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12 Toxicological Review of 

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

2 www.epa.gov/iris

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11 Toxicological Review of Ammonia 

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13 (CAS No. 7664-41-7) 

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17 February 2012

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25 NOTICE

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27 This document is an Interagency Science Consultation draft. This information is distributed 

28 solely for the purpose of pre-dissemination peer review under applicable information quality 

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

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

31 of its technical accuracy and science policy implications. 

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41 National Center for Environmental Assessment 

42 Office of Research and Development 

43 U.S. Environmental Protection Agency 

44 Washington, DC 

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1 DISCLAIMER

2 

3 

4 This document is a preliminary draft for review purposes only. This information is 

5 distributed solely for the purpose of pre-dissemination peer review under applicable 

6 information quality guidelines. It has not been formally disseminated by EPA. It does not 

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

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

9 recommendation for use. 
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1 

2 CONTENTS

3 

4 PREAMBLE .............................................................................................................................................. vi 

5 AUTHORS | CONTRIBUTORS | REVIEWERS ........................................................................................... xv 

6 PREFACE .............................................................................................................................................. xvii 

7 EXECUTIVE SUMMARY .......................................................................................................................... xx 

8 LITERATURE SEARCH STRATEGY & STUDY EVALUATION FOR HAZARD IDENTIFICATION .................. xxiv 

9 1. HAZARD IDENTIFICATION ................................................................................................................... 1 

10 1.1. Synthesis of Major Toxicological Effects ............................................................................. 1 

11 1.1.1. Respiratory Effects ............................................................................................ 1 

12 1.1.2. Gastrointestinal Effects ................................................................................... 14 

13 1.1.3. Reproductive and Developmental Effects ...................................................... 17 

14 1.1.4. Immune System Effects .................................................................................. 18 

15 1.1.5. Other Systemic Effects .................................................................................... 22 

16 1.1.6. Cancer ............................................................................................................. 31 

17 1.1.7. Susceptible Populations and Life Stages ......................................................... 33 

18 1.2. Weight of Evidence Evaluation for Toxicological Effects .................................................. 33 

19 2. DOSE-RESPONSE ANALYSIS .............................................................................................................. 40 

20 2.1. Oral Reference Dose for Effects other than Cancer.......................................................... 40 

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

22 2.2.1. Methods of Analysis ........................................................................................ 44 

23 2.2.2. Derivation of Reference Concentration .......................................................... 45 

24 2.2.3. Uncertainties in the Derivation of the RfC ...................................................... 46 

25 2.2.4. Confidence Statement .................................................................................... 47 

26 2.2.5. Previous IRIS Assessment: Reference Concentration ..................................... 48 

27 2.3. Cancer Risk Estimates ....................................................................................................... 48 

28 3. REFERENCES ..................................................................................................................................... 49 

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1 LIST OF TABLES 

2 

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

4 Table 1-2. Respiratory effects in animals following inhalation exposure .............................................. 9 

5 Table 1-3. Gastrointestinal effects in animals following oral exposure ............................................... 16 

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

7 Table 1-5. Immune system effects in animals following inhalation exposure ..................................... 20 

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

9 Table 1-7. Systemic effects in animals following oral exposure .......................................................... 24 

10 Table 1-8. Systemic effects in animals following inhalation exposure ................................................ 24 

11 Table 1-9. Cancer bioassays following oral exposure .......................................................................... 32 

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15 LIST OF FIGURES 

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17 Figure 1-1. Exposure-response array for respiratory effects following inhalation exposure .............. 13 

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

19 Figure 1-3. Exposure-response array for systemic effects following inhalation exposure .................. 30 

20 Figure 1-4. Exposure-response array for toxicological effects following inhalation exposure ........... 38 

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1 ABBREVIATIONS

2 

3 

4 ACGIH American Conference of Governmental 

5 Industrial Hygienists 

6 ALP alkaline phosphatase 

7 ALT alanine aminotransferase 

8 AST aspartate aminotransferase 

9 ATSDR Agency for Toxic Substances and Disease 

10 Registry 

11 ATSG acid-treated silica gel 

12 BAL bronchioalveolar lavage 

13 BMD benchmark dose 

14 BMI body mass index 

15 BrDU 5-bromo-2-deoxyuridine 

16 BUN blood urea nitrogen 

17 CAC cumulative ammonia concentration 

18 CASRN Chemical Abstracts Service Registry 

19 Number 

20 CI confidence interval 

21 EPA Environmental Protection Agency 

22 EU endotoxin unit 

23 FEF forced expiratory flow 

24 FEV1 forced expiratory volume in 1 second 

25 FVC forced vital capacity 

26 GABA γ-amino butyric acid 

27 IgE immunoglobin E 

28 IgG immunoglobin G 

29 IRIS Integrated Risk Information System 

30 LOAEL lowest-observed-adverse-effect level 

31 MAO monoamine oxidase 

32 MMEF mean midexpiratory flow 

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

34 MRM murine respiratory mycoplasmosis 

35 NH3 ammonia 

NH4

+

36 ammonium ion 

37 NIOSH National Institute for Occupational Safety 

38 and Health 

39 NOAEL no-observed-adverse-effect level 

40 NOx nitrogen oxide 

41 NRC National Research Council 

42 OR odds ratio 

43 PBPK physiologically based pharmacokinetic 

44 PEF peak expiratory flow 

45 PEFR peak expiratory flow rate 

46 PHA phytohemagglutin 

47 POD point of departure 

48 PPD purified protein derivative 

49 RfC inhalation reference concentration 

50 RfD oral reference dose 

51 RNA ribonucleic acid 

52 SD standard deviation 

53 SIFT-MS selected ion flow tube mass spectrometry 

54 TLV threshold limit value 

55 TWA time-weighted average 

56 UF uncertainty factor 

57 UFA interspecies uncertainty factor 

58 UFH intraspecies uncertainty factor 

59 UFL LOAEL to NOAEL uncertainty factor 

60 UFS subchronic-to-chronic uncertainty factor 

61 UFD database deficiencies uncertainty factor 

62 

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1 

2 

3 PREAMBLE 

4 

5 

6 1. Scope of the IRIS Program 

7 Soon after EPA was established in 1970, it was at 

8 the forefront of developing risk assessment as a 

9 science and applying it in decisions to protect human 

10 health and the environment. The Clean Air Act, for 

11 example, mandates that EPA provide “an ample 

12 margin of safety to protect public health”; the Safe 

13 Drinking Water Act, that “no adverse effects on the

14 health of persons may reasonably be anticipated to 

15 occur, allowing an adequate margin of safety.” 

16 Accordingly, EPA relies on health assessments to 

17 identify adverse effects and exposure levels below 

18 which these effects are not anticipated to occur. 

19 IRIS assessments critically review the publicly 

20 available studies to identify adverse health effects of 

21 chemicals and to characterize exposure-response 

22 relationships. Exceptions are chemicals currently used 

23 exclusively as pesticides, ionizing and non-ionizing 

24 radiation, and criteria air pollutants listed under

25 section 108 of the Clean Air Act (carbon monoxide, 

26 lead, nitrogen oxides, ozone, particulate matter, and 

27 sulfur oxides; EPA evaluates these in Integrated 

28 Science Assessments). An assessment may cover a 

29 single chemical, a group of structurally or 

30 toxicologically related chemicals, or a complex 

31 mixture. 

32 Once a year, the IRIS Program asks EPA 

33 programs and regions, other federal agencies, state

34 governments, and the general public to nominate 

35 chemicals and mixtures for future assessment or 

36 reassessment. These agents may be found in air, water, 

37 soil, or sediment. Selection is based on program and 

38 regional office priorities and on availability of 

39 adequate information to evaluate the potential for 

40 adverse effects. IRIS can assess other agents as an

41 urgent public health need arises. IRIS also reassesses 

42 agents as significant new data are published. 

43 2. Process for developing and peer44 reviewing IRIS assessments 

45 The process for developing IRIS assessments 

46 (revised in May 2009) involves systematic review of

47 the pertinent studies, opportunities for public input, 

48 and multiple levels of scientific review. EPA revises 

49 draft assessments after each review, and external drafts 

50 and comments become part of the public record (U.S.

51 EPA, 2009). 

52 Step 1. Development of a draft Toxicological 

53 Review (usually about 11-1/2 months duration). 

54 The draft assessment considers all pertinent 

55 publicly available studies and applies consistent 

56 criteria to evaluate the studies, identify health 

57 effects, weigh the evidence of causation for each 

58 effect, identify mechanistic events and pathways, 

59 and derive toxicity values. 

60 Step 2. Internal review by scientists in EPA 

61 programs and regions (2 months). The draft 

62 assessment is revised to address comments from 

63 within EPA. 

64 Step 3. Interagency science consultation with other

65 federal agencies and White House offices (1-1/2 

66 months). The draft assessment is revised to 

67 address the interagency comments. The science 

68 consultation draft, interagency comments, and 

69 EPA’s response to major comments become part 

70 of the public record. 

71 Step 4. External peer review, after public review 

72 and comment (3-1/2 months or more, depending 

73 on the review process). EPA releases the draft 

74 assessment for public review and comment, 

75 followed by external peer review. The peer review 

76 meeting is open to the public and includes time 

77 for oral public comments. The peer reviewers also 

78 receive the written public comments. The peer 

79 reviewers assess whether the evidence has been 

80 assembled and evaluated according to guidelines 

81 and whether the conclusions are justified by the 

82 evidence. The peer review draft, peer review 

83 report, and written public comments become part 

84 of the public record. 

85 Step 5. Revision of draft Toxicological Review and 

86 development of draft IRIS summary

87 (2 months). The draft assessment is revised to 

88 reflect the peer review comments, public 

89 comments, and newly available studies. The 

90 disposition of peer review comments and public 

91 comments becomes part of the public record. 

92 Step 6. Final EPA review and interagency science 

93 discussion with other federal agencies and 

94 White House offices (1-1/2 months). The draft 

95 assessment and summary are revised to address 

96 EPA and interagency comments. The science 

97 discussion draft, written interagency comments, 

98 and EPA’s response to major comments become 

99 part of the public record. 
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1 Step 7. Completion and posting (1 month). The 

2 Toxicological Review and IRIS summary are 

3 posted on the IRIS website (http://www.epa.gov/

4 iris/). 

5 

6 The remainder of this Preamble addresses step 1, 

7 the development of a draft Toxicological Review. IRIS 

8 assessments follow standard practices of evidence 

9 evaluation and peer review, many of which are 

10 discussed in EPA guidelines (U.S. EPA, 1986a, 

11 1986b, 1991, 1996, 1998, 2000a, 2005a, 2005b, 

12 2006a) and other descriptions of “best practices” (U.S. 

13 EPA, 1994, 2000b, 2002, 2006b, 2011a). Transparent 

14 application of scientific judgment is of paramount 

15 importance. To provide a harmonized approach across

16 IRIS assessments, this Preamble summarizes concepts

17 from these guidelines and emphasizes principles of 

18 general applicability. 

19 3. Identifying and selecting pertinent 

20 studies 

21 3.1 Identifying studies 

22 Before beginning an assessment, EPA conducts a 

23 comprehensive search of the primary scientific 

24 literature. The literature search follows standard 

25 practices and includes the PubMed and ToxNet 

26 databases of the National Library of Medicine and 

27 other databases listed in EPA’s HERO system (Health

28 and Environmental Research Online, http://

29 hero.epa.gov/). Each assessment specifies the search 

30 strategies, keywords, and cut-off dates of its literature 

31 searches. EPA posts the results of the literature search 

32 on the IRIS website and requests information from the 

33 public on additional studies and ongoing research. 

34 Each assessment also considers studies received 

35 through the IRIS Submission Desk and studies 

36 (typically unpublished) submitted to EPA under the 

37 Toxic Substances Control Act. If a study that may be 

38 critical to the conclusions of the assessment has not 

39 been peer-reviewed, EPA will have it peer-reviewed.

40 EPA also examines the toxicokinetics of the agent 

41 to identify other chemicals (for example, major 

42 metabolites of the agent) to include in the assessment 

43 if adequate information is available, in order to more 

44 fully explain the toxicity of the agent and to suggest 

45 dose metrics for subsequent modeling. 

46 In assessments of chemical mixtures, mixture 

47 studies are preferred for their ability to reflect 

48 interactions among components (U.S. EPA, 1986a, 

49 2000a). The literature search seeks, in decreasing 

50 order of preference: 

51 – Studies of the mixture being assessed. 

52 – Studies of a sufficiently similar mixture. In 

53 evaluating similarity, the assessment considers the

54 alteration of mixtures in the environment through 

55 partitioning and transformation. 

56 – Studies of individual chemical components of the 

57 mixture, if there are not adequate studies of 

58 sufficiently similar mixtures. 

59 3.2 Selecting pertinent epidemiologic studies 

60 Study design is the key consideration for selecting

61 pertinent epidemiologic studies from the results of the 

62 literature search. 

63 – Cohort studies and case-control studies provide 

64 the strongest epidemiologic evidence, as they 

65 collect information about individual exposures 

66 and disease. 

67 – Cross-sectional studies provide useful evidence if 

68 they relate exposures and disease at the individual

69 level and it is clear that exposure preceded the 

70 onset of disease. 

71 – Ecologic studies (geographic correlation studies)

72 relate exposures and disease by geographic area. 

73 They can provide strong evidence if there are 

74 large exposure contrasts between geographic 

75 areas, relatively little exposure variation within 

76 study areas, and population migration is limited. 

77 – Case reports of high or accidental exposure lack 

78 definition of the population at risk and the 

79 expected number of cases. They can provide 

80 information about a rare disease or about the 

81 relevance of analogous results in animals. 

82 The assessment briefly reviews ecologic studies 

83 and case reports but includes details only if they 

84 suggest effects not identified by other epidemiologic 

85 studies. 

86 3.3 Selecting pertinent experimental studies 

87 Exposure route is a key design consideration for 

88 selecting pertinent experimental studies from the 

89 results of the literature search. 

90 – Studies of oral, inhalation, or dermal exposure 

91 involve passage through an absorption barrier and 

92 are considered most pertinent to human 

93 environmental exposure. 

94 – Injection or implantation studies are often 

95 considered less pertinent but may provide 

96 valuable toxicokinetic or mechanistic information. 

97 They also may be useful for identifying effects in 

98 animals if deposition or absorption is problematic 

99 (for example, for particles and fibers). 

100 Exposure duration is also a key design 

101 consideration for selecting pertinent experimental 

102 studies. 

103 – Studies of effects from chronic exposure are most

104 pertinent to lifetime human exposure. 

105 – Studies of effects from subchronic exposure are 

106 pertinent but less preferred than studies of chronic 

107 exposure. 
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1 – Short-term and acute studies are less pertinent but 

2 are useful for obtaining toxicokinetic or 

3 mechanistic information. The assessment reviews 

4 short-term and acute studies if they suggest 

5 distribution or effects at a site not identified by

6 longer-term studies. 

7 – For developmental toxicity and reproductive 

8 toxicity, irreversible effects may result from a 

9 brief exposure during a critical period of 

10 development. Accordingly, specialized study 

11 designs are used for these effects (U.S. EPA, 

12 1991, 1996, 1998). 

13 4. Evaluating the quality of individual 

14 studies 

15 4.1 Evaluating the quality of epidemiologic 

16 studies

17 The assessment evaluates design and 

18 methodologic aspects that can increase or decrease the 

19 weight given to each epidemiologic study in the 

20 overall evaluation (U.S. EPA, 1991, 1994, 1996a, 

21 1998, 2005a): 

22 – Documentation of study design, methods, 

23 population characteristics, and results. 

24 – Definition and selection of the study and 

25 comparison populations. 

26 – Ascertainment of exposure and the potential for 

27 misclassification. 

28 – Ascertainment of disease or effect and the 

29 potential for misclassification. 

30 – Duration of exposure and follow-up and adequacy 

31 for assessing the occurrence of effects, including 

32 latent effects. 

33 – Characterization of exposure during critical 

34 periods for the development of effects. 

35 – Sample size and statistical power to detect 

36 anticipated effects. 

37 – Participation rates and the resulting potential for 

38 selection bias. 

39 – Potential confounding and other sources of bias 

40 are identified and addressed in the study design or

41 in the analysis of results. The basis for 

42 consideration of confounding is a reasonable 

43 expectation that the confounder is prevalent in the

44 population and is related to both exposure and 

45 outcome. 

46 For developmental toxicity, reproductive toxicity, 

47 neurotoxicity, and cancer there is further guidance on 

48 the nuances of evaluating epidemiologic studies of 

49 these effects (U.S. EPA, 1991, 1996, 1998, 2005a). 

50 4.2 Evaluating the quality of experimental 

51 studies

52 The assessment evaluates design and 

53 methodologic aspects that can increase or decrease the 

54 weight given to each experimental study in the overall 

55 evaluation (U.S. EPA, 1991, 1994, 1996, 1998, 

56 2005a): 

57 – Documentation of study design, animals or study 

58 population, methods, basic data, and results. 

59 – Relevance of the animal model or study 

60 population and the experimental methods. 

61 – Characterization of the nature and extent of 

62 impurities and contaminants of the administered 

63 chemical or mixture. 

64 – Characterization of dose and dosing regimen 

65 (including age at exposure) and their adequacy to 

66 elicit adverse effects, including latent effects. 

67 – Sample sizes and statistical power to detect dose68 related differences or trends. 

69 – Ascertainment of survival, vital signs, disease or 

70 effects, and cause of death. 

71 – Control of other variables that could influence the 

72 occurrence of effects. 

73 The assessment uses statistical tests to evaluate 

74 whether the observations may be due to chance. The 

75 standard for determining statistical significance of a 

76 response is a trend test or comparison of outcomes in 

77 the exposed groups against those of concurrent 

78 controls. In some situations, examination of historical 

79 control data from the same laboratory within a few 

80 years of the study may improve the analysis. For an

81 uncommon effect that is not statistically significant 

82 compared with concurrent controls, historical controls 

83 may show that the effect is unlikely to be due to 

84 chance. For a response that appears significant against 

85 a concurrent control response that is unusual, historical 

86 controls may offer a different interpretation (U.S.

87 EPA, 2005a). 

88 For developmental toxicity, reproductive toxicity, 

89 neurotoxicity, and cancer there is further guidance on 

90 the nuances of evaluating experimental studies of 

91 these effects (U.S. EPA, 1991, 1996, 1998, 2005a). In 

92 multi-generation studies, agents that produce 

93 developmental effects at doses that are not toxic to the 

94 maternal animal are of special concern. Effects that 

95 occur at doses associated with mild maternal toxicity 

96 are not assumed to result only from maternal toxicity. 

97 Moreover, maternal effects may be reversible, while

98 effects on the offspring may be permanent (U.S. EPA, 

99 1991, 1998). 

100 4.3 Reporting study results 

101 The assessment uses evidence tables to report 

102 details of the design and key results of pertinent 
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1 studies. There may be separate tables for each site of 

2 toxicity or type of study. 

3 If a large number of studies observe the same 

4 effect, the assessment considers the study 

5 characteristics in this section to identify the strongest 

6 studies or types of study. The tables report details 

7 from these studies, and the assessment explains the

8 reasons for not reporting details of other studies or 

9 groups of studies that do not add new information. 

10 Supplemental material provides references to all 

11 studies considered, including those not summarized in 

12 the tables. 

13 The assessment discusses strengths and 

14 limitations that affect the interpretation of each study. 

15 If the interpretation of a study in the assessment differs 

16 from that of the study authors, the assessment 

17 discusses the basis for the difference. 

18 As a check on the selection and evaluation of 

19 pertinent studies, EPA asks peer reviewers to identify 

20 studies that were not adequately considered. 

21 5. Weighing the overall evidence of 

22 each effect 

23 5.1 Weighing epidemiologic evidence 

24 For each effect, the assessment evaluates the 

25 evidence from the epidemiologic studies as a whole to 

26 determine the extent to which any observed 

27 associations may be causal. Positive, negative, and

28 null results are given weight according to study 

29 quality. This evaluation considers aspects of an 

30 association that suggest causality, discussed by Hill 

31 (1965) and elaborated by Rothman and Greenland 

32 (1998) (U.S. EPA, 1994, 2002, 2005a; DHHS, 2004). 

33 Strength of association: The finding of a large 

34 relative risk with narrow confidence intervals 

35 strongly suggests that an association is not due to

36 chance, bias, or other factors. Modest relative 

37 risks, however, may reflect a small range of 

38 exposures, an agent of low potency, an increase in 

39 a disease that is common, exposure 

40 misclassification, or other sources of bias. 

41 Consistency of association: An inference of causality 

42 is strengthened if elevated risks are observed in 

43 independent studies of different populations and 

44 exposure scenarios. Reproducibility of findings 

45 constitutes one of the strongest arguments for 

46 causality. Discordant results sometimes reflect 

47 differences in exposure or in confounding factors. 

48 Specificity of association: As originally intended, this 

49 refers to one cause associated with one disease. 

50 Current understanding that many agents cause 

51 multiple diseases and many diseases have 

52 multiple causes make this a less informative 

53 aspect of causality, unless the effect is rare or 

54 unlikely to have multiple causes. 

55 Temporal relationship: A causal interpretation 

56 requires that exposure precede development of the 

57 disease. 

58 Biologic gradient (exposure-response relationship):

59 Exposure-response relationships strongly suggest 

60 causality. A monotonic increase is not the only 

61 pattern consistent with causality. The presence of 

62 an exposure-response gradient also weighs against 

63 bias and confounding as the source of an 

64 association. 

65 Biologic plausibility: An inference of causality is 

66 strengthened by data demonstrating plausible 

67 biologic mechanisms, if available. 

68 Coherence: An inference of causality is strengthened 

69 by supportive results from animal experiments, 

70 toxicokinetic studies, and short-term tests. 

71 Coherence may also be found in other lines of 

72 evidence, such as changing disease patterns in the 

73 population. 

74 “Natural experiments”: A change in exposure that 

75 brings about a change in disease frequency 

76 provides strong evidence of causality. 

77 Analogy: Information on structural analogues or on 

78 chemicals that induce similar mechanistic events 

79 can provide insight into causality. 

80 These considerations are consistent with 

81 contemporary guidelines that evaluate the quality and 

82 weight of evidence. Confidence is increased if the 

83 magnitude of effect is large, if there is evidence of an 

84 exposure-response relationship, or if an association 

85 was observed and the plausible biases would tend to

86 decrease the magnitude of the reported effect. 

87 Confidence is decreased for study limitations, 

88 inconsistency of results, indirectness of evidence,

89 imprecision, or reporting bias (Guyatt et al., 2008a,b). 

90 To make clear how much the epidemiologic 

91 evidence contributes to the overall weight of the 

92 evidence, the assessment may choose a descriptor such 

93 as sufficient evidence, suggestive evidence, inadequate 

94 evidence, or evidence suggestive of no causal 

95 relationship to characterize the epidemiologic 

96 evidence of each effect (DHHS, 2004). 

97 5.2 Weighing experimental evidence 

98 For each effect, the assessment evaluates the 

99 evidence from the animal experiments as a whole to 

100 determine the extent to which they indicate a potential 

101 for effects in humans. Consistent results across various 

102 species and strains increase confidence that similar 

103 results would occur in humans. Although causality is 

104 not at issue in controlled experiments, several concepts 

105 discussed by Hill (1965) affect the weight of 

106 experimental results: consistency of response, dose107 response relationships, strength of response, biologic 

108 plausibility, and coherence (U.S. EPA, 1994, 2002, 

109 2005a). 
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1 In weighing evidence from multiple experiments, 

2 EPA (2005a) distinguishes 

3 Conflicting evidence (that is, mixed positive and 

4 negative results in the same sex and strain using a

5 similar study protocol) from 

6 Differing results (that is, positive results and negative 

7 results are in different sexes or strains or use 

8 different study protocols). 

9 Negative or null results do not invalidate positive

10 results in a different experimental system. EPA 

11 regards all as valid observations and looks to 

12 mechanistic information, if available, to reconcile

13 differing results. 

14 It is well established that there are critical periods 

15 for some developmental and reproductive effects. 

16 Accordingly, the assessment determines whether 

17 critical periods have been adequately investigated 

18 (U.S. EPA, 1991, 1996, 1998, 2005a, 2005b). 

19 Similarly, the assessment determines whether the 

20 database is adequate to evaluate other critical sites and 

21 effects. 

22 5.3 Characterizing modes of action 

23 For each effect, the assessment discusses the 

24 available information on its modes of action and 

25 associated key events (key events being empirically 

26 observable, necessary precursor steps or biologic 

27 markers of such steps; mode of action being a series of 

28 key events involving interaction with cells, operational 

29 and anatomic changes, and resulting in disease). 

30 Pertinent information may also come from studies of

31 metabolites or of compounds that are structurally 

32 similar or that act through similar mechanisms. The

33 assessment addresses several questions about each 

34 hypothesized mode of action (U.S. EPA, 2005a). 

35 (a) Is the hypothesized mode of action sufficiently 

36 supported in test animals? Strong support for a 

37 key event being necessary to a mode of action can 

38 come from experimental challenge to the 

39 hypothesized mode of action, where suppressing a 

40 key event suppresses the disease. Support for a 

41 mode of action is meaningfully strengthened by 

42 consistent results in different experimental 

43 models, but not by replicate experiments in the 

44 same model. The assessment may consider 

45 various aspects of causality in addressing this 

46 question. 

47 (b) Is the hypothesized mode of action relevant to 

48 humans? The assessment reviews the key events 

49 to identify critical similarities and differences 

50 between the test animals and humans. Site 

51 concordance is not assumed between animals and 

52 humans, though it may hold for certain modes of 

53 action. Information suggesting quantitative 

54 differences is considered in dose-response 

55 analyses but is not used to determine relevance. 

56 Similarly, anticipated levels of human exposure 

57 are not used to determine relevance. 

58 (c) Which populations or life-stages can be 

59 particularly susceptible to the hypothesized 

60 mode of action? The assessment reviews the key 

61 events to identify populations and life-stages that

62 might be susceptible to their occurrence. 

63 Quantitative differences may result in separate 

64 toxicity values for susceptible populations or life65 stages. 

66 The assessment discusses the likelihood that an 

67 agent operates through multiple modes of action. An

68 uneven level of support for different modes of action 

69 can reflect disproportionate resources spent 

70 investigating them (U.S. EPA, 2005a). It should be 

71 noted that in clinical reviews, the quality of evidence 

72 may be reduced if evidence is limited to studies 

73 funded by one interested sector (Guyatt et al., 2008b). 

74 Studies of genetic toxicity are often available, and 

75 the assessment evaluates the evidence of a mutagenic 

76 mode of action. 

77 – Demonstration of gene mutations, chromosome 

78 aberrations, or aneuploidy in humans or 

79 experimental mammals (in vivo) provides the 

80 strongest evidence. 

81 – This is followed by positive results in lower 

82 organisms or in cultured cells (in vitro) or for 

83 other genetic events. 

84 – Negative results carry less weight, partly because 

85 they cannot exclude the possibility of effects in 

86 other tissues (IARC, 2006). 

87 For germ-cell mutagenicity, EPA has defined 

88 categories of evidence, ranging from positive results of 

89 human germ-cell mutagenicity to negative results for 

90 all effects of concern (U.S. EPA, 1986b). 

91 5.4 Characterizing the overall weight of the 

92 evidence 

93 After weighing the epidemiologic and 

94 experimental studies pertinent to each effect, the 

95 assessment may select a standard descriptor to 

96 characterize the overall weight of the evidence. For 

97 example, the following standard descriptors combine

98 epidemiologic, experimental, and mechanistic 

99 evidence of carcinogenicity (U.S. EPA, 2005a). 

100 Carcinogenic to humans: There is convincing 

101 epidemiologic evidence of a causal association 

102 (that is, there is reasonable confidence that the 

103 association cannot be fully explained by chance, 

104 bias, or confounding); or there is strong human 

105 evidence of cancer or its precursors, extensive 

106 animal evidence, identification of key precursor 

107 events in animals, and strong evidence that they 

108 are anticipated to occur in humans. 
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1 Likely to be carcinogenic to humans: The evidence 

2 demonstrates a potential hazard to humans but 

3 does not meet the criteria for carcinogenic. There 

4 may be a plausible association in humans, 

5 multiple positive results in animals, or a 

6 combination of human, animal, or other 

7 experimental data. 

8 Suggestive evidence of carcinogenic potential: The 

9 data raise concern for effects in humans but are 

10 not sufficient for a stronger conclusion. This 

11 descriptor covers a range of evidence, from a 

12 positive result in the only available study to a 

13 single positive result in an extensive database that 

14 includes negative results in other species. 

15 Inadequate information to assess carcinogenic 

16 potential: No other descriptors apply. Conflicting 

17 evidence can be classified as inadequate 

18 information if all positive results are opposed by 

19 negative studies of equal quality in the same sex 

20 and strain. Differing results, however, can be 

21 classified as suggestive evidence or as likely to be 

22 carcinogenic. 

23 Not likely to be carcinogenic to humans: There are 

24 robust data for concluding that there is no basis 

25 for concern. There may be no effects in both sexes 

26 of at least two appropriate animal species; positive 

27 animal results and strong, consistent evidence that

28 each mode of action in animals does not operate 

29 in humans; or convincing evidence that effects are 

30 not likely by a particular exposure route or below 

31 a defined dose. 

32 6. Selecting studies for derivation of 

33 toxicity values 

34 For each effect associated with an agent, the 

35 assessment derives toxicity values if there are suitable 

36 epidemiologic or experimental data. The derivation of 

37 toxicity values may be linked to the weight-of38 evidence descriptor. For example, EPA typically 

39 derives toxicity values for agents classified as 

40 carcinogenic to humans or likely to be carcinogenic, 

41 but not for agents with inadequate information or that 

42 are not likely to be carcinogenic (U.S. EPA, 2005a). 

43 Dose-response analysis requires quantitative 

44 measures of dose and response. Then, other factors 

45 being equal (U.S. EPA, 1994, 2005a): 

46 – Epidemiologic studies are preferred over animal 

47 studies, if quantitative measures of exposure are 

48 available and effects can be attributed to the 

49 agent. 

50 – Among experimental animal models, those that 

51 respond most like humans are preferred, if the 

52 comparability of response can be determined. 

53 – Studies by a route of human environmental 

54 exposure are preferred, although a validated 

55 toxicokinetic model can be used to extrapolate 

56 across exposure routes. 

57 – Studies of longer exposure duration and follow-up

58 are preferred, to minimize uncertainty about 

59 whether effects are representative of lifetime 

60 exposure. 

61 – Studies with multiple exposure levels are 

62 preferred for their ability to provide information 

63 about the shape of the exposure-response curve. 

64 – Studies that show an exposure-response gradient 

65 are preferred, as long as lack of a monotonic 

66 relationship at higher exposure levels can be 

67 satisfactorily explained by factors such as 

68 competing toxicity, saturation of absorption or 

69 metabolism, misclassification bias, or selection 

70 bias. 

71 – Among studies that show an exposure-response 

72 gradient, those with adequate power to detect 

73 effects at lower exposure levels are preferred, to 

74 minimize the extent of extrapolation to levels 

75 found in the environment. 

76 If a large number of studies are suitable for dose77 response analysis, the assessment considers the study 

78 characteristics in this section to focus on the most 

79 informative data. The assessment explains the reasons 

80 for not analyzing other groups of studies. As a check 

81 on the selection of studies for dose-response analysis, 

82 EPA asks peer reviewers to identify studies that were 

83 not adequately considered. 

84 7. Deriving toxicity values 

85 7.1 General framework for dose-response 

86 analysis 

87 EPA uses a two-step approach that distinguishes 

88 analysis of the observed dose-response data from 

89 inferences about lower doses (U.S. EPA, 2005a). 

90 Within the observed range, the preferred approach 

91 is to use modeling to incorporate a wide range of data 

92 into the analysis. The modeling yields a point of 

93 departure (an exposure level near the lower end of the 

94 observed range, without significant extrapolation to 

95 lower doses) (sections 7.2-7.3). 

96 Extrapolation to lower doses considers what is 

97 known about the modes of action for each effect 

98 (sections 7.4-7.5). An alternative to low-dose 

99 extrapolation is derivation of reference values, which 

100 are calculated by adjusting the point of departure by 

101 factors that account for several sources of uncertainty 

102 and variability (section 7.6). 

103 Increasingly, EPA is making use of multiple data 

104 sets or combining multiple responses in deriving 

105 toxicity values. EPA also considers multiple dose106 response approaches when they can be supported by 

107 robust data. 
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1 7.2 Modeling dose 

2 The preferred approach for analysis of dose is 

3 toxicokinetic modeling because of its ability to 

4 incorporate a wide range of data. The preferred dose 

5 metric would refer to the active agent at the site of its 

6 biologic effect or to a close, reliable surrogate 

7 measure. The active agent may be the administered 

8 chemical or a metabolite. Confidence in the use of a 

9 toxicokinetic model depends on the robustness of its 

10 validation process and on the results of sensitivity 

11 analyses (U.S. EPA, 1994, 2005a, 2006b). 

12 Because toxicokinetic modeling can require many 

13 parameters and more data than are typically available, 

14 EPA has developed standard approaches that can be 

15 applied to typical data sets. These standard approaches 

16 also facilitate comparison across exposure patterns and 

17 species. 

18 – Intermittent study exposures are standardized to a 

19 daily average over the duration of exposure. For 

20 chronic effects, daily exposures are averaged over 

21 the lifespan. Exposures during a critical period, 

22 however, are not averaged over a longer duration 

23 (U.S. EPA, 1991, 1996, 1998, 2005a). 

24 – Doses are standardized to equivalent human terms 

25 to facilitate comparison of results from different 

26 species. 

27 – Oral doses are scaled allometrically using 

mg/kg3/4 28 -d as the equivalent dose metric 

29 across species. As allometric scaling is 

30 typically based on adult body weight, it is not 

31 used for early-life exposure or for 

32 developmental effects (U.S. EPA, 2005a, 

33 2011a). 

34 – Inhalation exposures are scaled using 

35 dosimetry models that apply species-specific 

36 physiologic and anatomic factors and 

37 consider whether the effect occurs at the site 

38 of first contact or after systemic circulation 

39 (U.S. EPA, 1994). 

40 It can be informative to convert doses across 

41 exposure routes. If this is done, the assessment 

42 describes the underlying data, algorithms, and 

43 assumptions (U.S. EPA, 2005a). 

44 7.3 Modeling response in the range of 

45 observation 

46 Toxicodynamic (“biologically based”) modeling 

47 can incorporate data on biologic processes leading to a 

48 disease. Such models require sufficient data to 

49 ascertain a mode of action and to quantitatively 

50 support model parameters associated with its key 

51 events. Because different models may provide 

52 equivalent fits to the observed data but diverge 

53 substantially at lower doses, critical biologic 

54 parameters should be measured from laboratory 

55 studies, not by model fitting. Confidence in the use of 

56 a toxicodynamic model depends on the robustness of 

57 its validation process and on the results of sensitivity 

58 analyses. Peer review of the scientific basis and 

59 performance of a model is essential (U.S. EPA, 

60 2005a). 

61 Because toxicodynamic modeling can require 

62 many parameters and more knowledge and data than 

63 are typically available, EPA has developed a standard 

64 set of empirical (“curve-fitting”) models (http://

65 www.epa.gov/ncea/bmds/) that can be applied to 

66 typical data sets, including those that are nonlinear. 

67 EPA has also developed guidance on modeling dose68 response data, assessing model fit, selecting suitable 

69 models, and reporting modeling results (U.S. EPA, 

70 2000b). Additional judgment or alternative analyses

71 are used when the procedure fails to yield reliable

72 results, for example, if the fit is poor, modeling may 

73 be restricted to the lower doses, especially if there is 

74 competing toxicity at higher doses (U.S. EPA, 2005a). 

75 Modeling is used to derive a point of departure 

76 (U.S. EPA, 2000b, 2005a). (See section 7.6 for 

77 alternatives if a point of departure cannot be derived 

78 by modeling.) 

79 – For dichotomous responses, the point of departure

80 is the 95% lower bound on the dose associated 

81 with a small increase of a biologically significant

82 effect. 

83 – If linear extrapolation to lower doses will be 

84 used, a standard value near the low end of the 

85 observable range is used (10% response for 

86 animal data, 1% for epidemiologic data, 

87 depending on the observed response rates). 

88 – If nonlinear extrapolation will be used, both 

89 statistical and biologic factors are considered 

90 (10% response for minimally adverse effects, 

91 5% or lower for more severe effects or for 

92 developmental toxicity data on individual 

93 offspring). 

94 – For continuous responses, the point of departure is 

95 ideally a level where the effect is considered 

96 minimally adverse. In the absence of such 

97 definition, both statistical and biologic factors are 

98 considered in selecting a response level. 

99 7.4 Extrapolating to lower doses 

100 The purpose of extrapolating to lower doses is to 

101 estimate responses at exposures below the observed 

102 data. Low-dose extrapolation is typically used for 

103 known and likely carcinogens. Low-dose extrapolation 

104 considers what is known about modes of action (U.S.

105 EPA, 2005a). 

106 (1) If a biologically based model has been developed 

107 and validated for the agent, extrapolation may use 

108 the fitted model beyond the observed range if 

109 significant model uncertainty can be ruled out 

110 with reasonable confidence. Below the range 
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1 where confidence bounds on the predictions are 

2 reasonably precise, extrapolation may continue 

3 using a linear model. 

4 (2) Linear extrapolation is used if the dose-response 

5 curve is expected to have a linear component 

6 below the point of departure. This includes: 

7 – Agents or their metabolites that are DNA8 reactive and have direct mutagenic activity. 

9 – Agents or their metabolites for which human 

10 exposures or body burdens are near doses 

11 associated with key events leading to an 

12 effect. 

13 Linear extrapolation is also used if the evidence is 

14 insufficient to establish a mode of action. 

15 The result of linear extrapolation is described by

16 an oral slope factor or an inhalation unit risk, 

17 which is the slope of the dose-response curve at 

18 lower doses. 

19 (3) Nonlinear extrapolation is used if there are 

20 sufficient data to ascertain the mode of action and

21 to conclude that it is not linear at lower doses, and 

22 the agent does not demonstrate mutagenic or other 

23 activity consistent with linearity at lower doses. If 

24 nonlinear extrapolation is appropriate but no 

25 model is developed, a default is to calculate 

26 reference values. 

27 If linear extrapolation is used, the assessment 

28 develops a candidate slope factor or unit risk for each 

29 suitable data set. These results are arrayed, using

30 common dose metrics, to show the distribution of 

31 relative potency across various effects and 

32 experimental systems. The assessment then derives an 

33 overall slope factor and an overall unit risk for the 

34 agent, considering the various dose-response analyses, 

35 the study preferences discussed in section 6, and the 

36 possibility of basing a more robust result on multiple 

37 data sets. 

38 7.5 Considering susceptible populations and 

39 life-stages 

40 The assessment analyzes the available information 

41 on populations and life-stages that may be particularly 

42 susceptible to each effect. A tiered approach is used 

43 (U.S. EPA, 2005a). 

44 (1) If an epidemiologic or experimental study reports 

45 quantitative results for a susceptible population or 

46 life-stage, these data are analyzed to derive 

47 separate toxicity values for susceptible 

48 individuals. 

49 (2) If data on risk-related parameters allow 

50 comparison of the general population and 

51 susceptible individuals, these data are used to 

52 adjust the general-population toxicity values for 

53 application to susceptible individuals. 

54 (3) In the absence of chemical-specific data, 

55 application of age-dependent adjustment factors is 

56 recommended for early-life exposure to suspected 

57 carcinogens. There is evidence of early-life 

58 susceptibility to various carcinogenic agents, but 

59 most epidemiologic studies and cancer bioassays 

60 do not include early-life exposure. To address the 

61 potential for early-life susceptibility, EPA 

62 recommends: 

63 – 10-fold adjustment for exposures before age 2 

64 years. 

65 – 3-fold adjustment for exposures between ages 

66 2 and 16 years. 

67 These adjustments are generally applied only for a

68 mutagenic mode of action, though early-life 

69 susceptibility has been observed for several 

70 carcinogens that are not mutagenic (U.S. EPA, 

71 2005b). 

72 7.6 Reference values and uncertainty factors 

73 An oral reference dose or an inhalation reference 

74 concentration is an estimate of an exposure (including 

75 in susceptible subgroups) that is likely to be without 

76 an appreciable risk of adverse health effects over a 

77 lifetime (U.S. EPA, 2002). Reference values are 

78 typically calculated for effects other than cancer and 

79 for suspected carcinogens if a well characterized mode 

80 of action indicates that a threshold can be based on 

81 prevention of an early key event. Reference values 

82 provide no information about risks at exposures above 

83 the reference value. 

84 The assessment characterizes effects that form the 

85 basis for reference values as adverse, considered to be 

86 adverse, or a precursor to an adverse effect. For 

87 developmental, reproductive, and neurotoxicity there 

88 is guidance on adverse effects and their biologic 

89 markers (U.S. EPA, 1991, 1996, 1998). 

90 To account for uncertainty and variability in the 

91 derivation of a lifetime human exposure where effects 

92 are not anticipated to occur, reference values are 

93 calculated by adjusting the point of departure by a

94 series of uncertainty factors. If a point of departure 

95 cannot be derived by modeling, a no-observed96 adverse-effect level or a lowest-observed-adverse97 effect level is substituted. The assessment discusses 

98 scientific considerations involving several areas of 

99 variability or uncertainty. 

100 Human variation. A factor of 10 is applied to account 

101 for variation in susceptibility across the human 

102 population and the possibility that the available 

103 data may not be representative of individuals who 

104 are most susceptible to the effect. This factor is 

105 reduced only if the point of departure is derived 

106 specifically for susceptible individuals (not for a

107 general population that includes both susceptible 

108 and non-susceptible individuals) (U.S. EPA, 1991, 

109 1994, 1996, 1998, 2002). 
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1 Animal-to-human extrapolation. A factor of 10 is 

2 applied if animal results are used to make 

3 inferences about humans. This factor is often 

4 regarded as comprising toxicokinetics and 

5 toxicodynamics in equal parts. Accordingly, if the 

6 point of departure is based on toxicokinetic 

7 modeling, dosimetry modeling, or allometric 

scaling across species, a factor of 101/2 8 (rounded 

9 to 3) is applied to account for the remaining 

10 uncertainty involving toxicodynamic differences. 

11 An animal-to-human factor is not applied if a 

12 biologically based model adjusts fully for 

13 toxicokinetic and toxicodynamic differences and 

14 residual uncertainty across species (U.S. EPA, 

15 1991, 1994, 1996, 1998, 2002). 

16 Adverse-effect level to no-observed-adverse-effect 

17 level. If a point of departure is based on a lowest18 observed-adverse-effect level, the assessment 

19 must infer a dose where such effects are not 

20 expected. This can be a matter of great 

21 uncertainty, especially if there is no evidence 

22 available at lower doses. A factor of 10 is applied

23 to account for the uncertainty in making this 

24 inference. A factor other than 10 may be used, 

25 depending on the magnitude and nature of the 

26 response and the shape of the dose-response curve 

27 (U.S. EPA, 1991, 1994, 1996, 1998, 2002). 

28 Subchronic-to-chronic exposure. If a point of 

29 departure is based on subchronic studies, the 

30 assessment considers whether lifetime exposure 

31 would have effects at lower levels. A factor of 10 

32 is applied to account for the uncertainty in using 

33 subchronic studies to make inferences about 

34 lifetime exposure. This factor may also be applied 

35 for developmental or reproductive effects if 

36 exposure covered less than the full critical period. 

37 A factor other than 10 may be used, depending on 

38 the duration of the studies and the nature of the 

39 response (U.S. EPA, 1994, 1998, 2002). 

40 Incomplete database. If an incomplete database 

41 raises concern that further studies might identify a 

42 more sensitive effect, organ system, or life-stage,

43 the assessment may apply a database uncertainty 

44 factor (U.S. EPA, 1991, 1994, 1996, 1998, 2002). 

45 EPA typically follows the suggestion that a factor 

46 of 10 be applied if both a prenatal toxicity study 

47 and a two-generation reproduction study are 

missing, and a factor of 101/2 48 if either is missing 

49 (U.S. EPA, 2002). 

50 In this way, the assessment derives candidate 

51 reference values for each suitable data set and effect 

52 that is plausibly associated with the agent. These 

53 results are arrayed, using common dose metrics, to 

54 show where effects occur across a range of exposures 

55 (U.S. EPA, 1994). The assessment then selects an 

56 overall reference dose and an overall reference 

57 concentration for the agent to represent lifetime human 

58 exposure levels where effects are not anticipated to 

59 occur. 

60 The assessment may also report reference values 

61 for each effect. This would facilitate subsequent 

62 cumulative risk assessments, where it may be 

63 important to consider the combined effect of 

64 chemicals acting at a common site or operating 

65 through common mechanisms (U.S. EPA, 2002). 

66 7.7 Confidence and uncertainty in the 

67 reference values 

68 The assessment selects a standard descriptor to 

69 characterize the level of confidence in each reference 

70 value, based on the likelihood that the value would

71 change with further testing. Confidence in reference 

72 values is based on quality of the studies used and 

73 completeness of the database, with more weight given 

74 to the latter. The level of confidence is increased for 

75 reference values based on human data supported by 

76 animal data (U.S. EPA, 1994). 

77 High confidence: The reference value is not likely to 

78 change with further testing, except for 

79 mechanistic studies that might affect the 

80 interpretation of prior test results. 

81 Medium confidence: This is a matter of judgment, 

82 between high and low confidence. 

83 Low confidence: The reference value is especially 

84 vulnerable to change with further testing. 

85 These criteria are consistent with contemporary 

86 guidelines that evaluate the quality of evidence. These 

87 also focus on whether further research would be likely 

88 to change confidence in the estimate of effect (Guyatt 

89 et al 2008a). 

90 All assessments discuss the significant 

91 uncertainties encountered in the analysis. EPA 

92 provides guidance on characterization of uncertainty 

93 (U.S. EPA, 2005a). For example, the discussion 

94 distinguishes model uncertainty (lack of knowledge 

95 about the most appropriate experimental or analytic

96 model), parameter uncertainty (lack of knowledge 

97 about the parameters of a model), and human variation 

98 (interpersonal differences in biologic susceptibility or 

99 in exposures that modify the effects of the agent).

100 

101 For other general information about this assessment or 

102 other questions relating to IRIS, the reader is referred 

103 to EPA’s IRIS Hotline at (202) 566-1676 (phone), 

104 (202) 566-1749 (fax), or hotline.iris@epa.gov (email 

105 address).

106 
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1 

2 AUTHORS | CONTRIBUTORS | REVIEWERS 

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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1 Reviewers 

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

3 Comments were submitted by the Programs and Regions listed below. 

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 

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

7 federal agencies and White House offices that commented are listed below. Comments 

8 submitted by the agencies listed below are available on the IRIS website. 

9 

Agency for Toxic Substances and Disease Registry, Public Health Service, Department of 

 Health & Human Services 

Office of Public Health Service, Food Safety Inspection Service, U.S. Department of 

 Agriculture 

10 

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

12 EPA are listed below. 

13 

NAME 

NAME 

NAME 

NAME

Affiliation 

Affiliation 

Affiliation 

Affiliation

14 

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

16 period ended on [month] [date], [year]. Comments were received from the following entities. 

17 

NAME 

NAME 

NAME 

NAME

Affiliation, Location 

Affiliation, Location 

Affiliation, Location 

Affiliation, Location

18 

19 This assessment was peer-reviewed by independent expert scientists external to EPA (specify 

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

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

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

23 website. 

24 

NAME 

NAME 

NAME 

NAME

Affiliation, Location 

Affiliation, Location 

Affiliation, Location 

Affiliation, Location

25 

26 

27 
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1 

2 PREFACE 

3 

4 

5 The purpose of this Toxicological Review is to provide scientific support and rationale 

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

7 document presents background information and justification for the Integrated Risk Information 

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

9 appendices to this document include information addressing chemical and physical properties, 

10 ammonium salts, toxicokinetics, toxicity study summaries, and external peer review, and are 

11 included in a separate volume: the Supplemental Information for the Toxicological Review of 

12 Ammonia. 

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

14 ammonia posted to the IRIS database in 1991. The previous assessment included an inhalation 

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

16 emissions generated from its use in selective catalytic reduction-based diesel engine 

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

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

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

20 Portions of this Toxicological Review were developed under a Memorandum of 

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

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

23 effort in the development of human health toxicological assessments for the purposes of making 

24 more efficient use of available resources and to share scientific information. 

25 

26 Background 

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

soluble in water (4.82 × 10528 mg/L) and is a weak base (Lide, 2008; Eggeman, 2001; Dean, 1985). 

29 When ammonia (NH3) is present in water at environmental pH, a pKa of 9.25 indicates that the 

equilibrium will favor the formation of the conjugate acid, the ammonium ion (NH4

+

30 ) (Lide, 

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

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

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

34 ammonium ion and the anion. At physiological pH (7.4), the equilibrium between NH3 and 

NH4

+

 favors the formation of NH4

+

35 . Additional chemical and physical properties information for 

36 ammonia is provided in Appendix A, Section A.1. 
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1 Low levels of ammonia occur naturally in the environment in air, soil, and water. 

2 Ammonia is a major component of the geochemical nitrogen cycle and is essential for many 

3 biological processes (Rosswall, 1981). Nitrogen-fixing bacteria convert atmospheric nitrogen 

4 into ammonia available for plant uptake (Socolow, 1999; Rosswall, 1981). Organic nitrogen 

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

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

7 nitrification (Rosswall, 1981). Ammonia is endogenously produced in humans and animals, is 

8 an essential mammalian metabolite used in nucleic acid and protein synthesis, is necessary for 

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

10 2008). 

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

12 the agricultural application of fertilizers, which represents about 80-85% of commercially13 produced ammonia in the form of urea, ammonium nitrate, ammonium sulfate, ammonium 

14 phosphate, and other nitrogen compounds (Eggeman, 2001). Ammonia is also used as a 

15 corrosion inhibitor, in the purification of water supplies, as a component of household cleaners, 

16 as a refrigerant, as a chemical intermediate in pharmaceuticals, explosives and other chemicals, 

17 as a stabilizer in the rubber industry, and as a hydrogen source for the hydrogenation of fats and 

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

19 reduction of NOx emissions from the exhaust of diesel vehicles and stationary combustion 

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

21 2009; Johnson et al., 2009). 

22 

23 Scope of the Assessment 

24 This assessment presents a review of hazard and dose-response information for ammonia, 

25 including gaseous ammonia (NH3) and ammonia dissolved in water (ammonium hydroxide, 

26 NH4OH). Because ammonium salts (e.g., ammonium acetate, chloride, and sulfate) readily 

dissolve in water through disassociation into the ammonium ion (NH4

+

27 ) and the anion, EPA 

28 considered whether or not the literature on ammonium salts could inform the toxicity of 

29 ammonia. The toxicology literature for ammonium salts includes several oral toxicity studies of 

30 ammonium chloride and ammonium sulfate. No inhalation toxicity studies of ammonium salts 

31 are available. The toxicity data for ammonium chloride and ammonium sulfate demonstrate that 

32 these two salts present distinctly different toxicity profiles, suggesting that the anion can 

33 influence the toxicity of the ammonium compound, and that the toxicity of the salts cannot 

necessarily be attributed to the cation (i.e., NH4

+

34 ) only (for detailed ammonium salts information 

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

36 ammonium salts was not used to characterize the toxicity of ammonia or ammonium hydroxide 

37 in this assessment. 

38 
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1 Other Agency and International Assessments 

2 Assessments and regulatory limits for ammonia developed by other health agencies, 

3 including the Agency for Toxic Substances and Disease Registry (ATSDR), the National 

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

5 (ACGIH), the National Institute of Occupational Safety and Health (NIOSH), and the Food and 

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

7 
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1 

2 EXECUTIVE SUMMARY 

3 

4 

5 Effects other than cancer observed following oral exposure 

6 The oral toxicity database for ammonia is very limited. Gastric toxicity is identified as a 

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

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

9 gastrointestinal effects from ingesting household cleaning solutions containing ammonia or 

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

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

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

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

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

15 Given the limited number of studies available and the small number of toxicological 

16 evaluations, there are uncertainties associated with the oral database for ammonia and a RfD for 

17 ammonia was not derived. 

18 

19 Effects other than cancer observed following inhalation exposure 

20 Respiratory effects have been identified as a hazard following inhalation exposure to 

21 ammonia. Evidence for respiratory toxicity associated with exposure to ammonia comes from 

22 studies in humans and animals. Cross-sectional occupational studies involving chronic exposure 

23 to ammonia have consistently demonstrated an increased prevalence of respiratory effects and 

24 decreased lung function. Cross-sectional studies of livestock farmers exposed to ammonia, 

25 controlled human volunteer studies of ammonia inhalation, and case reports of injury in humans 

26 with inhalation exposure to ammonia provide additional and consistent support for the 

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

28 observed in several animal species following subchronic and short-term exposures to ammonia. 

29 The experimental toxicology literature for ammonia also provides evidence that inhaled 

30 ammonia may be associated with toxicity to target organs other than the respiratory system, 

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

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

33 
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1 Inhalation reference concentration (RfC) for effects other than cancer 

2 Table ES-1. Reference Concentration

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 

NOAELADJ: 3.1 mg/m3

 

10 0.3 mg/m3

*Because the POD (NOAEL = 8.8 mg/m3) involved workplace exposure conditions, the NOAEL was 

adjusted for continuous exposure based on the ratio of VEho (human occupational default minute 

volume of 10 m3

 breathed during an 8-hour workday) to VEh (human ambient default minute volume 

of 20 m3

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

4 

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

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

7 effects, characterized as increased respiratory irritation and decreased lung function, observed in 

8 workers exposed to ammonia were selected as the critical effect. In the evaluation of the 

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

ammonia (Holness et al., 1989), a NOAELADJ of 3.1 mg/m310 (adjusted for continuous exposure 

from 8.8 mg/m311 ; see calculation below) was identified based on the absence of statistically 

12 significant increases in the prevalence of the respiratory effects. BMD modeling was not utilized 

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

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

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

a NOAEL approach and the NOAELADJ of 3.1 mg/m316 was used as the POD for RfC 

17 derivation. 

18 The RfC was calculated by dividing the POD (i.e., NOAELADJ) by a composite 

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

20 of data evaluating variability of response to inhaled ammonia in the human population. 

21 

22 Confidence in the chronic inhalation RfC 

23 Study – medium 

24 Database – medium 

25 RfC – medium 

26 

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

28 principal study (adequate design, conduct, and reporting of the principal study; limited by small 
xxii DRAFT - DO NOT CITE OR QUOTE

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

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

3 duration. Although there are no studies of developmental toxicity and studies of reproductive 

4 and other systemic endpoints are limited, reproductive, developmental, and other systemic 

5 effects are not expected at the RfC because it is well documented that ammonia is endogenously 

6 produced in humans and animals, ammonia concentrations in blood are homeostatically 

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

8 to alter homeostasis. 

9 

10 Evidence for human carcinogenicity 

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

12 “inadequate information to assess the carcinogenic potential” of ammonia based on the 

13 absence of ammonia carcinogenicity studies in humans and a single lifetime drinking water study 

14 of ammonia in mice that showed no evidence of carcinogenic potential. There is limited 

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

16 gastric cancer. The available studies of ammonia genotoxicity are inadequate to characterize the 

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

18 not conducted.

19 

20 Susceptible Populations and Life Stages 

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

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

23 These elevated ammonia levels can predispose an individual to encephalopathy due to the ability 

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

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

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

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

28 animals compared to other life stages that would support an evaluation of childhood 

29 susceptibility have not been conducted. 

30 

31 Key issues addressed in assessment 

32 Endogenous ammonia 

33 Ammonia, which is produced endogenously, has been detected in the expired air of 

34 healthy volunteers. Higher and more variable ammonia concentrations are reported in breath 

exhaled from the mouth or oral cavity (0.09 to 2.1 mg/m335 ). These levels are largely attributed to 

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

37 gastrointestinal tract, and can be influenced by factors such as diet, oral hygiene, age, and living 
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1 conditions (i.e., urban vs. rural setting). In contrast, ammonia concentrations measured in breath 

exhaled from the nose and trachea are lower (0.013–0.078 mg/m32 ) and more likely reflect levels 

of ammonia circulating in the blood. These levels are lower than the ammonia RfC of 0.3 mg/m33 

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

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

6 be rapidly diluted in the much larger volume of ambient air. 

7 
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1 

2 LITERATURE SEARCH STRATEGY & STUDY 

3 EVALUATION FOR HAZARD IDENTIFICATION 

4 

5 Literature Search Strategy and Study Selection 

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

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

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

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

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

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

12 independent analyses of the health effects data were retrieved and may be included in the 

13 assessment where appropriate.

14 

Table LS-1. Details of the search strategy employed for ammonia 

Databases Limits Keywords 

Pubmed 

Toxcenter 

Toxline 

Current Contents (2008 

& 2010 only) 

Search constraints: 2003-currentb

Pre-2003—ATSDR (2004) was 

used as the source of references 

published before 2003 

Last search: November 11, 2011 

Chemical name and synonymsa: 

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 ammonia salts (i.e., ammonium nitrate [6484-52-2], ammonium fluoride 

[12125-01-8], ammonium sulfate [7783-20-2], ammonium persulfate [7727-54-0], and ammonium chloride [12125-02-9]) as 

keywords. Once the determination was made not to include data on ammonium salts in the assessment, updated searches 

focused on ammonia and ammonium hydroxide only. 

b

The search using search terms related to concentrations of ammonia in exhaled breath was conducted for the period 

1/1/2002–11/11/2011.

15 
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1 Approximately 4,900 references were identified in the literature search for ammonia 

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

can be found on the EPA’s HERO website.13 From this list, approximately 250 references were

4 identified that provided information relevant to the human health effects of ammonia or 

5 information on the physical and chemical properties of ammonia. 

6 The references cited in this document, as well as those that were considered but not 

7 included in the Toxicological Review of Ammonia, can be found on the HERO website 

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

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

10 Toxicological Review of Ammonia. 

11 

12 Study Evaluation for Hazard Identification 

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

14 toxicological nature of ammonia. In general, the quality and relevance of health effects studies 

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

16 Reference Dose and Reference Concentration Processes (U.S. EPA, 2002) and Methods for 

17 Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry

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

19 studies. 

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

21 available epidemiology and toxicity studies of ammonia and ammonium hydroxide were 

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

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

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

25 the ammonium compound. Approximately 100 case reports involving acute ammonia exposure 

26 were identified; because case reports generally provide little information that would be useful for 

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

28 this literature are provided as supplemental materials in Appendix A. Human studies that 

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

30 raised concerns of ethical conduct were excluded from consideration; two human studies fell into 

31 this category. 

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

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

34 an association between ammonia exposure and the health effects. The available studies 

 

1HERO (Health and Environmental Research On-line) is a database of scientific studies and other references used to 

develop EPA’s risk assessments aimed at understanding the health and environmental effects of pollutants and 

chemicals. It is developed and managed in EPA’s Office of Research and Development (ORD) by the National 

Center for Environmental Assessment (NCEA). The database includes more than 300,000 scientific articles from 

the peer-reviewed literature. New studies are added continuously to HERO. 
xxvi DRAFT - DO NOT CITE OR QUOTE

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

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

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

4 and of the collection of studies noted. The evaluation of the effects seen in experimental animal 

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

6 study. Chronic toxicity studies were limited to oral exposure studies that did not adequately 

7 evaluate the noncancer effects of ammonia. 

8 
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1 

2 1. HAZARD IDENTIFICATION 

3 

4 1.1. Synthesis of Major Toxicological Effects 

5 1.1.1. Respiratory Effects 

6 Respiratory Irritation 

7 The respiratory system is the primary target of toxicity of inhaled ammonia in humans 

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

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

2007; Ballal et al., 1998) (see Table 1-1). Rahman et al. (2007)210 found a higher prevalence, by 

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

12 sneezing) in urea fertilizer factory workers exposed to a mean ammonia concentration of 18.5 

mg/m313 (high-exposure group) for about 16 years compared to a control group (staff workers); the 

14 prevalences of cough and chest tightness were statistically significantly elevated in the high15 exposure ammonia group compared to the control group. Respiratory irritation prevalence 

between the low-exposure group exposed to a mean ammonia concentration of 4.9 mg/m316 was 

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

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

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

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

limit value [TLV] of 18 mg/m3 21 [25 ppm]) compared with workers exposed to levels below the 

22 TLV. Distribution of respiratory irritation effects by cumulative ammonia concentration (CAC, 

mg/m323 -years) also showed significantly higher relative risk for these respiratory irritation effect 

among workers with higher CAC (>50 mg/m324 -years) compared to those with a lower CAC (< 50 

mg/m325 -years) (Ballal et al., 1998). Only Ballal et al. (1998) evaluated respiratory endpoints in 

26 terms of cumulative exposure. 

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

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

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

either as one group or when stratified into three exposure categories: high = >8.8 mg/m330 , 

medium = 4.4–8.8 mg/m3, or low = <4.4 mg/m331 . Although respiratory irritation prevalence was 

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

 

2

 Rahman et al. (2007) examined respiratory effects in workers from two plants in a urea fertilizer factory. 

Workers in the urea plant were exposed to higher concentrations of ammonia (arithmetic mean ammonia 

concentration of 18.5 mg/m3) than workers in the ammonia plant (arithmetic mean ammonia concentration of 4.9 

mg/m3). Therefore, the urea plant workers represented the high-exposure group, and the ammonia plant workers 

represented the low-exposure group. 
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1 of their reported respiratory symptoms (cough, sputum, chronic bronchitis, wheeze, chest 

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

3 and inorganic gases such as nitrogen dioxide and sulfur dioxide were possible in these cross4 sectional studies; however, except for the low levels of nitrogen dioxide identified in the Rahman 

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

6 Overall, these cross-sectional occupational epidemiology studies provide consistent 

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

observed that exposure to 18.5 mg/m38 ammonia increased the prevalence of respiratory effects. 

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

ammonia concentrations exceeding the TLV of 18 mg/m310 had significantly higher relative risks 

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

following occupational exposures at lower workplace concentrations (i.e., >8.8 mg/m312 ammonia 

[Holness et al., 1989] and 4.9 mg/m313 [Rahman et al., 2007]). 

14 Respiratory irritation, indicated by elevated prevalences of respiratory symptoms, 

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

16 reported in livestock farmers and stable workers compared to controls (Melbostad and Eduard, 

17 2001; Preller et al., 1995; Choudat et al., 1994; Zejda et al., 1994; Crook et al., 1991; Heederik et 

18 al., 1990). Additionally, bronchial hyperreactivity to methacholine or histamine challenge was 

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

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

21 farm settings may contribute to chronic airway inflammation. In addition to ammonia, these 

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

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

24 livestock farms is likely, but measurements for other volatile chemicals were not conducted. 

25 Therefore, while several studies have reported associations between ammonia exposure in 

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

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

28 exposure and the respiratory effects observed in the study populations. 

29 Support for ammonia as a respiratory irritant is also provided by reports of irritation and 

30 hyperventilation in volunteers acutely exposed to ammonia at concentrations ranging from 

11−354 mg/m331 ammonia for durations up to 4 hours under controlled exposure conditions 

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

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

34 reported habituation to eye, nose, and throat irritation in volunteers after several weeks of 

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

36 the acute respiratory effects of inhaled ammonia, ranging from mild symptoms (including nasal 

37 and throat irritation and perceived tightness in the throat) to moderate effects (including 

38 pharyngitis, tachycardia, dyspnea, rapid and shallow breathing, cyanosis, transient 
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1 bronchospasm, and rhonchi in the lungs) to severe effects (including burns of the nasal passages, 

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

3 dyspnea, persistent, productive cough, bilateral diffuse rales and rhonchi, mucous production, 

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

5 for more detailed information and references). 

6 Experimental studies in laboratory animals also provide consistent evidence that 

7 ammonia exposure for 35 days or more can produce respiratory irritation. Histopathological 

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

mg/m3 ammonia or 35 days of exposure to 177 mg/m39 ammonia, with respiratory and olfactory 

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

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

to 71 mg/m312 ammonia continuously for 1–6 weeks (Doig and Willoughby, 1971). Nonspecific 

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

Longs-Evans rats continuously exposed to 127 mg/m314 ammonia for 90 days and rats and guinea 

pigs intermittently exposed to 770 mg/m3 ammonia (or 183 mg/m315 , adjusted to continuous 

exposure316 ) (Coon et al., 1970). Focal or diffuse interstitial pneumonitis was observed in all 

17 Princeton-derived guinea pigs, New Zealand white rabbits, beagle dogs, and squirrel monkeys 

exposed to 470 mg/m318 ammonia that were examined (Coon et al., 1970). Additionally, under 

19 these exposure conditions, dogs exhibited nasal discharge and other signs of irritation (marked 

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

262 mg/m321 ammonia for 90 days (Coon et al., 1970). 

At lower concentrations, approximately 50 mg/m322 and below, the majority of studies of 

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

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

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

representative of those found in commercial pig farms (26 mg/m326 ammonia) for 5 weeks (Done 

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

were observed after continuous exposure to 53 mg/m328 ammonia for up to 109 days (Curtis et al., 

1975). No signs of toxicity in rats were observed after continuous exposure to 40 mg/m329 

ammonia for 114 days or after intermittent exposure to 155 mg/m3 ammonia (or 36.9 mg/m330 , 

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

32 

33 Lung Function 

34 Decreased lung function in ammonia-exposed workers has been reported in two cross35 sectional studies of industrial worker populations (Rahman et al., 2007; Ali et al., 2001) of three 

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

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

 

3Cadjusted = C × 8 hours/24 hours × 5 days/7 days, where C is the exposure concentration. 
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1 course of a work shift (cross-shift) as measured by forced vital capacity (FVC) and forced 

2 expiratory volume in one second (FEV1) in the high-exposure worker group (mean ammonia 

concentration of 18.5 mg/m33 ) in a fertilizer factory (Rahman et al., 2007). In a second study (Ali 

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

5 than in controls (4.6% increase, p ≤ 0.002); FEV1 was higher (1.5%) in the exposed workers but 

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

measures of cumulative exposure, workers with cumulative exposure >50 mg/m37 -years had 

8 significantly lower FVC% predicted (5.4% decrease, p ≤ 0.030) and FEV1% predicted (7.4% 

decrease, p < 0.006) than workers with cumulative exposure ≤50 mg/m39 -years, but similar 

10 FEV1/FVC%. The authors did not explain the inconsistent findings across the analyses of 

11 noncumulative and cumulative exposures. 

12 Lung function did not appear to be affected in worker populations chronically exposed to 

ammonia at concentrations below approximately 18 mg/m313 . Baseline lung function, based on 

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

control in workers exposed to ammonia concentrations ranging from <4.4 mg/m3 to >8.8 mg/m315 

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

17 statistically significant. Additionally, no changes in lung function were observed over either 

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

19 Similarly, measures of lung function (FVC, FEV1, and PEFR [peak expiratory flow rate]) in 

workers exposed to a mean concentration of 4.9 mg/m320 ammonia in a urea fertilizer factory 

21 showed no significant cross-shift changes (Rahman et al., 2007). 

22 Decreased lung function (e.g., measured as decreased FEV1, FVC) was reported in 

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

24 al., 1998; Reynolds et al., 1996; Preller et al., 1995; Crook et al., 1991; Heederik et al., 1990). 

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

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

27 the association between exposure to ammonia and decreased lung function observed in the study 

28 populations. 

29 Changes in lung function following acute exposure to ammonia have been observed in 

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

31 reduced lung function as measured by reduced expiratory minute volume and changes in exercise 

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

≥106 mg/m3 but not at 71 mg/m333 . Bronchioconstriction was reported in volunteers exposed to 

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

60 mg/m335 (Douglas and Coe, 1987); however, there were no bronchial symptoms reported in 

volunteers exposed to ammonia at concentrations of up to 35 mg/m336 for 10 minutes in an 

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

38 lung function (as measured by forced vital capacity and FEV1) were reported in healthy 
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volunteers exposed to ammonia at concentrations up to 18 mg/m31 for 1.5 hours during exercise 

2 (Sundblad et al., 2004). There were no changes in lung function as measured by FEV1 in 25 

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

concentrations up to 354 mg/m34 ammonia for up to 2.5 hours (Petrova et al., 2008), or in six 

healthy volunteers and eight mildly asthmatic volunteers exposed to 11–18 mg/m35 ammonia for 

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

7 Lung function effects following ammonia exposure were not evaluated in the available 

8 animal studies. 

9 The evidence of respiratory effects in humans and experimental animals exposed to 

10 ammonia is provided in Tables 1-1 and 1-2, respectively, and presented visually as an exposure11 response array in Figure 1-1.

12 

13 
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Table 1-1. Respiratory effects in humans following inhalation exposure 

Health Effect Study Design and Reference Results 

NOAEL/ LOAELa

(mg/m3) 

Respiratory 

irritation 

Cross-sectional occupational study 

of soda ash plant workers in 

Canada; 58 exposed workers and 

31 controls (from stores and office 

areas of plant)b

Low (<4.4 mg/m3), medium (4.4–

8.8 mg/m3), high (>8.8 mg/m3); 

adjustedc concentration ranges 

<1.6 mg/m3, 1.6–3.1 mg/m3 and 

>3.1 mg/m3

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/m3 d

(1.8 mg/m3

 adjustedc) 

Urea plant: 18.5 mg/m3 d

(6.6 mg/m3

 adjustedc) 

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)a28 (0.05, 

0.41)b

Chest 

tightness

8 17 (0.42)a33 (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 controlse

Exposures were stratified > or < the 

ACGIH TLV of 18 mg/m3

Mean of employment duration: 

51.8 mo (exposed workers) and 

73.1 mo (controls) 

Ballal et al., 1998 

Higher relative risks for those exposed to 

ammonia at concentrations >TLV as 

compared to those exposed at levels ≤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 
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Table 1-1. Respiratory effects in humans following inhalation exposure 

Health Effect Study Design and Reference Results 

NOAEL/ LOAELa

(mg/m3) 

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/m3), medium (4.4–

8.8 mg/m3), high (>8.8 mg/m3) 

adjustedc concentration ranges 

<1.6 mg/m3, 1.6–3.1 mg/m3 and 

>3.1 mg/m3

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 

FEV1 94.1 95.1 0.3520 

FEV1/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 

FEV1 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/m3 d

(1.8 mg/m3

 adjustedc) 

Urea plant: 18.5 mg/m3 d

(6.6 mg/m3 adjustedc) 

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 

 FEV1 2.627 2.705 0.24 

 PEFR 8.081 8.313 0.22 

Urea plant 

 FVC 3.362 3.258 0.01 

 FEV1 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/m3

Mean of employment duration: not 

reported 

Ali et al., 2001 

Lung function results based on exposure 

concentration and cumulative exposure: 

 Control Exposed p-value 

FVC1% 

predicted 

96.6 98.1 NS 

FVC% 

predicted 

101.0 105.6 0.002 

FEV1/FVC% 83.0 84.2 NS 

≤50 

mg/m3-y

>50 

mg/m3-y p-value 

FVC1% 

predicted 

100.7 93.4 0.006 

FVC% 

predicted 

105.6 100.2 0.03 

FEV1/FVC% 84.7 83.4 NS 

NS = not significant 

NOAEL and 

LOAEL values 

were not 

identified 

because 

exposures were 

not adequately 

characterized 
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a

The NOAEL and LOAEL values presented were identified by EPA. 

b

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

which in turn was reacted with salt to produce sodium bicarbonate and subsequently processed to form sodium 

carbonate. Ammonia and carbon dioxide were recovered in the process and reused. 

c

Adjusted to continuous exposure based on the ratio of VEho (human occupational default minute volume of

10 m3 breathed during an 8-hour workday) to VEh (human ambient default minute volume of 20 m3 breathed 

during the entire day) and an exposure of 5 days out of 7 days (i.e., measured concentration × 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 the ammonia and urea plants were 17.7 and 88.1 mg/m3

, 

respectively; using the Dräger PAC III method, ammonia concentrations were 4.9 and 18.5 mg/m3 (Rahman et al., 

2007). The study authors observed that their measurements indicated only relative differences in exposures 

between workers and production areas, and that the validity of the exposure measures could not be evaluated 

based on their results. Based on communication with technical support at Dräger (telephone conversations and emails dated June 22, 2010, from Michael Yanosky, Dräger Safety Inc., Technical Support Detection Products to 

Amber Bacom, SRC, Inc., contractor to NCEA, ORD, U.S. EPA), EPA considered the PAC III instrument to be a more 

sensitive monitoring technology than the Dräger tubes. Therefore, more confidence is attributed to the PAC III air 

measurements of ammonia for the Rahman et al. (2007) study. 

e

The process of fertilizer production involved synthesis of ammonia from natural gas, followed by reaction of the 

ammonia and carbon dioxide to form ammonium carbamide, which was then converted to urea.

1 

2 
9 DRAFT - DO NOT CITE OR QUOTE

Table 1-2. Respiratory effects in animals following inhalation exposure

Health Effect Study Design and Reference Results

NOAEL/ LOAELa

(mg/m3) 

Pulmonary 

inflammation 

and congestion

0, 155, or 770 mg/m3 8 hrs/d, 5 d/wk for 

6 wks; (36.9, 183 mg/m3 adjustedb) 

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/m3. 

Nonspecific lung inflammation 

observed in guinea pigs and rats 

but not in other species at 

183 mg/m3

NOAEL: 36.9 

LOAEL: 183 

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

Squirrel monkey (Saimiri sciureus); male; 

3/group and beagle dog; male; 2/group and 

New Zealand albino rabbit; male; 3/group 

and Princeton-derived guinea pig; male and 

female; 15/group 

Coon et al., 1970 

Focal or interstitial pneumonitis 

in all animals, calcification of 

bronchial epithelium was 

observed in several animals. 

Hemorrhagic lung lesion in 1 of 2 

dogs. Moderate lung congestion 

in 2 of 3 rabbits. 

NOAEL: 40 

LOAEL: 470 

0 or 40 mg/m3 for 114 d or 127, 262 or 

470 mg/m3

 for 90 d of 455 mg/m3 for 65 d 

Sprague-Dawley or Long-Evans rat; male 

and female; 15-51/group 

Coon et al., 1970 

Dyspnea (mild) at 455 mg/m3

.

Focal or interstitial pneumonitis 

in all animals, calcification of 

bronchial epithelium observed in 

several animals at 470 mg/m3. 

(Exposure to 455 and 470 mg/m3

ammonia increased mortality in 

rats.) 

NOAEL: 262 

LOAEL: 455 

0 or 14 for 7-42 days or 35 mg/m3 for 

42 days 

Guinea pig (strain not specified); male and 

female; 2/group 

Anderson et al., 1964 

Pulmonary congestion, edema 

and hemorrhage were observed 

at 14 and 35 mg/m3 after 42 d. 

NOAEL: NA 

LOAEL:14 

0 or 14 mg/m3 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/m3 after 42 d. 

NOAEL: not 

identified 

LOAEL: 14 
10 DRAFT - DO NOT CITE OR QUOTE

Table 1-2. Respiratory effects in animals following inhalation exposure

Health Effect Study Design and Reference Results

NOAEL/ LOAELa

(mg/m3) 

0, 0.4, 7, 13.3, or 26 mg/m3 and 1.2, 2.7, 5.1, 

or 9.9 mg/m3 inhalable dust for 5 wks 

Exposure to ammonia and inhalable dust at 

concentrations commonly found at pig 

farms 

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/m3 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/m3 from bedding for 75 d 

Sherman rat; 5/sex/group 

Broderson et al., 1976c

Thickening of the nasal 

epithelium (3-4 times) and nasal 

lesions. 

NOAEL: 7 

LOAEL:106 

0 or 177 mg/m3 in an inhalation chamber 

for 35 d 

F344 rat; 6/sex/group 

Broderson et al., 1976 

Thickening of the nasal 

epithelium (3-4 times) and nasal 

lesions. 

NOAEL: not 

identified 

LOAEL:177 

0 or 71 mg/m3 for 6 wks 

Yorkshire-Landrace pig; sex not specified; 

6/group 

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 following inhalation exposure

Health Effect Study Design and Reference Results

NOAEL/ LOAELa

(mg/m3) 

Nasal 

inflammation 

and lesions 

0, 155, or 770 mg/m3 8 hrs/d, 5 d/wk for 

6 wks; (36.9, 183 mg/m3 adjustedb) 

Squirrel monkey (Saimiri sciureus); male; 

3/group and beagle dog; male; 2/group and 

New Zealand albino rabbit; male; 3/group 

and Princeton-derived guinea pig; male and 

female; 15/group and Sprague-Dawley and 

Long-Evans rat; male and female; 15-

51/group 

Coon et al., 1970 

No nasal irritation observed. NOAEL: 183 

LOAEL: not 

identified 

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

Beagle dog; male; 2/group 

 

Coon et al., 1970 

Nasal discharge. NOAEL: 40 

LOAEL: 470 

0 or 40 mg/m3 for 114 d or 127, 262 or 

470 mg/m3

 for 90 d or 455 mg/m3 for 65 d 

Sprague-Dawley or Long-Evans rat; male 

and female; 15-51/group 

Coon et al., 1970 

Nasal irritation in all animals at 

455 mg/m3. 

(Exposure to 455 and 470 mg/m3

ammonia increased mortality in 

rats.) 

NOAEL: 262 

LOAEL: 455 

7 or 106 mg/m3 from bedding for 75 d 

Sherman rat; 5/sex/group 

Broderson et al., 1976 

Nasal lesions at 106 mg/m3. NOAEL: 7 

LOAEL:106 

0 or 177 mg/m3 in an inhalation chamber 

for 35 d 

F344 rat; 6/sex/group 

Broderson et al., 1976 

Nasal lesions at 177 mg/m3. NOAEL: not 

identified 

LOAEL:177 

Ammonia vapor of 0 or 12% ammonia 

solution for 15 min/d, 6 d/wk, for 8 wks 

White albino mouse; male; 50 

Gaafar et al., 1992 

Histological changes in the nasal 

mucosa. 

NOAEL and 

LOAEL values 

were not 

identified 

because of 

inadequate 

reporting of 

exposure 

concentrations. 
12 DRAFT - DO NOT CITE OR QUOTE

Table 1-2. Respiratory effects in animals following inhalation exposure

Health Effect Study Design and Reference Results

NOAEL/ LOAELa

(mg/m3) 

8, 43, 73, or 103 mg/m3 for 5 wks 

Duroc pig; both sexes; 9/group 

Stombaugh et al., 1969 

Excessive nasal, lacrimal, and 

mouth secretions and increased 

frequency of cough at 73 and 

103 mg/m3. 

NOAEL and 

LOAEL values 

were not 

identified 

because of the 

absence of a 

control group. 

a

The NOAEL and LOAEL values presented were identified 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 laboratory cage environments plays a role in the 

pathogenesis of murine respiratory mycoplasmosis caused by the bacterium M. pulmonis. The experiments 

conducted without co-exposure to M. pulmonis are summarized in this table; the results of experiments involving 

co-exposure to M. pulmonis are discussed in Section 1.1.4, Immune System Effects. 

1 
13 DRAFT - DO 

1 2 Figure 1-1. Exposure-response array for respiratory effects following inhalation exposure.

0.1

1

10

100

1000

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)

Pulmonary inflammation and congestion Thickening of airway epithelium Nasal inflam

Exposure (mg/m3)

LOAELNOAELFELIntermediate dose

EXPERIMENTAL ANIMAL STUDIES

HUMANSTUDIES
14 DRAFT - DO NOT CITE OR QUOTE

1  Mode of Action Analysis – Respiratory Effects 

2 Data regarding the potential mode of action for respiratory effects associated with chronic 

3 exposure to ammonia are limited. However, it is well established that injury to respiratory 

4 tissues resulting from acute exposure to inhaled ammonia is primarily due to its alkaline 

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

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

7 the tissues. Ammonia directly denatures tissue proteins due to the production of alkaline 

8 proteinates. Specifically, ammonium hydroxide causes saponification of cell membrane lipids 

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

10 resulting in an inflammatory response, which further damages the surrounding tissues (Amshel et 

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

12 

13 1.1.2. Gastrointestinal Effects 

14 Reports of gastrointestinal effects of ammonia in humans are limited to case reports 

15 involving intentional or accidental ingestion of household cleaning solutions or ammonia 

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

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

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

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

20 laryngeal and epiglottal edema, erythmatous esophagus with severe corrosive injury, and 

21 hemorrhagic esophago-gastro-duodeno-enteritis. 

22 In animals following oral exposure, statistically significant decreases of 40−60% in the 

23 thickness of the gastric mucosa were reported in Sprague-Dawley rats administered 0.01% 

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

25 estimated doses were 22 mg/kg-day (Kawano et al., 1991) and 33 mg/kg-day (Tsujii et al., 1993). 

26 These studies were designed to investigate the hypothesis that the bacterium Helicobacter pylori, 

27 which produces a potent urease that increases ammonia production, plays a significant role in the 

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

29 decrease in gastric mucosal thickness increased with dose and duration of exposure and that the 

30 effect was more prominent in the mucosa of the antrum region of the stomach than in the body 

region of the stomach.431 As discussed further under Mode of Action – Gastrointestinal Effects 

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

33 stomach may be a function of differences in pH in these regions and resulting differences in the 

extent of ionization of ammonia to NH4

+

34 . Parietal cell number per oxyntic gland also decreased 

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

 

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

adjacent to the body. 
15 DRAFT - DO NOT CITE OR QUOTE

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

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

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

4 thickness of the body mucosa. Increased mucosal cell proliferation and migration (as measured 

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

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

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

8 proliferation. They further observed that the ammonia-related changes in rat stomach resembled 

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

10 A relationship between ammonia ingestion and gastrointestinal effects is supported by 

11 findings from two acute oral studies in rats following gavage administration of ammonium 

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

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

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

15 hemorrhagic mucosal lesions in female Sprague-Dawley rats 15 minutes after exposure to an 

16 estimated dose of 48 mg/kg ammonium hydroxide via gavage. 

17 The evidence of gastrointestinal effects in experimental animals following oral exposure 

18 to ammonia is provided in Table 1-3. 

19 

20 
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Table 1-3. Gastrointestinal effects in animals following oral exposures

Health Effect Study Design and References Results

NOAEL/LOAELa

(mg/kg-day)

Histopathologic 

changes of the 

gastric mucosa 

0, 22, or 220 mg/kg-day in 

drinking water for 2 or 4 weeksb

Sprague-Dawley rat; male; 

6/group 

Kawano et al., 1991 

Statistically significant decrease in the 

thickness of the gastric mucosa that 

was dose and duration related; effect 

was more prominent in the mucosa of 

the antrum region of the stomach than 

the body region. 

Thickness of mucosa relative to 

control: 

Antrum 

 Week 2: 96, 80d% 

 Week 4: 62d, 39d% 

Body 

 Week 2: 99, 103% 

 Week 4: 78, 71d% 

NOAEL: not 

identified 

LOAEL: 22 

0 or 33 mg/kg-day in drinking 

water for 3 days or 1, 2, 4, or 8 

weeks; tap water provided for 

the balance of the 8-week 

studyc 

Sprague-Dawley rat; male; 

36/group 

Tsujii et al., 1993 

Antral mucosal thickness decreased 

significantly at 4 and 8 weeks of 

exposure; there was no significant 

effect on the thickness of the body 

mucosa. Cell migration was 

significantly increased. 

Thickness of mucosa relative to control 

(d 3, wk 1, 2, 4, 8): 

Antrum: 108, 96, 106, 56d, 59d% 

Body: 105, 101, 104, 99, 95% 

(extracted from Figure 3 of Tsujii et al., 

1993) 

NOAEL: not 

identified 

LOAEL: 33 

a

The NOAEL and LOAEL values presented were identified by EPA. 

b

Ammonia was provided in drinking water at concentrations of 0, 0.01 or 0.1%. Doses were estimated based on a 

body weight of 230 g for male rats and estimated daily water intake of 50 mL/day. 

c

Ammonia was provided in drinking water at concentrations of 0 or 0.01%. Doses were estimated based on an 

initial body weight of 150 g and estimated daily water intake of 50 mL. 

d

Statistically significant from controls. 

1 

2  Mode of Action Analysis – Gastrointestinal Effects 

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

4 relevant mechanistic information that informs ammonia mode of action comes largely from 

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

6 produces urease, which breaks down urea that is normally present in the stomach into ammonia 

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

8 and stomach cancer in humans. 
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1 This literature suggests that the alkalinity of the ammonia solution does not play a direct 

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

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

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

5 ammonia to damage the gastric mucosa may be related to its ionization state. Ammonia (NH3) 

can easily penetrate cell membranes, subsequently reacting to form NH4

+

 and OH6 in the interior 

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

8 stomach mucosa responded differently following administration of 33 mg/kg-day ammonia in 

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

10 toxicity. The hydrogen chloride secreted by the mucosa in the body of the stomach resulted in a 

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

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

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

observed in the stomach body where there was relatively higher exposure to NH4

+

14 . 

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

16 gastric lesions by ammonia. Increased cell vacuolation and decreased viability of cells in vitro 

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

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

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

20 were associated with the rapid release and activation of cathepsins, mammalian cysteine 

21 proteases that are released from lysosomes or activated in the cytosol and that can be damaging 

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

23 mitochondrial respiration, possibly by elevating intracellular or intraorganelle pH or by 

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

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

26 gastric mucosa in ammonia-induced gastric mucosal injury based on findings in rats given 

27 ammonia intragastrically. Regardless of the specific mechanism(s) by which ammonia induces 

28 cellular toxicity, gastric injury appears to accelerate mucosal cell desquamation and stimulate 

29 cell proliferation via a compensatory mechanism (Tsujii et al., 1992a). 

30 

31 1.1.3. Reproductive and Developmental Effects 

32 No statistically significant differences in reproductive or developmental endpoints were 

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

mean concentrations of 5 or 25 mg/m334 and then mated (Diekman et al., 1993) in the only study of 

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

puberty did not differ significantly between the two groups. Gilts exposed to 25 mg/m336 ammonia 

weighed 7% less (p < 0.05) at puberty than those exposed to 5 mg/m337 ; however, body weights of 

38 the two groups were similar at gestation day 30. Conception rates in the mated females were 
18 DRAFT - DO NOT CITE OR QUOTE

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

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

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

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

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

6 pathogens. The evidence of reproductive and developmental effects in experimental animals 

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

8 

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

exposure 

Health Effect Study Design and Reference Results NOAEL/LOAELa (mg/m3)

Reproductive and 

developmental 

parameters 

5 mg/m3 (range, 3–8.5 

mg/m3) or 25 mg/m3 (range, 

18–32 mg/m3) for 6 weeksb 

Crossbred gilts (female pigs), 

4.5 months old, 40/group 

Diekman et al., 1993 

No effect on any of the 

reproductive or 

developmental parameters 

measured (age at puberty, 

conception rates, body weight 

of pregnant gilts, number of 

corpora lutea, number of live 

fetuses, and weight or length 

of fetuses). 

NOAEL: 5 

LOAEL: not identified 

a

The NOAEL and LOAEL values presented were identified by EPA. 

b

A control group was not included. Prior to exposure to ammonia, pigs were also exposed naturally in 

conventional grower units to Mycoplasma hypopneumoniae and Pasteurella multocida, which cause pneumonia 

and atrophic rhinitis, respectively. 

9 

10 1.1.4. Immune System Effects 

11 A limited number of studies have evaluated the immunotoxicity of ammonia in human 

12 populations and in experimental animal models. Immunological function was evaluated in two 

13 independent investigations of livestock farmers exposed to ammonia; immunoglobulin G (IgG) 

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

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

16 data are suggestive of immunostimulatory effects; however, the test subjects were also exposed 

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

18 and mold that are known to stimulate immune responses. Data in humans following exposure to 

19 ammonia only are not available.

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

21 inhalation exposures and three general types of immune assays. Immunotoxicity studies of 

22 ammonia using measures of host resistance provide the most relevant data for assessing immune 

23 function since they directly measure the immune system’s ability to control microorganism 

24 growth. Other available studies of ammonia employed assays that evaluated immune function. 
19 DRAFT - DO NOT CITE OR QUOTE

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

2 the least predictive and were excluded from the hazard identification for ammonia (Neumann et 

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

4 Evidence of immunosuppression was observed in several host resistance studies utilizing 

5 lung pathogens to measure reduced bacterial clearance following ammonia exposure. 

6 Inoculation with the respiratory pathogen Mycoplasma pulmonis causes murine respiratory 

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

8 microscopic, were positively correlated with ammonia concentration in F344 rats continuously 

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

(10810 colony forming units [CFU]) followed by up to 42 days of ammonia exposure post 

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

ammonia concentrations ≥35 mg/m312 , and suggests that ammonia exposure decreased bacterial 

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

14 ammonia concentration was not associated with increased CFU of M. pulmonis isolated from the 

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

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

17 immunocompromised animals. Conversely, significantly increased CFU of M. pulmonis bacteria 

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

exposed to 71 mg/m3 ammonia for 7 days prior to M. pulmonis (104−106 19 CFU) inoculation and 

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

21 colonization indicates a reduction in bacterial clearance following exposure to ammonia. 

Lesions were not assessed in this study. OF1 mice exposed to 354 mg/m322 ammonia for 7 days 

23 prior to inoculation with a 50% lethal dose (LD50) of Pasteurella multocida significantly 

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

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

26 the irritating action of ammonia destroyed the tracheobronchial mucosa and caused inflammatory 

27 lesions thereby increasing sensitivity to respiratory infection with prolonged ammonia exposure. 

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

29 following ammonia exposure. Using a delayed-type hypersensitivity (DTH) test to evaluate cell30 mediated immunity, Hartley guinea pigs were vaccinated with Mycobacterium bovis BCG and 

31 exposed to ammonia followed by intradermal challenge with purified protein derivative (PPD). 

Dermal lesion size was reduced in animals exposed to 64 mg/m332 indicating immunosuppression 

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

34 treated with the same 3 week ammonia exposure and stimulated with phytohaemagglutinin or 

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

36 activity in alveolar macrophages isolated from ammonia-exposed guinea pigs was not affected. 

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

38 in vitro showed reduced proliferation and bactericidal capacity only at concentrations that 
20 DRAFT - DO NOT CITE OR QUOTE

1 reduced viability, indicating nonspecific effects of ammonia-induced immunosuppression 

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

3 specific macrophage effects were not observed. 

4 The evidence of immune system effects in experimental animals exposed to ammonia is 

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

6 

Table 1-5. Immune system effects in animals following inhalation exposure 

Health Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3)

Host resistance ≤3.5 (control), 18, 35, 71, 177 mg/m3, 7 

days (continuous exposure) pre 

inoculation/28-42 days post inoculation 

with M. pulmonis

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

group 

Broderson et al., 1976 

Increased incidence of gross 

lung lesions; no effect on CFU. 

% of animals with gross 

lesions: 16 (control), 46, 66b, 

33, 83% 

NOAEL: 18 

LOAEL: 35c

<1.4 (control) or 71 mg/m3, 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/m3, 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/m3, 3 weeks 

(continuous exposure) 

Hartley guinea pig; 8/group (sex unknown) 

Targowski et al., 1984 

Reduced proliferation in blood 

and bronchial T cells. 

No quantitative data available. 

NOAEL: 35 

LOAEL: 64 

Delayed-type 

hypersensitivity 

<11 (control), 35 or 64 mg/m3, 3 weeks 

(continuous exposure) followed by PPD 

challenge in BCG immunized 

Hartley guinea pig; 8/group (sex unknown) 

Targowski et al., 1984 

Reduced dermal lesion size. 

Mean diameter (mm):12 

(control), 12.6 and 8.7b 

NOAEL: 35 

LOAEL: 64 

a

The NOAEL and LOAEL values presented were identified by EPA. 

b

Statistically significant from controls. 

c

Study did not find statistical significance despite a large increase in the response at the lowest dose measured.
21 DRAFT - DO 

1 2 Figure 1-2. Exposure-response array for immune system effects following inhalation exposure.

1

10

100

1000

Increased incidence of 

gross lung lesions (rat);Broderson et al. (1976)

Increased bacterial 

colonization (rat);

Schoeb et al. (1982)

Increased mortality (mouse);

Richard et al. (1978)

Reduced proliferation 

in blood and bronchial

lymphocytes (guinea pig);

Targowski et al. (1984)

Host resistance Mitogen stimulation De

Exposure (mg/m3)

LOAELNOAELFELIntermediate dose

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

1 1.1.5. Other Systemic Effects 

2 Although the majority of information for ammonia suggests that ammonia induces effects 

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

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

5 heart. Alterations in liver function, based on elevated mean levels of aspartate aminotransferase 

6 (AST), alanine aminotransferase (ALT), and blood urea, decreased hemoglobin, and inhibition of 

7 catalase and monoamine oxidase (MAO) activities were observed in workers exposed to 

8 ammonia over an average exposure duration of 12 years at an Egyptian urea production plant; 

9 measurements of workplace exposure concentrations were not provided (Hamid and El-Gazzar, 

10 1996). The authors suggested that inhibition of catalase can affect electrical stability, 

11 permeability, and fluidity of membranes, which may lead to hepatotoxicity in occupationally 

12 exposed workers (Hamid and El-Gazzar, 1996). 

13 Evidence of hepatotoxicity in animals comes from observations of histopathological 

14 alterations in the liver. Fatty changes in the liver were consistently reported at concentrations 

≥470 mg/m315 ammonia in rats, guinea pigs, rabbits, dogs, and monkeys following identical 

16 subchronic inhalation exposure regimens (Coon et al., 1970). Congestion of the liver was 

17 observed in guinea pigs following subchronic and short-term inhalation exposure to 35 and 

120 mg/m318 (Anderson et al., 1964; Weatherby, 1952); no liver effects were observed in similarly 

exposed mice at 14 mg/m319 (Anderson et al., 1964; Weatherby, 1952). No histopathological or 

20 hematological effects were observed in rats, guinea pigs, rabbits, dogs, or monkeys when these 

21 animals were repeatedly, but not continuously, exposed to ammonia even at high concentrations 

(e.g., 770 mg/m322 for 8 hours/day, 5 days/week), suggesting that mammals can recover from 

23 short-term exposure to elevated ammonia levels (Coon et al., 1970). In addition, no effects were 

observed in mice exposed to 14 mg/m324 for up to 6 weeks (Anderson et al., 1964). 

25 Adrenal effects were observed in animals following subchronic and short-term exposure 

26 to ammonia; data in humans were not found. Increased mean adrenal weights and fat content of 

27 the adrenal gland, as well as histological changes in the adrenal gland (enlarged cells of the zona 

28 fasiculata of the adrenal cortex that were rich in lipid) were observed in rabbits exposed orally 

29 via gavage to ammonium hydroxide for durations ranging from 5.5 days to 17 months (Fazekas, 

30 1939). While the strength of these findings is limited by inadequate reporting and study design, 

31 a separate study identified early degenerative changes in the adrenal glands of guinea pigs 

exposed to 120 mg/m332 ammonia by inhalation for 18 weeks (Weatherby, 1952), providing 

33 additional limited evidence for effects on the adrenal gland. 

34 Evidence that inhaled ammonia can affect the kidney and spleen is limited to studies in 

35 experimental animals. Nonspecific degenerative changes in the kidneys (not further described) 

of rats exposed to 262 mg/m336 were reported (Coon et al., 1970). Histopathological evaluation of 

other animal species in the same study exposed to 470 mg/m337 , a concentration that induced a 

38 high rate of mortality in rats, consistently showed alterations in the kidneys (calcification and 
23 DRAFT - DO NOT CITE OR QUOTE

1 proliferation of tubular epithelium; incidence not reported). Exposure of guinea pigs to inhaled 

ammonia at a concentration of 120 mg/m32 for 18 weeks (but not 6 or 12 weeks) resulted in 

3 histopathological alterations (congestion) of the kidneys and spleen, although incidence was not 

4 reported (Weatherby, 1952). Enlarged and congested spleens were reported in guinea pigs 

exposed to 35 mg/m35 ammonia for 6 weeks in a separate study (Anderson et al., 1964). 

6 Myocardial fibrosis was observed in monkeys, dogs, rabbits, guinea pigs, and rats 

following subchronic, inhalation exposure to 470 mg/m37 ammonia; no changes were observed at 

8 lower concentrations (Coon et al., 1970). At the same concentration, ocular irritation 

9 (characterized as heavy lacrimation, erythema, discharge and ocular opacity of the cornea) was 

10 also reported by Coon et al. (1970) in dogs and rabbits, but not observed in similarly treated 

11 monkeys and rats. Additionally, there is limited evidence of biochemical or metabolic effects of 

12 acute or short-term ammonia exposure. Acidosis, as evidenced by a decrease in blood pH and an 

13 increase in arterial blood carbon dioxide partial pressure (pCO2), occurred in rats exposed to 

212 mg/m314 ammonia for 5−15 days (Manninen et al., 1988). Blood pH and pCO2 did not change 

in rats exposed to ≤818 mg/m315 for up to 24 hours, although statistically significant increases in 

oxygen partial pressure (pO2) were reported in rats exposed to 10.6 and 22.6 mg/m316 ammonia, 

but not at 219 and 818 mg/m317 over the same time period (Schaerdel et al., 1983). 

18 Encephalopathy related to ammonia may occur following disruption of the body’s normal 

19 homeostatic regulation of the glutamine and urea cycles resulting in elevated ammonia levels in 

20 blood, e.g., as a result of severe liver or kidney disease (Miñana et al., 1995; Souba, 1987). 

21 Acute inhalation exposure studies have identified alterations in amino acid levels and 

22 neurotransmitter metabolism (including glutamine concentrations) in the brain of rats and mice 

23 (Manninen and Savolainen, 1989; Manninen et al., 1988; Sadasivudu et al., 1979; Sadasivudu 

24 and Murthy, 1978). It has been suggested that glutamate and γ-amino butyric acid (GABA) play 

25 a role in ammonia-induced neurotoxicity (Jones, 2002). There is no evidence, however, that 

26 ammonia is neurotoxic in humans or animals following chronic exposures. 

27 The evidence of systemic toxicity in humans and experimental animals exposed to 

28 ammonia is provided in Tables 1-6 to 1-8, and presented visually in an exposure-response array 

29 in Figure 1-3. 

30 
24 DRAFT - DO NOT CITE OR QUOTE

Table 1-6. Systemic effects in humans following inhalation 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 oral 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, respectivelya 

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 default body weight of 3.5−4.1 kg for adult rabbits (U.S. EPA, 

1988).

3 

Table 1-8. Systemic effects in animals following inhalation exposures

Health 

Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3) 

Liver 

toxicity 

0 or 120 mg/m3 6 h/day, 5 days/week for 6, 

12 or 18 weeks; (24.1 mg/m3

 adjustedb), 

Guinea pig (strain not specified); male; 6–12/ 

group 

Weatherby, 1952 

Congestion of the liver at 18 weeks, 

not observed at earlier times. 

NOAEL: not 

identified 

LOAEL: 24.1 

0 or 14 for 7-42 days or 35 mg/m3 for 42 days

Guinea pig (strain not specified); male and 

female; 2/group 

Anderson et al., 1964 

Congestion of the liver at 35 mg/m3

for 42 days. 

NOAEL: 14 

LOAEL: 35 
25 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following inhalation exposures

Health 

Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3) 

0 or 14 mg/m3 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/m3 8 hrs/d, 5 d/wk for 

6 wks; (36.9, 183 mg/m3

 adjustedb) 

Squirrel monkey (Saimiri sciureus); male; 

3/group and beagle dog; male; 2/group and 

New Zealand albino rabbit; male; 3/group 

and Princeton-derived guinea pig; male and 

female; 15/group and Sprague-Dawley and 

Long-Evans rat; male and female; 15-

51/group 

Coon et al., 1970 

No histopathologic changes 

observed.

NOAEL: 183 

LOAEL: not 

identified 

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

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/m3 for 114 d or 127, 262 or 

470 mg/m3 for 90 d; 

Sprague-Dawley or Long-Evans rat; male and 

female; 15-51/group 

Coon et al., 1970 

Fatty liver changes in plate cells. NOAEL: 262 

LOAEL: 470

Adrenal 

gland 

toxicity 

0 and 120 mg/m3 6 h/day, 5 days/week for 6, 

12 or 18 weeks; (24.1 mg/m3

 adjustedb) 

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 inhalation exposures

Health 

Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3) 

Kidney and 

spleen 

toxicity 

0, 155, or 770 mg/m3 8 hrs/d, 5 d/wk for 

6 wks; (36.9, 183 mg/m3 adjustedb) 

Squirrel monkey (Saimiri sciureus); male; 

3/group and beagle dog; male; 2/group and 

New Zealand albino rabbit; male; 3/group 

and Princeton-derived guinea pig; male and 

female; 15/group and Sprague-Dawley and 

Long-Evans rat; male and female; 15-

51/group 

Coon et al., 1970

No histopathologic changes 

observed.

NOAEL: 183 

LOAEL: not 

identified

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

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/m3 for 114 d or 127, 262 or 

470 mg/m3

 for 90 d; 

Sprague-Dawley or Long-Evans rat; male and 

female; 15-51/group 

Coon et al., 1970 

Calcification and proliferation of 

renal tubular epithelium. 

NOAEL: 262 

LOAEL: 470 

0 or 120 mg/m3 6 h/day, 5 days/week for 6, 

12 or 18 weeks; (24.1 mg/m3 adjustedb) 

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/m3 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/m3 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 inhalation exposures

Health 

Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3) 

Myocardial 

toxicity 

0, 155, or 770 mg/m3 8 hrs/d, 5 d/wk for 

6 wks; (36.9, 183 mg/m3 adjustedb) 

Squirrel monkey (Saimiri sciureus); male; 

3/group and beagle dog; male; 2/group and 

New Zealand albino rabbit; male; 3/group 

and Princeton-derived guinea pig; male and 

female; 15/group and Sprague-Dawley and 

Long-Evans rat; male and female; 15-

51/group 

Coon et al., 1970

No histopathologic changes 

observed.

NOAEL: 183 

LOAEL: not 

identified

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

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/m3 for 114 d or 127, 262 or 

470 mg/m3

 for 90 d; 

Sprague-Dawley or Long-Evans rat; male and 

female; 15-51/group 

Coon et al., 1970 

Myocardial fibrosis. NOAEL: 262

LOAEL: 470 

Ocular 

Irritation 

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

Beagle dog; male; 2/group 

Coon et al., 1970 

Heavy lacrimation. NOAEL: 40 

LOAEL: 470 

0 or 40 mg/m3 for 114 d or 470 mg/m3 for 

90 d 

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/m3 for 114 d or 470 mg/m3 for 

90 d 

Squirrel monkey (Saimiri sciureus); male; 

3/group and 3/group and Princeton-derived 

guinea pig; male and female; 15/group 

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 inhalation exposures

Health 

Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3) 

0 or 40 mg/m3 for 114 d or 127, 262 or 

470 mg/m3 for 90 d; 

Sprague-Dawley and Long-Evans rat; male 

and female; 15-51/group 

Coon et al., 1970 

No ocular irritation observed. NOAEL: 470 

LOAEL: not 

identified 

0, 155, or 770 mg/m3 8 hrs/d, 5 d/wk for 

6 wks; (36.9, 183 mg/m3 adjustedb) 

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/m3 6 h/day for 5, 10 or 15 

days; (4.5, 53 mg/m3 adjustedb)

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: 

0.09c and 0.07c

NOAEL: 53 

LOAEL: not 

identified 

10.6–818 mg/m3 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 

pO2 at 10.6 and 22.6 mg/m3

exposure at 8, 12 and 24 hours 

(p<0.05). No change at higher 

exposures. No change in blood pH or 

pCO2. 

Response relative to controld

: 16, 6, 

20% at 10.6 mg/m3

 and at 8, 12, 24 

hrs; 18, 26, 17% at 22.6 mg/m3 and 

at 8, 12, 24 hrs 

NOAEL: 818 

LOAEL: not 

identified 
29 DRAFT - DO NOT CITE OR QUOTE

Table 1-8. Systemic effects in animals following inhalation exposures

Health 

Effect Study Design and Reference Results

NOAEL/LOAELa

(mg/m3) 

Amino acid 

levels and 

neurotransmitter 

metabolism 

in the brain 

0, 18, or 212 mg/m3 6 h/day for 5 days; (4.5, 

53 mg/m3 adjustedb)

Wistar rat; female; 5/group 

Manninen and Savolainen, 1989 

Statistically significant increase in 

brain glutamine (p< 0.05). 

Response relative to control: 42c, 

40c% for 18 and 212 mg/m3, 

respectively 

NOAEL: not 

identified 

LOAEL: 4.5e

0, 18, or 212 mg/m3 6 h/day for 5, 10 or 15 

days; (4.5, 53 mg/m3

 adjustedb)

Wistar rat; female; 5/group 

Manninen et al., 1988 

Brain and blood glutamine 

statistically significantly increased 

(p< 0.05 and 0.01, respectively) at 

212 mg/m3 at 5 days, no statistically 

significant difference from control at 

10 and 15 days. 

Response relative to control at 

212 mg/m3: 44c, 13 and 14% 

increase in blood glutamine at 5, 10, 

15 days; 40c

, 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 identified 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.

dMeasurements at time zero were used as a control. The study did not include an unexposed, control group. 

1 

2 
30 DRAFT - DO 

1 2 3 Figure 1-3. Exposure-response array for systemic effects following inhalation exposure.

1

10

100

1000

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)

Liver toxicity Adrenal 

gland 

toxicity

Kidney and spleen toxicity Myocardial toxicity 

Exposure (mg/m3)

LOAELNOAELFELIntermediate dose

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

1 1.1.6. Cancer 

2 No information is available regarding the carcinogenic effects of ammonia in humans 

3 following oral or inhalation exposure. The carcinogenic potential of ammonia by the inhalation 

4 route has not been assessed in animals, and animal carcinogenicity data by the oral route of 

5 exposure are limited. Toth (1972) concluded that tumor incidence was not increased in Swiss 

6 mice exposed for their lifetime (not further specified) to ammonium hydroxide in drinking water 

7 at concentrations up to 0.3% (equivalent to 410 and 520 mg/kg-day in female and male mice, 

8 respectively) or in C3H mice exposed to ammonium hydroxide in drinking water at a 

9 concentration of 0.1% (equivalent to 214 and 191 mg/kg-day in female and male mice, 

10 respectively). With the exception of mammary gland tumors in female C3H mice (a tumor with a 

11 high background incidence), concurrent control tumor incidence data were not reported and 

12 comparison of tumor incidence in exposed and control mice could not be performed. The 

13 general lack of concurrent control data limits the ability to interpret the findings of this study. 

14 The incidence of gastric cancer and the number of gastric tumors per tumor-bearing rat 

15 were statistically significantly higher in rats exposed to 0.01% ammonia solution in drinking 

16 water (equivalent to 10 mg/kg-day) for 24 weeks following pretreatment (for 24 weeks) with the 

17 initiator N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) compared with rats receiving only 

18 MNNG and tap water (Tsujii et al., 1992a). In an almost identically designed study, reported by 

19 Tsujii et al. (1995), similar increases in the incidence of gastric tumors were observed in rats 

20 following exposure to MNNG and 10 mg/kg-day ammonia. Additionally, the size and 

21 penetration to deeper tissue layers of the MNNG-initiated gastric tumors were enhanced in the 

22 rats treated with ammonia (Tsujii et al., 1995). The investigators suggested that ammonia 

23 administered in drinking water may act as a cancer promoter (Tsujii et al., 1995, 1992a); 

24 however, in the absence of an ammonia-only exposure group in these studies, it is not possible to 

25 distinguish between possible promotion and initiator activity. 

26 The evidence of carcinogenicity in experimental animals exposed to ammonia is provided 

27 in Table 1-9. 

28 
32 DRAFT - DO NOT CITE OR QUOTE

Table 1-9. Cancer bioassays following oral exposure 

Health Effect Study Design and Reference Results

Tumor incidence 250, 440, and 520 mg/kg-day (males); 

240, 370, and 410 mg/kg-day (females) 

[0.1, 0.2, and 0.3% ammonium hydroxide 

in drinking watera] for their lifetime (not 

further specified) 

Swiss mouse, 50/sex/group 

Toth, 1972 

The authors reported that tumor incidence was 

not increased in ammonia-exposed mice; 

however, concurrent control tumor incidence 

data were not reported and comparison of 

tumor incidence in exposed and control mice 

could not be performed. 

191 (males) and 214 mg/kg-day (females) 

[0.1% ammonium hydroxide in drinking 

waterb

] for their lifetime (not further 

specified) 

C3H mouse, 40/sex/group 

Toth, 1972 

The authors reported that tumor incidence was 

not increased in ammonia-exposed mice; 

however, with the exception of mammary gland 

tumors in female mice, concurrent control tumor 

incidence data were not reported and 

comparison of tumor incidence in exposed and 

control mice could not be performed. 

Mammary gland adenocarcinoma: 76, 60% 

0 or 10 mg/kg-day [0 or 0.01% ammonia 

in drinking waterc

] for 24 weeks; both 

groups pretreated for 24 weeks with the 

tumor initiator MNNG 

Sprague Dawley rat, male; 40/group 

Tsujii et al.,1992a 

Statistically significantly increased incidence of 

gastric cancers and number of gastric tumors per 

tumor-bearing rat in ammonia + MNNG group 

compared to MNNG only group 

Gastric tumor incidence: 31, 70d% 

# of gastric tumors/tumor-bearing rat: 1.3, 2.1d

0 or 10 mg/kg-day [0 or 0.01% ammonia 

in drinking waterc] for 24 weeks; both 

groups pretreated for 24 weeks with the 

tumor initiator MNNG 

Sprague Dawley rat; male; 43-44/group 

Tsujii et al., 1995 

Statistically significantly increased incidence of 

gastric cancers, size, and penetration to deeper 

tissue layers in ammonia + MNNG group 

compared to MNNG only group 

Gastric tumor incidence: 30, 66d% 

Penetrated muscle layer or deeper: 12, 22d% 

Size (mm): 4.4, 5.3d

a

Ammonium hydroxide doses estimated based on reported average daily drinking water intakes of 9.2, 8.2, and 6.5 

mL/day for males and 8.3, 6.5, and 4.8 mL/day for females in the 0.1, 0.2, and 0.3% groups, respectively, and 

assumed average default body weights of 37.3 and 35.3 g for males and females, respectively (U.S. EPA, 1988). 

b

Ammonium hydroxide doses estimated based on reported average daily drinking water intakes of 7.9 and 8.4 

mL/day for males and females, respectively, and assumed average default body weights of 37.3 and 35.3 g for 

males and females, respectively (U.S. EPA, 1988). 

c

Ammonia doses estimated based on reported drinking water intake of 50 mL/day and assumed average default 

body weight of 523 g for male Sprague-Dawley rats during chronic exposure (U.S. EPA, 1988). 

d

Statistically significantly different from control.

1 

2 A limited number of genotoxicity studies are available for ammonia vapor, including one 

3 study in exposed fertilizer factory workers in India that reported chromosomal aberrations and 

4 sister chromatid exchanges in lymphocytes (Yadav and Kaushik, 1997), mutation assays in S. 
33 DRAFT - DO NOT CITE OR QUOTE

1 typhimurium and E. coli (Shimizu et al., 1985; Demerec et al., 1951), a micronucleus assay in 

2 mice (Yadav and Kaushik, 1997), studies in D. melanogaster (Auerbach and Robson, 1947; 

3 Lobasov and Smirnov, 1934), and a chromosomal aberration test in chick fibroblast cells in vitro 

4 (Rosenfeld, 1932) (see Appendix A, Section A.5). Four of the six available studies were 

5 published between 1932 and 1951, and the available genotoxicity database in general is 

6 inadequate to characterize the genotoxic potential of ammonia. 

7 

8 1.1.7. Susceptible Populations and Life Stages 

9 Studies of the toxicity of ammonia in children or young animals compared to other life 

10 stages that would support an evaluation of childhood susceptibility have not been conducted. 

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

12 in individuals with severe diseases of the liver or kidney, organs that biotransform and excrete 

13 ammonia, or with hereditary urea cycle disorders (Córdoba et al., 1998; Schubiger et al., 1991; 

14 Gilbert, 1988; Jeffers et al., 1988; Souba, 1987). The elevated ammonia levels that accompany 

15 human diseases such as acute liver or renal failure can predispose an individual to 

16 encephalopathy due to the ability of ammonia to cross the blood-brain barrier; these effects are 

17 especially marked in newborn infants (Miñana et al., 1995; Souba, 1987). Thus, individuals with 

18 disease conditions that lead to hyperammonemia may be more susceptible to the effects of 

19 ammonia from external sources, but there are no studies that specifically support this 

20 susceptibility. 

21 Because the respiratory system is a target of ammonia toxicity, individuals with 

22 respiratory disease (e.g., asthmatics) might be expected to be a susceptible population; however, 

23 controlled human studies that examined both healthy volunteers and volunteers with asthma 

24 exposed to ammonia as well as cross-sectional studies of livestock farmers exposed to ammonia 

25 (Petrova et al., 2008; Sigurdarson et al., 2004; Vogelzang et al., 2000, 1998, 1997; Preller et al., 

26 1995) generally did not observe a greater sensitivity to respiratory effects in populations with 

27 underlying respiratory disease.

28 

29 1.2. Weight of Evidence Evaluation for Toxicological Effects 

30 The available evidence for ammonia toxicity indicates that respiratory effects are 

31 associated with inhalation exposure and gastrointestinal effects are associated with oral exposure 

32 to ammonia. Ammonia exposure may not be associated with reproductive or developmental 

33 toxicity, at least at levels in which respiratory and gastrointestinal effects are observed. Immune 

34 system and other systemic effects (i.e., effects on the liver, kidney, heart, spleen, and adrenal 

35 gland) may be associated with exposure to ammonia but are not sensitive targets of ammonia 

36 toxicity. The evidence for these health effects are presented in more detail below. Figure 1-4 is 
34 DRAFT - DO NOT CITE OR QUOTE

1 an exposure-response array comparing effect levels for inhaled ammonia across a range of 

2 toxicological effect categories. 

3 

4 Respiratory Effects 

5 Evidence for respiratory toxicity associated with exposure to ammonia comes from 

6 studies in humans and animals. Cross-sectional occupational studies involving chronic exposure 

7 to ammonia have consistently demonstrated an increased prevalence of respiratory effects 

8 (Rahman et al., 2007; Ballal et al., 1998) and decreased lung function (Rahman et al., 2007; Ali., 

9 2001). Cross-sectional studies of livestock farmers exposed to ammonia, controlled human 

10 volunteer studies of ammonia inhalation, and case reports of injury in humans with inhalation 

11 exposure to ammonia provide additional and consistent support for the respiratory system as a 

12 target of ammonia toxicity. 

13 Short-term and subchronic animals studies show respiratory effects in several animal 

14 species (lung inflammation in guinea pigs and rats; focal or interstitial pneumonitis in monkeys, 

15 dogs, rabbits and guinea pigs; pulmonary congestion in mice; thickening of nasal epithelium in 

16 rats and pigs; nasal inflammation or lesions in rats and mice) across different dose regimens and 

17 show respiratory effects across ranges of concentrations suggesting a dose-response (Coon et al., 

18 1970; Anderson et al., 1964; Broderson et al., 1976; Doig and Willoughby, 1971; Gaafar et al., 

19 1992). EPA considers the respiratory effects associated with ammonia exposure to be 

20 biologically plausible and adverse. The evidence of observed respiratory effects seen across 

21 multiple human and animal studies identifies the respiratory system as a hazard for ammonia. 

22 

23 Gastrointestinal Effects 

24 Effects on gastric mucosa associated with oral exposure to ammonia are based on 

25 evidence in animals and, to a more limited extent, in humans. Acute gastric toxicity observed in 

26 case reports involving intentional or accidental ingestion of cleaning solutions or ammonia 

27 inhalant capsules appears to reflect the corrosive properties of ammonia. Whether these acute 

28 effects are relevant to toxicity following chronic low-level ammonia exposure is not known. 

29 Indirect evidence is provided by the association between the stomach bacterium H. pylori, which 

30 produces urease that catalyzes urea into ammonia, and human diseases of the upper 

31 gastrointestinal tract (including chronic gastritis, gastric ulcers, and stomach cancer). 

32 In vivo experimental evidence that ammonia is associated with gastric effects is provided 

33 by two short-term studies in male Sprague-Dawley rats (Tsujii et al., 1993; Kawano et al., 1991). 

34 These studies provide consistent findings of decreased gastric mucosal thickness that increased 

35 with ammonia dose (Kawano et al., 1991) and duration of exposure (Tsujii et al., 1993; Kawano 

36 et al., 1991); Tsujii et al. (1993) employed only one ammonia drinking water concentration and 

37 therefore did not provide information on dose-response. Evidence for ammonia-related gastric 

38 toxicity is limited to male rats of one strain and to investigations conducted by one research 
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1 group (Kawano et al. and Tsujii et al. were both affiliated with Osaka University Medical 

2 School). 

3 Mechanistic studies in rodent models support the biological plausibility that ammonia 

4 exposure may be associated with gastric effects. Conditions that favor the unionized form of 

5 ammonia facilitate the penetration of the cell membrane and induce greater gastric toxicity. 

6 Multiple specific mechanistic events have been proposed that may contribute to the induction of 

7 gastric lesions, including ammonia-induced release of proteases, inhibition of mitochondrial 

8 respiration, and increased release of endothelin-1 and thyrotropin-releasing hormone. EPA 

9 considers the gastric effects associated with ammonia exposure to be biologically plausible and 

10 adverse, and relevant to humans. Given the evidence from human, animal, and mechanistic 

11 studies, gastric effects are identified as a hazard for ammonia. 

12 

13 Reproductive/Developmental Effects 

14 No studies of the potential reproductive or developmental toxicity of ammonia in humans 

15 are available, and only one animal study that examined the reproductive effects of ammonia in 

16 the pig has been conducted. This study did not use a conventional test species and did not 

17 include a control group with no ammonia exposure. Further, animals were exposed naturally to 

18 bacterial and mycoplasm pathogens. Although the reproductive and developmental toxicity 

19 database for ammonia is limited, evidence on the endogenous formation of ammonia can inform 

20 the potential for ammonia to present a reproductive and developmental hazard. 

21 Ammonia is endogenously produced in humans and animals during fetal and adult life 

22 and concentrations in blood are homeostatically regulated to remain at low levels. Studies in 

23 humans and animals demonstrate that ammonia is present in fetal circulation. In vivo studies in 

24 several animal species and in vitro studies of human placenta suggest that ammonia is produced 

25 within the uteroplacenta and released into the fetal and maternal circulations (Bell et al., 1989; 

26 Johnson et al., 1986; Haugel et al., 1983; Meschia et al., 1980; Remesar et al., 1980; Holzman et 

27 al., 1979, 1977; Rubaltelli and Formentin, 1968; Luschinsky, 1951). Jόźwik et al. (2005) 

28 reported that ammonia levels in human fetal blood (specifically, umbilical arterial and venous 

29 blood) at birth were 1.0–1.4 μg/mL, compared to 0.5 μg/mL in the mothers’ venous blood. 

30 DeSanto et al. (1993) similarly collected human umbilical arterial and venous blood at delivery 

31 (range of 25–43 weeks of gestation). Ammonia was present in blood samples, with umbilical 

32 arterial ammonia concentrations significantly higher than venous concentrations; there was no 

33 correlation between umbilical ammonia levels and gestational age. In sheep, uteroplacental 

34 tissues are the main site of ammonia production, with outputs of ammonia into both the uterine 

35 and umbilical circulations (Jόźwik et al., 1999). In late-gestation pregnant sheep that were 

36 catheterized to allow measurement of ammonia exposure to the fetus, concentrations of ammonia 

37 in umbilical arterial and venous blood and uterine arterial and venous blood ranged from about 

38 0.39 to 0.60 μg/mL (Jόźwik et al., 2005, 1999). Thus, the developing fetus and reproductive 
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1 tissues are normally exposed to ammonia in blood, and external concentrations that do not alter 

2 homeostasis would not be expected to pose a developmental or reproductive hazard. 

3 

4 Immune System Effects 

5 The evidence for ammonia immunotoxicity is based on two epidemiological studies and 

6 four animal studies. Available epidemiological studies that addressed immunological function 

7 are confounded by exposures to a number of other respirable agents that have been demonstrated 

8 to be immunostimulatory. Single-exposure human studies of ammonia evaluating immune 

9 endpoints are not available. Therefore, human studies provide little support for ammonia 

10 immunotoxicity. 

11 Animal studies consistently provide evidence of elevated bacterial growth following 

12 ammonia exposure. This is supported by observations of lung lesions (Broderson et al., 1976), 

13 elevated CFU (Schoeb et al., 1982), and increased mortality (Richard et al., 1978a) in rats or 

14 mice exposed to ammonia; however, the findings from the Broderson et al. (1976) study (% of 

15 animals with gross lesions) were not dose-responsive, and the other studies used single 

16 concentrations of ammonia and therefore did not provide information on dose-response. A 

17 single study suggested that T cells are inhibited by ammonia, but the data were not dose 

18 responsive (Targowski et al, 1984). 

19 Mechanistic data are not available that would support a biologically plausible mechanism 

20 for immunosuppression. Because ammonia damages the protective mucosal epithelium of the 

21 respiratory tract, it is unclear if elevated bacterial colonization is the result of damage to this 

22 barrier or the result of suppressed immunity. Overall, the evidence in humans and animals 

23 indicates that ammonia exposure may be associated with these effects but does not support the 

24 immune system as a sensitive target for ammonia toxicity. 

25 

26 Systemic Effects 

27 Effects of ammonia exposure on organs distal from the portal of entry are based on 

28 evidence in animals and, to a more limited extent, in humans. One occupational epidemiology 

29 study of ammonia-exposed workers reported changes in serum enzymes indicative of altered 

30 liver function (Hamid and El-Gazzar, 1996). Because the study population was small and 

31 measurements of workplace ammonia concentrations were not provided, the evidence for liver 

32 toxicity in humans associated with ammonia exposure is weak. 

33 Effects on various organs, including liver, adrenal gland, kidney, spleen, and heart, were 

34 observed in several studies that examined responses to ammonia exposure in a number of 

35 laboratory species. While effects on many of these organs were observed in multiple species, 

36 including monkey, dog, rabbit, guinea pig, and rat, effects were not consistent across exposure 

37 protocols. For example, Coon et al. (1970) reported fatty liver and calcification and proliferation 

38 of renal tubular epithelium in monkeys, dogs, rabbits, and guinea pigs exposed continuously to 
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ammonia for 90 days at a concentration of 470 mg/m31 , but no histopathological changes in these 

2 organs were observed in the same species following intermittent exposure (8 hours/day, 5 

days/week for 6 weeks) to concentrations as high as 770 mg/m33 . It could be speculated that these 

4 differences in response reflect recovery from short-term (i.e., 8-hour exposures), but the reason 

5 for the inconsistent findings is not known. 

6 Studies of ammonia toxicity that examined systemic effects were all published in the 

7 older toxicological literature. The only oral study of ammonium hydroxide was published in 

8 1939 (Fazekas, 1939), and three subchronic inhalation studies were published between 1952 and 

9 1970 (Coon et al., 1970; Anderson et al., 1964; Weatherby, 1952). In general, the information 

10 from these studies is limited by small group sizes, minimal characterization of some of the 

11 reported responses (e.g., “congestion,” “enlarged,” “fatty liver”), insufficiently detailed reporting 

12 of study results, and incomplete if any incidence data. In addition, Weatherby (1952), Anderson 

13 et al. (1964), and some of the experiments reported by Coon et al. (1970) used only one ammonia 

14 concentration in addition to the control, so no dose-response information is available the majority 

15 of experimental studies to inform the evidence for systemic effects of ammonia. 

16 As discussed above, ammonia is endogenously produced in all human and animal tissues, 

17 and concentrations in all physiological fluids are homeostatically regulated to remain at low 

18 levels (Souba, 1987). Thus, tissues are normally exposed to ammonia, and external 

19 concentrations that do not alter homeostasis would not be expected to pose a hazard for systemic 

20 effects. Overall, the evidence in humans and animals indicates that ammonia exposure may be 

21 associated with these effects but does not support the liver, adrenal gland, kidney, spleen, or 

22 heart as sensitive targets for ammonia toxicity. 

23 
38 DRAFT - DO 

1 2 3 Figure 1-4. Exposure-response array for toxicological effects following inhalation exposure.

0.1

1

10

100

1000

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)

Respiratory effects Immunologic 

effects

Systemic effects

Exposure (mg/m3)

LOAELNOAELFELIntermediate dose

Vertical lirange of study. Cshow exptested in 

HUMAN STUDIES EXPERIMENTAL ANIMAL STUDIES
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1 Cancer 

2 The available information on carcinogenicity following exposure to ammonia is limited 

3 to oral animal studies. There was no evidence of carcinogenicity in Swiss or C3H mice 

4 administered ammonium hydroxide in drinking water for a lifetime (Toth, 1972). There is 

5 limited evidence that ammonia administered in drinking water may act as a cancer promoter 

6 based on the findings of studies designed to examine H. pylori-induced gastric cancer (Tsujii et 

7 al., 1995, 1992a). Additionally, the genotoxic potential cannot be characterized based on the 

8 available genotoxicity information. Thus, under the Guidelines for Carcinogen Risk Assessment

9 (U.S. EPA, 2005a), there is “inadequate information to assess the carcinogenic potential” of 

10 ammonia. 

11 

12 

13 
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1 

2 2. DOSE-RESPONSE ANALYSIS 

3 

4 2.1. Oral Reference Dose for Effects other than Cancer 

5 The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty 

6 spanning perhaps an order of magnitude) of a daily exposure to the human population (including 

7 sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a 

8 lifetime. It can be derived from a NOAEL, LOAEL, or benchmark dose (BMD), with 

9 uncertainty factors (UFs) generally applied to reflect limitations of the data used. 

10 The oral toxicity database for ammonia is very limited, although as noted in Section 1.2, 

11 gastric toxicity is identified as a hazard for ammonia based on evidence from case reports in 

12 humans, two animal studies, and mechanistic studies. Evidence in humans is limited to case 

13 reports of individuals suffering from gastrointestinal (e.g., stomach ache, nausea, diarrhea, 

14 distress, and burns along the digestive tract) effects from ingesting household cleaning solutions 

15 containing ammonia or biting into capsules of ammonia smelling salts. The data in humans were 

16 not considered for derivation of the RfD because although case reports can suggest the nature of 

17 acute endpoints in humans they are inadequate for dose-response analysis and derivation of a 

18 chronic reference value due to short duration of exposure and incomplete or missing quantitative 

19 exposure information. 

20 Two studies reported gastrointestinal effects, characterized as increased epithelial cell 

21 migration in the mucosa of the stomach (in particular the antrum) leading to a statistically 

22 significant decrease in the thickness of the antral mucosa, in rats following subchronic (Tsujii et 

23 al., 1993) and short-term (Kawano et al., 1991) oral exposure to ammonia. These studies are 

24 repeated dose studies that analyzed gastrointestinal effects of ammonia and did not evaluate a 

25 comprehensive array of endpoints. Additionally, although both studies included a control group, 

26 Tsujii et al. (1993) employed one dose group and Kawano et al. (1991) included two dose 

27 groups. However, the decreased gastric antral mucosal thickness was consistently observed 

28 across these two studies. Prevalence of this effect was observed to generally increase with dose 

29 and duration, and the magnitude of decreases in thickness was 40-60%. Tsujii et al. (1993) and 

30 Kawano et al. (1991) reported that the gastric mucosal effects observed in rats resemble mucosal 

31 changes in human atrophic gastritis; indicating this effect is biological plausible and relevant to 

32 humans. Therefore, decreased gastric antral mucosal thickness is an effect considered by EPA to 

33 be adverse. 

34 Given the limited number of studies available and the small number of toxicological 

35 evaluations, there are uncertainties associated with the oral database for ammonia. Although the 

36 oral database is limited, derivation of a RfD was considered due to the toxicological significance 
41 DRAFT - DO NOT CITE OR QUOTE

1 of the reported gastrointestinal effects. However, uncertainties with extrapolations from the 

2 available data (described below) were too high to support derivation of a chronic RfD; thus, in 

3 consideration of the limited oral database and associated uncertainties a RfD for ammonia was 

4 not derived. 

5 In considering the derivation of a RfD, the subchronic study by Tsujii et al. (1993) was 

6 considered as a potential principal study due to the relatively longer duration of exposure 

7 compared with the short-term study by Kawano et al. (1991). Decreased gastric antral mucosal 

8 thickness was considered as a potential critical effect. This effect was characterized as a portal9 of-entry effect based on the following. Tsujii et al. (1993) postulated that the difference in 

10 response of the mucosa in the stomach body versus the mucosa of the antrum relates to 

11 differences in pH in the two stomach regions. Most ammonia is transformed to ammonium ion 

12 in solution at physiological pH; the ratio of ammonia to ammonium ion increases 10-fold with 

13 each unit rise in pH. In the mucosa of the stomach body—an acid-secreting mucosa—ammonia 

14 is protonated to the ammonium ion, which reduces the cytotoxicity associated with nonionized 

15 ammonia. In the antral mucosa—a nonacid secreting area of the stomach—the pH is higher, 

16 resulting in a relatively higher concentration of ammonia and thus enhanced cytotoxicity. 

17 EPA identified a potential point of departure (POD) based on the LOAEL of 33 mg/kg18 day, for decreased gastric antral mucosal thickness in rats, from this study. BMD modeling was 

19 not utilized because the Tsujii et al. (1993) employed only one dose level and a control, a data set 

20 that is not amenable to dose-response analysis. 

In U.S. EPA’s Recommended Use of Body Weight3/4 21 as the Default Method in Derivation 

22 of the Oral Reference Dose (U.S. EPA, 2011a), the Agency endorses a hierarchy of approaches 

23 to derive human equivalent oral exposures from data from laboratory animal species, with the 

24 preferred approach being physiologically based pharmacokinetic modeling. Other approaches 

25 may include using some chemical-specific information, without a complete physiologically 

26 based pharmacokinetic model. In lieu of chemical-specific models or data to inform the 

27 derivation of human equivalent oral exposures, EPA endorses body weight scaling to the 3⁄4 

power (i.e., BW3/4 28 ) as a default to extrapolate toxicologically equivalent doses of orally 

29 administered agents from laboratory animals to humans for the purpose of deriving a RfD. More 

specifically, the use of BW3/4 30 scaling for deriving a RfD is recommended when the observed 

31 effects are associated with the parent compound or a stable metabolite, but not for portal-of-entry 

32 effects. 

33 No PBPK model or chemical-specific information exists to inform the generation of 

34 human equivalent oral exposures for ammonia. Furthermore, because ammonia oral toxicity 

35 appears to be a function of the physical/chemical environment at the mucosal surface (i.e., a 

36 portal-of-entry effect) and it is not clear if regions of the stomach scale allometrically across 

37 species, a surface area adjustment would be the most relevant for interspecies extrapolation; 

38 however, a dose scaling approach involving mass per unit surface area has not been developed 
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1 (U.S. EPA, 2011a). Therefore, because effects on the gastric antral mucosa are not expected to 

scale allometrically, a BW3/4 2 scaling approach (in combination with a reduced default UF for 

3 interspecies extrapolation) was not applied. 

4 The composite UF for ammonia that would be applied to the POD (LOAEL of 33 mg/kg5 day) from the Tsujii et al. (1993) study would be 10,000, consisting of four areas of uncertainty. 

6 These areas of uncertainty, and the UFs that address each, are based on EPA’s A Review of the 

7 Reference Dose and Reference Concentration Processes (U.S. EPA, 2002; Section 4.4.5) and 

8 include the following: uncertainties associated with intraspecies extrapolation (i.e., to account for 

9 human variability in susceptibility to ammonia; UFH = 10), uncertainties associated with 

10 extrapolation of data from the rat to humans in the absence of information on species differences 

11 in toxicokinetics and toxicodynamics (i.e., interspecies extrapolation; UFA = 10), uncertainties 

12 associated with extrapolation of data from a subchronic study (i.e., 8-week study) to a reference 

13 value for chronic exposure scenarios (UFS = 10), uncertainties associated with extrapolation

14 from a LOAEL to NOAEL (UFL = 10), and database deficiencies (UFD = 1; see Section 2.2.2 for 

15 the justification for this UF). 

16 In the report, A Review of the Reference Dose and Reference Concentration 

17 Processes (U.S. EPA, 2002), the RfD/RfC technical panel concluded that, in cases where 

18 maximum uncertainty exists in four or more areas of uncertainty, or when the total UF is 

19 ≥10,000, it is unlikely that the database is sufficient to derive a reference value. Therefore, 

20 consistent with the recommendations in U.S. EPA (2002), the available oral data for ammonia 

21 were considered insufficient to support reference value derivation and an RfD for ammonia was 

22 not derived. 

23 Route-to-route extrapolation from inhalation data was considered for deriving the oral 

24 RfD; however, in the absence of a PBPK model and because the critical effect from the 

25 inhalation literature is a portal-of-entry effect (respiratory irritation and decreased lung function), 

26 route-to-route extrapolation is not supported (U.S. EPA, 1994). 

27 

28 Previous IRIS Assessment: Reference Dose 

29 No RfD was derived in the previous IRIS assessment for ammonia 

30 

31 2.2. Inhalation Reference Concentration for Effects other than Cancer 

The RfC (expressed in units of mg/m332 ) is defined as an estimate (with uncertainty 

33 spanning perhaps an order of magnitude) of a continuous inhalation exposure to the human 

34 population (including sensitive subgroups) that is likely to be without an appreciable risk of 

35 deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or benchmark 

36 concentration (BMC), with UFs generally applied to reflect limitations of the data used. 
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1 As discussed in Section 1.2, respiratory effects have been identified as a hazard following 

2 inhalation exposure to ammonia. The studies in humans and animals examining inhalation 

3 exposure to ammonia provide evidence that inhaled ammonia is associated with toxicity to the 

4 respiratory system. The experimental toxicology literature for ammonia also provides evidence 

5 that inhaled ammonia may be associated with toxicity to target organs other than the respiratory 

6 system, including the liver, adrenal gland, kidney, spleen, heart, and immune system. The 

7 weight of evidence for these effects is less robust than for respiratory effects. Therefore, the 

8 respiratory system is the primary and most sensitive target of inhaled ammonia toxicity in 

9 humans and experimental animals. 

10 Human data are preferred over animal data for deriving reference values when possible 

11 because the use of human data is more relevant in the assessment of human health and avoids the 

12 uncertainty associated with interspecies extrapolation introduced when animal data serve as the 

13 basis for the RfC. Additionally, the respiratory effects in animals were observed at ammonia 

14 concentrations higher than those associated with respiratory effects in humans and represent 

15 much shorter durations (up to 114 days) of exposure, and thus were considered to carry less 

16 weight than the available human data. Therefore, data in humans were considered for derivation 

17 of the RfC and the respiratory effects in animals were not further considered. 

18 Of the available human data, two occupational studies—Rahman et al. (2007) and 

19 Holness et al. (1989)—provide information useful for examining the relationship between 

20 chronic ammonia exposure and respiratory irritation and decreased lung function (quantitative 

21 dose-response analysis of ammonia respiratory tract toxicity data). Both studies reported the 

22 presence or absence of respiratory effects in workers exposed to ammonia over a range of 

concentrations (approximately 1 to 7 mg/m323 ). Both studies provide consistent estimates of the 

effect level for ammonia, with the NOAELADJ of 3.1 mg/m324 identified from the Holness et al. 

(1989) study falling between the NOAELADJ and LOAELADJ values (1.8 and 6.6 mg/m325 , 

26 respectively) from the Rahman et al. (2007) study. These studies are considered as candidate 

27 principal studies for RfC derivation. Other occupational epidemiology studies (Ali et al., 2001; 

28 Ballal et al., 1998) did not provide exposure information adequate for dose-response analysis and 

29 thus were not useful for RfC derivation. 

30 Consideration of analytical methods suggests that higher confidence is associated with 

31 the exposure measures reported by Holness et al. (1989) than Rahman et al. (2007). Rahman et 

32 al. (2007) used two analytical methods for measuring ammonia concentrations in workplace air 

33 (Dräger PAC III and Dräger tube); concentrations measured by the two methods differed by 

34 four- to fivefold, indicating some uncertainty in these measurements, although ammonia 

35 concentrations measured by the two methods were strongly correlated. In contrast, the Holness 

36 et al. (1989) study used an established analytical method for measuring exposure to ammonia 

37 recommended by NIOSH that involved the collection of air samples on acid-treated silica gel 

38 (ATSG) absorption tubes. 
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1 Due to the greater confidence in the ammonia measurements in Holness et al. (1989) and 

2 considering the range of NOAELs and LOAELs reported in both studies (in which a higher 

3 NOAEL was reported by Holness et al. [1989]) the occupational exposure study of ammonia 

4 exposure in workers in a soda ash plant by Holness et al. (1989) was identified as the principal 

5 study for RfC derivation. Respiratory effects, characterized as increased respiratory irritation 

6 and decreased lung function, observed in workers exposed to ammonia concentrations ≥6.6 

mg/m37 were selected as the critical effect. Respiratory effects, including changes in measures of 

8 lung function and increased prevalence of wheezing, chest tightness, and cough/phlegm, have 

9 been identified as adverse respiratory health effects by the American Thoracic Society (2000), 

10 and are similarly noted as adverse in the EPA’s Methods for Derivation of Inhalation Reference 

11 Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994). 

12 

13 2.2.1. Methods of Analysis 

14 In the evaluation of the prevalence of increased respiratory irritation and decreased lung 

function in workers exposed to ammonia (Holness et al., 1989), a NOAELADJ of 3.1 mg/m315 

(adjusted for continuous exposure from 8.8 mg/m316 ; see calculation below) was identified based 

17 on the absence of statistically significant increases in the prevalence of the respiratory effects. 

18 BMD modeling was not utilized because ammonia concentrations in the Holness et al. (1989) 

19 study were not associated with changes in respiratory effects in the study population (i.e., data 

20 from Holness et al. could not be subjected to dose-response modeling). Thus, the Holness et al. 

(1989) data were analyzed using a NOAEL approach and the NOAELADJ of 3.1 mg/m321 was 

22 used as the POD for RfC derivation. 

23 Because the RfC is a measure that assumes continuous human exposure over a lifetime, 

24 the POD derived from an occupational exposure was adjusted to account for the noncontinuous 

25 exposure associated with occupational exposure (i.e., 8-hour workday and 5-day workweek). 

26 The duration-adjusted POD was calculated as follows: 

27 

28 NOAELADJ = NOAEL × VEho/VEh × 5 days/7 days 

 = 8.8 mg/m3 × 10 m3/20 m329 × 5 days/7 days 

 = 3.1 mg/m330 

31 

32 Where: 

VEho = human occupational default minute volume (10 m333 breathed during the 8-hour workday, 

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

VEh = human ambient default minute volume (20 m3

35 breathed during the entire day) 

36 
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1 2.2.2. Derivation of Reference Concentration 

2 The UFs, selected based on EPA’s A Review of the Reference Dose and Reference 

3 Concentration Processes (U.S. EPA, 2002; Section 4.4.5) and described in the Preamble of this 

4 document, address five areas of uncertainty resulting in a composite UF of 10. This composite 

UF was applied to the selected POD (3.1 mg/m35 ) to derive an RfC. 

6 

7 • An intraspecies uncertainty factor, UFH, of 10 was applied to account for potentially 

8 susceptible individuals in the absence of data evaluating variability of response to inhaled 

9 ammonia in the human population; 

10 

11 • An interspecies uncertainty factor, UFA, of 1 was applied to account for uncertainty in 

12 extrapolating from laboratory animals to humans because the POD was based on human 

13 data from an occupational study; 

14 

15 • A subchronic to chronic uncertainty factor, UFS, of 1 was applied because the 

16 occupational exposure period in the principal study (Holness et al., 1989), i.e., mean 

17 number of years at present job for exposed workers, of approximately 12 years was of 

18 chronic duration; 

19 

20 • A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because a NOAEL value 

21 was used as the POD; and 

22 

23 • A database uncertainty factor, UFD, of 1 was applied to account for deficiencies in the 

24 database. The ammonia inhalation database consists of studies of occupational exposure 

25 focused on effects of ammonia on respiratory irritation and lung function, studies in 

26 livestock farmers, controlled exposure studies involving volunteers exposed to ammonia 

27 vapors for short periods of time to evaluate irritation effects and changes in lung function, 

28 and a large number of case reports of acute exposure to high ammonia concentrations 

29 (e.g., accidental spills/releases). Studies of the toxicity of inhaled ammonia in 

30 experimental animals include subchronic studies in rats, guinea pigs, and pigs that 

31 examined respiratory and other systemic effects of ammonia and one limited, 

32 reproductive toxicity study in young female pigs. The database lacks developmental and 

33 multigeneration reproductive toxicity studies. 

34 

35 As noted in EPA’s A Review of the Reference Dose and Reference Concentration 

36 Processes (U.S. EPA, 2002), “the size of the database factor to be applied will depend on 

37 other information in the database and on how much impact the missing data may have on 

38 determining the toxicity of a chemical and, consequently, the POD.” Multigeneration 

39 reproductive and developmental toxicity studies would not be expected to impact the 

40 determination of ammonia toxicity at the POD, and therefore a database UF to account 

41 for the lack of these studies is not necessary. This determination was based on the 

42 observation that ammonia is endogenously produced and homeostatically regulated in 

43 humans and animals during fetal and adult life. Baseline blood levels in healthy 

44 individuals range from 0.1 to 1.0 μg/mL (Monsen, 1987; Conn, 1972; Brown et al., 

45 1957). The fetoplacental unit produces ammonia, and concentrations in human umbilical 

46 vein and artery blood (at term) have been shown to be higher than concentrations in 
46 DRAFT - DO NOT CITE OR QUOTE

1 maternal blood (Jόźwik et al., 2005), providing some assurance that developmental 

2 toxicity would not be associated with concentrations of ammonia at or below the POD. 

3 DeSanto et al. (1993) reported that human fetal umbilical blood levels of ammonia at 

4 birth were not influenced by gestational age based on deliveries ranging from gestation 

5 week 25–43. Finally, evidence in animals (Manninen et al., 1988; Schaerdel et al., 1983) 

suggests that exposure to ammonia at concentrations up to 18 mg/m36 does not alter blood 

7 ammonia levels (see Appendix A, Section A.3, for a more detailed discussion of 

ammonia distribution and elimination). Accordingly, exposure at the POD (3.1 mg/m38 ) 

9 would not be expected to alter ammonia homeostasis or result in measureable increases in 

10 blood ammonia concentrations. Thus, the concentration of ammonia at the POD for the 

11 RfC would not be expected to result in systemic toxicity, including reproductive or 

12 developmental toxicity. 

13 

14 The RfC for ammonia was calculated as follows: 

15 

16 RfC = NOAELADJ ÷ UF 

 = 3.1 mg/m317 ÷ 10 

 = 0.31 mg/m3 or 0.3 mg/m3 18 (rounded to one significant figure)

19 

20 2.2.3. Uncertainties in the Derivation of the RfC 

21 As presented earlier in this section and in the Preamble, EPA standard practices and RfC 

22 guidance (U.S. EPA, 2002, 1995, 1994a, b) were followed in applying a UF approach to a POD 

23 to derive the RfC. Specific uncertainties were accounted for by the application of UFs (i.e., in 

24 the case of the ammonia RfC, a factor to address the absence of data to evaluate the variability in 

25 response to inhaled ammonia in the human population). The following discussion identifies 

26 additional uncertainties associated with the quantification of the RfC for ammonia. 

27 

28 Use of a NOAEL as a POD 

29 Data sets that support BMD modeling are generally preferred for reference value 

30 derivation because the shape of the dose-response curve can be taken into account in establishing 

31 the POD. For the ammonia RfC, no decreases in lung function or respiratory irritation were 

32 observed in the worker population studied by Holness et al. (1989), i.e., the principal study used 

33 to derive the RfC, and as such the data from this study did not support dose-response modeling. 

34 Rather, a NOAEL from the Holness et al. (1989) study was used to estimate the POD. The 

35 availability of dose-response data from a single study of ammonia would increase the confidence 

36 in the estimation of the POD. 

37 

38 Endogenous ammonia 

39 Ammonia, which is produced endogenously, has been detected in the expired air of 

healthy volunteers at levels generally ranging from 0.013 to 2.1 mg/m340 (Boshier et al., 2010; 

41 Smith et al., 2008; Spanel et al., 2007a, b; Turner et al., 2006; Diskin et al., 2003; Kearney et al.,
47 DRAFT - DO NOT CITE OR QUOTE

1 2002; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977). The higher and more 

2 variable ammonia concentrations within this range are reported in breath exhaled from the mouth 

3 or oral cavity, with the majority of ammonia concentrations from these sources ranging from 

0.09 to 2.1 mg/m34 (Smith et al., 2008; Spanel et al., 2007a, b; Turner et al., 2006; Diskin et al., 

5 2003; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977). Ammonia in exhaled breath 

6 from the mouth or oral cavity is largely attributed to the production of ammonia via bacterial 

7 degradation of food protein in the oral cavity or gastrointestinal tract (Turner et al., 2006; Smith 

8 et al., 1999; Vollmuth and Schlesinger, 1984), and can be influenced by factors such as diet, oral 

9 hygiene, age, and living conditions (i.e., urban vs. rural setting). In contrast, ammonia 

10 concentrations measured in breath exhaled from the nose and trachea are lower (range: 0.013–

0.078 mg/m311 ; Smith et al., 2008; Larson et al., 1977) and more likely reflect systemic levels of 

12 ammonia (i.e., circulating levels in the blood) (Smith et al., 2008). 

13 Ammonia concentrations measured in breath exhaled from the nose and trachea, i.e., 

14 concentrations expected to more closely correlate with circulating levels of ammonia in blood, 

are lower than the ammonia RfC of 0.3 mg/m315 by a factor of approximately fourfold or more; 

16 however, the RfC does fall within the more variable range of breath concentrations collected 

17 from the mouth or oral cavity. Although the contribution of ammonia generated endogenously 

18 and expired through exhalation to ammonia present in ambient air is not known, this contribution 

19 is expected to be minimal considering the ammonia in expired air should rapidly mix with and be 

20 diluted in the much larger volume of ambient air. 

21 

22 2.2.4. Confidence Statement 

23 A confidence level of high, medium, or low is assigned to the study used to derive the 

24 RfC, the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA’s Methods 

25 for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry

26 (U.S. EPA, 1994b). Confidence in the principal study (Holness et al., 1989) is medium. The 

27 design, conduct, and reporting of this occupational exposure study were adequate, but the study 

28 was limited by a small sample size and by the fact that workplace ammonia concentrations to 

29 which the study population was exposed were below those associated with ammonia-related 

30 effects (i.e., only a NOAEL was identified). However, this study is supported in the context of 

31 the entire database, including the NOAEL and LOAEL values identified in the Rahman et al. 

32 (2007) occupational exposure study, multiple studies of acute ammonia exposure in volunteers, 

33 and the available inhalation data from animals. Confidence in the database is medium. The 

34 inhalation ammonia database includes limited studies of reproductive toxicity and no studies of 

35 developmental toxicity; however, reproductive, developmental, and other systemic effects are 

36 not expected at the RfC because it is well documented that ammonia is endogenously produced 

37 in humans and animals, ammonia concentrations in blood are homeostatically regulated to 

38 remain at low levels, and ammonia concentrations in air at the POD are not expected to alter 
48 DRAFT - DO NOT CITE OR QUOTE

1 homeostasis. Reflecting medium confidence in the principal study and medium confidence in 

2 the database, the overall confidence in the RfC is medium. 

3 

4 2.2.5. Previous IRIS Assessment: Reference Concentration 

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

RfC of 0.1 mg/m36 based on co-principal studies—the occupational exposure study of workers in 

7 a soda ash plant by Holness et al. (1989) and the subchronic study by Broderson et al. (1976) that 

8 examined the effects of ammonia exposure in F344 rats inoculated on day 7 of the study with the 

bacterium M. pulmonis. The NOAEL of 6.4 mg/m39 (estimated as the mean concentration of the 

10 entire exposed group) from the Holness et al. (1989) study (duration adjusted: NOAELADJ = 

2.3 mg/m3) was used as the POD.511 

12 The previous RfC was derived by dividing the POD by a composite UF of 30: 10 to 

13 account for the protection of sensitive individuals and 3 for database deficiencies to account for 

14 the lack of chronic data, the proximity of the LOAEL from the subchronic inhalation study in the 

15 rat (Broderson et al., 1976) to the NOAEL, and the lack of reproductive and developmental 

16 toxicology studies. A UFD of 3 (rather than 10) was applied because studies in rats (Schaerdel et 

17 al., 1983) showed no increase in blood ammonia levels at an inhalation exposure to 32 ppm (22.6 

mg/m3) and only minimal increases at 300–1,000 ppm (212–707 mg/m318 ), suggesting that no 

19 significant distribution is likely to occur at the human equivalent concentration. In this 

20 document, a UFD of one was selected because a more thorough investigation of the literature on 

21 ammonia homeostasis and literature published since 1991 on fetoplacental ammonia levels 

22 provides further support that exposure to ammonia at the POD would not result in a measureable 

23 increase in blood ammonia, including fetal blood levels. 

24 2.3. Cancer Risk Estimates 

25 The carcinogenicity assessment provides information on the carcinogenic hazard 

26 potential of the substance in question and quantitative estimates of risk from oral and inhalation 

27 exposure may be derived. Quantitative risk estimates may be derived from the application of a 

28 low-dose extrapolation procedure. If derived, and unless otherwise stated, the oral slope factor is 

29 a plausible upper bound on the estimate of risk per mg/kg-day of oral exposure. Similarly, an 

inhalation unit risk is a plausible upper bound on the estimate of risk per μg/m330 air breathed. 

31 As discussed in Section 1.2, there is “inadequate information to assess the 

32 carcinogenic potential” of ammonia. Therefore, a quantitative cancer assessment was not 

33 conducted and cancer risk estimates were not derived for ammonia. The previous IRIS 

34 assessment did not include a carcinogenicity assessment. 

 

5

In this document, the lower bound of the high exposure category from the Holness et al. (1989) study (8.8 mg/m3, 

adjusted for continuous exposure to 3.1 mg/m3) was identified as the POD because workers in this high exposure 

category, as well as those in the two lower exposure categories, showed no statistically significant increase in 

respiratory irritation or decreases in pulmonary function. 
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55 Assessment Forum, Washington, DC; EPA/630/R-00/002. Available online at http://www.epa.gov/iris/backgrd.html 

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57 
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4 

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8 

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11 

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13 Assessment Forum, Washington, DC; EPA/630/R-03/003F. Available online at 

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15 

16 U.S. EPA. (2006a) A framework for assessing health risk of environmental exposures to children. National Center 

17 for Environmental Assessment, Washington, DC, EPA/600/R-05/093F. Available online at 

18 http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=158363 (accessed March 14, 2011). 

19 

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22 

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26 

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29 

30 U.S. EPA. (2009) IRIS Process. http://www.epa.gov/iris/process.htm. 

31 

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