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www.epa.gov/iris

 

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 BIOASSAYS 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-1

APPENDIX B. SUMMARY OF REPEAT DOSE TOXICITY INFORMATION FOR 

SELECTED AMMONIUM SALTS ......................................................................................B-1

APPENDIX C. SUPPLEMENTAL INFORMATION ON AMMONIA ...................................C-1

C.1. AMMONIA LEVELS MEASURED IN EXPIRED AIR IN HUMANS ....................C-1

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 ............................................................ 3

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 

mucosa of the rat 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 with M. 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-1. Summary of noncancer results of repeat dose studies of oral exposure of 

experimental animals to selected ammonium salts .............................................................. B-1

Table C-1. 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 American Conference of Governmental Industrial Hygienists 

ALP alkaline phosphatase 

ALT alanine aminotransferase 

AMP adenosine monophosphate 

AST aspartate aminotransferase 

ATSDR Agency for Toxic Substances and Disease Registry 

ATSG acid-treated silica gel

BMD benchmark dose 

BrDU 5-bromo-2-deoxyuridine

BUN blood urea nitrogen 

C3 complement 3 

CAC cumulative ammonia concentration 

CASRN Chemical Abstracts Service Registry Number 

CI confidence interval 

FEF forced expiratory flow 

FEV forced expiratory volume 

FVC forced vital capacity 

GABA γ-amino butyric acid 

IgE immunoglobin E 

IgG immunoglobin G 

IRIS Integrated Risk Information System 

LOAEL lowest-observed-adverse-effect level 

MAO monoamine oxidase 

MMEF mean midexpiratory flow 

MNNG N-methyl-N’-nitro-N-nitrosoguanidine 

MRM murine respiratory mycoplasmosis 

NAD+nicotineamide adenine dinucleotide, oxidized

NADH nicotineamide adenine dinucleotide, reduced 

NH3 ammonia 

NH4

+

 ammonium ion 

NIOSH National Institute for Occupational Safety and Health

NMDA N-methyl D-aspartate 

NOAEL no-observed-adverse-effect level 

NOx nitrogen oxides

NRC National Research Council 

OR odds ratio 

PARP poly(ADP-ribose) polymerase 

PEF peak expiratory flow 

PEFR peak expiratory flow rate 

PHA phytohemagglutin 

POD point of departure

PPD purified protein derivative 

PRRSV porcine reproductive and respiratory syndrome virus

RfC reference concentration 

RfD reference dose 

SIFT-MS 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 Bacom 

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

Ted Berner, 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 

2 

3 

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 

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m313 ) 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 

plausible upper bound on the estimate of risk per μg/m329 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. 

EPA, 2006b), and Recommended Use of Body Weight3/4 12 as the Default Method in Derivation of 

13 the Oral Reference Dose (U.S. EPA, 2011a). 

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. 

28 
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1 2. CHEMICAL AND PHYSICAL INFORMATION 

2 

3 

4 Ammonia is a corrosive gas with a very pungent odor (O’Neil et al., 2006). It is highly 

soluble in water (4.82 × 1055 mg/L) and is a weak base (Lide, 2008; Eggeman, 2001; Dean, 1985). 

When ammonia (NH3

+

6 ) 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

+

7 ) (Lide, 

8 2008). A solution of ammonia in water is sometimes referred to as ammonium hydroxide 

9 because the ammonia and water both ionize to form ammonium cations and hydroxide anions 

10 (Eggeman, 2001). Ammonium salts are easily dissolved in water and disassociate into the 

11 ammonium ion and the anion. 

12 Many physical and chemical properties of ammonia are related to the pH of ammonia in 

13 solution (ammonium hydroxide). Ammonium hydroxide is a weak base that is partially ionized 

in water with a dissociation constant of 1.77 x 10-5 14 at 25 °C that increases slightly with 

15 increasing temperature (Reed, 1982). At a pH of 8.25, 90% of ammonia will be protonated. At a 

16 pH of 7.25, 99% of ammonia will be protonated. Thus, a decrease in pH would result in an 

17 increase in the ammonium ion concentration and an increase in solubility of ammonia in water. 

At physiological pH (7.4), the equilibrium between NH3 and NH4

+

 favors the formation of NH4

+

18 . 

19 Chemical and physical properties of ammonia are listed in Table 2-1. 

20 

Table 2-1. Chemical and physical properties of ammonia 

Chemical name Ammoniaa

Synonym(s) AM-Fol; anhydrous ammonia; ammonia gas; 

Nitro-sil; R 717; Spirit of hartshorn 

ChemID Plus, 2009 

Structure 

N

H

H H

ChemID Plus, 2009 

Chemical formula NH3 ChemID Plus, 2009 

CASRN 7664-41-7a ChemID Plus, 2009 

Molecular weight 17.031 Lide, 2008 

Form Colorless gas; corrosive O’Neil et al., 2006 

Melting point -77.73°C Lide, 2008

Boiling point -33.33°C Lide, 2008 

Odor threshold 53 ppm O’Neil et al., 2006 

Density 0.7714 g/L at 25°C O’Neil et al., 2006 

Vapor density 0.5967 (air = 1) O’Neil et al., 2006 

pKa (ammonium ion) 9.25 Lide, 2008 

Solubility: 

 Water 

 Organic solvents 

4.82 × 105 mg/L at 24°C 

Soluble in ethanol, chloroform, and ether 

Dean, 1985 

Lide, 2008; O’Neil et al., 2006 
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Table 2-1. Chemical and physical properties of ammonia 

Vapor pressure 7.51 × 103 mm Hg at 25°C AIChE, 1999 

Henry’s law constant 1.61 × 10-5 atm-m3/mol at 25°C Betterton, 1992 

Conversion factors 

 ppm to mg/m3

 mg/m3 to ppm 

1 ppm = 0.707 mg/m3

1 mg/m3 = 1.414 ppm 

Verschueren, 2001 

Verschueren, 2001 

aAmmonia dissolved in water is sometimes referred to as ammonium hydroxide (CASRN 1336-21-6). Ammonium

hydroxide does not exist outside of solution. 

1 

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. TOXICOKINETICS11 

2 

3 

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 

ammonium ion (NH4

+

10 ). 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 

Experiments with volunteers225 show that ammonia, regardless of its tested concentration 

in air (range, 57–500 ppm or 40–354 mg/m326 ), 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 

(354 mg/m329 ) resulted in lower retention (4–30%), with expired breath concentrations of 350–400 

ppm (247–283 mg/m330 ) 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. 

 

1

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. 

Exposure to a common occupational limit of ammonia in air (25 ppm or 18 mg/m33 ), assuming 

4 30% uptake into blood, would yield an increase in blood ammonia concentration of 0.09 μg/mL 

5 (calculated by WHO, 1986). This calculated rise would likely be indistinguishable from the 

6 observed baseline levels of 0.1–1.0 μg/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; 

Dalhamn, 1963; Boyd et al., 1944). Continuous exposure of rats to up to 32 ppm (23 mg/m310 ) for 

11 24 hours did not result in a statistically significant increase in blood ammonia levels (0.1 μg/mL 

above preexposure levels), whereas exposures to 310–1,157 ppm (219–818 mg/m312 ) 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 (Schaerdel et al., 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 μg/mL in the blood (Klendshoj and Rejent, 1966). This blood concentration is about 30-

23 fold higher than the concentration of 1 μg/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 
7 DRAFT - DO NOT CITE OR QUOTE 

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 μg/mL in arterial blood and 0.2 to 1.7 μg/mL in venous 

11 blood (Huizenga et al., 1994). Other baseline levels observed in experimental volunteers range 

12 from 1 to 5.5 μg/mL (Conn, 1972; Brown et al., 1957). Ammonia is homeostatically regulated to 

remain at low concentrations, with 95–98% existing in the blood (at physiological pH) as NH4

+

13 

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). Jόźwik 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 μg/mL, compared 

21 to 0.5 μg/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 (Jόźwik 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 μg/mL (Jόźwik 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). 

Rats inhaling 300 ppm (212 mg/m37 ) 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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1 

2 

3 

4 

5 Adapted from: Nelson and Cox (2008). 

6 

7 Figure 3-1. Glutamine cycle. 

8 

Glutamate

γ-Glutamyl

phosphate

glutamine

synthetase

glutamine

synthetase

Glutamine

glutaminase

(in liver 

mitochondria)

NH4

+

H2O

ATP

ADP

NH4

+ Pi
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1 

2 

3 Adapted from: Nelson and Cox (2008). 

4 

5 Figure 3-2. The urea cycle showing the compartmentalization of its steps 

6 within liver cells. 

7 

8 Given its important metabolic role, ammonia exists in a homeostatically regulated 

9 equilibrium in the body. In particular, free ammonia has been shown to be homeostatically 

10 regulated to remain at low concentrations, with 95–98% of body burden existing in the blood (at 

physiological pH) as NH4

+

11 ion (da Fonseca-Wolhheim, 1995; Souba, 1987). Two studies in rats 

12 (Manninen et al., 1988; Schaerdel et al., 1983) provided evidence that ammonia concentrations 

in air below 25 ppm (18 mg/m313 ) do not alter blood ammonia concentrations. Schaerdel et al. 

14 (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/m315 ) ammonia did not increase blood ammonia 

16 concentrations after 24 hours; concentrations of ≥32 ppm caused an exposure-released increase 

17 in blood ammonia, but concentrations at 12- and 24-hour sampling periods were lower than at 8 

18 hours, suggesting compensation by increasing ammonia metabolism through conversion to urea, 

19 pyrimidine and polyamine synthesis, incorporation into amino acid substrates, and metabolism in 

nervous system tissue. Rats inhaling 25 ppm (18 mg/m320 ) ammonia for 6 hours/day for 15 days 

Carbamoyl

phosphate 

synthase I

H2O

2 ATP

2 ADP

Pi

3 H+

CO2 + NH4

+

Pi

Citrulline

Ornithine

Aspartate

Arginine Argininosuccinate

H2O

Urea

Ornithine

Ornithine

transcarbamoylase

Citrulline

Carbamoyl

phosphate

Argininosuccinate

synthase

Argininosuccinate

lyase

Arginase

MITOCHONDRIAL

MATRIX

Fumarate

ATP

AMP + PPi

CYTOSOL
11 DRAFT - DO NOT CITE OR QUOTE 

1 did not exhibit blood or brain ammonia or glutamine levels that were different from controls; 

however, rats inhaling 300 ppm (212 mg/m32 ) 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 

result in a shift in the NH4

+

16 /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 

encephalopathy, the observed trapping of [13 18 N]-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 

outer medullary collecting duct via H+32 secretion and parallels ammonia diffusion (Flessner et al., 

1992). The inner medullary collecting duct exhibits a Na+- and K+-independent NH4

+

/H+33 

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 

volunteers (with samples collected daily over a 4-week period) of 0.059–0.078 mg/m32 ; these 

3 concentrations were similar to or slightly higher than the mean laboratory air level of ammonia 

of 0.056 mg/m34 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 

to 0.046 mg/m36 . One sample collected from the trachea (via a tube inserted through the nose of 

one subject) was 0.029 mg/mg 7 3—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 

from 0.085 to 2.1 mg/m312 (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) 

reported decreases in baseline ammonia levels (0.085–0.905 mg/m321 ) 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 

mg/m327 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.1 in Table C-1. 

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 

of rats showed concentrations ranging from 0.01 to 0.353 ppm (0.007–0.250 mg/m34 ) 

(mean = 0.08 ppm or 0.06 mg/m35 ) in nose-breathing animals (Barrow and Steinhagen, 1980). 

6 Larson et al. (1980) reported ammonia concentrations measured in the larynx of dogs exposed to 

sulfuric acid ranging between 0.03 and 0.225 ppm (0.02 and 0.16 mg/m37 ) following mouth 

breathing, and between 0.05 and 0.220 ppm (0.04 and 0.16 mg/m38 ) 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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1 4. HAZARD IDENTIFICATION 

2 

3 

4 As noted in Section 2, ammonium salts (e.g., ammonium acetate, chloride, and sulfate) 

readily dissolve in water through disassociation into the ammonium ion (NH4

+

5 ) and the anion. 

At physiological pH (7.4), the equilibrium favors the formation of NH4

+

6 . 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 dose15 related depression in body weight gain at exposure to ammonium chloride doses ≥1,590 mg/kg16 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-1, 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 

>15 ppm (11 mg/m3), with odor detection thresholds at <20 ppm (14 mg/m36 ). In another study, 

12 healthy volunteers exposed to 5 and 25 ppm (4 and 18 mg/m37 ) ammonia on three different 

8 occasions for 1.5 hours in an exposure chamber while exercising on a stationary bike reported 

discomfort in the eyes and odor detection at 5 ppm (4 mg/m39 ) (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 

140 ppm (35, 57, 78, and 99 mg/m3); ammonia concentrations of 140 ppm (99 mg/m312 ) caused 

13 severe and intolerable irritation (Verberk, 1977). The lachrymatory threshold was determined to 

be 55 ppm (39 mg/m314 ) 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 

ppm (64 mg/m316 ) 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 

al., 1970). Exposure to 500 ppm (354 mg/m318 ) 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 

eyes and nose at concentrations ranging from 2 to 500 ppm (1–354 mg/m324 ) 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 

129 ppm (91 mg/m3) and 175 ppm (124 mg/m327 ), respectively. Smeets et al. (2007) investigated

28 odor and irritation thresholds for ammonia vapor in 24 healthy female volunteers at 

concentrations ranging from 0.03 to 615 ppm (0.02 to 435 mg/m329 ). This study observed a mean 

odor detection threshold of 2.6 ppm (2 mg/m330 ) and a mean irritation threshold of 31.7 or 60.9 

ppm (22 or 43 mg/m331 ), 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, 

14, and 35 mg/m336 ) 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 

group, but at a concentration of 20 ppm (14 mg/m338 ), there were no differences in perceived 
17 DRAFT - DO NOT CITE OR QUOTE 

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 

to ammonia concentrations of 25, 50, and 100 ppm (18, 35, and 71 mg/m35 ) 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 

breaths of gas experienced bronchioconstriction at a concentration of 85 ppm (60 mg/m310 ) 

11 (Douglas and Coe, 1987); however, there were no bronchial symptoms reported in seven 

volunteers exposed to ammonia at concentrations of 30, 50, or 90 ppm (21, 35, and 64 mg/m312 )

13 for 10 minutes in an exposure chamber (MacEwen et al., 1970). Similarly, 12 healthy volunteers 

exposed to ammonia on three separate occasions to 5 and 25 ppm (4 and 18 mg/m314 ) 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 FEV1 (Sundblad et al., 2004). In another study, 18 healthy 

18 servicemen volunteers were placed in an exposure chamber for 3 consecutive half-day sessions. 

Exposure to ammonia at concentrations of 50–344 mg/m

3

 (70–486 ppm) occurred on the 2nd 19 

20 session, with sessions 1 and 3 acting as controls (Cole et al., 1977). The no-effect concentration 

was determined to be 71 mg/m3 (100 ppm). Exercise tidal volume was increased at 106 mg/m3 21 

22 (150 ppm), but then decreased at higher concentrations in a concentration-dependent manner 

23 (Cole et al., 1977). Decreased FEV1 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 

mg/m325 ) 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 FEV1 in 25 healthy volunteers and 15 mild/moderate persistent asthmatic volunteers after 

ocular and nasal exposure to 2–500 ppm (1–354 mg/m332 ) ammonia at durations lasting up to 

33 2.5 hours (Petrova et al., 2008). In another study, six healthy volunteers and eight mildly 

asthmatic volunteers were exposed to 16–25 ppm (11–18 mg/m334 ) 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 FEV1 in the healthy 

37 volunteers for any exposure. A decrease in FEV1 was reported in asthmatics exposed to dust and 

38 ammonia, but not ammonia alone; similarly, increased bronchial hyperreactivity was reported in 
18 DRAFT - DO NOT CITE OR QUOTE 

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 

exposure to 2 mg/m36 (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-

oxycorticosteroids in plasma. Ammonia concentrations of 5 mg/m39 (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 

concentration of 5 mg/m313 (7.2 ppm) (Kalandarov et al., 1984). 

14 In summary, volunteer studies demonstrate that eye irritation can occur following acute 

exposure to ammonia at concentrations as low as 5 ppm (4 mg/m315 ). 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 

acutely exposed to ammonia concentrations as high as 71 mg/m318 (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 FEV1, 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 
19 DRAFT - DO NOT CITE OR QUOTE 

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 

carbonate) plant315 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) 

was 9.2 ppm (6.5 mg/m3), compared to 0.3 ppm (0.2 mg/m320 ) 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. 
20 DRAFT - DO NOT CITE OR QUOTE 

1 in linear regression analysis. Exposed workers were grouped into three exposure categories 

(high = >12.5 ppm [>8.8 mg/m3], medium = 6.25–12.5 ppm [4.4–8.8 mg/m32 ], and low = 

<6.25 ppm [<4.4 mg/m33 ]) 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, FEV1, FEV1/FVC, forced expiratory flow [FEF50], 

7 and FEF75). 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 

exposure category (<4.4 mg/m310 ) 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 

mg/m322 (12.5 ppm) as the no-observed-adverse-effect level (NOAEL). A lowest-observed23 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 3/31(10)a 6/34 (18) 1/12 (8) 2/12 (17) 

Sputum 5/31 (16) 9/34 (26) 3/12 (25) 1/12 (8) 

Wheeze 3/31 (10) 5/34 (15) 1/12 (8) 0/12 (0) 

Chest tightness 2/31 (6) 2/34 (6) 0/12 (0) 0/12 (0)

Shortness of breath 4/31 (13) 3/34 (9) 1/12 (8) 0/12 (0) 

Nasal complaints 6/31 (19) 4/34 (12) 2/12 (17) 0/12 (0) 

Eye irritation 6/31 (19) 2/34 (6) 2/12 (17) 1/12 (8) 

Throat irritation 1/31 (3) 2/34 (6) 1/12 (8) 1/12 (8) 

Skin problems 2/31 (6) 10/34b (29) 1/12 (8) 1/12 (8) 

Lung function (% predicted)

FVC 98.6 96.7 96.9 96.8 

FEV1 95.1 93.7 93.9 95.3 

FEF50 108.4 106.9 106.2 111.2 

FEF75 65.2 71.0 67.8 78.8 

FVC = forced vital capacity; FEV1=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. 

b

Significantly different from controls, p < 0.05, by Fisher’s exact test performed for this review. 

Source: Holness et al. (1989). 

1 

2 Ballal et al. (1998) conducted a cross-sectional study of male workers at two urea 

fertilizer factories in Saudi Arabia43 . 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 

exposed to air ammonia levels of 2–130 mg/m35 (2.8–184.4 ppm), and workers in factory B were 

exposed to 0.02–7 mg/m3 6 (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. 
22 DRAFT - DO NOT CITE OR QUOTE 

[TLV] of 18 mg/m3 1 [25 ppm]) showed that those exposed to ammonia concentrations higher than 

2 the TLV had significantly higher relative risks for cough, phlegm, wheezing, dyspnea, and 

3 asthma than workers exposed to levels below the TLV (Table 4-2). The relative risk for 

4 wheezing was also elevated among those exposed to ammonia levels at or below the TLV. 

Distribution of symptoms by cumulative ammonia concentration (CAC, mg/m35 -years) also 

6 showed significantly higher relative risk for all the above symptoms among those with higher 

7 CAC (Table 4-2). Results of the logistic regression analysis showed that ammonia concentration 

8 was significantly related to cough, phlegm, wheezing with and without shortness of breath, and 

9 asthma (Table 4-3). 

10 

Table 4-2. The prevalence of respiratory symptoms and disease in urea 

fertilizer workers exposed to ammonia 

Respiratory 

symptom/disease 

Relative risk (95% CI) 

Exposure category 

Cumulative concentration 

(mg/m3 of air-yrs) 

≤ACGIH TLV 

(18 mg/m3) 

(n = 138) 

>ACGIH TLV 

(18 mg/m3) 

(n = 17) 

≤50 

(n = 130) 

>50 

(n = 30) 

Cough 0.86 (0.48–1.52) 3.48 (1.84–6.57) 0.72 (0.38–1.35) 2.82 (1.58–5.03) 

Wheezing 2.26 (1.32–3.88) 5.01 (2.38–10.57) 1.86 (1.04–3.32) 5.24 (2.85–9.52) 

Phlegm 0.79 (0.43–1.47) 3.75 (1.97–7.11) 0.63 (0.31–1.26) 3.03 (1.69–5.45) 

Dyspnea 1.13 (0.62–2.04) 4.57 (2.37–8.81) 1.19 (0.66–2.17) 2.59 (1.25–5.36) 

Chronic bronchitis 1.43 (0.49–4.19) 2.32 (0.31–17.28) 0.61 (0.13–2.77) 5.32 (1.72–16.08)

Bronchial asthma 1.15 (0.62–2.15) 4.32 (2.08–8.98) 1.22 (0.66–2.28) 2.44 (1.10–5.43) 

Chronic bronchitis and 

bronchial asthma 

2.57 (0.53–12.59) 6.96 (0.76–63.47) 1.82 (0.31–10.77) 8.38 (1.37–45.4) 

Source: Ballal et al. (1998).

11 

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 OR (95% CI) 

Cough 1.32 (1.08–1.62)a

Phlegm 1.36 (1.10–1.67)a

Shortness of breath with wheezing 1.26 (1.04–1.54)a

Wheezing alone 1.55 (1.17–2.06)a

Dyspnea on effort 0.83 (0.68–1.02) 

Diagnosis of asthma 1.33 (1.07–1.65)a 

a

p ≤ 0.05. 

Source: Ballal et al. (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 FEV1 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, 

workers with cumulative exposure ≥50 mg/m37 -years had significantly lower FEV1% predicted 

8 (7.4% decrease, p < 0.006) and FVC% predicted (5.4% decrease, p ≤ 0.030) than workers with 

cumulative exposure ≤50 mg/m39 -years. A comparison between symptomatic and asymptomatic 

10 exposed workers showed that FEV1% predicted and FEV1/FVC% were significantly lower 

11 among symptomatic workers (9.2% decrease in FEV1% predicted, p < 0.001 and 4.6% decrease 

12 in FEV1/FVC%, p < 0.02). Although Ballal et al. (1998) and Ali (2001) suggest that exposure to 

ammonia concentrations above 18 mg/m3 (50 mg/m313 -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 (Dräger PAC III and Dräger 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, FEV1, 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 FEV1 (∆FEV1%) while adjusting for current smoking. 

35 Mean exposure levels at the ammonia plant determined by the Dräger tube and Dräger 

PAC III methods were 25.0 and 6.9 ppm (17.7 and 4.9 mg/m336 ), respectively; the corresponding 

means in the urea plant were 124.6 and 26.1 ppm (88.1 and 18.5 mg/m337 ) (Rahman et al., 2007). 

38 Although the Dräger tube measurements indicated ammonia exposure about 4–5 times higher 
24 DRAFT - DO NOT CITE OR QUOTE 

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 Dräger tube (concentrations less than the measuring range of 2.5 to 

200 ppm [1.8 to 141 mg/m34 ]). Based on an evaluation of the two monitoring methods and 

communication with technical support at Dräger55 , EPA considered the PAC III instrument to be 

6 a more sensitive monitoring technology than the Dräger 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, FEV1, and PEFR did not differ between urea plant and ammonia plant 

17 workers. Significant cross-shift reductions in FVC and FEV1 were reported in the urea plant 

18 (2 and 3%, respectively, p ≤ 0.05), but not in the ammonia plant. When controlled for current 

19 smoking, a significant decrease in ∆FEV1% 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 FEV1 of 3.9% per unit of logtransformed ammonia concentration in ppm. EPA identified a NOAEL of 6.9 ppm (4.9 mg/m3

22 ) 

and a LOAEL of 26.1 ppm (18.5 mg/m323 ) in the Rahman et al. (2007) study based on increased 

24 prevalence of respiratory symptoms and an acute decrease in lung function. 

25 

 

5Telephone conversations and e-mails dated June 22, 2010, from Michael Yanosky, Dräger 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)

a

Administration building 

(concentration not 

determined)b

Respiratory symptoms 

 Cough 4/24 (17%)c 18/64 (28%)d 2/25 (8%) 

 Chest tightness 4/24 (17%) 21/64 (33%)d 2/25 (8%) 

 Stuffy nose 3/24 (12%) 10/64 (16%) 1/25 (4%) 

 Runny nose 1/24 (4%) 10/64 (16%) 1/25 (4%) 

 Sneeze 0/24 (0%) 14/64 (22%) 2/25 (8%) 

Lung function parameters (cross-shift percentage change)e, f

FVC 0.2 ± 9.3 -2.3 ± 8.8 ND 

FEV1 3.4 ± 13.3 -1.4 ± 8.9 ND 

PEFR 2.9 ± 11.1 -1.0 ± 16.2 ND 

ND: no data 

aMean ammonia concentrations measured by the Dräger PAC III method. 

bConcentrations in the administration building were rejected by study authors due to relatively large drift in the zero 

levels. 

cValues are presented as incidence (prevalence expressed as a percentage). 

d

p ≤ 0.05 by Fisher’s exact test, comparing exposed workers to administrators. 

eCalculated as ((Post shift-preshift)/preshift) x 100. 

fValues are presented as mean ± SD. 

Source: Rahman et al. (2007).

1 

2 Tepper et al. (1991) evaluated pulmonary function (FEV1) 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 FEV1 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. 
26 DRAFT - DO NOT CITE OR QUOTE 

1 Hamid and El-Gazzar (1996) evaluated changes in serum clinical chemistry as measures 

2 of neurochemical alterations and liver function among workers at a urea production plant in 

3 Alexandria, Egypt. The study group consisted of 60 male workers from the fertilizer plant, 

4 including 30 workers with known exposures to ammonia and 30 workers from the administrative 

5 departments with no known history of exposure to ammonia. The authors indicated that the 

6 exposed population had worked at the fertilizer plant on average for 12 years. The exposed and 

7 reference populations were matched on demographic characteristics including age, educational 

8 status, and socioeconomic status. No information is reported on exposure levels. Blood samples 

9 were collected from each subject and analyzed for aspartate aminotransferase (AST), alanine 

10 aminotransferase (ALT), hemoglobin, blood urea, and monoamine oxidase (MAO) and catalase 

11 activity. Table 4-5 shows statistically significant changes in hemoglobin and serum chemistry. 

12 Mean levels of AST, ALT, and blood urea were significantly elevated among exposed workers 

13 over controls. Mean levels of hemoglobin were significantly lower, and MAO and catalase 

14 enzyme activities were significantly depressed among exposed workers compared to controls. A 

15 correlation analysis showed a positive correlation between catalase activity and levels of 

16 hemoglobin, AST and ALT, and MAO activities. Hamid and El-Gazaar (1996) noted that 

17 inhibition of catalase can affect electrical stability, permeability, and fluidity of membranes, 

18 which may lead to hepatotoxic and neurotoxic alterations in occupationally exposed workers. 

19 NOAEL and LOAEL values were not identified in this study due to the absence of information 

20 on exposures at this fertilizer plant. 

21 

Table 4-5. Summary of significant changes in serum from workers 

occupationally exposed to ammonia at a fertilizer plant 

Parameter Controlsa

 Exposeda

ALT (U/mL) 16.0 ± 5.59 19.4 ± 5.69b

AST (U/mL) 14.5 ± 4.67 17.9 ± 4.14b

Hb (%) 14.8 ± 2.62 12.2 ± 2.29c

Blood urea (mg/mL) 0.203 ± 0.0512 0.319 ± 0.0755c

MAO (units) 31.9 ± 10.1 20.8 ± 4.30c

Catalase (IU/mL) 119.3 ± 4.76 80.9 ± 9.31c

aMean ± standard deviation. 

b

Significantly different from controls (p < 0.05). 

c

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 Helicobacter pylori

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 (μM); mean ± sem 

Control (tap water) 

Percent ammonia in drinking water 

0.01% 0.1% 

Antral mucosa 

2 weeks 270 ± 18 258 ± 22 217 ± 40a

4 weeks 276 ± 39 171 ± 22a 109 ± 12b,c

Body mucosa 

2 weeks 574 ± 116 568 ± 159 591 ± 183 

4 weeks 618 ± 154 484 ± 123 440 ± 80a,c

a 

p < 0.05 vs. control group

b 

p < 0.01 vs. control group 

 c 

p < 0.01 versus 2 week treatment group 

Source: Kawano et al. (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 

Thickness of mucosa (μM) 

Antral mucosa Body mucosa 

Control (tap water only) 283 ± 26 534 ± 27 

3 Days 305 ± 45 559 ± 50 

1 Week 272 ± 31 542 ± 28 

2 Weeks 299 ± 26 555 ± 37 

4 Weeks 159 ± 29b 531 ± 32 

8 Weeks 168 ± 26b 508 ± 29 

aExtracted from Figure 3 of Tsujii et al. (1993); mean ± SD 

b

p < 0.05 vs. control (tap water only) group 

Source: Tsujii et al. (1993) 

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 
29 DRAFT - DO NOT CITE OR QUOTE 

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 
30 DRAFT - DO NOT CITE OR QUOTE 

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 C3H 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 (C3H) 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 C3H 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 N37 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 
31 DRAFT - DO NOT CITE OR QUOTE 

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 

MNNG and tap water (p < 0.01, χ

2

14 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 

and 1.3 ± 0.6 among control rats (p < 0.01, χ

2

17 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+ammonia21 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 

albino mice of both sexes continuously to 20 ppm (14 mg/m338 ) ammonia vapors for up to 6 weeks 
32 DRAFT - DO NOT CITE OR QUOTE 

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 

guinea pigs was exposed to 50 ppm (35 mg/m33 ) 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 

effects were observed in animals exposed for up to 4 weeks, but exposure to 20 ppm (14 mg/m37 ) 

8 for 6 weeks caused darkening, edema, congestion, and hemorrhage in the lung. Exposure of 

guinea pigs to 50 ppm (35 mg/m39 ) 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 

period for the 20 ppm (14 mg/m312 ) 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 

monkeys, and purebred male beagle dogs to 0, 155, or 770 mg/m315 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. 

Exposure to 155 mg/m326 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 

mg/m330 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 

ammonia, >99% pure) at 0, 40, 127, 262, 455, or 470 mg/m337 (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 
33 DRAFT - DO NOT CITE OR QUOTE 

exposed continuously under similar conditions to ammonia at 40 mg/m3 (57 ppm) or 470 mg/m31 

(665 ppm). No significant effects were reported in any animals exposed to 40 mg/m32 and 

3 nonspecific inflammatory changes in the lungs and kidneys (not further described) were seen in 

rats exposed to 127 mg/m3 (180 ppm) ammonia. Exposure to 262 mg/m34 (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 

455 mg/m37 (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 

rats. Exposure to 470 mg/m39 (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 

significantly from controls in animals exposed to 470 mg/m312 (665 ppm) ammonia. 

Histopathological evaluation of animals exposed to 470 mg/m313 (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 

40 mg/m3 (57 ppm) and a LOAEL of 127 mg/m3 18 (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 

concentrations of 12, 61, 103, or 145 ppm ammonia (8, 43, 73, or 103 mg/m321 ) 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. 

Exposure to ≥103 ppm (>73 mg/m328 ) 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 

Yorkshire Landrace pigs to 0 or 100 ppm ammonia (0 or 71 mg/m335 ) continuously for up to 

6 weeks. The mean concentration of ammonia in the control chamber was 8 ppm (6 mg/m336 ). 

Additional groups of pigs were exposed to similar levels of ammonia as well as to 0.3 mg/ft337 of 

38 ground corn dust to simulate conditions on commercial farms. Pigs were monitored daily for 
34 DRAFT - DO NOT CITE OR QUOTE 

1 clinical signs and changes in behavior. Initial and terminal body weights were measured to 

2 determine body weight gain during the exposure period. Blood samples were collected prior to 

3 the start of each experiment and at study termination for hematology (packed cell volume, white 

4 blood cell, differential leukocyte percentage, and total serum lactate dehydrogenase). Two pigs 

5 (one exposed and one control) were necropsied at weekly intervals and tracheal swabs for 

6 bacterial and fungal culture were taken. Histological examination was conducted on tissue 

7 samples from the lung, trachea, and bronchial lymph nodes. 

8 During the first week of exposure, exposed pigs exhibited slight signs of conjunctival 

9 irritation including photophobia and excessive lacrimation. These irritation effects were not 

10 apparent beyond the first week. Measured air concentrations in the exposure chambers increased 

to more than 150 ppm (106 mg/m311 ) on two occasions. Doig and Willoughby (1971) reported 

12 that at this concentration, the signs of conjunctival irritation were more pronounced in all pigs. 

13 No adverse effects on body weight gain were apparent. Hematological parameters and gross 

14 pathology were comparable between exposed and control pigs. Histopathology revealed 

15 epithelial thickening in the trachea of exposed pigs and a corresponding decrease in the numbers 

16 of goblet cells as shown in Table 4-8. Tracheal thickening was characterized by thinning and 

17 irregularity of the ciliated brush border and an increased number of cell layers. Changes in 

18 bronchi and bronchioles characterized as lymphocytic cuffing were comparable between exposed 

19 and control pigs. Similarly, intraalveolar hemorrhage and lobular atelectasis were common 

20 findings in both exposed and control pigs. Pigs exposed to both ammonia and dust exhibited 

21 similar reactions as those pigs exposed only to ammonia although initial signs of conjunctival 

22 irritation were more severe in these pigs and these pigs demonstrated lesions in the nasal 

23 epithelium similar to those observed in the tracheal epithelium of pigs exposed only to ammonia. 

24 

Table 4-8. Summary of histological changes observed in rats exposed to 

ammonia for 6 weeks 

Duration of exposure 

(wks) 

Thickness of tracheal epithelium (μm)

Number of tracheal goblet cells (per 

500 μm) 

Control 71 mg/m3 NH3 Control 71 mg/m3 NH3

1 15.7 21.0 13.6 24.0 

2 20.4 29.3 22.7 10.3 

3 20.4 36.6 18.9 7.3 

4 21.8 36.2 18.3 10.7 

5 19.3 33.2 20.2 10.0 

6 18.9 41.6 20.0 1.3 

Mean ± standard deviation 19.4 ± 2.1 32.9 ± 7.2 18.9 ± 3.0 10.6 ± 7.5 

Source: Doig and Willoughby (1971). 

25 
35 DRAFT - DO NOT CITE OR QUOTE 

Doig and Willoughby (1971) concluded that ammonia exposure at 71 mg/m31 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 

identified a LOAEL of 71 mg/m35 (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 

10 or 150 ppm ammonia (7 or 106 mg/m38 , 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 

250 ppm (177 mg/m313 ) 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 

(106 mg/m3) for 75 days (from bedding) or 250 ppm (177 mg/m317 ) 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 

LOAEL from this study. EPA identified a NOAEL of 10 ppm (7 mg/m328 ) and a LOAEL of 150 

ppm (106 mg/m329 ) 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 Mycoplasma pulmonis. 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, 
36 DRAFT - DO NOT CITE OR QUOTE 

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 

mg/m3) and 1.2, 2.7, 5.1, or 9.9 mg/m325 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 × 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 
37 DRAFT - DO NOT CITE OR QUOTE 

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 

study. EPA identified a NOAEL of 26 mg/m316 (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 

concentration of 170 ppm (120 mg/m319 ) 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 

varied between 140 (99 mg/m3) and 200 ppm (141 mg/m321 ). 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 

ammonia concentration of 170 ppm (120 mg/m333 ) 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 

ammonia (0, 35, or 53 mg/m337 ) continuously for up to 109 days (anhydrous ammonia, >99.9% 

38 pure). Endpoints evaluated included clinical signs and body weight gain. At termination, all 
38 DRAFT - DO NOT CITE OR QUOTE 

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 

of 75 ppm (53 mg/m37 ) 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–

12 ppm) (5 mg/m3; range, 3–8.5 mg/m3) or 35 ppm (range, 26–45 ppm) (25 mg/m316 ; range, 18–32 

mg/m317 ) 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. 

Gilts exposed to 35 ppm (25 mg/m326 ) ammonia gained less weight than gilts exposed to 

7 ppm (5 mg/m327 ) 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, 

but gilts exposed to 35 ppm (25 mg/m331 ) ammonia weighed 7% less (p < 0.05) at puberty than 

those exposed to 7 ppm (5 mg/m332 ). 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 
39 DRAFT - DO NOT CITE OR QUOTE 

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 

rats and mice (rat LC50 values ≥11,590 mg/m3; mouse LC50 values ≥2,990 mg/m322 ) (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 

mg/m327 (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 

rate observed in rats at concentrations ≥848 mg/m330 (1,200 ppm) (Rejniuk et al., 2008, 2007), in 

mice at concentrations ≥214 mg/m331 (303 ppm) (Kane et al., 1979), and in rabbits at 

concentrations ≥35 mg/m332 (50 ppm) (Mayan and Merilan, 1972). Decreased airway pressure and 

pO2 were also observed in rabbits exposed to ammonia concentrations ≥24,745 mg/m333 

34 (35,000 ppm) for 4 minutes (Sjöblom et al., 1999). Histopathological changes in the respiratory 

tract of ammonia-exposed rats (≥141 mg/m3; 200 ppm) and mice (≥216 mg/m335 ; 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 
40 DRAFT - DO NOT CITE OR QUOTE 

1 (1980) showed that pulmonary function deficits measured 1, 7, 21, and 35 days postexposure 

were correlated with lung histopathology in cats exposed to ammonia (707 mg/m32 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 

ammonia exposure. Bradycardia was observed in rabbits exposed to 1,768 mg/m36 (2,500 ppm) 

for 180 minutes and arterial pressure variations were seen at 3,535 mg/m37 (5,000 ppm) (Richard 

8 et al., 1978b). Acidosis, as evidenced by a decrease in blood pH and an increase in arterial blood 

pCO2, occurred in rats exposed to 212 mg/m39 (300 ppm) ammonia for 5, 10, or 15 days 

(Manninen et al., 1988) and in rabbits exposed to ≥3,535 mg/m310 (5,000 ppm) for up to 

11 180 minutes (Richard et al., 1978b). No effects on blood pH, pO2, or pCO2 were seen in rats 

exposed to ≤818 mg/m312 (1,157 ppm) (Schaerdel et al., 1983) or cattle exposed to concentrations 

of <71 mg/m313 (100 ppm) (Mayan and Merilan, 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 γ-amino 

17 butyric acid (GABA) 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 

ammonia concentrations of 108 or 212 ppm ammonia (76 or 150 mg/m330 ), respectively (from 

31 soiled bedding), for 7 days prior to inoculation with M. pulmonis and for 30–35 days following 

32 inoculation. The matched control groups (bedding changed daily) were exposed to 11 ppm 

(8 mg/m3, Sherman rats) or 2 ppm (1 mg/m333 , 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, 71, or 177 mg/m335 , 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 with M. pulmonis in order to produce murine 

38 respiratory mycoplasmosis (MRM). Clinical observations were performed (frequency not 
41 DRAFT - DO NOT CITE OR QUOTE 

1 given). Rats were sacrificed at the end of the exposure period and tissues were prepared for 

2 histopathological examination of nasal passages, middle ear, trachea, lungs, liver, kidneys, 

3 adrenal, pancreas, testicle, spleen, mediastinal lymph nodes, and spleen. 

4 Typical signs of MRM (i.e., snuffling, head shaking) began in all rat groups 

5 approximately 10 days after inoculation. Rats exposed to ammonia exhibited dyspnea and 

6 hyperpnea, had rough coats, and sat in a hunched posture. All ammonia concentrations 

7 significantly increased the severity of rhinitis, otitis media, tracheitis, and pneumonia 

8 characteristic of M. pulmonis. Furthermore, there was a significant concentration response 

9 between observed respiratory lesions and increasing environmental ammonia concentration for 

10 gross and microscopic lesions (see Table 4-9). All lesions observed were characteristic of MRM. 

11 Gross bronchiectasis and/or pulmonary abscesses and the extent of gross atelectasis and 

12 consolidation were consistently more prevalent in exposed animals at all ammonia 

13 concentrations than in their corresponding controls. Increasing ammonia concentration was 

14 associated with an increasing rate of isolating of M. pulmonis from the respiratory tract. EPA 

identified a LOAEL of 18 mg/m315 (25 ppm) for increased severity of rhinitis, otitis media, 

16 tracheitis, and pneumonia, and increased incidence of respiratory lesions in F344 rats. A 

17 NOAEL was not identified in this study. 

18 

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 (%) Incidence of microscopic lesions (%) 

Ammonia Matched control Ammonia Matched control 

Soiled bedding 

76 20/24 (83%)a 10/23 (43%) 23/24 (96%)a 14/23 (61%) 

150 6/22 (27%) 2/24 (8%) 19/22 (86%)a 8/24 (33%) 

Inhalation chamberb

18 5/12 (42%) 1/12 (8%) 9/12 (75%) 6/12 (50%) 

18 6/12 (50%) 4/12 (33%) 9/12 (75%) 7/12 (58%) 

40 8/12 (66%) 2/12 (17%) 10/12 (83%) 9/12 (75%) 

70 4/12 (33%) 1/12 (8%) 12/12 (100%) 6/12 (50%) 

180 10/12 (83%) 2/12 (17%) 12/12 (100%) 2/12 (17%) 

a

p ≤ 0.01, χ

2

 test comparing ammonia-exposed group to matched controls. 

b

p ≤ 0.01, χ

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: Broderson et al. (1976). 

19 

20 Richard et al. (1978a) exposed groups of 99 male OFI mice continuously to ammonia 

concentrations of 0 or 500 ppm (0 or 354 mg/m321 ) for 8 hours and for 168 hours prior to infection 
42 DRAFT - DO NOT CITE OR QUOTE 

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 Controls Exposed (400 mg/m3) 

8 h 23/49 (47%)a 21/49 (43%) 

168 h 25/50 (50%) 42/49 (86%)b

aNumber of deaths/total number of animals (percentage mortality). 

b

Significantly 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 

ammonia concentrations of ≤2 ppm (≤1 mg/m312 ; controls, air concentration was usually not 

measureable and never exceeded 2 ppm) or 100 ppm (71 mg/m313 ) for either 7 days prior to 

14 inoculation with M. 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 of M. 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 

the first experiment to ammonia at 100 ppm (71 mg/m327 ) for 1 week were exposed under 

anesthesia to ammonia via face mask at ≤500 ppm (354 mg/m328 ) 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 
43 DRAFT - DO NOT CITE OR QUOTE 

tracheal air of any rats during exposure to <500 ppm (<354 mg/m31 ), and measured 10–20 ppm 

(7–14 mg/m3) in some rats (number not specified) at 500 ppm (354 mg/m32 ). These results 

3 indicate that virtually all ammonia is absorbed in the nasal passages in rats exposed to <500 ppm 

(<354 mg/m34 ) ammonia. In the other in vivo experiment, groups of 38 F344 rats were exposed 

to either 0 or 100 ppm (0 or 71 mg/m35 ) 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 μg of purified protein derivative (PPD) of 

17 tuberculin were continuously exposed to ammonia concentrations of 50 or 90 ppm (35 or 

64 mg/m318 ) for 3 weeks (Targowski et al., 1984). An additional eight PPD-positive guinea pigs 

exposed to a normal environment with ammonia concentrations <15 ppm (<11 mg/m319 ) 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 μg/mL phytohemagglutin [PHA] or 

26 concanavalin A) or 24 μg/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. 

Guinea pigs exposed to ammonia at 50 or 90 ppm (35 or 64 mg/m335 ) 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 at p < 0.05 (Student’s t-test) in guinea pigs exposed 
44 DRAFT - DO NOT CITE OR QUOTE 

to 64 mg/m31 at 24, 72, and 96 hours compared to controls (Table 4-11). No significant 

2 hematological changes were observed between exposed and control groups. The response to 

3 mitogens and PPD of blood lymphocytes and bronchial lymphocytes from animals exposed to 90 

ppm (64 mg/m34 ) ammonia was significantly less than the response from control animals (p < 

5 0.01) (shown graphically by authors). The lymphocyte response for guinea pigs exposed to 50 

ppm (35 mg/m36 ) ammonia was similar to control. In vitro exposure of blood lymphocytes to 

7 ammonia at 10 or 100 mg/L resulted in decreased viability and reduced response to PHA 

8 stimulation (significantly different from controls at p < 0.01). Lower concentrations of ammonia 

9 (≤1 mg/L) did not significantly affect viability or suppress the responsiveness of lymphocytes in 

10 vitro. Bactericidal and phagocytic activity of alveolar macrophages from animals exposed to 

11 ammonia was not significantly affected. Similarly, phagocytic activity was not significantly 

12 affected in vitro following treatment with ammonia at 1–100 mg/L. However, a significant 

13 inhibition in the macrophage bactericidal activity was observed in vitro at 0.01 and 0.05 mg/L (p

14 < 0.01). No significant changes in bactericidal activity were observed in cultures treated with 

15 ammonia at ≤1 mg/L. 

16 

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)

Ammonia concentration (mg/m3) 

Control 35 64 

24 17.6 ± 1.7a 15.2 ± 2.23 14.4 ± 2.2 b

48 15.5 ± 1.9 13.8 ± 1.6 14.7 ± 1.8 

72 12.0 ± 1.1 12.6 ± 1.0 8.7 ± 1.4 b

96 10.4 ± 1.5 12.6 ± 0.7 0.0b

aMean ± standard deviation. 

b

Significantly different from controls (p < 0.05, Student’s t-test). 

Source: Targowski et al. (1984). 

17 

18 Neumann et al. (1987) conducted three experiments to evaluate the ability of unweaned 

19 piglets exposed to ammonia to fight off infection following an intranasal challenge with P. 

20 multocida (Carter type A). In the first experiment (Trial 1), unweaned piglets were exposed to 

ammonia at 50 ppm (35 mg/m321 ) for 42 days either without additional treatment (22 piglets) or 

22 following intranasal challenge with P. multocida (10 piglets). Groups of 8–10 piglets were also 

23 exposed for 35 days during the second experiment (Trial 2) under these same conditions. A 

24 thermo-motor stress swim test (water temperature was 15°C) was also included as an additional 

25 condition during Trial 2. During the third experiment (Trial 3), all three conditions (i.e., 

26 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/m327 ) 
45 DRAFT - DO NOT CITE OR QUOTE 

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. 

Additionally, on the 23rd 5 day of exposure, a small test group (five test and five control pigs from 

Trial 2) was exposed to a radioactive aerosol (Tc99m 6 -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. 

Neumann et al. (1987) suggested that ammonia concentrations of 35 and 71 mg/m313 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 

globulin concentrations were not significantly affected at 35 mg/m3 16 ammonia, but in pigs 

inhaling 71 mg/m317 ammonia, gamma globulin concentrations were significantly lower than 

18 controls (α = 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. 

No differences in bronchoalveolar cells were observed in pigs exposed to either 35 or 71 mg/m320 

21 compared to controls. However, a significantly higher proportion of neutrophils was detected in 

lavage fluid from infected pigs exposed to 35 mg/m322 ammonia compared with infected controls 

23 at 7 days postinfection (α = 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 (α = 

26 0.10, Wilcoxon, Mann, and Whitney U-test). In addition, the proportion of alveolar 

macrophages in the lavage fluid of pigs exposed to 35 mg/m327 ammonia was significantly lower 

28 in infected pigs (α = 0.05, Wilcoxon, Mann, and Whitney U-test) and in infected pigs subjected 

29 to the swimming test (α = 0.10, Wilcoxon, Mann, and Whitney U-test) at postinfection day 7. 

Under these conditions, pigs exposed to 71 mg/m330 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 
46 DRAFT - DO NOT CITE OR QUOTE 

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 

ammonia concentrations of 0 or 100 ppm (0 or 71 mg/m

3

4 ) 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 

0, 25, 50, or 100 ppm (0, 18, 35, or 71 mg/m311 ) 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 

reaction was strongly altered in lungs from pigs exposed to 100 ppm (71 mg/m317 ) where the 

18 endotoxin-induced increase in total blood flow resistance was completely abolished. The 

vascular response to endotoxin was also reduced in pigs exposed to 50 ppm (35 mg/m319 ). In 

summary, concentrations of ammonia >50 ppm (35 mg/m320 ) 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 
47 DRAFT - DO NOT CITE OR QUOTE 

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 
48 DRAFT - DO NOT CITE OR QUOTE 

1 4.1.2 and Appendix C.3). Pulmonary function was not affected in volunteers acutely exposed to 

ammonia concentrations as high as 71 mg/m32 (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 

LOAELs did not vary greatly across species and strains. LOAELs ranged from 18 to 127 mg/m317 

and NOAELs ranged from 7 to 53 mg/m318 . 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 

exposure to ammonia concentrations up to 25 mg/m321 (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 LC50 values 

≥11,590 mg/m3; mouse LC50 values ≥2,990 mg/m324 ), decreased pulmonary function with 

decreased respiratory rate in rats at concentrations ≥848 mg/m325 , mice at concentrations 

≥214 mg/m3, and in rabbits at concentrations ≥35 mg/m326 . 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. 
49 DRAFT - DO NO

Table 4-12. Summary of noncancer results of human occupational studies and repeat-dose studies in e

animals involving inhalation exposure to ammonia 

Exposed 

population 

Exposure 

concentration 

(mg/m

3)/ duration

NOAEL 

(mg/m

3) 

LOAEL 

(mg/m

3) 

Effects at the 

LOAEL 

concentration Comment 

Occupational worker studies Industrial workers (58 male workers and 31 male controls from office area)

low (<4.4), medium (4.4–8.8), and high (>8.8) avg exposure: 12 y 

8.8 (adjusted: 3.1)

a

Not determined Cross-sectional study in soda ash plant. No 

differences in pulmonary function or subjesymptomology relative to the control group 

Industrial workers (63 ammonia plant and 77 urea plant workers; 25 controls from administration building) 

ammonia plant (mean): 4.9 urea plant (mean): 18.5 mean employment duration: 16 y 

4.9 18.5 Increased prevalence 

of respiratory symptoms and decrease in lung function 

Cross-sectional study in urea fertilizer factoConcentrations in this table were based on III method; concentrations measured using Dräger tube method were 4-5 times higher

Experimental animal studies Rat (SpragueDawley and Longs-Evans) (15/sex/group) 

0, 40, 127, 262, 455, or 470 for 90–114 d 

40 127 Nonspecific 

inflammatory changes in the lungs and kidneys 

Nasal discharge and nonspecific circulatory degenerative changes in the lungs and kidnobserved at 262 mg/m

3 (not further describ

incidence not reported). Frank-effect-level mg/m

3 based on 90–98% mortality 

Sherman rat (5/sex/group) 

7 or 106 for 75 d 7 106 Nasal lesions 

F344 rat (6/sex/group) 

0, 18, 35, 71, or 177for up to 49 d 

Not determined 18 Increased severity of 

rhinitis, otitis media, tracheitis, and pneumonia, and increased incidence of respiratory lesions 

Rats were exposed continuously to ammondays prior to inoculation with M. pulmonis 

42 days following inoculation 

Guinea pigs (12 guinea pigs, sex not specified) 

0 or 120, 6 hrs/d, 5 d/wk for 18 wks 

Not determined 120 Congestion of the 

spleen, liver, and kidneys and early degenerative changes in the adrenal gland 
50 DRAFT - DO NO

Table 4-12. Summary of noncancer results of human occupational studies and repeat-dose studies in e

animals involving inhalation exposure to ammonia 

Exposed 

population 

Exposure 

concentration 

(mg/m

3)/ duration

NOAEL 

(mg/m

3) 

LOAEL 

(mg/m

3) 

Effects at the 

LOAEL 

concentration Comment 

YorkshireLandrace pig (6/group, sex not specified) 

0 or 71 for 6 wks Not determined 71 Thickening of the 

tracheal epithelium with a concomitant decrease in the number of tracheal epithelial goblet cells

The mean concentration of ammonia in the chamber was measured at 5.6 mg/m

3

Pig (4–8/group, sex not specified) 

0, 35, or 53 for 109 d 

53 Not identified 

Pig (24 pigs/group, several breeds, sex not reported)

0, 0.4, 7, 13.3, or 26 for 5 wks 

26 Not identified Exposures were to ammonia at concentratio

0.4, 7, 13.3, or 26 mg/m

3 and to inhalable d

2.7, 5.1, or 9.9 mg/m

3; all pigs including co

demonstrated infections common to pigs on commercial farms 

aAdjusted to continuous exposure as follows: NOAELADJ = NOAEL × VEho/VEh × 5 days/7 days = 8.8 mg/m

3 × 10 m

3/20 m

3 × 5 days/7 da

VEho = human occupational default minute volume and VEh = human ambient default minute volume.
51 DRAFT - DO NOT CITE OR QUOTE 

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 (Mégraud 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 MNNG 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-1 (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 
52 DRAFT - DO NOT CITE OR QUOTE 

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 (Córdoba 

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 (Miñana 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). 
53 DRAFT - DO NOT CITE OR QUOTE 

1 5. DOSE-RESPONSE ASSESSMENTS 

2 

3 

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. 

In EPA’s guidance document entitled, Recommended Use of Body Weight3/4 30 as the 

31 Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 2011a), 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 

human equivalent oral exposures, EPA endorses body weight scaling to the 3⁄4 power (i.e., BW3/4 37 ) 

38 as a default to extrapolate toxicologically equivalent doses of orally administered agents from 
54 DRAFT - DO NOT CITE OR QUOTE 

laboratory animals to humans for the purpose of deriving an RfD. When BW3/4 1 scaling is used in 

2 deriving the RfD, EPA also advocates a reduction in the interspecies uncertainty factor (UFA) 

from 10 to 3, as BW3/4 3 scaling addresses predominantly toxicokinetic (and some toxicodynamic) 

4 aspects of the UFA. 

5 The guidance raises some important uncertainties in applying allometric (more 

specifically BW3/4 6 ) scaling when the critical effect used to derive the reference value is a portal7 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, 2011a). Therefore, 

23 because effects on the gastric antral mucosa are not expected to scale allometrically, a 

BW3/4 24 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 
55 DRAFT - DO NOT CITE OR QUOTE 

1 inhalation literature is a portal-of-entry effect (respiratory symptoms and lung function changes), 

2 route-to-route extrapolation is not supported. 

3 

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 dose21 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 

(6.5 mg/m328 ) and a nonexposed worker population. When the exposed workers were grouped into 

three exposure categories (high: ≥12.5 ppm [8.8 mg/m329 ], medium: 6.25–12.5 ppm [4.4–

8.8 mg/m3], and low: ≤6.25 ppm [4.4 mg/m330 ]), no statistically significant differences (p < 0.05) 

31 in symptoms or changes in lung function between the groups were evident. Based on lack of 

symptomology and changes in lung function, EPA identified a NOAEL of 12.5 ppm (8.8 mg/m332 ) 

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 

(18.5 mg/m336 ) ammonia in the urea plant of a fertilizer factory than in workers exposed to 

6.9 ppm (4.9 mg/m337 ) ammonia in the ammonia plant or nonexposed workers in the 

38 administration building. Furthermore, workers in the urea plant demonstrated significant cross-
56 DRAFT - DO NOT CITE OR QUOTE 

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 

not observed among workers in the ammonia plant. EPA identified a NOAEL (4.9 mg/m33 ) and a 

LOAEL (18.5 mg/m34 ), 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 

to ammonia concentrations >18 mg/m38 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. 
 57 DRAFT - DO 

1 2 Figure 5-1. Exposure-response array comparing workers occupationally exposed and human voluntee3 exposed to ammonia. 

0.01

0.1

1

10

100

1000

Prevalence 

of 

respiratory 

symptoms 

and 

lung 

function 

Holness 

et al. 

(1989)

Prevalence 

of 

respiratory 

symptoms 

and 

decreased 

lung 

function 

Rahman 

et al. 

(2007)

Cough, 

lacrimation, 

nose and 

throat 

irritation 

Silverman 

et al. 

(1949)

Eye 

irritation 

MacEwen 

et al. 

(1970)

No effect 

on lung 

function 

Cole 

et al. 

(1977)

Lacrimation, 

transient 

discomfort 

Ferguson 

et al. 

(1977)

Eye 

irritation, 

odor 

detection, 

headache, 

dizziness, 

feeling of 

intoxication 

Sundblad 

et al. 

(2004)

Eye 

irritation 

Verberk 

(1977)

Nasal 

irritation

Petrova

et al.

(2008)

Eye 

irritation

Petrova

et al.

(2008)

Severe eye, 

nose and 

throat 

irritation 

Verberk 

(1977)

Lung

function

Verberk

(1977)

Lung

function

Sigurdarson 

et al.,

(2004)

Lung

function

Sundblad 

et al.,

(2004)

INDUSTRIAL STUDIES CONTROLLED ACUTE EXPOSURE STUDIES

Exposure (mg/m3)

NOAELLOAEL

Vertical lines show range of exposures in study. Closed circles show exposures tested in study.
58 DRAFT - DO NOT CITE OR QUOTE 

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. 
59 DRAFT - DO 

1 2 Figure 5-2. Exposure-response array comparing noncancer effects in occupationally exposed work3 experimental animals exposed to ammonia by inhalation. 

0.01

0.1

1

10

100

1000

Prevalence of 

respiratory symptoms 

& lung function (male 

occupational); 

Holness et al. (1989)

Prevalence of 

respiratory symptoms 

& acute decreased 

lung function 

(occupational); 

Rahman et al. (2007)

Upper respiratory 

tract - structural 

damage (pig); Doig 

and Willoughby 

(1971)

Nasal lesions (rat); 

Broderson et al. 

(1976)

Respiratory lesions 

(rat); Broderson et al. 

(1976)

Respiratory infection 

in presence of 

inhalable dust (pig); 

Done et al. (2005)

Nonspecific 

inflammatory 

changes in lungs and 

kidneys (rat); Coon et 

al. (1970)

Congestion of sple

liver & kidneys, ea

degenerative 

changes in adren

gland (guinea pig

Weatherby (1952

HUMAN STUDIES EXPERIMENTAL ANIMAL STUDIES

Exposure (mg/m3)

LOAELNOAELFELIntermediate dose

Vertical lines show range of exposures in study. Closed circles show exposures tested in 
60 DRAFT - DO NOT CITE OR QUOTE 

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 

of the effect level for ammonia, with the NOAEL of 8.8 mg/m37 identified from the Holness et al. 

(1989) study falling between the NOAEL and LOAEL values (4.9 mg/m3 and 18.5 mg/m38 , 

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 (Dräger PAC III and Dräger 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 

EPA identified a NOAEL of 8.8 mg/m323 , 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 

effects at higher workplace ammonia concentrations (i.e., the LOAEL of 18.5 mg/m327 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) 

The NOAEL of 8.8 mg/m3 32 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 × VEho/VEh × 5 days/7 days 

= 8.8 mg/m3 × 10 m3/20 m337 × 5 days/7 days 

= 3.1 mg/m338 
61 DRAFT - DO NOT CITE OR QUOTE 

1 Where: 

VEho = human occupational default minute volume (10 m32 breathed during the 8-hour 

3 workday, corresponding to a light to moderate activity level [U.S. EPA, 2011b]) 

VEh = human ambient default minute volume (20 m34 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, UFH, 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 μg/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 

up to 18 mg/m338 does not alter blood ammonia levels (see Section 3). Therefore, 
62 DRAFT - DO NOT CITE OR QUOTE 

exposure at the POD (3.1 mg/m31 ) 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 two14 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 

 = 3.1 mg/m321 ÷ 10 

 = 0.3 mg/m322 

23 

24 5.2.4. Previous RfC Assessment 

25 The previous IRIS assessment for ammonia (posted to the database in 1991) presented an 

RfC of 0.1 mg/m326 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 with M.

pulmonis. The NOAEL of 6.4 mg/m329 (mean concentration of the entire exposed group) from the 

Holness et al. (1989) study (duration adjusted: NOAELADJ = 2.3 mg/m330 ) 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 

3 

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 

the blood as ammonium ion (NH4

+

23 ). 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. 

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 

 The RfC for ammonia of 0.3 mg/m310 was derived using a duration-adjusted NOAEL of 

3.1 mg/m311 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’s Methods 

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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1 APPENDIX B. SUMMARY OF REPEAT DOSE TOXICITY INFORMATION FOR SELECTED AM2 3 

Table B-1. Summary of noncancer results of repeat dose studies of oral exposure of experimental anim

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 

Subchronic studies 

Wistar rat (10/sex/group) 

Ammonium chloride 

0, 1,590, or 3,050 (males); 0, 1,800, or 3,700 (females) for 13 wks (administered in diet) 

Not determined 1,590 (males) 

1,800 (females) 

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) 

 

Wistar rat (5 females/ group) 

Ammonium acetate in diet and drinking water 

17.1 or 2,370 in diet; and 0 or 42.8 in water continuously for 90 d 

Not determined 2,410 (combined 

dose) 

Depression in body weight gain 

Control diet contained 0

ammonia. Authors suggthat the transient effects GFAP protein and compblood levels were indicatof an adaptive response. 
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Table B-1. Summary of noncancer results of repeat dose studies of oral exposure of experimental anim

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 

Chronic studies 

Sprague-Dawley rat(11 males/group)

Ammonium chloride

0 or 1,800 for 330 d(administered in drinking water)

Not determined 1,800 Depression in body weights 

(13-20% with regular and low-calcium diets, respectively), metabolic acidosis, and loss of bone (femur) tissue

Metabolic acidosis (redublood pH and plasma cardioxide).Combined effects of calcintake and observed that loss of bone tissue was independent of the level dietary calcium. 

Wistar rat (15/sex/group) 

Ammonium chloride 

0, 481, or 1,020 (males); 

0, 610, or 1,370 (females) for 18 mo 

Not determined 481 (males) 

610 (females) 

Metabolic acidosis Femur weight significan

increased (~17%) in high

males. Adrenal and kidnweights elevated (<15%)Increased incidence of zoglomerulosa hypertrophy the adrenal at high dose statistically significant). Severity of metabolic acincreased with dose. 

Wistar rat (50/sex/group) 

Ammonium chloride 

0, 455, or 1,000 (males); 

0, 551, or 1,200 (females) for 30 mo 

Not determined 455 (males) 

551 (females) 

Metabolic acidosis and an increased incidence of hypertrophy of the adrenal glomerulosa (males only) 

Increased incidence of zoglomerulosa hypertrophy the adrenal in high-dose females; attributed to chrstimulation of the adrenacortex by ammonium chinduced acidosis. There no evidence of a carcinoresponse. 
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Table B-1. Summary of noncancer results of repeat dose studies of oral exposure of experimental anim

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 

F344 rat (10/sex/group) 

Ammonium sulfate 

0, 42, 256, or 1,527 (males); 0, 48, 284, or 1,490 (females) for 52 wks (administered in diet) 

256 (males) 284 (females) 

1,527 (males) 1,490 (females) 

Elevated relative liver and kidney weights (7-10%) 

No significant effects on hematology, serum chemor histopathology. 

F344 rat (50/sex/group) 

Ammonium sulfate 

0, 564, or 1,288 (males); 0, 650, or 1,371 (females) for 104 wks (administered in diet) 

1,288 (males) 1,371 (females)

Not determined Clinical chemistry and 

hematology not evaluateorgan weights not measuIncidence of chronic nephropathy in male rats increased over control (15/49, 3/48 in the control, and high dose); increase statistically significant othe mid-dose and not dosrelated. There was no evidence of a carcinogenresponse. 

Developmental studies 

Wistar rat (group sizes of pregnant rats not reported) 

Ammonium acetate 

0 or 21,000 starting on d 1 of pregnancy through weaning to PND 21 

Not determined (maternal or developmental)

Maternal effects not reported 21,000 (developmental)

Reduction in pup body weight gain 

Pups exposed to ammonduring gestation and lactonly were resistant to acuammonia toxicity inducea single injection of 540 mg/kg ammonium acetat
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1 APPENDIX C. SUPPLEMENTAL INFORMATION ON AMMONIA 

2 

3 C.1. 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-1. 
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Table C-1. Ammonia levels in exhaled breath of volunteers

Breath samples from the nose and trachea 

Test subjects Breath samples 

Levels of ammonia in exhaled 

breath Methods Com

Three healthy male volunteers (>30 yrs of age) 

Ammonia levels measured in noseexhaled breath of test subjects each 

morning about 2 hrs after eating a regular breakfast; samples collected daily over a 4-wk period 

Volunteer A = 103 ± 1.2 ppb (0.0728 ± 0.000848 mg/m

3) 

Volunteer B = 110 ±1.3 ppb (0.0777 ± 0.000919 mg/m

3) 

Volunteer C = 83 ± 1.2 ppb (0.0587 ± 0.000848 mg/m

3) 

(median ammonia levels estimated as geometric mean ± geometric standard deviation) 

SIFT-MS analysis Mean ambien

ammonia was (0.056 ± 0.00

The authors iammonia meamouth-exhalebe generated cavity and suconcentrationexhaled breatrepresent systconditions (sumetabolic dis

Sixteen healthy subjects (9 males aged 25–63 yrs and 7 females aged 23–41 yrs); subgroups tested were all male 

Breath samples collected during quiet nose breathing, and direct sampling during a deep inspiration followed by breath-holding with the glottis closed 

Ammonia concentrations ranged from 0.013 to 0.046 mg/m

3 during nose 

breathing (median 0.025 mg/m

3) (5 

male subjects), and 0.029 mg/m

3 from 

an air sampled collected from the trachea (collected from a tube inserted into one male subject’s nose and into the trachea) 

Chemiluminescence 

1 
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Table C-1. Ammonia levels in exhaled breath of volunteers

Breath samples from the mouth and oral cavity 

Test subjects Breath samples 

Levels of ammonia in exhaled 

breath Methods Com

Three healthy male volunteers (>30 yrs of age) 

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 

via Mouth: Volunteer A = 1,088 ± 1.3 ppb (0.769 ± 0.000919 mg/m

3) 

Volunteer B = 885 ± 1.3 ppb (0.626 ± 0.000919 mg/m

3) 

Volunteer C = 855 ± 1.3 ppb (0.604 ± 0.000919 mg/m

3) 

via Oral Cavity: 

Volunteer A = 1,465 ± 1.4 ppb (1.04 ± 0.000990 mg/m

3) 

Volunteer B = 2,146 ± 1.5 ppb (1.52 ± 0.00106 mg/m

3) 

Volunteer C = 1,859 ± 1.3 ppb (1.31 ± 0.000919 mg/m

3) 

(median ammonia levels estimated as geometric mean ± geometric standard deviation) 

SIFT-MS analysis Mean ambien

ammonia was (0.056 ± 0.00

The authors iammonia meamouth-exhalebe generated cavity and suconcentrationexhaled breatrepresent systconditions (sumetabolic dis

Twenty-six secondary school students (10 males and 16 females, 17–18 yrs old and one 19 yr old) 

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) 

Median values reported for: 17 yr olds = 233 ppb (0.165 mg/m

3) 

18 yr olds = 346 ppb (0.245 mg/m

3) 

SIFT-MS analysis Significant di

ammonia levebreath betwee18 yr olds (p 

reported 
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Table C-1. Ammonia levels in exhaled breath of volunteers

Breath samples from the mouth and oral cavity 

Test subjects Breath samples 

Levels of ammonia in exhaled 

breath Methods Com

Four healthy children (2 males and 2 females, 4–6 yrs old) Thirteen senior volunteers (11 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 

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) 

Children = range 223–643 ppb (0.157–0.454 mg/m

3) 

Seniors = 317–2,091 ppb (0.224–1.48 mg/m

3) 

SIFT-MS analysis Ammonia bre

significantly age Some seniors diabetes Measured ambreath reportesubject 

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 

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 

Geometric mean and geometric standard deviation = 833 ± 1.62 ppb (0.589 ± 0.00114 mg/m

3) 

Median = 842 ppb (0.595 mg/m

3) 

Range = 248–2,935 ppb (0.175–2.08 mg/m

3) 

SIFT-MS analysis Ammonia bre

were shown twith age Background ltesting laboratypically arou(0.28 mg/m

3) 

Five subjects (2 females, 3 males; age range 27–65 yrs) 

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 

Ammonia concentrations ranged from 422 to 2,389 ppb (0.298–1.69 mg/m

3) 

SIFT-MS analysis Differences in 

breath levels individuals w(p < 0.001) 
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Table C-1. Ammonia levels in exhaled breath of volunteers

Breath samples from the mouth and oral cavity 

Test subjects Breath samples 

Levels of ammonia in exhaled 

breath Methods Com

Six normal nonsmoking male volunteers (24–61 yrs old), fasted for 12 hrs prior to testing 

Baseline breath sample obtained; breath samples collected 20, 40, and 60 min and 5 hrs following the ingestion of a liquid protein-calorie 

meal 

Premeal levels ranged from 300 to 600 ppb (0.2–0.4 mg/m

3); 

Postmeal levels at 30 min were 200 ppb (0.1 mg/m

3) increasing to 

maximum values at 5 hrs of 600–1,800 ppb (0.4–1.3 mg/m

3) 

SIFT-MS analysis A biphasic re

breath ammoconcentration after eating 

Fourteen healthy, nonsmoking subjects (age range 21–54 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 

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) 

Baseline levels varied from 120 to 1,280 ppb (0.085–0.905 mg/m

3) 

Nitrogen oxide analyzer with an ammonia conversion channel (similar to chemiluminescence) 

An 80–90% d

volatile ammwas seen withacid rinsing; l50% depletiowas seen follobrushing or drinse; gaseoulevels increasrinse procedu

Sixteen healthy subjects (9 males aged 25–63 yrs and 7 females aged 23–41 yrs); subgroups tested were all male 

Breath samples collected during quiet mouth breathing 

Ammonia concentrations ranged from 0.029 to 0.52 mg/m

3 during mouth 

breathing (median of 0.17 mg/m

3) 

Chemiluminescence The oral cavi

be a source oammonia; no made to contrsubjects or stinterval betwmeal and the 

1 
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Table C-1. 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 Com

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/m

3; 

median ± measurement error) Experiment 2: Nonstandardized = 1,007 ± 184 ppb (0.712 ± 0.130 mg/m

3; median ± 

standard deviation) Standardized = 1,433 ± 160 ppb (1.01 ± 0.113 mg/m

3; median ± 

standard deviation) Experiment 3: Mixed = 1,216 ± 827 ppb (0.860 ± 0.585 mg/m

3; median ± standard 

deviation) Alveolar = 1,301 ± 791 ppb (0.920 ± 0.559 mg/m

3; median ± standard 

deviation) 

SIFT-MS analysis This study establishes that SIFT-MS analysis is reliable and repeatable 

Relatively smhealthy subjeDoes not addof those with Intra-day and repeatability investigated 
C-7 DRAFT - DO NO

Table C-1. 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 Com

Eight healthy subjects (average age 39.8 ± 9.6 yrs) 

Subjects fasted for 6 hrs prior to samples being collected. Subjects breathed normally into collection device for 5 min 

Mean breath ammonia = 0.49 ± 0.24 ppm (0.35 ± 0.17 mg/m

3) 

Fiber optic sensor This study me

ammonia levevolunteers coHelicobacter positive indivsubjects) (datthe experimenincluded a ch300 mg urea evaluate the uof healthy vs. individuals (dshown); the aconcluded thaammonia meabe feasible as test for H. py

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) 

Subjects performed a 5-sec breathhold and exhaled slowly into collection device 

Asthmatic children from National Park = 5.6 ± 4.7 ppb (0.0040 ± 0.0033 mg/m

3) 

Asthmatic urban children: Mean NH3 = 14.3 ± 10.2 ppb 

(0.0101 ± 0.00721 mg/m

3) 

Urban control group: Mean NH3 = 14.8 ± 10.3 ppb (0.0105 ± 0.00728 mg/m

3) 

Chemiluminescence Both groups o

children had on glucocorticombined witantagonists anagonists, whileft untreatedconcentrationbreath appearcorrelated witurban air 

1 
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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–

11 mg/m330 ). 

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 effectsa Delayed effectsb Reference 

Oral exposure 

57-yr-old male Ingested unknown 

quantity of dilute 

ammonium hydroxide 

(2.4% ammonia) 

Hemorrhagic esophagogastro-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) Exposure conditions Immediate effectsa Delayed effectsb Reference 

15-yr-old male Ingestion of about 30 mL 

Parson’s sudsy ammonia 

(ammonia 3.6%; 

pH 11.5) 

White and blistered 

tongue and pharynx, 

esophageal burns with 

friable, boggy mucosa; no 

other complications 

Not applicable Klein et al., 

1985 

Middle-aged 

female 

Ingested “two gulps” 

Parson’s sudsy detergent 

ammonia 

Several small esophageal 

lesions with mild to 

moderate ulceration and 

some bleeding; no 

oropharyngeal burns 

Not applicable Klein et al., 

1985 

69-yr-old 

female 

Ingested an unknown 

quantity of Albertson’s 

lemon ammonia 

(ammonia concentration 

3%) 

Lethargy, gurgling 

respiratory sounds, 

laryngeal, and epiglottal 

edema, and a friable, 

erythmatous esophagus 

with severe corrosive 

injury 

Renal failure, death Klein et al., 

1985 

Eight young 

children (ages 

not given) 

Biting into an unbroken 

capsule of aromatic 

ammonia inhalant 

Vomiting, drooling, 

dysphagia, cough, and 

oropharyngeal burns 

Not applicable Lopez et al., 

1988 

Three children, 

<4 yrs old 

Ingestion of aromatic 

ammonia “smelling salt” 

capsules that contained 

0.33 mL of a mixture of 

18% ammonia and 36% 

alcohol 

Pain swallowing, 

reddened and blistered 

tongues, drooling, 

erythema, and swelling of 

lower lip 

Not applicable Wason et al., 

1990 

Eleven adults, 

≥15 yrs 

Intentional or accidental 

ingestion of ammonia 

water 

Two patients exhibited 

acute respiratory 

obstruction, no additional 

details were provided. 

One patient developed 

esophageal stricture 3 mo 

postinjury 

Christesen, 1995

3-yr-old female Biting into an ammonia 

inhalant capsule 

Drooling with multiple 

1–2 cm white, ulcerative 

lesions on the midposterior 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 

Not applicable 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 effectsa Delayed effectsb 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 ammoniacontaminated 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 Dworkin et al., 

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 et al., 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 et al., 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 effectsa Delayed effectsb Reference 

Inhalation/dermal/ocular exposure 

Dental patient 

(age not given)

A 10% ammonia solution 

spilled into eyes while 

trying to revive patient 

from a faint 

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 

Increase in corneal 

ulceration with hypopyon 

and perforation was seen 

after 8 d; severe 

conjunctivitis still evident 

4 mo after exposure 

Abramovicz, 

1924 

21-yr-old 

female 

Explosion in an ice 

cream factory; ammonia 

pipe burst 

Shock, second-degree 

burns of both feet, the 

right leg, and small area 

of the right cheek 

Residual bronchitis (not 

further described) 

Slot, 1938 

21-yr-old 

female 

Explosion in an ice 

cream factory; ammonia 

pipe burst 

Shock, conjunctivitis of 

the right eye, severe 

tracheitis 

Not applicable Slot, 1938 

23-yr-old 

female 

Explosion in an ice 

cream factory; ammonia 

pipe burst 

Severe shock, persistent, 

blood-stained vomiting, 

confusion 

Not applicable Slot, 1938 

21-yr-old 

female 

Explosion in an ice 

cream factory; ammonia 

pipe burst 

Shock, hemicranial 

headache with nausea 

Residual bronchitis, 

“fullness of the head”, and 

body pains; congestion of 

both lungs 

Slot, 1938 

36-yr-old 

female 

Explosion in an ice 

cream factory; ammonia 

pipe burst 

Burns and shock; existing 

bronchitis greatly 

aggravated 

Anxiety symptoms, 

insomnia, painful scar 

Slot, 1938 

25-yr-old 

female 

Explosion in an ice 

cream factory; ammonia 

pipe burst 

Extreme shock, grey 

pallor, burns to the face, 

eyes, neck, and both 

arms, difficulty/inability 

to swallow, labored 

breathing 

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 

Slot, 1938 

Nine shelter 

occupants (ages 

not given) 

Ammonia condenser leak 

in an air-raid shelter (low 

exposure group) 

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 

Not applicable Caplin, 1941 
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Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

Twenty seven 

shelter 

occupants (ages 

not given) 

Ammonia condenser leak 

in an air-raid shelter 

(moderate exposure 

group; victims in closer 

proximity to leak) 

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 

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 

Caplin, 1941 

Eleven shelter 

occupants (ages 

not given) 

Ammonia condenser leak 

in an air-raid shelter 

(high exposure group, 

victims closest to leak 

source with longest 

exposure time) 

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 

The remaining four cases 

improved without incident 

Caplin, 1941 

17-yr-old male Struck by a jet of 

anhydrous ammonia 

during crop dusting 

operations 

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 

Permanent loss of vision, 

with the exception of 

sustained light perception 

Levy et al., 

1964 

17-yr-old male Sprayed with anhydrous 

ammonia during crop 

fertilizing operations 

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 

Chronic persistent cough 

with a small amount of 

mucoid sputum, no chronic 

visual effects 

Levy et al., 

1964 
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Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

61-yr-old male Struck in the face with a 

blast of anhydrous 

ammonia while 

fertilizing crops 

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 

No chronic impairment of 

pulmonary function or of 

vision occurred 

Levy et al., 

1964 

28-yr-old male Struck in the face by a 

spray of anhydrous 

ammonia while working 

on a refrigeration unit 

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 

Pulmonary function tests 

3 yrs later were 

unremarkable 

Levy et al., 

1964 

Male employee 

(age not given)

Exposure for an 

unspecified length of 

time to ammonia fumes 

created when a tanker 

overflowed during a 

filling operation with 

25% ammonia water 

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 

Not applicable Mulder et al., 

1967 

Male victim at 

a bank robbery 

(age not given)

Ammonia thrown into 

face and forced down 

throat and up the nose 

Burns to the face, eyes, 

and mouth, with severe 

edema of the 

nasopharynx and glottis 

The right eye recovered 

within 4 d; the left eye 

showed gross chemosis, 

corneal staining, 

nonreactive pupil, intense 

uveitis with aqueous flare 

and cells 

Osmond and 

Tallents, 1968 
C-16 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

57-yr-old male Assaulted with ammonia 

squirted into the eyes 

during a robbery 

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 

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 

Highman, 1969 

46-yr-old male Assaulted with ammonia 

squirted into the eyes 

during a robbery 

Conjunctivitis in the right 

eye, the cornea was hazy 

with edema 

Four d following discharge, 

severe anterior uveitis 

developed, along with 

recurrent corneal ulceration 

and diminished corneal 

sensation 

Highman, 1969 

Female (age 

not given) 

Ammonia was thrown 

into eyes in the course of 

a robbery 

Two hrs postexposure, 

loss of corneal 

epithelium, 

conjunctivitis, stromal 

haze, fibrinous material 

in the anterior chamber, 

iris atrophy, vertical, 

oval-shaped pupil, and 

subcapsular lens opacities

Band-shaped corneal 

degeneration and 

heterochromia of the iris, 

but no progression of lens 

opacities (5 mo following 

exposure) 

McGuinness, 

1969 

40-yr-old male Sprayed on face and 

chest during a transfer 

operation involving 

anhydrous ammonia 

Facial burns (not 

extremely serious), 

pulmonary edema, and 

pneumonitis with 

inflammation and edema 

of the upper airways 

No residual lung damage Helmers et al., 

1971 

17-yr-old 

farmer 

Sprayed with several 

gallons of 25% ammonia 

in water during transfer 

operation 

Throat tightness during 

first few minutes 

following exposure; 

second-degree burns to 

the buttocks 

No residual effects Helmers et al., 

1971 

36-yr-old male Sprayed in the face 

during field repairs to 

fertilization equipment 

Immediate blepharospasm, second-degree 

facial burns, irritative 

conjunctivitis, superficial 

corneal ulceration, and 

palpebral edema of the 

left eye 

No known sequelae Helmers et al., 

1971 

45-yr-old 

farmer 

Sprayed on the left side 

of face during a transfer 

operation 

Immediate blepharospasm (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) 

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 

Helmers et al., 

1971 
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Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

20-yr-old male Safety valve release in a 

refrigeration unit due to 

accidental heating of the 

ammonia line 

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 

Six-mo followup: normal 

vision, mild cough 

White, 1971 

20-yr-old 

female 

Exposure (approximately 

30 min) to anhydrous 

ammonia following train 

derailment 

Chemical burns to the 

soft palate, oropharynx, 

and feet, unable to speak, 

blood-streaked sputum 

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 

Kass et al., 1972

22-yr-old 

female 

Exposure (approximately 

90 min) to anhydrous 

ammonia following train 

derailment 

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 

Re-hospitalized 1 yr later 

with bilateral pneumonia, 

peripheral edema, and acute 

right heart failure; marked 

deterioration of vision in the 

left eye, bilateral corneal 

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

Kass et al., 1972
C-18 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

24-yr-old male, 

instrument 

artificer, 

smoker 

Vent stack overflow from 

100 feet above work area, 

resulting in complete 

drenching 

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 

26 mo following exposure, 

dyspnea on exertion, 

excessive collagenization in 

the bronchial submucosal 

layer 

Walton, 1973 

39-yr-old male, 

smoker 

Vent stack overflow from 

100 feet above work area, 

resulting in complete 

drenching 

Dyspnea, chest pain, 

blepharospasm, burning 

throat, extensive 

blistering, tachycardia, 

moist sounds from both 

lung bases, corneal burns, 

aphonia (lasting about 

10 d) 

Continued to smoke at 

follow up; lung function 

tests (FVC and expiratory 

volume in 1 sec) well below 

normal after 2 yrs 

Walton, 1973 

42-yr-old male, 

maintenance 

fitter, smoker 

Exposure due to the 

inadvertent resumption of 

ammonia gas in a high 

pressure line during valve 

maintenance 

Burns to the left eye (no 

further details) 

Five-yr followup indicated 

normal lung function 

Walton, 1973 

39-yr-old male, 

assistant 

process 

foreman 

Doused with ammonia 

liquid from 

approximately 130 feet 

above 

Pink frothy sputum, 

cyanosis, bronchospasm, 

severe burns and 

blistering on the face, 

mouth and hands 

Recurring bronchitis, 

dyspnea on exertion; in lung 

function tests, FVC reached 

near normal 1 yr following 

exposure; FEV1 test 

indicated moderate 

obstructive airways disease 

and the gas transfer figure 

remained below normal 

Walton, 1973 

39-yr-old male, 

process 

foreman, 

smoker 

Doused with ammonia 

liquid from 

approximately 130 feet 

above 

Pain in eyes and throat, 

blepharospasm, burns to 

the eyes, mouth, face, and 

throat 

Continued to smoke at 

follow up; lung function 

tests showed progressive 

improvement in ventilation 

but consistent depression of 

gas transfer factor 

Walton, 1973 

47-yr-old male, 

process worker

In vicinity of a high 

pressure compressor 

pump burst 

Pain and tightness of the 

chest, blepharospasm, 

bloodstained sputum, 

burns and blistering of 

the face, mouth, eyes, 

throat, and left heel 

Lung function tests indicate 

gradual improvement in 

ventilatory ability, but 

below normal in gas 

transfer factor 

Walton, 1973 
C-19 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

Male, process 

worker (age not 

given) 

In vicinity of a high 

pressure compressor 

pump burst 

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 

Not applicable Walton, 1973 

28-yr-old male Sprayed in the face when 

a hose coupling came 

loose 

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 

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 

Taplin et al., 

1976 

25-yr-old male Tank explosion 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 

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 

Sobonya, 1977 

6-mo-old male; 

two 12-yr-old 

males; 17-yrold female 

Road accident involving 

a tank truck rolling from 

an overpass to the road 

below, resulting in a 

thick cloud of ammonia 

vapor 

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 

Measurement of several 

urinary metabolites of 

hydroxylysine indicated that 

considerable collagen 

degradation occurred after 

inhaling concentrated 

ammonia vapors 

Hatton et al., 

1979 
C-20 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

17-yr-old 

female 

Direct exposure to high 

concentrations resulting 

from a tanker truck 

explosion 

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 fullthickness burns of the 

larynx, diffuse 

parenchymal densities in 

both lungs, inspiratory 

wheezing, rhonchi, and 

rales were present 

Pulmonary function studies 

(1 yr following discharge) 

indicated moderate 

obstructive and restrictive 

abnormalities 

Close et al., 

1980 

6-mo-old male Direct exposure to high 

concentrations resulting 

from a tanker truck 

explosion 

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 

No remarkable findings in 

2-mo followup 

Close et al., 

1980 

26-yr-old male Direct exposure 

(approximately 20 min) 

to high concentrations 

resulting from a tanker 

truck explosion 

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 

Rapid degeneration and 

fatality followed a massive 

hemorrhage around the 

tracheostomy tube 

(innominate artery erosion); 

autopsy revealed fullthickness burns to the entire 

respiratory tract, acute 

necrotizing tracheabronchitis, and bilateral 

bronchopneumonia 

Close et al., 

1980 
C-21 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

23-yr-old 

female 

Within a few hundred 

feet of the tanker truck 

explosion, exposure time 

>30 min 

Mild facial erythema and 

bilateral conjunctival 

irritation, first- and 

second-degree burns of 

the oral cavity, 

oropharynx, and larynx, 

bilateral parenchymal 

infiltrate, mild 

hypoxemia 

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 

Close et al., 

1980 

30-yr-old 

female 

In the vicinity of the 

tanker truck explosion, 

exposure time 

approximately 30 min 

Mild facial erythema, 

conjunctival irritation of 

the eyes, diffuse bilateral 

rales and rhonchi, 

bilateral infiltrates, 

hypoxemia (requiring 

mechanical ventilation) 

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 

Close et al., 

1980 
C-22 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

Fourteen male 

fishermen, 18–

39 yrs old 

Refrigeration system leak 

below decks 

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 

48 hrs 

Not applicable Montague and 

Macneil, 1980 

58-yr-old 

female 

Leaking pipe near the 

offices for a grocery 

distributor 

Loss of consciousness, 

severe eye irritation with 

bilateral corneal burns, 

dyspnea, pharyngeal 

edema, bilateral diffuse 

rhonchi, and rales 

Two yrs following 

exposure, patient developed 

severe pulmonary 

insufficiency requiring a 

1-yr hospitalization with a 

permanent tracheostomy 

and continuous oxygen 

Stroud, 1981 

30-yr-old 

female 

Exposure to fumes 

released when a tanker 

truck overturned and 

exploded 

Initial intense eye and 

nasopharyngeal irritation 

developing to 

breathlessness and 

nonproductive cough, 

initial transient infiltrate 

of pulmonary edema 

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, bronchopneumonia; fatality 

Hoeffler et al., 

1982 
C-23 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

50-yr-old male Splashed with liquid 

ammonia when a 

refrigeration coolant tank 

exploded 

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 

Progressively worsening 

respiratory distress 

(mechanical ventilation was 

required for 10 d), extensive 

pseudomembrane formation 

throughout the tracheabronchial tree, gastrointestinal bleeding, 

persistent tachypnea and 

expiratory wheezing 

(following removal of 

tracheostomy tube), 

pneumonitis complications; 

5-yr followup: asymptomatic except during very 

strenuous activity 

Flury et al., 

1983 

Two male 

factory 

workers, 

smokers, 

24 and 40 yrs 

old 

Sudden release of 

ammonia gas to the 

factory floor when the 

pressure valve on a 

refrigeration unit released

Eye and throat irritation, 

slightly red throat; one 

patient had burns on the 

legs and genitals 

 O’Kane, 1983 

Three male 

factory 

workers, 

smokers, 20–

30 yrs old, with 

histories of 

asthma, 

smoker’s 

cough, and 

chronic 

bronchitis 

Sudden release of 

ammonia gas to the 

factory floor when the 

pressure valve on a 

refrigeration unit released

Moderate dyspnea, 

labored breathing, sore 

eyes, moderate to severe 

throat pain, cough, 

nausea 

 O’Kane, 1983 

30-yr-old male 

factory worker, 

smoker, no 

history of lung 

problems 

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 

Severe cough, massive 

nasal discharge, very sore 

throat, constant retching, 

development of 

inspiratory and expiratory 

rhonchi 

Hoarseness and productive 

cough persisting for several 

months 

O’Kane, 1983 

28-yr-old male 

factory worker, 

smoker, no 

history of lung 

problems 

Sudden release of 

ammonia gas to the 

factory floor when the 

pressure valve on a 

refrigeration unit 

released; directly in line 

with escaping gas 

Severely inflamed eyes, 

lips, tongue, edematous 

pharynx and hoarseness, 

high-pitched inspiratory 

and expiratory rhonchi 

Not applicable O’Kane, 1983 
C-24 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

44-yr-old male 

factory worker, 

nonsmoker, no 

history of lung 

problems 

Sudden release of 

ammonia gas to the 

factory floor when the 

pressure valve on a 

refrigeration unit 

released; 4–5-min 

exposure 

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 

Following initial 

improvement, the patient 

began deteriorating the 

13th d following exposure, 

lungs developed multiple 

cavitating lesions, 

secondary infection, and 

finally necrotizing 

pneumonia; patient survived 

with chronic infective lung 

disease 

O’Kane, 1983 

25-yr-old male 

refrigeration 

technician 

Sudden, massive leak 

from the refrigeration 

plant 

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 

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 

Price, 1983 

Two males, 

26 and 40 yrs 

old 

Pipe valve failure 

allowing ammonia gas to 

fill a factory area 

Mild pharyngitis and 

conjunctivitis; the 40-yrold male also had burns 

to legs and genitals 

Followup every 6 mo for up 

to 2 yrs: no additional 

findings 

Ward et al., 

1983 

Three males, 

26, 36, and 

45 yrs old 

Pipe valve failure 

allowing ammonia gas to 

fill a factory area 

Moderate conjunctivitis, 

moderate to severe 

pharyngitis, tachycardia, 

and tachypnea, frothy 

sputum, moderate 

dyspnea, nausea 

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 

Ward et al., 

1983 

30-yr-old male Pipe valve failure 

allowing ammonia gas to 

fill a factory area 

Severe dyspnea, sore 

eyes, nausea, generalized 

rhonchi, leukocytosis, 

and marked hypoxemia 

Improvement in lung 

function tests was seen 

during followup; however, 

patient experienced dyspnea 

upon exertion and chronic 

productive cough 

Ward et al., 

1983 

27-yr-old male Pipe valve failure 

allowing ammonia gas to 

fill a factory area 

Severe dyspnea, sore 

eyes, nausea, generalized 

rhonchi, leukocytosis, 

and marked hypoxemia 

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 

Ward et al., 

1983 
C-25 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

44-yr-old male Pipe valve failure 

allowing ammonia gas to 

fill a factory area 

Severe dyspnea, sore 

eyes, nausea, generalized 

rhonchi, leukocytosis, 

and marked hypoxemia 

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 

Ward et al., 

1983 

33-yr-old male Explosion while 

transferring ammonia 

from one tank to another 

Burns over 12% of total 

body surface area and 

oropharynx, bilateral 

corneal clouding, severe 

respiratory distress 

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 

Arwood et al., 

1985 

51-yr-old male 

smoker 

Explosion while 

transferring ammonia 

from one tank to another 

Burns over 50% total 

body surface area (eyes, 

back, legs, and 

oropharynx), severe 

respiratory distress 

(bilateral wheezing and 

bilateral lower lobe 

infiltrates) 

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 

Arwood et al., 

1985 

Five males, 26–

48 yrs old 

An overturned tanker 

truck released a dense 

cloud of ammonia gas 

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 

One delayed fatality 

(2.5 wks after exposure) due 

to bronchopneumonia and 

necrotizing 

tracheobronchitis 

Burns et al., 

1985 
C-26 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

Twenty two 

seafood 

processing 

plant workers 

Liquefied ammonia tank 

explosion within an 

enclosed area 

Fourteen cases with 

irritative symptoms only; 

six workers exhibited 

transient bronchospasm 

and edema of the lips and 

oral mucosa 

Five workers exhibited eye 

pain and photophobia, four 

of which developed corneal 

damage; one fatality due to 

respiratory failure (2 wks 

following exposure); one 

worker developed 

restrictive lung disease and 

chronic bronchitis (6-mo 

followup) 

Yang, 1987 

37-yr-old male 

with myotonic 

dystrophy 

Ammonia intoxication 

37 yrs after 

ureterosigmoidostomy 

Lethargy, restlessness, 

irritability, and confusion, 

both lungs were 

underinflated due to high 

diaphragm, severe 

metabolic encephalopathy, right-sided 

hydronephrosis (left 

kidney absent), blood 

ammonia levels were 

markedly elevated. 

None Gilbert, 1988 

Four males, all 

smokers (ages 

not given) 

Acute exposure (no 

additional details 

provided) 

Skin blisters, ocular and 

nasal burning, dyspnea, 

chest pain, chest 

tightness, nonproductive 

cough, and hemoptysis 

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 

Bernstein and 

Bernstein, 1989 

28-yr-old male Equipment failure while 

spraying anhydrous 

ammonia on farmland 

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 

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 

Millea et al., 

1989 
C-27 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

17-yr-old male, 

farm supply 

store employee

A connector valve 

opened on a hose 

carrying ammonia 

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, corneal burns to the 

right eye, hoarseness, 

erythema and edema of 

the oral mucosa and 

uvula 

On postexposure d 5, a 

pneumomediastinum and 

subcutaneous emphysema 

were noted, as well as vocal 

cord and tracheobronchial 

edema 

Millea et al., 

1989 

22-yr-old male 

farmer 

Sprayed while applying 

anhydrous ammonia to a 

field 

Partial thickness burns 

over 12% total body 

surface area of the chest, 

abdomen, and upper 

extremities; no 

pulmonary effects 

Patient was readmitted 6 d 

following discharge for an 

infected burn wound on the 

right arm 

Millea et al., 

1989 

21-yr-old male 

refrigeration 

plant worker 

Directly below 

refrigeration pipe rupture 

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 

Fatality 13 d after exposure 

due to development of 

treatment-resistant 

bronchopneumonia 

Weiser and 

Mackenroth, 

1989 

35-yr-old male 

refrigeration 

plant worker 

In vicinity of 

refrigeration pipe rupture

Second-degree burns over 

10% of total body surface 

area, dyspnea, nausea, 

severe eye irritation, nose 

and throat irritation, 

pulmonary edema 

Not applicable Weiser and 

Mackenroth, 

1989 

Six 

refrigeration 

plant workers, 

male and 

female, 19–

24 yrs old 

In vicinity of 

refrigeration pipe rupture

Erythema, small burn 

areas, irritation of the 

eyes, nose, and throat, 

dyspnea and nausea to 

varying degrees 

Not applicable Weiser and 

Mackenroth, 

1989 

Workers at a 

fertilizer plant 

Leak and explosion of an 

ammonia tank at a 

fertilizer plant 

Seven fatalities, 

57 injuries (no further 

details provided) 

Not applicable Andersson, 

1991 
C-28 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

28-yr-old male Tank explosion in an 

industrial butter plant 

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 

At 1 wk followup: 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 followup, severe 

fixed airway obstruction 

remained, with coexisting 

bronchiectasis 

Leduc et al., 

1992 

30-yr-old male, 

smoker 

Occupational exposure to 

a refrigeration gas leak in 

adjacent room; 

approximately 15-min 

exposure 

Immediate burning of the 

eyes, upper airways, and 

skin, with cough and 

pleuritic chest pain, 

conjunctivitis and 

rhinopharyngitis 

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 

de la Hoz et al., 

1996 

27-yr-old male, 

smoker 

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 

Difficulty breathing; 

irritation of the eyes, 

nose, throat, burning 

sensation on the skin; 

respiratory failure 

requiring mechanical 

ventilation 

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 

de la Hoz et al., 

1996 

46-yr-old male Sprayed while unloading 

tanks of ammonium 

hydroxide (29.4% 

ammonia) 

Mucocutaneous burns 

(unspecified), productive 

cough, dyspnea and 

wheezing 

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 

de la Hoz et al., 

1996 
C-29 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

41-yr-old male, 

fish-processing 

plant 

Several hours spent in the 

vicinity of an ongoing 

ammonia leak from a 

refrigeration unit, without 

respiratory protection 

Eye and nasal irritation 

and mild facial burning 

Nasal congestion and 

intermittent epistaxis, which 

resolved in about 2 wks, 

hyposmia (decreased sense 

of smell), which persisted 

without additional abnormal 

findings 

Prudhomme et 

al., 1998 

51-yr-old 

maintenance 

man 

Pressurized liquid 

ammonia explosion 

during inspection for 

maintenance and repair 

on a refrigerator coolant 

storage system 

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 

None Sotiropoulos et 

al., 1998 

57-yr-old male, 

supervisor to 

above 

Pressurized liquid 

ammonia explosion 

during inspection for 

maintenance and repair 

on a refrigerator coolant 

storage system 

Pain in the genital area 

was first noted an hour 

after explosion, with 

significant blistering on 

the scrotum and distal 

penis 

Healing without incident or 

sequelae 

Sotiropoulos et 

al., 1998 

28-yr-old male Anhydrous ammonia 

explosion at a fertilizer 

factory 

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) 

No effects observed at 

18-mo followup 

Amshel et al., 

2000 

47-yr-old male Burst pipe in a liquefied 

ammonia production 

plant, with over 45 min 

of exposure 

Mixed burns on the face, 

neck, chest, genitalia, 

both lower limbs, and full 

thickness burns of the 

back (38% total body 

surface area), frostbite 

injury to the back, the 

eyes showed chemosis 

and abrasions, congestion 

and corneal edema 

increasing with 

subconjunctival 

hemorrhaging, harsh 

breathing sounds with 

crepitations, intermittent 

bradycardia 

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 

George et al., 

2000 
C-30 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

25-yr-old male Liquid ammonia spill 

during transfer from a 

barge tank to a dockside 

tank 

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 

Not applicable Latenser and 

Lucktong, 2000 

32-yr-old male Liquid ammonia spill 

during transfer from a 

barge tank to a dockside 

tank 

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 

Not applicable Latenser and 

Lucktong, 2000 

40-yr-old male Exposed to liquid 

ammonia while emptying 

a refrigeration unit 

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 

Bilateral infrahilar 

consolidations and apical 

infiltrates were noted in 

postburn d 3; there was no 

long-term followup 

Kerstein et al., 

2001 

Two food 

processing 

plant workers, 

ages not 

specified 

Discharge of pressurized 

ammonia during 

maintenance 

One immediate fatality 

(pulmonary edema, focal 

lung hemorrhage); one 

worker permanently 

blinded (no additional 

details) 

Not applicable Morton, 2005 
C-31 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

32-yr-old male Warehouse explosion 

resulting in an ammonia 

tank rupture 

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 

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 

White et al., 

2007 

22-yr-old male Warehouse explosion 

resulting in an ammonia 

tank rupture 

0.5% total body surface 

area burns to the scrotum, 

bilateral corneal injury, 

tracheobronchitis 

At 4-wk followup, bilateral 

uveitis with stromal 

vascularization, haze and 

scarring, and pigmented 

keratic precipitates resulting 

in legal blindness (after 

unsuccessful attempt at 

corneal transplants) 

White et al., 

2007 

19-yr-old male Warehouse explosion 

resulting in an ammonia 

tank rupture 

0.5% total body surface 

area burns to the face and 

scrotum; bilateral corneal 

injury; tracheobronchitis 

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 

White et al., 

2007 

21-yr-old male Warehouse explosion 

resulting in an ammonia 

tank rupture 

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 

Extensive peribronchiolar 

edema, necrosis and 

sloughing were observed 

down to the tertiary 

bronchioles; fatality at 

100 d postinjury 

White et al., 

2007 

33-yr-old male Warehouse explosion 

resulting in an ammonia 

tank rupture 

Respiratory difficulty, but 

no burns to the skin and 

eyes; peribronchiolar 

edema limited to the right 

lung 

Not applicable White et al., 

2007 

Patients and 

hospital staff in 

the critical care 

unit 

Ammonia leak from a 

refrigerator 

Respiratory and ocular 

irritation 

Not applicable Price and Watts, 

2008 
C-32 DRAFT - DO NOT CITE OR QUOTE 

Table C-2. Human case studies and reports of human exposure to ammonia 

Case(s) Exposure conditions Immediate effectsa Delayed effectsb Reference 

57-yr-old male, 

nonsmoker 

At age 25, massive 

accidental exposure to 

anhydrous ammonia 

while working in a 

refrigeration unit 

Loss of consciousness, 

corneal scarring, and 

burns to the face and 

chest 

Chronic hypoxemic 

respiratory failure, bilateral 

inspiratory crackles, and 

prolonged expiration, 

central cylindrical, varicose 

and cystic bronchiectasis 

with mild interstitial fibrosis

Tonelli and 

Pham, 2009 

20-yr-old male Forklift caught an 

ammonia pipe causing a 

sudden burst of a large 

quantity of ammonia 

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 

Continued hospital care for 

respiratory support until 

transplantation of both 

lungs performed 6 mo after 

exposure 

Lalic et al., 

2009; Pirjavec 

et al., 2009 

53-yr-old male Forklift caught an 

ammonia pipe causing a 

sudden burst of a large 

quantity of ammonia 

Loss of consciousness, 

acute respiratory distress 

syndrome 

Developed obliterating 

bronchiolitis and restrictive 

and obstructive ventilation 

disturbances, unable to 

work, breathless at rest, 

lung transplant candidate 

Lalic et al., 

2009 

Fifteen males, 

31.1 ± 8.8 yrs 

old 

Resulted from events 

during illicit 

methamphetamine 

production, 14/15 due to 

explosions 

Burns on body, 

respiratory distress 

One fatality, ventilator 

assistance and skin grafting 

required 

Bloom et al., 

2008 

Five males, 

49.4 ± 23.0 yrs 

old 

Resulted from farming 

accidents, 2/5 due to 

explosions 

Burns on body, 

respiratory distress 

Ventilator assistance and 

skin grafting required 

Bloom et al., 

2008 

aEffects occurring within 48 hrs of exposure. 

bEffects occurring longer than 48 hrs after exposure. 

1 

2 
C-33 DRAFT - DO NOT CITE OR QUOTE 

1 C.3. CONTROLLED HUMAN EXPOSURE STUDIES OF AMMONIA INHALATION 

2 Controlled human exposure studies of inhaled ammonia are summarized in Table C-3. 

3 

Table C-3. Controlled human exposure studies of ammonia inhalation 

Subjects Exposure conditions Results Reference 

Seven male 

volunteers 

500 ppm (354 mg/m3) 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/m3 (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 

Habituation 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 et al., 

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, FEV1 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 
C-34 DRAFT - DO NOT CITE OR QUOTE 

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/m3

(3.0 ppm) for 37 d; 

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 shortterm 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-oxycorticosteroids 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-oxycorticosteroid 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 FEV1 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 
C-35 DRAFT - DO NOT CITE OR QUOTE 

Table C-3. Controlled human exposure studies of ammonia inhalation 

Subjects Exposure conditions Results Reference 

Healthy male and 

female volunteers 

grouped by age, 18–

35 and 45–65 yrs 

old 

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 

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 naïve 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 concentrationdependent in both groups 

Ihrig et al., 

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 (FEV1) for subjects in either 

group 

Petrova et al., 

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 et al., 

2007 

1 

2 
C-36 DRAFT - DO NOT CITE OR QUOTE 

1 C.4. CROSS SECTIONAL STUDIES OF LIVESTOCK FARMERS EXPOSED TO 

2 AMMONIA 

3 Cross sectional studies of livestock farmers exposed to ammonia are summarized in 

4 Table C-4. 

5 

Table C-4. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

27 pig farmers (mean 

age of 29 yrs) 

Environmental and 

personal exposures 

were analyzed; lung 

function was 

measured on 

Monday, Tuesday, 

and Friday 

Mean exposure to dust = 

1.57 mg/m3; endotoxin = 

24 ng/m3, and 

ammonia = 5.60 mg/m3

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 

Heederik et 

al., 1990 

29 farm workers; 

48 electronic factory 

workers (controls) 

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 

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

Respiratory symptoms 

included chest tightness, 

wheeze, nasal and eye 

irritation (23/29 farm 

workers); 3/29 farm workers 

had impaired lung function 

(decreased FEV1 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 

Crook et al., 

1991 
C-37 DRAFT - DO NOT CITE OR QUOTE 

Table C-4. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

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; 51 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) 

Pulmonary function 

tests were given to 

subjects before and 

after a methacholine 

challenge; respiratory 

symptoms were 

determined by 

questionnaire 

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

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 

Choudat et al., 

1994 

54 male swine 

producers (mean age 

= 36.3 yrs; mean 

duration of 

employment = 

10.7 yrs) 

Assessment of 

respiratory symptoms 

with questionnaire 

and lung function 

tests 

Mean contaminant 

levels: carbon dioxide = 

2,632 ppm (1,861 mg/ 

m

3

); ammonia = 

11.3 ppm (8 mg/m3); 

total dust = 2.93 mg/m3

; 

respirable dust = 

0.13 mg/m3; endotoxin = 

11,332 units/m3

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 

Zejda et al., 

1994 

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) 

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 

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) 

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 

in FEV1; the correlation 

between exposure and 

decreased pulmonary 

function was stronger after 

6 yrs of exposure 

Donham et al., 

1995 
C-38 DRAFT - DO NOT CITE OR QUOTE 

Table C-4. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

194 Dutch pig 

farmers (94 with 

chronic respiratory 

symptoms, 100 

without symptoms) 

Cross-sectional study 

evaluating exposure 

response relations of 

exposures to dust, 

endotoxins, 

ammonia, and 

disinfection 

procedures 

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 = 

112 ng/m3, ammonia = 

2 mg/m3

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 

FEV1, MMEF, and PEF) 

Preller et al., 

1995 

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) 

Followup study from 

Donham et al. (1995) 

previously described; 

followup 

measurements taken 

48 mo from the 

initial measurements 

Mean personal air 

exposure for all subjects:

total dust = 3.45 mg/m3; 

respirable dust = 

0.26 mg/m3

; total 

endotoxin = 

176.12 EU/m3; 

respirable endotoxin = 

11.86 EU/m3; 

ammonia = 5.15 ppm 

(4 mg/m3

) 

Swine workers had a mean 

cross-shift 2% decrease in 

FEV1 that was correlated 

with personal exposure to 

total dust, total endotoxin, 

respirable endotoxin, and 

ammonia 

Reynolds et 

al., 1996 

196 pig farmers 

(96 with chronic 

respiratory 

symptoms, 100 

without symptoms) 

Pig farmers tested for 

lung function and 

bronchial 

responsiveness to 

histamine challenge 

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

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 

Vogelzang et 

al., 1997 

171 pig farmers 

(82 with chronic 

respiratory 

symptoms, 89 

without) 

Longitudinal study 

for cohort of pig 

farmers observed 

over 3 yrs; subjects 

examined for lung 

function and tested 

for bronchial 

responsiveness to 

histamine challenge 

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

Decreased lung function 

(FEV1 and FVC) was 

observed over time; longterm exposure to ammonia 

was associated with 

increased bronchial 

responsiveness to histamine; 

exposure to respirable dust 

also caused increased 

bronchial responsiveness to 

histamine 

Vogelzang et 

al., 2000, 1998
C-39 DRAFT - DO NOT CITE OR QUOTE 

Table C-4. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

Eight healthy male 

volunteers (23–

28 yrs old) 

Exposed for 4 hrs at 

1-wk intervals to 

swine confinement 

buildings 

Mean airborne ammonia 

concentration of 

20.7 ppm (15 mg/m3); 

also exposed to airborne 

dust, bacteria, 

endotoxin, and molds 

Decreased expiratory flows 

(FEV1), increased 

neutrophils in the nasal wash 

and increased white blood 

cell count 

Cormier et al., 

2000 

257 poultry workers 

(30% women, 70% 

men); 63 women and 

87 men nonexposed 

blue-collar workers 

served as control 

subjects 

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 

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 μg/m3); respirable 

endotoxin = 58.9 EU/m3

(0.006 μg/m3) 

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 

μg/m3) 

Donham et al., 

2000 

Survey of 

8,482 farmers and 

spouses; exposure 

study conducted in 

102 farmers 

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 

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 μg/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 

Melbostad and 

Eduard, 2001 

13 stable workers 

(6 males, 7 females) 

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 

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 

1,3-β-glucan (85 ng/m3

) 

and horse allergen 

(18,300 U/m3

) were 

highest during the winter

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) 

Elfman et al., 

2009 

EU = endotoxin unit (10 EU/ng); MMEF = mean midexpiratory flow 

1 

2 
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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 γ-amino butyric acid (GABA) play a role in ammonia-induced neurotoxicity. 

14 
C-41 DRAFT - DO N

Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Rats

Female Porton rats (16/group) 

0 or 141 (0 or 200 ppm) 

Continuous exposure for 4, 8, or 12 d 

Histology of the trachea 4 d: transitional-stratified appearance of the 

epithelium; 8 d: gross change with disappearance of ciland stratification on luminal surface; 12 d: increased epithelial thickness 

Male OFA rats (27/group) 

0 or 354 (0 or 500 ppm) 

Continuous exposure for 1–8 wks 

Body weight, organ weights, airway structure, cell population, alveolar macrophages 

No deaths occurred; decreased food consumption and body weight gain; increaslung and kidney weights; at 3 wks nasal irritation and upper respiratory tract inflammation, but no effect on lower airwayslight decrease in alveolar macrophages; no histopathological effects seen at 8 wks, suggesting adaptation to exposure 

Male and female Wistar rats (5/sex/group) 

9,898–37,825 (14,000–53,500 ppm) (no mention of control group) 

10, 20, 40, or 60 min 

Clinical signs, pathology, LC50

Eye irritation, eye and nasal discharge, dysphemorrhagic lungs on necropsy; 10-min LC50 = 28,492 mg/m

3 (40,300 ppm) 

20-min LC50 = 20,217 mg/m

3 (28, 595 ppm

40-min LC50 = 14,352 mg/m

3 (20,300 ppm) 

60-min LC50 = 11,736 mg/m

3 (16,600 ppm) 

Male Crl:COBS CD (Sprague-Dawley) rats (8/group) 

11, 23, 219, and 818 (15, 32, 310, and 1,157 ppm); arterial blood collected prior to exposure served as control 

24 hrs Clinical signs, histology, 

blood pH, blood gas measurement 

No clinical signs of toxicity, no histologic differences in tracheal or lung sections, no change in blood pH or pCO2, minor change

pO2

Male Crl:COBS CD (Sprague-Dawley) rats (14/group) 

3, 17, 31, 117, and 505 (4, 24, 44, 165, and 714 ppm); arterial blood collected prior to exposure served as control 

3 and 7 d Hepatic cytochrome P450 

content and ethylmorphine–Ndemethylase activity 

No dose-related change in P450 content or enzyme activity 
C-42 DRAFT - DO N

Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Male Long Evans rats (4/group) 

70 and 212 (100 and 300 ppm); results were compared 

to “control”, but it was not clear if the authors were referring to historical or concurrent controls 

6 hrs Clinical signs, behavioral 

observation 

Decreased running, decreased activity 

Female Wistar rats (5/group) 

0, 18, or 212 (0, 25, or 300 ppm) 

6 hrs/d for 5, 10, or 15 d 

Blood ammonia, urea, glutamine, and pH; brain ammonia, glutamine; histopathology of lungs, heart, liver, and kidneys (light and electron microscopy) 

Brain and blood glutamine increased; slight acidosis (i.e., decreased blood pH) 212 mg/

(300 ppm); lung hemorrhage observed in soexposed rats 

Female Wistar rats (5/group) 

0, 18, or 212 (0, 25, or 300 ppm) 

6 hrs/d for 5 d Plasma and brain ammonia 

and amino acid analysis 

Increase in brain and plasma glutamine concentrations, increased brain/plasma ratio threonine 

Female albino rats (8/group) 

0, 848–1,068 (1,200–1,510 ppm) 

3 hrs Mortality, respiratory 

movement and O2

consumption 

No deaths reported; inhibition of external respiration and decreased O2 consumption 

Male SpragueDawley rats (number/group not given) 

Air concentration not given; ammonia vapor added to inspiratory line of ventilator; controls exposed to same volume of room air 

20 sec Activity of upper thoracic 

spinal neurons 

Lower airway irritation, activation of vagal pulmonary afferents and upper thoracic spinneurons receiving pulmonary sympathetic i
C-43 DRAFT - DO N

Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Male Wistar rats (4/group) 

0, 92–1,243 (130–1,758 ppm); the preexposure period was used as the control for each animal 

45 min Airway reflexes by the 

changes in respiratory patterns elicited by ammonia in either dry, steam-humidified or 

aqueous aerosol containing atmospheres 

Ammonia-induced upper respiratory tract sensory irritation is not affected to any appreciable extent by wet atmospheres (witwithout aerosol) up to 1,243 mg/m

3

Male rats (10/group) 0, 848–1,068 (0, 

1,200–1,510 ppm) at the beginning and end of the exposure period) 

3 hrs Oxygen consumption Decreased O2 consumption 

Mice

Mice (20/group, species, sex not specified) 

6,080–7,070 (8,600–10,000 ppm); no controls 

10 min LC50 LC50 = 7,056 mg/m

3

(9,980 ppm) 

Male Swiss albino mice (4/group) 

5,050–20,199 (7,143–28,571 ppm); no controls 

30–120 min LC50 LC50 (30 min) = 15,151 mg/m

3 (21,430 ppm

Albino mice (sex not specified; 6/dose) 

Air concentration not measured; results were compared to “control”, but it was not clear if the authors were referring to historical or concurrent controls 

Continuously for 2 or 5 d 

Regional brain metabolism (cerebral cortex, cerebellum, brainstem); MAO, enzymes of glutamate and ã-amino butyric acid (GABA) metabolism, and (Na

+-K

+)-

ATPase; amino acid levels in the brain 

Altered activities of MAO, glutamate decarboxylase, alanine amino transferase, GABA-transaminase, and (Na

+-K

+)-ATPase

increased alanine and decreased glutamate 
C-44 DRAFT - DO N

Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Male Swiss-Webster mice (4/group) 

Concentrations not given; baseline levels established prior to exposure 

10 min 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 

RD50 = 214 mg/m

3

(303 ppm) 

Male albino ICR mice (12/dose) 

0–3,436 (0–4,860 ppm) 

1 hr (14-d follow 

up) 

Clinical signs, body weight, organ weight, histopathology, LC50

Eye and nose irritation, dyspnea, ataxia, seizures, coma, and death; decreased body weight and increased liver to body weight rin mice surviving to 14 d; effects in the lung included focal pneumonitis, atelectasis, and intralveolar hemorrhage; liver effects includhepatocellular swelling and necrosis, vasculcongestion; LC50 = 2,990 mg/m

3 (4,230 ppm

Male Swiss-Webster mice (16–24/group) 

0 or 216 (0 or 305 ppm) 

6 hrs/d for 5 d Respiratory tract 

histopathology 

Lesions in the nasal respiratory epithelium (moderate inflammation, minimal necrosis, exfoliation, erosion, or ulceration); no lesiotrachea or lungs 

Male albino ICR mice (12/dose) 

0, 954, 3,097, or 3,323 (0, 1,350, 4,380, or 4,700 ppm) 

4 hrs Hexobarbitol sleeping 

time, microsomal protein content, liver microsomal enzyme activity 

Increased hexobarbitol sleeping time (3,097 mg/m

3), increased microsomal prote

content, aminopyrene-N-deethylase and anihydroxylase activities (3,323 mg/m

3) 

Male albino ICR mice (12/dose) 

0, 81, or 233 (0, 115, or 330 ppm) 

4 hrs/d for 4 d Microsomal protein 

content, liver microsomal enzyme activity 

No dose-dependent effects on microsomal enzymes 

Male Swiss mice (6/dose) 

71 and 212 (100 and 300 ppm); data collected during the 2 d separating each ammonia exposure served as the control baseline 

6 hrs Clinical signs, behavioral 

observation 

Decreased running, decreased activity 
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Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Mice (4/group) 3, 21, 40, or 78 (4, 30, 

56, or 110 ppm), lowest measured concentration was the nominal control group

2 d Responses to atmospheric 

ammonia in an environmental preference chamber with four chambers of different concentrations of ammonia

No distinguishable preference for, or aversito, different NH3 concentrations 

Male OF1 mice (4/group) 

0, 92–1,243 (130–1,758 ppm); the preexposure period was used as the control for each animal 

45 min Airway reflexes by the 

changes in respiratory patterns elicited by ammonia in either dry, steam-humidified, or 

aqueous aerosol containing atmospheres 

Ammonia-induced upper respiratory tract sensory irritation is not affected to any appreciable extent by wet atmospheres (witwithout aerosol) up to 1,243 mg/m

3

Rabbits

Female New Zealand White rabbits (7–9/dose) 

0, 35, or 71 (0, 50, or 100 ppm) 

2.5–3.0 hrs Pulmonary function Decreased respiratory rate at both 

concentrations 

Rabbits (species, sex, number/dose not specified) 

0, 707–14,140 (0, 1,000–20,000 ppm) 

15–180 min Pulmonary function, death Bradycardia at 1,768 mg/m

3 (2,500 ppm); 

arterial pressure variations and blood gas modifications (acidosis indicated by decreapH and increased pCO2) at 3,535 mg/m

3

(5,000 ppm); death occurred at 4,242 mg/m

(6,000 ppm)

New Zealand White rabbits (16 total; 8/dose) 

Peak concentrations: 24,745–27,573 mg/m

3

(35,000–39,000 ppm); concurrent controls tested 

4 min Pulmonary function, heart 

rate, blood pressure, blood gases 

Lung injury was evident after 2–3 min (decreased pO2, increased airway pressure) 
C-46 DRAFT - DO N

Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

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) 

Pigs

Young pigs (2/group) 

0, 35, 71, or 106 (0, 50, 100, or 150 ppm) 

Continuous exposure for 4 wks 

Clinical signs, food 

consumption, body weight, gross necropsy, organ weight, histopathology 

Lethargy and histopathological alterations itracheal and nasal epithelium were observed 71 and 106 mg/m

3; decreased body weight 

occurred at all concentrations (7–19% decrefrom control) 

Male and Female Belgian Landrace pigs (4/group) 

0, 18, 35, or 71 (0, 25, 50, or 100 ppm) 

6 d Clinical signs, body 

weight, pulmonary function 

Lethargy and decreased body weight gain (aconcentrations); no effect on pulmonary microvascular hemodynamics and permeab

Belgian Landrace pigs (4/group) 

0, 18, 35, or 71 (0, 25, 50, or 100 ppm) 

6 d Clinical signs, body 

weight, neutrophil count, and albumin in nasal lavage fluid 

Nasal irritation (increased neutrophils in nalavage fluid) and decreased body weight gaall concentrations 

Landrace-Yorkshire pigs (4/group) 

0 or 42 (0 or 60 ppm) 15 min/d for 

8 wks 

Thromboxane A2 (TXA2), leukotriene C4 (LTC4), and prostaglandin (PGI2) production 

Significant increases in TXA2 and LTC4, nsignificant effect on PGI2 production 

Hybrid gilts (White synthetic Pietrain, white Duroc, Landrace, Large White) (14 pigs/group) 

<4 (control) or 14 (<5 or 20 ppm) 

15 wks Salivary cortisol, adrenal 

morphometry, body weight, food conversion efficiency, general health scores, play behavior; reaction to light and noise intensity tested concurrently 

Decreased salivary cortisol, larger adrenal cortices, less play behavior, no measurable impact on productivity or physiological parameters 
C-47 DRAFT - DO N

Table C-5. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Cattle

Male Holestein calves (number/group not specified) 

0, 35, or 71 (0, 50, or 100 ppm) 

2.5 hrs Respiration rate, clinical 

chemistry 

No significant effect on respiration, BUN, ppO2, or pCO2

Brahman/Charolais (group size not reported) 

<6 (control), 11, 23, or 

34 (<8 [control], 16, 32, or 48 ppm) 

12 d 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) 

Increased lacrimation, nasal secretions, coughing, increased standing (as opposed to lying down), dose related increases in macrophage activity and neutrophil percentin BAL fluid indicating pulmonary inflammation, no effect on hematological variables or body weight 

Holstein Friesian and Brown Swiss (10 of each breed) 

~0, 4, and 15, (0.3 × 10-6, 6, and 

21 ppm) 

10 d at each concentration 

Respiration and pulse rate, blood gas parameters 

Respiration and pulse rates were higher in inadequately ventilated barns (elevated ammonia and CO2) 

1 
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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 of H. pylori on the stomach. 

20 H. pylori produces urease, which breaks down urea that is normally present in the stomach into 

21 ammonia (Mégraud 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 (Mégraud 

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 of H. 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 oC. 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 α-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 α-ketoglutaric acid or adenosine diphosphate. Oxygen 

31 consumption was significantly lower with α-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 
C-50 DRAFT - DO NOT CITE OR QUOTE 

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 

concentrations of 88.2 μg/m39 ; 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 Endpoint Test conditions Resultsa

Dose or 

concentrationb Reference 

Human 

lymphocytes 

Chromosome 

aberrations

22 healthy workers 

occupationally exposed to 

ammonia in fertilizer factory 

(ambient concentration of 

88.28 μg/m3); nonexposed 

factory staff served as control 

subjects

+ 88.28 μg/m3 Yadav and 

Kaushik, 1997

Human 

lymphocytes 

Sister chromatid 

exchanges

+ 88.28 μg/m3 Yadav and 

Kaushik, 1997

Swiss albino 

mice

Micronucleus Intraperitoneal injections for 

24−48 hr expression times

+ 12.5-50 mg/kg Yadav and 

Kaushik, 1997

Drosophila 

melanogaster 

Dominant lethal 

mutations

Dominant lethal assay; 

inhalation exposure up to 

450 ppm (318 mg/m3

) ammonia, 

6 hrs/d for 5 d

+ (T) NA Lobasov and 

Smirnov, 1934

D. melanogaster Sex-linked 

recessive lethal 

mutations

Inhalation exposure to ammonia

as vapor at a concentration 

killing the majority of flies

– (T) NA Auerbach and 

Robson, 1947

D. melanogaster Dominant lethal 

mutations

Inhalation exposure to ammonia

as vapor at a concentration 

killing the majority of flies

– (T) NA Auerbach and 

Robson, 1947

a

+ = positive; − = negative; (T) = toxicity reported; NA = not available. 

bLowest effective dose for positive results, highest dose tested for negative or equivocal results. 

1 

2 Positive results were obtained in a single micronucleus assay with mice (Yadav and 

3 Kaushik, 1997). One study in Drosophila exposed to ammonia gas was positive for 

4 mutagenicity; however, survival after exposure was <2% (Lobasov and Smirnov, 1934). Results 

5 from another study in Drosophila were negative for sex-linked recessive lethal and dominant 

6 lethal mutagenicity; however, the majority of Drosophila was killed by ammonia treatment 

7 (Auerbach and Robson, 1947). 

8 In vitro tests of the genotoxic effects of ammonia have been performed in bacteria and in 

9 chick fibroblasts (Table C-7). Ammonia (administered as vapor) did not induce reverse 

10 mutations in Salmonella typhimurium or E. coli either with or without metabolic activation 

11 (Shimizu et al., 1985). Demerec et al. (1951) reported an increase in reverse mutations in E. coli; 

12 however, positive findings were only reported for levels of ammonia that were toxic, with 98% 

13 lethality. Chick fibroblasts immersed in buffered ammonia solution were found to have 

14 increased frequencies of chromosomal aberrations (Rosenfeld, 1932). 

15 
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Table C-7. In vitro genotoxicity studies of ammonia 

Test system Endpoint Test conditions 

Resultsa

Dosec

 Reference 

Without 

activation

With 

activationb

S. typhimurium

(TA98, TA100, 

TA1535, TA1537, 

TA1538); E. coli 

(WP2 uvrA) 

Reverse 

mutation 

Plate incorporation 

assay with ammonia 

vapor 

– – 25,000 ppm 

(17,675 mg/m3) 

ammonia vapor 

Shimizu et al., 

1985 

E. coli (B/SD-4 

strains)

Reverse 

mutation, 

streptomycin 

resistance 

Plate incorporation 

assay 

+ (T) ND 0.25% 

ammoniad

Demerec et al., 

1951 

Chick fibroblasts Chromosomal 

aberrations 

Cultures immersed in 

buffered ammonia 

solution 

+ ND NA Rosenfeld, 

1932 

a

+ = positive; − = negative; (T) = toxicity reported; NA = not available; ND = no data. 

bExogenous metabolic activation used; S9 liver fractions from male Sprague-Dawley rats pretreated with 

pentachlorobiphenyl (KC500). 

cLowest effective dose for positive results, highest dose tested for negative or equivocal results. 

dOnly positive in treatments using toxic levels of NH3 (98% lethality). 

1