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APPENDIX A: SUPPLEMENTAL INFORMATION FOR 

THE TOXICOLOGICAL REVIEW OF AMMONIA 

A.1. Chemical and Physical Information 

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

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

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

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

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

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

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

+

 favors the formation of NH4

+

. 

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

Table A-1. Chemical and physical properties of ammonia 

Chemical name Ammoniaa

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

Nitro-sil; R 717; Spirit of hartshorn 

ChemID Plus, 2009 

Structure 

N

H

H H

ChemID Plus, 2009 

Chemical formula NH3 ChemID Plus, 2009 

CASRN 7664-41-7a ChemID Plus, 2009 

Molecular weight 17.031 Lide, 2008 

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

Melting point -77.73°C Lide, 2008

Boiling point -33.33°C Lide, 2008 

Odor threshold 53 ppm (37 mg/m3) 

<20 ppm (14 mg/m3) 

2.6 ppm (2 mg/m3) 

O’Neil et al., 2006 

Altmann et al., 2006 

Smeets et al., 2007 

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

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

pKa (ammonium ion) 9.25 Lide, 2008 

Solubility: 

 Water 

 Organic solvents 

4.82 × 105

 mg/L at 24°C 

Soluble in ethanol, chloroform, and ether 

Dean, 1985 

Lide, 2008; O’Neil et al., 2006 

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

Henry’s law constant 1.61 × 10-5 atm-m3/mol at 25°C Betterton, 1992 
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Table A-1. Chemical and physical properties of ammonia 

Conversion factors 

 ppm to mg/m3

 mg/m3 to ppm 

1 ppm = 0.707 mg/m3

1 mg/m3 = 1.414 ppm 

Verschueren, 2001 

Verschueren, 2001 

a

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

hydroxide does not exist outside of solution. 

A.2. AMMONIUM SALTS 

The toxicology literature for ammonium salts includes studies of ammonium chloride (3-, 

18-, and 30-month dietary studies in male and female Wistar rats [Lina and Kuijpers, 2004] and a 

330-day drinking water study in Sprague-Dawley rats [Barzel et al., 1969]) and ammonium 

sulfate (52- and 104-week dietary studies in male and female F344 rats [Ota et al., 2006]). No 

inhalation toxicity studies of ammonium salts are available. Ammonium chloride in the diet or 

drinking water of rats consistently altered the acid-base balance in the body (Lina and Kuijpers, 

2004; Barzel et al., 1969) causing a dose-related hyperchloremic metabolic acidosis in rats as 

evidenced by decreases in blood pH, base excess, and bicarbonate concentration, and increased 

plasma chloride levels. Ammonium chloride administered in the diet for 3, 18 or 30 months was 

also associated with zona glomerulosa hypertrophy of the adrenal gland (Lina and Kuijpers, 

2004). In contrast, dietary administration of ammonium sulfate to rats did not induce metabolic 

acidosis at 52 weeks (Ota et al., 2006) or histopathologic changes in the adrenal gland at 104 

weeks (Ota et al., 2006). The only dose-related effects in male and female rats associated with 

52-week exposure to ammonium sulfate were increased liver and kidney weights (Ota et al., 

2006). In the 104-week study, the incidence of chronic nephropathy was statistically 

significantly increased in low-dose, but not in high-dose male rats (Ota et al., 2006). See Table 

A-2 for study details. 

The toxicity data for ammonium chloride and ammonium sulfate demonstrate that these two 

salts present distinctly different toxicity profiles, suggesting that the anion can influence the 

toxicity of the ammonium compound, and that the toxicity of the salts cannot necessarily be 

attributed to the cation (i.e., NH4

+

) only. Accordingly, information on the toxicity of ammonium 

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

assessment.
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Table A-2. Summary of repeat dose studies of selected ammonium salts following oral exposure 

Strain/ 

species/sex 

Ammonia 

species 

Dose (mg/kgd)/ duration NOAEL (mg/kg-d) LOAEL (mg/kg-d) Effects at the LOAEL Comment 

Subchronic studies 

Wistar rat (10/sex/group) 

Ammonium chloride 

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

Not determined 1,590 (males) 

1,800 (females) 

Decreased body weights (6-17% in males; 11-19% in females), changes in serum chemistry (increased plasma chloride and ALP activity), increased relative kidney weights (both dose levels, 7-28%) and adrenal weights (high-dose males, 18%), metabolic acidosis (males and females) and subsequent hypertrophy of the adrenal zona glomerulosa (males only) 

 

Chronic studies Sprague-Dawley rat (11 males/group)

Ammonium chloride

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

Not determined 1,800 Depression in body weights 

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

Metabolic acidosis (redublood pH and plasma cadioxide).Combined effects of calcintake and observed thaloss of bone tissue was independent of the levedietary calcium. 
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Table A-2. Summary of repeat dose studies of selected ammonium salts following oral exposure 

Strain/ 

species/sex 

Ammonia 

species 

Dose (mg/kgd)/ duration NOAEL (mg/kg-d) LOAEL (mg/kg-d) Effects at the LOAEL Comment 

Wistar rat (15/sex/group) 

Ammonium chloride 

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

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

Not determined 481 (males) 

610 (females) 

Metabolic acidosis Femur weight significant

increased (~17%) in highmales. Adrenal and kidnweights elevated (<15%)Increased incidence of zoglomerulosa hypertrophthe adrenal at high dose statistically significant). Severity of metabolic acincreased with dose. 

Wistar rat (50/sex/group) 

Ammonium chloride 

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

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

Not determined 455 (males) 

551 (females) 

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

Increased incidence of zoglomerulosa hypertrophthe adrenal in high-dose females; attributed to chstimulation of the adren

cortex by ammonium chinduced acidosis. There no evidence of a carcinoresponse. 

F344 rat (10/sex/group) 

Ammonium sulfate 

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

256 (males) 284 (females) 

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

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

No significant effects on hematology, serum chemistry, or histopatho
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Table A-2. Summary of repeat dose studies of selected ammonium salts following oral exposure 

Strain/ 

species/sex 

Ammonia 

species 

Dose (mg/kgd)/ duration NOAEL (mg/kg-d) LOAEL (mg/kg-d) Effects at the LOAEL Comment 

F344 rat (50/sex/group) 

Ammonium sulfate 

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

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

Not determined Clinical chemistry and 

hematology not evaluateorgan weights not measIncidence of chronic nephropathy in male ratincreased over control (15/49, 3/48 in the controand high dose); increase statistically significant onthe mid-dose and not dorelated. There was no evidence of a carcinogenresponse. 
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A.3. TOXICOKINETICS 

Overview 

 Ammonia can be absorbed by the inhalation and oral routes of exposure. There is less 

certainty regarding absorption through the skin, although absorption through the eye has been 

documented. Most of the inhaled ammonia is retained in the upper respiratory tract and is 

subsequently eliminated in expired air. Ammonia that reaches systemic circulation is widely 

distributed to all body compartments, although substantial first-pass metabolism occurs in the 

liver, where biotransformation into urea and glutamine occurs. Ammonia exists in the blood as 

ammonium ion (NH4

+

). Ammonia is transported in the circulatory system primarily via 

glutamine and alanine, amino acids that are used to transport ammonia to and from tissues. 

When transported to the liver and kidney, the amide moiety is hydrolyzed via glutaminase 

forming glutamatic acid (glutamate) and ammonium ion, which is synthesized into urea and 

excreted in the urine. Ammonia or ammonium ion reaching the tissues is utilized for glutamate 

production, which participates in transamination and other reactions. The principal means of 

excretion of absorbed ammonia in mammals is as urinary urea; minimal amounts are excreted in 

the feces and in expired air. 

 Ammonia is endogenously produced in humans and animals. It is an essential 

mammalian metabolite used in nucleic acid and protein synthesis, is necessary for maintaining 

acid-base balance, and is an integral part of nitrogen homeostasis. Given its important metabolic 

role, ammonia exists in a homeostatically regulated equilibrium in the body. 

Absorption 

Inhalation Exposure 

 Experiments with volunteers1

 show that ammonia, regardless of its tested concentration 

in air (range, 57–500 ppm or 40–354 mg/m3

), is almost completely retained in the nasal mucosa 

(83–92%) during short-term acute exposure, i.e., up to 120 seconds (Landahl and Hermann, 

1950). However, longer-term acute exposure (10–27 minutes) to a concentration of 500 ppm 

(354 mg/m3

) resulted in lower retention (4–30%), with expired breath concentrations of 350–

400 ppm (247–283 mg/m3

) observed by the end of the exposure period (Silverman et al., 1949), 

suggesting saturation of absorption into the nasal mucosa. Nasal and pharyngeal irritation, but 

not tracheal irritation, suggests that ammonia is retained in the upper respiratory tract. 

Unchanged levels of blood urea nitrogen (BUN), nonprotein nitrogen, urinary urea, and urinary 

ammonia following these acute exposures are evidence of low absorption into the blood. 

 

1The human toxicokinetic studies cited in this section did not provide information on the human subjects’ research 

ethics procedures undertaken in the studies; however, there is no evidence that the conduct of the research was 

fundamentally unethical or significantly deficient relative to the ethical standards prevailing at the time the research 

was conducted. 
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Exposure to a common occupational limit of ammonia in air (25 ppm or 18 mg/m3), assuming 

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

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

observed baseline levels of 0.1–1.0 μg/mL (Monsen, 1987; Conn, 1972; Brown et al., 1957) for 

healthy controls. 

 Data in rabbits and dogs provide supporting evidence for high-percentage nasal retention, 

resulting in a lower fraction of the inhaled dose reaching the lower respiratory tract (Egle, 1973; 

Dalhamn, 1963; Boyd et al., 1944). Continuous exposure of rats to up to 32 ppm (23 mg/m3

) for 

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

above preexposure levels), whereas exposures to 310–1,157 ppm (219–818 mg/m3) led to 

significantly increased blood concentrations of ammonia within 8 hours of exposure initiation; 

blood ammonia returned to preexposure values within 12 hours of continuous exposure 

(Schaerdel et al., 1983). 

Oral Exposure 

 Case reports of human ingestion of household ammonia (ammonium hydroxide) provide 

evidence of oral absorption, but few quantitative data are available. For example, in a fatal case 

of a man who drank an unknown amount of a 2.4% solution of ammonium hydroxide, analysis of 

the contents of the stomach and blood showed ammonium ion levels of 15.3 mg in the stomach 

and 33 μg/mL in the blood (Klendshoj and Rejent, 1966). This blood concentration is about 

30-fold higher than the concentration of 1 μg/mL in fasting volunteers, as reported by Conn 

(1972). 

 Ammonium ion is endogenously produced in the human digestive tract, much of it arising 

from the bacterial degradation of nitrogenous compounds from ingested food. Approximately 

4,200 mg of ammonia are produced each day with >70% of that amount liberated from fecal 

contents within the colon (Summerskill and Wolpert, 1970). About 99% of the total amount 

produced (4,150 mg) is systemically absorbed. Evidence suggests that fractional absorption of 

ammonia increases as the lumen pH increases, and that active transport occurs at the lower pH 

levels (absorption has been detected at a pH as low as 5) (Castell and Moore, 1971; Mossberg 

and Ross, 1967). Ammonium ion absorbed from the gastrointestinal tract travels via the hepatic 

portal vein directly to the liver, where in healthy individuals, most of it is converted to urea and 

glutamine. 

Dermal Exposure 

 Quantitative data on absorption from exposure by the dermal route are not available. One 

report of five case histories of workers exposed to anhydrous ammonia via a burst gas pipe 

indicated that there was systemic toxicity (vomiting, renal congestion, and delirium), suggesting 
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dermal absorption; however, the fractional dose from dermal exposure could not be determined 

(Slot, 1938). WHO (1986) concluded that systemic effects from skin and eye exposure are not 

quantitatively important. Ammonia is readily absorbed into the eye, and it was found to diffuse 

within seconds into the cornea, lens, drainage system, and retina (Beare et al., 1988; Jarudi and 

Golden, 1973). However, amounts absorbed were not quantified, and absorption into systemic 

circulation was not investigated. 

Distribution 

 The range of mean ammonia concentrations in humans as a result of endogenous 

production was reported as 0.1–0.6 μg/mL in arterial blood and 0.2–1.7 μg/mL in venous blood 

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

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

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

+

ion (da Fonseca-Wolhheim, 1995; Souba, 1987). 

 Ammonia is present in fetal circulation. In vivo studies in several animal species and in 

vitro studies of human placenta suggest that ammonia is produced within the uteroplacenta and 

released into the fetal and maternal circulations (Bell et al., 1989; Johnson et al., 1986; Haugel et 

al., 1983; Meschia et al., 1980; Remesar et al., 1980; Holzman et al., 1979, 1977; Rubaltelli and 

Formentin, 1968; Luschinsky, 1951). Jόźwik et al. (2005) reported that ammonia levels in 

human fetal blood (specifically, umbilical arterial and venous blood) at birth were 1.0–

1.4 μg/mL, compared to 0.5 μg/mL in the mothers’ venous blood. DeSanto et al. (1993) 

similarly collected human umbilical arterial and venous blood at delivery, and found that 

umbilical arterial ammonia concentrations were significantly higher than venous concentrations; 

there was no correlation between umbilical ammonia levels and gestational age (range of 25–

43 weeks of gestation). In sheep, uteroplacental tissues are the main site of ammonia production, 

with outputs of ammonia into both the uterine and umbilical circulations (Jόźwik et al., 1999). 

In late-gestation pregnant sheep that were catheterized to allow measurement of ammonia 

exposure to the fetus, concentrations of ammonia in umbilical arterial and venous blood and 

uterine arterial and venous blood ranged from about 0.39 to 0.60 μg/mL (Jόźwik et al., 2005, 

1999). 

 Ammonia is present in human breast milk as one of the sources of nonprotein nitrogen 

(Atkinson et al., 1980). 

Inhalation Exposure 

 Little information was found in the available literature for distribution of inhaled 

ammonia. Information on the distribution of endogenously produced ammonia suggests that any 

ammonia absorbed through inhalation would be distributed to all body compartments via the 
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blood, where it would be used in protein synthesis, as a buffer, reduced to normal concentrations 

by urinary excretion, or converted by the liver to glutamine and urea (Takagaki et al., 1961). 

Rats inhaling 300 ppm (212 mg/m3) ammonia for 6 hours/day for 15 days exhibited increased 

blood ammonia (200%) and brain (28%) glutamine levels at 5 days of exposure, but not at 10 or 

15 days (Manninen et al., 1988), demonstrating transient distribution of ammonia to the brain 

(metabolic adaptation). 

Oral Exposure 

 Human oral exposure data indicate that ammonia readily enters the portal circulation and 

is delivered to the liver, as has been shown to be the case for endogenously produced ammonia 

(Pitts, 1971; Summerskill and Wolpert, 1970). Un-ionized ammonia is freely diffusible, whereas 

the ammonium ion is less so, and is relatively confined to the extracellular compartment 

(Stabenau et al., 1958). 

Dermal Exposure 

 No quantitative data on distribution of ammonia from dermal exposure were located in 

the available literature. 

Metabolism 

 Endogenously, ammonia is produced by catabolism of amino acids by glutamate 

dehydrogenase primarily in the liver and renal cortex, but also in the brain and heart (Souba, 

1987). In skeletal muscle, ammonia may be produced by metabolism of adenosine 

monophosphate via adenylate deaminase. Information on the metabolism of exogenouslyintroduced ammonia was not found in the available literature. Ammonia and ammonium ion are 

metabolized to glutamine mainly in the liver via glutamine synthetase in the glutamine cycle 

(Figure A-1), or incorporated into urea as part of the urea cycle as observed in the hepatic 

mitochondria and cytosol (Figure A-2) (Souba, 1987). Ammonia can be rapidly converted to 

glutamine in the brain as well (Takagaki et al., 1961). Van de Poll (2008) reported that the liver 

removes an amount of ammonia from circulation equal to the amount added by the intestines at 

metabolic steady state, indicating that the gut does not contribute significantly to systemic 

ammonia release. 
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Adapted from: Nelson and Cox (2008). 

Figure A-1. Glutamine cycle. 

Adapted from: Nelson and Cox (2008). 

Figure A-2. The urea cycle showing the compartmentalization of its steps within liver 

cells. 

Glutamate

γ-Glutamyl

phosphate

glutamine

synthetase

glutamine

synthetase

Glutamine

glutaminase

(in liver 

mitochondria)

NH4

+

H2O

ATP

ADP

NH4

+ Pi

Carbamoyl

phosphate 

synthase I

H2O

2 ATP

2 ADP

Pi

3 H+

CO2 + NH4

+

Pi

Citrulline

Ornithine

Aspartate

Arginine Argininosuccinate

H2O

Urea

Ornithine

Ornithine

transcarbamoylase

Citrulline

Carbamoyl

phosphate

Argininosuccinate

synthase

Argininosuccinate

lyase

Arginase

MITOCHONDRIAL

MATRIX

Fumarate

ATP

AMP + PPi

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

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

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

physiological pH) as NH4

+

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

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

in air below 25 ppm (18 mg/m3) do not alter blood ammonia concentrations. Schaerdel et al. 

(1983) exposed rats to ammonia for 24 hours at concentrations ranging from 15 to 1,157 ppm 

(11–818 mg/m3). Exposure to 15 ppm (11 mg/m3) ammonia did not increase blood ammonia 

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

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

8 hours, suggesting compensation by increasing ammonia metabolism through conversion to 

urea, pyrimidine and polyamine synthesis, incorporation into amino acid substrates, and 

metabolism in nervous system tissue. Rats inhaling 25 ppm (18 mg/m3) ammonia for 

6 hours/day for 15 days did not exhibit blood or brain ammonia or glutamine levels that were 

different from controls; however, rats inhaling 300 ppm (212 mg/m3) for the same duration 

exhibited statistically significantly increased levels of blood ammonia (threefold) and brain 

glutamine (approximately 40%) at 5 days of exposure, but not at 10 or 15 days (Manninen et al., 

1988). The return of blood and brain ammonia and glutamine levels to control levels with time 

is consistent with metabolic adaptation, and these data suggest that animals have a large capacity 

to handle high concentrations of inhaled ammonia. 

 Various disease states can affect the rate of glutamine uptake and catabolism and, 

thereby, affect the blood and tissue levels of ammonia. Abnormally elevated levels of ammonia 

are indicative of end-stage renal failure (Davies et al., 1997). Acute renal failure can result in 

increased renal glutamine consumption and ammonia production with a decreased capability of 

eliminating urea in the urine (Souba, 1987). End-stage liver failure due to fulminant hepatitis or 

hepatic cirrhosis may result in decreased ureagenesis and increased levels of ammonia in blood 

(hyperammonemia), leading to increased uptake into the brain and the onset of hepatic 

encephalopathy. The increased metabolic alkalosis associated with hepatic encephalopathy may 

result in a shift in the NH4

+

/NH3 ratio in the direction of ammonia, which could pass through the 

blood-brain barrier (Katayama, 2004). In patients with liver cirrhosis and acute clinical hepatic 

encephalopathy, the observed trapping of [13N]-ammonia in the brain appeared to be related to a

fivefold increase, relative to healthy controls, of ammonia permeability across the blood-brain 

barrier (Keiding et al., 2010, 2006). Furthermore, Sorensen et al. (2009) demonstrated greater 

unidirectional clearance of ammonia from the blood to brain cells than metabolic clearance of 

ammonia from the blood in both healthy controls and in cirrhotic patients with and without 

hepatic encephalopathy. 
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Elimination 

 Absorbed ammonia, as well as endogenously produced ammonia, is excreted by the 

kidneys as urea (Summerskill and Wolpert, 1970; Gay et al., 1969; Muntwyler et al., 1956; 

Davies and Yudkin, 1952; Van Slyke et al., 1943) and is a component of sweat (Guyton, 1981; 

Wands, 1981). Lee and colleagues observed that acidosis-stimulated renal excretion of ammonia 

is mediated by intercalated cell-specific Rh B glycoprotein expression in mice (Bishop et al., 

2010; Lee et al., 2010, 2009). In rat kidney, ammonium ion is secreted into the lumen of the 

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

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

+

/H+

exchange activity that may be mediated by an Rh C glycoprotein (Handlogten et al., 2005), 

which is also expressed in human kidneys (Han et al., 2006). 

 Additionally, ammonia is known to be excreted through expired air and is present in the 

expired air of all humans (Manolis, 1983). Two investigators specifically measured ammonia in 

breath exhaled from the nose (Smith et al., 2008; Larson et al., 1977). Smith et al. (2008) 

reported median ammonia concentrations in exhaled breath from the nose of three healthy 

volunteers (with samples collected daily over a 4-week period) of 0.059–0.078 mg/m3; these 

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

of 0.056 mg/m3 reported in this study. Larson et al. (1977) reported that the median 

concentration of ammonia collected from air samples exhaled from the nose ranged from 

0.013 to 0.046 mg/m3. One sample collected from the trachea (via a tube inserted through the 

nose of one subject) was 0.029 mg/mg3—a concentration within the range of concentrations in 

breath exhaled through the nose (Larson et al., 1977). 

 Higher and more variable ammonia concentrations are reported in breath exhaled from 

the mouth or oral cavity than from air exhaled from the nose. In studies that reported ammonia 

in breath samples from the mouth or oral cavity, the majority of ammonia concentrations ranged 

from 0.085 to 2.1 mg/m3

 (Smith et al., 2008; Spanel et al., 2007a, b; Turner et al., 2006; Diskin 

et al., 2003; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977). These higher 

concentrations are largely attributed to the production of ammonia by bacterial degradation of 

food protein in the oral cavity or gastrointestinal tract (Turner et al., 2006; Smith et al., 1999; 

Vollmuth and Schlesinger, 1984). This source of ammonia in breath was demonstrated by Smith 

et al. (1999), who observed elevated ammonia concentrations in the expired air of six healthy 

volunteers following the ingestion of a protein-rich meal. 

 Other factors that can affect ammonia levels in breath exhaled from the mouth or oral 

cavity include diet, oral hygiene, age, living conditions, and disease state. Norwood et al. (1992) 

reported decreases in baseline ammonia levels (0.085–0.905 mg/m3

) in exhaled breath following 

tooth brushing (<50% depletion), a distilled water oral rinse (<50% depletion), and an acid oral 

rinse (80–90% depletion). These findings are consistent with ammonia generation in the oral 
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cavity by bacterial and/or enzymatic activity. Several investigators have reported that ammonia 

in breath from the mouth and oral cavity increases with age (Spanel et al., 2007a, b; Turner et al., 

2006), with ammonia concentrations increasing, on average, by about 0.1 mg/m3 for each 

10 years of life (Spanel et al., 2007b). Turner et al. (2006) reported that the age of the individual

accounts for about 25% of the variation in observed mean breath ammonia levels and the 

remaining 75% is due to factors other than age. Certain disease states can also influence 

ammonia levels in exhaled breath. Ammonia is greatly elevated in the breath of patients in renal 

failure (Spanel et al., 2007b; Davies, 1997). Increases in breath ammonia are associated with 

increasing age (Diskin et al., 2003). These studies are further described below in Table A-2.
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Table A-3. Ammonia levels in exhaled breath of volunteers

Test subjects Breath samples Levels of ammonia in exhaled breath Methods Commen

Breath samples from the nose and trachea

Three healthy male volunteers (>30 yrs of age) 

Ammonia levels measured in nose-exhaled breath of test subjects each morning about 2 hrs after eating a regular breakfast; samples collected daily over a 4-wk period 

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

3) 

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

3) 

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

3) 

(median ammonia levels estimated as geometric mean ± geometric SD) 

SIFT-MS analysis Mean ambient air le

ammonia was 80 ± 1(0.056 ± 0.0071 mg/

The authors indicateammonia measured exhaled breath may generated in the orasuggested that concin nose-exhaled brebetter represent sysconditions (such as disease) 

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

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

Ammonia concentrations ranged from 0.013 to 0.046 mg/m

3 during nose 

breathing (median 0.025 mg/m

3) 

(5 male subjects), and 0.029 mg/m

3

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

Chemiluminescence 
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Table A-3. Ammonia levels in exhaled breath of volunteers

Test subjects Breath samples Levels of ammonia in exhaled breath Methods Commen

Breath samples from the mouth and oral cavity 

Three healthy male volunteers (>30 yrs of age) 

Ammonia levels measured in mouth-exhaled breath and in the closed mouth cavity of test subjects each morning about 2 hrs after eating a regular breakfast; samples collected daily over a 4-wk period 

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

3) 

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

3) 

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

3) 

Via oral cavity: Volunteer A = 1,465 ± 1.4 ppb (1.04 ± 0.000990 mg/m

3) 

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

3) 

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

3) 

(median ammonia levels estimated as geometric mean ± geometric SD) 

SIFT-MS analysis Mean ambient air le

ammonia was 80 ± 1(0.056 ± 0.0071 mg/

The authors indicateammonia measured exhaled breath may generated in the orasuggested that concin nose-exhaled brebetter represent sysconditions (such as disease) 

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

Three sequential breath exhalations collected over 5 min following the students listening to a 1-hr presentation (at least 1 hr following breakfast and before lunch); alveolar portion measured (identified using humidity) 

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

3) 

18 yr olds = 346 ppb (0.245 mg/m

3) 

SIFT-MS analysis Significant differenc

ammonia levels in ebreath between 17 olds (p < 10-8) were 
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Table A-3. Ammonia levels in exhaled breath of volunteers

Test subjects Breath samples Levels of ammonia in exhaled breath Methods Commen

Four healthy children (two males and two females, 4–6 yrs old) Thirteen senior volunteers (11 males and 2 females, 60–83 yrs old); four had type2 diabetes mellitus with onset at ages between 50 and 70 yrs, and controlled by diet All subjects had their regular breakfast without any specific restrictions 

Breath samples collected in morning at least 1 hr after breakfast and at least 1 hr prior to lunch; each volunteer performed two exhalation/inhalation cycles (both about 5–10 sec in duration) 

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

3) 

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

3) 

SIFT-MS analysis Ammonia breath lev

significantly increasSome seniors report

Measured ammonia breath reported for subject 

Thirty healthy volunteers (19 males and 11 females, 24–59 yrs, 28 Caucasian, 1 African, and 1 mixed race); volunteers were instructed to maintain their normal daily routines and to not rinse out their mouths prior to providing a breath sample 

Breath samples collected in the 

morning prior to lunch at approximately weekly intervals for about 6 mo; some volunteers provided samples more frequently than others; 480 samples collected and analyzed for ammonia 

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

3) 

Median = 842 ppb (0.595 mg/m

3) 

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

3) 

SIFT-MS analysis Ammonia breath lev

shown to increase wBackground levels in laboratory were typaround 400 ppb (0.2
A-17 DRAFT – DO 

Table A-3. Ammonia levels in exhaled breath of volunteers

Test subjects Breath samples Levels of ammonia in exhaled breath Methods Commen

Five subjects (two females, three males; age range 27–65 yrs) 

Breath samples collected between 8 and 9 AM in three sequential breath exhalations on multiple days (12–30 d) over the course of a month 

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

3) 

SIFT-MS analysis Differences in ammo

levels between indivsignificant (p < 0.00

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

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

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

3); 

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

3) increasing to 

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

3) 

SIFT-MS analysis A biphasic response 

ammonia concentraobserved after eatin

Fourteen healthy, nonsmoking subjects (age range 21–54 yrs) performed one or more of the following hygiene maneuvers: (1) acidic oral rinse (pH 2.5) (2) tooth brushing followed by acidic oral rinse (3) tooth brushing followed by distilled water rinse (4) distilled water rinse 

Subjects fasted for 8 hrs prior to baseline measurement, refrained from oral hygiene after their most recent meal, refrained from heavy exercise for 12 hrs, and had no liquid intake for several hours; initial breath ammonia was measured between 8 and 10 am, then subjects performed one or more of the hygiene measures listed (at 30-min intervals for a total 90-min period; samples collected over 5 min) 

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

3) 

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

An 80–90% depletioammonia emissions within 10 min of aci<50% depletion of awas seen following t

brushing or distilled gaseous ammonia leincreased after all riprocedures over tim
A-18 DRAFT – DO 

Table A-3. Ammonia levels in exhaled breath of volunteers

Test subjects Breath samples Levels of ammonia in exhaled breath Methods Commen

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

Breath samples collected during quiet mouth breathing 

Ammonia concentrations ranged from 0.029 to 0.52 mg/m

3 during mouth 

breathing (median of 0.17 mg/m

3) 

Chemiluminescence The oral cavity appe

source of breath amattempt was made tthe diet of subjects standardize the intebetween the last memeasurement 

Breath samples: source (nose/mouth/oral cavity) not specified 

Sixteen healthy, nonsmoking subjects (4 females and 12 males, 29 ± 7 yrs); no significant differences in mean age, height, weight, BMI, or time since last oral intake; 10 subjects tested in each experiment 

Experiment 1: single wholebreath samples collected from each subject (same samples immediately reanalyzed within <10 sec to assess instrument specific variability) Experiment 2: three repeat breath samples collected from each subject (to evaluate intrasubject differences); this experiment evaluated differences based on standardization of expiratory pressure and flow Experiment 3: two mixed breath samples and two bag alveolar breath samples collected in short succession from each subject 

Experiment 1: 1,192 ± 85 ppb (0.843 ± 0.0601 mg/m

3

; 

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

3; median ± SD) 

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

3; median ± SD) 

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

3; median ± SD) 

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

3; median ± SD) 

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

Relatively small numhealthy subjects useDoes not address ththose with disease Intra- and inter-day repeatability were ninvestigated 
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Table A-3. Ammonia levels in exhaled breath of volunteers

Test subjects Breath samples Levels of ammonia in exhaled breath Methods Commen

Eight healthy subjects (average age 39.8 ± 9.6 yrs) 

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

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

3) 

Fiber optic sensor This study measured 

levels in healthy volcompared to H. pyloindividuals (five subnot shown); the expalso included a chal300 mg urea capsule the urease activity oversus infected indivnot shown); the autconcluded that breameasurement may bas a diagnostic test 

Three groups of children were used as test subjects: (1) 68 asthmatic children residing in a National Park in the mountains (mean age 10 yrs, 48 boys, 20 girls)(2) 52 asthmatic children in an urban area (mean age 9 yrs, 35 boys, 17 girls) (3) 20 healthy children from the same urban area as a control group (mean age 10 yrs, 12 boys, 8 girls) 

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

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

3) 

Asthmatic urban children: Mean NH3 = 14.3 ± 10.2 ppb (0.0101 ± 

0.00721 mg/m

3) 

Urban control group: Mean NH3 = 14.8 ± 10.3 ppb (0.0105 ± 

0.00728 mg/m

3) 

Chemiluminescence Both groups of asth

children had some sglucocorticoids, oftewith histamine anta

and/or b2 agonists, were left untreatedconcentrations in exbreath appeared to correlated with expurban air 

BMI = body mass index; SIFT-MS = selected ion flow tube mass spectrometry 
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Because ammonia measured in samples of breath exhaled from the mouth or oral cavity can be 

generated in the oral cavity and may thus be substantially influenced by diet and other factors, 

ammonia levels measured in mouth or oral cavity breath samples do not likely reflect systemic 

(blood) levels of ammonia. Ammonia concentrations in breath exhaled from the nose appear to 

better represent systemic or background levels (Smith et al., 2008). 

 Ammonia has also been detected in the expired air of animals. Whittaker et al. (2009) 

observed a significant association between ambient ammonia concentrations and increases in 

exhaled ammonia in stabled horses. Analysis of endogenous ammonia levels in the expired air 

of rats showed concentrations ranging from 0.01 to 0.353 ppm (0.007–0.250 mg/m3) 

(mean = 0.08 ppm or 0.06 mg/m3) in nose-breathing animals (Barrow and Steinhagen, 1980). 

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

sulfuric acid ranging between 0.03 and 0.225 ppm (0.02 and 0.16 mg/m3) following mouth 

breathing, and between 0.05 and 0.220 ppm (0.04 and 0.16 mg/m3) following nose breathing. 

Physiologically Based Pharmacokinetic Models 

 No physiologically based pharmacokinetic (PBPK) models have been developed for 

ammonia. An expanded one-compartment toxicokinetic model in rats was developed by Diack 

and Bois (2005), which used physiological values to represent first-order uptake and elimination 

of inhaled ammonia (and other chemicals). The model is not useful for dose-response 

assessment of ammonia because: (1) it cannot specify time-dependent amounts or concentrations 

of ammonia in specific target tissues, (2) it has not been verified against experimental data for 

ammonia, glutamate, or urea levels in tissues, and (3) it cannot extrapolate internal doses of 

ammonia between animals and humans. 

A.4. HUMAN STUDIES 

Occupational Studies in Industrial Worker Populations 

Holness et al. (1989) 

 Holness et al. (1989) conducted a cross-sectional study of workers in a soda ash (sodium 

carbonate) plant2 who had chronic, low-level exposure to ammonia. The cohort consisted of 

58 workers and 31 controls from stores and office areas of the plant. All workers were males 

(average age, 40.5 years) and the average exposure duration for the exposed workers at the plant 

was 12.2 years. The mean time-weighted average (TWA) ammonia exposure of the exposed 

group based on personal sampling over one work shift (mean sample collection time, 8.4 hours) 

was 9.2 ppm (6.5 mg/m3

), compared to 0.3 ppm (0.2 mg/m3) for the control group. The average 

 

2At this plant, ammonia, carbon dioxide, and water were the reactants used to form ammonium bicarbonate, which 

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

Ammonia and carbon dioxide were recoved in the process and reused. 
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concentrations of ammonia to which workers were exposed were determined using the procedure 

recommended by the National Institute for Occupational Safety and Health (NIOSH), which 

involves the collection of air samples on sulfuric acid-treated silica gel (ATSG) adsorption tubes 

(NIOSH, 1979). 

 No statistically significant differences were observed in age, height, years worked, 

percentage of smokers, or pack-years smoked for exposed versus control workers. Exposed 

workers weighed approximately 8% (p < 0.05) more than control workers. Information 

regarding past occupational exposures, working conditions, and medical and smoking history, as 

well as respiratory symptoms and eye and skin complaints was obtained by means of a 

questionnaire that was based on an American Thoracic Society questionnaire (Ferris, 1978). 

Each participant’s sense of smell was evaluated at the beginning and end of the work week using 

several concentrations of pyridine (0.4, 0.66, or 10 ppm). Lung function tests were conducted at 

the beginning and end of the work shift on the first and last days of their work week (four tests 

administered). Differences in reported symptoms and lung function between groups were 

evaluated using the actual exposure values with age, height, and pack-years smoked as covariates 

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

(high = >12.5 ppm [>8.8 mg/m3

], medium = 6.25–12.5 ppm [4.4–8.8 mg/m3], and 

low = <6.25 ppm [<4.4 mg/m3]) for analysis of symptom reporting and lung function data. 

Endpoints evaluated in the study included sense of smell, prevalence of respiratory symptoms 

(cough, bronchitis, wheeze, dyspnea, and others), eye and throat irritation, skin problems, and 

lung function parameters (FVC, FEV1, FEV1/FVC, forced expiratory flow [FEF50], and FEF75). 

No statistical differences in the prevalence of respiratory irritation or eye irritation were evident 

between the exposed and control groups (Table A-4). There was a statistically significant 

increase (p < 0.05) in the prevalence of skin problems in workers in the lowest exposure category 

(<4.4 mg/m3

) compared to controls; however, the prevalence was not increased among workers 

in the two higher exposure groups. Workers also reported that exposure at the plant had 

aggravated specific symptoms including coughing, wheezing, nasal complaints, eye irritation, 

throat discomfort, and skin problems. Odor detection threshold and baseline lung functions were 

similar in the exposed and control groups. No changes in lung function were demonstrated over 

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

controls. No relationship was demonstrated between chronic ammonia exposure and baseline 

lung function changes either in terms of the level or duration of exposure. Study investigators 

noted that this finding was limited by the lack of adequate exposure data collected over time, 

precluding development of a meaningful index accounting for both level and length of exposure. 

Based on the lack of exposure-related differences in subjective symptomatology, sense of smell, 

and measures of lung function, EPA identified 8.8 mg/m3

 (12.5 ppm) as the no-observed-
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adverse-effect level (NOAEL). A lowest-observed-adverse-effect level (LOAEL) was not 

identified for this study. 

Table A-4. Symptoms and lung function results of workers exposed to 

different levels of TWA ammonia concentrations 

Parameter 

Ammonia concentration 

Control 

0.2 mg/m3

(0.3 ppm)

Exposed 

<4.4 mg/m3

(<6.25 ppm) 

Exposed 

4.4–8.8 mg/m3

(6.25–12.5 ppm) 

Exposed 

>8.8 mg/m3

(>12.5 ppm) 

Symptom

Cough 3/31(10)a 6/34 (18) 1/12 (8) 2/12 (17) 

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

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

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

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

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

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

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

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

Lung function (% predicted)

FVC 98.6 96.7 96.9 96.8 

FEV1 95.1 93.7 93.9 95.3 

FEF50 108.4 106.9 106.2 111.2 

FEF75 65.2 71.0 67.8 78.8 

a

Number affected/number examined. The percentage of workers reporting symptoms is indicated in parentheses.

b

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

Source: Holness et al. (1989). 

Ballal et al. (1998) 

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

fertilizer factories in Saudi Arabia3

. The cohort consisted of 161 exposed subjects (84 from 

factory A and 77 from factory B) and 355 unexposed controls. Workers in factory A were 

exposed to air ammonia levels of 2–130 mg/m3

 (2.8–184.4 ppm), and workers in factory B were 

exposed to 0.02–7 mg/m3 (0.03–9.9 ppm). Mean duration of employment was 51.8 months for 

exposed workers and 73.1 months for controls. Exposure levels were estimated by analyzing a 

total of 97 air samples collected over 8-hour shifts close to the employee’s work site. The 

 

3

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. 
A-23 DRAFT – DO NOT CITE OR QUOTE 

prevalence of respiratory symptoms and diseases was determined by administration of a 

questionnaire. The authors stated that there were no other chemical pollutants in the workplace 

that might have affected the respiratory system. Smoking habits were similar for exposed 

workers and controls. Stratifying the workers by ammonia exposure levels (above or below the 

American Conference of Governmental Industrial Hygienists [ACGIH] threshold limit value 

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

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

asthma than workers exposed to levels below the TLV (Table A-5). The relative risk for 

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

Distribution of symptoms by cumulative ammonia concentration (CAC, mg/m3-years) also 

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

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

concentration was significantly related to cough, phlegm, wheezing with and without shortness 

of breath, and asthma (Table A-6). 

 

Table A-5. The prevalence of respiratory symptoms and disease in urea 

fertilizer workers exposed to ammonia 

Respiratory 

symptom/disease 

Relative risk (95% CI) 

Exposure category 

CAC 

(mg/m3 of air-yrs) 

≤ACGIH TLV 

(18 mg/m3

) 

(n = 138) 

>ACGIH TLV 

(18 mg/m3) 

(n = 17) 

≤50 

(n = 130) 

>50 

(n = 30) 

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

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

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

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

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

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

Chronic bronchitis and 

bronchial asthma 

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

CI = confidence interval 

Source: Ballal et al. (1998).
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Table A-6. Logistic regression analysis of the relationship between ammonia 

concentration and respiratory symptoms or disease in exposed urea fertilizer 

workers 

Respiratory symptom/disease OR (95% CI) 

Cough 1.32 (1.08–1.62)a

Phlegm 1.36 (1.10–1.67)a

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

Wheezing alone 1.55 (1.17–2.06)a

Dyspnea on effort 0.83 (0.68–1.02) 

Diagnosis of asthma 1.33 (1.07–1.65)a 

a

p ≤ 0.05. 

OR = odds ratio 

Source: Ballal et al. (1998).

Ali (2001) 

 Results from limited spirometry testing of workers from factory A were reported in a 

followup study (Ali, 2001). The lung function indices measured in 73 ammonia workers and 343 

control workers included FEV1 and FVC. Prediction equations for these indices were developed 

for several nationalities (Saudis, Arabs, Indians, and other Asians) and corrected values were 

expressed as the percentage of the predicted value for age and height. The FVC% predicted was 

higher in exposed workers than in controls (4.6% increase, p ≤ 0.002); however, workers with 

cumulative exposure ≥50 mg/m3-years had significantly lower FEV1% predicted (7.4% decrease, 

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

exposure ≤50 mg/m3

-years. A comparison between symptomatic and asymptomatic exposed 

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

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

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

ammonia concentrations >18 mg/m3 (50 mg/m3-years) is associated with respiratory irritation 

and altered lung function, NOAEL and LOAEL values could not be identified by EPA from 

these studies due to inadequate reporting of exposure concentrations. 

Rahman et al. (2007) 

 Rahman et al. (2007) conducted a cross-sectional study of workers at a urea fertilizer 

factory in Bangladesh that consisted of an ammonia plant and a urea plant. The exposed group 

consisted of 63 operators in the ammonia plant and 77 in the urea plant; 25 individuals from the 

administration building served as a control group. Mean duration of employment exceeded 

16 years in all groups. Personal ammonia exposures were measured by two different methods 

(Dräger PAC III and Dräger tube) in five to nine exposed workers per day for 10 morning shifts 
A-25 DRAFT – DO NOT CITE OR QUOTE 

in the urea plant (for a total of 64 workers) and in five to nine exposed workers per day for 

4 morning shifts from the ammonia plant (for a total of 24 workers). Four to seven volunteer 

workers per day were selected from the administration building as controls for a total of 

25 workers over a 5-day period. Questionnaires were administered to inquire about 

demographics, past chronic respiratory disease, past and present occupational history, smoking 

status, respiratory symptoms (cough, chest tightness, runny nose, stuffy nose, and sneezing), and 

use of protective devices. Lung function tests (FVC, FEV1, and peak expiratory flow rate 

[PEFR]) were administered preshift and postshift (8-hour shifts) to the 88 exposed workers after 

exclusion of workers who planned to have less than a 4-hour working day; lung function was not 

tested in the control group. Personal ammonia exposure and lung function were measured on the 

same shift for 28 exposed workers. Linear multiple regression was used to analyze the 

relationship between workplace and the percentage cross-shift change in FEV1 (∆FEV1%) while 

adjusting for current smoking. 

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

PAC III methods were 25.0 and 6.9 ppm (17.7 and 4.9 mg/m3), respectively; the corresponding 

means in the urea plant were 124.6 and 26.1 ppm (88.1 and 18.5 mg/m3

) (Rahman et al., 2007). 

Although the Dräger tube measurements indicated ammonia exposure about 4–5 times higher 

than those obtained with the PAC III instrument, there was a significant correlation between the 

ammonia concentrations measured by the two methods (p = 0.001). No ammonia was detected 

in the control area using the Dräger tube (concentrations less than the measuring range of 2.5–

200 ppm [1.8–141 mg/m3

]). Based on an evaluation of the two monitoring methods and 

communication with technical support at Dräger4, EPA considered the PAC III instrument to be 

a more sensitive monitoring technology than the Dräger tubes. Therefore, the PAC III air 

measurements were considered the more reliable measurement of exposure to ammonia for the 

Rahman et al. (2007) study. The study authors, however, 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. 

 The prevalence of respiratory irritation and decreased lung function was higher in the 

urea plant than in the ammonia plant or in the administration building. Comparison between the 

urea plant and the administration building showed that cough and chest tightness were 

statistically higher in the former; a similar comparison of the ammonia plant and the 

administration building showed no statistical difference in symptom prevalence between the two 

groups (Table A-7). Preshift measurement of FVC, FEV1, and PEFR did not differ between urea 

plant and ammonia plant workers. Significant cross-shift reductions in FVC and FEV1 were 

reported in the urea plant (2 and 3%, respectively, p ≤ 0.05), but not in the ammonia plant. 

 

4

Telephone conversations and e-mails dated June 22, 2010, from Michael Yanosky, Dräger Safety Inc., Technical 

Support Detection Products to Amber Bacom, SRC, Inc. [contractor to National Center for Environmental 

Assessment, Office of Research and Development, U.S. EPA]. 
A-26 DRAFT – DO NOT CITE OR QUOTE 

When controlled for current smoking, a significant decrease in ∆FEV1% was observed in the 

urea plant (p ≤ 0.05). Among 23 workers with concurrent measurements of ammonia and lung 

function on the same shift, ammonia exposure was correlated with a cross-shift decline in FEV1

of 3.9% per unit of log-transformed ammonia concentration in ppm. EPA identified a NOAEL 

of 6.9 ppm (4.9 mg/m3) and a LOAEL of 26.1 ppm (18.5 mg/m3) in the Rahman et al. (2007) 

study based on increased prevalence of respiratory symptoms and a decrease in lung function. 

Table A-7. Prevalence of respiratory symptoms and cross-shift changes in lung 

function among workers exposed to ammonia in a urea fertilizer factory 

Parameter 

Ammonia plant 

(4.9 mg/m3)

a

Urea plant 

(18.5 mg/m3)

a

Administration building 

(concentration not 

determined)b

Respiratory symptoms 

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

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

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

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

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

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

FVC 0.2 ± 9.3 -2.3 ± 8.8 No data 

FEV1 3.4 ± 13.3 -1.4 ± 8.9 No data 

PEFR 2.9 ± 11.1 -1.0 ± 16.2 No data 

aMean ammonia concentrations measured by the Dräger PAC III method. 

b

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

zero levels. 

c

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

d

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

e

Calculated as ([post shift-preshift]/preshift) × 100. 

f

Values are presented as mean ± standard deviation (SD). 

Source: Rahman et al. (2007).

Hamid and El-Gazzar (1996) 

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

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

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

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

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

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

reference populations were matched on demographic characteristics including age, educational 
A-27 DRAFT – DO NOT CITE OR QUOTE 

status, and socioeconomic status. No information was reported on exposure levels. Blood 

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

alanine aminotransferase (ALT), hemoglobin, and blood urea levels, and for monoamine oxidase 

(MAO) and catalase activity. Table A-8 shows statistically significant changes in hemoglobin 

and serum chemistry. Mean levels of AST, ALT, and blood urea were significantly elevated 

among exposed workers over controls. Mean levels of hemoglobin were significantly lower, and 

MAO and catalase enzyme activities were significantly depressed among exposed workers 

compared to controls. A correlation analysis showed a positive correlation between catalase 

activity and levels of hemoglobin, AST, and ALT, and MAO activities. Hamid and El-Gazaar 

(1996) noted that inhibition of catalase can affect electrical stability, permeability, and fluidity of 

membranes, which may lead to hepatotoxic and neurotoxic alterations in occupationally exposed 

workers. NOAEL and LOAEL values were not identified in this study due to the absence of 

information on exposures at this fertilizer plant. 

Table A-8. Summary of significant changes in serum from workers 

occupationally exposed to ammonia at a fertilizer plant 

Parameter Controlsa Exposeda

ALT (U/mL) 16.0 ± 5.59 19.4 ± 5.69b

AST (U/mL) 14.5 ± 4.67 17.9 ± 4.14b

Hemoglobin (%) 14.8 ± 2.62 12.2 ± 2.29c

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

MAO (units) 31.9 ± 10.1 20.8 ± 4.30c

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

aMean ± SD. 

b

Significantly different from controls (p < 0.05). 

c

Significantly different from controls (p < 0.01). 

Source: Hamid and El-Gazzar (1996). 

Cross-Sectional Studies in Farmers Exposed to Inhaled Ammonia 

 Several studies have evaluated respiratory symptoms and changes in lung function in 

livestock farmers and stable workers exposed to ammonia (see Table A-9). In addition to 

ammonia, these studies also documented exposures to airborne dust, bacteria, fungal spores, 

endotoxin, and mold. The release of other volatiles on livestock farms is likely, but 

measurements for other volatile chemicals were not conducted. Although studies of farm 

workers summarized here focused on exposure to ammonia, these and other studies have also 

demonstrated respiratory effects associated with exposure to other constituents in farm worker 

air (e.g., respirable dust, endotoxin). 
A-28 DRAFT – DO NOT CITE OR QUOTE 

 Swine and dairy farmers had a higher prevalence of respiratory symptoms including 

cough, phlegm, wheezing, chest tightness, and eye, nasal and throat irritation compared to 

controls (Melbostad and Eduard, 2001; Preller et al., 1995; Choudat et al., 1994; Zejda et al., 

1994; Crook et al., 1991; Heederik et al., 1990). Impaired respiratory function (e.g., decreased 

FEV1, FVC) in farmers was associated with ammonia exposure in several studies (Cormier et al., 

2000; Donham et al., 2000, 1995; Vogelzang et al., 1998; Reynolds et al., 1996; Preller et al., 

1995; Crook et al., 1991; Heederik et al., 1990). Bronchial hyperreactivity to methacholine or 

histamine challenge was increased in farmers exposed to ammonia compared to control workers 

(Vogelzang et al., 2000, 1997; Choudat et al., 1994). Stable workers showed signs of bronchial 

obstruction with increased peak expiratory flow (PEF) variability as well as increased pulmonary 

inflammation related to allergies (Elfman et al., 2009). Other findings that suggest an allergic or 

inflammatory response in livestock farmers exposed to ammonia include the presence of 

immunoglobin E (IgE) and immunoglobin G (IgG) antibodies to pig squames and urine in blood 

(Crook et al., 1991), increased neutrophils in the nasal wash (Cormier et al., 2000), and increased 

white blood cell count (Cormier et al., 2000). In summary, several studies have demonstrated an 

association between ammonia exposure in livestock farmers and respiratory symptoms and 

impaired respiratory function; however, farmers are additionally exposed to several constituents 

that likely contribute to these effects, including respirable dust, endotoxin, bacteria, fungi, and 

mold. 
A-29 DRAFT – DO NOT CITE OR QUOTE 

Table A-9. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

27 pig farmers (mean 

age of 29 yrs) 

Environmental and 

personal exposures 

were analyzed; lung 

function was 

measured on 

Monday, Tuesday, 

and Friday 

Mean exposure to 

dust = 1.57 mg/m3; 

endotoxin = 24 ng/m3, 

and ammonia = 

5.60 mg/m3

There was no significant 

correlation with lung 

function and exposure to 

dust or endotoxins; there 

was a correlation with 

decreased lung function (5–

10%) and exposure to 

ammonia on the Tuesday 

testing, but not the Monday 

or Friday testing; reported 

respiratory symptoms 

included cough, phlegm, and 

wheezing 

Heederik et al., 

1990 

29 farm workers; 

48 electronic factory 

workers (controls) 

20 pig houses were 

monitored for dust 

and ammonia 

concentrations; 

respiratory 

symptoms were 

determined by 

questionnaire; lung 

function tests were 

performed; 

24 subjects provided 

blood samples to 

determine IgE and 

IgG antibody levels 

Mean airborne 

ammonia 

concentrations ranged 

from 1.5 to 13.23 ppm 

(1–9 mg/m3

) and mean 

dust concentrations 

ranged from 

approximately 2 to 

21 mg/m3

Respiratory symptoms 

included chest tightness, 

wheeze, nasal and eye 

irritation (23/29 farm 

workers); 3/29 farm workers 

had impaired lung function 

(decreased FEV1 and FVC); 

3 farmers had IgE antibodies 

to pig squames or urine; 

specific IgG antibodies were 

found in 14 workers to pig 

squames, and 9 to pig urine, 

suggesting an allergic 

response 

Crook et al., 

1991 

102 pig farmers 

(mean age 39.7 yrs; 

mean duration of 

employment of 

15.7 yrs) who 

worked at least halftime in a swine 

confinement 

building; 51 male 

dairy farmers (mean 

age 40.1 yrs; mean 

duration of 

employment of 

20.3 yrs) and 81 

male dairy industry 

workers (controls; 

mean age 38.5 yrs; 

mean duration of 

employment of 

15.7 yrs) 

Lung function tests 

were given to 

subjects before and 

after a methacholine 

challenge; 

respiratory 

symptoms were 

determined by 

questionnaire 

Mean total dust level of 

2.41 mg/m3

; mean 

airborne ammonia 

concentration of 

8.5 mg/m3; mean 

personal ammonia 

exposure of 3.23 mg/m3

Pig and dairy farmers had 

higher prevalence of 

reported cough and morning 

phlegm; bronchial 

hyperreactivity to 

methacholine was higher for 

pig and dairy farmers 

compared to controls 

Choudat et al., 

1994 
A-30 DRAFT – DO NOT CITE OR QUOTE 

Table A-9. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

54 male swine 

producers (mean age 

= 36.3 yrs; mean 

duration of 

employment = 

10.7 yrs) 

Assessment of 

respiratory 

symptoms with 

questionnaire and 

lung function tests 

Mean contaminant 

levels: carbon dioxide = 

2,632 ppm (1,861 mg/ 

m

3

); ammonia = 

11.3 ppm (8 mg/m3); 

total dust = 2.93 mg/m3; 

respirable dust = 

0.13 mg/m3; endotoxin 

= 11,332 units/m3

Exposure to high 

concentrations of ammonia 

was associated with chronic 

cough and bronchitis; 

incidence of chronic cough 

was dependent on 

interaction of ammonia with 

endotoxin, and respirable 

dust; ammonia 

concentrations were not 

correlated with changes in 

lung function parameters 

Zejda et al., 

1994 

207 males ≥18 yrs of 

age employed at 

swine farms and 

spent time in swine 

confinement 

buildings (mean 

years of employment 

= 9.6); a farm 

comparison group 

(nonconfinement 

production) was 

included (number 

not given) 

Lung function tests 

were performed 

before shift 

(baseline) and then 

after a minimum of 

2 hrs of exposure; 

environmental and 

personal air samples 

were made for 

ammonia, carbon 

dioxide, hydrogen 

sulfide, carbon 

monoxide, and total 

and respirable dust 

Mean personal air 

exposure for all 

subjects: 

total dust = 4.53 mg/m3

; 

respirable dust = 

0.23 mg/m3

; total 

endotoxin = 

202.35 EU/m3

; 

respirable endotoxin = 

16.59 EU/m3

; 

ammonia = 5.64 ppm 

(4 mg/m3) 

Positive correlations were 

associated with lung 

function and exposure to 

total dust, respirable dust, 

respirable endotoxin, and 

ammonia; exposure to 

ammonia concentrations of 

≥7.5 ppm (5 mg/m3) were 

predictive of a ≥3% decrease 

in FEV1; the correlation 

between exposure and 

decreased lung function was 

stronger after 6 yrs of 

exposure 

Donham et al., 

1995 

194 Dutch pig 

farmers (94 with 

chronic respiratory 

symptoms, 

100 without 

symptoms) 

Cross-sectional study 

evaluating exposure 

response relations of 

exposures to dust, 

endotoxins, 

ammonia, and 

disinfection 

procedures 

Estimates of long-term 

exposure based on two 

personal exposure 

samples (one winter 

sample, one summer 

sample); Mean 

estimated exposure to 

dust = 2.7 mg/m3

, 

endotoxin = 112 ng/m3

, 

ammonia = 2 mg/m3

Chronic respiratory 

symptoms included cough, 

phlegm, chest tightness, and 

wheezing; exposure to dust, 

endotoxins, and ammonia 

was not correlated to 

chronic respiratory 

symptoms; ammonia 

exposure and duration of 

disinfection were correlated 

with impairment of baseline 

lung function (decreased 

FEV1, MMEF, and PEF) 

Preller et al., 

1995 
A-31 DRAFT – DO NOT CITE OR QUOTE 

Table A-9. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

151 males ≥18 yrs of 

age employed at 

swine farms and 

spent time in swine 

confinement 

buildings (mean 

years of employment 

= 12.4); a farm 

comparison group 

(nonconfinement 

production) was 

included (number 

not given) 

Followup study from 

Donham et al. (1995) 

previously described; 

followup 

measurements taken 

48 mo from the 

initial measurements

Mean personal air 

exposure for all 

subjects: 

total dust = 3.45 mg/m3

; 

respirable dust = 

0.26 mg/m3; total 

endotoxin = 

176.12 EU/m3; 

respirable endotoxin = 

11.86 EU/m3; 

ammonia = 5.15 ppm 

(4 mg/m3

) 

Swine workers had a mean 

cross-shift 2% decrease in 

FEV1 that was correlated 

with personal exposure to 

total dust, total endotoxin, 

respirable endotoxin, and 

ammonia 

Reynolds et al., 

1996 

196 pig farmers 

(96 with chronic 

respiratory 

symptoms, 

100 without 

symptoms) 

Pig farmers tested 

for lung function and 

bronchial 

responsiveness to 

histamine challenge 

Estimates of long-term 

exposure based on two 

personal exposure 

samples (one winter 

sample, one summer 

sample); mean 

estimated exposure to 

respirable dust = 

2.7 mg/m3

, endotoxin = 

111 ng/m3

, ammonia = 2 

mg/m3

No association between 

bronchial responsiveness 

and exposure to respirable 

dust, endotoxins, or 

ammonia; mild bronchial 

responsiveness was 

associated with the 

disinfectant use of 

quaternary ammonia 

Vogelzang et 

al., 1997 

171 pig farmers 

(82 with chronic 

respiratory 

symptoms, 

89 without 

symptoms) 

Longitudinal study 

for cohort of pig 

farmers observed 

over 3 yrs; subjects 

examined for lung 

function and tested 

for bronchial 

responsiveness to 

histamine challenge 

Estimates of long-term 

exposure based on two 

personal exposure 

samples (one winter 

sample, one summer 

sample); mean 

estimated exposure to 

respirable dust = 

2.63 mg/m3, endotoxin 

= 105 ng/m3, ammonia = 

2 mg/m3

Decreased lung function 

(FEV1 and FVC) was observed 

over time; long-term 

exposure to ammonia was 

associated with increased 

bronchial responsiveness to 

histamine; exposure to 

respirable dust also caused 

increased bronchial 

responsiveness to histamine

Vogelzang et 

al., 2000, 1998 

Eight healthy male 

volunteers (23–

28 yrs old) 

Exposed for 4 hrs at 

1-wk intervals to 

swine confinement 

buildings 

Mean airborne 

ammonia concentration 

of 20.7 ppm (15 mg/m3

); 

also exposed to airborne 

dust, bacteria, 

endotoxin, and molds 

Decreased expiratory flows 

(FEV1), increased neutrophils 

in the nasal wash, and 

increased white blood cell 

count 

Cormier et al., 

2000 
A-32 DRAFT – DO NOT CITE OR QUOTE 

Table A-9. Cross sectional studies of livestock farmers exposed to ammonia 

Subjects Methods Exposure conditions Results Reference 

257 poultry workers 

(30% women, 70% 

men); 63 women and 

87 men nonexposed 

blue-collar workers 

served as control 

subjects 

Personal sampling 

conducted for total 

and respirable dust, 

total and respirable 

endotoxin, and 

ammonia; medical 

evaluations included 

lung function tests 

given before and 

after a work period 

Mean exposure levels of 

poultry workers: 

ammonia = 18.4 ppm 

(13 mg/m3); total dust = 

6.5 mg/m3; respirable 

dust = 0.63 mg/m3; total 

endotoxin 1,589 EU/m3

(0.16 μg/m3); respirable 

endotoxin = 58.9 EU/m3

(0.006 μg/m3

) 

Significant cross-shift 

declines in lung function 

were reported for poultry 

workers; concentrations 

associated with significant 

lung function deficits were 

12 ppm ammonia (8 mg/m3), 

2.4 mg/m3 total dust, 

0.16 mg/m3

 respirable dust, 

and 614 EU/m3

 endotoxin 

(0.614 μg/m3

) 

Donham et al., 

2000 

Survey of 

8,482 farmers and 

spouses; exposure 

study conducted in 

102 farmers 

Exposure study with 

survey of respiratory 

symptoms; personal 

exposures to total 

dust, fungal spores, 

bacteria, endotoxin, 

and ammonia in 

12 tasks were 

measured in 

102 farmers 

Ammonia 

concentrations ranged 

from 0 to 8.2 ppm (0–

6 mg/m3

) over the 

12 tasks; total dust (0.4–

5.1 mg/m3

), fungal 

spores (0.02–

2.0 106/m3), bacteria 

(0.2–48 106

/m3), 

endotoxin (0.5–

28/103

 EU/m3 [0.05–

2.8 μg/m3

) 

There was a significant 

positive correlation between 

task mean exposures to total 

dust, fungal spores, and 

endotoxins and task-specific 

symptoms; there was no 

association between 

exposures to bacteria and 

ammonia and task-specific 

symptoms; symptoms 

included eye, nose, and 

throat irritation, cough, 

chest tightness, and 

wheezing 

Melbostad and 

Eduard, 2001 

13 stable workers 

(6 males, 7 females) 

Stable workers were 

tested for lung 

function and nasal 

lavage was 

performed to 

analyze for 

inflammation 

markers; tests were 

performed during 

two consecutive 

winters and the 

interjacent summer 

Ammonia concentration 

was 20–27 ppm (14–

19 mg/m3) in late 

summer, but was not 

detected in winter; 

levels of endotoxin were 

highest during late 

summer (15 ng/m3) 

while levels of 

1,3-β-glucan (85 ng/m3) 

and horse allergen 

(18,300 U/m3

) were 

highest during the 

winter 

Increased PEF-variability in 

2/13 workers; eosinophil 

cationic protein in 

3/13 (indicative of bronchial 

obstruction and allergic 

inflammation equivalent to 

allergic asthma); increased 

myeloperoxidase and 

lysozyme levels in 

9/13 (indicating enhanced 

activity of neutrophil 

granulocytes in the airways 

and enhanced mucosal 

secretion) 

Elfman et al., 

2009 

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

Controlled Human Inhalation Exposure Studies 

 Controlled exposure studies conducted in volunteers to evaluate irritation effects and 

changes in lung function following acute inhalation exposure to ammonia are summarized in 

Table A-10. 
A-33 DRAFT – DO NOT CITE OR QUOTE 

Table A-10. Controlled human exposure studies of ammonia inhalation 

Subjects Exposure conditions Results Reference 

Seven male 

volunteers 

500 ppm (354 mg/m3) for 

30 min from masked breathing 

apparatus for nose and throat 

inhalation; there was no 

mention of preexposure 

examinations 

Hyperventilation (50–250% increase above 

controls) characterized by increased 

breathing rate and expiratory minute 

volume (i.e., volume of air exhaled in 1 min); 

no coughing was induced, excessive 

lacrimation occurred in two subjects; two 

subjects reported nose and throat irritation 

that lasted 24 hrs after exposure; no 

changes were reported in nitrogen 

metabolism or in blood or urine urea, 

ammonia, or nonprotein nitrogen 

Silverman et 

al., 1949b

Seven male 

volunteers with an 

average age of 

31 yrs 

30, 50, and 90 ppm (21, 35, 

and 64 mg/m3

) for 10 min in an 

inhalation chamber; physical 

and neurological examinations 

were conducted prior to 

exposure 

Increased eye erythema at 90 ppm 

(64 mg/m3) compared to 30 and 50 ppm 

(21 and 35 mg/m3) exposure; 90 ppm 

(64 mg/m3

) did not produce significant 

bronchiospasm or severe lacrimation; 

intensity of odor perception was reported as 

higher at 30 and 50 ppm (21 and 35 mg/m3) 

than at 90 ppm (64 mg/m3

) 

MacEwen et 

al., 1970a

18 healthy 

servicemen 

volunteers, 18–

39 yrs old 

50–344 mg/m3 (70–486 ppm) 

for a half-day (session day 2); 

sessions on days 1 and 3 acted 

as controls; all volunteers 

underwent a preliminary 

examination prior to exposure 

No effect at concentrations of 71 mg/m3

(100 ppm); reduced expiratory minute 

volume at concentrations ranging from 

106 to 235 mg/m3 (150–332 ppm) 

compared to controls (not dose dependent); 

exercise tidal volume was increased at 106 

mg/m3 (150 ppm), but reduced at higher 

concentrations in a dose-dependent manner

Cole et al., 

1977a

Six male and female 

volunteers, 24–

46 yrs old 

25, 50, and 100 ppm (18, 35, 

and 71 mg/m3

) ammonia for 

6 hrs/d 1 time/wk over 6 wks; 

occasional brief exposure to 

150–200 ppm (106–

141 mg/m3

); there was no 

mention of preexposure 

examinations 

Habituation to eye, nose, and throat 

irritation after 2–3 wks with short-term 

adaption; there were no significant 

differences for common biological 

indicators, physical exams, or in normal job 

performance when compared to control 

subjects; continuous exposure to 100 ppm 

(71 mg/m3

) became easily tolerated and had 

no effect on general health after acclimation 

occurred; brief exposure to 150–200 ppm 

(106–141 mg/m3) produced lacrimation and 

transient discomfort 

Ferguson et al., 

1977b

15 volunteers, 18–

53 yrs old 

50, 80, 110, and 140 ppm (35, 

57, 78, and 99 mg/m3

) for 2 hrs 

in an exposure chamber; there 

was no mention of 

preexposure examinations 

No effect on vital capacity or FEV1; 140 ppm 

(99 mg/m3

) caused severe irritation and 

could not be tolerated; reported eye 

irritation increased with concentration 

Verberk, 1977b
A-34 DRAFT – DO NOT CITE OR QUOTE 

Table A-10. Controlled human exposure studies of ammonia inhalation 

Subjects Exposure conditions Results Reference 

20 male volunteers; 

groups of four were 

exposed to 

ammonia at various 

concentrations and 

durations 

Group 1: exposed to 2 mg/m3

(3.0 ppm) for 37 d; 

Group 2: exposed to 5 mg/m3

(7.2 ppm) for 17 d; 

Group 3: exposed to 2 mg/m3

(3.0 ppm) for 35 d with shortterm increases to 10 mg/m3

(14 ppm); 

Groups 4 and 5: exposed to 

2 and 5 mg/m3 (3.0 and 

7.2 ppm), respectively, for 20 d 

with variations in temperature 

and humidity; exposure 

duration each day was not 

specified; there was no 

mention of preexposure 

examinations 

Significantly elevated adrenalin levels in 

urine at 2.1 mg/m3

 (3.0 ppm); dopamine 

and DOPA levels in urine were not 

significantly affected at any concentration; 

significant increase of adrenalin and 7-oxycorticosteroids in urine, and 

11-oxycorticosteroids free fractions in 

plasma at 5.1 mg/m3

 (7.2 ppm); increased 

temperature and humidity resulted in 

increased urine adrenalin, urine 7-oxycorticosteroids and free 

11-oxycorticosteroid levels in plasma at 5.1 

mg/m3 (7.2 ppm) 

Kalandarov et 

al., 1984b

Unspecified number 

of volunteer 

subjects 

Acute exposure up to 15 sec, 

1 time/d at unspecified 

concentrations; also a separate 

exposure of 10 inhaled breaths 

via mouthpiece at unspecified 

concentrations; there was no 

mention of preexposure 

examinations 

The lachrymatory threshold was 55 ppm 

(39 mg/m3

) and bronchoconstriction was 

seen at 85 ppm (60.1 mg/m3

) 

Douglas and 

Coe, 1987b

Six healthy 

volunteers (two 

males and four 

females, 25–45 yrs 

old) and eight 

volunteers with 

mild asthma (four 

males and four 

females, 18–52 yrs 

old) 

16–25 ppm (11–18 mg/m3) for 

30-min sessions with 1 wk 

between sessions; lung 

function was measured before 

and after exposure 

No significant changes in lung function in 

healthy subjects at any concentration; 

decreased FEV1 and increased bronchial 

hyperreactivity were reported in asthmatics 

exposed to dust and ammonia, but not to 

ammonia alone; exposure to dust alone 

caused similar effects, suggesting that dust 

was responsible for the effects 

Sigurdarson et 

al., 2004a

12 healthy 

volunteers 

(7 females, 5 males) 

21–28 yrs old 

5 and 25 ppm (4 and 18 

mg/m3) for three separate 

exposures in inhalation 

chamber for 1.5 hrs resting 

and 1.5 hrs exercising on a 

stationary bike; 1–4 volunteers 

were exposed on each 

occasion; lung function and 

nasal lavage were performed 

before and after exposure 

Reported discomfort in eyes, detection of 

solvent smell, headache, dizziness, and 

feeling of intoxication were significantly 

increased at 5 ppm (4 mg/m3); there were 

no changes in lung function or exhaled nitric 

oxide levels in exposed individuals; exposure 

did not result in upper-airway inflammation 

or bronchial responsiveness 

Sundblad et al., 

2004a
A-35 DRAFT – DO NOT CITE OR QUOTE 

Table A-10. Controlled human exposure studies of ammonia inhalation 

Subjects Exposure conditions Results Reference 

Healthy male and 

female volunteers 

grouped by age, 

18–35 and 45–65 

yrs old 

Repeated 2-sec exposures at 

increasing concentrations 

ranging from 0.9 to 228 ppm 

(0.6–161 mg/m3

) by dynamic 

olfactometry; there was no 

mention of preexposure 

examinations 

Mean odor detection threshold <20 ppm 

(14 mg/m3), mean irritation (lateralization) 

threshold well above 20 ppm (14 mg/m3), 

dose-response for odor annoyance and 

irritation; strong olfactory and moderate to 

strong irritating sensations at >15 ppm 

(11 mg/m3) 

Altmann et al., 

2006b

43 healthy male 

volunteers age 21–

47 yrs; one group of 

30 men not familiar 

with the smell of 

ammonia and 

10 men exposed to 

ammonia regularly 

at the workplace 

0, 10, 20, 20 + 2 peak 

exposures at 40, and 50 ppm 

(0, 7, 14, 14 + 2 peak 

exposures at 28, and 35 

mg/m3) on 5 consecutive days 

for 4 hrs/d in an exposure 

chamber 

Subjects familiar to ammonia reported 

fewer symptoms than naïve subjects; at 

concentrations ≤20 ppm (14 mg/m3), there 

were no significant differences in symptoms 

reported between the groups; the perceived 

intensity of symptoms was concentrationdependent in both groups 

Ihrig et al., 

2006a

25 healthy 

volunteers (mean 

age 29.7 yrs), and 

15 mild/moderate 

persistent 

asthmatic 

volunteers (mean 

age 29.1 yrs) 

2–500 ppm (1–354 mg/m3) 

(ocular and nasal exposure) for 

various durations lasting up to 

2.5 hrs; baseline lung function 

was recorded prior to 

exposure 

Irritation threshold, odor intensity, and 

annoyance were not significantly different 

between healthy volunteers and asthmatics; 

nasal irritation threshold = 129 ppm 

(91 mg/m3

); ocular irritation threshold = 

175 ppm (124 mg/m3); there were no 

changes in lung function (FEV1) for subjects 

in either group 

Petrova et al., 

2008a

24 healthy female 

volunteers age 18–

45 yrs (mean age 

29.9 yrs) 

0.03–615.38 ppm (0.02–

435 mg/m3

) (nasal exposure) 

for a maximum of 2 sec; 

preexposure measurements 

included rhinoscopic exam, 

screening for chemical 

sensitivities, allergies, 

respiratory disease, general 

health, and prior chemical 

exposure by personal interview

Both the static and dynamic methods 

showed similar averages for detection 

thresholds for the odor and irritancy of 

ammonia; mean odor detection threshold of 

2.6 ppm (2 mg/m3) (both static and 

dynamic) and mean irritation thresholds of 

31.7 or 60.9 ppm (22 or 43 mg/m3

) for static 

and dynamic methods, respectively 

Smeets et al., 

2007a

a

Investigators reported that informed consent by volunteers and/or study approval by local boards/officials 

regarding ethical conduct was obtained. 

b

This controlled-exposure study did not provide information on the human subjects research ethics procedures 

undertaken in the study; however, there is no evidence that the conduct of the research was fundamentally 

unethical or significantly deficient relative to the ethical standards prevailing at the time the research was 

conducted. 

 Altmann et al. (2006) showed a dose-dependent increase in the intensity of odor 

annoyance and irritation in healthy male and female volunteers during inhalation exposure to 

ammonia; strong olfactory and moderate to strong irritation sensations occurred at concentrations 

>15 ppm (11 mg/m3

), with odor detection thresholds at <20 ppm (14 mg/m3). In another study, 
A-36 DRAFT – DO NOT CITE OR QUOTE 

12 healthy volunteers exposed to 5 and 25 ppm (4 and 18 mg/m3) ammonia on three different 

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

discomfort in the eyes and odor detection at 5 ppm (4 mg/m3) (Sundblad et al., 2004). Eye 

irritation was also shown to increase in a concentration-dependent manner in 15 volunteers 

exposed to ammonia for 2 hours in an exposure chamber at concentrations of 50, 80, 110, and 

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

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

be 55 ppm (39 mg/m3) in volunteers exposed to ammonia gas inside tight-fitting goggles for an 

acute duration of up to 15 seconds (Douglas and Coe, 1987). In contrast, exposures to up to 

90 ppm (64 mg/m3) ammonia gas did not produce severe lacrimation in seven volunteers after 

10 minutes in an exposure chamber, although increased eye erythema was reported (MacEwen et 

al., 1970). Exposure to 500 ppm (354 mg/m3) of ammonia gas for 30 minutes through a masked 

nose and throat inhalation apparatus resulted in two of seven volunteers reporting lacrimation, 

and two of seven reporting nose and throat irritation that lasted up to 24 hours after exposure 

(Silverman et al., 1949). 

 Petrova et al. (2008) investigated irritation threshold differences between 25 healthy 

volunteers and 15 mild-to-moderate persistent asthmatic volunteers exposed to ammonia via the 

eyes and nose at concentrations ranging from 2 to 500 ppm (1–354 mg/m3) for durations lasting 

up to 2.5 hours. Irritation threshold, odor intensity, and annoyance were not significantly 

different between the two groups. The nasal and eye irritation thresholds were reported to be 

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

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

concentrations ranging from 0.03 to 615 ppm (0.02–435 mg/m3). This study found a mean odor 

detection threshold of 2.6 ppm (2 mg/m3) and a mean irritation threshold of 31.7 or 60.9 ppm 

(22 or 43 mg/m3), depending on the olfactometry methodology followed (static versus dynamic, 

respectively). Irritation thresholds may be higher in people who have had prior experience with 

ammonia exposure (Ihrig et al., 2006). Thirty male volunteers who had not experienced the 

smell of ammonia and 10 male volunteers who had regular workplace exposure to ammonia were 

exposed to ammonia vapors at concentrations of 0, 10, 20, and 50 ppm (0, 7, 14, and 35 mg/m3) 

on 5 consecutive days (4 hours/day) in an exposure chamber; volunteers in the group familiar to 

the smell of ammonia reported fewer symptoms than the nonhabituated group, but at a 

concentration of 20 ppm (14 mg/m3), there were no differences in perceived symptoms between 

the groups. However, the perceived intensity of symptoms was concentration-dependent in both 

groups, but was only significant in the group of volunteers not familiar with ammonia exposure 

(Ihrig et al., 2006). Ferguson et al. (1977) reported habituation to eye, nose, and throat irritation

in six male and female volunteers after 2–3 weeks of exposure to ammonia concentrations of 25, 

50, and 100 ppm (18, 35, and 71 mg/m3

) during a 6-week study (6 hours/day, 1 time/week). 
A-37 DRAFT – DO NOT CITE OR QUOTE 

Continuous exposure to even the highest concentration tested became easily tolerated with no 

general health effects occurring after acclimation.

 Several studies evaluated lung functions following acute inhalation exposure to ammonia. 

Volunteers exposed to ammonia (lung only) through a mouthpiece for 10 inhaled breaths of gas 

experienced bronchioconstriction at a concentration of 85 ppm (60 mg/m3) (Douglas and Coe, 

1987); however, there were no bronchial symptoms reported in seven volunteers exposed to 

ammonia at concentrations of 30, 50, or 90 ppm (21, 35, and 64 mg/m3) for 10 minutes in an 

exposure chamber (MacEwen et al., 1970). Similarly, 12 healthy volunteers exposed to 

ammonia on three separate occasions to 5 and 25 ppm (4 and 18 mg/m3) for 1.5 hours in an 

exposure chamber while exercising on a stationary bike did not have changes in bronchial 

responsiveness, upper airway inflammation, exhaled nitric oxide levels, or lung function as 

measured by vital capacity and forced expiratory volume in 1 second (FEV1) (Sundblad et al., 

2004). In another study, 18 healthy servicemen volunteers were placed in an exposure chamber 

for 3 consecutive half-day sessions. Exposure to ammonia at concentrations of 50–344 mg/m3

(70–486 ppm) occurred on the second session, with sessions 1 and 3 acting as controls (Cole et 

al., 1977). The no-effect concentration was determined to be 71 mg/m3

 (100 ppm). Exercise 

tidal volume was increased at 106 mg/m3 (150 ppm), but then decreased at higher concentrations 

in a concentration-dependent manner (Cole et al., 1977). Decreased FEV1 and forced vital 

capacity (FVC) were reported in eight healthy male volunteers exposed to a mean airborne 

ammonia concentration of 20.7 ppm (15 mg/m3) in swine confinement buildings for 4 hours at 

1-week intervals; however, swine confinement buildings also include confounding exposures to 

dust, bacteria, endotoxin, and molds, thereby making measurement of effects due to ammonia 

uncertain in this study (Cormier et al., 2000). 

 Differences in lung function between healthy and asthmatic volunteers exposed to 

ammonia were evaluated in several studies. There were no changes in lung function as measured 

by FEV1 in 25 healthy volunteers and 15 mild/moderate persistent asthmatic volunteers after 

ocular and nasal exposure to 2–500 ppm (1–354 mg/m3) ammonia at durations lasting up to 

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

asthmatic volunteers were exposed to 16–25 ppm (11–18 mg/m3

) ammonia, ammonia and dust, 

and dust alone for 30-minute sessions, with 1 week between sessions (Sigurdarson et al., 2004). 

There were no significant changes in lung function as measured by FEV1 in the healthy 

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

ammonia, but not to ammonia alone; similarly, increased bronchial hyperreactivity was reported 

in asthmatics after exposure to dust and ammonia, but not to ammonia alone. Exposure to dust 

alone caused similar effects, suggesting that dust was responsible for decreased lung function 

(Sigurdarson et al., 2004). 
A-38 DRAFT – DO NOT CITE OR QUOTE 

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

exposure to ammonia at concentrations as low as 5 ppm (4 mg/m3). Irritation thresholds may be 

higher in people who have had prior experience with ammonia exposure, and habituation to eye, 

nose, and throat irritation occurs over time. Lung function was not affected in workers acutely 

exposed to ammonia concentrations as high as 71 mg/m

3

 (100 ppm). Studies comparing the lung 

function of asthmatics and healthy volunteers exposed to ammonia do not suggest that asthmatics 

are more sensitive to the lung effects of ammonia. 

Case Studies and Reports of Human Exposure to Ammonia 

 Oral exposure to ammonia most commonly involved ingestion of household cleaning 

solutions or biting into the capsules of ammonia smelling salts, which are commonly found in 

first aid kits. Young children, generally <4 years old, have been reported as “biting into” or 

ingesting smelling salts capsules. The acute effects included drooling, erythematous and 

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

(Rosenbaum et al., 1998; Wason et al., 1990; Lopez et al., 1988). Delayed effects were not noted 

in these cases. Gilbert (1988) reported ammonia intoxication characterized by lethargy, 

restlessness, irritability, and confusion in a 37-year-old man following surgery. Most other cases 

of ammonia ingestion involved household cleaning solutions and detergents. Many cases were 

intentional; however, not all were fatal. Klein et al. (1985) described two cases of ingestion of 

approximately 30 mL and “two gulps” of Parson’s sudsy ammonia (ammonia 3.6%; pH 11.5), 

respectively. The first case resulted in a white and blistered tongue and pharynx, and esophageal 

burns with friable, boggy mucosa; and in the second case, several small esophageal lesions with 

mild to moderate ulceration and some bleeding were reported. There were no oropharyngeal 

burns in the second case and no delayed complications in either case. Christesen (1995) reported 

that of the 11 cases involving accidental or intentional ingestion of ammonia water by adults 

(≥15 years old), 2 cases exhibited acute respiratory obstruction and 1 case developed an 

esophageal stricture 3 months postinjury. In cases involving fatalities, evidence of laryngeal and 

epiglottal edema, erythmatous esophagus with severe corrosive injury, and hemorrhagic 

esophago-gastro-duodeno-enteritis was noted (Klein et al., 1985; Klendshoj and Rejent, 1966). 

Dworkin et al. (2004) reported a case of ingestion of contaminated chicken tenders, prepared and 

served in a school cafeteria, by approximately 157 students and 6 teachers. The onset of acute 

symptoms occurred within an hour of ingestion, and included headache, nausea, vomiting, 

dizziness, diarrhea, and burning mouth. In a case of forced ingestion of an unknown quantity of 

dilute ammonia, a 14-year-old boy presented with difficulty speaking, ataxic gait, isochoric 

pupils, and evidence of brain edema. There were no burns to the eyes or mouth and no 

indication of gastric pathology. It was only after the patient was able to communicate that 
A-39 DRAFT – DO NOT CITE OR QUOTE 

ammonia was involved that appropriate treatment, followed by a satisfactory outcome, was 

achieved. 

 Inhalation is the most frequently reported route of exposure and cause of morbidity and 

fatality, and often occurs in conjunction with dermal and ocular exposures. Acute effects from 

inhalation have been reported to range from mild to severe, with mild symptoms consisting of 

nasal and throat irritation, sometimes with perceived tightness in the throat (Price and Watts, 

2008; Prudhomme et al., 1998; Weiser and Mackenroth, 1989; Yang, 1987; O’Kane, 1983; Ward 

et al., 1983; Caplin, 1941). Moderate effects are described as moderate to severe pharyngitis; 

tachycardia; frothy, often blood-stained sputum; moderate dyspnea; rapid, shallow breathing; 

cyanosis; some vomiting; transient bronchospasm; edema and some evidence of burns to the lips 

and oral mucosa, and localized to general rhonchi in the lungs (Weiser and Mackenroth, 1989; 

Yang, 1987; O’Kane, 1983; Ward et al., 1983; Counturier et al., 1971; Caplin, 1941). Severe 

effects include second- and third-degree burns to the nasal passages, soft palate, posterior 

pharyngeal wall, and larynx, upper airway obstruction, loss of consciousness, bronchospasm, 

dyspnea, persistent, productive cough, bilateral diffuse rales and rhonchi, production of large 

amounts of mucous, pulmonary edema, marked hypoxemia, local necrosis of the lung, 

deterioration of the whole lung, and fatality. Delayed effects of acute exposure to high 

concentrations of ammonia include bronchiectasis, bronchitis, bronchospasm/asthma, dyspnea 

upon exertion and chronic productive cough, bronchiolitis, severe pulmonary insufficiency, and 

chronic obstructive pulmonary disease (Lalic et al., 2009; Leduc et al., 1992; Bernstein and 

Bernstein, 1989; Flury et al., 1983; Ward et al., 1983; Stroud, 1981; Close, 1980; Taplin, et al., 

1976; Walton, 1973; Kass et al., 1972; Slot, 1938).

 Respiratory effects were also observed following chronic occupational exposure to 

ammonia. After 18 months and 1 year on the job, respectively, both men developed cough, chest 

tightness, and wheezing, typically after 2–6 hours from the beginning of each work day, but not 

on weekends or holidays. In another case, progressive deterioration of the clinical condition of a 

68-year-old male was documented for 4 years, and development of diffuse interstitial and severe 

restrictive lung disease was reported following long-term repetitive occupational exposure to 

ammonia at or above the odor recognition level (Brautbar et al., 2003). Lee et al. (1993) 

reported a case of a 39-year-old man who developed occupational asthma 5 months after 

beginning a job requiring the polishing of silverware. The room in which he worked was poorly 

ventilated. The product used contained ammonia and isopropyl alcohol and the measured 

ammonia concentration in the breathing zone when using this product was found to be 8–15 ppm 

(6–11 mg/m3

). 

 Acute dermal exposure to anhydrous (liquid) ammonia and ammonia vapor has resulted 

in caustic burns of varying degrees to the skin and eyes. There are numerous reports of 

exposures from direct contact with anhydrous ammonia in which first-, second-, and third-degree 
A-40 DRAFT – DO NOT CITE OR QUOTE 

burns occurred over as much as 50% of the total body surface (Lalic et al., 2009; Pirjavec et al., 

2009; Arwood et al., 1985). Frostbite injury has also been reported in conjunction with exposure 

to sudden decompression of liquefied ammonia, which is typically stored at -33°F (George et al., 

2000; Sotiropoulos et al., 1998; Arwood et al., 1985). However, direct contact is not a 

prerequisite for burn injury. Several reports have indicated that burns to the skin occurred with 

exposure to ammonia gas or vapor. Kass et al. (1972) reported one woman with chemical burns 

to her abdomen, left knee, and forearm and another with burns to the feet when exposed to 

anhydrous ammonia gas released from a derailed train in the vicinity. Several victims at or near 

the scene of an overturned truck that had been carrying 8,000 gallons of anhydrous ammonia 

were reported as having second- and third-degree burns over exposed portions of the body 

(Burns et al., 1985; Close et al., 1980; Hatton et al., 1979). In a case involving a refrigeration 

leak in a poorly ventilated room, workers located in an adjacent room reported a “burning skin” 

sensation (de la Hoz et al., 1996) while in another case involving the sudden release of ammonia 

from a pressure valve in a refrigeration unit, one victim received burns to the leg and genitalia 

(O’Kane, 1983). 

 In addition to the skin, the eyes are particularly vulnerable to ammonia burns due to the 

highly water-soluble nature of the chemical and the ready dissociation of ammonium hydroxide 

to release hydroxyl ions. When ammonia or ammonia in solution has been splashed or sprayed 

into the face (accidently or intentionally), immediate effects include temporary blindness, 

blepharospasm, conjunctivitis, corneal burns, ulceration, edema, chemosis, and loss of corneal 

epithelium (George et al., 2000; Helmers et al., 1971; Highman, 1969; McGuinness, 1969; Levy 

et al., 1964; Abramovicz, 1924). The long-term effects included photophobia, progressive loss 

of sensation, formation of bilateral corneal opacities and cataracts, recurrent corneal ulcerations, 

nonreactive pupil, and gradual loss of vision (Yang, 1987; Kass et al., 1972; Helmers et al., 

1971; Highman, 1969; Osmond and Tallents, 1968; Levy et al., 1964; Abramovicz, 1924). 

White et al. (2007) reported a case with acute bilateral corneal injury that developed into bilateral 

uveitis with stromal vascularization and stromal haze and scarring, and pigmented keratic 

precipitates that resulted in legal blindness. An increase in intraocular pressure, resembling 

acute-angle closure glaucoma, was reported by Highman (1969) following ammonia 

intentionally sprayed into the eyes during robbery attempts. 

A.5. ANIMAL STUDIES 

Oral Exposure 

Kawano et al. (1991); Tsujii et al. (1993) 

Kawano et al. (1991) investigated the hypothesis that the bacterium Helicobacter pylori, 

which produces a potent urease that increases ammonia production, plays a significant role in the 
A-41 DRAFT – DO NOT CITE OR QUOTE 

etiology of chronic atrophic gastritis. Male Sprague-Dawley rats (6/group) were given tap water 

or 0.01 or 0.1% ammonia ad libitum for 2 or 4 weeks. The daily dose of 0.01 and 0.1% 

ammonia in drinking water, based on a weight of 230 g for male rats and a water consumption of 

50 mL/day, was estimated to be 22 and 220 mg/kg-day, respectively. The effect of ammonia on 

the antral mucosa was estimated by three measurements of the thickness of the mucosa about 

175 μM from the pyloric ring in the antral mucosa. The parietal cell number per gland was 

determined at three locations in the oxyntic glandular area. Mucosal lesions were not observed 

macro- or microscopically. There was a statistically significant decrease in mean antral mucosal 

thickness with increasing dose and duration of exposure (Table A-11). Parietal cell number per 

oxyntic gland decreased in a statistically significant dose- and time-dependent fashion. The 

index of periodic acid-Schiff Alician blue positive intracellular mucin was significantly lower in 

the antral and body mucosa with 0.1% ammonia; the index was significantly lower only for the 

antral mucosa with 0.01% ammonia. The authors suggested that administration of ammonia in 

drinking water causes gastric mucosal atrophy. Based on the reduction in antral mucosal 

thickness, EPA identified a LOAEL of 22 mg/kg-day; a NOAEL was not identified. 

Table A-11. Effect of ammonia in drinking water on the thickness of the gastric 

antral and body mucosa of the rat stomach

Length of treatment 

Thickness of mucosa (μM); mean ± standard error of the mean 

Control (tap water) 

Percent ammonia in drinking water 

0.01% 0.1% 

Antral mucosa 

2 wks 270 ± 18 258 ± 22 217 ± 40a

4 wks 276 ± 39 171 ± 22a 109 ± 12b,c

Body mucosa 

2 wks 574 ± 116 568 ± 159 591 ± 183 

4 wks 618 ± 154 484 ± 123 440 ± 80a,c

a

p < 0.05 versus control group.

b

p < 0.01 versus control group. 

c

p < 0.01 versus 2-week treatment group. 

Source: Kawano et al. (1991). 

In a follow-up study of the effect of ammonia produced from H. pylori, Tsujii et al. 

(1993) studied the subchronic effect of ammonia in drinking water on the cell kinetics of the 

gastric mucosa of the stomach. Six groups of male Sprague-Dawley rats (36 rats/group) were 

given 0.01% ammonia in drinking water for 3 days, or 1, 2, 4, or 8 weeks; ammonia solutions 

were changed daily. Tap water was provided for the balance of the 8-week study. A control 

group was given tap water for 8 weeks. Based on the initial body weight (150 g) and estimated 
A-42 DRAFT – DO NOT CITE OR QUOTE 

daily water intake (50 mL), the daily dose at a drinking water concentration of 0.01% ammonia 

was estimated to be 33 mg/kg-day. Cellular migration was measured by labeling cells with 

5-bromo-2-deoxyuridine (BrDU) at different time periods and measuring the incorporation of 

this modified nucleoside with a histochemical technique using anti BrDU monoclonal antibodies. 

Antral and body mucosa thickness was measured as described in Kawano et al. (1991). The 

measurement of cell proliferation in the gastric mucosa was estimated using the labeling index in 

gastric pits (ratio of labeled nuclei to total nuclei in the proliferation zone). The antral mucosal 

thickness decreased significantly at 4 and 8 weeks of treatment (Table A-12), but there was no 

effect on the body mucosa. Cell migration preceded the decrease in thickness of the antral 

mucosa. The rate of cell migration (cells/day) toward the mucosal surface was significantly 

greater for 0.01% ammonia-treated rats compared to the control at 4 and 8 weeks of treatment. 

Cell proliferation, as estimated from the labeling index, was significantly increased after 1 week 

for the antral and body mucosa. The authors concluded that 0.01% ammonia increased epithelial 

cell migration in the antrum leading to mucosal atrophy. The EPA identified a LOAEL of 

33 mg/kg-day based on decreased thickness of the gastric antrum; a NOAEL was not identified. 

Table A-12. Effect of ammonia in drinking water on gastric antral and body 

mucosa in the stomach of Sprague-Dawley rats administered 0.01% ammonia 

in drinking water 

Length of treatment 

Thickness of mucosa (μM) 

Antral mucosa Body mucosa 

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

3 D 305 ± 45 559 ± 50 

1 Wk 272 ± 31 542 ± 28 

2 Wks 299 ± 26 555 ± 37 

4 Wks 159 ± 29b 531 ± 32 

8 Wks 168 ± 26b 508 ± 29 

a

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

b

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

Source: Tsujii et al. (1993). 
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Fazekas (1939) 

Fazekas (1939) administered ammonium hydroxide to 51 rabbits (strain and sex not 

specified) via gavage every other day initially and later daily in increasing amounts of 50–80 mL 

as either a 0.5 or 1.0% solution over a long period of time. The daily dose (mg/kg-day) was 

estimated using the weight of adult rabbits from standard growth curve for rabbits (3.5–4.1 kg) 

(U.S. EPA, 1988). Based on a daily gavage of 50–80 mL, daily doses for the rabbits receiving 

0.5 and 1.0% ammonia solutions were approximately 61–110 and 120–230 mg/kg-day, 

respectively. The exact duration of the study is not reported, but it is clear from the data that by 

the end of the experiment, some rabbits received only three or four doses before dying as a result 

of intoxication in 5.5 days and other rabbits received over 80 doses and survived for up to 17 

months. Toxicological endpoints evaluated included fluctuations in body weights, changes in 

blood pressure measured at the central artery of the ear in 10 rabbits after lengthy treatment, and 

changes in the weight, fat, and cholesterol content of adrenals. For comparison purposes, the 

weight of the adrenals from 41 healthy rabbits of similar age and body weight were also 

determined. The average weight of adrenals from these 41 control rabbits was 400.0 ± 13.4 mg. 

Fazekas (1939) reported that differences in mean adrenal weight in ammonium 

hydroxide-treated animals were significant, although there was no description of the statistical 

analysis performed in this study. Chemical evaluation of the adrenals from treated rabbits 

revealed fat content 4.5 times greater and cholesterol content 6.5 times greater than controls. At 

the beginning of the experiment, a greater weight loss was observed among those rabbits 

receiving ammonium hydroxide more frequently (daily) at higher doses. Body weights 

fluctuated among treated rabbits and generally decreased initially and gradually increased in the 

later months only to drop again a few weeks before death. Body weights for controls were not 

reported. Thirteen rabbits exhibited weight increases after the initial loss that persisted until the

end of the experiment. Dissection of these rabbits revealed enlarged adrenals (800–1,340 mg), 

and fatty tissue surrounding the kidneys, mesentery, and the pericardium. This fat accumulation 

was not observed in untreated controls. Histology revealed enlarged cells of the zona fasciculata 

of the adrenal cortex that were rich in lipid. The blood pressure of rabbits before dosing ranged 

from 60 to 74 mm Hg and dropped with initial exposure (during the first 5–10 minutes that lasted 

up to 7 hours) to 20–30 mm Hg. Following several months of ammonium hydroxide treatment, a 

moderate elevation in blood pressure of 10–30 mm Hg was found in 8/10 rabbits. In the other 

two rabbits, the blood pressure increased from the initial values of 62 and 65–90 mm Hg during 

the first 7 months of treatment and remained almost unchanged at this level until sacrifice. 

In summary, Fazekas (1939) concluded that initial decreases in blood pressure and effects 

of emaciation in rabbits following gavage treatment with ammonium hydroxide is associated 

with the hypofunction of the cortical or medullary substance of the adrenal gland. The authors 

also concluded that the subsequent increases in blood pressure and body weight could be 
A-44 DRAFT – DO NOT CITE OR QUOTE 

attributed to hypertrophy of the adrenal cortex. This study is limited by lack of reporting detail 

and inadequate study design. The EPA did not identify a NOAEL or LOAEL from this study. 

Toth (1972) 

Toth (1972) evaluated whether hydrazine, methylhydrazines, and ammonium hydroxide 

play a role in tumorigenesis in mice. Solutions of hydrazine (0.001%), methyl hydrazine 

(0.01%), methyl hydrazine sulfate (0.001%), and ammonium hydroxide (0.1, 0.2, and 0.3%) 

were administered continuously in the drinking water of 5- and 6-week-old randomly bred Swiss 

mice (50/sex) for their entire lifetime. For ammonium hydroxide, the study authors reported the 

average daily drinking water intakes for the 0.1, 0.2, and 0.3% groups as 9.2, 8.2, and 

6.5 mL/day for males, respectively, and 8.3, 6.5, and 4.8 mL/day for females, respectively. 

Given these rates and assuming average default body weights of 37.3 and 35.3 g for males and 

females, respectively (U.S. EPA, 1988), the approximate continuous doses for ammonium 

hydroxide are 250, 440, and 520 mg/kg-day for males and 240, 370, and 410 mg/kg-day for 

females. Additionally, groups of C3H mice (40/sex) were exposed to ammonium hydroxide in 

the drinking water at a concentration of 0.1% for their lifetime. Average daily water 

consumption for these mice was reported as 7.9 and 8.4 mL/day for males and females, 

respectively. The approximate equivalent doses for these mice assuming the same default body 

weights as above (U.S. EPA, 1988) are 191 and 214 mg/kg-day for males and females, 

respectively. Data were not reported for a concurrent control group. Mice were monitored 

weekly for changes in body weights and gross pathological changes were recorded. The animals 

were either allowed to die or were killed when found in poor condition. Complete necropsies 

were performed on all mice, and the liver, kidney, spleen, lung, and organs with gross lesions 

were processed for histopathological examination. Data on body weights were not reported. 

For Swiss mice, tumor incidence at the 0.3% ammonium hydroxide concentration was as 

follows: malignant lymphomas: 3/50 (males), 9/50 (females); and lung adenoma or 

adenocarcinoma: 7/50 (males), 4/50 (females). Tumor incidence at the 0.2% ammonium 

hydroxide concentration was: malignant lymphomas: 7/50 (males), 10/50 (females); lung 

adenoma or adenocarcinoma: 5/50 (males), 8/50 (females); and breast tumors: 4/50 (females). 

Tumor incidence at the 0.1% ammonium hydroxide concentration was: malignant lymphomas: 

4/50 (males), 10/50 (females); lung adenoma or adenocarcinoma: 5/50 (males), 12/50 (females); 

and breast tumors: 1/50 (females). The denominators were not adjusted for survival, and 

concurrent control data were not provided. For a second strain of mice (C3H) that received 0.1% 

ammonium hydroxide in drinking water, the incidence of adenocarcinomas of the mammary 

gland in female mice was 60%. The incidence of breast tumors in the corresponding untreated 

control mice was 76%. Other tumors were identified in treated mice, but were of low incidence. 

Toth (1972) concluded that ammonium hydroxide was not carcinogenic in either strain of mouse. 
A-45 DRAFT – DO NOT CITE OR QUOTE 

Because concurrent control tumor incidence was not provided other than the incidence of breast 

tumors in C3H female mice, the incidence of tumors in treated mice cannot be independently 

compared to control tumor incidence. 

Tsujii et al. (1995, 1992a) 

Tsujii et al. (1995, 1992a) evaluated the role of ammonia in H. pylori-related gastric 

carcinogenesis. H. pylori is a bacterium that produces a potent urease, which generates ammonia 

from urea in the stomach, and has been implicated in the development of gastric cancer. Tsujii et 

al. (1995, 1992a) pretreated groups of 40–44 male Sprague-Dawley rats with the initiator 

N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in the drinking water for 24 weeks before 

administering 0.01% ammonium solution as a drinking fluid for 24 weeks. Based on an average 

body weight of 523 g for male Sprague-Dawley rats during chronic exposure (U.S. EPA, 1988) 

and a reported water consumption rate of 0.05 L/day, the approximate continuous dose 

administered to these rats is 10 mg/kg-day. In each study, an additional group of 40–43 rats 

given tap water for 24 weeks following pretreatment with MNNG served as controls. The study 

protocol did not include a dose group that received ammonia only in drinking water. Stomachs 

from rats surviving beyond 45 weeks were examined histologically for evidence of ulcers, 

lesions, and tumors. Tsujii et al. (1995) also evaluated serum gastrin levels from blood collected 

at 30 and 46 weeks and mucosal cell proliferation in animals surviving to 48 weeks by 

calculating the labeling index (percentage ratio of labeled nuclei to total number of nuclei in the 

proliferation zone) and the proliferation zone index (fraction of the gastric pit occupied by the 

proliferation zone). 

Tsujii et al. (1995, 1992a) observed a significantly greater incidence of gastric cancers 

among rats receiving ammonia after pretreatment with MNNG compared to rats receiving only 

MNNG and tap water (p < 0.01, χ

2

 test). Seventy percent of MNNG+ammonia-treated rats 

versus 31% of control rats developed gastric tumors in the first study (Tsujii et al., 1992a). The 

number of gastric cancers per tumor-bearing rat in this study was 2.1 ± 1.4 among treated rats 

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

2

 test). 

In the second study, 66% of rats dosed with ammonia and pretreated with MNNG 

developed gastric cancers compared to 30% of the control rats (Tsujii et al., 1995). The numbers 

of gastric tumors per rat in this study were also significantly higher among MNNG+ammoniaexposed rats than controls (p < 0.001, Mann-Whitney test), suggesting that ammonia was a 

promoter. In the absence of an ammonia-only treatment group, however, it is not possible to 

distinguish with certainty between possible promotion and initiator activity. The degree of 

differentiation of adenocarcinomas in control and ammonia-treated rats was significantly 

different. Ammonia-treated rats also demonstrated a significantly higher incidence of larger 

tumors (5.3 mm compared to 4.4 mm for controls) and of gastric cancers penetrating the 
A-46 DRAFT – DO NOT CITE OR QUOTE 

muscularis propria or deeper (p < 0.01, 22% compared to controls 12%). In this study, the 

labeling index and the proliferation zone index were statistically significantly elevated in 

ammonia exposed rats compared to controls in the fundic mucosa and antral mucosa. 

Tsujii et al. (1995) explored the hypothesis that ammonia might increase intragastric pH, 

leading to an increase in serum gastrin, a trophic hormone in the gastric fundus mucosa, and a 

possible proliferating factor in gastric epithelial cells. The investigators found no significant 

effects on serum gastrin levels and concluded that serum gastrin does not appear to play a 

significant role in ammonia-induced promotion. 

Inhalation Exposure 

Anderson et al. (1964) 

Anderson et al. (1964) exposed a group of 10 guinea pigs (strain not given) and 10 Swiss 

albino mice of both sexes continuously to 20 ppm (14 mg/m3) ammonia vapors for up to 6 weeks 

(anhydrous ammonia, purity not reported). Controls (number not specified) were maintained 

under identical conditions except for the exposure to ammonia. An additional group of six 

guinea pigs was exposed to 50 ppm (35 mg/m3

) for 6 weeks. The animals were observed daily 

for abnormal signs or lesions. At termination, the mice and guinea pigs were sacrificed (two per 

group at 1, 2, 3, 4, and 6 weeks of exposure) and selected tissues (lungs, trachea, turbinates, 

liver, and spleen) were examined for gross and microscopic pathological changes. No significant 

effects were observed in animals exposed for up to 4 weeks, but exposure to 20 ppm (14 mg/m3

) 

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

guinea pigs to 50 ppm (35 mg/m3) ammonia for 6 weeks caused grossly enlarged and congested 

spleens, congested livers and lungs, and pulmonary edema. NOAEL and LOAEL values were 

not identified in this study because only two animals per group were examined at a single time 

period for the 20 ppm (14 mg/m3

) exposure groups (mice and guinea pigs). 

Coon et al. (1970) 

Coon et al. (1970) exposed groups of male and female Sprague-Dawley and Long-Evans 

rats, male and female Princeton-derived guinea pigs, male New Zealand rabbits, male squirrel 

monkeys, and purebred male beagle dogs to 0, 155, or 770 mg/m3

 ammonia (0, 219, or 

1,089 ppm) 8 hours/day, 5 days/week for 6 weeks (anhydrous ammonia, >99% pure). The 

investigators stated that a typical loaded chamber contained 15 rats, 15 guinea pigs, 3 rabbits, 

3 monkeys, and 2 dogs. Blood samples were taken before and after the exposures for 

determination of hemoglobin concentration, packed erythrocyte volume, and total leukocyte 

counts. Animals were routinely checked for clinical signs of toxicity. At termination, sections 

of the heart, lung, liver, kidney, and spleen were processed for microscopic examination in 

approximately half of the surviving rats and guinea pigs and all of the surviving dogs and 
A-47 DRAFT – DO NOT CITE OR QUOTE 

monkeys. Sections of the brain, spinal cord, and adrenals from dogs and monkeys were also 

retained, as were sections of the thyroid from the dogs. The nasal passages were not examined in 

this study. 

Exposure to 155 mg/m3 ammonia did not result in any deaths or adverse clinical signs of 

toxicity in any of the animals. Hematological values were within normal limits for the laboratory 

and there were no significant gross alterations in the organs examined. Microscopic examination 

showed evidence of focal pneumonitis in the lung of one of three monkeys. Exposure to 

770 mg/m3 caused initial mild to moderate lacrimation and dyspnea in rabbits and dogs. 

However, these clinical signs disappeared by the second week of exposure. No significant 

alterations were observed in hematology tests or upon gross or microscopic examinations at the 

highest dose. However, consistent nonspecific inflammatory changes (not further described) 

were observed in the lungs from rats and guinea pigs in the high-dose group that were more 

extensive than in control animals (incidence not reported). 

Coon et al. (1970) also exposed rats (15–51/group) continuously to ammonia (anhydrous 

ammonia, >99% pure) at 0, 40, 127, 262, 455, or 470 mg/m3

 (0, 57, 180, 371, 644, or 665 ppm) 

for 90–114 days. Fifteen guinea pigs, three rabbits, two dogs, and three monkeys were also 

exposed continuously under similar conditions to ammonia at 40 mg/m3

 (57 ppm) or 470 mg/m3

(665 ppm). No significant effects were reported in any animals exposed to 40 mg/m3 and 

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

rats exposed to 127 mg/m3 (180 ppm) ammonia. Exposure to 262 mg/m3 (371 ppm) caused 

nasal discharge in 25% of the rats and nonspecific circulatory and degenerative changes in the 

lungs and kidneys (not further described, incidence not reported). A frank effect level at 

455 mg/m3 (644 ppm) was observed due to high mortality in the rats (50/51). Thirty-two of 

51 rats died by day 25 of exposure; no histopathological examinations were conducted in these 

rats. Exposure to 470 mg/m3 (665 ppm) caused death in 13/15 rats and 4/15 guinea pigs and 

marked eye irritation in dogs and rabbits. Dogs experienced heavy lacrimation and nasal 

discharge, and corneal opacity was noted in rabbits. Hematological values did not differ 

significantly from controls in animals exposed to 470 mg/m3

 (665 ppm) ammonia. 

Histopathological evaluation of animals exposed to 470 mg/m3 (665 ppm) consistently showed 

focal or diffuse interstitial pneumonitis in all animals and also alterations in the kidneys 

(calcification and proliferation of tubular epithelium), heart (myocardial fibrosis), and liver (fatty

change) in several animals of each species (incidence not reported). The study authors did not 

determine a NOAEL or LOAEL concentration from this study. EPA identified a NOAEL of 

40 mg/m3

 (57 ppm) and a LOAEL of 127 mg/m3 (180 ppm) based on nonspecific inflammatory 

changes in the lungs and kidneys in rats exposed to ammonia for 90 days. 
A-48 DRAFT – DO NOT CITE OR QUOTE 

Stombaugh et al. (1969) 

Stombaugh et al. (1969) exposed groups of Duroc pigs (9/group) to measured 

concentrations of 12, 61, 103, or 145 ppm ammonia (8, 43, 73, or 103 mg/m3

) continuously for 

5 weeks (anhydrous ammonia, purity not reported). Endpoints evaluated included clinical signs, 

food consumption (measured 3 times/week), weight gain (measured weekly), and gross and 

microscopic examination of the respiratory tract at termination. A control group was not 

included. In general, exposure to ammonia reduced food consumption and body weight gain, but 

since a control group was not used, it is impossible to determine whether this reduction was 

statistically significant. Food efficiency (food consumed/kg body weight gain) was not affected. 

Exposure to ≥103 ppm (>73 mg/m3

) ammonia appeared to cause excessive nasal, lacrimal, and 

mouth secretions and increased the frequency of cough (incidence data for these effects were not 

reported). Examination of the respiratory tract did not reveal any significant exposure-related 

alterations. The study authors did not identify a NOAEL or LOAEL concentration from this 

study. EPA did not identify a NOAEL or LOAEL value for this study due to the absence of a 

control group. 

Doig and Willoughby (1971) 

Doig and Willoughby (1971) exposed groups of six specific-pathogen-free derived 

Yorkshire Landrace pigs to 0 or 100 ppm ammonia (0 or 71 mg/m3) continuously for up to 

6 weeks. The mean concentration of ammonia in the control chamber was 8 ppm (6 mg/m3). 

Additional groups of pigs were exposed to similar levels of ammonia as well as to 0.3 mg/ft3 of 

ground corn dust to simulate conditions on commercial farms. Pigs were monitored daily for 

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

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

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

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

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

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

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

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

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

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

to more than 150 ppm (106 mg/m3) on two occasions. Doig and Willoughby (1971) reported 

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

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

pathology were comparable between exposed and control pigs. Histopathology revealed 

epithelial thickening in the trachea of exposed pigs and a corresponding decrease in the numbers 
A-49 DRAFT – DO NOT CITE OR QUOTE 

of goblet cells as shown in Table A-13. Tracheal thickening was characterized by thinning and 

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

bronchi and bronchioles characterized as lymphocytic cuffing were comparable between exposed 

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

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

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

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

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

Table A-13. Summary of histological changes observed in rats exposed to 

ammonia for 6 weeks 

Duration of exposure (wks)

Thickness of tracheal epithelium (μm) 

Number of tracheal goblet cells (per 

500 μm) 

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

1 15.7 21.0 13.6 24.0 

2 20.4 29.3 22.7 10.3 

3 20.4 36.6 18.9 7.3 

4 21.8 36.2 18.3 10.7 

5 19.3 33.2 20.2 10.0 

6 18.9 41.6 20.0 1.3 

Mean ± SD 19.4 ± 2.1 32.9 ± 7.2 18.9 ± 3.0 10.6 ± 7.5 

Source: Doig and Willoughby (1971). 

Doig and Willoughby (1971) concluded that ammonia exposure at 71 mg/m3 may be 

detrimental to young pigs. The authors suggested that although the structural damage to the 

upper respiratory epithelium was slight, such changes may cause severe functional impairment. 

The study authors did not identify a NOAEL or LOAEL concentration from this study. EPA 

identified a LOAEL of 71 mg/m3 (100 ppm), based on damage to the upper respiratory 

epithelium. A NOAEL could not be identified from this single-concentration study. 

Broderson et al. (1976) 

Broderson et al. (1976) exposed groups of Sherman rats (5/sex/dose) continuously to 

10 or 150 ppm ammonia (7 or 106 mg/m3, respectively) for 75 days (anhydrous ammonia, purity 

not reported). The 10-ppm exposure level represented the background ammonia concentration 

resulting from cage bedding that was changed 3 times/week. The 150-ppm concentration 

resulted from cage bedding that was replaced occasionally, but never completely changed. F344 

rats (6/sex/group) were exposed to ammonia in an inhalation chamber at concentrations of 0 or 

250 ppm (177 mg/m3

) continuously for 35 days. Rats were sacrificed at the end of the exposure 
A-50 DRAFT – DO NOT CITE OR QUOTE 

period, and tissues were prepared for histopathological examination of nasal passages, middle 

ear, trachea, lungs, liver, kidneys, adrenal, pancreas, testicle, mediastinal lymph nodes, and 

spleen. 

Histopathological changes were observed in the nasal passage of rats exposed to 150 ppm 

(106 mg/m3) for 75 days (from bedding) or 250 ppm (177 mg/m3) for 35 days (inhalation 

chamber). Nasal lesions were most extensive in the anterior portions of the nose compared with 

posterior sections of the nasal cavity. The respiratory and olfactory mucosa was similarly 

affected with a three- to fourfold increase in the thickness of the epithelium. Pyknotic nuclei and 

eosinophilic cytoplasm were observed in epithelial cells located along the basement membrane. 

Epithelial cell hyperplasia and formation of glandular crypts were observed and neutrophils were 

located in the epithelial layer, the lumina of submucosal glands, and the nasal passages. Dilation 

of small blood vessels and edema were observed in the submucosa of affected areas. Collagen 

replacement of submucosal glands and the presence of lymphocytes and neutrophils were also 

observed. No histopathological alterations were seen in control rats (10 ppm from bedding or 

0 ppm from the inhalation chamber). Broderson et al. (1976) did not identify a NOAEL or 

LOAEL from this study. EPA identified a NOAEL of 10 ppm (7 mg/m3) and a LOAEL of 

150 ppm (106 mg/m3) based on nasal lesions in rats exposed to ammonia (from bedding) for 

75 days. Broderson et al. (1976) also studied the effect of ammonia inhalation on the incidence 

and severity of murine respiratory plasmosis in mice inoculated with Mycoplasma pulmonis. 

These results are presented in Section 4.4.3. 

 

Gaafar et al. (1992) 

 Gaafar et al. (1992) exposed 50 adult male white albino mice under unspecified 

conditions to ammonia vapor derived from a 12% ammonia solution (air concentrations were not 

reported) 15 minutes/day, 6 days/week for up to 8 weeks. Twenty-five additional mice served as 

controls. Starting the fourth week, 10 exposed and 5 control mice were sacrificed weekly. 

Following sacrifice, the nasal mucosa was removed and examined histologically. Frozen 

sections of the nasal mucosa were subjected to histochemical analysis (succinic dehydrogenase, 

nonspecific estrase, acid phosphatase, and alkaline phosphatase [ALP]). Histological 

examination revealed a progression of changes in the nasal mucosa from exposed rats from the 

formation of crypts and irregular cell arrangements at 4 and 5 weeks, epithelial hyperplasia, 

patches of squamous metaplasia, and loss of cilia at 6 weeks, and dysplasia in the nasal 

epithelium at 7 weeks. Similar changes were exaggerated in the nasal mucosa from rats 

sacrificed at 8 weeks. Neoplastic changes included a carcinoma in situ in the nostril of one rat 

sacrificed at 7 weeks that presented with loss of polarity of the epithelium, hyperchromatism and 

mitotic figures with an intact basement membrane, and an invasive adenocarcinoma in one rat 

sacrificed at 8 weeks. Histochemical results revealed changes in succinic dehydrogenase, acid 
A-51 DRAFT – DO NOT CITE OR QUOTE 

phosphatase, and ALP in exposed mice compared to controls (magnitude of change not 

reported), especially in areas of the epithelium characterized by dysplasia. Succinic 

dehydrogenase and acid phosphatase changes were largest in the superficial layer of the 

epithelium, although the acid phosphatase reaction was stronger in the basal and intermediate 

layers in areas of squamous metaplasia. ALP was strongest in the goblet cells from the basal part 

of the epithelium and basement membrane. 

 In summary, Gaafar et al. (1992) observed that ammonia exposure induces histological 

changes in the nasal mucosa of male mice that increase in severity over longer exposure periods. 

Corresponding abnormalities in histochemistry suggest altered cell metabolism and energy 

production, cell injury, cell proliferation, and possible chronic inflammation and neoplastic 

transformation. The study authors did not determine a NOAEL or LOAEL concentration from 

this study. EPA did not identify a NOAEL or LOAEL because air concentrations were not 

reported in the study. 

Done et al. (2005) 

 Done et al. (2005) continuously exposed groups of 24 weaned pigs of several breeds in an 

experimental facility to atmospheric ammonia at 0, 0.6, 10, 18.8, or 37 ppm (0, 0.4, 7, 13.3, or 

26 mg/m3

) and 1.2, 2.7, 5.1, or 9.9 mg/m3 inhalable dust for 5 weeks (16 treatment 

combinations). The concentrations of ammonia and dust used were representative of those found 

commercially. A split-plot design was used in which one dust concentration was allocated to a 

“batch” (which involved five lots of 24 pigs each) and the four ammonia concentrations were 

allocated to the four lots within that batch. The fifth lot served as a control. Each batch was 

replicated. In other words, there were four dust concentrations × four ammonia concentrations 

plus four controls each replicated once, giving 40 lots in total. In total, 960 pigs (460 males and 

500 females) were used in the study; 560 pigs were given postmortem examinations. Blood was 

collected from 15 sows before the start of the experiment and tested for porcine reproductive and 

respiratory syndrome virus and swine influenza. Five sentinel pigs were sacrificed at the start of 

each batch and lung, nasal cavity, and trachea, together with material from any lesions, were 

examined postmortem and subjected to bacteriological examination. Postmortem examination 

involved examining the pigs’ external surfaces for condition and abnormalities, examination of 

the abdomen for peritonitis and lymph node size, internal gross examination of the stomach for 

abnormalities, and gross examination of the nasal turbinates, thorax, larynx, trachea, 

tracheobronchial lymph nodes, and lung. Pigs were monitored for clinical signs (daily), growth 

rate, feed consumption, and feed conversion efficiency (frequency of observations not specified). 

After 37 days of exposure, eight pigs from each lot were sacrificed. Swabs of the nasal cavity 

and trachea were taken immediately after death for microbiological analysis, and the pigs were 

grossly examined postmortem. On day 42, the remaining pigs were removed from the exposure 
A-52 DRAFT – DO NOT CITE OR QUOTE 

facility and transferred to a naturally ventilated building for a recovery period of 2 weeks. Six 

pigs from each lot were assessed for evidence of recovery and the remaining 10 pigs were 

sacrificed and examined postmortem. 

 The pigs in this study demonstrated signs of respiratory infection and disease common to 

young pigs raised on a commercial farm (Done et al., 2005). The different concentrations of 

ammonia and dust did not have a significant effect on the pathological findings in pigs or on the 

incidence of pathogens. In summary, exposure to ammonia and inhalable dust at concentrations 

commonly found at pig farms was not associated with increase in the incidence of respiratory or 

other disease. The study authors did not identify a NOAEL or LOAEL concentration from this 

study. EPA identified a NOAEL of 26 mg/m3 (37 ppm), based on the lack of respiratory or other 

disease following exposure to ammonia in the presence of respirable dust. 

 

Weatherby (1952) 

 Weatherby (1952) exposed a group of 12 guinea pigs (strain not reported) to a target 

concentration of 170 ppm (120 mg/m3) 6 hours/day, 5 days/week for up to 18 weeks (anhydrous 

ammonia, purity not reported). The actual concentration measured in the exposure chamber 

varied between 140 (99 mg/m3

) and 200 ppm (141 mg/m3). A control group of six guinea pigs 

was exposed to room air. All animals were weighed weekly. Interim sacrifices were conducted 

at intervals of 6 weeks (four exposed and two control guinea pigs) and the heart, lungs, liver, 

stomach and small intestine, spleen, kidneys, and adrenal glands were removed for microscopic 

examination; the upper respiratory tract was not examined. No exposure-related effects were 

observed in guinea pigs sacrificed after 6 or 12 weeks of exposure. However, guinea pigs 

exposed to ammonia for 18 weeks showed considerable congestion of the spleen, liver, and 

kidneys, and early degenerative changes in the adrenal gland. The most severe changes occurred 

in the spleen and the least severe changes occurred in the liver. The spleen of exposed guinea 

pigs contained a large amount of hemosiderin and kidney tubules showed cloudy swelling with 

precipitated albumin in the lumens and some urinary casts (cylindrical structures indicative of 

disease). The incidence of histopathological lesions was not reported. EPA identified the 

ammonia concentration of 170 ppm (120 mg/m3

) to be a LOAEL based on congestion of the 

spleen, liver, and kidneys and early degenerative changes in the adrenal gland. A NOAEL could 

not be identified in this single-concentration study. 

 

Curtis et al. (1975) 

 Curtis et al. (1975) exposed groups of crossbred pigs (4–8/group) to 0, 50, or 75 ppm 

ammonia (0, 35, or 53 mg/m3) continuously for up to 109 days (anhydrous ammonia, >99.9% 

pure). Endpoints evaluated included clinical signs and body weight gain. At termination, all 

pigs were subjected to a complete gross examination and representative tissues from the 
A-53 DRAFT – DO NOT CITE OR QUOTE 

respiratory tract, the eye and its associated structures, and the visceral organs (not specified) 

were taken for subsequent microscopic examination. Weight gain was not significantly affected 

by exposure to ammonia and the results of the evaluations of tissues and organs were 

unremarkable. The turbinates, trachea, and lungs of all pigs were classified as normal. The 

study authors did not identify a NOAEL or LOAEL from this study. EPA identified a NOAEL 

of 75 ppm (53 mg/m3) based on the absence of effects occurring in pigs exposed to ammonia; a 

LOAEL was not identified from this study. 

Reproductive/Developmental Studies 

Diekman et al. (1993) 

 Diekman et al. (1993) reared 80 crossbred gilts (young female pigs) in a conventional 

grower from 2 to 4.5 months of age; pigs were exposed naturally during that time to Mycoplasma 

hypopneumoniae and Pasteurella multocida, which causes pneumonia and atrophic rhinitis, 

respectively. At 4.5 months of age, the pigs were transferred to environmentally regulated rooms 

where they were exposed continuously to a mean concentration of ammonia of 7 ppm (range, 4–

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

32 mg/m3) for 6 weeks (Diekman et al., 1993). A control group was not included in this study. 

The low concentration of ammonia was obtained by the flushing of manure pits weekly and the 

higher concentration of ammonia was maintained by adding anhydrous ammonia (purity not 

reported) to manure pits that were not flushed. After 6 weeks of exposure, 20 gilts from each 

group were sacrificed and sections of the lungs and snout were examined for gross lesions. In 

addition, the ovaries, uterus, and adrenal glands were weighed. The remaining 20 gilts/group 

were mated with mature boars and continued being exposed to ammonia until gestation day 30, 

at which time they were sacrificed. Fetuses were examined for viability, weight, and length, and 

the number of copora lutea were counted. 

Gilts exposed to 35 ppm (25 mg/m3

) ammonia gained less weight than gilts exposed to 

7 ppm (5 mg/m3

) during the first 2 weeks of exposure (7% decrease, p < 0.01), but growth rate 

recovered thereafter. Mean scores for lesions in the lungs and snout were not statistically 

different between the two exposure groups and there were no differences in the weight of the 

ovaries, uterus, and adrenals. Age at puberty did not differ significantly between the two groups, 

but gilts exposed to 35 ppm (25 mg/m3) ammonia weighed 7% less (p < 0.05) at puberty than 

those exposed to 7 ppm (5 mg/m3). In gilts that were mated, conception rates were similar 

between the two groups (94.1 versus 100% in low versus high exposure). At sacrifice on day 30 

of gestation, body weights were not significantly different between the two groups. In addition, 

there were no significant differences between the two groups regarding percentage lung tissue 

with lesions and mean snout grade. Number of corpora lutea, number of live fetuses, and weight 

and length of the fetuses on day 30 of gestation were not significantly different between 
A-54 DRAFT – DO NOT CITE OR QUOTE 

treatment groups. Diekman et al. (1993) did not identify NOAEL or LOAEL concentrations for 

maternal or fetal effects in this study. The EPA did not identify NOAEL or LOAEL values from 

this study due to the absence of a control group and due to confounding by exposure to bacterial 

and mycoplasm pathogens. 

Acute and Short-term Inhalation Toxicity Studies 

(see Table A-14) 
A-55 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Rats

Female Porton rats (16/group) 

0 or 141 (0 or 200 ppm) 

Continuous exposure for 4, 8, or 12 d 

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

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

Male OFA rats (27/group) 

0 or 354 (0 or 500 ppm) 

Continuous exposure for 1–8 wks 

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

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

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

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

10, 20, 40, or 60 min 

Clinical signs, pathology, LC50

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

3 (40,300 ppm) 

20-min LC50 = 20,217 mg/m

3 (28, 595 ppm) 

40-min LC50 = 14,352 mg/m

3 (20,300 ppm) 

60-min LC50 = 11,736 mg/m

3 (16,600 ppm) 

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

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

24 hrs Clinical signs, histology, 

blood pH, blood gas measurement 

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

pO2

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

3, 17, 31, 117, and 505 (4, 24, 44, 165, 

and 714 ppm); arterial blood collected prior to exposure served as control 

3 and 7 d Hepatic cytochrome P450 

content and ethylmorphineN-demethylase activity 

No dose-related change in P450 content or enzyme activity 
A-56 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Male Long-Evans rats (4/group) 

70 and 212 (100 and 300 ppm); results were compared to “control”, but it was not clear if the authors were referring to historical or concurrent controls

6 hrs Clinical signs, behavioral 

observation 

Decreased running, decreased activity 

Female Wistar rats (5/group) 

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

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

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

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

(300 ppm); lung hemorrhage observed in soexposed rats 

Female Wistar rats (5/group) 

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

6 hrs/d for 5 d Plasma and brain 

ammonia and amino acid analysis 

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

Female albino rats (8/group) 

0, 848–1,068 (1,200–1,510 ppm) 

3 hrs Mortality, respiratory 

movement, and O2

consumption 

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

Male SpragueDawley rats (number/group not given) 

Air concentration not 

given; ammonia vapor added to inspiratory line of ventilator; controls exposed to same volume of room air 

20 sec Activity of upper thoracic 

spinal neurons 

Lower airway irritation, activation of vagal pulmonary afferents and upper thoracic spinneurons receiving pulmonary sympathetic in
A-57 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Male Wistar rats (4/group) 

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

45 min Airway reflexes by the 

changes in respiratory patterns elicited by ammonia in either dry, steam-humidified, or aqueous aerosol containing atmospheres 

Ammonia-induced upper respiratory tract sensory irritation is not affected to any appreciable extent by wet atmospheres (witor without aerosol) up to 1,243 mg/m

3

Male rats (10/group) 

0, 848–1,068 (0, 1,200–1,510 ppm) at 

the beginning and end of the exposure period) 

3 hrs Oxygen consumption Decreased O2 consumption 

Mice

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

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

10 min LC50 LC50 = 7,056 mg/m

3

(9,980 ppm) 

Male Swiss albino mice (4/group) 

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

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

3 (21,430 ppm) 

Albino mice (sex not specified; 6/dose) 

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

Continuously for 2 or 5 d 

Regional brain metabolism (cerebral cortex, cerebellum, brainstem); MAO, enzymes of glutamate and GABA metabolism, and (Na

+-K

+)-

ATPase; amino acid levels in the brain 

Altered activities of MAO, glutamate decarboxylase, ALT, GABA-transaminase, an(Na

+-K

+)-ATPase; increased alanine and 

decreased glutamate 
A-58 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Male Swiss-Webster mice (4/group) 

Concentrations not given; baseline levels established prior to exposure 

10 min Reflex decrease in 

respiratory rate was used as an index of sensory irritation; RD50 = the 

concentration associated 

with a 50% decrease in the respiratory rate 

RD50 = 214 mg/m

3

(303 ppm) 

Male albino ICR mice (12/dose) 

0–3,436 (0–4,860 ppm) 

1 hr (14-d followup) 

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

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

3 (4,230 ppm) 

Male Swiss-Webster mice (16–24/group)

0 or 216 (0 or 305 ppm) 

6 hrs/d for 5 d Respiratory tract 

histopathology 

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

trachea or lungs 

Male albino ICR mice (12/dose) 

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

4 hrs Hexobarbitol sleeping 

time, microsomal protein content, liver microsomal enzyme activity 

Increased hexobarbitol sleeping time (3,097 mg/m

3), increased microsomal prote

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

3) 

Male albino ICR mice (12/dose) 

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

4 hrs/d for 4 d Microsomal protein 

content, liver microsomal enzyme activity 

No dose-dependent effects on microsomal enzymes 

Male Swiss mice (6/dose) 

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

6 hrs Clinical signs, behavioral 

observation 

Decreased running, decreased activity 
A-59 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

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

56, or 110 ppm), lowest measured 

concentration was the nominal control group

2 d Responses to atmospheric 

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

No distinguishable preference for, or aversioto, different NH3 concentrations 

Male OF1 mice (4/group) 

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

45 min Airway reflexes by the 

changes in respiratory patterns elicited by ammonia in either dry, steam-humidified, or aqueous aerosol containing atmospheres 

Ammonia-induced upper respiratory tract sensory irritation is not affected to any appreciable extent by wet atmospheres (witor without aerosol) up to 1,243 mg/m

3

Rabbits

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

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

2.5–3.0 hrs Lung function Decreased respiratory rate at both 

concentrations 

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

0, 707–14,140 (0, 1,000–20,000 ppm) 

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

3 (2,500 ppm); 

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

3

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

(6,000 ppm)

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

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

3

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

4 min Lung function, heart rate, 

blood pressure, blood gases 

Lung injury was evident after 2–3 min (decreased pO2 increased airway pressure) 
A-60 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

CatsMixed breed stray cats (5/group) 

0 or 707 (0 or 1,000 ppm) 

10 min Lung function, lung 

histopathology on 1, 7, 21, and 35 d postexposure 

Lung function deficits were correlated with 

histopathology; acute effects were followed chronic respiratory dysfunction (secondary bronchitis, bronchiolitis, and bronchopneumonia) 

Pigs

Young pigs (2/group) 

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

Continuous exposure for 4 wks 

Clinical signs, food consumption, body weight, gross necropsy, organ weight, histopathology 

Lethargy and histopathological alterations in the tracheal and nasal epithelium were observed at 71 and 106 mg/m

3; decreased b

weight occurred at all concentrations (7–19% decrease from control) 

Male and female Belgian Landrace pigs (4/group) 

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

6 d Clinical signs, body weight, 

lung function 

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

Belgian Landrace pigs (4/group) 

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

6 d Clinical signs, body weight, 

neutrophil count, and albumin in nasal lavage fluid 

Nasal irritation (increased neutrophils in naslavage fluid) and decreased body weight gaiall concentrations 

Landrace-Yorkshire pigs (4/group) 

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

8 wks 

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

Significant increases in TXA2 and LTC4, no significant effect on PGI2 production 

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

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

15 wks Salivary cortisol, adrenal 

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

Decreased salivary cortisol, larger adrenal cortices, less play behavior, no measurable impact on productivity or physiological parameters 
A-61 DRAFT – DO NO

Table A-14. Acute and short-term inhalation toxicity studies of ammonia in animals 

Animal 

Concentration 

(mg/m

3) Duration 

Parameter 

examined Results 

Cattle

Male Holestein calves (number/group not specified) 

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

2.5 hrs Respiration rate, clinical 

chemistry 

No significant effect on respiration, BUN, pHpO2, or pCO2

Brahman/Charolais (group size not reported) 

<6 (control), 11, 23, or 34 (<8 [control], 16, 32, or 48 ppm) 

12 d Behavioral activity, body 

weight, analysis of bronchioalveolar lavage (BAL) fluid, hematological variables (hemoglobin, mean cell volume, platelet volume, eosinophils, neutrophils, total white cell count, monocytes) 

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

Holstein Friesian and Brown Swiss (10 of each breed) 

~0, 4, and 15, (0.3 × 10-6, 6, and 

21 ppm) 

10 d at each concentration 

Respiration and pulse rate, blood gas parameters 

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

Table A-15. Summary of in vitro studies of ammonia genotoxicity 

End point Test system Concentrationa

Resultsb

Comments Reference 

Without 

activation 

With 

activation 

Genotoxicity studies in prokaryotic organisms 

Reverse 

mutation 

S. typhimurium 

(TA98, TA100, 

TA1535, 

TA1537, 

TA1538); E. coli 

(WP2 uvrA) 

25,000 ppm 

(17,675 mg/m3) 

ammonia vapor 

– –

c

Plate 

incorporation 

assay with 

ammonia vapor 

Shimizu et 

al., 1985 

Reverse 

mutation, 

streptomycin 

resistance 

E. coli (B/SD-4 

strains) 

0.25% ammonia + (T)d No data Plate 

incorporation 

assay 

Demerec et 

al., 1951 

Genotoxicity studies in nonmammalian eukaryotic organisms 

Chromosomal 

aberrations 

Chick 

fibroblasts 

Not available + No data Cultures 

immersed in 

buffered 

ammonia 

solution 

Rosenfeld, 

1932 

a

Lowest effective dose for positive results; highest dose tested for negative or equivocal results. 

b

+ = positive; − = negative; (T) = toxicity reported. 

c

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

pentachlorobiphenyl (KC500). 

d

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

Table A-16. Summary of in vivo studies of ammonia genotoxicity 

End point Test system 

Dose/ 

concentrationa

 Resultsb Comments Reference 

Genotoxicity studies in mammalian systems in vivo

Chromosomal 

aberrations 

Human 

lymphocytes 

88.28 μg/m3 +c 22 healthy workers 

occupationally exposed 

to ammonia in an Indian 

fertilizer factory (ambient 

concentration of 

0.0883 mg/m3); 42 

nonexposed factory staff 

served as control 

subjects 

Yadav and 

Kaushik, 1997 

Sister chromatid 

exchange 

Human 

lymphocytes 

88.28 μg/m3 +c Yadav and 

Kaushik, 1997 
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Table A-16. Summary of in vivo studies of ammonia genotoxicity 

End point Test system 

Dose/ 

concentrationa Resultsb Comments Reference 

Micronucleus 

formation 

Swiss albino mice 12.5–50 mg/kg + Intraperitoneal injections 

for 24−48 hr expression 

times 

Yadav and 

Kaushik, 1997 

Sex-linked 

recessive lethal 

mutations 

Drosophila 

melanogaster 

Not available – (T) Inhalation exposure to 

ammonia as vapor at a 

concentration killing the 

majority of flies 

Auerbach and 

Robson, 1947 

Dominant lethal 

mutations 

D. melanogaster Not available – (T) Inhalation exposure to 

ammonia as vapor at a 

concentration killing the 

majority of flies 

Auerbach and 

Robson, 1947 

Dominant lethal 

mutations 

D. melanogaster Not available + (T)d Dominant lethal assay; 

inhalation exposure up to 

318 mg/m3

 ammonia, 6 

hrs/d for 5 d 

Lobasov and 

Smirnov, 1934 

a

Lowest effective dose for positive results; highest dose tested for negative or equivocal results. 

b

+ = positive; − = negative; (T) = toxicity reported. 

c

Frequencies of chromosomal aberrations, sister chromatid exchanges, and mitotic index all increased with 

increased duration of exposure. This study is difficult to interpret because of small samples sizes and confounding 

by smoking and alcohol consumption. In addition, the levels of ammonia in the plant seemed low compared to 

other fertilizer plant studies (see, for example, Section 1.1; Rahman et al., 2007; Ali, 2001; Ballal et al., 1998); the 

accuracy and reliability of the sampling and measurement could not be determined. 

d

Survival after exposure was <2%. 

 
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APPENDIX B: OTHER AGENCY AND 

INTERNATIONAL ASSESSMENTS

 Toxicity values and other health-related regulatory limits developed by government 

agencies and expert organizations are summarized in Table B-1. 

Table B-1. Other Agency Assessments 

Agency Toxicity value

Agency for Toxic Substances and 

Disease Registry (ATSDR, 2004) 

Chronic inhalation miminal risk levels (MRL) = 0.1 ppm (0.07 mg/m3), based 

on lack of significant alterations in lung function in chronically exposed 

workers (Holness et al., 1989) and a composite uncertainty factor (UF) of 30 

(10 for human variability and a modifying factor of 3 for the lack of 

reproductive and developmental studies) 

National Advisory Committee 

for Acute Exposure Guideline 

Levels for Hazardous Substances 

(NRC, 2007) 

AEGL-1 (nondisabling) – 30 ppm (21 mg/m3) for exposures ranging from 10 

minutes to 8 hours to protect against mild irritation, based on mild irritation 

in human subjects 

AEGL-2 (disabling) – 220 ppm (154 mg/m3) for a 10-minute exposure to 110 

ppm (77 mg/m3

) for an 8-hour exposure, based on irritation in human 

subjects 

AEGL-3 (lethal) – 2,700 ppm (1,888 mg/m3) for a 10-minute exposure to 390 

ppm (273 mg/m3

) for an 8-hour exposure, based on lethality in the mouse 

American Conference of 

Governmental Industrial 

Hygienists (ACGIH, as cited in 

NIOSH, 2002) 

Threshold Limit Value (TLV) – 25 ppm (17 mg/m3) time weighted average 

(TWA) for an 8-hour workday and a 40-hour work week

National Institue of 

Occupational Safety and Health 

(NIOSH, 2011) 

Recommended Exposure Limit (REL) – 25 ppm (18 mg/m3) TWA for up to a 10-

hour workday and a 40-hour work week 

Occupational Safety and Health 

(OSHA, 2011) 

Permissible Exposure Limit (PEL) for general industry – 50 ppm (35 mg/m3) 

TWA for an 8-hour workday 

Food and Drug Admistration 

(FDA, 2011a, b) 

Ammonium hydroxide: direct food substance affirmed as generally recognized 

as safe (21 CFR 184.1139); substance generally recognized as safe when used 

in accordance with good manufacturing or feeding practices (21 CFR 

582.1139) 

 
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APPENDIX C: SUMMARY OF EXTERNAL PEER 

REVIEW AND PUBLIC COMMENTS AND 

DISPOSITION

To be added