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# Toxicological Review of 12 

# Ammonia

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### EPA/635/R-11/013C-2

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### www.epa.gov/iris 2
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## Toxicological Review of Ammonia 11

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### (CAS No. 7664-41-7) 13
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### February 2012 17
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### NOTICE 25
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### This document is an Interagency Science Consultation draft . This information is distributed 27 

### solely for the purpose of pre-dissemination peer re view under applicable information quality 28 

### guidelines. It has not been formally disseminated by EPA. It does not represent and should not 29 

### be construed to represent any Agency determination or policy. It is being circulated for review 30 

### of its technical accuracy and science policy implic ations. 31
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### National Center for Environmental Assessment 41 

### Office of Research and Development 42 

### U.S. Environmental Protection Agency 43 

### Washington, DC 44

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## DISCLAIMER 1

2

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### This document is a preliminary draft for review pur poses only. This information is 4 

### distributed solely for the purpose of pre-dissemina tion peer review under applicable 5 

### information quality guidelines. It has not been fo rmally disseminated by EPA. It does not 6 

### represent and should not be construed to represent any Agency determination or policy. 7 

### Mention of trade names or commercial products does not constitute endorsement of 8


### recommendation for use. 9

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# CONTENTS 2

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### PREAMBLE ..........................................

................................................... ................................................. v i 4


### AUTHORS | CONTRIBUTORS | REVIEWERS ................

................................................... ........................ xv 5



### PREFACE ...........................................

................................................... ................................................ xv ii 6



### EXECUTIVE SUMMARY .................................

................................................... ...................................... xx 7



### LITERATURE SEARCH STRATEGY & STUDY EVALUATION FOR H AZARD IDENTIFICATION .................. xxiv 8



### 1. HAZARD IDENTIFICATION ..........................

................................................... ...................................... 1 9


### 1.1. Synthesis of Major Toxicological Effects .....

................................................... ..................... 1 10


### 1.1.1. Respiratory Effects .......................

................................................... .................. 1 11



### 1.1.2. Gastrointestinal Effects ..................

................................................... .............. 14 12



### 1.1.3. Reproductive and Developmental Effects ....

.................................................. 17 13


### 1.1.4. Immune System Effects .....................

................................................... .......... 18 14


### 1.1.5. Other Systemic Effects ....................

................................................... ............. 22 15



### 1.1.6. Cancer ....................................

................................................... ...................... 31 16


### 1.1.7. Susceptible Populations and Life Stages ...

................................................... ... 33 17


### 1.2. Weight of Evidence Evaluation for Toxicologica l Effects ......................................... ......... 33 18



### 2. DOSE-RESPONSE ANALYSIS ........................

................................................... ................................... 40 19



### 2.1. Oral Reference Dose for Effects other than Can cer................................................ .......... 40 20



### 2.2. Inhalation Reference Concentration for Effects other than Cancer ................................ . 42 21



### 2.2.1. Methods of Analysis .......................

................................................... .............. 44 22



### 2.2.2. Derivation of Reference Concentration .....

................................................... .. 45 23



### 2.2.3. Uncertainties in the Derivation of the RfC

................................................... ... 46 24



### 2.2.4. Confidence Statement ......................

................................................... ........... 47 25



### 2.2.5. Previous IRIS Assessment: Reference Concent ration ..................................... 48 26



### 2.3. Cancer Risk Estimates ........................

................................................... ............................ 48 27



### 3. REFERENCES ....................................

................................................... .............................................. 49 28

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## LIST OF TABLES 1

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### Table 1-1. Respiratory effects in humans following inhalation exposure ............................. ................ 6 3 

### Table 1-2. Respiratory effects in animals followin g inhalation exposure ............................. ................. 9 4


### Table 1-3. Gastrointestinal effects in animals fol lowing oral exposure .............................. ................. 16 5 

### Table 1-4. Reproductive and developmental effects in animals following inhalation exposure ......... 18 6 

### Table 1-5. Immune system effects in animals follow ing inhalation exposure ........................... .......... 20 7 

### Table 1-6. Systemic effects in humans following in halation exposure ................................. ............... 24 8 

### Table 1-7. Systemic effects in animals following o ral exposure ...................................... .................... 24 9 

### Table 1-8. Systemic effects in animals following i nhalation exposure ................................ ................ 24 10 

### Table 1-9. Cancer bioassays following oral exposur e ................................................. ......................... 32 11

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## LIST OF FIGURES 15

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### Figure 1-1. Exposure-response array for respirator y effects following inhalation exposure ........... ... 13 17 

### Figure 1-2. Exposure-response array for immune sys tem effects following inhalation exposure ...... 21 18 

### Figure 1-3. Exposure-response array for systemic e ffects following inhalation exposure .............. .... 30 19 

### Figure 1-4. Exposure-response array for toxicologi cal effects following inhalation exposure ........ ... 38 20

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## ABBREVIATIONS 1

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ACGIH American Conference of Governmental 4 Industrial Hygienists 5 ALP alkaline phosphatase 6 ALT alanine aminotransferase 7 AST aspartate aminotransferase 8 ATSDR Agency for Toxic Substances and Disease 9 Registry 10 ATSG acid-treated silica gel 11 BAL bronchioalveolar lavage 12 BMD benchmark dose 13 BMI body mass index 14 BrDU 5-bromo-2-deoxyuridine 15 BUN blood urea nitrogen 16 CAC cumulative ammonia concentration 17 CASRN Chemical Abstracts Service Registry 18 Number 19 CI confidence interval 20 EPA Environmental Protection Agency 21 EU endotoxin unit 22 FEF forced expiratory flow 23 FEV 1 forced expiratory volume in 1 second 24 FVC forced vital capacity 25 GABA γ -amino butyric acid 26 IgE immunoglobin E 27 IgG immunoglobin G 28 IRIS Integrated Risk Information System 29 LOAEL lowest-observed-adverse-effect level 30 MAO monoamine oxidase 31 MMEF mean midexpiratory flow 32 MNNG N-methyl-N’-nitro-N-nitrosoguanidine 33 MRM murine respiratory mycoplasmosis 34 NH 3 ammonia 35 NH 4 + ammonium ion 36 NIOSH National Institute for Occupational Safety 37 and Health 38 NOAEL no-observed-adverse-effect level 39 NOx nitrogen oxide 40 NRC National Research Council 41 OR odds ratio 42 PBPK physiologically based pharmacokinetic 43 PEF peak expiratory flow 44 PEFR peak expiratory flow rate 45 PHA phytohemagglutin 46 POD point of departure 47 PPD purified protein derivative 48 RfC inhalation reference concentration 49 RfD oral reference dose 50 RNA ribonucleic acid 51 SD standard deviation 52 SIFT-MS selected ion flow tube mass spectrometry 53 TLV threshold limit value 54 TWA time-weighted average 55 UF uncertainty factor 56 UF A interspecies uncertainty factor 57 UF H intraspecies uncertainty factor 58 UF L LOAEL to NOAEL uncertainty factor 59 UF S subchronic-to-chronic uncertainty factor 60 UF D database deficiencies uncertainty factor 61 62 63

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1

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# PREAMBLE 3

4 5


### 1. Scope of the IRIS Program 6

Soon after EPA was established in 1970, it was at 7 the forefront of developing risk assessment as a 8 science and applying it in decisions to protect hum an 9 health and the environment. The Clean Air Act, for 10 example, mandates that EPA provide “an ample 11 margin of safety to protect public health”; the Saf e 12 Drinking Water Act, that “no adverse effects on the 13 health of persons may reasonably be anticipated to 14 occur, allowing an adequate margin of safety.” 15 Accordingly, EPA relies on health assessments to 16 identify adverse effects and exposure levels below 17 which these effects are not anticipated to occur. 18 IRIS assessments critically review the publicly 19 available studies to identify adverse health effect s of 20 chemicals and to characterize exposure-response 21 relationships. Exceptions are chemicals currently u sed 22 exclusively as pesticides, ionizing and non-ionizin g 23 radiation, and criteria air pollutants listed under 24 section 108 of the Clean Air Act (carbon monoxide, 25 lead, nitrogen oxides, ozone, particulate matter, a nd 26 sulfur oxides; EPA evaluates these in Integrated 27 Science Assessments). An assessment may cover a 28 single chemical, a group of structurally or 29 toxicologically related chemicals, or a complex 30 mixture. 31 Once a year, the IRIS Program asks EPA 32 programs and regions, other federal agencies, state 33 governments, and the general public to nominate 34 chemicals and mixtures for future assessment or 35 reassessment. These agents may be found in air, wat er, 36 soil, or sediment. Selection is based on program an d 37 regional office priorities and on availability of 38 adequate information to evaluate the potential for 39 adverse effects. IRIS can assess other agents as an 40 urgent public health need arises. IRIS also reasses ses 41 agents as significant new data are published. 42



### 2. Process for developing and peer43 

### reviewing IRIS assessments 44
The process for developing IRIS assessments 45 (revised in May 2009) involves systematic review of 46 the pertinent studies, opportunities for public inp ut, 47 and multiple levels of scientific review. EPA revis es 48 draft assessments after each review, and external d rafts 49 and comments become part of the public record (U.S. 50 EPA, 2009). 51 Step 1. Development of a draft Toxicological 52 Review (usually about 11-1/2 months duration). 53 The draft assessment considers all pertinent 54 publicly available studies and applies consistent 55 criteria to evaluate the studies, identify health 56 effects, weigh the evidence of causation for each 57 effect, identify mechanistic events and pathways, 58 and derive toxicity values. 59
Step 2. Internal review by scientists in EPA 60 programs and regions (2 months). The draft 61 assessment is revised to address comments from 62 within EPA. 63
Step 3. Interagency science consultation with other 64 federal agencies and White House offices (1-1/2 65 months). The draft assessment is revised to 66 address the interagency comments. The science 67 consultation draft, interagency comments, and 68 EPA’s response to major comments become part 69 of the public record. 70
Step 4. External peer review, after public review 71 and comment (3-1/2 months or more, depending 72 on the review process). EPA releases the draft 73 assessment for public review and comment, 74 followed by external peer review. The peer review 75 meeting is open to the public and includes time 76 for oral public comments. The peer reviewers also 77 receive the written public comments. The peer 78 reviewers assess whether the evidence has been 79 assembled and evaluated according to guidelines 80 and whether the conclusions are justified by the 81 evidence. The peer review draft, peer review 82 report, and written public comments become part 83 of the public record. 84
Step 5. Revision of draft Toxicological Review and 85 development of draft IRIS summary 86 (2 months). The draft assessment is revised to 87 reflect the peer review comments, public 88 comments, and newly available studies. The 89 disposition of peer review comments and public 90 comments becomes part of the public record. 91
Step 6. Final EPA review and interagency science 92 discussion with other federal agencies and 93 White House offices (1-1/2 months). The draft 94 assessment and summary are revised to address 95 EPA and interagency comments. The science 96 discussion draft, written interagency comments, 97 and EPA’s response to major comments become 98 part of the public record. 99

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Step 7. Completion and posting (1 month). The 1 Toxicological Review and IRIS summary are 2 posted on the IRIS website (http://www.epa.gov/ 3 iris/). 4

5 The remainder of this Preamble addresses step 1, 6 the development of a draft Toxicological Review. IR IS 7 assessments follow standard practices of evidence 8 evaluation and peer review, many of which are 9 discussed in EPA guidelines (U.S. EPA, 1986a, 10 1986b, 1991, 1996, 1998, 2000a, 2005a, 2005b, 11 2006a) and other descriptions of “best practices” ( U.S. 12 EPA, 1994, 2000b, 2002, 2006b, 2011a). Transparent 13 application of scientific judgment is of paramount 14 importance. To provide a harmonized approach across 15 IRIS assessments, this Preamble summarizes concepts 16 from these guidelines and emphasizes principles of 17 general applicability. 18



### 3. Identifying and selecting pertinent 19 

### studies 20
3.1 Identifying studies 21
Before beginning an assessment, EPA conducts a 22 comprehensive search of the primary scientific 23 literature. The literature search follows standard 24 practices and includes the PubMed and ToxNet 25 databases of the National Library of Medicine and 26 other databases listed in EPA’s HERO system (Health 27 and Environmental Research Online, http:// 28 hero.epa.gov/). Each assessment specifies the searc h 29 strategies, keywords, and cut-off dates of its lite rature 30 searches. EPA posts the results of the literature s earch 31 on the IRIS website and requests information from t he 32 public on additional studies and ongoing research. 33 Each assessment also considers studies received 34 through the IRIS Submission Desk and studies 35 (typically unpublished) submitted to EPA under the 36 Toxic Substances Control Act. If a study that may b e 37 critical to the conclusions of the assessment has n ot 38 been peer-reviewed, EPA will have it peer-reviewed. 39 EPA also examines the toxicokinetics of the agent 40 to identify other chemicals (for example, major 41 metabolites of the agent) to include in the assessm ent 42 if adequate information is available, in order to m ore 43 fully explain the toxicity of the agent and to sugg est 44 dose metrics for subsequent modeling. 45 In assessments of chemical mixtures, mixture 46 studies are preferred for their ability to reflect 47 interactions among components (U.S. EPA, 1986a, 48 2000a). The literature search seeks, in decreasing 49 order of preference: 50
– Studies of the mixture being assessed. 51
– Studies of a sufficiently similar mixture. In 52 evaluating similarity, the assessment considers the 53 alteration of mixtures in the environment through 54 partitioning and transformation. 55 – Studies of individual chemical components of the 56 mixture, if there are not adequate studies of 57 sufficiently similar mixtures. 58 3.2 Selecting pertinent epidemiologic studies 59
Study design is the key consideration for selecting 60 pertinent epidemiologic studies from the results of the 61 literature search. 62
– Cohort studies and case-control studies provide 63 the strongest epidemiologic evidence, as they 64 collect information about individual exposures 65 and disease. 66
– Cross-sectional studies provide useful evidence i f 67 they relate exposures and disease at the individual 68 level and it is clear that exposure preceded the 69 onset of disease. 70
– Ecologic studies (geographic correlation studies) 71 relate exposures and disease by geographic area. 72 They can provide strong evidence if there are 73 large exposure contrasts between geographic 74 areas, relatively little exposure variation within 75 study areas, and population migration is limited. 76
– Case reports of high or accidental exposure lack 77 definition of the population at risk and the 78 expected number of cases. They can provide 79 information about a rare disease or about the 80 relevance of analogous results in animals. 81
The assessment briefly reviews ecologic studies 82 and case reports but includes details only if they 83 suggest effects not identified by other epidemiolog ic 84 studies. 85 3.3 Selecting pertinent experimental studies 86
Exposure route is a key design consideration for 87 selecting pertinent experimental studies from the 88 results of the literature search. 89
– Studies of oral, inhalation, or dermal exposure 90 involve passage through an absorption barrier and 91 are considered most pertinent to human 92 environmental exposure. 93
– Injection or implantation studies are often 94 considered less pertinent but may provide 95 valuable toxicokinetic or mechanistic information. 96 They also may be useful for identifying effects in 97 animals if deposition or absorption is problematic 98 (for example, for particles and fibers). 99
Exposure duration is also a key design 100 consideration for selecting pertinent experimental 101 studies. 102
– Studies of effects from chronic exposure are most 103 pertinent to lifetime human exposure. 104
– Studies of effects from subchronic exposure are 105 pertinent but less preferred than studies of chroni c 106
exposure. 107

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– Short-term and acute studies are less pertinent b ut 1 are useful for obtaining toxicokinetic or 2 mechanistic information. The assessment reviews 3 short-term and acute studies if they suggest 4 distribution or effects at a site not identified by 5 longer-term studies. 6
– For developmental toxicity and reproductive 7 toxicity, irreversible effects may result from a 8 brief exposure during a critical period of 9 development. Accordingly, specialized study 10 designs are used for these effects (U.S. EPA, 11 1991, 1996, 1998). 12



### 4. Evaluating the quality of individual 13 

### studies 14
4.1 Evaluating the quality of epidemiologic 15 studies 16
The assessment evaluates design and 17 methodologic aspects that can increase or decrease the 18 weight given to each epidemiologic study in the 19 overall evaluation (U.S. EPA, 1991, 1994, 1996a, 20 1998, 2005a): 21
– Documentation of study design, methods, 22 population characteristics, and results. 23
– Definition and selection of the study and 24 comparison populations. 25
– Ascertainment of exposure and the potential for 26 misclassification. 27

– Ascertainment of disease or effect and the 28 potential for misclassification. 29
– Duration of exposure and follow-up and adequacy 30 for assessing the occurrence of effects, including 31 latent effects. 32
– Characterization of exposure during critical 33 periods for the development of effects. 34
– Sample size and statistical power to detect 35 anticipated effects. 36
– Participation rates and the resulting potential f or 37 selection bias. 38
– Potential confounding and other sources of bias 39 are identified and addressed in the study design or 40 in the analysis of results. The basis for 41 consideration of confounding is a reasonable 42 expectation that the confounder is prevalent in the 43 population and is related to both exposure and 44

outcome. 45
For developmental toxicity, reproductive toxicity, 46 neurotoxicity, and cancer there is further guidance on 47 the nuances of evaluating epidemiologic studies of 48 these effects (U.S. EPA, 1991, 1996, 1998, 2005a). 49 4.2 Evaluating the quality of experimental 50 studies 51
The assessment evaluates design and 52 methodologic aspects that can increase or decrease the 53 weight given to each experimental study in the over all 54 evaluation (U.S. EPA, 1991, 1994, 1996, 1998, 55 2005a): 56
– Documentation of study design, animals or study 57 population, methods, basic data, and results. 58
– Relevance of the animal model or study 59 population and the experimental methods. 60

– Characterization of the nature and extent of 61 impurities and contaminants of the administered 62 chemical or mixture. 63
– Characterization of dose and dosing regimen 64 (including age at exposure) and their adequacy to 65 elicit adverse effects, including latent effects. 66
– Sample sizes and statistical power to detect dose

67 related differences or trends. 68
– Ascertainment of survival, vital signs, disease o r 69 effects, and cause of death. 70

– Control of other variables that could influence t he 71 occurrence of effects. 72

The assessment uses statistical tests to evaluate 73 whether the observations may be due to chance. The 74 standard for determining statistical significance o f a 75 response is a trend test or comparison of outcomes in 76 the exposed groups against those of concurrent 77 controls. In some situations, examination of histor ical 78 control data from the same laboratory within a few 79 years of the study may improve the analysis. For an 80 uncommon effect that is not statistically significa nt 81 compared with concurrent controls, historical contr ols 82 may show that the effect is unlikely to be due to 83 chance. For a response that appears significant aga inst 84 a concurrent control response that is unusual, hist orical 85 controls may offer a different interpretation (U.S. 86 EPA, 2005a). 87 For developmental toxicity, reproductive toxicity, 88 neurotoxicity, and cancer there is further guidance on 89 the nuances of evaluating experimental studies of 90 these effects (U.S. EPA, 1991, 1996, 1998, 2005a). In 91 multi-generation studies, agents that produce 92 developmental effects at doses that are not toxic t o the 93 maternal animal are of special concern. Effects tha t 94 occur at doses associated with mild maternal toxici ty 95 are not assumed to result only from maternal toxici ty. 96 Moreover, maternal effects may be reversible, while 97 effects on the offspring may be permanent (U.S. EPA

, 98 1991, 1998). 99
4.3 Reporting study results 100
The assessment uses evidence tables to report 101 details of the design and key results of pertinent 102

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studies. There may be separate tables for each site of 1 toxicity or type of study. 2 If a large number of studies observe the same 3 effect, the assessment considers the study 4 characteristics in this section to identify the str ongest 5 studies or types of study. The tables report detail s 6 from these studies, and the assessment explains the 7 reasons for not reporting details of other studies or 8 groups of studies that do not add new information. 9 Supplemental material provides references to all 10 studies considered, including those not summarized in 11 the tables. 12 The assessment discusses strengths and 13 limitations that affect the interpretation of each study. 14 If the interpretation of a study in the assessment differs 15 from that of the study authors, the assessment 16 discusses the basis for the difference. 17 As a check on the selection and evaluation of 18 pertinent studies, EPA asks peer reviewers to ident ify 19 studies that were not adequately considered. 20



### 5. Weighing the overall evidence of 21 

### each effect 22
5.1 Weighing epidemiologic evidence 23

For each effect, the assessment evaluates the 24 evidence from the epidemiologic studies as a whole to 25 determine the extent to which any observed 26 associations may be causal. Positive, negative, and 27 null results are given weight according to study 28 quality. This evaluation considers aspects of an 29 association that suggest causality, discussed by Hi ll 30 (1965) and elaborated by Rothman and Greenland 31 (1998) (U.S. EPA, 1994, 2002, 2005a; DHHS, 2004). 32
Strength of association: The finding of a large 33 relative risk with narrow confidence intervals 34 strongly suggests that an association is not due to 35 chance, bias, or other factors. Modest relative 36 risks, however, may reflect a small range of 37 exposures, an agent of low potency, an increase in 38 a disease that is common, exposure 39 misclassification, or other sources of bias. 40
Consistency of association: An inference of causality 41 is strengthened if elevated risks are observed in 42 independent studies of different populations and 43 exposure scenarios. Reproducibility of findings 44 constitutes one of the strongest arguments for 45 causality. Discordant results sometimes reflect 46 differences in exposure or in confounding factors. 47
Specificity of association: As originally intended, this 48 refers to one cause associated with one disease. 49 Current understanding that many agents cause 50 multiple diseases and many diseases have 51 multiple causes make this a less informative 52 aspect of causality, unless the effect is rare or 53 unlikely to have multiple causes. 54 Temporal relationship: A causal interpretation 55 requires that exposure precede development of the 56 disease. 57
Biologic gradient (exposure-response relationship): 58 Exposure-response relationships strongly suggest 59 causality. A monotonic increase is not the only 60 pattern consistent with causality. The presence of 61 an exposure-response gradient also weighs against 62 bias and confounding as the source of an 63 association. 64
Biologic plausibility: An inference of causality is 65 strengthened by data demonstrating plausible 66 biologic mechanisms, if available. 67

Coherence: An inference of causality is strengthened 68 by supportive results from animal experiments, 69 toxicokinetic studies, and short-term tests. 70 Coherence may also be found in other lines of 71 evidence, such as changing disease patterns in the 72 population. 73
“Natural experiments”: A change in exposure that 74 brings about a change in disease frequency 75 provides strong evidence of causality. 76
Analogy: Information on structural analogues or on 77 chemicals that induce similar mechanistic events 78 can provide insight into causality. 79

These considerations are consistent with 80 contemporary guidelines that evaluate the quality a nd 81 weight of evidence. Confidence is increased if the 82 magnitude of effect is large, if there is evidence of an 83 exposure-response relationship, or if an associatio n 84 was observed and the plausible biases would tend to 85 decrease the magnitude of the reported effect. 86 Confidence is decreased for study limitations, 87 inconsistency of results, indirectness of evidence, 88 imprecision, or reporting bias (Guyatt et al., 2008 a,b). 89 To make clear how much the epidemiologic 90 evidence contributes to the overall weight of the 91 evidence, the assessment may choose a descriptor su ch 92

as sufficient evidence

, suggestive evidence

, inadequate 93 evidence , or evidence suggestive of no causal 94 relationship to characterize the epidemiologic 95 evidence of each effect (DHHS, 2004). 96
5.2 Weighing experimental evidence 97

For each effect, the assessment evaluates the 98 evidence from the animal experiments as a whole to 99 determine the extent to which they indicate a poten tial 100 for effects in humans. Consistent results across va rious 101 species and strains increase confidence that simila r 102 results would occur in humans. Although causality i s 103 not at issue in controlled experiments, several con cepts 104 discussed by Hill (1965) affect the weight of 105 experimental results: consistency of response, dose

106 response relationships, strength of response, biolo gic 107 plausibility, and coherence (U.S. EPA, 1994, 2002, 108 2005a). 109

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In weighing evidence from multiple experiments, 1 EPA (2005a) distinguishes 2
Conflicting evidence (that is, mixed positive and 3 negative results in the same sex and strain using a 4 similar study protocol) from 5
Differing results (that is, positive results and negative 6 results are in different sexes or strains or use 7 different study protocols). 8
Negative or null results do not invalidate positive 9 results in a different experimental system. EPA 10 regards all as valid observations and looks to 11 mechanistic information, if available, to reconcile 12 differing results. 13 It is well established that there are critical peri ods 14 for some developmental and reproductive effects. 15 Accordingly, the assessment determines whether 16 critical periods have been adequately investigated 17 (U.S. EPA, 1991, 1996, 1998, 2005a, 2005b). 18 Similarly, the assessment determines whether the 19 database is adequate to evaluate other critical sit es and 20 effects. 21 5.3 Characterizing modes of action 22

For each effect, the assessment discusses the 23 available information on its modes of action and 24 associated key events ( key events being empirically 25 observable, necessary precursor steps or biologic 26 markers of such steps; mode of action being a series of 27 key events involving interaction with cells, operat ional 28 and anatomic changes, and resulting in disease). 29 Pertinent information may also come from studies of 30 metabolites or of compounds that are structurally 31 similar or that act through similar mechanisms. The 32 assessment addresses several questions about each 33 hypothesized mode of action (U.S. EPA, 2005a). 34

(a) Is the hypothesized mode of action sufficiently 35 supported in test animals? Strong support for a 36 key event being necessary to a mode of action can 37 come from experimental challenge to the 38 hypothesized mode of action, where suppressing a 39 key event suppresses the disease. Support for a 40 mode of action is meaningfully strengthened by 41 consistent results in different experimental 42 models, but not by replicate experiments in the 43 same model. The assessment may consider 44 various aspects of causality in addressing this 45 question. 46
(b) Is the hypothesized mode of action relevant to 47 humans? The assessment reviews the key events 48 to identify critical similarities and differences 49 between the test animals and humans. Site 50 concordance is not assumed between animals and 51 humans, though it may hold for certain modes of 52 action. Information suggesting quantitative 53 differences is considered in dose-response 54 analyses but is not used to determine relevance. 55 Similarly, anticipated levels of human exposure 56 are not used to determine relevance. 57

(c) Which populations or life-stages can be 58 particularly susceptible to the hypothesized 59 mode of action? The assessment reviews the key 60 events to identify populations and life-stages that 61 might be susceptible to their occurrence. 62 Quantitative differences may result in separate 63 toxicity values for susceptible populations or life

64 stages. 65

The assessment discusses the likelihood that an 66 agent operates through multiple modes of action. An 67 uneven level of support for different modes of acti on 68 can reflect disproportionate resources spent 69 investigating them (U.S. EPA, 2005a). It should be 70 noted that in clinical reviews, the quality of evid ence 71 may be reduced if evidence is limited to studies 72 funded by one interested sector (Guyatt et al., 200 8b). 73 Studies of genetic toxicity are often available, an d 74 the assessment evaluates the evidence of a mutageni c 75 mode of action. 76
– Demonstration of gene mutations, chromosome 77 aberrations, or aneuploidy in humans or 78 experimental mammals ( in vivo ) provides the 79 strongest evidence. 80
– This is followed by positive results in lower 81 organisms or in cultured cells ( in vitro ) or for 82 other genetic events. 83
– Negative results carry less weight, partly becaus e 84 they cannot exclude the possibility of effects in 85 other tissues (IARC, 2006). 86
For germ-cell mutagenicity, EPA has defined 87 categories of evidence, ranging from positive resul ts of 88 human germ-cell mutagenicity to negative results fo r 89 all effects of concern (U.S. EPA, 1986b). 90 5.4 Characterizing the overall weight of the 91 evidence 92
After weighing the epidemiologic and 93 experimental studies pertinent to each effect, the 94 assessment may select a standard descriptor to 95 characterize the overall weight of the evidence. Fo r 96 example, the following standard descriptors combine 97 epidemiologic, experimental, and mechanistic 98 evidence of carcinogenicity (U.S. EPA, 2005a). 99
Carcinogenic to humans: There is convincing 100 epidemiologic evidence of a causal association 101 (that is, there is reasonable confidence that the 102 association cannot be fully explained by chance, 103 bias, or confounding); or there is strong human 104 evidence of cancer or its precursors, extensive 105 animal evidence, identification of key precursor 106 events in animals, and strong evidence that they 107 are anticipated to occur in humans. 108

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Likely to be carcinogenic to humans: The evidence 1 demonstrates a potential hazard to humans but 2 does not meet the criteria for carcinogenic . There 3 may be a plausible association in humans, 4 multiple positive results in animals, or a 5 combination of human, animal, or other 6 experimental data. 7
Suggestive evidence of carcinogenic potential: The 8 data raise concern for effects in humans but are 9 not sufficient for a stronger conclusion. This 10 descriptor covers a range of evidence, from a 11 positive result in the only available study to a 12 single positive result in an extensive database tha t 13 includes negative results in other species. 14
Inadequate information to assess carcinogenic 15 potential: No other descriptors apply. Conflicting 16 evidence can be classified as inadequate 17 information if all positive results are opposed by 18 negative studies of equal quality in the same sex 19 and strain. Differing results , however, can be 20 classified as suggestive evidence or as likely to be 21 carcinogenic

. 22
Not likely to be carcinogenic to humans: There are 23 robust data for concluding that there is no basis 24 for concern. There may be no effects in both sexes 25 of at least two appropriate animal species; positiv e 26 animal results and strong, consistent evidence that 27 each mode of action in animals does not operate 28 in humans; or convincing evidence that effects are 29 not likely by a particular exposure route or below 30 a defined dose. 31


### 6. Selecting studies for derivation of 32 

### toxicity values 33
For each effect associated with an agent, the 34 assessment derives toxicity values if there are sui table 35 epidemiologic or experimental data. The derivation of 36 toxicity values may be linked to the weight-of37 evidence descriptor. For example, EPA typically 38 derives toxicity values for agents classified as 39 carcinogenic to humans or likely to be carcinogenic

, 40 but not for agents with inadequate information or that 41

are not likely to be carcinogenic (U.S. EPA, 2005a). 42 Dose-response analysis requires quantitative 43 measures of dose and response. Then, other factors 44 being equal (U.S. EPA, 1994, 2005a): 45
– Epidemiologic studies are preferred over animal 46 studies, if quantitative measures of exposure are 47 available and effects can be attributed to the 48 agent. 49
– Among experimental animal models, those that 50 respond most like humans are preferred, if the 51 comparability of response can be determined. 52
– Studies by a route of human environmental 53 exposure are preferred, although a validated 54 toxicokinetic model can be used to extrapolate 55 across exposure routes. 56
– Studies of longer exposure duration and follow-up 57 are preferred, to minimize uncertainty about 58 whether effects are representative of lifetime 59
exposure. 60
– Studies with multiple exposure levels are 61 preferred for their ability to provide information 62 about the shape of the exposure-response curve. 63
– Studies that show an exposure-response gradient 64 are preferred, as long as lack of a monotonic 65 relationship at higher exposure levels can be 66 satisfactorily explained by factors such as 67 competing toxicity, saturation of absorption or 68 metabolism, misclassification bias, or selection 69 bias. 70
– Among studies that show an exposure-response 71 gradient, those with adequate power to detect 72 effects at lower exposure levels are preferred, to 73 minimize the extent of extrapolation to levels 74 found in the environment. 75
If a large number of studies are suitable for dose76 response analysis, the assessment considers the stu dy 77 characteristics in this section to focus on the mos t 78 informative data. The assessment explains the reaso ns 79 for not analyzing other groups of studies. As a che ck 80 on the selection of studies for dose-response analy sis, 81 EPA asks peer reviewers to identify studies that we re 82 not adequately considered. 83



### 7. Deriving toxicity values 84
7.1 General framework for dose-response 85 analysis 86
EPA uses a two-step approach that distinguishes 87 analysis of the observed dose-response data from 88 inferences about lower doses (U.S. EPA, 2005a). 89 Within the observed range, the preferred approach 90 is to use modeling to incorporate a wide range of d ata 91 into the analysis. The modeling yields a point of 92 departure (an exposure level near the lower end of the 93 observed range, without significant extrapolation t o 94 lower doses) (sections 7.2-7.3). 95 Extrapolation to lower doses considers what is 96 known about the modes of action for each effect 97 (sections 7.4-7.5). An alternative to low-dose 98 extrapolation is derivation of reference values, wh ich 99 are calculated by adjusting the point of departure by 100 factors that account for several sources of uncerta inty 101 and variability (section 7.6). 102 Increasingly, EPA is making use of multiple data 103 sets or combining multiple responses in deriving 104 toxicity values. EPA also considers multiple dose105 response approaches when they can be supported by 106 robust data. 107

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7.2 Modeling dose 1
The preferred approach for analysis of dose is 2 toxicokinetic modeling because of its ability to 3 incorporate a wide range of data. The preferred dos e 4 metric would refer to the active agent at the site of its 5 biologic effect or to a close, reliable surrogate 6 measure. The active agent may be the administered 7 chemical or a metabolite. Confidence in the use of a 8 toxicokinetic model depends on the robustness of it s 9 validation process and on the results of sensitivit y 10 analyses (U.S. EPA, 1994, 2005a, 2006b). 11 Because toxicokinetic modeling can require many 12 parameters and more data than are typically availab le, 13 EPA has developed standard approaches that can be 14 applied to typical data sets. These standard approa ches 15 also facilitate comparison across exposure patterns and 16 species. 17
– Intermittent study exposures are standardized to a 18 daily average over the duration of exposure. For 19 chronic effects, daily exposures are averaged over 20 the lifespan. Exposures during a critical period, 21 however, are not averaged over a longer duration 22 (U.S. EPA, 1991, 1996, 1998, 2005a). 23
– Doses are standardized to equivalent human terms 24 to facilitate comparison of results from different 25 species. 26
– Oral doses are scaled allometrically using 27 mg/kg 3/4 -d as the equivalent dose metric 28 across species. As allometric scaling is 29 typically based on adult body weight, it is not 30 used for early-life exposure or for 31 developmental effects (U.S. EPA, 2005a, 32 2011a). 33
– Inhalation exposures are scaled using 34 dosimetry models that apply species-specific 35 physiologic and anatomic factors and 36 consider whether the effect occurs at the site 37 of first contact or after systemic circulation 38 (U.S. EPA, 1994). 39

It can be informative to convert doses across 40 exposure routes. If this is done, the assessment 41 describes the underlying data, algorithms, and 42 assumptions (U.S. EPA, 2005a). 43
7.3 Modeling response in the range of 44 observation 45
Toxicodynamic (“biologically based”) modeling 46 can incorporate data on biologic processes leading to a 47 disease. Such models require sufficient data to 48 ascertain a mode of action and to quantitatively 49 support model parameters associated with its key 50 events. Because different models may provide 51 equivalent fits to the observed data but diverge 52 substantially at lower doses, critical biologic 53 parameters should be measured from laboratory 54 studies, not by model fitting. Confidence in the us e of 55 a toxicodynamic model depends on the robustness of 56 its validation process and on the results of sensit ivity 57 analyses. Peer review of the scientific basis and 58 performance of a model is essential (U.S. EPA, 59 2005a). 60 Because toxicodynamic modeling can require 61 many parameters and more knowledge and data than 62 are typically available, EPA has developed a standa rd 63 set of empirical (“curve-fitting”) models (http:// 64 www.epa.gov/ncea/bmds/) that can be applied to 65 typical data sets, including those that are nonline ar. 66 EPA has also developed guidance on modeling dose67 response data, assessing model fit, selecting suita ble 68 models, and reporting modeling results (U.S. EPA, 69 2000b). Additional judgment or alternative analyses 70 are used when the procedure fails to yield reliable 71 results, for example, if the fit is poor, modeling may 72 be restricted to the lower doses, especially if the re is 73 competing toxicity at higher doses (U.S. EPA, 2005a ). 74 Modeling is used to derive a point of departure 75 (U.S. EPA, 2000b, 2005a). (See section 7.6 for 76 alternatives if a point of departure cannot be deri ved 77 by modeling.) 78
– For dichotomous responses, the point of departure 79 is the 95% lower bound on the dose associated 80 with a small increase of a biologically significant 81 effect. 82
– If linear extrapolation to lower doses will be 83 used, a standard value near the low end of the 84 observable range is used (10% response for 85 animal data, 1% for epidemiologic data, 86 depending on the observed response rates). 87
– If nonlinear extrapolation will be used, both 88 statistical and biologic factors are considered 89 (10% response for minimally adverse effects, 90 5% or lower for more severe effects or for 91 developmental toxicity data on individual 92 offspring). 93
– For continuous responses, the point of departure is 94 ideally a level where the effect is considered 95 minimally adverse. In the absence of such 96 definition, both statistical and biologic factors a re 97 considered in selecting a response level. 98
7.4 Extrapolating to lower doses 99
The purpose of extrapolating to lower doses is to 100 estimate responses at exposures below the observed 101 data. Low-dose extrapolation is typically used for 102 known and likely carcinogens. Low-dose extrapolatio n 103 considers what is known about modes of action (U.S. 104 EPA, 2005a). 105
(1) If a biologically based model has been develope d 106 and validated for the agent, extrapolation may use 107 the fitted model beyond the observed range if 108 significant model uncertainty can be ruled out 109 with reasonable confidence. Below the range 110

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where confidence bounds on the predictions are 1 reasonably precise, extrapolation may continue 2 using a linear model. 3
(2) Linear extrapolation is used if the dose-respon se 4 curve is expected to have a linear component 5 below the point of departure. This includes: 6
– Agents or their metabolites that are DNA7 reactive and have direct mutagenic activity. 8
– Agents or their metabolites for which human 9 exposures or body burdens are near doses 10 associated with key events leading to an 11 effect. 12
Linear extrapolation is also used if the evidence is 13 insufficient to establish a mode of action. 14
The result of linear extrapolation is described by 15

an oral slope factor or an inhalation unit risk

, 16 which is the slope of the dose-response curve at 17 lower doses. 18
(3) Nonlinear extrapolation is used if there are 19 sufficient data to ascertain the mode of action and 20 to conclude that it is not linear at lower doses, a nd 21 the agent does not demonstrate mutagenic or other 22 activity consistent with linearity at lower doses. If 23 nonlinear extrapolation is appropriate but no 24 model is developed, a default is to calculate 25 reference values. 26
If linear extrapolation is used, the assessment 27 develops a candidate slope factor or unit risk for each 28 suitable data set. These results are arrayed, using 29 common dose metrics, to show the distribution of 30 relative potency across various effects and 31 experimental systems. The assessment then derives a n 32 overall slope factor and an overall unit risk for t he 33 agent, considering the various dose-response analys es, 34 the study preferences discussed in section 6, and t he 35 possibility of basing a more robust result on multi ple 36 data sets. 37 7.5 Considering susceptible populations and 38 life-stages 39
The assessment analyzes the available information 40 on populations and life-stages that may be particul arly 41 susceptible to each effect. A tiered approach is us ed 42 (U.S. EPA, 2005a). 43
(1) If an epidemiologic or experimental study repor ts 44 quantitative results for a susceptible population o r 45 life-stage, these data are analyzed to derive 46 separate toxicity values for susceptible 47 individuals. 48
(2) If data on risk-related parameters allow 49 comparison of the general population and 50 susceptible individuals, these data are used to 51 adjust the general-population toxicity values for 52 application to susceptible individuals. 53 (3) In the absence of chemical-specific data, 54 application of age-dependent adjustment factors is 55 recommended for early-life exposure to suspected 56 carcinogens. There is evidence of early-life 57 susceptibility to various carcinogenic agents, but 58 most epidemiologic studies and cancer bioassays 59 do not include early-life exposure. To address the 60 potential for early-life susceptibility, EPA 61 recommends: 62
– 10-fold adjustment for exposures before age 2 63
years. 64
– 3-fold adjustment for exposures between ages 65 2 and 16 years. 66
These adjustments are generally applied only for a 67 mutagenic mode of action, though early-life 68 susceptibility has been observed for several 69 carcinogens that are not mutagenic (U.S. EPA, 70 2005b). 71
7.6 Reference values and uncertainty factors 72

An oral reference dose or an inhalation reference 73 concentration is an estimate of an exposure (including 74 in susceptible subgroups) that is likely to be with out 75 an appreciable risk of adverse health effects over a 76 lifetime (U.S. EPA, 2002). Reference values are 77 typically calculated for effects other than cancer and 78 for suspected carcinogens if a well characterized m ode 79 of action indicates that a threshold can be based o n 80 prevention of an early key event. Reference values 81 provide no information about risks at exposures abo ve 82 the reference value. 83 The assessment characterizes effects that form the 84 basis for reference values as adverse, considered t o be 85 adverse, or a precursor to an adverse effect. For 86 developmental, reproductive, and neurotoxicity ther e 87 is guidance on adverse effects and their biologic 88 markers (U.S. EPA, 1991, 1996, 1998). 89 To account for uncertainty and variability in the 90 derivation of a lifetime human exposure where effec ts 91 are not anticipated to occur, reference values are 92 calculated by adjusting the point of departure by a 93 series of uncertainty factors . If a point of departure 94 cannot be derived by modeling, a no-observed95 adverse-effect level or a lowest-observed-adverse96 effect level is substituted. The assessment discuss es 97 scientific considerations involving several areas o f 98 variability or uncertainty. 99

Human variation. A factor of 10 is applied to account 100 for variation in susceptibility across the human 101 population and the possibility that the available 102 data may not be representative of individuals who 103 are most susceptible to the effect. This factor is 104 reduced only if the point of departure is derived 105 specifically for susceptible individuals (not for a 106 general population that includes both susceptible 107 and non-susceptible individuals) (U.S. EPA, 1991, 108 1994, 1996, 1998, 2002). 109

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Animal-to-human extrapolation. A factor of 10 is 1 applied if animal results are used to make 2 inferences about humans. This factor is often 3 regarded as comprising toxicokinetics and 4 toxicodynamics in equal parts. Accordingly, if the 5 point of departure is based on toxicokinetic 6 modeling, dosimetry modeling, or allometric 7 scaling across species, a factor of 10 1/2 (rounded 8 to 3) is applied to account for the remaining 9 uncertainty involving toxicodynamic differences. 10 An animal-to-human factor is not applied if a 11 biologically based model adjusts fully for 12 toxicokinetic and toxicodynamic differences and 13 residual uncertainty across species (U.S. EPA, 14 1991, 1994, 1996, 1998, 2002). 15

Adverse-effect level to no-observed-adverse-effect 16 level. If a point of departure is based on a lowest17 observed-adverse-effect level, the assessment 18 must infer a dose where such effects are not 19 expected. This can be a matter of great 20 uncertainty, especially if there is no evidence 21 available at lower doses. A factor of 10 is applied 22 to account for the uncertainty in making this 23 inference. A factor other than 10 may be used, 24 depending on the magnitude and nature of the 25 response and the shape of the dose-response curve 26 (U.S. EPA, 1991, 1994, 1996, 1998, 2002). 27
Subchronic-to-chronic exposure . If a point of 28 departure is based on subchronic studies, the 29 assessment considers whether lifetime exposure 30 would have effects at lower levels. A factor of 10 31 is applied to account for the uncertainty in using 32 subchronic studies to make inferences about 33 lifetime exposure. This factor may also be applied 34 for developmental or reproductive effects if 35 exposure covered less than the full critical period

. 36 A factor other than 10 may be used, depending on 37 the duration of the studies and the nature of the 38 response (U.S. EPA, 1994, 1998, 2002). 39
Incomplete database. If an incomplete database 40 raises concern that further studies might identify a 41 more sensitive effect, organ system, or life-stage, 42 the assessment may apply a database uncertainty 43 factor (U.S. EPA, 1991, 1994, 1996, 1998, 2002). 44 EPA typically follows the suggestion that a factor 45 of 10 be applied if both a prenatal toxicity study 46 and a two-generation reproduction study are 47 missing, and a factor of 10 1/2 if either is missing 48 (U.S. EPA, 2002). 49
In this way, the assessment derives candidate 50 reference values for each suitable data set and eff ect 51 that is plausibly associated with the agent. These 52 results are arrayed, using common dose metrics, to 53 show where effects occur across a range of exposure s 54 (U.S. EPA, 1994). The assessment then selects an 55 overall reference dose and an overall reference 56 concentration for the agent to represent lifetime h uman 57 exposure levels where effects are not anticipated t o 58

occur. 59 The assessment may also report reference values 60 for each effect. This would facilitate subsequent 61 cumulative risk assessments, where it may be 62 important to consider the combined effect of 63 chemicals acting at a common site or operating 64 through common mechanisms (U.S. EPA, 2002). 65
7.7 Confidence and uncertainty in the 66 reference values 67
The assessment selects a standard descriptor to 68 characterize the level of confidence in each refere nce 69 value, based on the likelihood that the value would 70 change with further testing. Confidence in referenc e 71 values is based on quality of the studies used and 72 completeness of the database, with more weight give n 73 to the latter. The level of confidence is increased for 74 reference values based on human data supported by 75 animal data (U.S. EPA, 1994). 76
High confidence: The reference value is not likely to 77 change with further testing, except for 78 mechanistic studies that might affect the 79 interpretation of prior test results. 80

Medium confidence: This is a matter of judgment, 81 between high and low confidence. 82

Low confidence: The reference value is especially 83 vulnerable to change with further testing. 84
These criteria are consistent with contemporary 85 guidelines that evaluate the quality of evidence. T hese 86 also focus on whether further research would be lik ely 87 to change confidence in the estimate of effect (Guy att 88 et al 2008a). 89 All assessments discuss the significant 90 uncertainties encountered in the analysis. EPA 91 provides guidance on characterization of uncertaint y 92 (U.S. EPA, 2005a). For example, the discussion 93 distinguishes model uncertainty (lack of knowledge 94 about the most appropriate experimental or analytic 95 model), parameter uncertainty (lack of knowledge 96 about the parameters of a model), and human variati on 97 (interpersonal differences in biologic susceptibili ty or 98 in exposures that modify the effects of the agent). 99 100 For other general information about this assessment or 101 other questions relating to IRIS, the reader is ref erred 102 to EPA’s IRIS Hotline at (202) 566-1676 (phone), 103 (202) 566-1749 (fax), or hotline.iris@epa.gov (emai l 104 address). 105 106

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1 

# AUTHORS | CONTRIBUTORS | REVIEWERS 2

3 4
5 

### Authors

Audrey Galizia, Dr. PH (Chemical Manager)

James Ball, Ph.D. Christopher Brinkerhoff, Ph.D. Keith Salazar, Ph.D. U.S. EPA Office of Research and Development National Center for Environmental Assessment Edison, New Jersey

U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC
6
7 

### Contributors

Louis D’Amico, Ph.D. Christopher Sheth, Ph.D. U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC

8 

### Technical Support

Maureen Johnson U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC
9
10 

### Contractor Support

SRC, Inc., Chemical and Biological Center, Syracuse , NY Amber Bacon Fernando Llados Julie Stickney

11 

### Executive Direction

Vincent Cogliano, Ph.D. Samantha Jones, Ph.D. Susan Rieth, MPH U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC
12 

### EPA Internal Reviewers

Ted Berner, M.S Marian Rutigliano, MD John Whalan

Amanda S. Persad, Ph.D. Paul Reinhart, Ph.D. U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC

U.S. EPA Office of Research and Development National Center for Environmental Assessment Research Triangle Park, NC
13

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## Reviewers 1



### This assessment was provided for review to scientis ts in EPA’s Program and Regional Offices. 2 

### Comments were submitted by the Programs and Regions listed below. 3
4 Rebecca Dzubow

Joyce Donohue Marian Olsen Office of Children’s Health Protection, Washington DC Office of Water, Washington, DC Region 2, New York, New York

5 

### This assessment was provided for review to other fe deral agencies and White House offices. The 6 

### federal agencies and White House offices that comme nted are listed below. Comments 7 

### submitted by the agencies listed below are availabl e on the IRIS website. 8
9 Agency for Toxic Substances and Disease Registry, P ublic Health Service, Department of

Health & Human Services Office of Public Health Service, Food Safety Inspec tion Service, U.S. Department of Agriculture
10 

### A public listening session was held by EPA on [mont h] [date], [year]. Attendees external to the 11 

### EPA are listed below. 12
13 NAME

NAME NAME NAME Affiliation Affiliation Affiliation Affiliation
14 

### This assessment was released for public comment on [month] [date], 2012; the public comment 15 

### period ended on [month] [date], [year]. Comments w ere received from the following entities. 16
17 NAME

NAME NAME NAME Affiliation, Location Affiliation, Location Affiliation, Location Affiliation, Location
18 

### This assessment was peer-reviewed by independent ex pert scientists external to EPA (specify 19 

### SAB or NAS panel) and a peer review meeting was hel d on [month] [date], [year]. The external 20 

### peer review comments are available on the IRIS webs ite. EPA’s response to the external peer 21 

### review and public comments is included in Appendix C and is also available on the IRIS 22 

### website. 23
24 NAME

NAME NAME NAME Affiliation, Location Affiliation, Location Affiliation, Location Affiliation, Location
25
26

27

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1 

# PREFACE 2

3

4 

### The purpose of this Toxicological Review is to prov ide scientific support and rationale 5 

### for the hazard and dose-response assessment in IRIS pertaining to exposure to ammonia. This 6 

### document presents background information and justif ication for the Integrated Risk Information 7 

### System (IRIS) Summary of the hazard and dose-respon se assessment of ammonia. The 8 

### appendices to this document include information add ressing chemical and physical properties, 9 

### ammonium salts, toxicokinetics, toxicity study summ aries, and external peer review, and are 10 

### included in a separate volume: the Supplemental Information for the Toxicological Revi ew of 11


### Ammonia

. 12 

### The Toxicological Review of Ammonia is an update of a previous IRIS assessment for 13 

### ammonia posted to the IRIS database in 1991. The p revious assessment included an inhalation 14 

### RfC only. A reassessment of ammonia was conducted because of concerns related to ammonia 15 

### emissions generated from its use in selective catal ytic reduction-based diesel engine 16 

### aftertreatment technology to reduce nitrogen oxide (NOx) to N2 gas and the presence of 17


### ammonia at hazardous waste National Priorities List (NPL) sites. Ammonia is found in over 8% 18 

### of the hazardous waste NPL sites (ATSDR, 2004). 19 

### Portions of this Toxicological Review were develope d under a Memorandum of 20 

### Understanding with the Agency for Toxic Substances and Disease Registry (ATSDR) and were 21 

### adapted from the Toxicological Profile for Ammonia (ATSDR, 2004) as part of a collaborative 22 

### effort in the development of human health toxicolog ical assessments for the purposes of making 23


### more efficient use of available resources and to sh are scientific information. 24

25 

## Background 26 

### Ammonia is a corrosive gas with a very pungent odor (O’Neil et al., 2006). It is highly 27 

### soluble in water (4.82 × 10 5 

### mg/L) and is a weak base (Lide, 2008; Eggeman, 200 1; Dean, 1985). 28 

### When ammonia (NH 3 

### ) is present in water at environmental pH, a pKa of 9.25 indicates that the 29 

### equilibrium will favor the formation of the conjuga te acid, the ammonium ion (NH 4 + 

### ) (Lide, 30 

### 2008). A solution of ammonia in water is sometimes referred to as ammonium hydroxide 31


### because the ammonia and water both ionize to form a mmonium cations and hydroxide anions 32 

### (Eggeman, 2001). Ammonium salts are easily dissolv ed in water and disassociate into the 33 

### ammonium ion and the anion. At physiological pH (7 .4), the equilibrium between NH 3 

### and 34



### NH 4 + 

### favors the formation of NH 4 + 

### . Additional chemical and physical properties info rmation for 35 

### ammonia is provided in Appendix A, Section A.1. 36

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Low levels of ammonia occur naturally in the enviro nment in air, soil, and water. 1 

### Ammonia is a major component of the geochemical nit rogen cycle and is essential for many 2 

### biological processes (Rosswall, 1981). Nitrogen-fi xing bacteria convert atmospheric nitrogen 3 

### into ammonia available for plant uptake (Socolow, 1 999; Rosswall, 1981). Organic nitrogen 4


### released from biota is converted into ammonia throu gh nitrogen mineralization (Rosswall, 1981). 5 

### Ammonia in water and soil is naturally converted in to nitrite and nitrate through the process of 6 

### nitrification (Rosswall, 1981). Ammonia is endogen ously produced in humans and animals, is 7


### an essential mammalian metabolite used in nucleic a cid and protein synthesis, is necessary for 8 

### maintaining acid-base balance, and is an integral p art of nitrogen homeostasis (Nelson and Cox, 9 

### 2008). 10 

### With regard to exogenous exposure, the largest and most significant use of ammonia is 11 

### the agricultural application of fertilizers, which represents about 80-85% of commercially12 

### produced ammonia in the form of urea, ammonium nitr ate, ammonium sulfate, ammonium 13 

### phosphate, and other nitrogen compounds (Eggeman, 2 001). Ammonia is also used as a 14 

### corrosion inhibitor, in the purification of water s upplies, as a component of household cleaners, 15 

### as a refrigerant, as a chemical intermediate in pha rmaceuticals, explosives and other chemicals, 16 

### as a stabilizer in the rubber industry, and as a hy drogen source for the hydrogenation of fats and 17 

### oils. Ammonia (generated from urea injected into t he exhaust stream) is also used in the 18


### reduction of NOx emissions from the exhaust of dies el vehicles and stationary combustion 19 

### sources such as industrial and municipal boilers an d power generators (Eggeman, 2001; HSDB, 20 

### 2009; Johnson et al., 2009). 21

22 

## Scope of the Assessment 23 

### This assessment presents a review of hazard and dos e-response information for ammonia, 24 

### including gaseous ammonia (NH 3 

### ) and ammonia dissolved in water (ammonium hydroxid e, 25



### NH 4 

### OH). Because ammonium salts (e.g., ammonium acetat e, chloride, and sulfate) readily 26 

### dissolve in water through disassociation into the a mmonium ion (NH 4 + 

### ) and the anion, EPA 27


### considered whether or not the literature on ammoniu m salts could inform the toxicity of 28 

### ammonia. The toxicology literature for ammonium sa lts includes several oral toxicity studies of 29


### ammonium chloride and ammonium sulfate. No inhalat ion toxicity studies of ammonium salts 30 

### are available. The toxicity data for ammonium chlo ride and ammonium sulfate demonstrate that 31 

### these two salts present distinctly different toxici ty profiles, suggesting that the anion can 32 

### influence the toxicity of the ammonium compound, an d that the toxicity of the salts cannot 33 

### necessarily be attributed to the cation (i.e., NH 4 + 

### ) only (for detailed ammonium salts information 34 

### see Appendix A, Section A.2 and Table A-2). Accord ingly, information on the toxicity of 35


### ammonium salts was not used to characterize the tox icity of ammonia or ammonium hydroxide 36


### in this assessment. 37

38

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## Other Agency and International Assessments 1 

### Assessments and regulatory limits for ammonia deve loped by other health agencies, 2 

### including the Agency for Toxic Substances and Disea se Registry (ATSDR), the National 3 

### Research Council (NRC), the American Conference of Governmental Industrial Hygienists 4 

### (ACGIH), the National Institute of Occupational Saf ety and Health (NIOSH), and the Food and 5 

### Drug Administration (FDA), are identified in Append ix B of the Supplemental Material. 6

7

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1 

# EXECUTIVE SUMMARY 2

3

4 

## Effects other than cancer observed following oral e xposure 5 

### The oral toxicity database for ammonia is very limi ted. Gastric toxicity is identified as a 6 

### hazard for ammonia based on evidence from case repo rts in humans, two animal studies, and 7


### mechanistic studies. Evidence in humans is limited to case reports of individuals suffering from 8 

### gastrointestinal effects from ingesting household c leaning solutions containing ammonia or 9 

### biting into capsules of ammonia smelling salts. In rats, gastrointestinal effects, characterized as 10 

### increased epithelial cell migration in the mucosa o f the stomach and decreased thickness of the 11 

### gastric mucosa, were reported following subchronic and short-term exposure to ammonia. These 12 

### gastric mucosal effects observed in rats resemble m ucosal changes in human atrophic gastritis; 13 

### indicating this effect is biological plausible and relevant to humans. 14


### Given the limited number of studies available and t he small number of toxicological 15 

### evaluations, there are uncertainties associated wit h the oral database for ammonia and a RfD for 16


### ammonia was not derived

. 17

18 

## Effects other than cancer observed following inhala tion exposure 19 

### Respiratory effects have been identified as a hazar d following inhalation exposure to 20 

### ammonia. Evidence for respiratory toxicity associa ted with exposure to ammonia comes from 21


### studies in humans and animals. Cross-sectional occ upational studies involving chronic exposure 22 

### to ammonia have consistently demonstrated an increa sed prevalence of respiratory effects and 23 

### decreased lung function. Cross-sectional studies o f livestock farmers exposed to ammonia, 24


### controlled human volunteer studies of ammonia inhal ation, and case reports of injury in humans 25 

### with inhalation exposure to ammonia provide additio nal and consistent support for the 26 

### respiratory system as a target of ammonia toxicity. Additionally, respiratory effects were 27 

### observed in several animal species following subchr onic and short-term exposures to ammonia. 28 

### The experimental toxicology literature for ammoni a also provides evidence that inhaled 29 

### ammonia may be associated with toxicity to target o rgans other than the respiratory system, 30 

### including the liver, adrenal gland, kidney, spleen, heart, and immune system. The weight of 31


### evidence for these effects is less robust than for respiratory effects. 32

33

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## Inhalation reference concentration (RfC) for effect s other than cancer 1


## Table ES-1. Reference Concentration 2

3


### Critical Effect Point of Departure* UF Chronic RfC

Lack of decreased lung function and increased respiratory irritation

Occupational epidemiology study

Holness et al., 1989 NOAEL ADJ 

### : 3.1 mg/m 3 

### 10 0.3 mg/m 3



### * Because the POD (NOAEL = 8.8 mg/m 3 

### ) involved workplace exposure conditions, the NOAEL was adjusted for continuous exposure based on 

### the ratio of VE ho 

### (human occupational default minute volume of 10 m 3 

### breathed during an 8-hour workday) to VE h 

### (human ambient default minute volume of 20 m 3 

### breathed during the entire day) and an exposure of 5 days out of 7 days.

4 

### The occupational exposure study of ammonia exposure in workers in a soda ash plant by 5 

### Holness et al. (1989) was identified as the princip al study for RfC derivation . Respiratory 6 

### effects, characterized as increased respiratory irr itation and decreased lung function, observed in 7 

### workers exposed to ammonia were selected as the cri tical effect. In the evaluation of the 8 

### prevalence of increased respiratory irritation and decreased lung function in workers exposed to 9 

### ammonia (Holness et al., 1989), a NOAEL ADJ 

### of 3.1 mg/m 3 

### (adjusted for continuous exposure 10 

### from 8.8 mg/m 3 

### ; see calculation below) was identified based on th e absence of statistically 11 

### significant increases in the prevalence of the resp iratory effects. BMD modeling was not utilized 12


### because ammonia concentrations in the Holness et al . (1989) study were not associated with 13 

### changes in respiratory effects in the study populat ion (i.e., data from Holness et al. could not be 14 

### subjected to dose-response modeling). Thus, the Ho lness et al. (1989) data were analyzed using 15 

### a NOAEL approach and the NOAEL ADJ 

### of 3.1 mg/m 3 

### was used as the POD for RfC 16


### derivation

. 17 

### The RfC was calculated by dividing the POD (i.e., N OAEL ADJ 

### ) by a composite 18 

### uncertainty factor (UF) of 10 to account for potentially susceptible individuals in the absence 19 

### of data evaluating variability of response to inhal ed ammonia in the human population. 20

21


## Confidence in the chronic inhalation RfC 22 

### Study – medium 23


### Database – medium 24


### RfC – medium 25

26


### The overall confidence in the RfC is medium and reflects medium confidence in the 27 

### principal study (adequate design, conduct, and repo rting of the principal study; limited by small 28

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sample size and identification of a NOAEL only) and medium confidence in the database, which 1 

### includes occupational and volunteer studies and stu dies in animals that are mostly of subchronic 2 

### duration. Although there are no studies of develop mental toxicity and studies of reproductive 3 

### and other systemic endpoints are limited, reproduct ive, developmental, and other systemic 4 

### effects are not expected at the RfC because it is w ell documented that ammonia is endogenously 5 

### produced in humans and animals, ammonia concentrati ons in blood are homeostatically 6 

### regulated to remain at low levels, and ammonia conc entrations in air at the POD are not expected 7


### to alter homeostasis. 8

9 

## Evidence for human carcinogenicity 10



### Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 11



### “ inadequate information to assess the carcinogenic p otential ” of ammonia based on the 12 

### absence of ammonia carcinogenicity studies in human s and a single lifetime drinking water study 13


### of ammonia in mice that showed no evidence of carci nogenic potential. There is limited 14 

### evidence that ammonia may act as a cancer promoter based on the findings of H. pylori- induced 15 

### gastric cancer. The available studies of ammonia g enotoxicity are inadequate to characterize the 16 

### genotoxic potential of this compound. A quantitative cancer assessment for ammonia was 17


### not conducted. 18

19 

## Susceptible Populations and Life Stages 20 

### Hyperammonemia is a condition of elevated levels of circulating ammonia that can occur 21


### in individuals with severe diseases of the liver or kidney or with hereditary urea cycle disorders. 22 

### These elevated ammonia levels can predispose an ind ividual to encephalopathy due to the ability 23 

### of ammonia to cross the blood-brain barrier; these effects are especially marked in newborn 24 

### infants. Thus, individuals with disease conditions that lead to hyperammonemia may be more 25 

### susceptible to the effects of ammonia from external sources, but there are no studies that 26 

### specifically support this susceptibility. Studies of the toxicity of ammonia in children or young 27 

### animals compared to other life stages that would su pport an evaluation of childhood 28 

### susceptibility have not been conducted. 29

30 

## Key issues addressed in assessment 31 

### Endogenous ammonia 32 

### Ammonia, which is produced endogenously, has been d etected in the expired air of 33 

### healthy volunteers. Higher and more variable ammon ia concentrations are reported in breath 34 

### exhaled from the mouth or oral cavity (0.09 to 2.1 mg/m 3 

### ). These levels are largely attributed to 35 

### the production of ammonia via bacterial degradation of food protein in the oral cavity or 36 

### gastrointestinal tract, and can be influenced by fa ctors such as diet, oral hygiene, age, and living 37

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conditions (i.e., urban vs. rural setting). In con trast, ammonia concentrations measured in breath 1 

### exhaled from the nose and trachea are lower (0.013– 0.078 mg/m 3 

### ) and more likely reflect levels 2 

### of ammonia circulating in the blood. These levels are lower than the ammonia RfC of 0.3 mg/m 3 3 

### by a factor of approximately fourfold or more. Alt hough the RfC falls within the range of breath 4


### concentrations collected from the mouth or oral cav ity, ammonia in exhaled breath is expected to 5 

### be rapidly diluted in the much larger volume of amb ient air. 6

7

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1 

# LITERATURE SEARCH STRATEGY & STUDY 2 

# EVALUATION FOR HAZARD IDENTIFICATION 3

4 

## Literature Search Strategy and Study Selection 5 

### The literature search strategy employed for ammonia was conducted with the keywords 6 

### listed in Table LS-1. Primary, peer-reviewed liter ature was identified through a literature search 7 

### using the databases listed in Table LS-1. The lite rature search was last conducted on November 8 

### 11, 2011. A data call-in was announced by EPA on D ecember 21, 2007 (U.S. EPA, 2007); no 9 

### submissions in response to the data call-in were re ceived. Other peer-reviewed information, 10 

### including health assessments developed by other hea lth agencies, review articles, and 11 

### independent analyses of the health effects data wer e retrieved and may be included in the 12 

### assessment where appropriate. 13

14 

### Table LS-1. Details of the search strategy employe d for ammonia

Databases Limits Keywords

Pubmed
Toxcenter Toxline Current Contents (2008 & 2010 only) Search constraints: 2003-current b Pre-2003—ATSDR (2004) was used as the source of references published before 2003

Last search: November 11, 2011 Chemical name and synonyms a : ammonia (7664-41-7); ammonium hydroxide (1336-21-6); ammonium; spirit of hartshorn; aquammonia

Other keywords: toxicity (including duration, effects to children and occupational exposure); development; reproduction; teratogenicity; exposure routes; pharmacokinetics; toxicokinetics; metabolism; body fluids; endocrinology; carcinogenicity; genotoxicity; antagonists; inhibitors; respiration; metabolism; breath tests; inhalation; air; breath; exhalation; biological markers; analysis

TSCATS ChemID Chemfinder
CCRIS
HSDB
GENETOX
RTECS 2011
2011
2011
2011
2011
2008
2011 NA
NA
NA
NA
NA
NA
NA

a The initial search conducted in 2008 included ammon ia salts (i.e., ammonium nitrate [6484-52-2], ammon ium fluoride [12125-01-8], ammonium sulfate [7783-20-2], ammoniu m persulfate [7727-54-0], and ammonium chloride [12 125-02-9]) as keywords. Once the determination was made not to i nclude data on ammonium salts in the assessment, up dated searches focused on ammonia and ammonium hydroxide only.
b The search using search terms related to concentrat ions of ammonia in exhaled breath was conducted for the period 1/1/2002–11/11/2011.

15

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Approximately 4,900 references were identified in t he literature search for ammonia 1 

### using the literature search strategy identified in Table LS-1; the references captured in this search 2


### can be found on the EPA’s HERO website. 1 

### From this list, approximately 250 references were 3 

### identified that provided information relevant to th e human health effects of ammonia or 4 

### information on the physical and chemical properties of ammonia. 5 

### The references cited in this document, as well as t hose that were considered but not 6 

### included in the Toxicological Review of Ammonia, ca n be found on the HERO website 7 

### ( http://hero.epa.gov/{chemical} ). This site contains HERO links to lists of refer ences, including 8 

### bibliographic information and abstracts, which were considered for inclusion in the 9 

### Toxicological Review of Ammonia. 10

11 

## Study Evaluation for Hazard Identification 12 

### This document is not intended to be a comprehensive treatise on the chemical or 13 

### toxicological nature of ammonia. In general, the q uality and relevance of health effects studies 14


### were evaluated as outlined in the Preamble to this assessment. In addition, A Review of the 15 

### Reference Dose and Reference Concentration Processe s (U.S. EPA, 2002) and Methods for 16 

### Derivation of Inhalation Reference Concentrations a nd Application of Inhalation Dosimetry 17 

### (U.S. EPA, 1994) were consulted for guidance in eva luating the scientific quality of the available 18


### studies. 19


### The health effects literature for ammonia is not ex tensive; therefore, essentially all of the 20 

### available epidemiology and toxicity studies of ammo nia and ammonium hydroxide were 21 

### considered in the characterization of the potential health hazards associated with ammonia 22 

### exposure. As discussed in the preface, literature on ammonium salts were not included in this 23 

### review because the available data suggest that the anion of the salt can influence the toxicity of 24 

### the ammonium compound. Approximately 100 case repo rts involving acute ammonia exposure 25 

### were identified; because case reports generally pro vide little information that would be useful for 26 

### characterizing chronic health hazard, these studies were only briefly reviewed and citations to 27 

### this literature are provided as supplemental materi als in Appendix A. Human studies that 28 

### provided unreliable measures of exposure (e.g., sel f-reporting) or intentional dosing studies that 29


### raised concerns of ethical conduct were excluded fr om consideration; two human studies fell into 30 

### this category. 31 

### The hazard identification analysis for each health endpoint in Chapter 1 includes a 32 

### synthesis of the relevant health effects literature and an analysis of the weight of the evidence for 33 

### an association between ammonia exposure and the hea lth effects. The available studies 34

1 HERO (Health and Environmental Research On-line) is a database of scientific studies and other referen ces used to develop EPA’s risk assessments aimed at understandi ng the health and environmental effects of pollutan ts and chemicals. It is developed and managed in EPA’s Of fice of Research and Development (ORD) by the Natio nal Center for Environmental Assessment (NCEA). The da tabase includes more than 300,000 scientific articl es from the peer-reviewed literature. New studies are adde d continuously to HERO.

### xxvi DRAFT - DO NOT CITE OR QUOTE

examining health effects of ammonia exposure in hum ans (four cross-sectional occupational 1 

### exposure studies, studies in livestock farmers and stable workers, and acute controlled-exposure 2 

### studies in volunteers) are discussed and evaluated, with specific limitations of individual studies 3


### and of the collection of studies noted. The evalua tion of the effects seen in experimental animal 4


### studies focuses on the available subchronic toxicit y studies and a single reproductive toxicity 5 

### study. Chronic toxicity studies were limited to or al exposure studies that did not adequately 6


### evaluate the noncancer effects of ammonia. 7

8

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1 

# 1. HAZARD IDENTIFICATION 2

3 

## 1.1. Synthesis of Major Toxicological Effects 4 

## 1.1.1. Respiratory Effects 5 

### Respiratory Irritation 6 

### The respiratory system is the primary target of tox icity of inhaled ammonia in humans 7 

### and experimental animals. Symptoms consistent with respiratory irritation were reported in two 8 

### cross-sectional studies of industrial worker popula tions exposed to ammonia (Rahman et al., 9 

### 2007; Ballal et al., 1998) (see Table 1-1). Rahman et al. (2007) 2 

### found a higher prevalence, by 10 

### up to 20%, of respiratory irritation (cough, chest tightness, runny nose, stuffy nose, and 11 

### sneezing) in urea fertilizer factory workers expose d to a mean ammonia concentration of 18.5 12 

### mg/m 3 

### (high-exposure group) for about 16 years compared to a control group (staff workers); the 13 

### prevalences of cough and chest tightness were stati stically significantly elevated in the high14 

### exposure ammonia group compared to the control grou p. Respiratory irritation prevalence 15 

### between the low-exposure group exposed to a mean am monia concentration of 4.9 mg/m 3 

### was 16 

### not statistically significantly different from the control group. Significantly higher relative risks 17 

### for cough, phlegm, wheezing, dyspnea, and bronchial asthma were also observed in workers 18 

### from another cross-sectional study (Ballal et al., 1998) with ammonia exposure concentrations 19 

### higher than the American Conference of Governmental Industrial Hygienists [ACGIH] threshold 20 

### limit value [TLV] of 18 mg/m 3 

### [25 ppm]) compared with workers exposed to levels b elow the 21 

### TLV. Distribution of respiratory irritation effect s by cumulative ammonia concentration (CAC, 22 

### mg/m 3 

### -years) also showed significantly higher relative r isk for these respiratory irritation effect 23 

### among workers with higher CAC (>50 mg/m 3 

### -years) compared to those with a lower CAC (< 50 24 

### mg/m 3 

### -years) (Ballal et al., 1998). Only Ballal et al. (1998) evaluated respiratory endpoints in 25 

### terms of cumulative exposure. 26 

### In a third cross-sectional study of male ammonia-ex posed workers, no differences were 27 

### observed in the prevalence of respiratory irritatio n, eye irritation, or odor detection threshold 28 

### between any of the ammonia-exposed workers and the control group (Holness et al., 1989), 29 

### either as one group or when stratified into three e xposure categories: high = >8.8 mg/m 3



### , 30 

### medium = 4.4–8.8 mg/m 3 

### , or low = <4.4 mg/m 3 

### . Although respiratory irritation prevalence was 31 

### similar across groups, the exposed workers reported that exposure in the plant aggravated some 32

2 Rahman et al. (2007) examined respiratory effects in workers from two plants in a urea fertilizer fac tory. Workers in the urea plant were exposed to higher co ncentrations of ammonia (arithmetic mean ammonia concentration of 18.5 mg/m 3 ) than workers in the ammonia plant (arithmetic mea n ammonia concentration of 4.9 mg/m 3 ). Therefore, the urea plant workers represented t he high-exposure group, and the ammonia plant worke rs represented the low-exposure group.

### 2 DRAFT - DO NOT CITE OR QUOTE

of their reported respiratory symptoms (cough, sput um, chronic bronchitis, wheeze, chest 1 

### tightness, dyspnea, chest pain, rhinitis) (no furth er information provided). Co-exposures to dust 2 

### and inorganic gases such as nitrogen dioxide and su lfur dioxide were possible in these cross3 

### sectional studies; however, except for the low leve ls of nitrogen dioxide identified in the Rahman 4 

### et al. (2007) study, these workplace exposures were not measured or reported. 5 

### Overall, these cross-sectional occupational epidemi ology studies provide consistent 6 

### estimates of the effect level for respiratory irrit ation by ammonia. Rahman et al. (2007) 7 

### observed that exposure to 18.5 mg/m 3 

### ammonia increased the prevalence of respiratory ef fects. 8 

### This is consistent with the observation by Ballal e t al. (1998) that workers in a factory with 9 

### ammonia concentrations exceeding the TLV of 18 mg/m 3 

### had significantly higher relative risks 10 

### for respiratory irritation effects. The prevalence of respiratory effects was not increased 11 

### following occupational exposures at lower workplace concentrations (i.e., >8.8 mg/m 3 

### ammonia 12 

### [Holness et al., 1989] and 4.9 mg/m 3 

### [Rahman et al., 2007]). 13 

### Respiratory irritation, indicated by elevated preva lences of respiratory symptoms, 14 

### including cough, phlegm, wheezing, chest tightness, and eye, nasal and throat irritation, has been 15 

### reported in livestock farmers and stable workers co mpared to controls (Melbostad and Eduard, 16 

### 2001; Preller et al., 1995; Choudat et al., 1994; Z ejda et al., 1994; Crook et al., 1991; Heederik et 17 

### al., 1990). Additionally, bronchial hyperreactivit y to methacholine or histamine challenge was 18 

### increased in farmers exposed to ammonia compared to control workers (Vogelzang et al., 2000, 19 

### 1997; Choudat et al., 1994), indicating that exposu re to ammonia and other air contaminants in 20 

### farm settings may contribute to chronic airway infl ammation. In addition to ammonia, these 21 

### studies also documented exposures to airborne dust, bacteria, fungal spores, endotoxin, and 22 

### mold—agents that could also induce respiratory effe cts. The release of other volatiles on 23 

### livestock farms is likely, but measurements for oth er volatile chemicals were not conducted. 24 

### Therefore, while several studies have reported asso ciations between ammonia exposure in 25 

### livestock farmers or stable workers and respiratory irritation, these findings are limited by 26 

### exposures to other constituents in air that likely confound the association between ammonia 27 

### exposure and the respiratory effects observed in th e study populations. 28 

### Support for ammonia as a respiratory irritant is al so provided by reports of irritation and 29 

### hyperventilation in volunteers acutely exposed to a mmonia at concentrations ranging from 30



### 11

− 354 mg/m 3 

### ammonia for durations up to 4 hours under controll ed exposure conditions 31 

### (Petrova et al., 2008; Smeets et al., 2007; Altmann et al., 2006; Ihrig et al., 2006; Verberk, 1977; 32 

### Silverman et al., 1949) (see Appendix A, Section A. 4). Two controlled-exposure studies 33 

### reported habituation to eye, nose, and throat irrit ation in volunteers after several weeks of 34 

### ammonia exposure (Ihrig et al., 2006; Ferguson et a l., 1977). Numerous case reports document 35 

### the acute respiratory effects of inhaled ammonia, r anging from mild symptoms (including nasal 36 

### and throat irritation and perceived tightness in th e throat) to moderate effects (including 37 

### pharyngitis, tachycardia, dyspnea, rapid and shallo w breathing, cyanosis, transient 38

### 3 DRAFT - DO NOT CITE OR QUOTE

bronchospasm, and rhonchi in the lungs) to severe e ffects (including burns of the nasal passages, 1 

### soft palate, posterior pharyngeal wall, and larynx, upper airway obstruction, bronchospasm, 2 

### dyspnea, persistent, productive cough, bilateral di ffuse rales and rhonchi, mucous production, 3 

### pulmonary edema, marked hypoxemia, and necrosis of the lung) (see Appendix A, Section A.4, 4 

### for more detailed information and references). 5 

### Experimental studies in laboratory animals also pro vide consistent evidence that 6 

### ammonia exposure for 35 days or more can produce re spiratory irritation. Histopathological 7 

### changes in the nasal passages were observed in Sher man rats after 75 days of exposure to 106 8 

### mg/m 3 

### ammonia or 35 days of exposure to 177 mg/m 3 

### ammonia, with respiratory and olfactory 9 

### epithelium thickness increased three- to four times that of normal thickness (Broderson et al., 10 

### 1976). Thickening of nasal and tracheal epithelium (50 to 100%) was observed in pigs exposed 11 

### to 71 mg/m 3 

### ammonia continuously for 1–6 weeks (Doig and Willo ughby, 1971). Nonspecific 12 

### inflammatory changes (not further described) were r eported in the lungs of Sprague-Dawley and 13 

### Longs-Evans rats continuously exposed to 127 mg/m 3 

### ammonia for 90 days and rats and guinea 14 

### pigs intermittently exposed to 770 mg/m 3 

### ammonia (or 183 mg/m 3 

### , adjusted to continuous 15



### exposure 3 

### ) (Coon et al., 1970). Focal or diffuse interstiti al pneumonitis was observed in all 16 

### Princeton-derived guinea pigs, New Zealand white ra bbits, beagle dogs, and squirrel monkeys 17 

### exposed to 470 mg/m 3 

### ammonia that were examined (Coon et al., 1970). A dditionally, under 18 

### these exposure conditions, dogs exhibited nasal dis charge and other signs of irritation (marked 19 

### eye irritation, heavy lacrimation). Nasal discharg e was observed in 25% of rats exposed to 20 

### 262 mg/m 3 

### ammonia for 90 days (Coon et al., 1970). 21 

### At lower concentrations, approximately 50 mg/m 3 

### and below, the majority of studies of 22 

### inhaled ammonia show that ammonia does not produce respiratory irritation effects in laboratory 23 

### animals. No increase in the incidence of respirato ry or other diseases common to young pigs 24 

### were observed after continuous exposure to ammonia and inhalable dust at concentrations 25 

### representative of those found in commercial pig far ms (26 mg/m 3 

### ammonia) for 5 weeks (Done 26 

### et al., 2005). No gross or histopathological chang es in the turbinates, trachea, and lungs of pigs 27 

### were observed after continuous exposure to 53 mg/m 3 

### ammonia for up to 109 days (Curtis et al., 28 

### 1975). No signs of toxicity in rats were observed after continuous exposure to 40 mg/m 3 29 

### ammonia for 114 days or after intermittent exposure to 155 mg/m 3 

### ammonia (or 36.9 mg/m 3



### , 30 

### adjusted to continuous exposure) for 6 weeks (Coon et al., 1970). 31

32 

### Lung Function 33 

### Decreased lung function in ammonia-exposed workers has been reported in two cross34 

### sectional studies of industrial worker populations (Rahman et al., 2007; Ali et al., 2001) of three 35 

### such studies that measured lung function (Rahman et al., 2007; Ali et al., 2001; Holness et al., 36 

### (1989). Ammonia exposure was correlated with a sig nificant decline in lung function over the 37

3 C adjusted = C × 8 hours/24 hours × 5 days/7 days, where C is the exposure concentration.

### 4 DRAFT - DO NOT CITE OR QUOTE

course of a work shift (cross-shift) as measured by forced vital capacity (FVC) and forced 1 

### expiratory volume in one second (FEV 1 

### ) in the high-exposure worker group (mean ammonia 2 

### concentration of 18.5 mg/m 3 

### ) in a fertilizer factory (Rahman et al., 2007). I n a second study (Ali 3 

### et al., 2001), the FVC% predicted was higher in fer tilizer factory workers exposed to ammonia 4 

### than in controls (4.6% increase, p ≤ 0.002); FEV 1 

### was higher (1.5%) in the exposed workers but 5 

### the difference was not statistically significant. When Ali et al. (2001) based their analysis on 6 

### measures of cumulative exposure, workers with cumul ative exposure >50 mg/m 3 

### -years had 7 

### significantly lower FVC% predicted (5.4% decrease, p ≤ 0.030) and FEV 1 

### % predicted (7.4% 8 

### decrease, p < 0.006) than workers with cumulative exposure ≤ 50 mg/m 3 

### -years, but similar 9


### FEV 1 

### /FVC%. The authors did not explain the inconsisten t findings across the analyses of 10 

### noncumulative and cumulative exposures. 11 

### Lung function did not appear to be affected in work er populations chronically exposed to 12 

### ammonia at concentrations below approximately 18 mg /m 3 

### . Baseline lung function, based on 13 

### spirometry conducted at the beginning and end of th e work shift, differed very slightly relative to 14 

### control in workers exposed to ammonia concentration s ranging from <4.4 mg/m 3 

### to >8.8 mg/m 3 15 

### in a cross-sectional study of male workers in a sod a ash plant (Holness et al., 1989), but was not 16 

### statistically significant. Additionally, no change s in lung function were observed over either 17 

### work shift (days 1 or 2) or over the work week in t he exposed group compared with controls. 18 

### Similarly, measures of lung function (FVC, FEV 1 

### , and PEFR [peak expiratory flow rate]) in 19 

### workers exposed to a mean concentration of 4.9 mg/m 3 

### ammonia in a urea fertilizer factory 20 

### showed no significant cross-shift changes (Rahman e t al., 2007). 21 

### Decreased lung function (e.g., measured as decrease d FEV 1 

### , FVC) was reported in 22 

### farmers with ammonia exposure (Cormier et al., 2000 ; Donham et al., 2000, 1995; Vogelzang et 23 

### al., 1998; Reynolds et al., 1996; Preller et al., 1 995; Crook et al., 1991; Heederik et al., 1990). 24 

### These findings are limited by exposures to other co nstituents in air (including respirable dust, 25 

### bacteria, fungal spores, endotoxin, and mold) that can affect lung function, and likely confound 26 

### the association between exposure to ammonia and dec reased lung function observed in the study 27 

### populations. 28 

### Changes in lung function following acute exposure t o ammonia have been observed in 29 

### some but not all controlled exposure studies conduc ted in volunteers. Cole et al. (1977) reported 30 

### reduced lung function as measured by reduced expira tory minute volume and changes in exercise 31 

### tidal volume in volunteers exposed for a half-day i n a chamber at ammonia concentrations 32



### ≥ 106 mg/m 3 

### but not at 71 mg/m 3 

### . Bronchioconstriction was reported in volunteers exposed to 33 

### ammonia through a mouthpiece for 10 inhaled breaths of ammonia gas at a concentration of 34 

### 60 mg/m 3 

### (Douglas and Coe, 1987); however, there were no br onchial symptoms reported in 35 

### volunteers exposed to ammonia at concentrations of up to 35 mg/m 3 

### for 10 minutes in an 36 

### exposure chamber (MacEwen et al., 1970). Similarly , no changes in bronchial responsiveness or 37 

### lung function (as measured by forced vital capacity and FEV 1 

### ) were reported in healthy 38

### 5 DRAFT - DO NOT CITE OR QUOTE

volunteers exposed to ammonia at concentrations up to 18 mg/m 3 

### for 1.5 hours during exercise 1 

### (Sundblad et al., 2004). There were no changes in lung function as measured by FEV 1 

### in 25 2 

### healthy volunteers and 15 mild/moderate persistent asthmatic volunteers exposed to ammonia 3 

### concentrations up to 354 mg/m 3 

### ammonia for up to 2.5 hours (Petrova et al., 2008) , or in six 4 

### healthy volunteers and eight mildly asthmatic volun teers exposed to 11–18 mg/m 3 

### ammonia for 5 

### 30-minute sessions (Sigurdarson et al., 2004). 6 

### Lung function effects following ammonia exposure we re not evaluated in the available 7


### animal studies. 8 

### The evidence of respiratory effects in humans and e xperimental animals exposed to 9 

### ammonia is provided in Tables 1-1 and 1-2, respecti vely, and presented visually as an exposure10 

### response array in Figure 1-1. 11

12

13

### 6 DRAFT - DO NOT CITE OR QUOTE

Table 1-1. Respiratory effects in humans following inhalation exposure

Health Effect Study Design and Reference Results NOAEL/ LOAEL a (mg/m 3 )
Respiratory
irritation Cross-sectional occupational study

of soda ash plant workers in Canada; 58 exposed workers and 31 controls (from stores and office areas of plant) b

Low (<4.4 mg/m 3 ), medium (4.4– 8.8 mg/m 3 ), high (>8.8 mg/m 3 ); adjusted c concentration ranges <1.6 mg/m 3 , 1.6–3.1 mg/m 3 and >3.1 mg/m 3
Average exposure: 12 y

Holness et al., 1989 No statistically significant differences in subjective symptomology relative to the control.

Control Exposed p-value
Flu 3 7 0.6299 Cough 10 16 0.5289 Sputum 16 22 0.9770 Bronchitis 19 22 0.6938 Wheeze 10 10 0.9068 Chest tightness 6 3 0.6221 Dyspnea 13 7 0.0470 Chest pain 6 2 0.1563 Rhinitis 19 10 0.1185 Throat 3 7 0.5296 NOAEL: 3.1
LOAEL: not identified

Cross-sectional occupational study of urea fertilizer factory in Bangladesh; 63 ammonia plant workers, 77 urea plant workers, and 25 controls (from administration building)

Ammonia plant: 4.9 mg/m 3 d (1.8 mg/m 3 adjusted c ) Urea plant: 18.5 mg/m 3 d (6.6 mg/m 3 adjusted c ) Mean employment duration: 16 y

Rahman et al., 2007 Exposure-related increase in respiratory symptoms.

Respiratory symptom prevalence (%): Control

(admin) Ammonia

plant Urea

plant Cough 8 17 (0.42) a 28 (0.05, 0.41) b

Chest tightness 8 17 (0.42) a 33 (0.02, 0.19) b Stuffy

nose 4 12 (0.35) a 16 (0.17, 1.0) b
Runny

nose 4 4 (1.0) a 16 (0.17, 0.28) b Sneeze 8 0 (0.49) a 22 (0.22, 0.01) b

a p-value for ammonia plant compared to control

b p-value for urea plant compared to control and urea plant compared to ammonia plant NOAEL: 1.8
LOAEL: 6.6

Cross-sectional study of two urea fertilizer factories in Saudi Arabia; 161 exposed workers and 355 unexposed controls e

Exposures were stratified > or < the ACGIH TLV of 18 mg/m 3 Mean of employment duration: 51.8 mo (exposed workers) and 73.1 mo (controls)

Ballal et al., 1998 Higher relative risks for those exposed to
ammonia at concentrations >TLV as compared to those exposed at levels ≤TLV:

Cough: 4-fold Phlegm: 4.7-fold Wheezing: 2.2-fold Dyspnea: 4-fold Chronic bronchitis:1.6-fold Asthma: 3.7-fold NOAEL and LOAEL values

were not identified because

exposures were not adequately characterized

### 7 DRAFT - DO NOT CITE OR QUOTE

Table 1-1. Respiratory effects in humans following inhalation exposure

Health Effect Study Design and Reference Results NOAEL/ LOAEL a (mg/m 3 )
Lung function Cross-sectional occupational study

of soda ash plant workers in Canada; 58 exposed workers and 31 controls (from stores and office areas of plant) b

Low (<4.4 mg/m 3 ), medium (4.4– 8.8 mg/m 3 ), high (>8.8 mg/m 3 ) adjusted c concentration ranges <1.6 mg/m 3 , 1.6–3.1 mg/m 3 and >3.1 mg/m 3
Average exposure: 12 y

Holness et al., 1989 No statistically significant differences in lung function relative to the control.

Exposed Control p value Lung function (% predicted values):
FVC 96.8 98.6 0.0944
FEV 1 94.1 95.1 0.3520
FEV 1 /FVC 97.1 96.5 0.4801 Change in lung function over work shift: FVC day1 -0.8 -0.9 0.9940 day 2 -0.0 +0.1 0.8378
FEV 1 day 1 -0.2 -0.2 0.9363 day 2 +0.7 +0.5 0.8561 NOAEL: 3.1
LOAEL: not identified

Cross-sectional occupational study of urea fertilizer factory in Bangladesh; 63 ammonia plant workers, 77 urea plant workers, and 25 controls (from administration building)

Ammonia plant: 4.9 mg/m 3 d (1.8 mg/m 3 adjusted c ) Urea plant: 18.5 mg/m 3 d (6.6 mg/m 3 adjusted c ) Mean employment duration: 16 y

Rahman et al., 2007 Dose-related decrease in lung function parameters.

Pre-shift Post-shift p-value Ammonia plant
FVC 3.308 3.332 0.67
FEV 1 2.627 2.705 0.24
PEFR 8.081 8.313 0.22 Urea plant
FVC 3.362 3.258 0.01
FEV 1 2.701 2.646 0.05
PEFR 7.805 7.810 0.97 NOAEL: 1.8
LOAEL: 6.6

Cross-sectional study of a urea fertilizer factory in Saudi Arabia— follow-up of Ballal et al. (1998); 73 exposed workers and 343 unexposed controls

Exposures were stratified < or > the ACGIH TLV of 18 mg/m 3 Mean of employment duration: not reported

Ali et al., 2001 Lung function results based on exposure concentration and cumulative exposure:

Control Exposed p-value
FVC 1 % predicted 96.6 98.1 NS

FVC% predicted 101.0 105.6 0.002

FEV 1 /FVC% 83.0 84.2 NS

≤50 mg/m 3 -y >50

mg/m 3 -y p-value
FVC 1 % predicted 100.7 93.4 0.006

FVC% predicted 105.6 100.2 0.03

FEV 1 /FVC% 84.7 83.4 NS NS = not significant NOAEL and LOAEL values

were not identified because

exposures were not adequately characterized

### 8 DRAFT - DO NOT CITE OR QUOTE a The NOAEL and LOAEL values presented were identifie d by EPA.

b At this plant, ammonia, carbon dioxide, and water w ere the reactants used to form ammonium bicarbonate

, which in turn was reacted with salt to produce sodi um bicarbonate and subsequently processed to form s odium carbonate. Ammonia and carbon dioxide were recover ed in the process and reused.

c Adjusted to continuous exposure based on the ratio of VEho (human occupational default minute volume of
10 m 3 breathed during an 8-hour workday) to VEh (human am bient default minute volume of 20 m 3 breathed during the entire day) and an exposure of 5 days ou t of 7 days (i.e., measured concentration × 10/20 × 5/7).

d Exposure concentrations were determined by both the Dräger tube and Dräger PAC III methods. Using the Dräger tube method, concentrations of ammonia in th e ammonia and urea plants were 17.7 and 88.1 mg/m 3

, respectively; using the Dräger PAC III method, ammo nia concentrations were 4.9 and 18.5 mg/m 3 (Rahman et al., 2007). The study authors observed that their measu rements indicated only relative differences in expo sures between workers and production areas, and that the validity of the exposure measures could not be eval uated based on their results. Based on communication wit h technical support at Dräger (telephone conversati ons and emails dated June 22, 2010, from Michael Yanosky, Dr äger Safety Inc., Technical Support Detection Produ cts to Amber Bacom, SRC, Inc., contractor to NCEA, ORD, U. S. EPA), EPA considered the PAC III instrument to b e a more sensitive monitoring technology than the Dräger tub es. Therefore, more confidence is attributed to th e PAC III air measurements of ammonia for the Rahman et al. (2007 ) study.

e The process of fertilizer production involved synth esis of ammonia from natural gas, followed by react ion of the ammonia and carbon dioxide to form ammonium carbami de, which was then converted to urea.
1
2

### 9 DRAFT - DO NOT CITE OR QUOTE

Table 1-2. Respiratory effects in animals followin g inhalation exposure

Health Effect Study Design and Reference Results NOAEL/ LOAEL a (mg/m 3 )

Pulmonary inflammation and congestion 0, 155, or 770 mg/m 3 8 hrs/d, 5 d/wk for

6 wks; (36.9, 183 mg/m 3 adjusted b )

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group and Sprague-Dawley and Long-Evans rat; male and female; 15- 51/group

Coon et al., 1970 No visible signs of toxicity, gross necropsies were normal, focal pneumonitis in 1 of 3 monkeys at 36.9 mg/m 3

.

Nonspecific lung inflammation observed in guinea pigs and rats but not in other species at 183 mg/m 3 NOAEL: 36.9
LOAEL: 183

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for
90 d

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group

Coon et al., 1970 Focal or interstitial pneumonitis in all animals, calcification of bronchial epithelium was observed in several animals. Hemorrhagic lung lesion in 1 of 2 dogs. Moderate lung congestion in 2 of 3 rabbits. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 127, 262 or 470 mg/m 3 for 90 d of 455 mg/m 3 for 65 d

Sprague-Dawley or Long-Evans rat; male and female; 15-51/group

Coon et al., 1970 Dyspnea (mild) at 455 mg/m 3

. Focal or interstitial pneumonitis in all animals, calcification of bronchial epithelium observed in several animals at 470 mg/m 3

. (Exposure to 455 and 470 mg/m 3 ammonia increased mortality in rats.) NOAEL: 262
LOAEL: 455

0 or 14 for 7-42 days or 35 mg/m 3 for 42 days

Guinea pig (strain not specified); male and female; 2/group

Anderson et al., 1964 Pulmonary congestion, edema and hemorrhage were observed at 14 and 35 mg/m 3 after 42 d. NOAEL: NA
LOAEL:14

0 or 14 mg/m 3 for 7-42 days

Swiss albino mouse; male and female; 4/group

Anderson et al., 1964 Pulmonary congestion, edema and hemorrhage were observed at 14 mg/m 3 after 42 d. NOAEL: not identified
LOAEL: 14

### 10 DRAFT - DO NOT CITE OR QUOTE

Table 1-2. Respiratory effects in animals followin g inhalation exposure

Health Effect Study Design and Reference Results NOAEL/ LOAEL a (mg/m 3 ) 0, 0.4, 7, 13.3, or 26 mg/m 3 and 1.2, 2.7, 5.1, or 9.9 mg/m 3 inhalable dust for 5 wks

Exposure to ammonia and inhalable dust at concentrations commonly found at pig farms

Pig (several breeds); sex not specified; 24/group

Done et al., 2005 No increase in the incidence of respiratory or other disease. NOAEL: 26
LOAEL: not identified

0, 35, or 53 mg/m 3 for 109 d

Pig (crossbred); sex not specified; 4–8/group

Curtis et al., 1975 Turbinates, trachea, and lungs of all pigs were classified as normal. NOAEL: 53
LOAEL: not identified

Thickening of airway epithelium 7 or 106 mg/m 3 from bedding for 75 d

Sherman rat; 5/sex/group

Broderson et al., 1976 c Thickening of the nasal epithelium (3-4 times) and nasal
lesions. NOAEL: 7
LOAEL:106

0 or 177 mg/m 3 in an inhalation chamber for 35 d

F344 rat; 6/sex/group

Broderson et al., 1976 Thickening of the nasal epithelium (3-4 times) and nasal
lesions. NOAEL: not identified
LOAEL:177

0 or 71 mg/m 3 for 6 wks

Yorkshire-Landrace pig; sex not specified; 6/group

Doig and Willoughby, 1971 Thickening of nasal and tracheal epithelium (50-100% increase). NOAEL: not identified
LOAEL:71

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Table 1-2. Respiratory effects in animals followin g inhalation exposure

Health Effect Study Design and Reference Results NOAEL/ LOAEL a (mg/m 3 )

Nasal inflammation and lesions 0, 155, or 770 mg/m 3 8 hrs/d, 5 d/wk for

6 wks; (36.9, 183 mg/m 3 adjusted b )

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group and Sprague-Dawley and Long-Evans rat; male and female; 15- 51/group

Coon et al., 1970 No nasal irritation observed. NOAEL: 183
LOAEL: not identified

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for
90 d

Beagle dog; male; 2/group

Coon et al., 1970 Nasal discharge. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 127, 262 or 470 mg/m 3 for 90 d or 455 mg/m 3 for 65 d

Sprague-Dawley or Long-Evans rat; male and female; 15-51/group

Coon et al., 1970 Nasal irritation in all animals at 455 mg/m 3

. (Exposure to 455 and 470 mg/m 3 ammonia increased mortality in rats.) NOAEL: 262
LOAEL: 455

7 or 106 mg/m 3 from bedding for 75 d

Sherman rat; 5/sex/group

Broderson et al., 1976 Nasal lesions at 106 mg/m 3 . NOAEL: 7
LOAEL:106

0 or 177 mg/m 3 in an inhalation chamber for 35 d

F344 rat; 6/sex/group

Broderson et al., 1976 Nasal lesions at 177 mg/m 3 . NOAEL: not identified
LOAEL:177

Ammonia vapor of 0 or 12% ammonia solution for 15 min/d, 6 d/wk, for 8 wks

White albino mouse; male; 50

Gaafar et al., 1992 Histological changes in the nasal

mucosa. NOAEL and LOAEL values

were not identified because of inadequate reporting of

exposure concentrations.

### 12 DRAFT - DO NOT CITE OR QUOTE

Table 1-2. Respiratory effects in animals followin g inhalation exposure

Health Effect Study Design and Reference Results NOAEL/ LOAEL a (mg/m 3 ) 8, 43, 73, or 103 mg/m 3 for 5 wks

Duroc pig; both sexes; 9/group

Stombaugh et al., 1969 Excessive nasal, lacrimal, and mouth secretions and increased frequency of cough at 73 and 103 mg/m 3

. NOAEL and LOAEL values

were not identified because of the absence of a control group.

a The NOAEL and LOAEL values presented were identifie d by EPA.

b Adjusted to continuous exposure based on the ratio of hours exposed per day and days exposed per week (i.e., measured concentration × 8/24 × 5/7).

c The Broderson et al. (1976) paper includes a number of experiments in rats designed to examine whether ammonia at concentrations commonly encountered in l aboratory cage environments plays a role in the pathogenesis of murine respiratory mycoplasmosis ca used by the bacterium M. pulmonis . The experiments conducted without co-exposure to M. pulmonis are summarized in this table; the results of exper iments involving co-exposure to M. pulmonis are discussed in Section 1.1.4, Immune System Effe cts.

113 DRAFT - DO NOT CITE OR QUOTE1Figure 1-1. Exposure-response array for respiratory effects following inhalation exposure.20.11101001000 Prevalence of respiratory symptoms & decreased lung function (male occupational); Holness et al. (1989) Prevalence of respiratory symptoms & decreased lung function (occupational); Rahman et al. (2007) Focal or interstitial pneumonitis & calcification of bronchial epithelia (monkey, dog, rabbit); Coon et al. (1970) Nonspecific lung inflammation (guinea pig); Coon et al. (1970) Nonspecific lung inflammation dyspnea, focal or interstitial pneumonitis & calcification of bronchial epithelia (rat); Coon et al. (1970) Pulmonary congestion edema hemorrhage (guinea pig); Anderson et al. (1964) Pulmonary congestion edema hemorrhage (mouse); Anderson et al. (1964) No increase in respiratory infection (pig); Done et al. (2005) no pulmonary inflammation (pig); Curtis et al. (1975) thickening tracheal epithelium (pig); Doig & Willoughby (1971) thickening nasal epithelium (Sherman rat); Broderson et al. (1976) thickening nasal epithelium (F344 rat); Broderson et al. (1976) Nasal discharge (dog); Coon et al. (1970) Nasal irritation (rat); Coon et al. (1970) Nasal irritation (monkey, rabbit, guinea pig); Coon et al. (1970) Nasal lesions (Sherman rat); Broderson et al. (1976) Nasal lesions (F344 rat); Broderson et al. (1976)Pulmonary inflammation and congestion Thickening ofairway epithelium Nasal inflammation and lesions

Exposure (mg/m 3 )LOAELNOAELFELIntermediate doseVertical lines showrange of exposures instudy. Closed circlesshow exposurestested in study.EXPERIMENTAL ANIMAL STUDIESHUMANSTUDIES

### 14 DRAFT - DO NOT CITE OR QUOTE



##  Mode of Action Analysis – Respiratory Effects 1 

### Data regarding the potential mode of action for res piratory effects associated with chronic 2 

### exposure to ammonia are limited. However, it is we ll established that injury to respiratory 3 

### tissues resulting from acute exposure to inhaled am monia is primarily due to its alkaline 4 

### properties and its solubility. Given its high solu bility, ammonia readily dissolves in the moisture 5 

### on the mucous membranes, forming ammonium hydroxide , which causes liquefaction necrosis of 6 

### the tissues. Ammonia directly denatures tissue pro teins due to the production of alkaline 7 

### proteinates. Specifically, ammonium hydroxide caus es saponification of cell membrane lipids 8 

### that leads to cell disruption and death (necrosis). As cell proteins break down, water is extracted, 9 

### resulting in an inflammatory response, which furthe r damages the surrounding tissues (Amshel et 10 

### al, 2000; Mellea, 1989; Jarudi and Golden, 1973). 11

12


## 1.1.2. Gastrointestinal Effects 13 

### Reports of gastrointestinal effects of ammonia in h umans are limited to case reports 14 

### involving intentional or accidental ingestion of ho usehold cleaning solutions or ammonia 15 

### inhalant capsules (Dworkin et al., 2004; Rosenbaum et al., 1998; Christesen, 1995; Wason et al., 16 

### 1990; Lopez et al., 1988; Klein et al., 1985; Klend shoj and Rejent, 1966). Clinical signs reported 17 

### in these case studies include stomachache, nausea, dizziness, diarrhea, drooling, erythematous 18 

### and edematous lips, reddened and blistered tongues, dysphagia, vomiting, oropharyngeal burns, 19 

### laryngeal and epiglottal edema, erythmatous esophag us with severe corrosive injury, and 20 

### hemorrhagic esophago-gastro-duodeno-enteritis. 21 

### In animals following oral exposure, statistically s ignificant decreases of 40

− 60% in the 22 

### thickness of the gastric mucosa were reported in Sp rague-Dawley rats administered 0.01% 23 

### ammonia in drinking water for durations of 2–8 week s (Tsujii et al., 1993; Kawano et al., 1991); 24 

### estimated doses were 22 mg/kg-day (Kawano et al., 1 991) and 33 mg/kg-day (Tsujii et al., 1993). 25 

### These studies were designed to investigate the hypo thesis that the bacterium Helicobacter pylori

, 26 

### which produces a potent urease that increases ammon ia production, plays a significant role in the 27 

### etiology of chronic atrophic gastritis. Kawano et al. (1991) reported that the magnitude of the 28 

### decrease in gastric mucosal thickness increased wit h dose and duration of exposure and that the 29 

### effect was more prominent in the mucosa of the antr um region of the stomach than in the body 30 

### region of the stomach. 4 

### As discussed further under Mode of Action – Gastr ointestinal Effects 31 

### (see below), the difference in response to ammonia in drinking water in the two regions of the rat 32 

### stomach may be a function of differences in pH in t hese regions and resulting differences in the 33


### extent of ionization of ammonia to NH 4 + 

### . Parietal cell number per oxyntic gland also decr eased 34 

### in a statistically significant dose- and time-depen dent fashion, up to approximately 35% at 0.01% 35

4 The body is the main, central region of the stomach . The antrum is located in the distal part of the stomach adjacent to the body.

### 15 DRAFT - DO NOT CITE OR QUOTE

ammonia in drinking water after 4 weeks. In a foll ow-up study (Tsujii et al., 1993), antral 1 

### mucosal thickness decreased significantly (by 56–59 % of the tap water control) at 4 and 8 weeks 2 

### of exposure to 0.01% ammonia in drinking water, but there was no significant effect on the 3 

### thickness of the body mucosa. Increased mucosal ce ll proliferation and migration (as measured 4 

### by 5-bromo-2’-deoxyuridine [BrDU] labeling) were si gnificantly increased. The authors 5


### observed that it was not clear whether mucosal cell proliferation was primarily stimulated 6 

### directly by ammonia or indirectly by increased cell loss followed by compensatory cell 7 

### proliferation. They further observed that the ammo nia-related changes in rat stomach resembled 8 

### mucosal changes in human atrophic gastritis (Tsujii et al., 1993; Kawano et al., 1991). 9 

### A relationship between ammonia ingestion and gastro intestinal effects is supported by 10 

### findings from two acute oral studies in rats follow ing gavage administration of ammonium 11 

### hydroxide (Takeuchi et al., 1995; Nagy et al., 1996 ). Takeuchi et al. (1995) reported 12 

### hemorrhagic necrosis of the gastric mucosa in male Sprague-Dawley rats that received a single 13 

### gavage dose of ammonium hydroxide (concentration ≥ 1%). Nagy et al. (1996) observed severe 14 

### hemorrhagic mucosal lesions in female Sprague-Dawle y rats 15 minutes after exposure to an 15 

### estimated dose of 48 mg/kg ammonium hydroxide via g avage. 16 

### The evidence of gastrointestinal effects in experim ental animals following oral exposure 17 

### to ammonia is provided in Table 1-3. 18

19

20

### 16 DRAFT - DO NOT CITE OR QUOTE

Table 1-3. Gastrointestinal effects in animals foll owing oral exposures

Health Effect Study Design and References Results NOAEL/LOAEL a (mg/kg-day)
Histopathologic changes of the gastric mucosa 0, 22, or 220 mg/kg-day in

drinking water for 2 or 4 weeks b

Sprague-Dawley rat; male; 6/group

Kawano et al., 1991 Statistically significant decrease in the thickness of the gastric mucosa that was dose and duration related; effect was more prominent in the mucosa of the antrum region of the stomach than the body region.

Thickness of mucosa relative to control : Antrum Week 2: 96, 80 d % Week 4: 62 d , 39 d % Body Week 2: 99, 103% Week 4: 78, 71 d % NOAEL: not identified
LOAEL: 22

0 or 33 mg/kg-day in drinking water for 3 days or 1, 2, 4, or 8 weeks; tap water provided for the balance of the 8-week study c

Sprague-Dawley rat; male; 36/group

Tsujii et al., 1993 Antral mucosal thickness decreased significantly at 4 and 8 weeks of exposure; there was no significant effect on the thickness of the body mucosa. Cell migration was significantly increased.

Thickness of mucosa relative to control (d 3, wk 1, 2, 4, 8) : Antrum: 108, 96, 106, 56 d , 59 d % Body: 105, 101, 104, 99, 95% (extracted from Figure 3 of Tsujii et al., 1993) NOAEL: not identified
LOAEL: 33

a The NOAEL and LOAEL values presented were identifie d by EPA.

b Ammonia was provided in drinking water at concentra tions of 0, 0.01 or 0.1%. Doses were estimated bas ed on a body weight of 230 g for male rats and estimated da ily water intake of 50 mL/day.

c Ammonia was provided in drinking water at concentra tions of 0 or 0.01%. Doses were estimated based on an initial body weight of 150 g and estimated daily wa ter intake of 50 mL.

d Statistically significant from controls.

1 

##  Mode of Action Analysis – Gastrointestinal Effects 2 

### The mode of action for the gastric effects of ammo nia has not been established; however, 3


### relevant mechanistic information that informs ammon ia mode of action comes largely from 4 

### investigation of the action of the bacterium Helicobacter pylori on the stomach. H. pylori 5 

### produces urease, which breaks down urea that is nor mally present in the stomach into ammonia 6 

### (Mégraud et al. 1992; Tsujii et al. 1992a), and has been linked to chronic gastritis, gastric ulcers, 7


### and stomach cancer in humans. 8

### 17 DRAFT - DO NOT CITE OR QUOTE

This literature suggests that the alkalinity of th e ammonia solution does not play a direct 1 

### role in the gastric effects associated with ammonia . An ammonia solution (pH 10.3) produced 2 

### dose-related acute macroscopic mucosal lesions, whe reas a glycine-sodium hydroxide buffer (pH 3 

### 10.3) or ammonium chloride (pH 4.5) did not (Tsujii et al., 1992b). Rather, the ability of 4 

### ammonia to damage the gastric mucosa may be related to its ionization state. Ammonia (NH 3 

### ) 5 

### can easily penetrate cell membranes, subsequently r eacting to form NH 4 + 

### and OH 

### in the interior 6 

### of the membrane (Tsujii et al., 1992b). The findin g that antral and body regions of the rat 7 

### stomach mucosa responded differently following admi nistration of 33 mg/kg-day ammonia in 8 

### drinking water for 8 weeks (Tsujii et al., 1993) is consistent with the influence of ionization on 9 

### toxicity. The hydrogen chloride secreted by the mu cosa in the body of the stomach resulted in a 10 

### decrease in pH and a corresponding decrease in the ratio of ammonia to ammonium ion. In 11 

### contrast, in the antral mucosa (a nonacid-secreting area), pH is higher, the ratio of ammonia to 12 

### ammonium ion is increased, and measures of gastric atrophy were increased compared to those 13 

### observed in the stomach body where there was relati vely higher exposure to NH 4 +



### . 14 

### Several specific events have been identified that may contribute to the induction of 15 

### gastric lesions by ammonia. Increased cell vacuola tion and decreased viability of cells in vitro 16 

### were associated with increasing ammonia concentrati on in an in vitro system (Mégraud et al., 17 

### 1992); the effect was not linked to pH change becau se of the high buffering properties of the 18 

### medium. Using an in situ rat stomach model, hemorr hagic mucosal lesions induced by ammonia 19 

### were associated with the rapid release and activati on of cathepsins, mammalian cysteine 20 

### proteases that are released from lysosomes or activ ated in the cytosol and that can be damaging 21 

### to cells, tissues, or organs (Nagy et al., 1996). Ammonia also appears to inhibit cellular and 22 

### mitochondrial respiration, possibly by elevating in tracellular or intraorganelle pH or by 23 

### impairing adenosine triphosphate (ATP) synthesis (T sujii et al., 1992b). Mori et al. (1998) 24 

### proposed a role for increased release of endothelin -1 and thyrotropin releasing hormone from the 25 

### gastric mucosa in ammonia-induced gastric mucosal i njury based on findings in rats given 26 

### ammonia intragastrically. Regardless of the specif ic mechanism(s) by which ammonia induces 27 

### cellular toxicity, gastric injury appears to accele rate mucosal cell desquamation and stimulate 28 

### cell proliferation via a compensatory mechanism (Ts ujii et al., 1992a). 29

30 

## 1.1.3. Reproductive and Developmental Effects 31 

### No statistically significant differences in reprodu ctive or developmental endpoints were 32 

### found between two groups of female pigs (crossbred gilts) exposed to ammonia for 6 weeks at 33 

### mean concentrations of 5 or 25 mg/m 3 

### and then mated (Diekman et al., 1993) in the only study of 34 

### the reproductive and developmental toxicity potenti al of ammonia (see Table 1-4). Age at 35 

### puberty did not differ significantly between the tw o groups. Gilts exposed to 25 mg/m 3 

### ammonia 36 

### weighed 7% less (p < 0.05) at puberty than those ex posed to 5 mg/m 3 

### ; however, body weights of 37 

### the two groups were similar at gestation day 30. C onception rates in the mated females were 38

### 18 DRAFT - DO NOT CITE OR QUOTE

similar between the two groups (94.1 versus 100% in low- versus high-exposure groups). At 1 

### sacrifice on day 30 of gestation, there were no sig nificant differences between the two exposed 2 

### groups in body weights of the pregnant gilts, numbe r of corpora lutea, number of live fetuses, or 3 

### weight and length of the fetuses. The strength of the findings from this study are limited by the 4 

### absence of a control group and possible confounding by exposures to bacterial and mycoplasm 5 

### pathogens. The evidence of reproductive and develo pmental effects in experimental animals 6 

### exposed to ammonia is provided in Table 1-4. 7

8 

### Table 1-4. Reproductive and developmental effects in animals following inhalation

exposure

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 )
Reproductive and developmental parameters 5 mg/m 3 (range, 3–8.5

mg/m 3 ) or 25 mg/m 3 (range, 18–32 mg/m 3 ) for 6 weeks b

Crossbred gilts (female pigs), 4.5 months old, 40/group

Diekman et al., 1993 No effect on any of the reproductive or developmental parameters measured (age at puberty, conception rates, body weight of pregnant gilts, number of corpora lutea, number of live fetuses, and weight or length of fetuses). NOAEL: 5 LOAEL: not identified

a The NOAEL and LOAEL values presented were identifie d by EPA.

b A control group was not included. Prior to exposur e to ammonia, pigs were also exposed naturally in conventional grower units to Mycoplasma hypopneumoniae and Pasteurella multocida , which cause pneumonia and atrophic rhinitis, respectively.

9 

## 1.1.4. Immune System Effects 10



### A limited number of studies have evaluated the immu notoxicity of ammonia in human 11 

### populations and in experimental animal models. Imm unological function was evaluated in two 12 

### independent investigations of livestock farmers exp osed to ammonia; immunoglobulin G (IgG) 13 

### and IgE-specific antibodies for pig skin and urine (Crook et al., 1991), elevated neutrophils from 14 

### nasal washes, and increased white blood cell counts (Cormier et al., 2000) were reported. These 15 

### data are suggestive of immunostimulatory effects; h owever, the test subjects were also exposed 16 

### to a number of other respirable agents in addition to ammonia such as endotoxin, bacteria, fungi, 17 

### and mold that are known to stimulate immune respons es. Data in humans following exposure to 18 

### ammonia only are not available. 19 

### Animal studies that examined ammonia immunotoxicity were conducted using short-term 20 

### inhalation exposures and three general types of imm une assays. Immunotoxicity studies of 21 

### ammonia using measures of host resistance provide t he most relevant data for assessing immune 22 

### function since they directly measure the immune sys tem’s ability to control microorganism 23 

### growth. Other available studies of ammonia employe d assays that evaluated immune function. 24

### 19 DRAFT - DO NOT CITE OR QUOTE

Changes in immune cell populations without correspo nding functional data are considered to be 1 

### the least predictive and were excluded from the haz ard identification for ammonia (Neumann et 2 

### al, 1987; Gustin et al, 1994). 3 

### Evidence of immunosuppression was observed in sever al host resistance studies utilizing 4 

### lung pathogens to measure reduced bacterial clearan ce following ammonia exposure. 5 

### Inoculation with the respiratory pathogen Mycoplasma pulmonis causes murine respiratory 6 

### mycoplasmosis (MRM) characterized by lung lesions. Lung lesions, both gross and 7 

### microscopic, were positively correlated with ammoni a concentration in F344 rats continuously 8 

### exposed to ammonia in an inhalation chamber for 7 d ays prior to inoculation with M. pulmonis 9 

### (10 8 

### colony forming units [CFU]) followed by up to 42 d ays of ammonia exposure post 10 

### inoculation (Broderson et al., 1976). The incidenc e of lesions was significantly increased at 11


### ammonia concentrations ≥ 35 mg/m 3 

### , and suggests that ammonia exposure decreased bact erial 12 

### clearance resulting in the development of M. pulmonis -induced MRM. However, the increasing 13


### ammonia concentration was not associated with incre ased CFU of M. pulmonis isolated from the 14 

### respiratory tract. The high number of inoculating CFU could have overwhelmed the immune 15 

### response and elicited a maximal response that could not be further magnified in 16 

### immunocompromised animals. Conversely, significant ly increased CFU of M. pulmonis bacteria 17 

### isolated in the trachea, nasal passages, lungs, and larynx was observed in F344 rats continuously 18 

### exposed to 71 mg/m 3 

### ammonia for 7 days prior to M. pulmonis (10 4



### − 10 6 

### CFU) inoculation and 19 

### continued for 28 days post inoculation (Schoeb et a l., 1982). This increase in bacterial 20


### colonization indicates a reduction in bacterial cle arance following exposure to ammonia. 21 

### Lesions were not assessed in this study. OF1 mice exposed to 354 mg/m 3 

### ammonia for 7 days 22 

### prior to inoculation with a 50% lethal dose (LD 50 

### ) of Pasteurella multocida significantly 23 

### increased mortality compared to controls (86% versu s 50%, respectively); however, an 8-hour 24 

### exposure was insufficient to affect mortality (Rich ard et al., 1978a). The authors suggested that 25 

### the irritating action of ammonia destroyed the trac heobronchial mucosa and caused inflammatory 26 

### lesions thereby increasing sensitivity to respirato ry infection with prolonged ammonia exposure. 27 

### Suppressed cell-mediated immunity and decreased T c ell proliferation was also observed 28 

### following ammonia exposure. Using a delayed-type h ypersensitivity (DTH) test to evaluate cell29 

### mediated immunity, Hartley guinea pigs were vaccina ted with Mycobacterium bovis BCG and 30 

### exposed to ammonia followed by intradermal challeng e with purified protein derivative (PPD). 31 

### Dermal lesion size was reduced in animals exposed t o 64 mg/m 3 

### indicating immunosuppression 32 

### (Targowski et al., 1984). Blood and bronchial lymp hocytes harvested from naïve guinea pigs 33 

### treated with the same 3 week ammonia exposure and s timulated with phytohaemagglutinin or 34 

### concanavalin A demonstrated reduced T cell prolifer ation (Targowski et al., 1984). Bactericidal 35 

### activity in alveolar macrophages isolated from ammo nia-exposed guinea pigs was not affected. 36 

### Lymphocytes and macrophages isolated from unexposed guinea pigs and treated with ammonia 37 

### in vitro showed reduced proliferation and bacterici dal capacity only at concentrations that 38

### 20 DRAFT - DO NOT CITE OR QUOTE

reduced viability, indicating nonspecific effects o f ammonia-induced immunosuppression 1 

### (Targowski et al., 1984). These data suggest that T cells may be the target of ammonia since 2 

### specific macrophage effects were not observed. 3 

### The evidence of immune system effects in experiment al animals exposed to ammonia is 4 

### provided in Table 1-5, and presented visually in an exposure-response array in Figure 1-2. 5

6 

### Table 1-5. Immune system effects in animals follow ing inhalation exposure

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 )

Host resistance ≤ 3.5 (control), 18, 35, 71, 177 mg/m 3 , 7

days (continuous exposure) pre inoculation/28-42 days post inoculation
with M. pulmonis

F344 rat; male and female; 11-12/sex/

group

Broderson et al., 1976 Increased incidence of gross lung lesions; no effect on CFU.

% of animals with gross lesions: 16 (control), 46, 66 b

, 33, 83% NOAEL: 18
LOAEL: 35 c

<1.4 (control) or 71 mg/m 3 , 7 days (continuous exposure) pre inoculation/ 28 days post inoculation with M. pulmonis

F344 rat; 5-15/group (sex unknown)

Schoeb et al., 1982 Increased bacterial colonization (as a result of reduced bacterial clearance).

No quantitative data available. NOAEL: not identified
LOAEL: 71

0 or 354 mg/m 3 , 8 hours or 7 days (continuous exposure), prior to infection
with P. multocida

OF1 mouse; male; 99/group

Richard et al., 1978 Increased mortality.

Mouse mortality: 50% (control) and 86% b NOAEL: not identified
LOAEL: 354

T cell proliferation <11 (control), 35 or 64 mg/m 3 , 3 weeks

(continuous exposure)

Hartley guinea pig; 8/group (sex unknown)

Targowski et al., 1984 Reduced proliferation in blood and bronchial T cells.

No quantitative data available. NOAEL: 35
LOAEL: 64

Delayed-type hypersensitivity <11 (control), 35 or 64 mg/m 3 , 3 weeks

(continuous exposure) followed by PPD challenge in BCG immunized

Hartley guinea pig; 8/group (sex unknown)

Targowski et al., 1984 Reduced dermal lesion size.

Mean diameter (mm): 12 (control), 12.6 and 8.7 b NOAEL: 35
LOAEL: 64

a The NOAEL and LOAEL values presented were identifie d by EPA.

b Statistically significant from controls.

c Study did not find statistical significance despite a large increase in the response at the lowest dos e measured.21 DRAFT - DO NOT CITE OR QUOTE1Figure 1-2. Exposure-response array for immune system effects following inhalation exposure.21101001000Increased incidence ofgross lung lesions (rat);Broderson et al. (1976)Increased bacterialcolonization (rat);Schoeb et al. (1982)Increased mortality (mouse);Richard et al. (1978)Reduced proliferationin blood and bronchiallymphocytes (guinea pig);Targowski et al. (1984)Reduced dermallesion size (guinea pig);Targowski et al. (1984)Host resistanceMitogen stimulation Delayed-type hypersensitivity

Exposure (mg/m 3 )LOAELNOAELFELIntermediate doseVertical lines showrange of exposures instudy. Closed circlesshow exposurestested in study.

### 22 DRAFT - DO NOT CITE OR QUOTE



## 1.1.5. Other Systemic Effects 1 

### Although the majority of information for ammonia su ggests that ammonia induces effects 2 

### in and around the portal of entry, there is limited evidence that ammonia can produce effects on 3 

### organs distal from the portal of entry, including t he liver, adrenal gland, kidney, spleen, and 4 

### heart. Alterations in liver function, based on ele vated mean levels of aspartate aminotransferase 5 

### (AST), alanine aminotransferase (ALT), and blood ur ea, decreased hemoglobin, and inhibition of 6 

### catalase and monoamine oxidase (MAO) activities wer e observed in workers exposed to 7 

### ammonia over an average exposure duration of 12 yea rs at an Egyptian urea production plant; 8 

### measurements of workplace exposure concentrations w ere not provided (Hamid and El-Gazzar, 9 

### 1996). The authors suggested that inhibition of ca talase can affect electrical stability, 10 

### permeability, and fluidity of membranes, which may lead to hepatotoxicity in occupationally 11 

### exposed workers (Hamid and El-Gazzar, 1996). 12 

### Evidence of hepatotoxicity in animals comes from ob servations of histopathological 13 

### alterations in the liver. Fatty changes in the liv er were consistently reported at concentrations 14



### ≥ 470 mg/m 3 

### ammonia in rats, guinea pigs, rabbits, dogs, and m onkeys following identical 15 

### subchronic inhalation exposure regimens (Coon et al ., 1970). Congestion of the liver was 16 

### observed in guinea pigs following subchronic and sh ort-term inhalation exposure to 35 and 17 

### 120 mg/m 3 

### (Anderson et al., 1964; Weatherby, 1952); no liver effects were observed in similarly 18 

### exposed mice at 14 mg/m 3 

### (Anderson et al., 1964; Weatherby, 1952). No hist opathological or 19 

### hematological effects were observed in rats, guinea pigs, rabbits, dogs, or monkeys when these 20 

### animals were repeatedly, but not continuously, expo sed to ammonia even at high concentrations 21 

### (e.g., 770 mg/m 3 

### for 8 hours/day, 5 days/week), suggesting that mam mals can recover from 22 

### short-term exposure to elevated ammonia levels (Coo n et al., 1970). In addition, no effects were 23 

### observed in mice exposed to 14 mg/m 3 

### for up to 6 weeks (Anderson et al., 1964). 24 

### Adrenal effects were observed in animals following subchronic and short-term exposure 25 

### to ammonia; data in humans were not found. Increas ed mean adrenal weights and fat content of 26 

### the adrenal gland, as well as histological changes in the adrenal gland (enlarged cells of the zona 27


### fasiculata of the adrenal cortex that were rich in lipid) were observed in rabbits exposed orally 28 

### via gavage to ammonium hydroxide for durations rang ing from 5.5 days to 17 months (Fazekas, 29 

### 1939). While the strength of these findings is lim ited by inadequate reporting and study design, 30 

### a separate study identified early degenerative chan ges in the adrenal glands of guinea pigs 31 

### exposed to 120 mg/m 3 

### ammonia by inhalation for 18 weeks (Weatherby, 195 2), providing 32


### additional limited evidence for effects on the adre nal gland. 33 

### Evidence that inhaled ammonia can affect the kidney and spleen is limited to studies in 34 

### experimental animals. Nonspecific degenerative cha nges in the kidneys (not further described) 35 

### of rats exposed to 262 mg/m 3 

### were reported (Coon et al., 1970). Histopathologi cal evaluation of 36 

### other animal species in the same study exposed to 4 70 mg/m 3 

### , a concentration that induced a 37 

### high rate of mortality in rats, consistently showed alterations in the kidneys (calcification and 38

### 23 DRAFT - DO NOT CITE OR QUOTE

proliferation of tubular epithelium; incidence not reported). Exposure of guinea pigs to inhaled 1 

### ammonia at a concentration of 120 mg/m 3 

### for 18 weeks (but not 6 or 12 weeks) resulted in 2 

### histopathological alterations (congestion) of the k idneys and spleen, although incidence was not 3 

### reported (Weatherby, 1952). Enlarged and congested spleens were reported in guinea pigs 4 

### exposed to 35 mg/m 3 

### ammonia for 6 weeks in a separate study (Anderson et al., 1964). 5 

### Myocardial fibrosis was observed in monkeys, dogs, rabbits, guinea pigs, and rats 6 

### following subchronic, inhalation exposure to 470 mg /m 3 

### ammonia; no changes were observed at 7 

### lower concentrations (Coon et al., 1970). At the s ame concentration, ocular irritation 8 

### (characterized as heavy lacrimation, erythema, disc harge and ocular opacity of the cornea) was 9 

### also reported by Coon et al. (1970) in dogs and rab bits, but not observed in similarly treated 10 

### monkeys and rats. Additionally, there is limited e vidence of biochemical or metabolic effects of 11 

### acute or short-term ammonia exposure. Acidosis, as evidenced by a decrease in blood pH and an 12 

### increase in arterial blood carbon dioxide partial p ressure (pCO 2 

### ), occurred in rats exposed to 13 

### 212 mg/m 3 

### ammonia for 5

− 15 days (Manninen et al., 1988). Blood pH and pCO 2 

### did not change 14 

### in rats exposed to ≤ 818 mg/m 3 

### for up to 24 hours, although statistically signifi cant increases in 15 

### oxygen partial pressure (pO 2 

### ) were reported in rats exposed to 10.6 and 22.6 mg /m 3 

### ammonia, 16 

### but not at 219 and 818 mg/m 3 

### over the same time period (Schaerdel et al., 1983)

. 17 

### Encephalopathy related to ammonia may occur followi ng disruption of the body’s normal 18 

### homeostatic regulation of the glutamine and urea cy cles resulting in elevated ammonia levels in 19 

### blood, e.g., as a result of severe liver or kidney disease (Miñana et al., 1995; Souba, 1987). 20 

### Acute inhalation exposure studies have identified a lterations in amino acid levels and 21 

### neurotransmitter metabolism (including glutamine co ncentrations) in the brain of rats and mice 22 

### (Manninen and Savolainen, 1989; Manninen et al., 19 88; Sadasivudu et al., 1979; Sadasivudu 23 

### and Murthy, 1978). It has been suggested that glut amate and γ -amino butyric acid (GABA) play 24 

### a role in ammonia-induced neurotoxicity (Jones, 200 2). There is no evidence, however, that 25


### ammonia is neurotoxic in humans or animals followin g chronic exposures. 26 

### The evidence of systemic toxicity in humans and exp erimental animals exposed to 27 

### ammonia is provided in Tables 1-6 to 1-8, and prese nted visually in an exposure-response array 28 

### in Figure 1-3. 29

30

### 24 DRAFT - DO NOT CITE OR QUOTE

Table 1-6. Systemic effects in humans following in halation exposure

Health Effect Study Design and Reference Results NOAEL/LOAEL

Serum clinical chemistry; liver function Occupational study workers in an

Egyptian urea plant; 30 exposed and 30 control subjects

No measurement of exposure
concentrations Average employment time: 12 y

Hamid and El-Gazzar, 1996 Elevated AST, ALT and blood urea in exposed workers; lower hemoglobin and inhibition of catalase and
MAO. Not identified because the study did not report measurements of

exposure.

1

2 

### Table 1-7. Systemic effects in animals following o ral exposure

Health Effect Study Design and Reference Results NOAEL/LOAEL

Adrenal effects 50–80 mL of a 0.5 or 1.0%

ammonium hydroxide solution by gavage; initially every other day, later daily; duration ranged from 5.5 days to 17 months; estimated dose: 61–110 mg/kg-day and 120–230 mg/kg-day, respectively a

Rabbits (strain and sex not specified); 16-33/group

Fazekas, 1939 Increased mean adrenal weights and fat content of the adrenal gland.

Response relative to control (adrenal weight): 95%
increase

Response relative to control (fat): 4.5-fold increase Not identified.

a Ammonia doses estimated using assumed average defau lt body weight of 3.5

− 4.1 kg for adult rabbits (U.S. EPA, 1988).

3 

### Table 1-8. Systemic effects in animals following i nhalation exposures

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 )

Liver toxicity 0 or 120 mg/m 3 6 h/day, 5 days/week for 6,

12 or 18 weeks; (24.1 mg/m 3 adjusted b ),

Guinea pig (strain not specified); male; 6–12/

group

Weatherby, 1952 Congestion of the liver at 18 weeks, not observed at earlier times. NOAEL: not identified
LOAEL: 24.1

0 or 14 for 7-42 days or 35 mg/m 3 for 42 days

Guinea pig (strain not specified); male and female; 2/group

Anderson et al., 1964 Congestion of the liver at 35 mg/m 3 for 42 days. NOAEL: 14
LOAEL: 35

### 25 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following i nhalation exposures

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 ) 0 or 14 mg/m 3 for 7-42 days

Swiss albino mouse; male and female; 4/group

Anderson et al., 1964 No visible signs of liver toxicity. NOAEL: 14
LOAEL: not identified

0, 155, or 770 mg/m 3 8 hrs/d, 5 d/wk for 6 wks; (36.9, 183 mg/m 3 adjusted b )

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group and Sprague-Dawley and Long-Evans rat; male and female; 15- 51/group

Coon et al., 1970 No histopathologic changes observed. NOAEL: 183
LOAEL: not identified

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for
90 d

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group

Coon et al., 1970 Fatty liver changes in plate cells. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 127, 262 or 470 mg/m 3 for 90 d; Sprague-Dawley or Long-Evans rat; male and female; 15-51/group

Coon et al., 1970 Fatty liver changes in plate cells. NOAEL: 262
LOAEL: 470

Adrenal gland toxicity 0 and 120 mg/m 3 6 h/day, 5 days/week for 6,

12 or 18 weeks; (24.1 mg/m 3 adjusted b )

Guinea pig (strain not specified); male; 6–12/

group

Weatherby, 1952 “Early” degenerative changes in the adrenal gland (swelling of cells, degeneration of the cytoplasm with loss of normal granular structure) at 18 weeks, not observed at earlier
times. NOAEL: not identified
LOAEL: 24.1

### 26 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following i nhalation exposures

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 )
Kidney and spleen toxicity 0, 155, or 770 mg/m 3 8 hrs/d, 5 d/wk for

6 wks; (36.9, 183 mg/m 3 adjusted b )

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group and Sprague-Dawley and Long-Evans rat; male and female; 15- 51/group

Coon et al., 1970 No histopathologic changes observed. NOAEL: 183
LOAEL: not identified

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for
90 d

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group

Coon et al., 1970 Calcification and proliferation of renal tubular epithelium. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 127, 262 or 470 mg/m 3 for 90 d;

Sprague-Dawley or Long-Evans rat; male and female; 15-51/group

Coon et al., 1970 Calcification and proliferation of renal tubular epithelium. NOAEL: 262
LOAEL: 470

0 or 120 mg/m 3 6 h/day, 5 days/week for 6, 12 or 18 weeks; (24.1 mg/m 3 adjusted b )

Guinea pig (strain not specified); male; 6–12/

group

Weatherby, 1952 Congestion of the spleen and kidneys. NOAEL: not identified
LOAEL: 24.1

0 or 14 for 7-42 days or 35 mg/m 3 for 42 days

Guinea pig (strain not specified); male and female; 2/group

Anderson et al., 1964 Enlarged and congested spleens. NOAEL: 14
LOAEL: 35

0 or 14 mg/m 3 for 7-42 days

Swiss albino mouse; male and female; 4/group

Anderson et al., 1964 No visible signs of toxicity. NOAEL: 14
LOAEL: not identified

### 27 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following i nhalation exposures

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 )
Myocardial toxicity 0, 155, or 770 mg/m 3 8 hrs/d, 5 d/wk for

6 wks; (36.9, 183 mg/m 3 adjusted b )

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group and Sprague-Dawley and Long-Evans rat; male and female; 15- 51/group

Coon et al., 1970 No histopathologic changes observed. NOAEL: 183
LOAEL: not identified

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for
90 d

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group

Coon et al., 1970 Myocardial fibrosis. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 127, 262 or 470 mg/m 3 for 90 d;

Sprague-Dawley or Long-Evans rat; male and female; 15-51/group

Coon et al., 1970 Myocardial fibrosis. NOAEL: 262
LOAEL: 470

Ocular
Irritation 0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for

90 d

Beagle dog; male; 2/group

Coon et al., 1970 Heavy lacrimation. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for
90 d

New Zealand albino rabbit; male; 3/group

Coon et al., 1970 Erythema, discharge and ocular opacity over 1⁄4 to 1⁄2 of cornea. NOAEL: 40
LOAEL: 470

0 or 40 mg/m 3 for 114 d or 470 mg/m 3 for 90 d

Squirrel monkey ( Saimiri sciureus ); male; 3/group and 3/group and Princeton-derived guinea pig; male and female; 15/group

Coon et al., 1970 No ocular irritation observed. NOAEL: 470
LOAEL: not identified

### 28 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following i nhalation exposures

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 ) 0 or 40 mg/m 3 for 114 d or 127, 262 or 470 mg/m 3 for 90 d;

Sprague-Dawley and Long-Evans rat; male and female; 15-51/group

Coon et al., 1970 No ocular irritation observed. NOAEL: 470
LOAEL: not identified

0, 155, or 770 mg/m 3 8 hrs/d, 5 d/wk for 6 wks; (36.9, 183 mg/m 3 adjusted b )

Squirrel monkey ( Saimiri sciureus ); male; 3/group and beagle dog; male; 2/group and New Zealand albino rabbit; male; 3/group and Princeton-derived guinea pig; male and female; 15/group and Sprague-Dawley and Long-Evans rat; male and female; 15- 51/group

Coon et al., 1970 No ocular irritation observed. NOAEL: 183
LOAEL: not identified

Blood pH changes 0, 18, or 212 mg/m 3 6 h/day for 5, 10 or 15

days; (4.5, 53 mg/m 3 adjusted b )

Wistar rat; female; 5/group

Manninen et al., 1988 Statistically significant decrease in blood pH at 5 days. pH differences “leveled off at later time points (data not shown)”.

Response difference from control : 0.09 c and 0.07 c NOAEL: 53
LOAEL: not identified

10.6–818 mg/m 3 for 0, 8, 12, 24 hours, 3 and 7 days

Crl:COBS CD(SD) rat; male; 32 and 70

Schaerdel et al., 1983 Statistically significant increase in pO 2 at 10.6 and 22.6 mg/m 3 exposure at 8, 12 and 24 hours (p<0.05). No change at higher exposures. No change in blood pH or pCO 2 .

Response relative to control d : 16, 6, 20% at 10.6 mg/m 3 and at 8, 12, 24 hrs; 18, 26, 17% at 22.6 mg/m 3 and at 8, 12, 24 hrs NOAEL: 818
LOAEL: not identified

### 29 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following i nhalation exposures

Health Effect Study Design and Reference Results NOAEL/LOAEL a (mg/m 3 )

Amino acid levels and

neurotransmitter metabolism in the brain 0, 18, or 212 mg/m 3 6 h/day for 5 days; (4.5,

53 mg/m 3 adjusted b )

Wistar rat; female; 5/group

Manninen and Savolainen, 1989 Statistically significant increase in brain glutamine (p< 0.05).

Response relative to control : 42 c

, 40 c % for 18 and 212 mg/m 3

, respectively NOAEL: not identified
LOAEL: 4.5 e

0, 18, or 212 mg/m 3 6 h/day for 5, 10 or 15 days; (4.5, 53 mg/m 3 adjusted b )

Wistar rat; female; 5/group

Manninen et al., 1988 Brain and blood glutamine statistically significantly increased (p< 0.05 and 0.01, respectively) at 212 mg/m 3 at 5 days, no statistically significant difference from control at 10 and 15 days.

Response relative to control at 212 mg/m 3 : 44 c , 13 and 14% increase in blood glutamine at 5, 10, 15 days; 40 c , 4 and 2% increase in brain glutamine at 5, 10, 15 days NOAEL: 53
LOAEL: not identified

a The NOAEL and LOAEL values presented were identifie d by EPA.

b Adjusted to continuous exposure based on the ratio of hours exposed per day and days exposed per week (i.e., measured concentration × 8/24 × 5/7).

c Statistically significant difference from controls.

d Measurements at time zero were used as a control. T he study did not include an unexposed, control grou p.

1

230 DRAFT - DO NOT CITE OR QUOTE12Figure 1-3. Exposure-response array for systemic effects following inhalation exposure.31101001000 Congestion of the liver (guinea pig); Weatherby (1952) Congestion of liver (guinea pig); Anderson et al. (1964) Fatty liver changes in plate cells (monkey, dog, rabbit, guinea pig); Coon et al. (1970) Fatty liver changes in plate cells (rat); Coon et al. (1970) Early degenerative changes in the adrenal gland (guinea pig); Weatherby (1952) Calcification & proliferation renal tubular epithelia (monkey, dog, rabbit, guinea pig); Coon et al. (1970) calcification & proliferation of renal tubular epithelia (rat); Coon et al. (1970) Congestion of the spleen (guinea pig); Weatherby (1952) Enlarged & congested spleen (guinea pig); Anderson et al. (1964) Myocardial fibrosis (monkey, dog, rabbit, guinea pig); Coon et al. (1970) Myocardial fibrosis (rat); Coon et al. (1970) Heavy lacrimation (dog); Coon et al. (1970) Erythema, discharge & ocular opacity 1⁄4 - 1⁄2 of cornea (rabbit); Coon et al. (1970) No irritation observed (monkey, guinea pig, rat); Coon et al. (1970)Liver toxicity AdrenalglandtoxicityKidney and spleen toxicity Myocardial toxicity OcularIrritation

Exposure (mg/m 3 )LOAELNOAELFELIntermediate doseVertical lines showrange of exposures instudy. Closed circlesshow exposurestested in study.

### 31 DRAFT - DO NOT CITE OR QUOTE



## 1.1.6. Cancer 1 

### No information is available regarding the carcinoge nic effects of ammonia in humans 2 

### following oral or inhalation exposure. The carcino genic potential of ammonia by the inhalation 3 

### route has not been assessed in animals, and animal carcinogenicity data by the oral route of 4 

### exposure are limited. Toth (1972) concluded that t umor incidence was not increased in Swiss 5 

### mice exposed for their lifetime (not further specif ied) to ammonium hydroxide in drinking water 6 

### at concentrations up to 0.3% (equivalent to 410 and 520 mg/kg-day in female and male mice, 7 

### respectively) or in C 3 

### H mice exposed to ammonium hydroxide in drinking wa ter at a 8 

### concentration of 0.1% (equivalent to 214 and 191 mg /kg-day in female and male mice, 9 

### respectively). With the exception of mammary gland tumors in female C 3 

### H mice (a tumor with a 10 

### high background incidence), concurrent control tumo r incidence data were not reported and 11 

### comparison of tumor incidence in exposed and contro l mice could not be performed. The 12 

### general lack of concurrent control data limits the ability to interpret the findings of this study. 13 

### The incidence of gastric cancer and the number of g astric tumors per tumor-bearing rat 14 

### were statistically significantly higher in rats exp osed to 0.01% ammonia solution in drinking 15 

### water (equivalent to 10 mg/kg-day) for 24 weeks fol lowing pretreatment (for 24 weeks) with the 16 

### initiator N-methyl-N’-nitro-N-nitrosoguanidine (MNN G) compared with rats receiving only 17 

### MNNG and tap water (Tsujii et al., 1992a). In an a lmost identically designed study, reported by 18 

### Tsujii et al. (1995), similar increases in the inci dence of gastric tumors were observed in rats 19 

### following exposure to MNNG and 10 mg/kg-day ammonia . Additionally, the size and 20 

### penetration to deeper tissue layers of the MNNG-ini tiated gastric tumors were enhanced in the 21 

### rats treated with ammonia (Tsujii et al., 1995). T he investigators suggested that ammonia 22 

### administered in drinking water may act as a cancer promoter (Tsujii et al., 1995, 1992a); 23 

### however, in the absence of an ammonia-only exposure group in these studies, it is not possible to 24 

### distinguish between possible promotion and initiato r activity. 25 

### The evidence of carcinogenicity in experimental ani mals exposed to ammonia is provided 26


### in Table 1-9. 27

28

### 32 DRAFT - DO NOT CITE OR QUOTE

Table 1-9. Cancer bioassays following oral exposur e

Health Effect Study Design and Reference Results Tumor incidence 250, 440, and 520 mg/kg-day (males) ;

240, 370, and 410 mg/kg-day (females) [0.1, 0.2, and 0.3% ammonium hydroxide in drinking water a ] for their lifetime (not further specified)

Swiss mouse, 50/sex/group

Toth, 1972 The authors reported that tumor incidence was not increased in ammonia-exposed mice; however, concurrent control tumor incidence data were not reported and comparison of tumor incidence in exposed and control mice could not be performed.

191 (males) and 214 mg/kg-day (females) [0.1% ammonium hydroxide in drinking

water b ] for their lifetime (not further specified)

C 3 H mouse, 40/sex/group

Toth, 1972 The authors reported that tumor incidence was not increased in ammonia-exposed mice; however, with the exception of mammary gland tumors in female mice, concurrent control tumor incidence data were not reported and comparison of tumor incidence in exposed and control mice could not be performed.

Mammary gland adenocarcinoma : 76, 60% 0 or 10 mg/kg-day [0 or 0.01% ammonia in drinking water c ] for 24 weeks; both groups pretreated for 24 weeks with the
tumor initiator MNNG

Sprague Dawley rat, male; 40/group

Tsujii et al.,1992a Statistically significantly increased incidence of gastric cancers and number of gastric tumors per tumor-bearing rat in ammonia + MNNG group compared to MNNG only group

Gastric tumor incidence: 31, 70 d % # of gastric tumors/tumor-bearing rat: 1.3, 2.1 d

0 or 10 mg/kg-day [0 or 0.01% ammonia in drinking water c ] for 24 weeks; both groups pretreated for 24 weeks with the
tumor initiator MNNG

Sprague Dawley rat; male; 43-44/group

Tsujii et al., 1995 Statistically significantly increased incidence of gastric cancers, size, and penetration to deeper tissue layers in ammonia + MNNG group compared to MNNG only group

Gastric tumor incidence: 30, 66 d % Penetrated muscle layer or deeper : 12, 22 d % Size (mm): 4.4, 5.3 d

a Ammonium hydroxide doses estimated based on reporte d average daily drinking water intakes of 9.2, 8.2, and 6.5 mL/day for males and 8.3, 6.5, and 4.8 mL/day for f emales in the 0.1, 0.2, and 0.3% groups, respective ly, and assumed average default body weights of 37.3 and 35 .3 g for males and females, respectively (U.S. EPA, 1988).

b Ammonium hydroxide doses estimated based on reporte d average daily drinking water intakes of 7.9 and 8 .4 mL/day for males and females, respectively, and ass umed average default body weights of 37.3 and 35.3 g for males and females, respectively (U.S. EPA, 1988).

c Ammonia doses estimated based on reported drinking water intake of 50 mL/day and assumed average defau lt body weight of 523 g for male Sprague-Dawley rats d uring chronic exposure (U.S. EPA, 1988).

d Statistically significantly different from control.

1 

### A limited number of genotoxicity studies are availa ble for ammonia vapor, including one 2 

### study in exposed fertilizer factory workers in Indi a that reported chromosomal aberrations and 3 

### sister chromatid exchanges in lymphocytes (Yadav an d Kaushik, 1997), mutation assays in S. 4

### 33 DRAFT - DO NOT CITE OR QUOTE

typhimurium and E. coli (Shimizu et al., 1985; Demerec et al., 1951), a mi cronucleus assay in 1 

### mice (Yadav and Kaushik, 1997), studies in D. melanogaster (Auerbach and Robson, 1947; 2 

### Lobasov and Smirnov, 1934), and a chromosomal aberr ation test in chick fibroblast cells in vitro 3 

### (Rosenfeld, 1932) (see Appendix A, Section A.5). F our of the six available studies were 4 

### published between 1932 and 1951, and the available genotoxicity database in general is 5 

### inadequate to characterize the genotoxic potential of ammonia. 6

7 

## 1.1.7. Susceptible Populations and Life Stages 8 

### Studies of the toxicity of ammonia in children or y oung animals compared to other life 9 

### stages that would support an evaluation of childhoo d susceptibility have not been conducted. 10 

### Hyperammonemia is a condition of elevated levels of circulating ammonia that can occur 11


### in individuals with severe diseases of the liver or kidney, organs that biotransform and excrete 12 

### ammonia, or with hereditary urea cycle disorders (C órdoba et al., 1998; Schubiger et al., 1991; 13 

### Gilbert, 1988; Jeffers et al., 1988; Souba, 1987). The elevated ammonia levels that accompany 14


### human diseases such as acute liver or renal failure can predispose an individual to 15 

### encephalopathy due to the ability of ammonia to cro ss the blood-brain barrier; these effects are 16 

### especially marked in newborn infants (Miñana et al. , 1995; Souba, 1987). Thus, individuals with 17 

### disease conditions that lead to hyperammonemia may be more susceptible to the effects of 18 

### ammonia from external sources, but there are no stu dies that specifically support this 19 

### susceptibility. 20 

### Because the respiratory system is a target of ammon ia toxicity, individuals with 21 

### respiratory disease (e.g., asthmatics) might be exp ected to be a susceptible population; however, 22 

### controlled human studies that examined both healthy volunteers and volunteers with asthma 23 

### exposed to ammonia as well as cross-sectional studi es of livestock farmers exposed to ammonia 24 

### (Petrova et al., 2008; Sigurdarson et al., 2004; Vo gelzang et al., 2000, 1998, 1997; Preller et al., 25 

### 1995) generally did not observe a greater sensitivi ty to respiratory effects in populations with 26 

### underlying respiratory disease. 27

28 

## 1.2. Weight of Evidence Evaluation for Toxicologica l Effects 29 

### The available evidence for ammonia toxicity indicat es that respiratory effects are 30 

### associated with inhalation exposure and gastrointes tinal effects are associated with oral exposure 31 

### to ammonia. Ammonia exposure may not be associated with reproductive or developmental 32 

### toxicity, at least at levels in which respiratory a nd gastrointestinal effects are observed. Immune 33 

### system and other systemic effects (i.e., effects on the liver, kidney, heart, spleen, and adrenal 34 

### gland) may be associated with exposure to ammonia b ut are not sensitive targets of ammonia 35 

### toxicity. The evidence for these health effects ar e presented in more detail below. Figure 1-4 is 36

### 34 DRAFT - DO NOT CITE OR QUOTE

an exposure-response array comparing effect levels for inhaled ammonia across a range of 1 

### toxicological effect categories. 2

3 

### Respiratory Effects 4 

### Evidence for respiratory toxicity associated with e xposure to ammonia comes from 5


### studies in humans and animals. Cross-sectional occ upational studies involving chronic exposure 6 

### to ammonia have consistently demonstrated an increa sed prevalence of respiratory effects 7 

### (Rahman et al., 2007; Ballal et al., 1998) and decr eased lung function (Rahman et al., 2007; Ali., 8 

### 2001). Cross-sectional studies of livestock farmer s exposed to ammonia, controlled human 9 

### volunteer studies of ammonia inhalation, and case r eports of injury in humans with inhalation 10 

### exposure to ammonia provide additional and consiste nt support for the respiratory system as a 11 

### target of ammonia toxicity. 12 

### Short-term and subchronic animals studies show resp iratory effects in several animal 13 

### species (lung inflammation in guinea pigs and rats; focal or interstitial pneumonitis in monkeys, 14 

### dogs, rabbits and guinea pigs; pulmonary congestion in mice; thickening of nasal epithelium in 15 

### rats and pigs; nasal inflammation or lesions in rat s and mice) across different dose regimens and 16 

### show respiratory effects across ranges of concentra tions suggesting a dose-response (Coon et al., 17 

### 1970; Anderson et al., 1964; Broderson et al., 1976 ; Doig and Willoughby, 1971; Gaafar et al., 18 

### 1992). EPA considers the respiratory effects assoc iated with ammonia exposure to be 19 

### biologically plausible and adverse. The evidence o f observed respiratory effects seen across 20 

### multiple human and animal studies identifies the re spiratory system as a hazard for ammonia. 21

22 

### Gastrointestinal Effects 23 

### Effects on gastric mucosa associated with oral expo sure to ammonia are based on 24 

### evidence in animals and, to a more limited extent, in humans. Acute gastric toxicity observed in 25 

### case reports involving intentional or accidental in gestion of cleaning solutions or ammonia 26 

### inhalant capsules appears to reflect the corrosive properties of ammonia. Whether these acute 27 

### effects are relevant to toxicity following chronic low-level ammonia exposure is not known. 28 

### Indirect evidence is provided by the association be tween the stomach bacterium H. pylori , which 29 

### produces urease that catalyzes urea into ammonia, a nd human diseases of the upper 30 

### gastrointestinal tract (including chronic gastritis , gastric ulcers, and stomach cancer). 31 

### In vivo experimental evidence that ammonia is assoc iated with gastric effects is provided 32 

### by two short-term studies in male Sprague-Dawley ra ts (Tsujii et al., 1993; Kawano et al., 1991). 33 

### These studies provide consistent findings of decrea sed gastric mucosal thickness that increased 34 

### with ammonia dose (Kawano et al., 1991) and duratio n of exposure (Tsujii et al., 1993; Kawano 35 

### et al., 1991); Tsujii et al. (1993) employed only o ne ammonia drinking water concentration and 36 

### therefore did not provide information on dose-respo nse. Evidence for ammonia-related gastric 37 

### toxicity is limited to male rats of one strain and to investigations conducted by one research 38

### 35 DRAFT - DO NOT CITE OR QUOTE

group (Kawano et al. and Tsujii et al. were both af filiated with Osaka University Medical 1 

### School). 2 

### Mechanistic studies in rodent models support the bi ological plausibility that ammonia 3 

### exposure may be associated with gastric effects. C onditions that favor the unionized form of 4 

### ammonia facilitate the penetration of the cell memb rane and induce greater gastric toxicity. 5 

### Multiple specific mechanistic events have been prop osed that may contribute to the induction of 6 

### gastric lesions, including ammonia-induced release of proteases, inhibition of mitochondrial 7 

### respiration, and increased release of endothelin-1 and thyrotropin-releasing hormone. EPA 8 

### considers the gastric effects associated with ammon ia exposure to be biologically plausible and 9 

### adverse, and relevant to humans. Given the evidenc e from human, animal, and mechanistic 10 

### studies, gastric effects are identified as a hazard for ammonia. 11

12 

### Reproductive/Developmental Effects 13 

### No studies of the potential reproductive or develo pmental toxicity of ammonia in humans 14 

### are available, and only one animal study that exami ned the reproductive effects of ammonia in 15 

### the pig has been conducted. This study did not use a conventional test species and did not 16 

### include a control group with no ammonia exposure. Further, animals were exposed naturally to 17 

### bacterial and mycoplasm pathogens. Although the re productive and developmental toxicity 18 

### database for ammonia is limited, evidence on the en dogenous formation of ammonia can inform 19 

### the potential for ammonia to present a reproductive and developmental hazard. 20 

### Ammonia is endogenously produced in humans and ani mals during fetal and adult life 21 

### and concentrations in blood are homeostatically reg ulated to remain at low levels. Studies in 22 

### humans and animals demonstrate that ammonia is pres ent in fetal circulation. In vivo studies in 23 

### several animal species and in vitro studies of huma n placenta suggest that ammonia is produced 24 

### within the uteroplacenta and released into the feta l and maternal circulations (Bell et al., 1989; 25 

### Johnson et al., 1986; Haugel et al., 1983; Meschia et al., 1980; Remesar et al., 1980; Holzman et 26 

### al., 1979, 1977; Rubaltelli and Formentin, 1968; Lu schinsky, 1951). J όź wik et al. (2005) 27 

### reported that ammonia levels in human fetal blood ( specifically, umbilical arterial and venous 28 

### blood) at birth were 1.0–1.4 μg/mL, compared to 0.5 μg/mL in the mothers’ venous blood. 29 

### DeSanto et al. (1993) similarly collected human umb ilical arterial and venous blood at delivery 30 

### (range of 25–43 weeks of gestation). Ammonia was p resent in blood samples, with umbilical 31 

### arterial ammonia concentrations significantly highe r than venous concentrations; there was no 32 

### correlation between umbilical ammonia levels and ge stational age. In sheep, uteroplacental 33 

### tissues are the main site of ammonia production, wi th outputs of ammonia into both the uterine 34 

### and umbilical circulations (J όź wik et al., 1999). In late-gestation pregnant shee p that were 35 

### catheterized to allow measurement of ammonia exposu re to the fetus, concentrations of ammonia 36


### in umbilical arterial and venous blood and uterine arterial and venous blood ranged from about 37 

### 0.39 to 0.60 μg/mL (J όź wik et al., 2005, 1999). Thus, the developing fetu s and reproductive 38

### 36 DRAFT - DO NOT CITE OR QUOTE

tissues are normally exposed to ammonia in blood, a nd external concentrations that do not alter 1 

### homeostasis would not be expected to pose a developmental or reproductive hazard. 2

3 

### Immune System Effects 4 

### The evidence for ammonia immunotoxicity is based on two epidemiological studies and 5 

### four animal studies. Available epidemiological stu dies that addressed immunological function 6 

### are confounded by exposures to a number of other re spirable agents that have been demonstrated 7 

### to be immunostimulatory. Single-exposure human stu dies of ammonia evaluating immune 8 

### endpoints are not available. Therefore, human stud ies provide little support for ammonia 9 

### immunotoxicity. 10 

### Animal studies consistently provide evidence of el evated bacterial growth following 11 

### ammonia exposure. This is supported by observation s of lung lesions (Broderson et al., 1976), 12 

### elevated CFU (Schoeb et al., 1982), and increased m ortality (Richard et al., 1978a) in rats or 13 

### mice exposed to ammonia; however, the findings from the Broderson et al. (1976) study (% of 14 

### animals with gross lesions) were not dose-responsiv e, and the other studies used single 15 

### concentrations of ammonia and therefore did not pro vide information on dose-response. A 16 

### single study suggested that T cells are inhibited b y ammonia, but the data were not dose 17 

### responsive (Targowski et al, 1984). 18 

### Mechanistic data are not available that would suppo rt a biologically plausible mechanism 19 

### for immunosuppression. Because ammonia damages the protective mucosal epithelium of the 20 

### respiratory tract, it is unclear if elevated bacter ial colonization is the result of damage to this 21 

### barrier or the result of suppressed immunity. Over all, the evidence in humans and animals 22 

### indicates that ammonia exposure may be associated w ith these effects but does not support the 23 

### immune system as a sensitive target for ammonia tox icity. 24

25 

### Systemic Effects 26 

### Effects of ammonia exposure on organs distal from t he portal of entry are based on 27 

### evidence in animals and, to a more limited extent, in humans. One occupational epidemiology 28 

### study of ammonia-exposed workers reported changes i n serum enzymes indicative of altered 29 

### liver function (Hamid and El-Gazzar, 1996). Becaus e the study population was small and 30 

### measurements of workplace ammonia concentrations we re not provided, the evidence for liver 31 

### toxicity in humans associated with ammonia exposure is weak. 32 

### Effects on various organs, including liver, adrenal gland, kidney, spleen, and heart, were 33 

### observed in several studies that examined responses to ammonia exposure in a number of 34 

### laboratory species. While effects on many of these organs were observed in multiple species, 35 

### including monkey, dog, rabbit, guinea pig, and rat, effects were not consistent across exposure 36 

### protocols. For example, Coon et al. (1970) reporte d fatty liver and calcification and proliferation 37 

### of renal tubular epithelium in monkeys, dogs, rabbi ts, and guinea pigs exposed continuously to 38

### 37 DRAFT - DO NOT CITE OR QUOTE

ammonia for 90 days at a concentration of 470 mg/m 3 

### , but no histopathological changes in these 1 

### organs were observed in the same species following intermittent exposure (8 hours/day, 5 2 

### days/week for 6 weeks) to concentrations as high as 770 mg/m 3 

### . It could be speculated that these 3 

### differences in response reflect recovery from short -term (i.e., 8-hour exposures), but the reason 4 

### for the inconsistent findings is not known. 5 

### Studies of ammonia toxicity that examined systemic effects were all published in the 6 

### older toxicological literature. The only oral stud y of ammonium hydroxide was published in 7 

### 1939 (Fazekas, 1939), and three subchronic inhalati on studies were published between 1952 and 8 

### 1970 (Coon et al., 1970; Anderson et al., 1964; Wea therby, 1952). In general, the information 9 

### from these studies is limited by small group sizes, minimal characterization of some of the 10 

### reported responses (e.g., “congestion,” “enlarged,” “fatty liver”), insufficiently detailed reporting 11 

### of study results, and incomplete if any incidence d ata. In addition, Weatherby (1952), Anderson 12 

### et al. (1964), and some of the experiments reported by Coon et al. (1970) used only one ammonia 13 

### concentration in addition to the control, so no dos e-response information is available the majority 14 

### of experimental studies to inform the evidence for systemic effects of ammonia. 15 

### As discussed above, ammonia is endogenously produce d in all human and animal tissues, 16 

### and concentrations in all physiological fluids are homeostatically regulated to remain at low 17 

### levels (Souba, 1987). Thus, tissues are normally e xposed to ammonia, and external 18


### concentrations that do not alter homeostasis would not be expected to pose a hazard for systemic 19 

### effects. Overall, the evidence in humans and anima ls indicates that ammonia exposure may be 20 

### associated with these effects but does not support the liver, adrenal gland, kidney, spleen, or 21 

### heart as sensitive targets for ammonia toxicity. 22

2338 DRAFT - DO NOT CITE OR QUOTE12Figure 1-4. Exposure-response array for toxicological effects following inhalation exposure.30.11101001000 Prevalence of respiratory symptoms & decreased lung function (male occupational); Holness et al. (1989) Prevalence of respiratory symptoms & decreased lung function (occupational); Rahman et al. (2007) Nonspecific lung inflammation dyspnea, focal or interstitial pneumonitis & calcification of bronchial epithelia (rat); Coon et al. (1970) thickening tracheal epithelium (pig); Doig & Willoughby (1971) Increased incidence of gross lung lesions (rat); Broderson et al. (1976) Congestion of the liver (guinea pig); Weatherby (1952) Fatty liver changes in plate cells (rat); Coon et al. (1970) calcification & proliferation of renal tubular epithelia (rat); Coon et al. (1970) Myocardial fibrosis (rat); Coon et al. (1970) Ocular irritation (dog, rabbit); Coon et al. (1970)Respiratory effectsImmunologiceffectsSystemic effects

Exposure (mg/m 3 )LOAELNOAELFELIntermediate doseVertical lines showrange of exposures instudy. Closed circlesshow exposurestested in study.EXPERIMENTAL ANIMAL STUDIESHUMAN STUDIES

### 39 DRAFT - DO NOT CITE OR QUOTE

Cancer 1 

### The available information on carcinogenicity follow ing exposure to ammonia is limited 2


### to oral animal studies. There was no evidence of c arcinogenicity in Swiss or C 3 

### H mice 3 

### administered ammonium hydroxide in drinking water f or a lifetime (Toth, 1972). There is 4


### limited evidence that ammonia administered in drink ing water may act as a cancer promoter 5 

### based on the findings of studies designed to examin e H. pylori- induced gastric cancer (Tsujii et 6 

### al., 1995, 1992a). Additionally, the genotoxic pot ential cannot be characterized based on the 7 

### available genotoxicity information. Thus, under th e Guidelines for Carcinogen Risk Assessment 8 

### (U.S. EPA, 2005a), there is “inadequate information to assess the carcinogenic potential” of 9


### ammonia. 10

11

12

13

### 40 DRAFT - DO NOT CITE OR QUOTE

1 

# 2. DOSE-RESPONSE ANALYSIS 2

3



## 2.1. Oral Reference Dose for Effects other than Can cer 4 

### The RfD (expressed in units of mg/kg-day) is define d as an estimate (with uncertainty 5 

### spanning perhaps an order of magnitude) of a daily exposure to the human population (including 6 

### sensitive subgroups) that is likely to be without a n appreciable risk of deleterious effects during a 7 

### lifetime. It can be derived from a NOAEL, LOAEL, o r benchmark dose (BMD), with 8 

### uncertainty factors (UFs) generally applied to refl ect limitations of the data used. 9 

### The oral toxicity database for ammonia is very limi ted, although as noted in Section 1.2, 10 

### gastric toxicity is identified as a hazard for ammo nia based on evidence from case reports in 11 

### humans, two animal studies, and mechanistic studies . Evidence in humans is limited to case 12 

### reports of individuals suffering from gastrointesti nal (e.g., stomach ache, nausea, diarrhea, 13 

### distress, and burns along the digestive tract) effe cts from ingesting household cleaning solutions 14 

### containing ammonia or biting into capsules of ammon ia smelling salts. The data in humans were 15


### not considered for derivation of the RfD because al though case reports can suggest the nature of 16 

### acute endpoints in humans they are inadequate for d ose-response analysis and derivation of a 17


### chronic reference value due to short duration of ex posure and incomplete or missing quantitative 18 

### exposure information. 19 

### Two studies reported gastrointestinal effects, char acterized as increased epithelial cell 20 

### migration in the mucosa of the stomach (in particul ar the antrum) leading to a statistically 21 

### significant decrease in the thickness of the antral mucosa, in rats following subchronic (Tsujii et 22 

### al., 1993) and short-term (Kawano et al., 1991) ora l exposure to ammonia. These studies are 23 

### repeated dose studies that analyzed gastrointestina l effects of ammonia and did not evaluate a 24 

### comprehensive array of endpoints. Additionally, al though both studies included a control group, 25 

### Tsujii et al. (1993) employed one dose group and Ka wano et al. (1991) included two dose 26 

### groups. However, the decreased gastric antral muco sal thickness was consistently observed 27


### across these two studies. Prevalence of this effec t was observed to generally increase with dose 28 

### and duration, and the magnitude of decreases in thi ckness was 40-60%. Tsujii et al. (1993) and 29 

### Kawano et al. (1991) reported that the gastric muco sal effects observed in rats resemble mucosal 30 

### changes in human atrophic gastritis; indicating thi s effect is biological plausible and relevant to 31 

### humans. Therefore, decreased gastric antral mucosa l thickness is an effect considered by EPA to 32


### be adverse. 33


### Given the limited number of studies available and t he small number of toxicological 34 

### evaluations, there are uncertainties associated wit h the oral database for ammonia. Although the 35 

### oral database is limited, derivation of a RfD was c onsidered due to the toxicological significance 36

### 41 DRAFT - DO NOT CITE OR QUOTE

of the reported gastrointestinal effects. However, uncertainties with extrapolations from the 1 

### available data (described below) were too high to s upport derivation of a chronic RfD; thus, in 2


### consideration of the limited oral database and asso ciated uncertainties a RfD for ammonia was 3


### not derived

. 4 

### In considering the derivation of a RfD, the subchro nic study by Tsujii et al. (1993) was 5 

### considered as a potential principal study due to th e relatively longer duration of exposure 6 

### compared with the short-term study by Kawano et al. (1991). Decreased gastric antral mucosal 7 

### thickness was considered as a potential critical effect. This effect was characterized as a portal8 

### of-entry effect based on the following. Tsujii et al. (1993) postulated that the difference in 9 

### response of the mucosa in the stomach body versus t he mucosa of the antrum relates to 10 

### differences in pH in the two stomach regions. Most ammonia is transformed to ammonium ion 11 

### in solution at physiological pH; the ratio of ammon ia to ammonium ion increases 10-fold with 12 

### each unit rise in pH. In the mucosa of the stomach body—an acid-secreting mucosa—ammonia 13 

### is protonated to the ammonium ion, which reduces th e cytotoxicity associated with nonionized 14 

### ammonia. In the antral mucosa—a nonacid secreting area of the stomach—the pH is higher, 15 

### resulting in a relatively higher concentration of a mmonia and thus enhanced cytotoxicity. 16 

### EPA identified a potential point of departure (POD) based on the LOAEL of 33 mg/kg17 

### day, for decreased gastric antral mucosal thickness in rats, from this study. BMD modeling was 18 

### not utilized because the Tsujii et al. (1993) emplo yed only one dose level and a control, a data set 19 

### that is not amenable to dose-response analysis. 20


### In U.S. EPA’s Recommended Use of Body Weight 3/4 

### as the Default Method in Derivation 21 

### of the Oral Reference Dose (U.S. EPA, 2011a), the Agency endorses a hierarchy of approaches 22 

### to derive human equivalent oral exposures from data from laboratory animal species, with the 23 

### preferred approach being physiologically based phar macokinetic modeling. Other approaches 24 

### may include using some chemical-specific informatio n, without a complete physiologically 25 

### based pharmacokinetic model. In lieu of chemical-s pecific models or data to inform the 26 

### derivation of human equivalent oral exposures, EPA endorses body weight scaling to the 3⁄4 27 

### power (i.e., BW 3/4 

### ) as a default to extrapolate toxicologically equiv alent doses of orally 28 

### administered agents from laboratory animals to huma ns for the purpose of deriving a RfD. More 29 

### specifically, the use of BW 3/4 

### scaling for deriving a RfD is recommended when the observed 30 

### effects are associated with the parent compound or a stable metabolite, but not for portal-of-entry 31


### effects. 32 

### No PBPK model or chemical-specific information exis ts to inform the generation of 33 

### human equivalent oral exposures for ammonia. Furth ermore, because ammonia oral toxicity 34 

### appears to be a function of the physical/chemical e nvironment at the mucosal surface (i.e., a 35 

### portal-of-entry effect) and it is not clear if regi ons of the stomach scale allometrically across 36 

### species, a surface area adjustment would be the mos t relevant for interspecies extrapolation; 37 

### however, a dose scaling approach involving mass per unit surface area has not been developed 38

### 42 DRAFT - DO NOT CITE OR QUOTE

(U.S. EPA, 2011a). Therefore, because effects on t he gastric antral mucosa are not expected to 1 

### scale allometrically, a BW 3/4 

### scaling approach (in combination with a reduced de fault UF for 2 

### interspecies extrapolation) was not applied. 3 

### The composite UF for ammonia that would be applied to the POD (LOAEL of 33 mg/kg4 

### day) from the Tsujii et al. (1993) study would be 1 0,000, consisting of four areas of uncertainty. 5 

### These areas of uncertainty, and the UFs that addres s each, are based on EPA’s A Review of the 6 

### Reference Dose and Reference Concentration Processe s (U.S. EPA, 2002; Section 4.4.5) and 7 

### include the following: uncertainties associated wit h intraspecies extrapolation (i.e., to account for 8 

### human variability in susceptibility to ammonia; UF H 

### = 10), uncertainties associated with 9 

### extrapolation of data from the rat to humans in the absence of information on species differences 10 

### in toxicokinetics and toxicodynamics (i.e., intersp ecies extrapolation; UF A 

### = 10), uncertainties 11 

### associated with extrapolation of data from a subchr onic study (i.e., 8-week study) to a reference 12 

### value for chronic exposure scenarios (UF S 

### = 10), uncertainties associated with extrapolation 13 

### from a LOAEL to NOAEL (UF L 

### = 10), and database deficiencies (UF D 

### = 1; see Section 2.2.2 for 14 

### the justification for this UF). 15 

### In the report, A Review of the Reference Dose and Reference Concen tration 16



### Processes (U.S. EPA, 2002), the RfD/RfC technical panel conc luded that, in cases where 17 

### maximum uncertainty exists in four or more areas of uncertainty, or when the total UF is 18



### ≥ 10,000, it is unlikely that the database is suffici ent to derive a reference value. Therefore, 19 

### consistent with the recommendations in U.S. EPA (20 02), the available oral data for ammonia 20 

### were considered insufficient to support reference v alue derivation and an RfD for ammonia was 21


### not derived. 22 

### Route-to-route extrapolation from inhalation data w as considered for deriving the oral 23 

### RfD; however, in the absence of a PBPK model and be cause the critical effect from the 24 

### inhalation literature is a portal-of-entry effect ( respiratory irritation and decreased lung function)

, 25 

### route-to-route extrapolation is not supported (U.S. EPA, 1994). 26

27 

### Previous IRIS Assessment: Reference Dose 28 

### No RfD was derived in the previous IRIS assessment for ammonia 29

30



## 2.2. Inhalation Reference Concentration for Effects other than Cancer 31 

### The RfC (expressed in units of mg/m 3 

### ) is defined as an estimate (with uncertainty 32 

### spanning perhaps an order of magnitude) of a contin uous inhalation exposure to the human 33 

### population (including sensitive subgroups) that is likely to be without an appreciable risk of 34 

### deleterious effects during a lifetime. It can be d erived from a NOAEL, LOAEL, or benchmark 35 

### concentration (BMC), with UFs generally applied to reflect limitations of the data used. 36

### 43 DRAFT - DO NOT CITE OR QUOTE

As discussed in Section 1.2, respiratory effects ha ve been identified as a hazard following 1 

### inhalation exposure to ammonia. The studies in hum ans and animals examining inhalation 2 

### exposure to ammonia provide evidence that inhaled a mmonia is associated with toxicity to the 3 

### respiratory system. The experimental toxicology li terature for ammonia also provides evidence 4 

### that inhaled ammonia may be associated with toxicit y to target organs other than the respiratory 5 

### system, including the liver, adrenal gland, kidney, spleen, heart, and immune system. The 6 

### weight of evidence for these effects is less robust than for respiratory effects. Therefore, the 7 

### respiratory system is the primary and most sensitiv e target of inhaled ammonia toxicity in 8 

### humans and experimental animals. 9 

### Human data are preferred over animal data for deriv ing reference values when possible 10


### because the use of human data is more relevant in t he assessment of human health and avoids the 11 

### uncertainty associated with interspecies extrapolat ion introduced when animal data serve as the 12 

### basis for the RfC. Additionally, the respiratory e ffects in animals were observed at ammonia 13 

### concentrations higher than those associated with re spiratory effects in humans and represent 14 

### much shorter durations (up to 114 days) of exposure , and thus were considered to carry less 15 

### weight than the available human data. Therefore, d ata in humans were considered for derivation 16 

### of the RfC and the respiratory effects in animals w ere not further considered. 17 

### Of the available human data, two occupational studi es—Rahman et al. (2007) and 18 

### Holness et al. (1989)—provide information useful fo r examining the relationship between 19 

### chronic ammonia exposure and respiratory irritation and decreased lung function (quantitative 20 

### dose-response analysis of ammonia respiratory tract toxicity data). Both studies reported the 21 

### presence or absence of respiratory effects in worke rs exposed to ammonia over a range of 22 

### concentrations (approximately 1 to 7 mg/m 3 

### ). Both studies provide consistent estimates of th e 23 

### effect level for ammonia, with the NOAEL ADJ 

### of 3.1 mg/m 3 

### identified from the Holness et al. 24 

### (1989) study falling between the NOAEL ADJ 

### and LOAEL ADJ 

### values (1.8 and 6.6 mg/m 3



### , 25 

### respectively) from the Rahman et al. (2007) study. These studies are considered as candidate 26 

### principal studies for RfC derivation. Other occupa tional epidemiology studies (Ali et al., 2001; 27 

### Ballal et al., 1998) did not provide exposure infor mation adequate for dose-response analysis and 28


### thus were not useful for RfC derivation. 29 

### Consideration of analytical methods suggests that h igher confidence is associated with 30 

### the exposure measures reported by Holness et al. (1 989) than Rahman et al. (2007). Rahman et 31 

### al. (2007) used two analytical methods for measurin g ammonia concentrations in workplace air 32 

### (Dräger PAC III and Dräger tube); concentrations me asured by the two methods differed by 33 

### four- to fivefold, indicating some uncertainty in t hese measurements, although ammonia 34 

### concentrations measured by the two methods were str ongly correlated. In contrast, the Holness 35 

### et al. (1989) study used an established analytical method for measuring exposure to ammonia 36 

### recommended by NIOSH that involved the collection o f air samples on acid-treated silica gel 37 

### (ATSG) absorption tubes. 38

### 44 DRAFT - DO NOT CITE OR QUOTE

Due to the greater confidence in the ammonia measur ements in Holness et al. (1989) and 1 

### considering the range of NOAELs and LOAELs reported in both studies (in which a higher 2 

### NOAEL was reported by Holness et al. [1989]) the oc cupational exposure study of ammonia 3 

### exposure in workers in a soda ash plant by Holness et al. (1989) was identified as the princip al 4 

### study for RfC derivation . Respiratory effects, characterized as increased respiratory irritation 5 

### and decreased lung function, observed in workers ex posed to ammonia concentrations ≥ 6.6 6 

### mg/m 3 

### were selected as the critical effect. Respiratory effects, including changes in measures of 7 

### lung function and increased prevalence of wheezing, chest tightness, and cough/phlegm, have 8 

### been identified as adverse respiratory health effec ts by the American Thoracic Society (2000), 9 

### and are similarly noted as adverse in the EPA’s Methods for Derivation of Inhalation Reference 10 

### Concentrations and Application of Inhalation Dosime try (U.S. EPA, 1994). 11

12 

## 2.2.1. Methods of Analysis 13 

### In the evaluation of the prevalence of increased re spiratory irritation and decreased lung 14 

### function in workers exposed to ammonia (Holness et al., 1989), a NOAEL ADJ 

### of 3.1 mg/m 3 15 

### (adjusted for continuous exposure from 8.8 mg/m 3 

### ; see calculation below) was identified based 16 

### on the absence of statistically significant increas es in the prevalence of the respiratory effects. 17 

### BMD modeling was not utilized because ammonia conce ntrations in the Holness et al. (1989) 18 

### study were not associated with changes in respirato ry effects in the study population (i.e., data 19 

### from Holness et al. could not be subjected to dose- response modeling). Thus, the Holness et al. 20 

### (1989) data were analyzed using a NOAEL approach an d the NOAEL ADJ 

### of 3.1 mg/m 3 

### was 21


### used as the POD for RfC derivation

. 22


### Because the RfC is a measure that assumes continuou s human exposure over a lifetime, 23 

### the POD derived from an occupational exposure was a djusted to account for the noncontinuous 24 

### exposure associated with occupational exposure (i.e ., 8-hour workday and 5-day workweek). 25 

### The duration-adjusted POD was calculated as follows : 26

27


### NOAEL ADJ 

### = NOAEL × VEho/VEh × 5 days/7 days 28 

### = 8.8 mg/m 3 

### × 10 m 3 

### /20 m 3 

### × 5 days/7 days 29 

### = 3.1 mg/m 3 30

31

Where: 32 VEho = human occupational default minute volume (10 m 3 breathed during the 8-hour workday, 33 corresponding to a light to moderate activity level [U.S. EPA, 2011b]) 34 VEh = human ambient default minute volume (20 m 3 breathed during the entire day) 35

36

### 45 DRAFT - DO NOT CITE OR QUOTE



## 2.2.2. Derivation of Reference Concentration 1


### The UFs, selected based on EPA’s A Review of the Reference Dose and Reference 2


### Concentration Processes (U.S. EPA, 2002; Section 4.4.5) and described in t he Preamble of this 3 

### document, address five areas of uncertainty resulti ng in a composite UF of 10 . This composite 4 

### UF was applied to the selected POD (3.1 mg/m 3 

### ) to derive an RfC. 5

6



### • An intraspecies uncertainty factor, UF H 

### , of 10 was applied to account for potentially 7 

### susceptible individuals in the absence of data eval uating variability of response to inhaled 8 

### ammonia in the human population; 9
10



### • An interspecies uncertainty factor, UF A 

### , of 1 was applied to account for uncertainty in 11 

### extrapolating from laboratory animals to humans bec ause the POD was based on human 12 

### data from an occupational study; 13
14



### • A subchronic to chronic uncertainty factor, UF S 

### , of 1 was applied because the 15 

### occupational exposure period in the principal study (Holness et al., 1989), i.e., mean 16 

### number of years at present job for exposed workers, of approximately 12 years was of 17 

### chronic duration; 18
19



### • A LOAEL to NOAEL uncertainty factor, UF L 

### , of 1 was applied because a NOAEL value 20 

### was used as the POD; and 21
22



### • A database uncertainty factor, UF D 

### , of 1 was applied to account for deficiencies in t he 23 

### database. The ammonia inhalation database consists of studies of occupational exposure 24 

### focused on effects of ammonia on respiratory irrita tion and lung function, studies in 25 

### livestock farmers, controlled exposure studies invo lving volunteers exposed to ammonia 26 

### vapors for short periods of time to evaluate irrita tion effects and changes in lung function, 27 

### and a large number of case reports of acute exposur e to high ammonia concentrations 28 

### (e.g., accidental spills/releases). Studies of the toxicity of inhaled ammonia in 29 

### experimental animals include subchronic studies in rats, guinea pigs, and pigs that 30 

### examined respiratory and other systemic effects of ammonia and one limited, 31 

### reproductive toxicity study in young female pigs. The database lacks developmental and 32 

### multigeneration reproductive toxicity studies. 33
34


### As noted in EPA’s A Review of the Reference Dose and Reference Conce ntration 35 

### Processes (U.S. EPA, 2002), “the size of the database factor to be applied will depend on 36 

### other information in the database and on how much i mpact the missing data may have on 37 

### determining the toxicity of a chemical and, consequ ently, the POD.” Multigeneration 38 

### reproductive and developmental toxicity studies wou ld not be expected to impact the 39 

### determination of ammonia toxicity at the POD, and t herefore a database UF to account 40 

### for the lack of these studies is not necessary. Th is determination was based on the 41 

### observation that ammonia is endogenously produced a nd homeostatically regulated in 42 

### humans and animals during fetal and adult life. Ba seline blood levels in healthy 43 

### individuals range from 0.1 to 1.0 μg/mL (Monsen, 19 87; Conn, 1972; Brown et al., 44 

### 1957). The fetoplacental unit produces ammonia, an d concentrations in human umbilical 45 

### vein and artery blood (at term) have been shown to be higher than concentrations in 46

### 46 DRAFT - DO NOT CITE OR QUOTE

maternal blood (J όź wik et al., 2005), providing some assurance that de velopmental 1 

### toxicity would not be associated with concentration s of ammonia at or below the POD. 2 

### DeSanto et al. (1993) reported that human fetal umb ilical blood levels of ammonia at 3 

### birth were not influenced by gestational age based on deliveries ranging from gestation 4 

### week 25–43. Finally, evidence in animals (Manninen et al., 1988; Schaerdel et al., 1983) 5 

### suggests that exposure to ammonia at concentrations up to 18 mg/m 3 

### does not alter blood 6 

### ammonia levels (see Appendix A, Section A.3, for a more detailed discussion of 7 

### ammonia distribution and elimination). Accordingly , exposure at the POD (3.1 mg/m 3 

### ) 8 

### would not be expected to alter ammonia homeostasis or result in measureable increases in 9 

### blood ammonia concentrations. Thus, the concentrat ion of ammonia at the POD for the 10 

### RfC would not be expected to result in systemic tox icity, including reproductive or 11 

### developmental toxicity. 12
13


### The RfC for ammonia was calculated as follows: 14

15 

### RfC = NOAEL ADJ 

### ÷ UF 16 

### = 3.1 mg/m 3 

### ÷ 10 17 

### = 0.31 mg/m 3 

### or 0.3 mg/m 3 

### (rounded to one significant figure) 18
19 

## 2.2.3. Uncertainties in the Derivation of the RfC 20 

### As presented earlier in this section and in the Pre amble, EPA standard practices and RfC 21 

### guidance (U.S. EPA, 2002, 1995, 1994a, b) were foll owed in applying a UF approach to a POD 22 

### to derive the RfC. Specific uncertainties were acc ounted for by the application of UFs (i.e., in 23 

### the case of the ammonia RfC, a factor to address th e absence of data to evaluate the variability in 24 

### response to inhaled ammonia in the human population ). The following discussion identifies 25 

### additional uncertainties associated with the quanti fication of the RfC for ammonia. 26

27 

### Use of a NOAEL as a POD 28 

### Data sets that support BMD modeling are generally p referred for reference value 29 

### derivation because the shape of the dose-response c urve can be taken into account in establishing 30 

### the POD. For the ammonia RfC, no decreases in lung function or respiratory irritation were 31 

### observed in the worker population studied by Holnes s et al. (1989), i.e., the principal study used 32 

### to derive the RfC, and as such the data from this s tudy did not support dose-response modeling. 33 

### Rather, a NOAEL from the Holness et al. (1989) stud y was used to estimate the POD. The 34 

### availability of dose-response data from a single st udy of ammonia would increase the confidence 35


### in the estimation of the POD. 36

37 

### Endogenous ammonia 38 

### Ammonia, which is produced endogenously, has been d etected in the expired air of 39 

### healthy volunteers at levels generally ranging from 0.013 to 2.1 mg/m 3 

### (Boshier et al., 2010; 40 

### Smith et al., 2008; Spanel et al., 2007a, b; Turner et al., 2006; Diskin et al., 2003; Kearney et al., 41

### 47 DRAFT - DO NOT CITE OR QUOTE

2002; Smith et al., 1999; Norwood et al., 1992; Lar son et al., 1977). The higher and more 1 

### variable ammonia concentrations within this range a re reported in breath exhaled from the mouth 2 

### or oral cavity, with the majority of ammonia concen trations from these sources ranging from 3 

### 0.09 to 2.1 mg/m 3 

### (Smith et al., 2008; Spanel et al., 2007a, b; Turn er et al., 2006; Diskin et al., 4 

### 2003; Smith et al., 1999; Norwood et al., 1992; Lar son et al., 1977). Ammonia in exhaled breath 5 

### from the mouth or oral cavity is largely attributed to the production of ammonia via bacterial 6 

### degradation of food protein in the oral cavity or g astrointestinal tract (Turner et al., 2006; Smith 7 

### et al., 1999; Vollmuth and Schlesinger, 1984), and can be influenced by factors such as diet, oral 8 

### hygiene, age, and living conditions (i.e., urban vs . rural setting). In contrast, ammonia 9


### concentrations measured in breath exhaled from the nose and trachea are lower (range: 0.013– 10 

### 0.078 mg/m 3 

### ; Smith et al., 2008; Larson et al., 1977) and more likely reflect systemic levels of 11 

### ammonia (i.e., circulating levels in the blood) (Sm ith et al., 2008). 12


### Ammonia concentrations measured in breath exhaled f rom the nose and trachea, i.e., 13 

### concentrations expected to more closely correlate w ith circulating levels of ammonia in blood, 14 

### are lower than the ammonia RfC of 0.3 mg/m 3 

### by a factor of approximately fourfold or more; 15 

### however, the RfC does fall within the more variable range of breath concentrations collected 16 

### from the mouth or oral cavity. Although the contri bution of ammonia generated endogenously 17 

### and expired through exhalation to ammonia present i n ambient air is not known, this contribution 18 

### is expected to be minimal considering the ammonia i n expired air should rapidly mix with and be 19 

### diluted in the much larger volume of ambient air. 20

21 

## 2.2.4. Confidence Statement 22 

### A confidence level of high, medium, or low is assig ned to the study used to derive the 23 

### RfC, the overall database, and the RfC itself, as d escribed in Section 4.3.9.2 of EPA’s Methods 24 

### for Derivation of Inhalation Reference Concentratio ns and Application of Inhalation Dosimetry 25 

### (U.S. EPA, 1994b). Confidence in the principal study (Holness et al., 1989) is medium . The 26 

### design, conduct, and reporting of this occupational exposure study were adequate, but the study 27 

### was limited by a small sample size and by the fact that workplace ammonia concentrations to 28 

### which the study population was exposed were below t hose associated with ammonia-related 29 

### effects (i.e., only a NOAEL was identified). Howev er, this study is supported in the context of 30 

### the entire database, including the NOAEL and LOAEL values identified in the Rahman et al. 31 

### (2007) occupational exposure study, multiple studie s of acute ammonia exposure in volunteers, 32


### and the available inhalation data from animals. Confidence in the database is medium . The 33


### inhalation ammonia database includes limited studie s of reproductive toxicity and no studies of 34 

### developmental toxicity; however, reproductive, deve lopmental, and other systemic effects are 35 

### not expected at the RfC because it is well document ed that ammonia is endogenously produced 36 

### in humans and animals, ammonia concentrations in bl ood are homeostatically regulated to 37 

### remain at low levels, and ammonia concentrations in air at the POD are not expected to alter 38

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homeostasis. Reflecting medium confidence in the p rincipal study and medium confidence in 1 

### the database, the overall confidence in the RfC is medium

. 2

3 

## 2.2.5. Previous IRIS Assessment: Reference Concent ration 4 

### The previous IRIS assessment for ammonia (posted to the database in 1991) presented an 5 

### RfC of 0.1 mg/m 3 

### based on co-principal studies—the occupational exp osure study of workers in 6 

### a soda ash plant by Holness et al. (1989) and the s ubchronic study by Broderson et al. (1976) that 7 

### examined the effects of ammonia exposure in F344 ra ts inoculated on day 7 of the study with the 8


### bacterium M. pulmonis . The NOAEL of 6.4 mg/m 3 

### (estimated as the mean concentration of the 9 

### entire exposed group) from the Holness et al. (1989 ) study (duration adjusted: NOAEL ADJ 

### = 10 

### 2.3 mg/m 3 

### ) was used as the POD. 5 11 

### The previous RfC was derived by dividing the POD by a composite UF of 30: 10 to 12 

### account for the protection of sensitive individuals and 3 for database deficiencies to account for 13 

### the lack of chronic data, the proximity of the LOAE L from the subchronic inhalation study in the 14 

### rat (Broderson et al., 1976) to the NOAEL, and the lack of reproductive and developmental 15 

### toxicology studies. A UF D 

### of 3 (rather than 10) was applied because studies in rats (Schaerdel et 16 

### al., 1983) showed no increase in blood ammonia leve ls at an inhalation exposure to 32 ppm (22.6 17 

### mg/m 3 

### ) and only minimal increases at 300–1,000 ppm (212– 707 mg/m 3 

### ), suggesting that no 18 

### significant distribution is likely to occur at the human equivalent concentration. In this 19 

### document, a UF D 

### of one was selected because a more thorough invest igation of the literature on 20 

### ammonia homeostasis and literature published since 1991 on fetoplacental ammonia levels 21 

### provides further support that exposure to ammonia a t the POD would not result in a measureable 22 

### increase in blood ammonia, including fetal blood le vels. 23



## 2.3. Cancer Risk Estimates 24 

### The carcinogenicity assessment provides information on the carcinogenic hazard 25 

### potential of the substance in question and quantita tive estimates of risk from oral and inhalation 26 

### exposure may be derived. Quantitative risk estimate s may be derived from the application of a 27 

### low-dose extrapolation procedure. If derived, and u nless otherwise stated, the oral slope factor is 28 

### a plausible upper bound on the estimate of risk per mg/kg-day of oral exposure. Similarly, an 29 

### inhalation unit risk is a plausible upper bound on the estimate of risk per μ g/m 3 

### air breathed. 30 

### As discussed in Section 1.2, there is “inadequate information to assess the 31 

### carcinogenic potential” of ammonia. Therefore, a quantitative cancer assessment was not 32


### conducted and cancer risk estimates were not derive d for ammonia . The previous IRIS 33 

### assessment did not include a carcinogenicity assess ment. 34

5 In this document, the lower bound of the high expos ure category from the Holness et al. (1989) study ( 8.8 mg/m 3

, adjusted for continuous exposure to 3.1 mg/m 3 ) was identified as the POD because workers in this high exposure category, as well as those in the two lower exposur e categories, showed no statistically significant i ncrease in respiratory irritation or decreases in pulmonary fu nction.

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1 

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