mucosa in male
10 Sprague-Dawley rats administered ammonium hydroxide once (by gavage) at >1% ammonium
11 hydroxide (dose could not be estimated because gavage volume was not reported). Nagy et al.
12 (1996) observed severe hemorrhagic mucosal lesions in female Sprague-Dawley rats (6/group)
13 15 minutes after exposure to 48 mg/kg ammonium hydroxide via gavage (dose estimated by EPA
14 assuming an average body weight of 0.185 kg).
15 Leakage of cysteine proteases into the gastric lumen occurred 15 minutes after exposure
16 and gastrointestinal effects were prevented by pretreatment with a sulfhydryl alkylating
17 compound (N-ethylmaleimide) (Nagy et al., 1996).
18
19 4.4.2. Acute and Short-Term Inhalation Studies
20 Several acute and short-term inhalation studies (exposure duration of <30 days) have
21 been conducted using ammonia. The lethality of ammonia inhalation has been investigated in
22 rats and mice (rat LCso values >11,590 mg/m3; mouse LCso values >2,990 mg/m3) (Appleman et
23 al., 1982; Kapeghian et al., 1982; Hilado et al., 1977; Silver and McGrath, 1948). Clinical signs
24 of acute ammonia inhalation in rats and mice included eye and nose irritation, dyspnea, ataxia,
25 seizures, coma, and death (Appleman et al., 1982; Kapeghian et al., 1982). Decreased body
26 weight gain was observed in rats, mice, and pigs to ammonia concentrations as low as 400
27 mg/m3 (500 ppm) for up to 30 days (Gustin et al., 1994; Urbain et al., 1994; Kapeghian et al.,
28 1982; Richard et al., 1978a).
29 Ammonia inhalation produced changes in pulmonary function, with decreased respiratory
30 rate observed in rats at concentrations >848 mg/m3 (1,200 ppm) (Rejniuk et al., 2008, 2007), in
31 mice at concentrations >214 mg/m3 (303 ppm) (Kane et al., 1979), and in rabbits at
32 concentrations >35 mg/m3 (50 ppm) (Mayan and Median, 1972). Decreased airway pressure and
33 pO2 were also observed in rabbits exposed to ammonia concentrations >24,745 mg/m3
34 (35,000 ppm) for 4 minutes (Sjoblom et al., 1999). Histopathological changes in the respiratory
35 tract of ammonia-exposed rats (>141 mg/m3; 200 ppm) and mice (>216 mg/m3; 305 ppm)
36 included alterations in the nasal and tracheal epithelium (i.e., irritation and inflammation) and
37 pneumonitis, atelectasis, and intralveolar hemorrhage of the lower lung (Buckley et al., 1984;
38 Kapeghian et al., 1982; Richard et al., 1978a; Gamble and Clough, 1976). Dodd and Gross
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1 (1980) showed that pulmonary function deficits measured 1, 7, 21, and 35 days postexposure
2 were correlated with lung histopathology in cats exposed to ammonia (707 mg/m3 or 1,000 ppm
3 for 10 minutes). Acute effects were followed by chronic respiratory dysfunction that was
4 characterized by secondary bronchitis, bronchiolitis, and bronchopneumonia.
5 Several studies evaluated cardiovascular and/or metabolic effects of acute or short-term
6 ammonia exposure. Bradycardia was observed in rabbits exposed to 1,768 mg/m3 (2,500 ppm)
7 for 180 minutes and arterial pressure variations were seen at 3,535 mg/m3 (5,000 ppm) (Richard
8 et al., 1978b). Acidosis, as evidenced by a decrease in blood pH and an increase in arterial blood
9 pCC>2, occurred in rats exposed to 212 mg/m3 (300 ppm) ammonia for 5, 10, or 15 days
10 (Manninen et al., 1988) and in rabbits exposed to >3,535 mg/m3 (5,000 ppm) for up to
11 180 minutes (Richard et al., 1978b). No effects on blood pH, pC>2, or pCC>2 were seen in rats
12 exposed to <818 mg/m3 (1,157 ppm) (Schaerdel et al., 1983) or cattle exposed to concentrations
13 of <71 mg/m3 (100 ppm) (Mayan and Median, 1976). Several studies also investigated amino
14 acid levels and neurotransmitter metabolism in the brain following acute inhalation exposure to
15 ammonia in rats and mice (Manninen and Savolainen, 1989; Manninen et al., 1988; Sadasivudu
16 et al., 1979; Sadasivudu and Murthy, 1978). It has been suggested that glutamate and y-amino
17 butyric acid (GAB A) play a role in ammonia-induced neurotoxicity. These acute and short-term
18 studies are further described in Appendix C.5.
19
20 4.4.3. Immunotoxicity
21 Secondary infection has been observed in humans who have received severe burns from
22 exposure to highly concentrated aerosols derived from ammonia (Sobonya, 1977; Taplin et al.,
23 1976). However, there is no evidence that the decreased resistance to infection in these cases
24 represents a primary impairment of the immune system in humans since necrosis of exposed
25 tissues facilitates infection by pathogenic organisms. Nevertheless, animal studies have shown
26 that exposure to airborne ammonia may impair immune function as described below (Gustin et
27 al., 1994; Neumann et al., 1987; Targowski et al., 1984; Schoeb et al., 1982; Richard et al.,
28 1978a; Broderson et al., 1976).
29 Broderson et al. (1976) exposed Sherman and F344 rats (11-12/sex/dose) continuously to
30 ammonia concentrations of 108 or 212 ppm ammonia (76 or 150 mg/m3), respectively (from
31 soiled bedding), for 7 days prior to inoculation withM pulmonis and for 30-35 days following
32 inoculation. The matched control groups (bedding changed daily) were exposed to 11 ppm
33 (8 mg/m3, Sherman rats) or 2 ppm (1 mg/m3, F344 rats). Additional groups of F344 rats
34 (6/sex/group) were exposed continuously to 25 (two groups), 50, 100, or 250 ppm ammonia (18,
35 35, 71, or 177 mg/m3, respectively) in an inhalation chamber for 7 days prior to inoculation with
36 M. pulmonis and for up to 42 days following inoculation. Each treatment group had a
37 corresponding control group that was inoculated withM pulmonis in order to produce murine
38 respiratory mycoplasmosis (MRM). Clinical observations were performed (frequency not
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given). Rats were sacrificed at the end of the exposure period and tissues were prepared for
histopathological examination of nasal passages, middle ear, trachea, lungs, liver, kidneys,
adrenal, pancreas, testicle, spleen, mediastinal lymph nodes, and spleen.
Typical signs of MRM (i.e., snuffling, head shaking) began in all rat groups
approximately 10 days after inoculation. Rats exposed to ammonia exhibited dyspnea and
hyperpnea, had rough coats, and sat in a hunched posture. All ammonia concentrations
significantly increased the severity of rhinitis, otitis media, tracheitis, and pneumonia
characteristic of M. pulmonis. Furthermore, there was a significant concentration response
between observed respiratory lesions and increasing environmental ammonia concentration for
gross and microscopic lesions (see Table 4-9). All lesions observed were characteristic of MRM.
Gross bronchiectasis and/or pulmonary abscesses and the extent of gross atelectasis and
consolidation were consistently more prevalent in exposed animals at all ammonia
concentrations than in their corresponding controls. Increasing ammonia concentration was
associated with an increasing rate of isolating of M pulmonis from the respiratory tract. EPA
identified a LOAEL of 18 mg/m3 (25 ppm) for increased severity of rhinitis, otitis media,
tracheitis, and pneumonia, and increased incidence of respiratory lesions in F344 rats. A
NOAEL was not identified in this study.
Table 4-9. Incidence of pulmonary lesions in rats inoculated with
M. pulmonis and exposed to ammonia (7 days later for 28-42 days)
Ammonia concentration
(mg/m3)
Incidence of gross lesions (%)
Ammonia
Matched control
Incidence of microscopic lesions (%)
Ammonia
Matched control
Soiled bedding
76
150
20/24 (83%)a
6/22 (27%)
10/23 (43%)
2/24 (8%)
23/24 (96%)a
19/22 (86%)a
14/23 (61%)
8/24 (33%)
Inhalation chamber1"
18
18
40
70
180
5/12 (42%)
6/12 (50%)
8/12 (66%)
4/12 (33%)
10/12 (83%)
1/12 (8%)
4/12 (33%)
2/12 (17%)
1/12 (8%)
2/12 (17%)
9/12 (75%)
9/12 (75%)
10/12 (83%)
12/12 (100%)
12/12 (100%)
6/12 (50%)
7/12 (58%)
9/12 (75%)
6/12 (50%)
2/12 (17%)
19
20
21
zp < 0.01, % test comparing ammonia-exposed group to matched controls.
bp < 0.01, %2 test comparing the sum of all ammonia group to the sum of matched controls. Regression analysis
showed that increasing concentrations of ammonia were related to the increased incidence of gross and microscopic
lesions.
Source: Brodersonetal. (1976).
Richard et al. (1978a) exposed groups of 99 male OFI mice continuously to ammonia
concentrations of 0 or 500 ppm (0 or 354 mg/m3) for 8 hours and for 168 hours prior to infection
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1 by aerosolized P. multocida (2-minute exposure to a predetermined 50% lethal concentration of
2 bacteria). Mice were observed for 12 days for mortality. At 168 hours, a significant increase in
3 mortality was observed among ammonia-exposed mice compared to controls (+36%)
4 (Table 4-10). Based on supporting findings in rat studies conducted by these same researchers
5 (summarized under Section 4.4.2), Richard et al. (1978) suggested that the irritating action of
6 ammonia destroys the tracheobronchial mucosa and causes inflammatory lesions so that the
7 sensitivity to respiratory infection increased with exposure to ammonia for 168 hours, while the
8 same exposure for 8 hours was not sufficient to increase this sensitivity.
9
Table 4-10. Mortality in P. multocida-infected mice exposed to ammonia for
8 or 168 hours
Exposure duration
8h
168 h
Controls
23/49 (47%)a
25/50 (50%)
Exposed (400 mg/m3)
21/49 (43%)
42/49 (86%)b
aNumber of deaths/total number of animals (percentage mortality).
bSignificantly different from controls (p < 0.05, Student's t-test).
Source: Richard et al. (1978a).
10
11 Schoeb et al. (1982) exposed pathogen-free F344 rats (5-15 rats/dose) continuously to
12 ammonia concentrations of <2 ppm (<1 mg/m3; controls, air concentration was usually not
13 measureable and never exceeded 2 ppm) or 100 ppm (71 mg/m3) for either 7 days prior to
14 inoculation withM pulmonis, or simultaneously with inoculation, for up to 28 days. Rats were
15 sacrificed in groups of five at unspecified intervals up to 28 days following inoculation. At
16 sacrifice, serum was collected from each rat for determination of IgG and IgM antibodies against
17 M. pulmonis by enzyme-linked immunosorbent assay. Nasal passages, larynges, tracheas, and
18 lungs were collected for quantitative assessment of M pulmonis growth. Growth of M pulmonis
19 increased most rapidly in the nasal passages and subsequently in the larynx, trachea, and lungs in
20 both control and exposed rats. However, overall growth of M pulmonis was much greater in
21 ammonia-exposed rats than controls. Serum immunoglobin antibody responses (IgG and IgM)
22 were also greater in ammonia-exposed rats than in controls. Schoeb et al. (1982) also conducted
23 two additional in vivo experiments to evaluate pulmonary absorption and clearance of ammonia
24 from the respiratory tract and one in vitro experiment to evaluate whether increased populations
25 ofM pulmonis in ammonia-exposed rats are the result of direct enhancement of growth of the
26 organism or indirect effects on the host. In one in vivo experiment, 11 rats that were exposed in
27 the first experiment to ammonia at 100 ppm (71 mg/m3) for 1 week were exposed under
28 anesthesia to ammonia via face mask at <500 ppm (354 mg/m3) for an unspecified length of
29 time. Tracheas were surgically exposed, cannulated, and attached to a gas detector for
30 measurement of ammonia concentration in the trachea. Ammonia was not detected in the
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1 tracheal air of any rats during exposure to <500 ppm (<354 mg/m3), and measured 10-20 ppm
2 (7-14 mg/m3) in some rats (number not specified) at 500 ppm (354 mg/m3). These results
3 indicate that virtually all ammonia is absorbed in the nasal passages in rats exposed to <500 ppm
4 (<354 mg/m3) ammonia. In the other in vivo experiment, groups of 38 F344 rats were exposed
5 to either 0 or 100 ppm (0 or 71 mg/m3) ammonia for 1 week prior to inoculation with
6 radiolabeled Staphylococcus epidermis (a bacterium commonly present on human skin) to
7 measure pulmonary bacterial clearance. Following inoculation, half of the exposed animals and
8 half of the controls were sacrificed immediately and the remaining rats were sacrificed 6 hours
9 later. There were no significant differences between ammonia-exposed and control rats
10 regarding the rate of pulmonary clearance (combined results showed 91 ± 6% clearance of
11 bacteria for both groups in 6 hours). In vitro, growth of M pulmonis was inhibited by 1 mM
12 ammonium ion added to a culture medium as ammonium hydroxide. Lower concentrations
13 (0.01, 0.1, and 0.5 mM) had no effect. The study authors suggest that these data indicate that
14 ammonia increases growth of M pulmonis indirectly through effects on the host.
15 Hartley guinea pigs (8/concentration, sex not specified) vaccinated with Mycobacterium
16 bovis BCG and challenged intradermally with 2.5 jig of purified protein derivative (PPD) of
17 tuberculin were continuously exposed to ammonia concentrations of 50 or 90 ppm (35 or
18 64 mg/m3) for 3 weeks (Targowski et al., 1984). An additional eight PPD-positive guinea pigs
19 exposed to a normal environment with ammonia concentrations <15 ppm (<11 mg/m3) served as
20 controls. Guinea pigs were again challenged intradermally with PPD following the exposure
21 period. Clinical observations were recorded daily and body weights were measured at the
22 beginning and end of the experiment. Blood samples collected at the beginning and end of the
23 experiment were submitted for determination of ammonia content and hematology (red blood
24 cells and white blood cells). Cultures of separated blood lymphocytes and bronchial leukocytes
25 were evaluated for responsiveness to mitogens (1 ug/mL phytohemagglutin [PHA] or
26 concanavalin A) or 24 |ig/mL PPD. Cultures of alveolar and bronchial macrophages from
27 control and exposed guinea pigs were measured for bactericidal and phagocytic capacities in the
28 presence of Staphylococcus aureus. Targowski et al. (1984) also evaluated the effect of
29 ammonia exposure on the in vitro immune response in lymphocytes and macrophages collected
30 from five normal Hartley guinea pigs. The percentage of viable lymphocytes (unstained) was
31 determined by trypan blue exclusion test at 0, 2, 4, 24, 48, and 72 hours following incubation in
32 medium alone or medium containing ammonia concentrations ranging from 1 to 100 mg/L
33 (ammonium hydroxide added to medium). Macrophages were incubated with concentrations of
34 ammonia ranging from 0.1 to 50 mg/L for 1 hour prior to the addition of S. aureus.
35 Guinea pigs exposed to ammonia at 50 or 90 ppm (35 or 64 mg/m3) did not exhibit any
36 significant changes in body weight and did not show any notable signs of distress, conjunctivitis,
37 or respiratory disease (Targowski et al., 1984). Dermal response to PPD measured as the
38 diameter of erythema was significantly less atp < 0.05 (Student's t-test) in guinea pigs exposed
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to 64 mg/m3 at 24, 72, and 96 hours compared to controls (Table 4-11). No significant
hematological changes were observed between exposed and control groups. The response to
mitogens and PPD of blood lymphocytes and bronchial lymphocytes from animals exposed to 90
ppm (64 mg/m3) ammonia was significantly less than the response from control animals (p <
0.01) (shown graphically by authors). The lymphocyte response for guinea pigs exposed to 50
ppm (35 mg/m3) ammonia was similar to control. In vitro exposure of blood lymphocytes to
ammonia at 10 or 100 mg/L resulted in decreased viability and reduced response to PHA
stimulation (significantly different from controls atp < 0.01). Lower concentrations of ammonia
(<1 mg/L) did not significantly affect viability or suppress the responsiveness of lymphocytes in
vitro. Bactericidal and phagocytic activity of alveolar macrophages from animals exposed to
ammonia was not significantly affected. Similarly, phagocytic activity was not significantly
affected in vitro following treatment with ammonia at 1-100 mg/L. However, a significant
inhibition in the macrophage bactericidal activity was observed in vitro at 0.01 and 0.05 mg/L (p
< 0.01). No significant changes in bactericidal activity were observed in cultures treated with
ammonia at <1 mg/L.
Table 4-11. Dermal response to the injection of tuberculin in animals
exposed to ammonia for 3 weeks (mean diameter of redness in mm)
Time of observation (hrs)
24
48
72
96
Ammonia concentration (mg/m3)
Control
17.6±1.7a
15.5 ±1.9
12.0 ±1.1
10.4 ±1.5
35
15.2 ±2.23
13. 8 ±1.6
12.6 ±1.0
12.6 ±0.7
64
14.4 ± 2.2 b
14.7 ±1.8
8.7±1.4b
0.0b
17
18
19
20
21
22
23
24
25
26
27
"Mean ± standard deviation.
bSignificantly different from controls (p < 0.05, Student's t-test).
Source: Targowskietal. (1984).
Neumann et al. (1987) conducted three experiments to evaluate the ability of unweaned
piglets exposed to ammonia to fight off infection following an intranasal challenge with P.
multocida (Carter type A). In the first experiment (Trial 1), unweaned piglets were exposed to
ammonia at 50 ppm (35 mg/m3) for 42 days either without additional treatment (22 piglets) or
following intranasal challenge with P. multocida (10 piglets). Groups of 8-10 piglets were also
exposed for 35 days during the second experiment (Trial 2) under these same conditions. A
thermo-motor stress swim test (water temperature was 15°C) was also included as an additional
condition during Trial 2. During the third experiment (Trial 3), all three conditions (i.e.,
ammonia inhalation, intranasal challenge with P. multocida and a thermo motor swim test) were
tested in groups of 8-10 piglets; however, the piglets were exposed to 100 ppm (71 mg/m3)
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1 ammonia for 31 days. The test groups were compared to corresponding controls during each
2 trial. Blood samples were collected on multiple days during each trial and analyzed for nucleolar
3 activity of peripheral lymphocytes and determination of gamma globulins. Lavage fluid from the
4 lungs was collected on multiple days to characterize differentiation of bronchoalveolar cells.
5 Additionally, on the 23r day of exposure, a small test group (five test and five control pigs from
6 Trial 2) was exposed to a radioactive aerosol (Tc99m-tagged sulfur colloid complex suspended in
7 NaCl) through a breathing mask for 10 minutes to evaluate pulmonary aerosol distribution
8 patterns. During this test, the dorsal pulmonary radioactivity was recorded using a gamma
9 camera.
10 Lymphocyte nucleolar activity increased in a dose-related manner following ammonia
11 exposure (data not shown) (Neumann et al., 1987). A more pronounced increase was observed
12 in the presence of infection and was further intensified under additional thermo-motor stress.
13 Neumann et al. (1987) suggested that ammonia concentrations of 35 and 71 mg/m3 can cause
14 activation of RNA synthesis in the cell nucleus of the peripheral lymphocytes of piglets, as
15 evidenced by an increase in the percentage of lymphocytes with compact nucleoli. Gamma
16 globulin concentrations were not significantly affected at 35 mg/m3 ammonia, but in pigs
17 inhaling 71 mg/m3 ammonia, gamma globulin concentrations were significantly lower than
18 controls (a = 0.01, Wilcoxon, Mann, and Whitney U-test) as early as 8 days after starting the
19 test. This effect was intensified in the presence of infection and additional thermo motor stress.
20 No differences in bronchoalveolar cells were observed in pigs exposed to either 35 or 71 mg/m3
21 compared to controls. However, a significantly higher proportion of neutrophils was detected in
22 lavage fluid from infected pigs exposed to 35 mg/m3 ammonia compared with infected controls
23 at 7 days postinfection (a = 0.02, Wilcoxon, Mann, and Whitney U-test). Under additional
24 stress, the proportion of neutrophils at 7 days post infection was slightly less than in pigs that
25 were not put under additional stress, but remained significantly elevated above controls (a =
26 0.10, Wilcoxon, Mann, and Whitney U-test). In addition, the proportion of alveolar
27 macrophages in the lavage fluid of pigs exposed to 35 mg/m3 ammonia was significantly lower
28 in infected pigs (a = 0.05, Wilcoxon, Mann, and Whitney U-test) and in infected pigs subjected
29 to the swimming test (a = 0.10, Wilcoxon, Mann, and Whitney U-test) at postinfection day 7.
30 Under these conditions, pigs exposed to 71 mg/m3 demonstrated similar differentiation in
31 bronchoalveolar cells with controls at postinfection day 7. However, significant differences from
32 controls in the proportions of alveolar macrophages and neutrophils were observed at
33 postinfection day 3 among these pigs. In summary, results of the experiments conducted by
34 Neumann et al. (1987) suggested that ammonia exposure in young pigs has the potential to
35 reduce systemic resistance to infection as evidenced by lower serum gamma globulin
36 concentrations and elevated nucleolar activity of peripheral lymphocytes among exposed piglets.
37 Local resistance to infection in the lungs measured by pulmonary accumulation of aerosol
38 particles and quantitative cytology of the broncho-alveolar space was also reduced in piglets
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1 exposed to ammonia. This study suggests that ammonia toxicity may play an important role in
2 infectious respiratory diseases of swine.
3 F344/N rats (48/group, sex not specified) inoculated with M. pulmonis were exposed to
4 ammonia concentrations of 0 or 100 ppm (0 or 71 mg/m3) for 3, 5, 7, or 9 days (Pinson et al.,
5 1986). Following the exposure periods, rats were killed and their respiratory organs were
6 collected for histological examination. Ammonia-exposed rats exhibited hyperplastic and
7 degenerative changes in the anterior nasal epithelium. Lesions of mycoplasmosis were more
8 severe in ammonia-exposed rats than controls.
9 Gustin et al. (1994) injected endotoxin (E. coli O127:B8) into the ventilated perfused
10 lungs collected from 7 to 23 male Belgian Landrace pigs exposed to ammonia concentrations of
11 0, 25, 50, or 100 ppm (0, 18, 35, or 71 mg/m3) ammonia for 6 days. Lethargy and decreased
12 body weight gain were observed at all ammonia concentrations. Blood samples taken on the first
13 and last day of the exposure period revealed no changes in the level of cortisol, total leukocyte
14 numbers, or in differential leukocyte percentages. No significant differences were observed on
15 the baseline pulmonary hemodynamics and microvascular permeability in perfused lungs from
16 any control or exposed pigs (see Section 4.4.2). However, the endotoxin-induced vascular
17 reaction was strongly altered in lungs from pigs exposed to 100 ppm (71 mg/m3) where the
18 endotoxin-induced increase in total blood flow resistance was completely abolished. The
19 vascular response to endotoxin was also reduced in pigs exposed to 50 ppm (35 mg/m3). In
20 summary, concentrations of ammonia >50 ppm (35 mg/m3) were shown to modulate the
21 pulmonary vascular response to endotoxins in pig lungs in vitro.
22
23 4.5. MECHANISTIC STUDIES
24 Studies have been conducted to investigate the mechanisms by which ammonia induces
25 irritation and effects on the gastric mucosa. A limited number of studies have looked at the
26 potential for ammonia to induce genetic changes, although the available studies of ammonia
27 genotoxicity are inadequate to characterize the genotoxic potential of this compound.
28 Studies conducted to examine the mechanisms of ammonia-related effects on the central
29 nervous system and kidney were largely conducted using ammonium salts (ATSDR, 2004);
30 however, as discussed in Section 4, because it is unclear the extent to which the anion can
31 influence the toxicity of the ammonium salt, these mechanistic studies are not further reviewed
32 in this assessment. A more detailed review of mechanistic studies of ammonia is provided as
33 supplemental information in Appendix C.6.
34
35 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
36 4.6.1. Oral
37 The only available data for oral exposure to ammonia in humans consists of case reports
38 of individuals involved in the ingestion of household cleaning solutions containing ammonia or
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1 biting into capsules of ammonia smelling salts (Dworkin et al., 2004; Rosenbaum et al., 1998;
2 Christesen, 1995; Wason et al., 1990; Lopez et al., 1988; Klein et al., 1985; Klendshoj and
3 Rejent, 1966). Clinical signs reported in the case studies included headache, stomachache,
4 nausea, dizziness, diarrhea, drooling, erythematous and edematous lips, reddened and blistered
5 tongues, dysphagia, vomiting, oropharyngeal burns, laryngeal and epiglottal edema, erythmatous
6 esophagus with severe corrosive injury, and hemorrhagic esophago-gastro-duodeno-enteritis.
7 Oral toxicity data in animals are limited to 4- and 8-week drinking water studies that
8 examined the effects of ammonia on rat gastric mucosa (Tsujii et al., 1993; Kawano et al. 1991),
9 a gavage study in rabbits exposed to ammonium hydroxide for up to 142 days (Fazekas, 1939),
10 and a lifetime drinking water study in mice (Toth, 1972). Kawano et al. (1991) reported that
11 ammonia administered to Sprague Dawley rats at concentrations of 0, 0.01 and 0.1% for two or
12 four weeks resulted in a statistically significant decrease in mean antral mucosal thickness; the
13 magnitude of the effect increased with dose and duration of exposure. In a follow-up study,
14 Tsujii et al. (1993) reported that treatment with 0.01% ammonia for increased epithelial cell
15 migration in the mucosa of the rat stomach, and in particular in the antrum, leading to a decrease
16 in mucosal thickness and to mucosal atrophy. Fazekas (1939) reported initial decreases in body
17 weights and blood pressure, and elevated adrenal weights in rabbits exposed to ammonium
18 hydroxide at 0.5 or 1% in solution for up to 142 days. Histological changes in the adrenal cortex
19 were also reported. Toth (1972) conducted the only chronic oral study of ammonia in
20 experimental animals, but did not evaluate noncancer endpoints.
21
22 4.6.2. Inhalation
23 Human and animal data indicate that the primary effects of ammonia inhalation are
24 irritation and burns and that the primary targets of ammonia toxicity are the respiratory tract,
25 eyes, and skin (Kerstein et al., 2001; George et al., 2000; Latenser and Lucktong, 2000; Morgan,
26 1997; de la Hoz et al., 1996; Leduc et al., 1992; Holness et al., 1989; Millea et al., 1989; Weiser
27 and Mackenroth, 1989; Burns et al., 1985; Flury et al., 1983; Price et al., 1983; Close et al.,
28 1980; Hatton et al., 1979; Sobonya, 1977; Verberk, 1977; Taplin et al., 1976; Couturier et al.,
29 1971; Heifer, 1971; Slot, 1938).
30 Studies documenting inhalation exposure to ammonia in humans include numerous case
31 reports following acute exposures to high concentrations of ammonia (i.e., accidental spill or
32 release), controlled exposure studies involving exposing volunteers to ammonia vapors to
33 evaluate irritation effects and changes in pulmonary function, studies in livestock farmers, and
34 studies of occupational exposures. Effects reported in case reports of human exposure included
35 acute point-of-contact irritation effects (e.g., burns to body surfaces, mouth, and respiratory tract)
36 and delayed effects including restricted pulmonary function (see Section 4.1.1 and Appendix
37 C.2). Inhalation effects in these studies were often accompanied by dermal and ocular irritation.
38 Primary effects reported in volunteer studies were eye, nose, and throat irritation (see Section
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1 4.1.2 and Appendix C.3). Pulmonary function was not affected in volunteers acutely exposed to
2 ammonia concentrations as high as 71 mg/m3 (100 ppm). Studies comparing the pulmonary
3 function of asthmatics and healthy volunteers exposed to ammonia do not suggest that asthmatics
4 are more sensitive to the pulmonary effects of ammonia. Studies in livestock farmers
5 demonstrated an association between ammonia exposure and impaired respiratory function, but
6 ammonia exposures in these studies were generally confounded by concomitant exposures to
7 airborne dust, bacteria, fungal spores, endotoxin, and mold (see Section 4.1.3 and Appendix
8 C.4). Studies of industrial exposures in workers reported pulmonary effects including coughing,
9 wheezing, phlegm, chest tightness, shortness of breath, and decreased respiratory function (Table
10 4-12) (Rahman et al., 2007; Ali, 2001; Ballal et al., 1998; Holness et al., 1989).
11 There are no chronic inhalation studies of ammonia in animals. The observed effects in
12 subchronic animal studies in which ammonia was administered via inhalation included
13 inflammatory changes in the nasal mucosa and tracheal epithelium of rats and pigs (Broderson et
14 al., 1976; Doig and Willoughby, 1971), and histological changes in the livers, kidneys, lung,
15 spleen, and adrenal gland of rats and guinea pigs (Coon et al., 1970; Weatherby, 1952)
16 (Table 4-12). Exposure durations in these studies ranged from 5 to 18 weeks. NOAELs and
17 LOAELs did not vary greatly across species and strains. LOAELs ranged from 18 to 127 mg/m3
18 and NOAELs ranged from 7 to 53 mg/m3. In three studies, effects were observed at the lowest
19 dose, and NOAELs could not be identified. In the only available study of developmental toxicity
20 of ammonia, no maternal or developmental effects were observed in pigs following gestational
21 exposure to ammonia concentrations up to 25 mg/m3 (Diekman et al., 1993).
22 Acute and short-term studies in animals following inhalation exposure demonstrate eye
23 and nose irritation, dyspnea, ataxia, seizures, coma, and death in rats and mice (rat LCso values
24 >11,590 mg/m3; mouse LCso values >2,990 mg/m3), decreased pulmonary function with
25 decreased respiratory rate in rats at concentrations >848 mg/m3, mice at concentrations
26 >214 mg/m3, and in rabbits at concentrations >35 mg/m3. Histopathological changes in the
27 respiratory tract following acute exposure to ammonia include irritation and inflammation of the
28 nasal and tracheal epithelium, pneumonitis, atelectasis, and intralveolar hemorrhage of the lower
29 lung.
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Table 4-12. Summary of noncancer results of human occupational studies and repeat-dose studies in experimental
animals involving inhalation exposure to ammonia
Exposed
population
Exposure
concentration
(mg/m3)/ duration
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Effects at the
LOAEL
concentration
Comment
Reference
Occupational worker studies
Industrial
workers (58 male
workers and 3 1
male controls
from office area)
Industrial
workers (63
ammonia plant
and 77 urea plant
workers; 25
controls from
administration
building)
low (<4.4), medium
(4.4-8.8), and high
(>8.8)
avg exposure: 12 y
ammonia plant
(mean): 4.9
urea plant (mean):
18.5
mean employment
duration: 16 y
8.8
(adjusted: 3. l)a
4.9
Not determined
18.5
Increased prevalence
of respiratory
symptoms and
decrease in lung
function
Cross-sectional study in soda ash plant. No
differences in pulmonary function or subjective
symptomology relative to the control group
Cross-sectional study in urea fertilizer factory.
Concentrations in this table were based on the PAC
III method; concentrations measured using the
Drager tube method were 4-5 times higher.
Holness et al.,
1989
Rahman etal.,
2007
Experimental animal studies
Rat (Sprague-
Dawley and
Longs-Evans)
(15/sex/group)
Sherman rat
(5/sex/group)
F344 rat
(6/sex/group)
Guinea pigs
(12 guinea pigs,
sex not
specified)
0, 40, 127, 262,
455, or 470 for 90-
114d
7 or 106 for 75 d
0, 18, 35, 71, or 177
for up to 49 d
0 or 120, 6 hrs/d,
5d/wkforl8wks
40
7
Not determined
Not determined
127
106
18
120
Nonspecific
inflammatory
changes in the lungs
and kidneys
Nasal lesions
Increased severity of
rhinitis, otitis media,
tracheitis, and
pneumonia, and
increased incidence
of respiratory lesions
Congestion of the
spleen, liver, and
kidneys and early
degenerative changes
in the adrenal gland
Nasal discharge and nonspecific circulatory and
degenerative changes in the lungs and kidneys were
observed at 262 mg/m3 (not further described,
incidence not reported). Frank-effect-level of 455
mg/m3 based on 90-98% mortality
Rats were exposed continuously to ammonia for 7
days prior to inoculation withM. pulmonis and 28-
42 days following inoculation
Coon etal., 1970
Broderson et al.,
1976
Broderson et al.,
1976
Weatherby, 1952
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Table 4-12. Summary of noncancer results of human occupational studies and repeat-dose studies in experimental
animals involving inhalation exposure to ammonia
Exposed
population
Yorkshire-
Landrace pig
(6/group, sex not
specified)
Pig
(4-8/group, sex
not specified)
Pig
(24 pigs/group,
several breeds,
sex not reported)
Exposure
concentration
(mg/m3)/ duration
Oor71for6wks
0,35, or 53 for 109
d
0,0.4,7, 13.3, or 26
for 5 wks
NOAEL
(mg/m3)
Not determined
53
26
LOAEL
(mg/m3)
71
Not identified
Not identified
Effects at the
LOAEL
concentration
Thickening of the
tracheal epithelium
with a concomitant
decrease in the
number of tracheal
epithelial goblet cells
Comment
The mean concentration of ammonia in the control
chamber was measured at 5.6 mg/m3
Exposures were to ammonia at concentrations of 0,
0.4, 7, 13.3, or 26 mg/m3 and to inhalable dust at 1.2,
2.7, 5.1, or 9.9 mg/m3; all pigs including controls
demonstrated infections common to pigs on
commercial farms
Reference
Doig and
Willoughby, 1971
Curtis etal., 1975
Done et al., 2005
""Adjusted to continuous exposure as follows:
VEho = human occupational default minute volume and
= NOAEL x VEho/VEh x 5 days/7 days = 8.8 mg/m3 x 10 m3/20 m3 x 5 days/7 days = 3.1 mg/m3, where:
VEh = human ambient default minute volume.
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1 4.6.3. Mode-of-Action Information
2 Inhalation exposure to ammonia is associated with upper respiratory irritation in humans
3 and animals. Ammonia interacts with moisture along the respiratory tract to form ammonium
4 hydroxide, which causes necrosis of tissue through saponification leading to inflammation
5 (Amshel et al., 2000; Jarudi and Golden, 1973).
6 Oral studies with H. pylori suggest possible mechanisms by which ammonia may cause
7 gastric mucosal damage, namely, increased cell vacuolation and decreased viability of the cells
8 (Megraud et al., 1992), increased release of endothelin-1 and thyrotropin releasing hormone from
9 the gastric mucosa (Mori et al., 1998), release of cysteine proteases in the stomach that
10 contribute to the development of gastric hemorrhagic mucosal lesions (Nagy et al., 1996) and
11 apoptosis directed by mitochondrial membrane destruction (Suzuki et al., 2000).
12
13 4.7. EVALUATION OF CARCINOGENICITY
14 4.7.1. Summary of Overall Weight-of-Evidence
15 Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is
16 "inadequate information to assess the carcinogenic potential" of ammonia based on the absence
17 of ammonia carcinogenicity studies in humans, and a single lifetime drinking water study of
18 ammonia in mice that showed no evidence of carcinogenic potential.
19
20 4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
21 Human data on the carcinogenic effects of ammonia are not available. Animal
22 carcinogenicity data are limited. Toth (1972) did not observe any evidence for carcinogenicity
23 of ammonium hydroxide administered orally to mice over a lifetime. Tsujii et al. (1995, 1992)
24 suggest that ammonia administered in drinking water may act as a cancer promoter in H. pylori
25 induced gastric cancer. In these studies (Tsujii et al., 1995, 1992), rats dosed with ammonia and
26 pretreated with MINING had a greater incidence of gastric cancer and number of tumors per
27 tumor-bearing rat than rats receiving only MNNG and tap water (Tsujii et al., 1995, 1992),
28 suggesting that ammonia can act as a tumor promoter. The available studies of ammonia
29 genotoxicity are inadequate to characterize the genotoxic potential of this compound.
30
31 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
32 4.8.1. Possible Childhood Susceptibility
33 There are no studies of the toxicity of ammonia in children comparing them to any other
34 life stages that would support an evaluation of childhood susceptibility. Case reports of acute
35 exposure of children (<18 years old) to ammonia are summarized in Table C-l (Dilli et al., 2005;
36 Dworkin et al., 2004; Rosenbaum et al., 1998; Wason et al., 1990; Millea et al., 1989; Lopez et
37 al., 1988; Klein et al., 1985; Close et al., 1980; Hatton et al., 1979; Helmers et al., 1971; Levy et
38 al., 1964); however, these studies do not provide information useful for evaluation of
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1 susceptibility from chronic low-level exposure to ammonia. No experimental animal studies of
2 ammonia were identified that compared effects in juvenile animals to adults that could be used to
3 inform childhood susceptibility in humans.
4
5 4.8.2. Possible Gender Differences
6 There are no studies of gender differences in susceptibility to the toxic effects of
7 ammonia; information from the available toxicity studies of ammonia provides no evidence of
8 gender differences.
9
10 4.8.3. Other Susceptible Populations
11 Persons who suffer from severe liver or kidney disease (including cirrhosis, acute renal
12 failure, or liver failure) may be more susceptible to ammonia intoxication (hyperammonemia), as
13 it is chiefly by the actions of these organs that ammonia is biotransformed and excreted (Cordoba
14 et al., 1998; Gilbert, 1988; Jeffers et al., 1988; Souba, 1987). Individuals with hereditary urea
15 cycle disorders are also at risk (Schubiger et al., 1991). In these disease conditions, it is the brain
16 where ammonia is most toxic, and the elevated ammonia levels that accompany human diseases
17 such as acute liver or renal failure can predispose an individual to encephalopathy; these effects
18 are especially marked in newborn infants (Mifiana et al., 1995; Souba, 1987).
19 Individuals with respiratory disease (e.g., asthma) also could represent a susceptible
20 population; however, controlled human studies that examined both healthy volunteers and
21 volunteers with asthma, as well as cross sectional studies of livestock farmers, exposed to
22 ammonia (Petrova et al., 2008; Sigurdarson et al., 2004; Vogelzang et al., 2000, 1998, 1997;
23 Preller et al., 1995) generally did not observe a greater sensitivity to respiratory effects in
24 populations with underlying respiratory disease. However, the findings from an epidemiological
25 study of a group of workers chronically exposed to airborne ammonia indicated that ammonia
26 inhalation can exacerbate existing symptoms, including cough, wheeze, nasal complaints, eye
27 irritation, throat discomfort, and skin irritation (Holness et al., 1989).
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5. DOSE-RESPONSE ASSESSMENTS
4 5.1. ORAL REFERENCE DOSE (RfD)
5 An RfD for ammonia was not derived because the available oral data for ammonia were
6 considered insufficient to support derivation of a chronic reference value. As discussed in
7 Sections 4.1.1, 4.2.1, and 4.6.1, oral toxicity data in humans are limited to case reports of
8 individuals suffering from acute neurological (e.g., headache, dizziness) and gastrointestinal
9 (e.g., stomach ache, nausea, diarrhea, distress and burns along the digestive tract) effects from
10 ingesting household cleaning solutions containing ammonia or biting into capsules of ammonia
11 smelling salts. In animals, the only chronic toxicity study of ammonia is the lifetime
12 carcinogenicity drinking water study in mice by Toth (1972) that reported tumor incidence only
13 and did not provide noncancer data to support development of an RfD. A subchronic gavage
14 study in rabbits exposed to ammonium hydroxide for up to 142 days (Fazekas, 1939) is available
15 but was inadequate for deriving an oral RfD because of limited reporting of study details and
16 results, as well as inadequate study design. The only remaining oral study is an eight-week
17 drinking water study in Sprague Dawley rats (Tsujii et al., 1993) that examined the effects of
18 ammonia on the gastric mucosa.
19 Tsujii et al. (1993) found that ammonia at a concentration of 0.01% (equivalent to a daily
20 dose of 33 mg/kg-day) increased epithelial cell migration in the mucosa of the stomach (in
21 particular the antrum) leading to a statistically significant decrease in the thickness of the antral
22 mucosa at 4 and 8 weeks of treatment; there was no effect on the body mucosa. The authors
23 reported that the gastric mucosal effects observed in rats resemble mucosal changes in human
24 atrophic gastritis. EPA identified a LOAEL for the Tsujii et al. (1993) study of 33 mg/kg-day
25 based on decreased gastric mucosal thickness, an effect considered by EPA to be adverse; a
26 NOAEL was not identified. The Tsujii et al. (1993) study and decreased antral mucosal
27 thickness were considered as a potential principal study and critical effect. EPA identified a
28 potential point of departure (POD) based on the LOAEL of 33 mg/kg-day; BMD modeling was
29 not utilized because this single-concentration study is not amenable to dose-response analysis.
30 In EPA's guidance document entitled, Recommended Use of Body Weight3'4 as the
31 Default Method in Derivation of the Oral Reference Dose (U. S. EPA, 2011 a), the Agency
32 endorses a hierarchy of approaches for converting doses administered orally to laboratory animal
33 species to human equivalent oral exposures in deriving the RfD, with the preferred approach
34 being physiologically-based toxicokinetic modeling. An alternate approach includes using
35 chemical-specific information in the absence of a complete physiologically-based toxicokinetic
36 model. In lieu of a toxicokinetic model or chemical-specific data to inform the generation of
37 human equivalent oral exposures, EPA endorses body weight scaling to the % power (i.e., BW3 4)
38 as a default to extrapolate lexicologically equivalent doses of orally administered agents from
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1 laboratory animals to humans for the purpose of deriving an RfD. When BW3/4 scaling is used in
2 deriving the RfD, EPA also advocates a reduction in the interspecies uncertainty factor (UFA)
3 from 10 to 3, as BW3/4 scaling addresses predominantly toxicokinetic (and some toxicodynamic)
4 aspects of the UFA.
5 The guidance raises some important uncertainties in applying allometric (more
6 specifically BW3/4) scaling when the critical effect used to derive the reference value is a portal-
7 of-entry effect. No physiologically-based pharmacokinetic model or chemical-specific
8 information exists to inform the generation of human equivalent oral exposures for ammonia.
9 Furthermore, the potential critical effect (i.e., decreased gastric antral mucosal thickness) is
10 considered a portal-of-entry effect based on the following. Tsujii et al. (1993) postulated that the
11 difference in response of the mucosa in the stomach body versus the mucosa of the antrum
12 relates to differences in pH in the two stomach regions. Most ammonia is transformed to
13 ammonium ion in solution at physiological pH; the ratio of ammonia to ammonium ion increases
14 10-fold with each unit rise in pH. In the mucosa of the stomach body—an acid-secreting
15 mucosa—ammonia is protonated to the ammonium ion, which reduces the cytotoxicity
16 associated with nonionized ammonia. In the antral mucosa—a nonacid secreting area of the
17 stomach—pH is higher, resulting in a relatively higher concentration of ammonia and thus
18 enhanced cytotoxicity. Because ammonia toxicity appears to be a function of the
19 physical/chemical environment at the mucosal surface (i.e., a portal-of-entry effect) and it is not
20 clear if regions of the stomach scale allometically across species, a surface area adjustment
21 would be the most relevant for interspecies extrapolation; however, a dose scaling approach
22 involving mass per unit surface area has not been developed (U.S. EPA, 201 la). Therefore,
23 because effects on the gastric antral mucosa are not expected to scale allometrically, a
24 BW3 4 scaling approach has not been applied as a default approach (in combination with a
25 reduced default UF for interspecies extrapolation).
26 An RfD is derived by dividing the POD by a composite uncertainty factor (UF). The
27 UFs, selected based on EPA's A Review of the Reference Dose and Reference Concentration
28 Processes (U.S. EPA, 2002; Section 4.4.5), address five areas of uncertainty. Considering the
29 available oral data, the composite UF for ammonia would be 10,000. In the report, A Review of
30 the Reference Dose and Reference Concentration Processes (U. S. EPA, 2002), the RfD/RfC
31 technical panel concluded that, in cases where maximum uncertainty exists in four or more areas
32 of uncertainty, or when the total uncertainty factor is 10,000 or more, it is unlikely that the
33 database is sufficient to derive a reference value. Therefore, consistent with the
34 recommendations in U.S. EPA (2002), the available oral data for ammonia were considered
35 insufficient to support reference value derivation and an RfD for ammonia was not derived.
36 Route-to-route extrapolation of the inhalation data was considered for deriving the oral
37 RfD; however, in the absence of a PBPK model and because the critical effect from the
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1 inhalation literature is a portal-of-entry effect (respiratory symptoms and lung function changes),
2 route-to-route extrapolation is not supported.
o
J
4 5.1.1. Previous RfD Assessment
5 No RfD was derived in the previous IRIS assessment for ammonia.
6
7 5.2. INHALATION REFERENCE CONCENTRATION (RfC)
8 5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
9 Inhalation studies of ammonia exposure in humans include numerous case reports
10 following acute exposures to high concentrations (e.g., accidental spills/releases), controlled
11 exposure studies involving volunteers exposed to ammonia vapors for short periods of time to
12 evaluate irritation effects and changes in pulmonary function, studies in livestock farmers, and
13 studies of industrial occupational exposures comparing the prevalence of acute respiratory
14 symptoms and changes in lung function between exposed and nonexposed worker populations.
15 Studies in experimental animals (including rats, guinea pigs, and pigs) have also examined
16 respiratory and other systemic effects of ammonia following subchronic inhalation exposures.
17 Acute exposure studies (i.e., case reports, controlled volunteer studies) involved
18 exposures too brief in duration to be used for derivation of a chronic RfC. Further, case reports
19 of acute exposure do not typically have the appropriate exposure information necessary for a
20 valid evaluation of dose response. Studies of livestock farmers are also not suitable for dose-
21 response assessment because of multiple and possibly confounding exposures to dusts,
22 endotoxins, bacteria, fungi, molds, and other chemicals (including potentially volatile chemicals
23 for which monitoring data have not been collected).
24 There are two occupational studies of ammonia exposure in workers for which
25 quantitative dose-response information is available. Holness et al. (1989) reported similar odor
26 detection thresholds, lung function, and prevalence in the reporting of acute respiratory
27 symptoms between workers exposed to a mean TWA ammonia concentration of 9.2 ppm
28 (6.5 mg/m3) and a nonexposed worker population. When the exposed workers were grouped into
29 three exposure categories (high: >12.5 ppm [8.8 mg/m3], medium: 6.25-12.5 ppm [4.4-
30 8.8 mg/m3], and low: <6.25 ppm [4.4 mg/m3]), no statistically significant differences (p < 0.05)
31 in symptoms or changes in lung function between the groups were evident. Based on lack of
32 symptomology and changes in lung function, EPA identified a NOAEL of 12.5 ppm (8.8 mg/m3)
33 for the Holness et al. (1989) study based on the grouping of workers into three exposure
34 categories. In a more recent occupational study, Rahman et al. (2007) reported a significantly
35 higher prevalence of acute respiratory symptoms among workers exposed to 26.1 ppm
36 (18.5 mg/m3) ammonia in the urea plant of a fertilizer factory than in workers exposed to
37 6.9 ppm (4.9 mg/m3) ammonia in the ammonia plant or nonexposed workers in the
38 administration building. Furthermore, workers in the urea plant demonstrated significant cross-
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1 shift (comparing preshift measurements to postshift measurements) changes in lung function
2 (i.e., FVC and FEV decreased significantly). A similar cross-shift change in lung function was
3 not observed among workers in the ammonia plant. EPA identified a NOAEL (4.9 mg/m3) and a
4 LOAEL (18.5 mg/m3), based on increased prevalence of respiratory symptoms and decrease in
5 lung function from the Rahman et al. (2007) study. The findings from Holness et al. (1989) and
6 Rahman et al. (2007) are supported by the results from the cross-sectional studies of fertilizer
7 factory workers by Ballal et al. (1998) and Ali (2001), which suggest that occupational exposure
8 to ammonia concentrations >18 mg/m3 are associated with respiratory symptoms and altered
9 pulmonary function.
10 Figure 5-1 compares effect levels from studies of workers occupationally exposed to
11 ammonia with those from studies of human volunteers acutely exposed to ammonia. As shown
12 in this figure, irritation effects in controlled human volunteer studies were generally observed at
13 concentrations higher than those that induced respiratory symptoms in industrially exposed
14 workers; however, Sundblad et al. (2004) reported eye irritation and other symptoms at a
15 concentration below effect levels reported in occupationally-exposed populations. Effects in the
16 Sundblad et al. study were transient, and the investigators noted a tendency towards adaptation.
17 Nevertheless, it is interesting to note that effect levels in acute inhalation studies may occur in
18 the same concentration range as those in occupational exposure settings.
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1000
100
10
O)
3
(A
o
Q.
X,
in
1
0.1
0.01
• I
1 1 .
1 x
•
ANOAEL Vertical lines show range of
exposures in study. Closed
circles show exposures tested in
"LOAEL stuc|y.
Prevalence Prevalence
of of
respiratory respiratory
symptoms symptoms
and and
ung decreased
function lung
Holness function
et al. Rahman
(1989) etal.
(2007)
INDUSTRIAL STUDIES
Cough, Eye No effect Lacrimation, Eye Eye Nasal Eye Severe eye,
lacrimation, irritation on lung transient rritation, irritation irritation irritation nose and
nose and MacEwen function discomfort odor Verberk Petrova Petrova throat
throat et al. Cole Ferguson detection, (1977) et al. et al. irritation
irritation (1970) et al. et al. headache, (2008) (2008) Verberk
Silverman (1 977) (1 977) d zziness, (1 977)
etal. feeling of
(1949) intoxication
Sundblad
etal.
(2004)
"T •
T A _
Lung Lung Lung Broncho- Habituation Odor
function function function constriction to acute detection,
Verberk Sigurdarson Sundblad Douglas irritation moderate to
(1977) et al., et al., and Coe and strong
(2004) (2004) (1987) respiratory irritation
symptoms Altmann
Ihrig et al. et al.
(2006) (2006)
CONTROLLED ACUTE EXPOSURE STUDIES
Figure 5-1. Exposure-response array comparing workers occupationally exposed and human volunteers acutely
exposed to ammonia.
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1 Animal studies of inhalation exposure to anhydrous ammonia include subchronic studies
2 that reported inflammatory changes in nasal mucosa and tracheal epithelium (Gaafar et al., 1992;
3 Broderson et al., 1976; Doig and Willoughby, 1971), and histological changes in liver and
4 kidneys (Coon et al., 1970; Weatherby, 1952). NOAEL and LOAEL values could not be
5 identified for a number of these studies. Studies for which effect levels could be derived are
6 summarized in Table 4-12. Figure 5-2 is an exposure-response array comparing inhalation
7 exposure to ammonia in humans (occupational exposure studies) and animals. As shown, effects
8 in animals are observed near or above the LOAEL observed in the human studies.
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1000
100
10
(A
o
Q.
X,
UJ
0.1
0.01
I
*p
,
• LOAEL
ANOAEL
XFEL
• Intermediatedosi
Vertical lines show
range of exposures
in study. Closed
circles show
exposures tested in
Prevalence of Prevalence of
respiratory symptoms respiratory symptoms
& lung function (male & acute decreased
occupational); lung function
Holnessetal. (1989) (occupational);
Rahman et al. (2007)
HUMAN STUDIES
Upper respiratory
tract - structural
damage (pig); Doig
and Willoughby
(1971)
Nasal lesions (rat);
Broderson et al.
(1976)
Respiratory lesions Respiratory infection
(rat); Broderson et al. in presence of
Nonspecific
inflammatory
Congestion of spleen, Weight gain &
liverS, kidneys, early histological lesions of
(1976) inhalable dust (pig); changes in lungs and degenerative respiratory tract &
Done et al. (2005) kidneys (rat); Coon et changes in adrenal visceral organs
al. (1970) gland (guinea pig); (pig); Curtis etal.
Weatherby(1952) (1975)
EXPERIMENTAL ANIMAL STUDIES
Figure 5-2. Exposure-response array comparing noncancer effects in occupationally exposed workers and
experimental animals exposed to ammonia by inhalation.
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1 As adequate data on the effects of inhalation exposure to ammonia are available in
2 humans for deriving an RfC, animal data are considered in this assessment as supportive. Use of
3 human data to derive the RfC also avoids the uncertainty associated with interspecies
4 extrapolation introduced when animal data are used as the basis for the RfC. The two
5 occupational exposure studies of ammonia by Holness et al. (1989) and Rahman et al. (2007)
6 which examined respiratory symptoms and effects on lung function provide consistent estimates
7 of the effect level for ammonia, with the NOAEL of 8.8 mg/m3 identified from the Holness et al.
8 (1989) study falling between the NOAEL and LOAEL values (4.9 mg/m3 and 18.5 mg/m3,
9 respectively) from the Rahman et al. (2007) study. The Holness et al. (1989) study was selected
10 as the principal study for deriving an RfC over the Rahman et al. (2007) study because it
11 identified the higher NOAEL of these two occupational studies. Consideration of analytical
12 methods also supports the selection of Holness et al. (1989) as the principal study. Rahman et al.
13 (2007) used two analytical methods for measuring ammonia concentrations in workplace air
14 (Drager PAC III and Drager tube); concentrations measured by the two methods differed by
15 four- to five-fold, indicating some uncertainty in these measurements. In contrast, the Holness et
16 al. (1989) study used an established analytical method of measuring exposure to ammonia
17 recommended by NIOSH that involved the collection of air samples on ATSG absorption tubes.
18 Therefore, the Holness et al. (1989) study was selected as the principal study, and
19 increased respiratory symptoms and decreased lung function, considered by EPA to be adverse,
20 were selected as the critical effect.
21
22 5.2.2. Methods of Analysis
23 EPA identified a NOAEL of 8.8 mg/m3, based on the lack of increased respiratory
24 symptoms and decreased lung function in Holness et al. (1989) and selected this as the point of
25 departure (POD) for RfC derivation. This NOAEL was identified as the POD in the context of
26 the entire ammonia database, including other occupational studies that reported pulmonary
27 effects at higher workplace ammonia concentrations (i.e., the LOAEL of 18.5 mg/m3 identified
28 by Rahman et al. (2007)). Additionally, effects in animals are observed near or above the
29 LOAEL observed in human studies, thereby supporting the occupational epidemiology studies.
30
31 5.2.3. RfC Derivation—Including Application of Uncertainty Factors (UFs)
32 The NOAEL of 8.8 mg/m3 identified in the Holness et al. (2007) study is used as the POD
33 for RfC derivation. Since this POD is based on occupational exposure, the value was first
34 adjusted for continuous exposure as follows:
35
36 NOAELADj = NOAEL x VEho/VEh x 5 days/7 days
37 =8.8 mg/m3 x 10 m3/20 m3 x 5 days/7 days
38 =3.1 mg/m3
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1 Where:
2 VEho = human occupational default minute volume (10 m3 breathed during the 8-hour
3 workday, corresponding to a light to moderate activity level [U.S. EPA, 201 lb])
4 VEh = human ambient default minute volume (20 m3 breathed during the entire day)
5
6 The UFs, selected based on EPA's A Review of the Reference Dose and Reference
7 Concentration Processes (U.S. EPA, 2002; Section 4.4.5), address five areas of uncertainty
8 resulting in a composite UF of 10. This composite uncertainty factor was applied to the selected
9 POD to derive an RfC.
10 • An intraspecies uncertainty factor, UFn, of 10 was applied to account for potentially
11 susceptible individuals in the absence of data evaluating variability of response to
12 inhaled ammonia in the human population.
13 • An interspecies uncertainty factor, UFA, of 1 was applied to account for uncertainty in
14 extrapolating from laboratory animals to humans because the POD was based on
15 human data from an occupational study.
16 • A subchronic to chronic uncertainty factor, UFs, of 1 was applied because the
17 occupational exposure period in the principal study (Holness et al., 1989), i.e., mean
18 years at present job for exposed workers, of approximately 12 years was of chronic
19 duration.
20 • A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because a NOAEL
21 value was used as the POD.
22 • A database uncertainty factor, UFD, of 1 was applied to account for deficiencies in the
23 database. The ammonia inhalation database consists of a large number of case reports
24 of acute exposure to high ammonia concentrations (e.g., accidental spills/releases),
25 controlled exposure studies involving volunteers exposed to ammonia vapors for
26 short periods of time to evaluate irritation effects and changes in pulmonary function,
27 studies in livestock farmers, and studies of occupational exposure focused on effects
28 of ammonia on respiratory symptoms and lung function. Studies of the toxicity of
29 inhaled ammonia in experimental animals include subchronic studies in rats, guinea
30 pigs, and pigs that examined respiratory and other systemic effects of ammonia and
31 one limited, reproductive toxicity study in young female gilts pigs. The database
32 lacks developmental and multigeneration reproductive toxicity studies.
33 Ammonia is endogenously produced in humans and animals during fetal and
34 adult life and concentrations in blood are homeostatically regulated to remain at low
35 levels. Baseline blood levels in healthy individuals range from 0.1-1.0 |ig/mL
36 (Monsen, 1987; Conn, 1972; Brown et al., 1957). Evidence in animals (Manninen et
37 al., 1988; Schaerdel et al., 1983) suggests that exposure to ammonia at concentrations
38 up to 18 mg/m3 does not alter blood ammonia levels (see Section 3). Therefore,
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1 exposure at the POD (3.1 mg/m3) associated with respiratory effects following
2 inhalation exposure would not be expected to alter ammonia homeostasis or result in
3 measureable increases in blood ammonia concentrations. Thus, the concentration of
4 ammonia at the POD for the RfC would not be expected to result in systemic toxicity,
5 including reproductive or developmental toxicity. The fact that the fetoplacental unit
6 produces ammonia and that concentrations in human umbilical vein and artery blood
7 (at term) have been shown to be higher than concentrations in maternal blood (see
8 Section 3) also provides assurance that developmental toxicity would not be
9 associated with concentrations of ammonia at or below the POD. As noted in EPA's
10 A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA,
11 2002), "the size of the database factor to be applied will depend on other information
12 in the database and on how much impact the missing data may have on determining
13 the toxicity of a chemical and, consequently, the POD." Because the lack of two-
14 generation reproductive and developmental toxicity studies in the ammonia toxicity
15 database should not impact the determination of ammonia toxicity at the POD, a
16 database UF to account for the lack of these studies was not considered necessary.
17
18 The RfC for ammonia was calculated as follows:
19
20 RfC = NOAELADJ - UF
21 =3.1 mg/m3 H-10
22 = 0.3 mg/m3
23
24 5.2.4. Previous RfC Assessment
25 The previous IRIS assessment for ammonia (posted to the database in 1991) presented an
26 RfC of 0.1 mg/m3 based on co-critical studies—the occupational exposure study of workers in a
27 soda ash plant by Holness et al. (1989) and the subchronic study by Broderson et al. (1976) that
28 examined the effects of ammonia exposure in F344 rats inoculated on day 7 of the study withM
29 pulmonis. The NOAEL of 6.4 mg/m3 (mean concentration of the entire exposed group) from the
30 Holness et al. (1989) study (duration adjusted: NOAELADJ = 2.3 mg/m3) was used to derive the
31 RfC by applying a composite UF of 30, 10 to account for the protection of sensitive individuals
32 and 3 for database deficiencies to account for the lack of chronic data, the proximity of the
33 LOAEL from the subchronic inhalation study in the rat (Broderson et al., 1976) to the NOAEL,
34 and the lack of reproductive and developmental toxicology studies. A database UF larger than 3
35 was not applied because studies in rats (Schaerdel et al., 1983) that showed no increase in blood
36 ammonia levels at an inhalation exposure to 32 ppm and only minimal increases at 300-1000
37 ppm suggested that no significant distribution is likely to occur at the human equivalent
38 concentration.
39
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1 5.3. CANCER ASSESSMENT
2 As discussed in Section 4.7, data are "inadequate for assessing the carcinogenic
3 potential" of ammonia, so no quantitative cancer assessment was conducted. Also, no
4 carcinogenicity assessment was presented in the previous IRIS assessment.
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1 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
2 RESPONSE
o
J
4
5 6.1. HUMAN HAZARD POTENTIAL
6 Ammonia is a colorless gas that occurs naturally in the environment. Ammonia is found
7 in water, soil, and air, and is a source of nitrogen for plants and animals. Most of the ammonia in
8 the environment comes from the natural breakdown of animal waste and dead plants and
9 animals. Ammonia may also be present in the environment as the result of its use as a fertilizer,
10 chemical intermediate, alkalizer, metal treating/extraction agent, water treatment chemical or as
11 emissions from urea selective catalytic reduction aftertreatment systems in diesel vehicles. If
12 compressed or in an aqueous solution, ammonia can occur as a liquid. Ammonia has a very
13 sharp odor that is familiar to most people because of its use in smelling salts and household
14 cleaners. In water, most of the ammonia is converted to the ionic form, ammonium ion.
15 Ammonium ions are not gaseous and have no odor.
16 Ammonia can be absorbed by both the inhalation and oral routes of exposure, but there is
17 less certainty regarding absorption through the skin. Absorption through the eye has been
18 documented. Most of the inhaled ammonia is retained in the upper respiratory tract and is
19 subsequently eliminated in expired air. Both exogenous and endogenously produced ammonia
20 are absorbed in the intestine. Ammonia that reaches the circulation is widely distributed to all
21 body compartments, although substantial first-pass metabolism occurs in the liver, where
22 biotransformation into urea and glutamine occur. In normal circumstances, ammonia exists in
23 the blood as ammonium ion (NH4+). Ammonia or ammonium ion reaching the tissues is utilized
24 for glutamate production, which participates in transamination and other reactions. The principal
25 means of excretion of absorbed ammonia in mammals is as urinary urea; minimal amounts are
26 excreted in the feces and in expired air.
27 As a liquid, ammonia is capable of burning the skin and causing permanent eye damage.
28 Case reports in humans following intentional or accidental ingestion of household cleaning
29 products or ammonia smelling salts observed neurological and gastrointestinal clinical symptoms
30 and burns along the digestive tract. As a gas, ammonia is capable of causing severe eye damage
31 and mild to severe pulmonary irritation and/or inflammation (see human case studies and case
32 reports presented in Appendix C.2). Occupational exposure studies and controlled human
33 exposure studies indicate respiratory symptoms (e.g., coughing, wheezing, etc.), altered lung
34 function, and respiratory irritation following inhalation exposure to ammonia. Animal studies
35 also reported respiratory irritation and inflammation following ammonia inhalation.
36 As discussed in Section 4.7, under EPA's Guidelines for Carcinogen Risk Assessment
37 (U.S. EPA, 2005a), there is "inadequate information to assess the carcinogenic potential" of
38 ammonia. A series of studies provided limited evidence that ammonia may act as a cancer
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1 promoter, and positive responses in a few genotoxicity studies have been reported, including one
2 study in fertilizer factory workers.
o
3
4 6.2. DOSE RESPONSE
5 6.2.1. Noncancer/Oral
6 The available oral data for ammonia were considered insufficient to support reference
7 value derivation. Therefore, an RfD for ammonia was not derived.
8
9 6.2.2. Noncancer/Inhalation
10 The RfC for ammonia of 0.3 mg/m3 was derived using a duration-adjusted NOAEL of
11 3.1 mg/m3 as the POD based on a lack of respiratory symptoms and the absence of changes in
12 lung function at this concentration in occupationally exposed workers at a soda ash plant
13 (Holness et al., 1989). To derive the RfC, the POD was divided by a total UF of 10 to account
14 for human interindividual variability. The default human variability factor was applied because
15 of the lack of quantitative information to assess toxicokinetic or toxicodynamic differences in the
16 range of susceptibilities to ammonia in the human population.
17 A confidence level of high, medium, or low is assigned to the study used to derive the
18 RfC, the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA"BMethods
19 for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry
20 (U.S. EPA, 1994b). The overall confidence in the RfC for ammonia is medium. Confidence in
21 the principal study (Holness et al., 1989) is medium. The design, conduct, and reporting of this
22 occupational exposure study were adequate, but the study was limited by a small sample size and
23 by the fact that workplace ammonia concentrations to which the study population was exposed
24 were below those associated with ammonia-related effects (i.e., only a NOAEL was identified).
25 However, this study is supported in the context of the entire database, including the NOAEL and
26 LOAEL values identified in the Rahman et al. (2007) occupational exposure study, multiple
27 studies of acute ammonia exposure in volunteers, and the available subchronic and chronic
28 inhalation data from animals. Confidence in the database is medium. The inhalation ammonia
29 database includes limited studies of reproductive toxicity and no studies of developmental
30 toxicity; however, reproductive, developmental, and other systemic effects are not expected at
31 the RfC because it is well documented that ammonia is endogenously produced in humans and
32 animals, because ammonia concentrations in blood are homeostatically regulated to remain at
33 low levels, and because ammonia concentrations in air at the POD are not expected to alter
34 homeostasis. Reflecting medium confidence in the principal study and medium confidence in
35 the database, the overall confidence in the RfC is medium.
36
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1 6.2.3. Cancer
2 No cancer assessment was conducted for ammonia due to the lack of adequate
3 carcinogenicity data in humans or animals.
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1 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
2 COMMENTS AND DISPOSITION
3
4
5
6 [To be added]
7
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APPENDIX B. SUMMARY OF REPEAT DOSE TOXICITY INFORMATION FOR SELECTED AMMONIUM SALTS
Table B-l. Summary of noncancer results of repeat dose studies of oral exposure of experimental animals to selected
ammonium salts
Strain/
species/sex
Ammonia
species
Dose
(mg/kg-d)/
duration
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
Effects at the LOAEL
dose
Comment
Reference
Subchronic studies
Wistar rat
(10/sex/group)
Wistar rat
(5 females/
group)
Ammonium
chloride
Ammonium
acetate in diet
and drinking
water
0, 1,590, or
3,050 (males);
0, 1,800, or
3,700 (females)
for 13 wks
(administered
in diet)
17. lor 2,370 in
diet; and 0 or
42.8 in water
continuously
for 90 d
Not determined
Not determined
1,590 (males)
1,800 (females)
2,410 (combined
dose)
Decreased body weights (6-
17% in males; 11-19% in
females), changes in serum
chemistry (increased plasma
chloride and ALP activity),
increased relative kidney
weights (both dose levels,
7-28%) and adrenal weights
(high-dose males, 18%),
metabolic acidosis (males
and females) and
subsequent hypertrophy of
the adrenal zona
glomerulosa (males only)
Depression in body weight
gain
Control diet contained 0.024%
ammonia. Authors suggested
that the transient effects on
GFAP protein and comparable
blood levels were indicative
of an adaptive response.
Lina and Kuijpers,
2004
Bodega etal., 1993
B-l
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Table B-l. Summary of noncancer results of repeat dose studies of oral exposure of experimental animals to selected
ammonium salts
Strain/
species/sex
Ammonia
species
Dose
(mg/kg-d)/
duration
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
Effects at the LOAEL
dose
Comment
Reference
Chronic studies
Sprague-Dawley
rat
(11 males/group)
Wistar rat
(15/sex/group)
Wistar rat
(50/sex/group)
Ammonium
chloride
Ammonium
chloride
Ammonium
chloride
0 or 1,800 for
330 d
(administered
in drinking
water)
0, 481, or
1,020 (males);
0, 610, or 1,370
(females) for
18 mo
0, 455, or
1,000 (males);
0,551, or 1,200
(females) for
30 mo
Not determined
Not determined
Not determined
1,800
481 (males)
610 (females)
455 (males)
551 (females)
Depression in body weights
(13-20% with regular and
low-calcium diets,
respectively), metabolic
acidosis, and loss of bone
(femur) tissue
Metabolic acidosis
Metabolic acidosis and an
increased incidence of
hypertrophy of the adrenal
glomerulosa (males only)
Metabolic acidosis (reduced
blood pH and plasma carbon
dioxide).
Combined effects of calcium
intake and observed that the
loss of bone tissue was
independent of the level of
dietary calcium.
Femur weight significantly
increased (-17%) in high-dose
males. Adrenal and kidney
weights elevated (<15%).
Increased incidence of zona
glomerulosa hypertrophy of
the adrenal at high dose (not
statistically significant).
Severity of metabolic acidosis
increased with dose.
Increased incidence of zona
glomerulosa hypertrophy of
the adrenal in high-dose
females; attributed to chronic
stimulation of the adrenal
cortex by ammonium chloride
induced acidosis. There was
no evidence of a carcinogenic
response.
Barzeletal., 1969
Lina and Kuijpers,
2004
Lina and Kuijpers,
2004
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Table B-l. Summary of noncancer results of repeat dose studies of oral exposure of experimental animals to selected
ammonium salts
Strain/
species/sex
F344 rat
(10/sex/group)
F344 rat
(50/sex/group)
Ammonia
species
Ammonium
sulfate
Ammonium
sulfate
Dose
(mg/kg-d)/
duration
0, 42, 256, or
1,527 (males);
0, 48, 284, or
1,490 (females)
for 52 wks
(administered
in diet)
0, 564, or 1,288
(males); 0, 650,
or 1,371
(females) for
104 wks
(administered
in diet)
NOAEL
(mg/kg-d)
256 (males)
284 (females)
1,288 (males)
1,371 (females)
LOAEL
(mg/kg-d)
1,527 (males)
1,490 (females)
Not determined
Effects at the LOAEL
dose
Elevated relative liver and
kidney weights (7-10%)
Comment
No significant effects on
hematology, serum chemistry,
or histopathology.
Clinical chemistry and
hematology not evaluated;
organ weights not measured.
Incidence of chronic
nephropathy in male rats
increased over control (1/48,
5/49, 3/48 in the control, mid
and high dose); increase was
statistically significant only at
the mid-dose and not dose-
related. There was no
evidence of a carcinogenic
response.
Reference
Ota et al., 2006
Ota et al., 2006
Developmental studies
Wistar rat
(group sizes of
pregnant rats not
reported)
Ammonium
acetate
Oor
21,000 starting
on d 1 of
pregnancy
through
weaning to
PND21
Not determined
(maternal or
developmental)
Maternal effects
not reported
21,000
(developmental)
Reduction in pup body
weight gain
Pups exposed to ammonia
during gestation and lactation
only were resistant to acute
ammonia toxicity induced by
a single injection of 540
mg/kg ammonium acetate.
Minana et al., 1995
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1 APPENDIX C. SUPPLEMENTAL INFORMATION ON AMMONIA
2
3 C.I. AMMONIA LEVELS MEASURED IN EXPIRED AIR IN HUMANS
4 Available data on ammonia levels in the expired air of volunteers are summaried in
5 Table C-l.
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Table C-l. Ammonia levels in exhaled breath of volunteers
Breath samples from the nose and trachea
Test subjects
Three healthy male
volunteers (>30 yrs of
age)
Sixteen healthy subjects
(9 males aged 25-63 yrs
and 7 females aged 23-
41 yrs); subgroups tested
were all male
Breath samples
Ammonia levels measured in nose-
exhaled breath of test subjects each
morning about 2 hrs after eating a
regular breakfast; samples
collected daily over a 4-wk period
Breath samples collected during
quiet nose breathing, and direct
sampling during a deep inspiration
followed by breath-holding with
the glottis closed
Levels of ammonia in exhaled
breath
Volunteer A = 103 ± 1.2 ppb (0.0728
± 0.000848 mg/m3)
Volunteer B = 110 ±1.3 ppb (0.0777 ±
0.000919 mg/m3)
Volunteer C = 83 ± 1.2 ppb (0.0587 ±
0.000848 mg/m3)
(median ammonia levels estimated as
geometric mean ± geometric standard
deviation)
Ammonia concentrations ranged from
0.013 to 0.046 mg/m3 during nose
breathing (median 0.025 mg/m3) (5
male subjects), and 0.029 mg/m3 from
an air sampled collected from the
trachea (collected from a tube inserted
into one male subject's nose and into
the trachea)
Methods
SIFT-MS analysis
Chemiluminescence
Comments
Mean ambient air level of
ammonia was 80 ± 10 ppb
(0.056 ±0.0071 mg/m3)
The authors indicated that
ammonia measured in
mouth-exhaled breath may
be generated in the oral
cavity and suggested that
concentrations in nose-
exhaled breath may better
represent systemic
conditions (such as
metabolic disease)
Reference
Smith et al.,
2008
Larson etal.,
1977
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Table C-l. Ammonia levels in exhaled breath of volunteers
Breath samples from the mouth and oral cavity
Test subjects
Three healthy male
volunteers (>30 yrs of
age)
Twenty-six secondary
school students (10 males
and 16 females, 17-18 yrs
old and one 19 yr old)
Breath samples
Ammonia levels measured in
mouth-exhaled breath and in the
closed mouth cavity of test
subjects each morning about 2 hrs
after eating a regular breakfast;
samples collected daily over a 4-
wk period
Three sequential breath exhalations
collected over 5 min following the
students listening to a 1-hr
presentation (at least 1 hr
following breakfast and before
lunch); alveolar portion measured
(identified using humidity)
Levels of ammonia in exhaled
breath
via Mouth:
Volunteer A = 1,088 ± 1.3 ppb (0.769
± 0.000919 mg/m3)
Volunteer B = 885 ± 1.3 ppb (0.626 ±
0.000919 mg/m3)
Volunteer C = 855 ± 1.3 ppb (0.604 ±
0.000919 mg/m3)
via Oral Cavity:
Volunteer A = 1,465 ± 1.4 ppb (1.04 ±
0.000990 mg/m3)
Volunteer B = 2,146 ± 1.5 ppb (1.52 ±
0.00106 mg/m3)
Volunteer C= 1,859 ± 1.3 ppb (1.31 ±
0.000919 mg/m3)
(median ammonia levels estimated as
geometric mean ± geometric standard
deviation)
Median values reported for:
17 yr olds = 233 ppb (0. 165 mg/m3)
18 yr olds = 346 ppb (0.245 mg/m3)
Methods
SIFT-MS analysis
SIFT-MS analysis
Comments
Mean ambient air level of
ammonia was 80 ± 10 ppb
(0.056 ±0.0071 mg/m3)
The authors indicated that
ammonia measured in
mouth-exhaled breath may
be generated in the oral
cavity and suggested that
concentrations in nose-
exhaled breath may better
represent systemic
conditions (such as
metabolic disease)
Significant differences in
ammonia levels in exhaled
breath between 17 and
18 yr olds (p
-------
Table C-l. Ammonia levels in exhaled breath of volunteers
Breath samples from the mouth and oral cavity
Test subjects
Four healthy children
(2 males and 2 females,
4-6 yrs old)
Thirteen senior volunteers
(1 1 males and 2 females,
60-83 yrs old); four had
type-2 diabetes mellitus
with onset at ages
between 50 and 70 yrs,
and controlled by diet
All subjects had their
regular breakfast without
any specific restrictions
Thirty healthy volunteers
(19 males and 11 females,
24-59 yrs, 28 Caucasian,
1 African, and 1 mixed
race); volunteers were
instructed to maintain
their normal daily
routines and to not rinse
out their mouths prior to
providing a breath sample
Five subjects (2 females,
3 males; age range 27-
65 yrs)
Breath samples
Breath samples collected in
morning at least 1 hr after
breakfast and at least 1 hr prior to
lunch; each volunteer performed
two exhalation/inhalation cycles
(both about 5-10 sec in duration)
Breath samples collected in the
morning prior to lunch at
approximately weekly intervals for
about 6 mo; some volunteers
provided samples more frequently
than others; 480 samples collected
and analyzed for ammonia
Breath samples collected between
8 and 9 am in three sequential
breath exhalations on multiple days
(12-30 d) over the course of a
month
Levels of ammonia in exhaled
breath
Children = range 223-643 ppb
(0. 157-0.454 mg/m3)
Seniors = 317-2,091 ppb (0.224-
1.48 mg/m3)
Geometric mean and geometric
standard deviation = 833 ± 1.62 ppb
(0.589 ±0.001 14 mg/m3)
Median = 842 ppb (0.595 mg/m3)
Range = 248-2,935 ppb (0.175-
2.08 mg/m3)
Ammonia concentrations ranged from
422 to 2,389 ppb (0.298-1.69 mg/m3)
Methods
SIFT-MS analysis
SIFT-MS analysis
SIFT-MS analysis
Comments
Ammonia breath levels
significantly increased with
age
Some seniors reported
diabetes
Measured ammonia level in
breath reported for each
subject
Ammonia breath levels
were shown to increase
with age
Background levels in the
testing laboratory were
typically around 400 ppb
(0.28 mg/m3)
Differences in ammonia
breath levels between
individuals were significant
(p< 0.001)
Reference
Spaneletal.,
2007b
Turner etal.,
2006
Diskin et al.,
2003
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Table C-l. Ammonia levels in exhaled breath of volunteers
Breath samples from the mouth and oral cavity
Test subjects
Six normal nonsmoking
male volunteers (24-
61 yrs old), fasted for
12 hrs prior to testing
Fourteen healthy,
nonsmoking subjects (age
range 2 1-54 yrs)
performed one or more of
the following hygiene
maneuvers:
(1) acidic oral rinse
(pH 2.5),
(2) tooth brushing
followed by acidic oral
rinse,
(3) tooth brushing
followed by distilled
water rinse, and
(4) distilled water rinse
Sixteen healthy subjects
(9 males aged 25-63 yrs
and 7 females aged 23-
41 yrs); subgroups tested
were all male
Breath samples
Baseline breath sample obtained;
breath samples collected 20, 40,
and 60 min and 5 hrs following the
ingestion of a liquid protein-calorie
meal
Subjects fasted for 8 hrs prior to
baseline measurement, refrained
from oral hygiene after their most
recent meal, refrained from heavy
exercise for 12 hrs, and had no
liquid intake for several hours;
initial breath ammonia was
measured between 8 and 10 am,
then subjects performed one or
more of the hygiene measures
listed (at 30-min intervals for a
total 90-min period; samples
collected over 5 min)
Breath samples collected during
quiet mouth breathing
Levels of ammonia in exhaled
breath
Premeal levels ranged from 300 to
600 ppb (0.2-0.4 mg/m3);
Postmeal levels at 30 min were
200 ppb (0. 1 mg/m3) increasing to
maximum values at 5 hrs of 600-
1,800 ppb (0.4-1.3 mg/m3)
Baseline levels varied from 120 to
1,280 ppb (0.085-0.905 mg/m3)
Ammonia concentrations ranged from
0.029 to 0.52 mg/m3 during mouth
breathing (median of 0. 17 mg/m3)
Methods
SIFT-MS analysis
Nitrogen oxide
analyzer with an
ammonia conversion
channel (similar to
chemilumine scence)
Chemiluminescence
Comments
A biphasic response in
breath ammonia
concentration was observed
after eating
An 80-90% depletion of
volatile ammonia emissions
was seen within 10 min of
acid rinsing; less than a
50% depletion of ammonia
was seen following tooth
brushing or distilled water
rinse; gaseous ammonia
levels increased after all
rinse procedures over time
The oral cavity appears to
be a source of breath
ammonia; no attempt was
made to control the diet of
subjects or standardize the
interval between the last
meal and the measurement
Reference
Smith et al.,
1999
Norwood et
al., 1992
Larson etal.,
1977
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Table C-l. Ammonia levels in exhaled breath of volunteers
Breath samples: source (nose/mouth/oral cavity) not specified
Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
Sixteen healthy,
nonsmoking subjects
(4 females and 12 males,
29 ± 7 yrs); no significant
differences in mean age,
height, weight, body mass
index (BMI), or time
since last oral intake;
10 subjects tested in each
experiment
Experiment 1: single whole-breath
samples collected from each
subject (same samples immediately
reanalyzed within <10 sec to assess
instrument specific variability)
Experiment 2: three repeat breath
samples collected from each
subject (to evaluate intra-subject
differences); this experiment
evaluated differences based on
standardization of expiratory
pressure and flow
Experiment 3: two mixed breath
samples and two bag alveolar
breath samples collected in short
succession from each subject
Experiment 1: 1,192 ± 85 ppb
(0.843 ± 0.0601 mg/m3;
median ± measurement error)
Experiment 2:
Nonstandardized = 1,007 ± 184 ppb
(0.712 ± 0.130 mg/m3; median ±
standard deviation)
Standardized = 1,433 ± 160 ppb
(1.01 ±0.113 mg/m3; median ±
standard deviation)
Experiment 3:
Mixed = 1,216 ± 827 ppb (0.860 ±
0.585 mg/m3; median ± standard
deviation)
Alveolar = 1,301 ± 791 ppb (0.920 ±
0.559 mg/m3; median ± standard
deviation)
SIFT-MS analysis
This study establishes
that SIFT-MS analysis
is reliable and
repeatable
Relatively small number of
healthy subjects used
Does not address the breath
of those with disease
Intra-day and inter-day
repeatability were not
investigated
Boshier et al..
2010
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Table C-l. Ammonia levels in exhaled breath of volunteers
Breath samples: source (nose/mouth/oral cavity) not specified
Test subjects
Eight healthy subjects
(average age 39.8 ±
9.6 yrs)
Three groups of children
were used as test subjects:
(1) 68 asthmatic children
residing in a National
Park in the mountains
(mean age 10 yrs,
48 boys, 20 girls),
(2) 52 asthmatic children
in an urban area (mean
age 9 yrs, 35 boys,
17 girls), and
(3) 20 healthy children
from the same urban area
as a control group (mean
age 10, 12 boys, 8 girls)
Breath samples
Subjects fasted for 6 hrs prior to
samples being collected. Subjects
breathed normally into collection
device for 5 min
Subjects performed a 5 -sec breath-
hold and exhaled slowly into
collection device
Levels of ammonia in exhaled
breath
Mean breath ammonia = 0.49 ±
0.24 ppm (0.35 ± 0.17 mg/m3)
Asthmatic children from National
Park = 5.6 ± 4.7 ppb (0.0040 ±
0.0033 mg/m3)
Asthmatic urban children:
Mean NH3= 14.3 ±10.2 ppb
(0.0101 ±0.00721 mg/m3)
Urban control group:
MeanNH3 = 14.8 ± 10.3 ppb
(0.0 105 ±0.00728 mg/m3)
Methods
Fiber optic sensor
Chemiluminescence
Comments
This study measured
ammonia levels in healthy
volunteers compared to
Helicobacter pylori
positive individuals (five
subjects) (data not shown);
the experiment also
included a challenge with a
300 mg urea capsule to
evaluate the urease activity
of healthy vs. infected
individuals (data not
shown); the authors
concluded that breath
ammonia measurement may
be feasible as a diagnostic
test for H. pylori
Both groups of asthmatic
children had some subjects
on glucocorticoids, often
combined with histamine
antagonists and/or b2
agonists, while others were
left untreated; ammonia
concentrations in exhaled
breath appeared to be
correlated with exposure to
urban air
Reference
Kearney et al.,
2002
Girouxetal.,
2002
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1 C.2. HUMAN CASE STUDIES AND REPORTS OF HUMAN EXPOSURE TO
2 AMMONIA
3 Case report findings of injury in adults and children due to exposure to ammonia via
4 inhalation of vapors, dermal contact, or ingestion of household cleaning solutions or ammonia
5 inhalant capsules are presented in Table C-2 below and are organized by exposure route.
6 Oral exposure to ammonia most commonly involved ingestion of household cleaning
7 solutions or biting into the capsules of ammonia smelling salts, which are commonly found in
8 first aid kits. Young children, generally <4 years old, have been reported as "biting into" or
9 ingesting smelling salts capsules. The acute effects included drooling, erythematous and
10 edematous lips, reddened and blistered tongues, dysphagia, vomiting, and oropharyngeal burns
11 (Rosenbaum et al., 1998; Wason et al., 1990; Lopez et al., 1988). Delayed effects were not noted
12 in these cases. Gilbert (1988) reported ammonia intoxication characterized by lethargy,
13 restlessness, irritability, and confusion in a 37-year-old man following surgery. Most other cases
14 of ammonia ingestion involved household cleaning solutions and detergents. Many cases were
15 intentional; however, not all were fatal. Klein et al. (1985) described two cases of ingestion of
16 approximately 30 mL and "two gulps" of Parson's sudsy ammonia (ammonia 3.6%; pH 11.5),
17 respectively. The first case resulted in a white and blistered tongue and pharynx, and esophageal
18 burns with friable, boggy mucosa; and in the second case, several small esophageal lesions with
19 mild to moderate ulceration and some bleeding were reported. There were no oropharyngeal
20 burns in the second case and no delayed complications in either case. Christesen (1995) reported
21 that of 11 cases involving accidental or intentional ingestion of ammonia water by adults
22 (15 years or older), 2 cases exhibited acute respiratory obstruction, and 1 case developed an
23 esophageal stricture 3 months postinjury. In cases involving fatalities, evidence of laryngeal and
24 epiglottal edema, erythmatous esophagus with severe corrosive injury, and hemorrhagic
25 esophago-gastro-duodeno-enteritis was noted (Klein et al., 1985; Klendshoj and Rejent, 1966).
26 Dworkin et al. (2004) reported a case of ingestion of contaminated chicken tenders, prepared and
27 served in a school cafeteria, by approximately 157 students and 6 teachers. The onset of acute
28 symptoms occurred within an hour of ingestion, and included headache, nausea, vomiting,
29 dizziness, diarrhea, and burning mouth. In a case of forced ingestion of an unknown quantity of
30 dilute ammonia, a 14-year-old boy presented with difficulty speaking, ataxic gait, isochoric
31 pupils, and evidence of brain edema. There were no burns to the eyes or mouth and no
32 indication of gastric pathology. It was only after the patient was able to communicate that
33 ammonia was involved that appropriate treatment, followed by a satisfactory outcome, was
34 achieved.
35 Inhalation is the most frequently reported route of exposure and cause of morbidity and
36 fatality, and often occurs in conjunction with dermal and ocular exposures. Acute effects from
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1 inhalation have been reported to range from mild to severe, with mild symptoms consisting of
2 nasal and throat irritation, sometimes with perceived tightness in the throat (Price and Watts,
3 2008; Prudhomme et al., 1998; Weiser and Mackenroth, 1989; Yang, 1987; O'Kane, 1983; Ward
4 et al., 1983; Caplin, 1941). Moderate effects are described as moderate to severe pharyngitis,
5 tachycardia, frothy, often blood-stained sputum, moderate dyspnea, rapid, shallow breathing,
6 cyanosis, some vomiting, transient bronchospasm, edema and some evidence of burns to the lips
7 and oral mucosa, and localized to general rhonchi in the lungs (Weiser and Mackenroth, 1989;
8 Yang, 1987; O'Kane, 1983; Ward et al., 1983; Counturier et al., 1971; Caplin, 1941). Severe
9 effects include second- and third-degree burns to the nasal passages, soft palate, posterior
10 pharyngeal wall, and larynx; upper airway obstruction, loss of consciousness, bronchospasm,
11 dyspnea, persistent, productive cough, bilateral diffuse rales and rhonchi, production of large
12 amounts of mucous, pulmonary edema, marked hypoxemia, local necrosis of the lung,
13 deterioration of the whole lung, and fatality. Delayed effects of acute exposure to high
14 concentrations of ammonia include bronchiectasis, bronchitis, bronchospasm/asthma, dyspnea
15 upon exertion and chronic productive cough, bronchiolitis/, severe pulmonary insufficiency, and
16 chronic obstructive pulmonary disease (Lalic et al., 2009; Leduc et al., 1992; Bernstein and
17 Bernstein, 1989; Flury et al., 1983; Ward et al., 1983; Stroud, 1981; Close, 1980; Taplin, et al.,
18 1976; Walton, 1973; Kass et al., 1972; Slot, 1938).
19 Respiratory effects were also observed following chronic occupational exposure to
20 ammonia. After 18 months and 1 year on the job, respectively, both men developed cough, chest
21 tightness, and wheezing, typically after 2-6 hours from the beginning of each work day, but not
22 on weekends or holidays. In another case, progressive deterioration of the clinical condition of a
23 68-year-old male was documented for 4 years and development of diffuse interstitial and severe
24 restrictive lung disease was reported following long-term repetitive occupational exposure to
25 ammonia at or above the odor recognition level (Brautbar et al., 2003). Lee et al. (1993) report a
26 case of a 39-year-old man who developed occupational asthma 5 months after beginning a job
27 requiring the polishing of silverware. The room in which he worked was poorly ventilated. The
28 product used contained ammonia and isopropyl alcohol and the measured ammonia
29 concentration in the breathing zone when using this product was found to be 8-15 ppm (6-
30 11 mg/m3).
31 Acute dermal exposure to anhydrous (liquid) ammonia and ammonia vapor has resulted
32 in caustic burns of varying degrees to the skin and eyes. There are numerous reports of
33 exposures from direct contact with anhydrous ammonia in which first-, second-, and third-degree
34 burns occurred over as much as 50% of the total body surface (Lalic et al., 2009; Pirjavec et al.,
35 2009; Arwood et al., 1985). Frostbite injury has also been reported in conjunction with exposure
36 to sudden decompression of liquefied ammonia, which is typically stored at -33°F (George et al.,
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1 2000; Sotiropoulos et al., 1998; Arwood et al., 1985). However, direct contact is not a
2 prerequisite for burn injury. Several reports have indicated that burns to the skin occurred with
3 exposure to ammonia gas or vapor. Kass et al. (1972) reported one woman with chemical burns
4 to her abdomen, left knee, and forearm and another with burns to the feet when exposed to
5 anhydrous ammonia gas released from a derailed train in the vicinity. Several victims at or near
6 the scene of an overturned truck that had been carrying 8,000 gallons of anhydrous ammonia
7 were reported as having second- and third-degree burns over exposed portions of the body
8 (Burns et al., 1985; Close et al., 1980; Hatton et al., 1979). In a case involving a refrigeration
9 leak in a poorly ventilated room, workers located in an adjacent room reported a "burning skin"
10 sensation (de la Hoz et al., 1996) while, in another case involving the sudden release of ammonia
11 from a pressure valve in a refrigeration unit, one victim received burns to the leg and genitalia
12 (O'Kane, 1983).
13 In addition to the skin, the eyes are particularly vulnerable to ammonia burns due to the
14 highly water-soluble nature of the chemical and the ready dissociation of ammonium hydroxide
15 to release hydroxyl ions. When ammonia or ammonia in solution has been splashed or sprayed
16 into the face (accidently or intentionally), immediate effects include temporary blindness,
17 blepharospasm, conjunctivitis, corneal burns, ulceration, edema, chemosis, and loss of corneal
18 epithelium (George et al., 2000; Helmers et al., 1971; Highman, 1969; McGuinness, 1969; Levy
19 et al., 1964; Abramovicz, 1924). The long-term effects included photophobia, progressive loss
20 of sensation, formation of bilateral corneal opacities and cataracts, recurrent corneal ulcerations,
21 nonreactive pupil, and gradual loss of vision (Yang, 1987; Kass et al., 1972; Helmers et al.,
22 1971; Highman, 1969; Osmond and Tallents, 1968; Levy et al., 1964; Abramovicz, 1924).
23 White et al. (2007) reported a case with acute bilateral corneal injury that developed into bilateral
24 uveitis with stromal vascularization and stromal haze and scarring, and pigmented keratic
25 precipitates that resulted in legal blindness. An increase in intraocular pressure, resembling
26 acute-angle closure glaucoma, was reported by Highman (1969) following ammonia
27 intentionally sprayed into the eyes during robbery attempts.
28
Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Exposure conditions
Immediate effects"
Delayed effects'"
Reference
Oral exposure
57-yr-old male
Ingested unknown
quantity of dilute
ammonium hydroxide
(2.4% ammonia)
Hemorrhagic esophago-
gastro-duodeno-enteritis,
death
Not applicable
Klendshoj and
Rejent, 1966
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
15-yr-old male
Middle-aged
female
69-yr-old
female
Eight young
children (ages
not given)
Three children,
<4 yrs old
Eleven adults,
>15 yrs
3 -yr-old female
Exposure conditions
Ingestion of about 30 mL
Parson's sudsy ammonia
(ammonia 3. 6%;
pH11.5)
Ingested "two gulps"
Parson's sudsy detergent
ammonia
Ingested an unknown
quantity of Albertson's
lemon ammonia
(ammonia concentration
3%)
Biting into an unbroken
capsule of aromatic
ammonia inhalant
Ingestion of aromatic
ammonia "smelling salt"
capsules that contained
0.33 mL of a mixture of
18% ammonia and 36%
alcohol
Intentional or accidental
ingestion of ammonia
water
Biting into an ammonia
inhalant capsule
Immediate effects"
White and blistered
tongue and pharynx,
esophageal burns with
friable, boggy mucosa; no
other complications
Several small esophageal
lesions with mild to
moderate ulceration and
some bleeding; no
oropharyngeal burns
Lethargy, gurgling
respiratory sounds,
laryngeal, and epiglottal
edema, and a friable,
erythmatous esophagus
with severe corrosive
injury
Vomiting, drooling,
dysphagia, cough, and
oropharyngeal burns
Pain swallowing,
reddened and blistered
tongues, drooling,
erythema, and swelling of
lower lip
Two patients exhibited
acute respiratory
obstruction, no additional
details were provided.
Drooling with multiple
1-2 cm white, ulcerative
lesions on the mid-
posterior upper surface of
the tongue, bilaterally on
the buccal mucosa, and
on the posterior
esophageal wall at the
junction of the middle
and upper thirds of the
esophagus
Delayed effects'"
Not applicable
Not applicable
Renal failure, death
Not applicable
Not applicable
One patient developed
esophageal stricture 3 mo
postinjury
Not applicable
Reference
Klein et al.,
1985
Klein et al.,
1985
Klein et al.,
1985
Lopez et al.,
1988
Wasonetal.,
1990
Christesen, 1995
Rosenbaum et
al., 1998
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Exposure conditions
Immediate effects"
Delayed effects
Reference
2-yr-old female
Biting into an ammonia
inhalant capsule
Drooling, edema, and
erythema on the upper
and lower lips with areas
of desquamation,
superficially ulcerative
lesions on the anterior
dorsum of the tongue;
edema and erythema of
the supraglottic structures
and upper trachea
Not applicable
Rosenbaum et
al., 1998
One hundred
fifty seven
students
(median age
10 yrs) and
six teachers
Ingestion of ammonia-
contaminated chicken
tenders (522-2,468 ppm
[369-1,749 mg/m3])
ammonia in uncooked
chicken tenders)
Stomach ache, headache,
nausea, vomiting,
dizziness, diarrhea, and
mouth burning
Not applicable
Dworkinetal.,
2004
14-yr-old male
Ingestion of an unknown
quantity of dilute
ammonia
Difficulty articulating,
ataxic gait, isochoric
pupils, cranial CT
indicating possible brain
edema; hematology, and
routine biochemical
analysis normal, no burns
of the eyes or mouth, and
no pathology of the
respiratory or
gastrointestinal tract
Not applicable
Dilli etal, 2005
Inhalation-only exposure
39-yr-old male
Fumes created during use
of a silver polishing
product containing
isopropyl alcohol and
ammonia (~12 ppm
8 mg/m3]) in a poorly
ventilated basement
Cough, breathlessness,
and wheezing, rhinitis,
and tearing
Occupational asthma
Lee etal., 1993
68-yr-old male
Long-term, repetitive
occupational exposure to
anhydrous ammonia at or
above odor recognition
threshold for 15-20 yrs
Examination found
persistent pulmonary
infiltrates, mainly in
upper portion of left
chest, diagnosed as
diffuse interstitial lung
disease
Progressive deterioration of
clinical condition over a
4-yr period, development of
diffuse interstitial lung
disease, and severe
restrictive lung disease,
with pulmonary function
tests indicating reduced
diffusion capacity at 47%
Brautbar et al..
2003
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Exposure conditions
Immediate effects"
Delayed effects'"
Reference
Inhalation/dermal/ocular exposure
Dental patient
(age not given)
21-yr-old
female
21-yr-old
female
23-yr-old
female
21-yr-old
female
36-yr-old
female
25-yr-old
female
Nine shelter
occupants (ages
not given)
A 10% ammonia solution
spilled into eyes while
trying to revive patient
from a faint
Explosion in an ice
cream factory; ammonia
pipe burst
Explosion in an ice
cream factory; ammonia
pipe burst
Explosion in an ice
cream factory; ammonia
pipe burst
Explosion in an ice
cream factory; ammonia
pipe burst
Explosion in an ice
cream factory; ammonia
pipe burst
Explosion in an ice
cream factory; ammonia
pipe burst
Ammonia condenser leak
in an air-raid shelter (low
exposure group)
Both eyes red, swollen,
with mucopurulent
secretions, swollen
conjunctiva; the lower
left cornea and sclera
showed loss of
epithelium and the entire
left cornea was dull
Shock, second-degree
burns of both feet, the
right leg, and small area
of the right cheek
Shock, conjunctivitis of
the right eye, severe
tracheitis
Severe shock, persistent,
blood-stained vomiting,
confusion
Shock, hemicranial
headache with nausea
Burns and shock; existing
bronchitis greatly
aggravated
Extreme shock, grey
pallor, burns to the face,
eyes, neck, and both
arms, difficulty /inability
to swallow, labored
breathing
Eye and mouth irritation,
pain on swallowing and
hoarseness, suffused
conjunctivae, swollen
eyelids; lips, mouth, and
tongue were
erythematous with edema
in the back of the throat,
and a strong smell of
ammonia on the breath
Increase in cornea!
ulceration with hypopyon
and perforation was seen
after 8 d; severe
conjunctivitis still evident
4 mo after exposure
Residual bronchitis (not
further described)
Not applicable
Not applicable
Residual bronchitis,
"fullness of the head", and
body pains; congestion of
both lungs
Anxiety symptoms,
insomnia, painful scar
Pulse often imperceptible,
Cheyne-Stokes respiration,
fatality; left lung was
congested, edematous, and a
small hemorrhage at the
base, hemorrhagic
bronchitis, desquamation,
and small epithelial ulcers
of the bronchi
Not applicable
Abramovicz,
1924
Slot, 1938
Slot, 1938
Slot, 1938
Slot, 1938
Slot, 1938
Slot, 1938
Caplin, 1941
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Twenty seven
shelter
occupants (ages
not given)
Eleven shelter
occupants (ages
not given)
17-yr-old male
17-yr-old male
Exposure conditions
Ammonia condenser leak
in an air-raid shelter
(moderate exposure
group; victims in closer
proximity to leak)
Ammonia condenser leak
in an air-raid shelter
(high exposure group,
victims closest to leak
source with longest
exposure time)
Struck by a jet of
anhydrous ammonia
during crop dusting
operations
Sprayed with anhydrous
ammonia during crop
fertilizing operations
Immediate effects"
Cough, blood-stained
sputum, hoarseness, chest
tightness, increased
respiration, hyperaemic
conjunctiva, lacrimation;
moist sounds in the chest;
lips, mouth, tongue, and
soft palate showed
erythema and edema,
with areas of denuded
epithelium scattered over
the buccal mucosa; three
cases showed no
improvement and fatality
occurred within 36 hrs
Slight cyanosis, intense
dyspnea, and persistent
cough with foamy
sputum; weak and rapid
pulse, generalized rales,
and rhonchi; seven cases
showed no improvement,
becoming increasingly
cyanotic and dysphonic
and died within 48 hrs of
exposure
Second-degree burns to
the face, marked edema
of the eyelids, and unable
to open eyes; lips, tongue,
and buccal mucosa were
hyperemic and burned,
swollen uvula (twice
normal size), and
edematous larynx
First- and second-degree
burns to the left arm,
anterior chest, face, and
neck, burning and edema
around the mouth and
eyes, conjunctiva
appeared inflamed and
the mouth was edematous
with the uvula swollen to
twice the normal size,
respiration was labored
and rhonchi were present
throughout the lungs
Delayed effects'"
Nine cases showed initial
improvement, with signs of
bronchopneumonia
appearing on d 2 and 3
postinjury; six recovered
while three died within 2 d
of onset of secondary
infection; autopsy on two
males revealed intensely
inflamed fauces, pharynx,
and larynx; trachea, and
bronchi denuded of
epithelium or inflamed and
filled with purulent exudate;
lungs deeply congested with
bronchopneumonia
The remaining four cases
improved without incident
Permanent loss of vision,
with the exception of
sustained light perception
Chronic persistent cough
with a small amount of
mucoid sputum, no chronic
visual effects
Reference
Caplin, 1941
Caplin, 1941
Levy et al.,
1964
Levy et al.,
1964
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
61-yr-oldmale
28-yr-old male
Male employee
(age not given)
Male victim at
a bank robbery
(age not given)
Exposure conditions
Struck in the face with a
blast of anhydrous
ammonia while
fertilizing crops
Struck in the face by a
spray of anhydrous
ammonia while working
on a refrigeration unit
Exposure for an
unspecified length of
time to ammonia fumes
created when a tanker
overflowed during a
filling operation with
25% ammonia water
Ammonia thrown into
face and forced down
throat and up the nose
Immediate effects"
First-degree burns of the
arms and axillae,
dyspnea, respiratory
distress, red and
edematous eyes, unable
to see clearly, moderate
edema of the mouth and
throat with no clear view
of the larynx, large
amounts of bronchial
secretions
First- and second-degree
burns to the eyelids, face,
anterior chest, ears,
forehead, and upper arms,
edema and erythema of
the pharynx, and
inspiratory and expiratory
rales could be heard over
both lung fields
Vomiting and coughing
initially, with some
difficulty breathing; after
3-hr delay in seeking
medical attention,
exhibited red and swollen
face, conjunctivitis, red
and raw mouth and throat
with a swollen tongue,
loss of speech, dyspnea,
with a weak rhonchi in
left lung, cardiac arrest
and fatality; autopsy
findings included
necrosis of the lung,
inflammation of the
bronchioli; the epithelial
layer of the trachea and
bronchial tubes were
denuded, with an absence
of secretions
Burns to the face, eyes,
and mouth, with severe
edema of the
nasopharynx and glottis
Delayed effects'"
No chronic impairment of
pulmonary function or of
vision occurred
Pulmonary function tests
3 yrs later were
unremarkable
Not applicable
The right eye recovered
within 4 d; the left eye
showed gross chemosis,
corneal staining,
nonreactive pupil, intense
uveitis with aqueous flare
and cells
Reference
Levy et al.,
1964
Levy et al.,
1964
Mulder etal.,
1967
Osmond and
Tallents, 1968
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
57-yr-old male
46-yr-old male
Female (age
not given)
40-yr-old male
17-yr-old
farmer
36-yr-old male
45-yr-old
farmer
Exposure conditions
Assaulted with ammonia
squirted into the eyes
during a robbery
Assaulted with ammonia
squirted into the eyes
during a robbery
Ammonia was thrown
into eyes in the course of
a robbery
Sprayed on face and
chest during a transfer
operation involving
anhydrous ammonia
Sprayed with several
gallons of 25% ammonia
in water during transfer
operation
Sprayed in the face
during field repairs to
fertilization equipment
Sprayed on the left side
efface during a transfer
operation
Immediate effects"
Decreased visual acuity
in the left eye,
conjunctivitis, necrosis
over the tarsus of the
lower lid; the pupil was
semi-dilated, oval, and
fixed, increased
interocular pressure
Conjunctivitis in the right
eye, the cornea was hazy
with edema
Two hrs postexposure,
loss of cornea!
epithelium,
conjunctivitis, stromal
haze, fibrinous material
in the anterior chamber,
iris atrophy, vertical,
oval-shaped pupil, and
subcapsular lens opacities
Facial burns (not
extremely serious),
pulmonary edema, and
pneumonitis with
inflammation and edema
of the upper airways
Throat tightness during
first few minutes
following exposure;
second-degree burns to
the buttocks
Immediate blepharo-
spasm, second-degree
facial burns, irritative
conjunctivitis, superficial
corneal ulceration, and
palpebral edema of the
left eye
Immediate blepharo-
spasm (30-min delay in
irrigating the exposed
areas with water); minor
skin burns and irritation
to the right eye; severe
damage to the left eye (no
details provided)
Delayed effects'"
Following initial
improvement, the left eye
became red, inflamed, rising
interocular pressure on
several occasions, each time
responding to surgery and
treatment, only to recur
after 1 or 2 mo of recovery
Four d following discharge,
severe anterior uveitis
developed, along with
recurrent corneal ulceration
and diminished corneal
sensation
Band-shaped corneal
degeneration and
heterochromia of the iris,
but no progression of lens
opacities (5 mo following
exposure)
No residual lung damage
No residual effects
No known sequelae
Diminished vision in left
eye at 3 d; loss of sensation
resulting in accidental
scratching of the sclera at
3 mo; 1 yr following
exposure, the left eye
perceived only light and the
cornea was vascularized and
opaque
Reference
Highman, 1969
Highman, 1969
McGuinness,
1969
Helmers etal.,
1971
Helmers etal.,
1971
Helmers etal.,
1971
Helmers etal.,
1971
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
20-yr-old male
20-yr-old
female
22-yr-old
female
Exposure conditions
Safety valve release in a
refrigeration unit due to
accidental heating of the
ammonia line
Exposure (approximately
30 min) to anhydrous
ammonia following train
derailment
Exposure (approximately
90 min) to anhydrous
ammonia following train
derailment
Immediate effects"
Loss of consciousness,
pinkish foaming at the
nose and mouth,
cyanotic, first- and
second-degree burns to
the neck, eyes, left arm,
glans penis, scrotum, and
both lower legs, spastic
extremities, both lungs
with fine and harsh rales,
constant and profuse
pulmonary excretions
Chemical burns to the
soft palate, oropharynx,
and feet, unable to speak,
blood-streaked sputum
Loss of consciousness,
convulsions, chemical
burns over the abdomen,
left knee and forearm,
both arms, soft palate,
and oropharynx, damage
to both corneas,
respiratory distress,
multiple areas of alveolar
type of infiltrate in both
lungs
Delayed effects'"
Six-mo folio wup: normal
vision, mild cough
Widespread infiltration
consistent with
bronchopneumonitis
developed shortly after
exposure; approximately
1.5-2 yrs following
exposure, patient developed
a productive cough and was
increasingly short-of-breath,
bronchiectatic changes
involving entire left lung,
mild changes in right lung
Re-hospitalized 1 yr later
with bilateral pneumonia,
peripheral edema, and acute
right heart failure; marked
deterioration of vision in the
left eye, bilateral cornea!
opacities and early cataract
changes were present; 2 yrs
later, progressive
development of infiltrations
in basilar section of the
lower left lung, generalized
varicose bronchiectatic
changes in lung segments,
hypoxemia, biopsies
revealed some mucosal
surface erosion, areas of
atelectasis, emphysema,
alveolar walls thickened
with monocellular infiltrates
Reference
White, 1971
Kassetal., 1972
Kassetal., 1972
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
24-yr-old male,
instrument
artificer,
smoker
39-yr-old male,
smoker
42-yr-old male,
maintenance
fitter, smoker
39-yr-old male,
assistant
process
foreman
39-yr-old male,
process
foreman,
smoker
47-yr-old male,
process worker
Exposure conditions
Vent stack overflow from
100 feet above work area,
resulting in complete
drenching
Vent stack overflow from
100 feet above work area,
resulting in complete
drenching
Exposure due to the
inadvertent resumption of
ammonia gas in a high
pressure line during valve
maintenance
Doused with ammonia
liquid from
approximately 130 feet
above
Doused with ammonia
liquid from
approximately 130 feet
above
In vicinity of a high
pressure compressor
pump burst
Immediate effects"
Dyspnea, chest pain,
blepharospasm, deep
cyanosis, burning throat,
blisters in the mouth and
throat, and congested
eyes, sloughs of oral
mucosa and exposed skin,
conjunctivitis, bronchitis
Dyspnea, chest pain,
blepharospasm, burning
throat, extensive
blistering, tachycardia,
moist sounds from both
lung bases, cornea! burns,
aphonia (lasting about
10 d)
Burns to the left eye (no
further details)
Pink frothy sputum,
cyanosis, bronchospasm,
severe burns and
blistering on the face,
mouth and hands
Pain in eyes and throat,
blepharospasm, burns to
the eyes, mouth, face, and
throat
Pain and tightness of the
chest, blepharospasm,
bloodstained sputum,
burns and blistering of
the face, mouth, eyes,
throat, and left heel
Delayed effects'"
26 mo following exposure,
dyspnea on exertion,
excessive collagenization in
the bronchial submucosal
layer
Continued to smoke at
follow up; lung function
tests (FVC and expiratory
volume in 1 sec) well below
normal after 2 yrs
Five -yr folio wup indicated
normal lung function
Recurring bronchitis,
dyspnea on exertion; in lung
function tests, FVC reached
near normal 1 yr following
exposure; FEVi test
indicated moderate
obstructive airways disease
and the gas transfer figure
remained below normal
Continued to smoke at
follow up; lung function
tests showed progressive
improvement in ventilation
but consistent depression of
gas transfer factor
Lung function tests indicate
gradual improvement in
ventilatory ability, but
below normal in gas
transfer factor
Reference
Walton, 1973
Walton, 1973
Walton, 1973
Walton, 1973
Walton, 1973
Walton, 1973
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Male, process
worker (age not
given)
28-yr-old male
25-yr-old male
6 -mo -old male;
two 12-yr-old
males; 17-yr-
old female
Exposure conditions
In vicinity of a high
pressure compressor
pump burst
Sprayed in the face when
a hose coupling came
loose
Tank explosion
Road accident involving
a tank truck rolling from
an overpass to the road
below, resulting in a
thick cloud of ammonia
vapor
Immediate effects"
Immediate fatality;
autopsy results indicate
extensive edema and
burns on the mouth, face,
trunk, arms, and upper
back; airway at the larynx
almost completely
blocked, greatly
distended and congested
lungs
First- and second-degree
burns to the face, nose,
and oropharynx mucosa
inflamed and sloughing,
rhonchi throughout both
lung fields, lung
congestion, and
segmental atelectasis
Initial effects: mild
bilateral conjunctival
edema, diffuse bilateral
wheezing, rhonchi, and
rales, burns to an
estimated 30% of the skin
surface area of the
extremities, chest, and
genitalia, pulmonary
edema; shortly after
admission, development
of severe respiratory
distress with production
of large amounts of
mucopurulent exudate
All four victims suffered
varying degrees of burns
to the face and body,
eyes, mouth, oral mucosa,
erythema, and edema of
the epiglottis and larynx;
two victims required
intubation for upper
airway obstruction
Delayed effects'"
Not applicable
Reassessment at 6 mo
revealed normal chest x-ray
films but abnormal
pulmonary arterial
perfusion and indication of
partial airway obstruction in
the left basal segment;
within 2 wks, this increased
to total obstruction of the
airways to the left basal
segment
Arterial hypoxemia
persisted despite respirator
inspired oxygen at 50-60%
and positive end expiratory
pressure; wheezing and
bronchospasm, bilateral
infiltrates, fatality occurred
at 60 d following exposure
with evidence of purulent
cavitary pneumonia
Measurement of several
urinary metabolites of
hydroxylysine indicated that
considerable collagen
degradation occurred after
inhaling concentrated
ammonia vapors
Reference
Walton, 1973
Taplinetal.,
1976
Sobonya, 1977
Hatton et al.,
1979
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
17-yr-old
female
6-mo-old male
26-yr-old male
Exposure conditions
Direct exposure to high
concentrations resulting
from a tanker truck
explosion
Direct exposure to high
concentrations resulting
from a tanker truck
explosion
Direct exposure
(approximately 20 min)
to high concentrations
resulting from a tanker
truck explosion
Immediate effects"
Second-degree burns of
the forehead and upper
extremities, total
epithelial loss of the
corneas, second- and
third-degree burns of the
nasal passages, soft
palate, posterior
pharyngeal wall and full-
thickness burns of the
larynx, diffuse
parenchyma! densities in
both lungs, inspiratory
wheezing, rhonchi, and
rales were present
Second- and third-degree
burns on the face, scalp,
upper extremities, and
buttocks, epithelial
defects in both corneas,
upper airway obstruction
(requiring intubation),
diffuse erythema and
edema of the lips, soft
palate, posterior
pharyngeal wall, and
epiglottis
Upper airway obstruction
(requiring mechanical
ventilation), extensive
second-degree burns of
the head, neck, and chest,
total loss of corneal
epithelium of both eyes,
extensive second- and
third-degree burns of the
oropharynx,
hypopharynx, and larynx,
diffuse rales, rhonchi, and
expiratory wheezing,
bilateral patchy
pulmonary infiltrates
Delayed effects'"
Pulmonary function studies
(1 yr following discharge)
indicated moderate
obstructive and restrictive
abnormalities
No remarkable findings in
2-mo followup
Rapid degeneration and
fatality followed a massive
hemorrhage around the
tracheostomy tube
(innominate artery erosion);
autopsy revealed full-
thickness burns to the entire
respiratory tract, acute
necrotizing trachea-
bronchitis, and bilateral
bronchopneumonia
Reference
Close etal.,
1980
Close etal.,
1980
Close etal.,
1980
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
23-yr-old
female
30-yr-old
female
Exposure conditions
Within a few hundred
feet of the tanker truck
explosion, exposure time
>30min
In the vicinity of the
tanker truck explosion,
exposure time
approximately 30 min
Immediate effects"
Mild facial erythema and
bilateral conjunctival
irritation, first- and
second-degree burns of
the oral cavity,
oropharynx, and larynx,
bilateral parenchymal
infiltrate, mild
hypoxemia
Mild facial erythema,
conjunctival irritation of
the eyes, diffuse bilateral
rales and rhonchi,
bilateral infiltrates,
hypoxemia (requiring
mechanical ventilation)
Delayed effects'"
After a period of stability,
progressive bilateral
pneumonia with progressive
dyspnea, and increased
secretions; subsequent
followup examinations
revealed accentuated
interstitial markings,
profuse, diffuse combined
bronchoalveolar and
perfusion abnormalities,
development of large
pneumatoceles, and bullous
changes to the right side of
the chest with herniation to
the left, facial edema, and
anasarca; ventilator
assistance through a
tracheostomy required
After initial improvement,
pneumonia developed in the
lower left lung,
accompanied by severe
progressive hoarseness,
moderate hypoxemia and
development of a combined
obstructive and restrictive
pulmonary disorder;
additional 2-yr followup
indicated scattered
parenchymal scarring, bleb
formation in the left lung
base and mild to moderate
hypoxemia
Reference
Close etal.,
1980
Close etal.,
1980
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Fourteen male
fishermen, 18-
39yrsold
58-yr-old
female
30-yr-old
female
Exposure conditions
Refrigeration system leak
below decks
Leaking pipe near the
offices for a grocery
distributor
Exposure to fumes
released when a tanker
truck overturned and
exploded
Immediate effects"
Inflammation of the
pharynx and conjunctiva
present in 12-14 victims,
with 2 showing corneal
burns; tachypnea was
present in 10 fisherman;
tachycardia was seen in
4 fishermen; 5 victims
had unremarkable chest
examinations upon
admission, while 9 were
found to have rales,
rhonchi, and wheezing,
productive cough; 1 of
the 9 moderately affected
victims developed
laryngeal edema
requiring tracheostomy,
while another had
persistent and progressive
airway obstruction for
48hrs
Loss of consciousness,
severe eye irritation with
bilateral corneal burns,
dyspnea, pharyngeal
edema, bilateral diffuse
rhonchi, and rales
Initial intense eye and
nasopharyngeal irritation
developing to
breathlessness and
nonproductive cough,
initial transient infiltrate
of pulmonary edema
Delayed effects'"
Not applicable
Two yrs following
exposure, patient developed
severe pulmonary
insufficiency requiring a
1-yr hospitalization with a
permanent tracheostomy
and continuous oxygen
Following apparent
recovery from initial acute
effects, development of
purulent cough with
progressive hypoxemia and
hypercapnia requiring
tracheotomy and
mechanically assisted
respiration for the next
3 yrs; development of
bilateral, severe, cylindrical,
and saccular bronchiectasis
with obliterative fibrous
adhesions of the right
pleural space, broncho-
pneumonia; fatality
Reference
Montague and
Macneil, 1980
Stroud, 1981
Hoeffleretal.,
1982
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
50-yr-old male
Two male
factory
workers,
smokers,
24 and 40 yrs
old
Three male
factory
workers,
smokers, 20-
30 yrs old, with
histories of
asthma,
smoker's
cough, and
chronic
bronchitis
30-yr-old male
factory worker,
smoker, no
history of lung
problems
28-yr-old male
factory worker,
smoker, no
history of lung
problems
Exposure conditions
Splashed with liquid
ammonia when a
refrigeration coolant tank
exploded
Sudden release of
ammonia gas to the
factory floor when the
pressure valve on a
refrigeration unit released
Sudden release of
ammonia gas to the
factory floor when the
pressure valve on a
refrigeration unit released
Sudden release of
ammonia gas to the
factory floor when the
pressure valve on a
refrigeration unit
released; exposure time
of 2-3 min with full
inhalation
Sudden release of
ammonia gas to the
factory floor when the
pressure valve on a
refrigeration unit
released; directly in line
with escaping gas
Immediate effects"
Burns to the right foot
and second-degree burns
to the thigh and groin,
progressive hypoxemia
and inspiratory stridor,
hyperemic, edematous
pharynx and vocal
chords, with the posterior
pharyngeal wall and the
entire tracheal wall below
the vocal chords covered
with a yellow purulent
pseudomembrane
Eye and throat irritation,
slightly red throat; one
patient had burns on the
legs and genitals
Moderate dyspnea,
labored breathing, sore
eyes, moderate to severe
throat pain, cough,
nausea
Severe cough, massive
nasal discharge, very sore
throat, constant retching,
development of
inspiratory and expiratory
rhonchi
Severely inflamed eyes,
lips, tongue, edematous
pharynx and hoarseness,
high-pitched inspiratory
and expiratory rhonchi
Delayed effects'"
Progressively worsening
respiratory distress
(mechanical ventilation was
required for 10 d), extensive
pseudomembrane formation
throughout the trachea-
bronchial tree, gastro-
intestinal bleeding,
persistent tachypnea and
expiratory wheezing
(following removal of
tracheostomy tube),
pneumonitis complications;
5-yr folio wup: asympto-
matic except during very
strenuous activity
Hoarseness and productive
cough persisting for several
months
Not applicable
Reference
Flury et al.,
1983
O'Kane, 1983
O'Kane, 1983
O'Kane, 1983
O'Kane, 1983
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
44-yr-old male
factory worker,
nonsmoker, no
history of lung
problems
25-yr-old male
refrigeration
technician
Two males,
26 and 40 yrs
old
Three males,
26, 36, and
45 yrs old
30-yr-old male
27-yr-old male
Exposure conditions
Sudden release of
ammonia gas to the
factory floor when the
pressure valve on a
refrigeration unit
released; 4-5 -min
exposure
Sudden, massive leak
from the refrigeration
plant
Pipe valve failure
allowing ammonia gas to
fill a factory area
Pipe valve failure
allowing ammonia gas to
fill a factory area
Pipe valve failure
allowing ammonia gas to
fill a factory area
Pipe valve failure
allowing ammonia gas to
fill a factory area
Immediate effects"
Cyanosed, respiratory
distress, bilateral
inspiratory and expiratory
rhonchi, inflamed eyes,
tongue, pharynx; 2 hrs
following admission,
developed severe
dyspnea, central cyanosis,
and an audible gurgle
from bronchial secretions
Loss of consciousness,
severe burns of the face,
eyes, mouth, and throat;
development of clinical
and radiologic features of
pulmonary edema with
audible crackles over the
lung fields and
production of large
amounts of mucous
Mild pharyngitis and
conjunctivitis; the 40-yr-
old male also had burns
to legs and genitals
Moderate conjunctivitis,
moderate to severe
pharyngitis, tachycardia,
and tachypnea, frothy
sputum, moderate
dyspnea, nausea
Severe dyspnea, sore
eyes, nausea, generalized
rhonchi, leukocytosis,
and marked hypoxemia
Severe dyspnea, sore
eyes, nausea, generalized
rhonchi, leukocytosis,
and marked hypoxemia
Delayed effects'"
Following initial
improvement, the patient
began deteriorating the
13 d following exposure,
lungs developed multiple
cavitating lesions,
secondary infection, and
finally necrotizing
pneumonia; patient survived
with chronic infective lung
disease
Respiratory failure with
tachypnea and arterial
hypoxemia developed;
death from respiratory
failure 12 wks following
exposure; intense
congestion of the mucosal
surfaces of the trachea and
major bronchi; the cut
surface of the left lung was
crepitant, and cylindrical
bronchiectasis was found in
the middle and lower lungs
Followup every 6 mo for up
to 2 yrs: no additional
findings
Followup every 6 mo for up
to 2 yrs: impaired
pulmonary function at
discharge (all three men);
two men showed
improvement while the third
did not; all three men had
preexisting lung disease
Improvement in lung
function tests was seen
during folio wup; however,
patient experienced dyspnea
upon exertion and chronic
productive cough
A right basal consolidation
developed requiring
prolonged treatment with
oxygen; improvement in
lung function was seen after
30 d; however, patient
experienced dyspnea upon
exertion and chronic
productive cough
Reference
O'Kane, 1983
Price, 1983
Ward et al.,
1983
Ward et al.,
1983
Ward et al.,
1983
Ward et al.,
1983
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
44-yr-old male
33-yr-old male
51-yr-oldmale
smoker
Five males, 26-
48 yrs old
Exposure conditions
Pipe valve failure
allowing ammonia gas to
fill a factory area
Explosion while
transferring ammonia
from one tank to another
Explosion while
transferring ammonia
from one tank to another
An overturned tanker
truck released a dense
cloud of ammonia gas
Immediate effects"
Severe dyspnea, sore
eyes, nausea, generalized
rhonchi, leukocytosis,
and marked hypoxemia
Burns over 12% of total
body surface area and
oropharynx, bilateral
corneal clouding, severe
respiratory distress
Burns over 50% total
body surface area (eyes,
back, legs, and
oropharynx), severe
respiratory distress
(bilateral wheezing and
bilateral lower lobe
infiltrates)
Four immediate fatalities;
autopsy findings:
second-degree burns on
exposed skin, hyperemic
tracheobronchial mucosal
surfaces, edematous and
congested parenchyma,
sloughing of the
tracheobronchial
epithelium and extensive
edema and congestion of
the lung parenchyma
Delayed effects'"
Following initial
improvement from
admission, a relapse
occurred, coincident with
development of pneumonia,
requiring artificial
respiration and
tracheostomy; during 2-yr
followup, patient remained
severely impaired in
pulmonary function
Fatality due to cardiac
arrest; autopsy findings:
tracheobronchial
ulcerations, denuded
epithelium, congested
lungs, and necrotizing
bronchitis with ulceration
and membrane formation,
micro abscesses of the
kidneys
Progressive
decline/respiratory distress
following initial
improvement, fatality due to
cardiac arrest; autopsy
findings: tracheobronchial
ulcerations, denuded
epithelium, congested
lungs, and necrotizing
bronchitis with ulceration
and membrane formation,
medullary fibrosis and tubal
necrosis of the kidneys
One delayed fatality
(2.5 wks after exposure) due
to bronchopneumonia and
necrotizing
tracheobronchitis
Reference
Ward et al.,
1983
Arwoodetal.,
1985
Arwoodetal.,
1985
Burns etal.,
1985
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
Twenty two
seafood
processing
plant workers
37-yr-old male
with myotonic
dystrophy
Four males, all
smokers (ages
not given)
28-yr-old male
Exposure conditions
Liquefied ammonia tank
explosion within an
enclosed area
Ammonia intoxication
37 yrs after
ureterosigmoidostomy
Acute exposure (no
additional details
provided)
Equipment failure while
spraying anhydrous
ammonia on farmland
Immediate effects"
Fourteen cases with
irritative symptoms only;
six workers exhibited
transient bronchospasm
and edema of the lips and
oral mucosa
Lethargy, restlessness,
irritability, and confusion,
both lungs were
underinflated due to high
diaphragm, severe
metabolic encephalo-
pathy, right-sided
hydronephrosis (left
kidney absent), blood
ammonia levels were
markedly elevated.
Skin blisters, ocular and
nasal burning, dyspnea,
chest pain, chest
tightness, nonproductive
cough, and hemoptysis
Skin burns of 12% total
body surface area
including face, chest,
neck, and right upper
extremity; periorbital
edema with the right eye
swollen shut; edema of
the uvula, soft palate,
tonsilar pillars, and
epiglottis with laryngeal
burns, thick mucoid
rhinorrhea, and bilateral
rhonchi
Delayed effects'"
Five workers exhibited eye
pain and photophobia, four
of which developed cornea!
damage; one fatality due to
respiratory failure (2 wks
following exposure); one
worker developed
restrictive lung disease and
chronic bronchitis (6-mo
followup)
None
Wheeze, productive cough,
dyspnea persisting for 12-
32 mo; pulmonary function
tests indicated reversible
airway obstruction in one
patient with persistent
hemoptysis; submucosal
inflammation, denuded
epithelium, and thickening
of the basement membrane
Thirteen mo following
exposure, patient presented
with perihilar adenopathy
and a right paratracheal
mass, as well as mild
obstructive disease; at 4 yrs
postexposure, there was no
evidence of paratracheal or
hilar masses
Reference
Yang, 1987
Gilbert, 1988
Bernstein and
Bernstein, 1989
Milleaetal.,
1989
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
17-yr-old male,
farm supply
store employee
22-yr-old male
farmer
21-yr-oldmale
refrigeration
plant worker
35-yr-old male
refrigeration
plant worker
Six
refrigeration
plant workers,
male and
female, 19-
24 yrs old
Workers at a
fertilizer plant
Exposure conditions
A connector valve
opened on a hose
carrying ammonia
Sprayed while applying
anhydrous ammonia to a
field
Directly below
refrigeration pipe rupture
In vicinity of
refrigeration pipe rupture
In vicinity of
refrigeration pipe rupture
Leak and explosion of an
ammonia tank at a
fertilizer plant
Immediate effects"
Partial thickness burns on
18% of total body surface
area including the face,
chest, and lower
extremities; full thickness
burns on approximately
3% of total body surface
area, cornea! burns to the
right eye, hoarseness,
erythema and edema of
the oral mucosa and
uvula
Partial thickness burns
over 12% total body
surface area of the chest,
abdomen, and upper
extremities; no
pulmonary effects
Cardiorespiratory arrest;
third-degree burns to
20% of the body surface,
conjunctivitis, burns of
the oral mucosa with
swelling of the epiglottis,
pulmonary edema and
bronchial inflammation
Second-degree burns over
10% of total body surface
area, dyspnea, nausea,
severe eye irritation, nose
and throat irritation,
pulmonary edema
Erythema, small burn
areas, irritation of the
eyes, nose, and throat,
dyspnea and nausea to
varying degrees
Seven fatalities,
57 injuries (no further
details provided)
Delayed effects'"
On postexposure d 5, a
pneumomediastinum and
subcutaneous emphysema
were noted, as well as vocal
cord and tracheobronchial
edema
Patient was readmitted 6 d
following discharge for an
infected burn wound on the
right arm
Fatality 13d after exposure
due to development of
treatment-resistant
bronchopneumonia
Not applicable
Not applicable
Not applicable
Reference
Milleaetal.,
1989
Milleaetal.,
1989
Weiser and
Mackenroth,
1989
Weiser and
Mackenroth,
1989
Weiser and
Mackenroth,
1989
Andersson,
1991
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
28-yr-old male
30-yr-old male,
smoker
27-yr-old male,
smoker
46-yr-old male
Exposure conditions
Tank explosion in an
industrial butter plant
Occupational exposure to
a refrigeration gas leak in
adjacent room;
approximately 15-min
exposure
Occupational exposure to
a refrigeration gas leak in
a poorly ventilated room;
approximately 1.5-2 min
of additional exposure
after detecting a strong
odor
Sprayed while unloading
tanks of ammonium
hydroxide (29.4%
ammonia)
Immediate effects"
Bilateral corneal scarring;
first- and second-degree
burns of the chest and
hands, pharyngeal and
laryngeal edema with
exudative lesions,
crackles and wheezes at
the lung bases;
production of copious
bronchial secretions
Immediate burning of the
eyes, upper airways, and
skin, with cough and
pleuritic chest pain,
conjunctivitis and
rhinopharyngitis
Difficulty breathing;
irritation of the eyes,
nose, throat, burning
sensation on the skin;
respiratory failure
requiring mechanical
ventilation
Mucocutaneous burns
(unspecified), productive
cough, dyspnea and
wheezing
Delayed effects'"
At 1 wk folio wup: severe
tracheobronchial damage
with diffuse erythema,
inflammation of the airway
walls, hemorrhagic areas
and abundant purulent
secretions, with severe
airflow blockage; during
10 yrs of folio wup, severe
fixed airway obstruction
remained, with coexisting
bronchiectasis
Following discharge from
hospital, persistent and
progressively worse
dyspnea, wheezing on
exertion, and atypical chest
pain; clinical diagnosis of
restrictive lung disease with
symptoms ongoing at 2 yrs
and beyond
At 26 mo following
exposure, victim had
persistent productive cough,
dyspnea on exertion and
expiratory wheezing;
pulmonary function testing
showed severe obstructive
impairment unresponsive to
bronchodilation
During 2 yrs following
initial treatment and
discharge, persistent
dyspnea at rest and exertion,
frequent episodes of dry
cough, wheezing, increasing
dyspnea and parethesia of
the hands and feet; tests
indicated a diagnosis of
laryngotracheobronchitis
and possible pneumonitis,
with evidence of
nonspecific bronchial
hyper-reactivity and small
airway disease
Reference
Leduc etal.,
1992
de la Hoz et al.,
1996
de la Hoz et al.,
1996
de la Hoz et al.,
1996
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
41-yr-oldmale,
fish-processing
plant
51-yr-old
maintenance
man
57-yr-old male,
supervisor to
above
28-yr-old male
47-yr-old male
Exposure conditions
Several hours spent in the
vicinity of an ongoing
ammonia leak from a
refrigeration unit, without
respiratory protection
Pressurized liquid
ammonia explosion
during inspection for
maintenance and repair
on a refrigerator coolant
storage system
Pressurized liquid
ammonia explosion
during inspection for
maintenance and repair
on a refrigerator coolant
storage system
Anhydrous ammonia
explosion at a fertilizer
factory
Burst pipe in a liquefied
ammonia production
plant, with over 45 min
of exposure
Immediate effects"
Eye and nasal irritation
and mild facial burning
Examination revealed
28% total body surface
area burns to the
extremities with some
torso involvement; there
were no facial burns,
erythema, mucosal
swelling, or signs of
respiratory distress
Pain in the genital area
was first noted an hour
after explosion, with
significant blistering on
the scrotum and distal
penis
Second- and third-degree
burns (45% total body
surface area) to posterior
trunk, buttocks, and
bilateral lower
extremities, as well as
inhalation injury (no
additional details
provided)
Mixed burns on the face,
neck, chest, genitalia,
both lower limbs, and full
thickness burns of the
back (3 8% total body
surface area), frostbite
injury to the back, the
eyes showed chemosis
and abrasions, congestion
and cornea! edema
increasing with
subconjunctival
hemorrhaging, harsh
breathing sounds with
crepitations, intermittent
bradycardia
Delayed effects'"
Nasal congestion and
intermittent epistaxis, which
resolved in about 2 wks,
hyposmia (decreased sense
of smell), which persisted
without additional abnormal
findings
None
Healing without incident or
sequelae
No effects observed at
18-mo folio wup
Eschar necrosis down to the
muscles on the back,
persistent intermittent
bradycardia and low blood
pressure, unusual muscle
weakness in all four limbs,
fatality due to severe
hemorrhage from the
endotracheal tube and
cardiac arrest
Reference
Prudhomme et
al., 1998
Sotiropoulos et
al., 1998
Sotiropoulos et
al., 1998
Amshel et al.,
2000
George etal.,
2000
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
25-yr-old male
32-yr-old male
40-yr-old male
Two food
processing
plant workers,
ages not
specified
Exposure conditions
Liquid ammonia spill
during transfer from a
barge tank to a dockside
tank
Liquid ammonia spill
during transfer from a
barge tank to a dockside
tank
Exposed to liquid
ammonia while emptying
a refrigeration unit
Discharge of pressurized
ammonia during
maintenance
Immediate effects"
Facial and neck
hyperemia, slightly
injected sclera,
erythematous petechiae
on the right ear,
edematous and peeling
lips, and bilateral corneal
abrasions; vision was
blurred and had difficulty
breathing
Asymptomatic at the
scene; within 90 min
developed swollen face
and lips, edematous
pharynx and vocal
compromising 50% of the
airway, partial thickness
burns on the face, neck,
anterior chest, left medial
upper arm, both hands,
and left anterior thigh, 8-
14% total body surface
area with full thickness
burns on the anterior
thigh
Skin burns of 15% total
body surface area to the
right neck, torso, arm,
and axilla, scattered
bilateral rhonchi but no
wheezing or stridor,
minimal edema and
erythema of the larynx
and proximal trachea
One immediate fatality
(pulmonary edema, focal
lung hemorrhage); one
worker permanently
blinded (no additional
details)
Delayed effects'"
Not applicable
Not applicable
Bilateral infrahilar
consolidations and apical
infiltrates were noted in
postburn d 3; there was no
long-term followup
Not applicable
Reference
Latenser and
Lucktong, 2000
Latenser and
Lucktong, 2000
Kersteinetal.,
2001
Morton, 2005
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
32-yr-old male
22-yr-old male
19-yr-old male
21-yr-oldmale
33-yr-old male
Patients and
hospital staff in
the critical care
unit
Exposure conditions
Warehouse explosion
resulting in an ammonia
tank rupture
Warehouse explosion
resulting in an ammonia
tank rupture
Warehouse explosion
resulting in an ammonia
tank rupture
Warehouse explosion
resulting in an ammonia
tank rupture
Warehouse explosion
resulting in an ammonia
tank rupture
Ammonia leak from a
refrigerator
Immediate effects"
Skin burn of 2% total
body surface area to the
face and scrotum;
perforated left tympanic
membrane and right
corneal injury; initial
artificial respiration
reinstated within 24 hrs
for hypoxia and
aspiration
0.5% total body surface
area burns to the scrotum,
bilateral corneal injury,
tracheobronchitis
0.5% total body surface
area burns to the face and
scrotum; bilateral corneal
injury; tracheobronchitis
1% total body surface
area burns to the face, left
axilla, and scrotum,
bilateral corneal injuries,
airway sloughing with
large amounts of debris
required performing a
tracheostomy and
mechanical ventilation
Respiratory difficulty, but
no burns to the skin and
eyes; peribronchiolar
edema limited to the right
lung
Respiratory and ocular
irritation
Delayed effects'"
Airway sloughing with
large amounts of debris
requiring tracheostomy;
after removal of
tracheostomy, patient
exhibited dysphagia and
hoarseness, evidence of
supraglottic and aryenoid
edema and left true vocal
cord paresis; pulmonary
function test at 8 mo
indicated obstructive
pulmonary disease
At 4-wk folio wup, bilateral
uveitis with stromal
vascularization, haze and
scarring, and pigmented
keratic precipitates resulting
in legal blindness (after
unsuccessful attempt at
corneal transplants)
Peribronchiolar edema was
noted 5 d after injury,
stridor developed and the
patient was reintubated and
given artificial respiration;
pulmonary function testing
at 6 mo showed a fixed
obstructive pattern
Extensive peribronchiolar
edema, necrosis and
sloughing were observed
down to the tertiary
bronchioles; fatality at
100 d postinjury
Not applicable
Not applicable
Reference
White etal.,
2007
White etal.,
2007
White etal.,
2007
White etal.,
2007
White etal.,
2007
Price and Watts,
2008
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Table C-2. Human case studies and reports of human exposure to ammonia
Case(s)
57-yr-old male,
nonsmoker
20-yr-old male
53-yr-old male
Fifteen males,
31.1±8.8yrs
old
Five males,
49.4 ± 23.0 yrs
old
Exposure conditions
At age 25, massive
accidental exposure to
anhydrous ammonia
while working in a
refrigeration unit
Forklift caught an
ammonia pipe causing a
sudden burst of a large
quantity of ammonia
Forklift caught an
ammonia pipe causing a
sudden burst of a large
quantity of ammonia
Resulted from events
during illicit
methamphetamine
production, 14/15 due to
explosions
Resulted from farming
accidents, 2/5 due to
explosions
Immediate effects"
Loss of consciousness,
corneal scarring, and
burns to the face and
chest
Loss of consciousness,
acute respiratory distress
syndrome (which
required percutaneous
tracheotomy), second and
third degree burns of his
face, anterior neck, left
axilla, left arm, parts of
anterior and posterior
chest wall, genitalia, and
both lower limbs
covering 50% total body
surface area,
symblepharon of both
eyes
Loss of consciousness,
acute respiratory distress
syndrome
Burns on body,
respiratory distress
Burns on body,
respiratory distress
Delayed effects'"
Chronic hypoxemic
respiratory failure, bilateral
inspiratory crackles, and
prolonged expiration,
central cylindrical, varicose
and cystic bronchiectasis
with mild interstitial fibrosis
Continued hospital care for
respiratory support until
transplantation of both
lungs performed 6 mo after
exposure
Developed obliterating
bronchiolitis and restrictive
and obstructive ventilation
disturbances, unable to
work, breathless at rest,
lung transplant candidate
One fatality, ventilator
assistance and skin grafting
required
Ventilator assistance and
skin grafting required
Reference
Tonelli and
Pham, 2009
Lalic et al.,
2009; Pirjavec
etal.,2009
Lalic et al.,
2009
Bloom et al.,
2008
Bloom et al.,
2008
aEffects occurring within 48 hrs of exposure.
bEffects occurring longer than 48 hrs after exposure.
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C.3. CONTROLLED HUMAN EXPOSURE STUDIES OF AMMONIA INHALATION
Controlled human exposure studies of inhaled ammonia are summarized in Table C-3.
Table C-3. Controlled human exposure studies of ammonia inhalation
Subjects
Exposure conditions
Results
Reference
Seven male
volunteers
500 ppm (354 mg/m ) for
30 min from masked breathing
apparatus for nose and throat
inhalation.
There was no mention of
preexposure examinations
Hyperventilation (50-250% increase above
controls) characterized by increased
breathing rate and expiratory minute volume
(i.e., volume of air exhaled in 1 min); no
coughing was induced, excessive lacrimation
occurred in two subjects; two subjects
reported nose and throat irritation that lasted
24 hrs after exposure; no changes were
reported in nitrogen metabolism or in blood
or urine urea, ammonia, or nonprotein
nitrogen.
Silverman et
al., 1949
Seven male
volunteers with an
average age of 31
yrs
30, 50, and 90 ppm (21, 35, and
64 mg/m3) for 10 min in an
inhalation chamber
Physical and neurological
examinations were conducted
prior to exposure
Increased eye erythema at 90 ppm (64
mg/m3) compared to 30 and 50 ppm (21 and
35 mg/m3) exposure; 90 ppm (64 mg/m3) did
not produce significant bronchiospasm or
severe lacrimation; intensity of odor
perception was reported as higher at 30 and
50 ppm (21 and 35 mg/m3) than at 90 ppm
(64 mg/m3)
MacEwen et
al., 1970
18 healthy
servicemen
volunteers, 18-39
yrs old
50-344 mg/m3 (70-486 ppm)
for a half-day (session day 2);
sessions on days 1 and 3 acted
as controls
All volunteers underwent a
preliminary examination prior
to exposure
No effect at concentrations of 71 mg/m (100
ppm); reduced expiratory minute volume at
concentrations ranging from 106 to
235 mg/m3 (150-332 ppm) compared to
controls (not dose dependent); exercise tidal
volume was increased at 106 mg/m3 (150
ppm), but reduced at higher concentrations
in a dose-dependent manner
Cole et al.,
1977
Six male and female
volunteers, 24-46
yrs old
25, 50, and 100 ppm (18, 35,
and 71 mg/m3) ammonia for 6
hrs/d 1 time/wk over 6 wks;
occasional brief exposure to
150-200 ppm (106-141
mg/m3)
There was no mention of
preexposure examinations
Habitation to eye, nose, and throat irritation
after 2-3 wks with short-term adaption; there
were no significant differences for common
biological indicators, physical exams, or in
normal job performance when compared to
control subjects; continuous exposure to 100
ppm (71 mg/m3) became easily tolerated and
had no effect on general health after
acclimation occurred; brief exposure to 150-
200 ppm (106-141 mg/m3) produced
lacrimation and transient discomfort
Ferguson etal.
1977
15 volunteers, 18-
53 yrs old
50,80, 110, and 140 ppm (35,
57, 78, and 99 mg/m3) for 2 hrs
in an exposure chamber
There was no mention of
preexposure examinations.
No effect on vital capacity, FEVi or forced
expiratory volume; 140 ppm (99 mg/m3)
caused severe irritation and could not be
tolerated; reported eye irritation increased
with concentration
Verberk, 1977
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Table C-3. Controlled human exposure studies of ammonia inhalation
Subjects
Exposure conditions
Results
Reference
20 male volunteers;
groups of four were
exposed to
ammonia at various
concentrations and
durations
Group 1: exposed to 2 mg/m
(3.0ppm)for37d;
Group 2: exposed to 5 mg/m3
(7.2 ppm) for 17 d;
Group 3: exposed to 2 mg/m3
(3.0 ppm) for 35 d with short-
term increases to 10 mg/m3
(14 ppm);
Groups 4 and 5: exposed to 2
and 5 mg/m3 (3.0 and 7.2 ppm),
respectively, for 20 d with
variations in temperature and
humidity; exposure duration
each day was not specified ;
there was no mention of
preexposure examinations
Significantly elevated adrenalin levels in
urine at 2.1 mg/m3 (3.0 ppm); dopamine and
DOPA levels in urine were not significantly
affected at any concentration; significant
increase of adrenalin and 7-oxy-
corticosteroids in urine, and
11-oxycorticosteroids free fractions in
plasma at 5.1 mg/m3 (7.2 ppm); increased
temperature and humidity resulted in
increased urine adrenalin, urine 7-
oxycorticosteroids and free 11-oxycortico-
steroid levels in plasma at 5.1 mg/m3
(7.2 ppm)
Kalandarov et
al., 1984
Unspecified number
of volunteer
subjects
Acute exposure up to 15 sec, 1
time/d at unspecified
concentrations; also a separate
exposure of 10 inhaled breaths
via mouthpiece at unspecified
concentrations; there was no
mention of preexposure
examinations
The lachrymatory threshold was 55 ppm
(39 mg/m3) and bronchoconstriction was
seen at 85 ppm (60.1 mg/m3)
Douglas and
Coe, 1987
Six healthy
volunteers (two
males and four
females, 25-45 yrs
old) and eight
volunteers with
mild asthma (four
males and four
females, 18-52 yrs
old)
16-25 ppm (11-18 mg/m3) for
30-min sessions with 1 wk
between sessions
Pulmonary function was
measured before and after
exposure
No significant changes in pulmonary
function in healthy subjects at any
concentration; a decrease in FEVi and
increased bronchial hyperreactivity was
reported in asthmatics exposed to dust and
ammonia, but not to ammonia alone;
exposure to dust alone caused similar effects,
suggesting that dust was responsible for the
effects.
Sigurdarson et
al., 2004
12 healthy
volunteers (7
females, 5 males)
21-28 yrs old
5 and 25 ppm (4 and 18 mg/m3)
for three separate exposures in
inhalation chamber for 1.5 hrs
resting and 1.5 hrs exercising
on a stationary bike; 1-4
volunteers were exposed on
each occasion
Lung function and nasal lavage
were performed before and
after exposure
Reported discomfort in eyes, detection of
solvent smell, headache, dizziness, and
feeling of intoxication were significantly
increased at 5 ppm (4 mg/m3); there were no
changes in lung function or exhaled nitric
oxide levels in exposed individuals;
exposure did not result in upper-airway
inflammation or bronchial responsiveness
Sundblad et al.,
2004
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Table C-3. Controlled human exposure studies of ammonia inhalation
Subjects
Repeated 2-sec exposures at
increasing concentrations
ranging from 0.9 to 228 ppm
(0.6-161 mg/m3) by dynamic
olfactometry
There was no mention of
preexposure examinations
Exposure conditions
Results
Reference
Healthy male and
female volunteers
grouped by age, 18-
35 and 45-65 yrs
old
Mean odor detection threshold <20 ppm
(14 mg/m3), mean irritation (lateralization)
threshold well above 20 ppm (14 mg/m3),
dose-response for odor annoyance and
irritation; strong olfactory and moderate to
strong irritating sensations at >15 ppm (11
mg/m3)
Altmann et al..
2006
43 healthy male
volunteers age 21-
47 yrs; one group of
30 men not familiar
with the smell of
ammonia and 10
men exposed to
ammonia regularly
at the workplace
0, 10, 20, 20+2 peak
exposures at 40, and 50 ppm
(0, 7, 14, 14+2 peak
exposures at 28, and 35 mg/m3)
on 5 consecutive days for
4 hrs/d in an exposure chamber
Subjects familiar to ammonia reported fewer
symptoms than naive subjects; at
concentrations <20 ppm (14 mg/m3), there
were no significant differences in symptoms
reported between the groups; the perceived
intensity of symptoms was concentration-
dependent in both groups
Ihrigetal..
2006
25 healthy
volunteers (mean
age 29.7 yrs), and
15 mild/moderate
persistent asthmatic
volunteers (mean
age 29.1 yrs)
2-500 ppm (1-354 mg/m3)
(ocular and nasal exposure) for
various durations lasting up to
2.5 hrs
Baseline lung function was
recorded prior to exposure
Irritation threshold, odor intensity and
annoyance were not significantly different
between healthy volunteers and asthmatics;
nasal irritation threshold = 129 ppm (91
mg/m3); ocular irritation threshold = 175
ppm (124 mg/m3); there were no changes in
lung function (FEVi) for subjects in either
group
PetrovaetaL
2008
24 healthy female
volunteers age 18-
45 yrs (mean age
29.9 yrs)
0.03-615.38 ppm (0.02-
435 mg/m3) (nasal exposure)
for a maximum of 2 sec
Preexposure measurements
included rhinoscopic exam,
screening for chemical
sensitivities, allergies,
respiratory disease, general
health, and prior chemical
exposure by personal interview
Both the static and dynamic methods showed
similar averages for detection thresholds for
the odor and irritancy of ammonia; mean
odor detection threshold of 2.6 ppm (2
mg/m3) (both static and dynamic) and mean
irritation thresholds of 31.7 or 60.9 ppm
(22 or 43 mg/m3) for static and dynamic
methods, respectively
Smeets etal.,
2007
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1
2
3
4
5
C.4. CROSS SECTIONAL STUDIES OF LIVESTOCK FARMERS EXPOSED TO
AMMONIA
Cross sectional studies of livestock fanners exposed to ammonia are summarized in
Table C-4.
Table C-4. Cross sectional studies of livestock farmers exposed to ammonia
Subjects
27 pig farmers (mean
age of 29 yrs)
29 farm workers;
48 electronic factory
workers (controls)
Methods
Environmental and
personal exposures
were analyzed; lung
function was
measured on
Monday, Tuesday,
and Friday
20 pig houses were
monitored for dust
and ammonia
concentrations;
respiratory symptoms
were determined by
questionnaire; lung
function tests were
performed;
24 subjects provided
blood samples to
determine IgE and
IgG antibody levels
Exposure conditions
Mean exposure to dust =
1.57 mg/m3; endotoxin =
24 ng/m3, and
ammonia = 5.60 mg/m3
Mean airborne ammonia
concentrations ranged
from 1.5 to 13.23 ppm
(1-9 mg/m3) and mean
dust concentrations
ranged from
approximately 2 to
21 mg/m3
Results
There was no significant
correlation with lung
function and exposure to dust
or endotoxins; there was a
correlation with decreased
lung function (5-10%) and
exposure to ammonia on the
Tuesday testing, but not the
Monday or Friday testing;
reported respiratory
symptoms included cough,
phlegm, and wheezing
Respiratory symptoms
included chest tightness,
wheeze, nasal and eye
irritation (23/29 farm
workers); 3/29 farm workers
had impaired lung function
(decreased FEVj and FVC);
3 farmers had IgE antibodies
to pig squames or urine;
specific IgG antibodies were
found in 14 workers to pig
squames, and 9 to pig urine,
suggesting an allergic
response
Reference
Heederik et
al., 1990
Crook et al.,
1991
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Table C-4. Cross sectional studies of livestock farmers exposed to ammonia
Subjects
102 pig farmers
(mean age 39.7 yrs;
mean duration of
employment of
15.7 yrs) who
worked at least half
time in a swine
confinement
building; 5 1 male
dairy farmers (mean
age 40.1 yrs; mean
duration of
employment of
20.3 yrs) and 81 male
dairy industry
workers (controls;
mean age 38.5 yrs;
mean duration of
employment of
15.7 yrs)
54 male swine
producers (mean age
= 36.3 yrs; mean
duration of
employment =
10.7 yrs)
207 males >18 yrs of
age employed at
swine farms and
spent time in swine
confinement
buildings (mean
years of employment
= 9.6); a farm
comparison group
(nonconfinement
production) was
included (number not
given)
Methods
Pulmonary function
tests were given to
subjects before and
after a methacholine
challenge; respiratory
symptoms were
determined by
questionnaire
Assessment of
respiratory symptoms
with questionnaire
and lung function
tests
Pulmonary function
tests were performed
before shift
(baseline) and then
after a minimum of 2
hrs of exposure;
environmental and
personal air samples
were made for
ammonia, carbon
dioxide, hydrogen
sulfide, carbon
monoxide, total and
respirable dust
Exposure conditions
Mean total dust level of
2.41 mg/m3; mean
airborne ammonia
concentration of
8.5 mg/m3; mean
personal ammonia
exposure of 3.23 mg/m3
Mean contaminant
levels: carbon dioxide =
2,632 ppm (1,861 mg/
m3); ammonia =
11.3 ppm (8 mg/m3);
total dust = 2. 93 mg/m3;
respirable dust =
0.13 mg/m3; endotoxin =
1 1,332 units/m3
Mean personal air
exposure for all subjects:
total dust = 4.53 mg/m3;
respirable dust =
0.23 mg/m3; total
endotoxin =
202.35 EU/m3;
respirable endotoxin =
16.59 EU/m3;
ammonia = 5.64 ppm
(4 mg/m3)
Results
Pig and dairy farmers had
higher prevalence of reported
cough and morning phlegm;
bronchial hyperreactivity to
methacholine was higher for
pig and dairy farmers
compared to controls
Exposure to high
concentrations of ammonia
was associated with chronic
cough and bronchitis;
incidence of chronic cough
was dependent on interaction
of ammonia with endotoxin,
and respirable dust; ammonia
concentrations were not
correlated with changes in
lung function parameters
Positive correlations were
associated with pulmonary
function and exposure to
total dust, respirable dust,
respirable endotoxin, and
ammonia; exposure to
ammonia concentrations of
>7.5 ppm (5 mg/m3) were
predictive of a >3% decrease
inFEVi; the correlation
between exposure and
decreased pulmonary
function was stronger after
6 yrs of exposure
Reference
Choudat et al.,
1994
Zejda et al.,
1994
Donham et al.,
1995
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Table C-4. Cross sectional studies of livestock farmers exposed to ammonia
Subjects
194 Dutch pig
farmers (94 with
chronic respiratory
symptoms, 100
without symptoms)
151 males >18 yrs of
age employed at
swine farms and
spent time in swine
confinement
buildings (mean
years of employment
= 12.4); a farm
comparison group
(nonconfinement
production) was
included (number not
given)
196 pig farmers
(96 with chronic
respiratory
symptoms, 100
without symptoms)
171 pig farmers
(82 with chronic
respiratory
symptoms, 89
without)
Methods
Cross-sectional study
evaluating exposure
response relations of
exposures to dust,
endotoxins,
ammonia, and
disinfection
procedures
Followup study from
Donhametal. (1995)
previously described;
followup
measurements taken
48 mo from the
initial measurements
Pig farmers tested for
lung function and
bronchial
responsiveness to
histamine challenge
Longitudinal study
for cohort of pig
farmers observed
over 3 yrs; subjects
examined for lung
function and tested
for bronchial
responsiveness to
histamine challenge
Exposure conditions
Estimates of long-term
exposure based on two
personal exposure
samples (one winter
sample, one summer
sample); Mean estimated
exposure to dust =
2.7 mg/m3, endotoxin =
1 12 ng/m3, ammonia =
2 mg/m3
Mean personal air
exposure for all subjects:
total dust =3. 45 mg/m3;
respirable dust =
0.26 mg/m3; total
endotoxin =
176.12EU/m3;
respirable endotoxin =
11.86EU/m3;
ammonia =5.15 ppm
(4 mg/m3)
Estimates of long-term
exposure based on two
personal exposure
samples (one winter
sample, one summer
sample); Mean estimated
exposure to respirable
dust = 2.7 mg/m3,
endotoxin =111 ng/m3,
ammonia = 2 mg/m3
Estimates of long-term
exposure based on two
personal exposure
samples (one winter
sample, one summer
sample); Mean estimated
exposure to respirable
dust = 2.63 mg/m3,
endotoxin = 105 ng/m3,
ammonia = 2 mg/m3
Results
Chronic respiratory
symptoms included cough,
phlegm, chest tightness, and
wheezing; exposure to dust,
endotoxins, and ammonia
were not correlated to
chronic respiratory
symptoms; ammonia
exposure and duration of
disinfection were correlated
with impairment of baseline
lung function (decreased
FEV!,MMEF, andPEF)
Swine workers had a mean
cross-shift 2% decrease in
FEVi that was correlated
with personal exposure to
total dust, total endotoxin,
respirable endotoxin, and
ammonia
No association between
bronchial responsiveness and
exposure to respirable dust,
endotoxins, or ammonia;
mild bronchial
responsiveness was
associated with the
disinfectant use of quaternary
ammonia
Decreased lung function
(FEVj and FVC) was
observed over time; long-
term exposure to ammonia
was associated with
increased bronchial
responsiveness to histamine;
exposure to respirable dust
also caused increased
bronchial responsiveness to
histamine
Reference
Preller et al.,
1995
Reynolds et
al., 1996
Vogelzang et
al., 1997
Vogelzang et
al., 2000, 1998
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Table C-4. Cross sectional studies of livestock farmers exposed to ammonia
Subjects
Eight healthy male
volunteers (23-
28 yrs old)
257 poultry workers
(30% women, 70%
men); 63 women and
87 men nonexposed
blue-collar workers
served as control
subjects
Survey of
8,482 farmers and
spouses; exposure
study conducted in
102 farmers
13 stable workers
(6 males, 7 females)
Methods
Exposed for 4 hrs at
1-wk intervals to
swine confinement
buildings
Personal sampling
conducted for total
and respirable dust,
total and respirable
endotoxin, and
ammonia; medical
evaluations included
pulmonary function
tests given before
and after a work
period
Exposure study with
survey of respiratory
symptoms; personal
exposure to total
dust, fungal spores,
bacteria, endotoxin,
and ammonia in
12 tasks were
measured in
102 farmers
Stable workers were
tested for lung
function and nasal
lavage was
performed to analyze
for inflammation
markers; tests were
performed during
two consecutive
winters and the
interjacent summer
Exposure conditions
Mean airborne ammonia
concentration of
20.7ppm(15mg/m3);
also exposed to airborne
dust, bacteria,
endotoxin, and molds
Mean exposure levels of
poultry workers:
ammonia = 18.4 ppm
(13 mg/m3); total dust =
6.5 mg/m3; respirable
dust = 0.63 mg/m3; total
endotoxin 1,589 EU/m3
(0.16 ug/m3); respirable
endotoxin =58.9 EU/m3
(0.006 ug/m3)
Ammonia
concentrations ranged
from 0 to 8.2 ppm (0-6
mg/m3) over the
12 tasks; total dust (0.4-
5.1 mg/m3), fungal
spores (0.02-
2.0 106/m3), bacteria
(0.2-48 106/m3),
endotoxin (0.5-
28/103 EU/m3 [0.05-
2.8 ug/m3)
Ammonia concentration
was 20-27 ppm (14-
19 mg/m3) in late
summer, but was not
detected in winter; levels
of endotoxin were
highest during late
summer (15 ng/m3)
while levels of
l,3-p-glucan(85ng/m3)
and horse allergen
(18,300 U/m3) were
highest during the winter
Results
Decreased expiratory flows
(FEVi), increased
neutrophils in the nasal wash
and increased white blood
cell count
Significant cross-shift
declines in pulmonary
function were reported for
poultry workers;
concentrations associated
with significant pulmonary
function deficits were 12
ppm ammonia (8 mg/m3), 2.4
mg/m3 total dust, 0.16 mg/m3
respirable dust, and
614 EU/m3 endotoxin (0.614
ug/m3)
There was a significant
positive correlation between
task mean exposures to total
dust, fungal spores, and
endotoxins and task-specific
symptoms; there was no
association between
exposures to bacteria and
ammonia and task specific
symptoms; symptoms
included eye, nose, and
throat irritation, cough, chest
tightness, and wheezing
Increased PEF -variability in
2/13 workers; eosinophil
cationic protein in
3/13 (indicative of bronchial
obstruction and allergic
inflammation equivalent to
allergic asthma); increased
myeloperoxidase and
lysozyme levels in
9/13 (indicating enhanced
activity of neutrophil
granulocytes in the airways
and enhanced mucosal
secretion)
Reference
Cormier et al.,
2000
Donham et al.,
2000
Melbostad and
Eduard, 2001
Elfmanetal.,
2009
EU = endotoxin unit (10 EU/ng); MMEF = mean midexpiratory flow
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1 C.5. ACUTE AND SHORT-TERM INHALATION TOXICITY STUDIES OF AMMONIA
2 IN EXPERIMENTAL ANIMALS
3 Acute and short-term inhalation studies (exposure duration of <30 days) of ammonia in
4 experimental animals are summarized in Table C-5. These studies demonstrate that ammonia
5 inhalation can produce changes in pulmonary function and histopathological changes in the
6 respiratory tract. Acute effects may be followed by chronic respiratory dysfunction
7 characterized by secondary bronchitis, bronchiolitis, and bronchopneumonia. In studies of
8 cardiovascular and/or metabolic effects of acute or short-term ammonia exposure, ammonia
9 exposure was associated with bradycardia, arterial pressure variations, and acidosis (as
10 evidenced by a decrease in blood pH and an increase in arterial blood pCO2). Several studies
11 have investigated amino acid levels and neurotransmitter metabolism in the brain of rats and
12 mice following acute inhalation exposure to ammonia. It has been suggested that glutamate and
13 y-amino butyric acid (GABA) play a role in ammonia-induced neurotoxicity.
14
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Concentration
(mg/m3)
Duration
Parameter
examined
Results
Reference
Rats
Female Porton rats
(16/group)
Male OFA rats
(27/group)
Male and female
Wistar rats
(5/sex/group)
Male CrLCOBS CD
(Sprague-Dawley)
rats (8/group)
Male CrLCOBS CD
(Sprague-Dawley)
rats (14/group)
0 or 141
(0 or 200 ppm)
0 or 354
(0 or 500 ppm)
9,898-37,825
(14,000-53,500 ppm)
(no mention of control
group)
11,23,219, and 818
(15, 32, 310, and
1,157 ppm); arterial
blood collected prior
to exposure served as
control
3, 17, 31, 117, and
505 (4, 24, 44, 165,
and 714 ppm); arterial
blood collected prior
to exposure served as
control
Continuous
exposure for 4,
8, or 12 d
Continuous
exposure for 1-
8wks
10, 20, 40, or
60 min
24hrs
3 and 7 d
Histology of the trachea
Body weight, organ
weights, airway structure,
cell population, alveolar
macrophages
Clinical signs, pathology,
LCso
Clinical signs, histology,
blood pH, blood gas
measurement
Hepatic cytochrome P450
content and
ethylmorphine-N-
demethylase activity
4 d: transitional -stratified appearance of the
epithelium;
8 d: gross change with disappearance of cilia
and stratification on luminal surface;
12 d: increased epithelial thickness
No deaths occurred; decreased food
consumption and body weight gain; increased
lung and kidney weights; at 3 wks nasal
irritation and upper respiratory tract
inflammation, but no effect on lower airways;
slight decrease in alveolar macrophages; no
histopathological effects seen at 8 wks,
suggesting adaptation to exposure
Eye irritation, eye and nasal discharge, dyspnea;
hemorrhagic lungs on necropsy;
lO-minLCso = 28,492 mg/m3 (40,300 ppm)
20-minLCso = 20,217 mg/m3 (28, 595 ppm)
40-minLCso = 14,352 mg/m3 (20,300 ppm)
60-minLC50 = 11,736 mg/m3 (16,600 ppm)
No clinical signs of toxicity, no histologic
differences in trachea! or lung sections, no
change in blood pH or pCO2, minor changes in
P02
No dose-related change in P450 content or
enzyme activity
Gamble and Clough,
1976
Richard etal., 1978a
Appelman et al., 1982
Schaerdel et al., 1983
Schaerdel et al., 1983
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Male Long Evans
rats (4/group)
Female Wistar rats
(5/group)
Female Wistar rats
(5/group)
Female albino rats
(8/group)
Male Sprague-
Dawley rats
(number/group not
given)
Concentration
(mg/m3)
70 and 2 12
(100 and 300 ppm);
results were compared
to "control", but it was
not clear if the authors
were referring to
historical or
concurrent controls
0, 18, or 212
(0, 25, or 300 ppm)
0, 18, or 212
(0, 25, or 300 ppm)
0, 848-1,068 (1,200-
1,5 10 ppm)
Air concentration not
given; ammonia vapor
added to inspiratory
line of ventilator;
controls exposed to
same volume of room
air
Duration
6 hrs
6 hrs/d for 5, 10,
or!5d
6 hrs/d for 5 d
3 hrs
20 sec
Parameter
examined
Clinical signs, behavioral
observation
Blood ammonia, urea,
glutamine, and pH; brain
ammonia, glutamine;
histopathology of lungs,
heart, liver, and kidneys
(light and electron
microscopy)
Plasma and brain ammonia
and amino acid analysis
Mortality, respiratory
movement and O2
consumption
Activity of upper thoracic
spinal neurons
Results
Decreased running, decreased activity
Brain and blood glutamine increased; slight
acidosis (i.e., decreased blood pH) 212 mg/m3
(300 ppm); lung hemorrhage observed in some
exposed rats
Increase in brain and plasma glutamine
concentrations, increased brain/plasma ratio of
threonine
No deaths reported; inhibition of external
respiration and decreased O2 consumption
Lower airway irritation, activation of vagal
pulmonary afferents and upper thoracic spinal
neurons receiving pulmonary sympathetic input
Reference
Tepperetal., 1985
Manninenet al., 1988
Manninen and
Savolainen, 1989
Rejniuketal.,2007
Qin et al., 2007a, b
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Male Wistar rats
(4/group)
Male rats (10/group)
Concentration
(mg/m3)
0, 92-1,243 (130-
1,758 ppm); the
preexposure period
was used as the
control for each
animal
0, 848-1,068 (0,
1,200-1,5 10 ppm) at
the beginning and end
of the exposure
period)
Duration
45 min
3hrs
Parameter
examined
Airway reflexes by the
changes in respiratory
patterns elicited by
ammonia in either dry,
steam-humidified or
aqueous aerosol containing
atmospheres
Oxygen consumption
Results
Ammonia-induced upper respiratory tract
sensory irritation is not affected to any
appreciable extent by wet atmospheres (with or
without aerosol) up to 1,243 mg/m3
Decreased O2 consumption
Reference
LiandPahluhn,2010
Rejniuketal., 2008
Mice
Mice (20/group,
species, sex not
specified)
Male Swiss albino
mice (4/group)
Albino mice (sex not
specified; 6/dose)
6,080-7,070
(8,600-10,000 ppm);
no controls
5,050-20,199
(7, 143-28,571 ppm);
no controls
Air concentration not
measured; results
were compared to
"control", but it was
not clear if the authors
were referring to
historical or
concurrent controls
10 min
30-120 min
Continuously for
2or5d
LCso
LCso
Regional brain metabolism
(cerebral cortex,
cerebellum, brainstem);
MAO, enzymes of
glutamate and a-amino
butyric acid (GABA)
metabolism, and (Na+-K+)-
ATPase; amino acid levels
in the brain
LC50 = 7,056 mg/m3
(9,980 ppm)
LC50 (30 min) = 15,151 mg/m3 (21,430 ppm)
Altered activities of MAO, glutamate
decarboxylase, alanine amino transferase,
GABA-transaminase, and (Na+-K+)-ATPase;
increased alanine and decreased glutamate
Silver and McGrath,
1948
Hiladoetal., 1977
Sadasivuduetal.,
1979; Sadasivudu and
Murthy, 1978
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Male Swiss-Webster
mice
(4/group)
Male albino ICR
mice (12/dose)
Male Swiss-Webster
mice (16-24/group)
Male albino ICR
mice (12/dose)
Male albino ICR
mice (12/dose)
Male Swiss mice
(6/dose)
Concentration
(mg/m3)
Concentrations not
given; baseline levels
established prior to
exposure
0-3,436
(0-4,860 ppm)
0 or 216
(0 or 305 ppm)
0, 954, 3,097, or 3,323
(0, 1,350, 4,380, or
4,700 ppm)
0,81, or 233
(0, 115, or 330 ppm)
71 and 212
(100 and 300 ppm);
data collected during
the 2 d separating
each ammonia
exposure served as the
control baseline
Duration
10 min
lhr(14-d follow
up)
6 hrs/d for 5 d
4hrs
4 hrs/d for 4 d
6 hrs
Parameter
examined
Reflex decrease in
respiratory rate was used
as an index of sensory
irritation; RD50 = the
concentration associated
with a 50% decrease in the
respiratory rate
Clinical signs, body
weight, organ weight,
histopathology, LC50
Respiratory tract
histopathology
Hexobarbitol sleeping
time, microsomal protein
content, liver microsomal
enzyme activity
Microsomal protein
content, liver microsomal
enzyme activity
Clinical signs, behavioral
observation
Results
RD50 = 214mg/m3
(303 ppm)
Eye and nose irritation, dyspnea, ataxia,
seizures, coma, and death; decreased body
weight and increased liver to body weight ratio
in mice surviving to 14 d; effects in the lung
included focal pneumonitis, atelectasis, and
intralveolar hemorrhage; liver effects included
hepatocellular swelling and necrosis, vascular
congestion; LC50 = 2,990 mg/m3 (4,230 ppm)
Lesions in the nasal respiratory epithelium
(moderate inflammation, minimal necrosis,
exfoliation, erosion, orulceration); no lesions in
trachea or lungs
Increased hexobarbitol sleeping time
(3,097 mg/m3), increased microsomal protein
content, aminopyrene-N-deethylase and aniline
hydroxylase activities (3,323 mg/m3)
No dose-dependent effects on microsomal
enzymes
Decreased running, decreased activity
Reference
Kaneetal., 1979
Kapeghian et al., 1982
Buckley etal., 1984
Kapeghian et al., 1985
Kapeghian et al., 1985
Tepperetal., 1985
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Mice (4/group)
Male OF1 mice
(4/group)
Concentration
(mg/m3)
3, 21, 40, or 78 (4, 30,
56, or HOppm),
lowest measured
concentration was the
nominal control group
0, 92-1,243 (130-
1,758 ppm); the
preexposure period
was used as the
control for each
animal
Duration
2d
45 min
Parameter
examined
Responses to atmospheric
ammonia in an
environmental preference
chamber with four
chambers of different
concentrations of ammonia
Airway reflexes by the
changes in respiratory
patterns elicited by
ammonia in either dry,
steam-humidified, or
aqueous aerosol containing
atmospheres
Results
No distinguishable preference for, or aversion
to, different NH3 concentrations
Ammonia-induced upper respiratory tract
sensory irritation is not affected to any
appreciable extent by wet atmospheres (with or
without aerosol) up to 1,243 mg/m3
Reference
Green etal., 2008
LiandPahluhn,2010
Rabbits
Female New
Zealand White
rabbits (7-9/dose)
Rabbits (species,
sex, number/dose
not specified)
New Zealand White
rabbits (16 total;
8/dose)
0,35, or 71
(0, 50, or 100 ppm)
0, 707-14,140
(0, 1,000-
20,000 ppm)
Peak concentrations:
24,745-27,573 mg/m3
(35,000-39,000 ppm);
concurrent controls
tested
2.5-3.0 hrs
15-180 min
4 min
Pulmonary function
Pulmonary function, death
Pulmonary function, heart
rate, blood pressure, blood
gases
Decreased respiratory rate at both
concentrations
Bradycardia at 1,768 mg/m3 (2,500 ppm);
arterial pressure variations and blood gas
modifications (acidosis indicated by decreased
pH and increased pCO2) at 3,535 mg/m3
(5,000 ppm); death occurred at 4,242 mg/m3
(6,000 ppm)
Lung injury was evident after 2-3 min
(decreased pO2j increased airway pressure)
Mayan and Merilan,
1972
Richard etal., 1978b
Sjoblometal, 1999
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Concentration
(mg/m3)
Duration
Parameter
examined
Results
Reference
Cats
Mixed breed stray
cats (5/group)
0 or 707
(0 or 1,000 ppm)
10 min
Pulmonary function, lung
histopathology on 1, 7, 21,
and 35 d postexposure
Pulmonary function deficits were correlated
with lung histopathology; acute effects are
followed by chronic respiratory dysfunction
(secondary bronchitis, bronchiolitis, and
bronchopneumonia)
Dodd and Gross, 1980
Pigs
Young pigs
(2/group)
Male and Female
Belgian Landrace
pigs (4/group)
Belgian Landrace
pigs (4/group)
Landrace-Yorkshire
pigs (4/group)
Hybrid gilts (White
synthetic Pietrain,
white Duroc,
Landrace, Large
White)
(14 pigs/group)
0,35, 71, or 106(0,
50, 100, or 150 ppm)
0, 18, 35, or 71
(0, 25, 50, or
100 ppm)
0, 18, 35, or 71
(0, 25, 50, or
100 ppm)
0 or 42 (0 or 60 ppm)
<4 (control) or
14 (<5 or 20 ppm)
Continuous
exposure for
4 wks
6d
6d
15 min/dfor
8 wks
15 wks
Clinical signs, food
consumption, body weight,
gross necropsy, organ
weight, histopathology
Clinical signs, body
weight, pulmonary
function
Clinical signs, body
weight, neutrophil count,
and albumin in nasal
lavage fluid
Thromboxane A2 (TXA2),
leukotriene C4 (LTC4),
and prostaglandin (PGI2)
production
Salivary cortisol, adrenal
morphometry, body
weight, food conversion
efficiency, general health
scores, play behavior;
reaction to light and noise
intensity tested
concurrently
Lethargy and histopathological alterations in the
tracheal and nasal epithelium were observed at
71 and 106 mg/m3; decreased body weight
occurred at all concentrations (7-19% decrease
from control)
Lethargy and decreased body weight gain (all
concentrations); no effect on pulmonary
microvascular hemodynamics and permeability
Nasal irritation (increased neutrophils in nasal
lavage fluid) and decreased body weight gain at
all concentrations
Significant increases in TXA2 and LTC4, no
significant effect on PGI2 production
Decreased salivary cortisol, larger adrenal
cortices, less play behavior, no measurable
impact on productivity or physiological
parameters
Drummondetal.,
1980
Gustinetal., 1994
Urbainetal., 1994
Chaung et al., 2008
O ' Connor etal., 20 10
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Concentration
(mg/m3)
Duration
Parameter
examined
Results
Reference
Cattle
Male Holestein
calves
(number/group not
specified)
Brahman/Charolais
(group size not
reported)
Holstein Friesian
and Brown Swiss
(10 of each breed)
0,35, or 71
(0, 50, or 100 ppm)
<6 (control), 11,23, or
34 (<8 [control], 16,
32, or 48 ppm)
~0, 4, and 15,
(0.3 x ID'6, 6, and
21 ppm)
2.5 hrs
12 d
10 d at each
concentration
Respiration rate, clinical
chemistry
Behavioral activity, body
weight, analysis of
bronchioalveolar lavage
(BAL) fluid, hematological
variables (hemoglobin,
mean cell volume, platelet
volume, eosinophils,
neutrophils, total white cell
count, monocytes)
Respiration and pulse rate,
blood gas parameters
No significant effect on respiration, BUN, pH,
P02,orpC02
Increased lacrimation, nasal secretions,
coughing, increased standing (as opposed to
lying down), dose related increases in
macrophage activity and neutrophil percentage
in BAL fluid indicating pulmonary
inflammation, no effect on hematological
variables or body weight
Respiration and pulse rates were higher in
inadequately ventilated barns (elevated
ammonia and CO2)
Mayan and Merilan,
1976
Phillips etal., 20 10
Sabuncuoglu et al.,
2008
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1 C.6. MECHANISTIC STUDIES
2 Portions of this appendix were adapted from the Mechanisms of Action and Genotoxicity
3 sections (Sections 3.3 and 3.5) of the Toxicological Profile for Ammonia (ATSDR, 2004) under
4 a Memorandum of Understanding with ATSDR.
5
6 C.6.1. Irritation
7 As described in Section 4.1, ammonia is an irritant in humans where the primary and
8 most immediate effect of ammonia exposure is burns to the skin, eyes, gastrointestinal tract, and
9 respiratory tract. Due to its high water solubility, ammonia interacts immediately upon contact
10 with available moisture in the skin, eyes, oral cavity, respiratory tract, and mucous membranes to
11 form ammonium hydroxide, which is a weak base. Ammonium hydroxide causes the necrosis of
12 tissues through disruption of cell membrane lipids (saponification) leading to cellular destruction
13 (Jarudi and Golden, 1973). As cell proteins break down, water is extracted, resulting in an
14 inflammatory response, which further damages the surrounding tissues (Amshel et al., 2000).
15 The severity of tissue damage is related to the concentration of the hydroxyl ions and the
16 duration of exposure (White et al., 2007; Welch, 2006; Millea et al., 1989).
17
18 C.6.2. Gastric Mucosal Damage
19 Ammonium ion may also contribute to adverse effects ofH. pylori on the stomach.
20 H. pylori produces urease, which breaks down urea that is normally present in the stomach into
21 ammonia (Megraud et al., 1992; Tsujii et al., 1992a). An in vitro study that examined the effects
22 of ammonia produced by H. pylori on HEp2 cells showed increased cell vacuolation and
23 decreased viability of the cells compared to a urease negative variant of the same cells (Megraud
24 et al., 1992). An in vivo study suggested that ammonia also causes macroscopic gastric lesions
25 and increases the release of endothelin-1 and thyrotropin releasing hormone from the gastric
26 mucosa, probably via an endothelin-A receptor, which exerts ulcerogenic action on the gastric
27 mucosa (Mori et al., 1998). Ammonia may also trigger the release of cysteine proteases in the
28 stomach that contribute to the development of gastric hemorrhagic mucosal lesions (Nagy et al.,
29 1996). Suzuki et al. (2000) reported findings of an in vitro assay with gastric surface mucous
30 cells from mice that demonstrated apoptosis directed by mitochondrial membrane destruction
31 and enhancing activities of caspase-3 and caspase-9 at concentrations of ammonia detected in
32 H. pylori infected patients. Neutrophils that migrate to the gastric mucosa in response to the
33 presence ofH. pylori may release hypochlorous acid, which can interact with ammonium ion to
34 produce the powerful cytotoxic oxidizing agent monochloramine (Murakami et al., 1995).
35 Igarashi et al. (2001) suggest that ammonia accelerates cytokine-induced apoptosis in gastric
36 epithelial cells.
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1 Tsujii et el. (1992b) studied the mechanism by which ammonia damages the gastric
2 mucosa by measuring the following: gastric mucosal damage, gastric mucosal hemodynamics,
3 the viability and oxygen consumption of cells separated from the gastric mucosa, and the effect
4 of ammonia on gastric mucosa mitochondrial oxygen consumption. For the measurement of
5 gastric mucosal damage, ammonia solutions (2 mL; 0, 125, 187.5, and 150 mM) were
6 administered to the stomach of male Sprague-Dawley rats (6 rats/group; 220-250 g). Thirty
7 minutes later, the ulcer index (defined as the ratio of the ulcerated area to that of the whole
8 stomach as measured using an image-analyzer system) was measured. Solutions >187.5 mM
9 ammonia induced significantly larger ulcers and the increase was dose dependent. The effect of
10 pH of the initial ammonia solution was measured by repeating the experiment with 250 mM
11 glycine-NaOH/250 mM sodium phosphate solution (pH 10.3); this solution did not cause any
12 significant damage to the mucosa suggesting that the effect was due to ammonia and not to
13 elevated pH.
14 The effects of ammonia on gastric mucosal hemodynamics were measured (5 rats/group)
15 after the surgical placement of an optical fiber bundle of a reflectance spectrometer in the
16 forestomach. Rats were administered 2 ml 250 mM ammonia/250 mM sodium phosphate
17 solution and their hemoglobin concentration and hemoglobin oxygen saturation were measured.
18 There was no significant effect of ammonia administration on indices of gastric hemoglobin
19 concentration or hemoglobin oxygen saturation.
20 Cells from the gastric mucosa were separated and isolated. Ammonia (0, 1, 5, or 10 mM
21 ammonia in sodium phosphate buffer, pH 7.4) was added to the cell cultures and incubated for 2
22 hours at 37 °C. Cell viability was measured using trypan blue exclusion. Cell viability and
23 oxygen consumption were significantly lower at 5 and 10 mM ammonia in sodium phosphate
24 buffer.
25 Mitochondria were obtained from the gastric mucosa of five rabbits (2 kg, the strain was
26 not specified) after removal of the stomach. Mitochondrial suspensions were treated with 0,
27 0.01, 0.1, 1, 5, or 10 mM ammonia/sodium phosphate in Tris buffer. Oxygen consumption was
28 measured after the addition of a-ketoglutaric acid followed by adenosine diphosphate. There
29 was significant inhibition of oxygen consumption at the lowest ammonia concentration tested
30 (0.01%) without the addition of a-ketoglutaric acid or adenosine diphosphate. Oxygen
31 consumption was significantly lower with a-ketoglutaric acid as the substrate at >1 mM
32 ammonia concentrations; with adenosine diphosphate as the substrate, ammonia concentrations
33 >0.1 mM were inhibitory.
34 In summary, Tsujii et al. (1992b) showed that millimolar concentrations of ammonia
35 inhibit cellular respiration and was cytotoxic to gastric mucosal cells. The effects were not due
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1 to the elevated pH of the ammonia solutions. The authors concluded that ammonia-induced
2 gastric mucosal damage may be due to impairment of cellular energy metabolism.
3
4 C.6.3. Genotoxicity Studies
5 A limited number of genotoxicity studies are available for ammonia vapor; four of the
6 available studies were published between 1932 and 1951. Studies examining in vivo
7 genotoxicity are described in Table C-6. Yadav and Kaushik (1997) examined the genotoxic
8 effects of ammonia exposure in 22 fertilizer factory workers exposed to ammonia at ambient
9 concentrations of 88.2 |ig/m3; 42 nonexposed workers served as control subjects. Increased
10 frequencies of chromosomal aberrations and sister chromatid exchanges in lymphocytes were
11 observed in exposed workers compared to control subjects. Frequencies of chromosomal
12 aberrations, sister chromatid exchanges, and mitotic index all increased with increased duration
13 of exposure. This study is difficult to interpret because of small samples sizes and confounding
14 by smoking and alcohol consumption. In addition, the levels of ammonia in the plant seemed
15 low compared to other fertilizer plant studies (see for example Section 4.1.4; Rahman et al.,
16 2007; Ali, 2001; Ballal et al., 1998); the accuracy and reliability of the sampling and
17 measurement could not be determined.
18
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Table C-6. In vivo genotoxicity studies of ammonia
Test system
Human
lymphocytes
Human
lymphocytes
Swiss albino
mice
Drosophila
melanogaster
D. melanogaster
D. melanogaster
Endpoint
Chromosome
aberrations
Sister chromatid
exchanges
Micronucleus
Dominant lethal
mutations
Sex-linked
recessive lethal
mutations
Dominant lethal
mutations
Test conditions
22 healthy workers
occupationally exposed to
ammonia in fertilizer factory
(ambient concentration of
88.28 ug/m3); nonexposed
factory staff served as control
subjects
Intraperitoneal injections for
24-48 hr expression times
Dominant lethal assay;
inhalation exposure up to
450 ppm (318 mg/m3) ammonia,
6 hrs/d for 5 d
Inhalation exposure to ammonia
as vapor at a concentration
killing the majority of flies
Inhalation exposure to ammonia
as vapor at a concentration
killing the majority of flies
Results"
+
+
+
+ (T)
-(T)
-(T)
Dose or
concentration1"
88.28 ug/m3
88.28 ug/m3
12.5-50 mg/kg
NA
NA
NA
Reference
Yadav and
Kaushik, 1997
Yadav and
Kaushik, 1997
Yadav and
Kaushik, 1997
Lobasov and
Smirnov, 1934
Auerbach and
Robson, 1947
Auerbach and
Robson, 1947
1
2
o
3
4
5
6
7
8
9
10
11
12
13
14
15
a+ = positive; - = negative; (T) = toxicity reported; NA = not available.
bLowest effective dose for positive results, highest dose tested for negative or equivocal results.
Positive results were obtained in a single micronucleus assay with mice (Yadav and
Kaushik, 1997). One study in Drosophila exposed to ammonia gas was positive for
mutagenicity; however, survival after exposure was <2% (Lobasov and Smirnov, 1934). Results
from another study in Drosophila were negative for sex-linked recessive lethal and dominant
lethal mutagenicity; however, the majority of Drosophila was killed by ammonia treatment
(Auerbach and Robson, 1947).
In vitro tests of the genotoxic effects of ammonia have been performed in bacteria and in
chick fibroblasts (Table C-7). Ammonia (administered as vapor) did not induce reverse
mutations in Salmonella typhimurium or E. coli either with or without metabolic activation
(Shimizu et al., 1985). Demerec et al. (1951) reported an increase in reverse mutations inE. coli;
however, positive findings were only reported for levels of ammonia that were toxic, with 98%
lethality. Chick fibroblasts immersed in buffered ammonia solution were found to have
increased frequencies of chromosomal aberrations (Rosenfeld, 1932).
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Table C-7. In vitro genotoxicity studies of ammonia
Test system
S. typhimurium
(TA98, TA100,
TA1535, TA1537,
TA1538); E. coli
(WP2 uvrA)
E. coli (B/SD-4
strains)
Chick fibroblasts
Endpoint
Reverse
mutation
Reverse
mutation,
streptomycin
resistance
Chromosomal
aberrations
Test conditions
Plate incorporation
assay with ammonia
vapor
Plate incorporation
assay
Cultures immersed in
buffered ammonia
solution
Results"
Without
activation
+ (T)
+
With
activation1"
ND
ND
Dosec
25,000 ppm
(17,675 mg/m3)
ammonia vapor
0.25%
ammoniad
NA
Reference
Shimizu et al.,
1985
Demerec et al.,
1951
Rosenfeld,
1932
a+ = positive; - = negative; (T) = toxicity reported; NA = not available; ND = no data.
Exogenous metabolic activation used; S9 liver fractions from male Sprague-Dawley rats pretreated with
pentachlorobiphenyl (KC500).
°Lowest effective dose for positive results, highest dose tested for negative or equivocal results.
dOnly positive in treatments using toxic levels of NH3 (98% lethality).
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