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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 ar e 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. Thu s, a decrease in pH would result in an

increase in the ammonium ion concentration and an i ncrease in solubility of ammonia in water. At physiological pH (7.4), the equilibrium between NH 3 

### and NH 4 + 

### favors the formation of NH 4 +



### . Chemical and physical properties of ammonia are lis ted in Table A-1.

Table A-1. Chemical and physical properties of amm onia

Chemical name Ammonia a

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 NH 3 ChemID Plus, 2009

CASRN 7664-41-7 a 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/m 3 ) <20 ppm (14 mg/m 3 ) 2.6 ppm (2 mg/m 3 ) 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

pK a (ammonium ion) 9.25 Lide, 2008 Solubility:

Water Organic solvents 4.82 × 10 5 mg/L at 24°C Soluble in ethanol, chloroform, and ether Dean, 1985 Lide, 2008; O’Neil et al., 2006

Vapor pressure 7.51 × 10 3 mm Hg at 25°C AIChE, 1999 Henry’s law constant 1.61 × 10 - 5 atm-m 3 /mol at 25°C Betterton, 1992

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Table A-1. Chemical and physical properties of amm onia

Conversion factors ppm to mg/m 3 mg/m 3 to ppm 1 ppm = 0.707 mg/m 3 1 mg/m 3 = 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 includ es studies of ammonium chloride (3-, 18-, and 30-month dietary studies in male and femal e 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 a nd female F344 rats [Ota et al., 2006]). No inhalation toxicity studies of ammonium salts are a vailable. Ammonium chloride in the diet or drinking water of rats consistently altered the aci d-base balance in the body (Lina and Kuijpers, 2004; Barzel et al., 1969) causing a dose-related h yperchloremic metabolic acidosis in rats as evidenced by decreases in blood pH, base excess, an d bicarbonate concentration, and increased plasma chloride levels. Ammonium chloride administ ered in the diet for 3, 18 or 30 months was also associated with zona glomerulosa hypertrophy o f the adrenal gland (Lina and Kuijpers, 2004). In contrast, dietary administration of ammo nium sulfate to rats did not induce metabolic acidosis at 52 weeks (Ota et al., 2006) or histopat hologic changes in the adrenal gland at 104 weeks (Ota et al., 2006). The only dose-related ef fects 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 chr onic nephropathy was statistically significantly increased in low-dose, but not in hig h-dose male rats (Ota et al., 2006). See Table A-2 for study details. The toxicity data for ammonium chloride and ammoniu m sulfate demonstrate that these two salts present distinctly different toxicity profile s, suggesting that the anion can influence the toxicity of the ammonium compound, and that the tox icity of the salts cannot necessarily be attributed to the cation (i.e., NH 4 + 

### ) only. Accordingly, information on the toxicity o f ammonium salts was not used to characterize the toxicity of ammonia or ammonium hydroxide in this

assessment.A-3 DRAFT – DO NOT CITE OR QUOTETable A-2. Summary of repeat dose studies of selected ammonium salts following oral exposureStrain/species/sexAmmoniaspeciesDose (mg/kgd)/ durationNOAEL(mg/kg-d)LOAEL(mg/kg-d) Effects at the LOAEL Comment ReferenceSubchronic studiesWistar rat(10/sex/group)Ammoniumchloride0, 1,590, or3,050 (males);0, 1,800, or3,700 (females)for 13 wks(administeredin diet)Not determined1,590 (males)1,800 (females)Decreased body weights (6-17% in males; 11-19% infemales), changes in serumchemistry (increasedplasma chloride and ALPactivity), increased relativekidney weights (both doselevels, 7-28%) and adrenalweights (high-dose males,18%), metabolic acidosis(males and females) andsubsequent hypertrophy ofthe adrenal zonaglomerulosa (males only)Lina and Kuijpers, 2004Chronic studiesSprague-Dawleyrat(11 males/group)Ammoniumchloride0 or 1,800 for330 d(administeredin drinkingwater)Not determined1,800Depression in body weights(13-20% with regular andlow-calcium diets,respectively), metabolicacidosis, and loss of bone(femur) tissueMetabolic acidosis (reducedblood pH and plasma carbondioxide).Combined effects of calciumintake and observed that theloss of bone tissue wasindependent of the level ofdietary calcium.Barzel et al., 1969A-4 DRAFT – DO NOT CITE OR QUOTETable A-2. Summary of repeat dose studies of selected ammonium salts following oral exposureStrain/species/sexAmmoniaspeciesDose (mg/kgd)/ durationNOAEL(mg/kg-d)LOAEL(mg/kg-d) Effects at the LOAEL Comment ReferenceWistar rat(15/sex/group)Ammoniumchloride0, 481, or1,020 (males);0, 610, or 1,370(females) for18 moNot determined481 (males)610 (females)Metabolic acidosis Femur weight significantlyincreased (~17%) in high-dosemales. Adrenal and kidneyweights elevated (<15%).Increased incidence of zonaglomerulosa hypertrophy ofthe adrenal at high dose (notstatistically significant).Severity of metabolic acidosisincreased with dose.Lina and Kuijpers, 2004Wistar rat(50/sex/group)Ammoniumchloride0, 455, or1,000 (males);0, 551, or 1,200(females) for30 moNot determined455 (males)551 (females)Metabolic acidosis and anincreased incidence ofhypertrophy of the adrenalglomerulosa (males only)Increased incidence of zonaglomerulosa hypertrophy ofthe adrenal in high-dosefemales; attributed to chronicstimulation of the adrenalcortex by ammonium chlorideinduced acidosis. There wasno evidence of a carcinogenicresponse.Lina and Kuijpers, 2004F344 rat(10/sex/group)Ammoniumsulfate0, 42, 256, or1,527 (males);0, 48, 284, or1,490 (females)for 52 wks(administeredin diet)256 (males)284 (females)1,527 (males)1,490 (females)Elevated relative liver andkidney weights (7-10%)No significant effects onhematology, serumchemistry, or histopathology.Ota et al., 2006A-5 DRAFT – DO NOT CITE OR QUOTETable A-2. Summary of repeat dose studies of selected ammonium salts following oral exposureStrain/species/sexAmmoniaspeciesDose (mg/kgd)/ durationNOAEL(mg/kg-d)LOAEL(mg/kg-d) Effects at the LOAEL Comment ReferenceF344 rat(50/sex/group)Ammoniumsulfate0, 564, or 1,288(males); 0, 650,or 1,371(females) for104 wks(administeredin diet)1,288 (males)1,371 (females)Not determinedClinical chemistry andhematology not evaluated;organ weights not measured.Incidence of chronicnephropathy in male ratsincreased over control (1/48,5/49, 3/48 in the control, midand high dose); increase wasstatistically significant only atthe mid-dose and not doserelated. There was noevidence of a carcinogenicresponse.Ota et al., 2006

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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, al though absorption through the eye has been

documented. Most of the inhaled ammonia is retaine d in the upper respiratory tract and is subsequently eliminated in expired air. Ammonia th at reaches systemic circulation is widely distributed to all body compartments, although subs tantial first-pass metabolism occurs in the liver, where biotransformation into urea and glutam ine occurs. Ammonia exists in the blood as ammonium ion (NH 4 + 

### ). Ammonia is transported in the circulatory syste m 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 io n, which is synthesized into urea and

excreted in the urine. Ammonia or ammonium ion rea ching the tissues is utilized for glutamate production, which participates in transamination an d other reactions. The principal means of

excretion of absorbed ammonia in mammals is as urin ary urea; minimal amounts are excreted in the feces and in expired air. Ammonia is endogenously produced in humans and ani mals. It is an essential mammalian metabolite used in nucleic acid and prote in synthesis, is necessary for maintaining acid-base balance, and is an integral part of nitro gen homeostasis. Given its important metabolic role, ammonia exists in a homeostatically regulated equilibrium in the body.



## Absorption

Inhalation Exposure Experiments with volunteers 1 

### show that ammonia, regardless of its tested concen tration in air (range, 57–500 ppm or 40–354 mg/m 3 

### ), is almost completely retained in the nasal mucos a (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/m 3 

### ) resulted in lower retention (4–30%), with expired breath concentrations of 350– 400 ppm (247–283 mg/m 3 

### ) observed by the end of the exposure period (Silve rman et al., 1949), suggesting saturation of absorption into the nasal mucosa. Nasal and pharyngeal irritation, but not tracheal irritation, suggests that ammonia is r etained in the upper respiratory tract. Unchanged levels of blood urea nitrogen (BUN), nonp rotein nitrogen, urinary urea, and urinary ammonia following these acute exposures are evidenc e of low absorption into the blood.

1 The human toxicokinetic studies cited in this secti on did not provide information on the human subject s’ research ethics procedures undertaken in the studies; howeve r, there is no evidence that the conduct of the res earch 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/m 3 

### ), assuming 30% uptake into blood, would yield an increase in b lood ammonia concentration of 0.09 μg/mL (calculated by WHO, 1986). This calculated rise wo uld 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 eviden ce for high-percentage nasal retention, resulting in a lower fraction of the inhaled dose r eaching the lower respiratory tract (Egle, 1973; Dalhamn, 1963; Boyd et al., 1944). Continuous expo sure of rats to up to 32 ppm (23 mg/m 3 

### ) for 24 hours did not result in a statistically signific ant increase in blood ammonia levels (0.1 μg/mL above preexposure levels), whereas exposures to 310 –1,157 ppm (219–818 mg/m 3 

### ) led to significantly increased blood concentrations of amm onia 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 ammon ia (ammonium hydroxide) provide evidence of oral absorption, but few quantitative d ata are available. For example, in a fatal case

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

the contents of the stomach and blood showed ammoni um ion levels of 15.3 mg in the stomach and 33 μg/mL in the blood (Klendshoj and Rejent, 19 66). 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 compo unds 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. Evid ence suggests that fractional absorption of ammonia increases as the lumen pH increases, and th at 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 ga strointestinal 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 t he 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 (vomitin g, renal congestion, and delirium), suggesting

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dermal absorption; however, the fractional dose fro m dermal exposure could not be determined (Slot, 1938). WHO (1986) concluded that systemic e ffects from skin and eye exposure are not quantitatively important. Ammonia is readily absor bed into the eye, and it was found to diffuse within seconds into the cornea, lens, drainage syst em, 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 arteria l blood and 0.2–1.7 μg/mL in venous blood (Huizenga et al., 1994). Other baseline levels obs erved in experimental volunteers range from 1 to 5.5 μg/mL (Conn, 1972; Brown et al., 1957). A mmonia is homeostatically regulated to remain at low concentrations, with 95–98% existing in the blood (at physiological pH) as NH 4 + 

### 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 ammoni a 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., 19 80; Holzman et al., 1979, 1977; Rubaltelli and Formentin, 1968; Luschinsky, 1951). J όź wik 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’ ve nous blood. DeSanto et al. (1993) similarly collected human umbilical arterial and ve nous blood at delivery, and found that umbilical arterial ammonia concentrations were sign ificantly 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 t issues are the main site of ammonia production, with outputs of ammonia into both the uterine and u mbilical circulations (J όź wik et al., 1999). In late-gestation pregnant sheep that were catheter ized 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 (J όź wik 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 lite rature for distribution of inhaled ammonia. Information on the distribution of endoge nously produced ammonia suggests that any ammonia absorbed through inhalation would be distri buted 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/m 3 

### ) ammonia for 6 hours/day for 15 days exhibited inc reased blood ammonia (200%) and brain (28%) glutamine leve ls at 5 days of exposure, but not at 10 or 15 days (Manninen et al., 1988), demonstrating tran sient distribution of ammonia to the brain (metabolic adaptation).

Oral Exposure Human oral exposure data indicate that ammonia rea dily 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-i onized ammonia is freely diffusible, whereas the ammonium ion is less so, and is relatively conf ined to the extracellular compartment (Stabenau et al., 1958).

Dermal Exposure No quantitative data on distribution of ammonia fr om 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 cort ex, 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 l iterature. Ammonia and ammonium ion are metabolized to glutamine mainly in the liver via gl utamine 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., 19 61). 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 doe s 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 compartment alization of its steps within liver cells. Glutamate

γ -Glutamyl phosphate glutamine synthetase

glutamine synthetase 

### Glutamine glutaminase (in liver mitochondria) NH 4 +

H 2 O ATP

ADP

NH 4 + P i

Carbamoyl phosphate synthase I H 2 O 2 ATP

2 ADP P i 3 H + CO 2 + NH 4 +

P i

Citrulline

Ornithine

Aspartate

Argininosuccinate Arginine H 2 O Urea Ornithine Ornithine transcarbamoylase

Citrulline Carbamoyl phosphate

Argininosuccinate

synthase

Argininosuccinate lyase Arginase M ITOCHONDRIAL

M ATRIX

Fumarate ATP

AMP + PP i

C YTOSOL

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Given its important metabolic role, ammonia exists in a homeostatically regulated equilibrium in the body. In particular, free ammon ia 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 NH 4 + 

### ion (da Fonseca-Wolhheim, 1995; Souba, 1987). Two studies in rats (Manninen et al., 1988; Schaerdel et al., 1983) pro vided evidence that ammonia concentrations in air below 25 ppm (18 mg/m 3 

### ) do not alter blood ammonia concentrations. Schae rdel et al. (1983) exposed rats to ammonia for 24 hours at conc entrations ranging from 15 to 1,157 ppm (11–818 mg/m 3 

### ). Exposure to 15 ppm (11 mg/m 3 

### ) 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 ammo nia metabolism through conversion to urea, pyrimidine and polyamine synthesis, incorpora tion into amino acid substrates, and metabolism in nervous system tissue. Rats inhaling 25 ppm (18 mg/m 3 

### ) ammonia for 6 hours/day for 15 days did not exhibit blood or br ain ammonia or glutamine levels that were different from controls; however, rats inhaling 300 ppm (212 mg/m 3 

### ) for the same duration exhibited statistically significantly increased lev els 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 g lutamine 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 glut amine uptake and catabolism and, thereby, affect the blood and tissue levels of ammo nia. Abnormally elevated levels of ammonia are indicative of end-stage renal failure (Davies e t al., 1997). Acute renal failure can result in increased renal glutamine consumption and ammonia p roduction with a decreased capability of eliminating urea in the urine (Souba, 1987). End-s tage liver failure due to fulminant hepatitis or hepatic cirrhosis may result in decreased ureagenes is 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 NH 4 + 

### /NH 3 

### ratio in the direction of ammonia, which could pas s through the blood-brain barrier (Katayama, 2004). In patients with liver cirrhosis and acute clinical hepatic encephalopathy, the observed trapping of [ 13 

### N]-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) an d is a component of sweat (Guyton, 1981; Wands, 1981). Lee and colleagues observed that acidosis-stimulated renal excretion o f ammonia is mediated by intercalated cell-specific Rh B glyc oprotein expression in mice (Bishop et al., 2010; Lee et al., 2010, 2009). In rat kidney, ammonium ion is secreted into the lu men of the outer medullary collecting duct via H + 

### secretion and parallels ammonia diffusion (Flessne r et al., 1992). The inner medullary collecting duct exhibit s a Na + 

### - and K + 

### -independent NH 4 + 

### /H + 

### exchange activity that may be mediated by an Rh C g lycoprotein (Handlogten et al., 2005), which is also expressed in human kidneys (Han et al ., 2006). Additionally, ammonia is known to be excreted thro ugh expired air and is present in the expired air of all humans (Manolis, 1983). Two inv estigators specifically measured ammonia in breath exhaled from the nose (Smith et al., 2008; L arson et al., 1977). Smith et al. (2008) reported median ammonia concentrations in exhaled b reath from the nose of three healthy volunteers (with samples collected daily over a 4-w eek period) of 0.059–0.078 mg/m 3 

### ; these concentrations were similar to, or slightly higher than, the mean laboratory air level of ammonia of 0.056 mg/m 3 

### reported in this study. Larson et al. (1977) repo rted that the median concentration of ammonia collected from air samples exhaled from the nose ranged from 0.013 to 0.046 mg/m 3 

### . One sample collected from the trachea (via a tub e inserted through the nose of one subject) was 0.029 mg/mg 3 

### —a concentration within the range of concentrations in breath exhaled through the nose (Larson et al., 197 7). Higher and more variable ammonia concentrations ar e 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, th e majority of ammonia concentrations ranged from 0.085 to 2.1 mg/m 3 

### (Smith et al., 2008; Spanel et al., 2007a, b; Turn er et al., 2006; Diskin et al., 2003; Smith et al., 1999; Norwood et al., 1 992; Larson et al., 1977). These higher concentrations are largely attributed to the produc tion 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 am monia in breath was demonstrated by Smith et al. (1999), who observed elevated ammonia concen trations in the expired air of six healthy volunteers following the ingestion of a protein-ric h meal.

Other factors that can affect ammonia levels in br eath exhaled from the mouth or oral cavity include diet, oral hygiene, age, living cond itions, and disease state. Norwood et al. (1992) reported decreases in baseline ammonia levels (0.08 5–0.905 mg/m 3 

### ) 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 consi stent with ammonia generation in the oral

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cavity by bacterial and/or enzymatic activity. Sev eral 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 a verage, by about 0.1 mg/m 3 

### 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. Ce rtain disease states can also influence ammonia levels in exhaled breath. Ammonia is great ly elevated in the breath of patients in renal failure (Spanel et al., 2007b; Davies, 1997). Incr eases in breath ammonia are associated with increasing age (Diskin et al., 2003). These studie s are further described below in Table A-2.A-14 DRAFT – DO NOT CITE OR QUOTETable A-3. Ammonia levels in exhaled breath of volunteersTest subjects Breath samples Levels of ammonia in exhaled breathMethods Comments ReferenceBreath samples from the nose and tracheaThree healthy malevolunteers (>30 yrs ofage)Ammonia levels measured innose-exhaled breath of testsubjects each morning about 2hrs after eating a regularbreakfast; samples collecteddaily over a 4-wk periodVolunteer A = 103 ± 1.2 ppb (0.0728 ±0.000848 mg/m3)Volunteer B = 110 ±1.3 ppb(0.0777 ± 0.000919 mg/m3)Volunteer C = 83 ± 1.2 ppb(0.0587 ± 0.000848 mg/m3)(median ammonia levels estimated asgeometric mean ± geometric SD)SIFT-MS analysisMean ambient air level ofammonia was 80 ± 10 ppb(0.056 ± 0.0071 mg/m3)The authors indicated thatammonia measured in mouthexhaled breath may begenerated in the oral cavity andsuggested that concentrationsin nose-exhaled breath maybetter represent systemicconditions (such as metabolicdisease)Smith et al.,2008Sixteen healthysubjects (9 males aged25–63 yrs and 7females aged 23–41 yrs); subgroupstested were all maleBreath samples collectedduring quiet nose breathing,and direct sampling during adeep inspiration followed bybreath-holding with the glottisclosedAmmonia concentrations ranged from0.013 to 0.046 mg/m3during nosebreathing (median 0.025 mg/m3)(5 male subjects), and 0.029 mg/m3from an air sampled collected from thetrachea (collected from a tube insertedinto one male subject’s nose and intothe trachea)ChemiluminescenceLarson etal., 1977A-15 DRAFT – DO NOT CITE OR QUOTETable A-3. Ammonia levels in exhaled breath of volunteersTest subjects Breath samples Levels of ammonia in exhaled breathMethods Comments ReferenceBreath samples from the mouth and oral cavityThree healthy malevolunteers (>30 yrs ofage)Ammonia levels measured inmouth-exhaled breath and inthe closed mouth cavity of testsubjects each morning about 2hrs after eating a regularbreakfast; samples collecteddaily over a 4-wk periodVia mouth:Volunteer A = 1,088 ± 1.3 ppb (0.769 ±0.000919 mg/m3)Volunteer B = 885 ± 1.3 ppb(0.626 ± 0.000919 mg/m3)Volunteer C = 855 ± 1.3 ppb(0.604 ± 0.000919 mg/m3)Via oral cavity:Volunteer A = 1,465 ± 1.4 ppb(1.04 ± 0.000990 mg/m3)Volunteer B = 2,146 ± 1.5 ppb(1.52 ± 0.00106 mg/m3)Volunteer C = 1,859 ± 1.3 ppb(1.31 ± 0.000919 mg/m3)(median ammonia levels estimated asgeometric mean ± geometric SD)SIFT-MS analysisMean ambient air level ofammonia was 80 ± 10 ppb(0.056 ± 0.0071 mg/m3)The authors indicated thatammonia measured in mouthexhaled breath may begenerated in the oral cavity andsuggested that concentrationsin nose-exhaled breath maybetter represent systemicconditions (such as metabolicdisease)Smith et al.,2008Twenty-six secondaryschool students (10males and 16 females,17–18 yrs old and one19 yr old)Three sequential breathexhalations collected over 5min following the studentslistening to a 1-hr presentation(at least 1 hr followingbreakfast and before lunch);alveolar portion measured(identified using humidity)Median values reported for:17 yr olds = 233 ppb (0.165 mg/m3)18 yr olds = 346 ppb (0.245 mg/m3)SIFT-MS analysisSignificant differences inammonia levels in exhaledbreath between 17 and 18 yrolds (p < 10-8) were reportedSpanel etal., 2007aA-16 DRAFT – DO NOT CITE OR QUOTETable A-3. Ammonia levels in exhaled breath of volunteersTest subjects Breath samples Levels of ammonia in exhaled breathMethods Comments ReferenceFour healthy children(two males and twofemales, 4–6 yrs old)Thirteen seniorvolunteers (11 malesand 2 females, 60–83yrs old); four had type2 diabetes mellitus withonset at ages between50 and 70 yrs, andcontrolled by dietAll subjects had theirregular breakfastwithout any specificrestrictionsBreath samples collected inmorning at least 1 hr afterbreakfast and at least 1 hrprior to lunch; each volunteerperformed twoexhalation/inhalation cycles(both about 5–10 sec induration)Children = range 223–643 ppb (0.157–0.454 mg/m3)Seniors = 317–2,091 ppb (0.224–1.48 mg/m3)SIFT-MS analysisAmmonia breath levelssignificantly increased with ageSome seniors reported diabetesMeasured ammonia level inbreath reported for eachsubjectSpanel etal., 2007bThirty healthyvolunteers (19 malesand 11 females, 24–59yrs, 28 Caucasian,1 African, and 1 mixedrace); volunteers wereinstructed to maintaintheir normal dailyroutines and to notrinse out their mouthsprior to providing abreath sampleBreath samples collected in themorning prior to lunch atapproximately weekly intervalsfor about 6 mo; somevolunteers provided samplesmore frequently than others;480 samples collected andanalyzed for ammoniaGeometric mean and geometricSD = 833 ± 1.62 ppb (0.589 ± 0.00114mg/m3)Median = 842 ppb (0.595 mg/m3)Range = 248–2,935 ppb (0.175–2.08 mg/m3)SIFT-MS analysisAmmonia breath levels wereshown to increase with ageBackground levels in the testinglaboratory were typicallyaround 400 ppb (0.28 mg/m3)Turner etal., 2006A-17 DRAFT – DO NOT CITE OR QUOTETable A-3. Ammonia levels in exhaled breath of volunteersTest subjects Breath samples Levels of ammonia in exhaled breathMethods Comments ReferenceFive subjects (twofemales, three males;age range 27–65 yrs)Breath samples collectedbetween 8 and 9 AM in threesequential breath exhalationson multiple days (12–30 d)over the course of a monthAmmonia concentrations ranged from422 to 2,389 ppb (0.298–1.69 mg/m3)SIFT-MS analysisDifferences in ammonia breathlevels between individuals weresignificant (p < 0.001)Diskin et al.,2003Six normal nonsmokingmale volunteers (24–61 yrs old), fasted for12 hrs prior to testingBaseline breath sampleobtained; breath samplescollected 20, 40, and 60 minand 5 hrs following theingestion of a liquid proteincalorie mealPremeal levels ranged from 300 to600 ppb (0.2–0.4 mg/m3);Postmeal levels at 30 min were200 ppb (0.1 mg/m3) increasing tomaximum values at 5 hrs of 600–1,800 ppb (0.4–1.3 mg/m3)SIFT-MS analysis A biphasic response in breathammonia concentration wasobserved after eatingSmith et al.,1999Fourteen healthy,nonsmoking subjects(age range 21–54 yrs)performed one or moreof the followinghygiene maneuvers:(1) acidic oral rinse(pH 2.5)(2) tooth brushingfollowed by acidic oralrinse(3) tooth brushingfollowed by distilledwater rinse(4) distilled water rinseSubjects fasted for 8 hrs priorto baseline measurement,refrained from oral hygieneafter their most recent meal,refrained from heavy exercisefor 12 hrs, and had no liquidintake for several hours; initialbreath ammonia wasmeasured between 8 and 10am, then subjects performedone or more of the hygienemeasures listed (at 30-minintervals for a total 90-minperiod; samples collected over5 min)Baseline levels varied from 120 to1,280 ppb (0.085–0.905 mg/m3)Nitrogen oxideanalyzer with anammoniaconversion channel(similar to chemiluminescence)An 80–90% depletion of volatileammonia emissions was seenwithin 10 min of acid rinsing;<50% depletion of ammoniawas seen following toothbrushing or distilled water rinse;gaseous ammonia levelsincreased after all rinseprocedures over timeNorwood etal., 1992A-18 DRAFT – DO NOT CITE OR QUOTETable A-3. Ammonia levels in exhaled breath of volunteersTest subjects Breath samples Levels of ammonia in exhaled breathMethods Comments ReferenceSixteen healthysubjects (9 males aged25–63 yrs and 7females aged 23–41 yrs); subgroupstested were all maleBreath samples collectedduring quiet mouth breathingAmmonia concentrations ranged from0.029 to 0.52 mg/m3during mouthbreathing (median of 0.17 mg/m3)ChemiluminescenceThe oral cavity appears to be asource of breath ammonia; noattempt was made to controlthe diet of subjects orstandardize the intervalbetween the last meal and themeasurementLarson etal., 1977Breath samples: source (nose/mouth/oral cavity) notspecifiedSixteen healthy,nonsmoking subjects(4 females and12 males, 29 ± 7 yrs);no significantdifferences in meanage, height, weight,BMI, or time since lastoral intake; 10 subjectstested in eachexperimentExperiment 1: single wholebreath samples collected fromeach subject (same samplesimmediately reanalyzed within<10 sec to assess instrumentspecific variability)Experiment 2: three repeatbreath samples collected fromeach subject (to evaluate intrasubject differences); thisexperiment evaluateddifferences based onstandardization of expiratorypressure and flowExperiment 3: two mixedbreath samples and two bagalveolar breath samplescollected in short successionfrom each subjectExperiment 1: 1,192 ± 85 ppb(0.843 ± 0.0601 mg/m3;median ± measurement error)Experiment 2:Nonstandardized = 1,007 ± 184 ppb(0.712 ± 0.130 mg/m3; median ± SD)Standardized = 1,433 ± 160 ppb(1.01 ± 0.113 mg/m3; median ± SD)Experiment 3:Mixed = 1,216 ± 827 ppb(0.860 ± 0.585 mg/m3; median ± SD)Alveolar = 1,301 ± 791 ppb(0.920 ± 0.559 mg/m3; median ± SD)SIFT-MS analysisThis studyestablishes thatSIFT-MS analysis isreliable andrepeatableRelatively small number ofhealthy subjects usedDoes not address the breath ofthose with diseaseIntra- and inter-dayrepeatability were notinvestigatedBoshier etal., 2010A-19 DRAFT – DO NOT CITE OR QUOTETable A-3. Ammonia levels in exhaled breath of volunteersTest subjects Breath samples Levels of ammonia in exhaled breathMethods Comments ReferenceEight healthy subjects(average age39.8 ± 9.6 yrs)Subjects fasted for 6 hrs priorto samples being collected;subjects breathed normallyinto collection device for 5 minMean breath ammonia =0.49 ± 0.24 ppm (0.35 ± 0.17 mg/m3)Fiber optic sensor This study measured ammonialevels in healthy volunteerscompared to H. pylori positiveindividuals (five subjects) (datanot shown); the experimentalso included a challenge with a300 mg urea capsule to evaluatethe urease activity of healthyversus infected individuals (datanot shown); the authorsconcluded that breath ammoniameasurement may be feasibleas a diagnostic test for H. pyloriKearney etal., 2002Three groups ofchildren were used astest subjects:(1) 68 asthmaticchildren residing in aNational Park in themountains (mean age10 yrs, 48 boys, 20 girls)(2) 52 asthmaticchildren in an urbanarea (mean age 9 yrs,35 boys, 17 girls)(3) 20 healthy childrenfrom the same urbanarea as a control group(mean age 10 yrs, 12boys, 8 girls)Subjects performed a 5-secbreath-hold and exhaledslowly into collection deviceAsthmatic children from National Park= 5.6 ± 4.7 ppb(0.0040 ± 0.0033 mg/m3)Asthmatic urban children:Mean NH3= 14.3 ± 10.2 ppb (0.0101 ±0.00721 mg/m3)Urban control group:Mean NH3= 14.8 ± 10.3 ppb (0.0105 ±0.00728 mg/m3)ChemiluminescenceBoth groups of asthmaticchildren had some subjects onglucocorticoids, often combinedwith histamine antagonistsand/or b2 agonists, while otherswere left untreated; ammoniaconcentrations in exhaledbreath appeared to becorrelated with exposure tourban airGiroux etal., 2002BMI = body mass index; SIFT-MS = selected ion flowtube mass spectrometry

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Because ammonia measured in samples of breath exhal ed from the mouth or oral cavity can be generated in the oral cavity and may thus be substa ntially influenced by diet and other factors, ammonia levels measured in mouth or oral cavity bre ath 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 (Smi th 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 end ogenous ammonia levels in the expired air of rats showed concentrations ranging from 0.01 to 0.353 ppm (0.007–0.250 mg/m 3 

### ) (mean = 0.08 ppm or 0.06 mg/m 3 

### ) in nose-breathing animals (Barrow and Steinhagen, 1980). Larson et al. (1980) reported ammonia concentration s measured in the larynx of dogs exposed to sulfuric acid ranging between 0.03 and 0.225 ppm (0 .02 and 0.16 mg/m 3 

### ) following mouth breathing, and between 0.05 and 0.220 ppm (0.04 and 0.16 mg/m 3 

### ) following nose breathing.



## Physiologically Based Pharmacokinetic Models

No physiologically based pharmacokinetic (PBPK) mo dels 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 mode l is not useful for dose-response assessment of ammonia because: (1) it cannot speci fy time-dependent amounts or concentrations of ammonia in specific target tissues, (2) it has n ot 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 Populatio ns Holness et al. (1989) Holness et al. (1989) conducted a cross-sectional study of workers in a soda ash (sodium carbonate) plant 2 

### who had chronic, low-level exposure to ammonia. T he cohort consisted of

58 workers and 31 controls from stores and office a reas 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 (TW A) ammonia exposure of the exposed group based on personal sampling over one work shif t (mean sample collection time, 8.4 hours) was 9.2 ppm (6.5 mg/m 3 

### ), compared to 0.3 ppm (0.2 mg/m 3 

### ) for the control group. The average

2 At this plant, ammonia, carbon dioxide, and water w ere the reactants used to form ammonium bicarbonate , which in turn was reacted with salt to produce sodium bic arbonate and subsequently processed to form sodium carbonate. Ammonia and carbon dioxide were recoved in the proc ess and reused.

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concentrations of ammonia to which workers were exp osed were determined using the procedure recommended by the National Institute for Occupatio nal 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 obse rved in age, height, years worked, percentage of smokers, or pack-years smoked for exp osed versus control workers. Exposed workers weighed approximately 8% ( p < 0.05) more than control workers. Information regarding past occupational exposures, working cond itions, and medical and smoking history, as well as respiratory symptoms and eye and skin compl aints was obtained by means of a questionnaire that was based on an American Thoraci c 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 1 0 ppm). Lung function tests were conducted at the beginning and end of the work shift on the firs t and last days of their work week (four tests administered). Differences in reported symptoms an d 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 wer e grouped into three exposure categories (high = >12.5 ppm [>8.8 mg/m 3 

### ], medium = 6.25–12.5 ppm [4.4–8.8 mg/m 3 

### ], and low = <6.25 ppm [<4.4 mg/m 3 

### ]) for analysis of symptom reporting and lung funct ion data. Endpoints evaluated in the study included sense of smell, prevalence of respiratory symptoms (cough, bronchitis, wheeze, dyspnea, and others), e ye and throat irritation, skin problems, and lung function parameters (FVC, FEV 1 

### , FEV 1 

### /FVC, forced expiratory flow [FEF 50 

### ], and FEF 75 

### ). No statistical differences in the prevalence of res piratory 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 work ers in the lowest exposure category (<4.4 mg/m 3 

### ) compared to controls; however, the prevalence was not increased among workers in the two higher exposure groups. Workers also re ported that exposure at the plant had aggravated specific symptoms including coughing, wh eezing, nasal complaints, eye irritation, throat discomfort, and skin problems. Odor detection threshold and baseline lung functio ns were similar in the exposed and control groups. No chan ges in lung function were demonstrated over either work shift (days 1 or 2) or over the work we ek 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 accoun ting for both level and length of exposure. Based on the lack of exposure-related differences i n subjective symptomatology, sense of smell, and measures of lung function, EPA identified 8.8 m g/m 3 

### (12.5 ppm) as the no-observed-

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adverse-effect level (NOAEL). A lowest-observed-ad verse-effect level (LOAEL) was not identified for this study.

Table A-4. Symptoms and lung function results of w orkers exposed to different levels of TWA ammonia concentrations

Parameter Ammonia concentration

Control 0.2 mg/m 3 (0.3 ppm) Exposed

<4.4 mg/m 3 (<6.25 ppm) Exposed

4.4–8.8 mg/m 3 (6.25–12.5 ppm) Exposed

>8.8 mg/m 3 (>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) 10/34 b (29) 1/12 (8) 1/12 (8) Lung function (% predicted)

FVC 98.6 96.7 96.9 96.8

FEV 1 95.1 93.7 93.9 95.3

FEF 50 108.4 106.9 106.2 111.2

FEF 75 65.2 71.0 67.8 78.8

a Number affected/number examined. The percentage of workers reporting symptoms is indicated in parenth eses. 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 s tudy of male workers at two urea

fertilizer factories in Saudi Arabia 3 

### . 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/m 3 

### (2.8–184.4 ppm), and workers in factory B were exposed to 0.02–7 mg/m 3 

### (0.03–9.9 ppm). Mean duration of employment was 5 1.8 months for exposed workers and 73.1 months for controls. Expo sure levels were estimated by analyzing a total of 97 air samples collected over 8-hour shift s close to the employee’s work site. The

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

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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. S moking habits were similar for exposed workers and controls. Stratifying the workers by a mmonia exposure levels (above or below the American Conference of Governmental Industrial Hygi enists [ACGIH] threshold limit value [TLV] of 18 mg/m 3 

### [25 ppm]) showed that those exposed to ammonia con centrations 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 a mmonia levels at or below the TLV. Distribution of symptoms by cumulative ammonia conc entration (CAC, mg/m 3 

### -years) also showed significantly higher relative risk for all o f the above symptoms among those with higher CAC (Table A-4). Results of the logistic regressio n analysis showed that ammonia concentration was significantly related to cough, p hlegm, 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/m 3 of air-yrs)

≤ACGIH TLV (18 mg/m 3 ) (n = 138) >ACGIH TLV

(18 mg/m 3 ) (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 (0.6 6–2.17) 2.59 (1.25–5.36) Chronic bronchitis 1.43 (0.49–4.19) 2.32 (0.31–17.2 8) 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 (0.31–10.7 7) 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 rel ationship between ammonia concentration and respiratory symptoms or disease i n 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 indi ces measured in 73 ammonia workers and 343

control workers included FEV 1 

### and FVC. Prediction equations for these indices w ere 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% in crease, p ≤ 0.002); however, workers with cumulative exposure ≥ 50 mg/m 3 

### -years had significantly lower FEV 1 

### % predicted (7.4% decrease,

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

exposure ≤ 50 mg/m 3 

### -years. A comparison between symptomatic and asymp tomatic exposed

workers showed that FEV 1 

### % predicted and FEV 1 

### /FVC% were significantly lower among symptomatic workers (9.2% decrease in FEV 1 

### % predicted, p < 0.001 and 4.6% decrease in

FEV 1 

### /FVC%, p < 0.02). Although Ballal et al. (1998) and Ali (2 001) suggest that exposure to ammonia concentrations >18 mg/m 3 

### (50 mg/m 3 

### -years) is associated with respiratory irritation and altered lung function, NOAEL and LOAEL values c ould not be identified by EPA from

these studies due to inadequate reporting of exposure concentrati ons.

Rahman et al. (2007) Rahman et al. (2007) conducted a cross-sectional s tudy 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 ex posed workers per day for 10 morning shifts

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in the urea plant (for a total of 64 workers) and i n five to nine exposed workers per day for 4 morning shifts from the ammonia plant (for a tota l of 24 workers). Four to seven volunteer workers per day were selected from the administrati on building as controls for a total of 25 workers over a 5-day period. Questionnaires wer e administered to inquire about demographics, past chronic respiratory disease, pas t and present occupational history, smoking status, respiratory symptoms (cough, chest tightnes s, runny nose, stuffy nose, and sneezing), and use of protective devices. Lung function tests (FV C, FEV 1 

### , 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 expo sure 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 c ross-shift change in FEV 1 

### ( ∆ FEV 1 

### %) while adjusting for current smoking. Mean exposure levels at the ammonia plant determin ed by the Dräger tube and Dräger PAC III methods were 25.0 and 6.9 ppm (17.7 and 4.9 mg/m 3 

### ), respectively; the corresponding means in the urea plant were 124.6 and 26.1 ppm (88 .1 and 18.5 mg/m 3 

### ) (Rahman et al., 2007). Although the Dräger tube measurements indicated amm onia exposure about 4–5 times higher than those obtained with the PAC III instrument, th ere 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 (concentr ations less than the measuring range of 2.5– 200 ppm [1.8–141 mg/m 3 

### ]). Based on an evaluation of the two monitoring m ethods and communication with technical support at Dräger 4 

### , EPA considered the PAC III instrument to be a more sensitive monitoring technology than the Drä ger tubes. Therefore, the PAC III air

measurements were considered the more reliable meas urement of exposure to ammonia for the Rahman et al. (2007) study. The study authors, how ever, observed that their measurements indicated only relative differences in exposures be tween 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 decre ased lung function was higher in the urea plant than in the ammonia plant or in the admi nistration building. Comparison between the urea plant and the administration building showed t hat cough and chest tightness were statistically higher in the former; a similar compa rison of the ammonia plant and the administration building showed no statistical diffe rence in symptom prevalence between the two groups (Table A-7). Preshift measurement of FVC, F EV 1 

### , and PEFR did not differ between urea plant and ammonia plant workers. Significant cross -shift reductions in FVC and FEV 1 

### 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., Tec hnical Support Detection Products to Amber Bacom, SRC, Inc . [contractor to National Center for Environmental Assessment, Office of Research and Development, U.S . EPA].

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When controlled for current smoking, a significant decrease in ∆ FEV 1 

### % was observed in the urea plant (

p ≤ 0.05). Among 23 workers with concurrent measureme nts of ammonia and lung function on the same shift, ammonia exposure was co rrelated with a cross-shift decline in FEV 1 

### of 3.9% per unit of log-transformed ammonia concent ration in ppm. EPA identified a NOAEL of 6.9 ppm (4.9 mg/m 3 

### ) and a LOAEL of 26.1 ppm (18.5 mg/m 3 

### ) 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/m 3 ) a Urea plant (18.5 mg/m 3 ) 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 ch ange) e,f

FVC 0.2 ± 9.3 -2.3 ± 8.8 No data

FEV 1 3.4 ± 13.3 -1.4 ± 8.9 No data

PEFR 2.9 ± 11.1 -1.0 ± 16.2 No data

a Mean ammonia concentrations measured by the Dräger PAC III method.

b Concentrations in the administration building were rejected by study authors due to relatively large d rift in the zero levels.

c Values are presented as incidence (prevalence expres sed as a percentage).

d p ≤ 0.05 by Fisher’s exact test, comparing exposed w orkers to administrators.

e Calculated as ([post shift-preshift]/preshift) × 10 0.

f Values are presented as mean ± standard deviation (S D).

Source: Rahman et al. (2007).



### Hamid and El-Gazzar (1996) Hamid and El-Gazzar (1996) evaluated changes in se rum clinical chemistry as measures

of neurochemical alterations and liver function amo ng 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 ammoni a and 30 workers from the administrative departments with no known history of exposure to am monia. The authors indicated that the exposed population had worked at the fertilizer pla nt on average for 12 years. The exposed and reference populations were matched on demographic c haracteristics including age, educational

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status, and socioeconomic status. No information w as reported on exposure levels. Blood samples were collected from each subject and analyz ed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), hemoglobin, and blo od urea levels, and for monoamine oxidase (MAO) and catalase activity. Table A-8 shows stati stically 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 o f hemoglobin were significantly lower, and MAO and catalase enzyme activities were significant ly depressed among exposed workers compared to controls. A correlation analysis showe d a positive correlation between catalase activity and levels of hemoglobin, AST, and ALT, an d MAO activities. Hamid and El-Gazaar (1996) noted that inhibition of catalase can affect electrical stability, permeability, and fluidity o f membranes, which may lead to hepatotoxic and neurot oxic alterations in occupationally exposed

workers. NOAEL and LOAEL values were not identifie d 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 p lant

Parameter Controls a Exposed a ALT (U/mL) 16.0 ± 5.59 19.4 ± 5.69 b

AST (U/mL) 14.5 ± 4.67 17.9 ± 4.14 b

Hemoglobin (%) 14.8 ± 2.62 12.2 ± 2.29 c Blood urea (mg/mL) 0.203 ± 0.0512 0.319 ± 0.0755 c

MAO (units) 31.9 ± 10.1 20.8 ± 4.30 c Catalase (IU/mL) 119.3 ± 4.76 80.9 ± 9.31 c

a Mean ± 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 Inhal ed Ammonia Several studies have evaluated respiratory symptom s and changes in lung function in livestock farmers and stable workers exposed to amm onia (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 volatile s 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 ammo nia, these and other studies have also demonstrated respiratory effects associated with ex posure to other constituents in farm worker air (e.g., respirable dust, endotoxin).

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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

FEV 1 

### , FVC) in farmers was associated with ammonia expos ure 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 expose d 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 (PE F) variability as well as increased pulmonary inflammation related to allergies (Elfman et al., 2 009). 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) antib odies 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 f armers 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.

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

Subjects Methods Exposure conditions Results Refere nce 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/m 3 ; endotoxin = 24 ng/m 3

, and ammonia = 5.60 mg/m 3 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/m 3 ) and mean dust concentrations ranged from approximately 2 to 21 mg/m 3 Respiratory symptoms included chest tightness, wheeze, nasal and eye irritation (23/29 farm workers); 3/29 farm workers had impaired lung function (decreased FEV 1 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) whoworked 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/m 3 ; mean airborne ammonia concentration of 8.5 mg/m 3 ; mean personal ammonia exposure of 3.23 mg/m 3 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

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

Subjects Methods Exposure conditions Results Refere nce

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/m 3 ); total dust = 2.93 mg/m 3 ; respirable dust = 0.13 mg/m 3 ; endotoxin = 11,332 units/m 3 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/m 3 ; respirable dust = 0.23 mg/m 3 ; total endotoxin = 202.35 EU/m 3 ; respirable endotoxin = 16.59 EU/m 3 ; ammonia = 5.64 ppm (4 mg/m 3 ) 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/m 3 ) were predictive of a ≥3% decrease

in FEV 1 ; 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/m 3

, endotoxin = 112 ng/m 3

, ammonia = 2 mg/m 3 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

FEV 1 , MMEF, and PEF) Preller et al.,

1995

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

Subjects Methods Exposure conditions Results Refere nce 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/m 3 ; respirable dust = 0.26 mg/m 3 ; total endotoxin = 176.12 EU/m 3 ; respirable endotoxin = 11.86 EU/m 3 ; ammonia = 5.15 ppm (4 mg/m 3 ) Swine workers had a mean cross-shift 2% decrease in

FEV 1 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/m 3 , endotoxin = 111 ng/m 3 , ammonia = 2 mg/m 3 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/m 3 , endotoxin = 105 ng/m 3 , ammonia = 2 mg/m 3 Decreased lung function (FEV 1 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/m 3 ); also exposed to airborne dust, bacteria, endotoxin, and molds Decreased expiratory flows (FEV 1 ), increased neutrophils in the nasal wash, and increased white blood cell

count Cormier et al.,

2000

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

Subjects Methods Exposure conditions Results Refere nce 257 poultry workers (30% women, 70% men); 63 w omen 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/m 3 ); total dust = 6.5 mg/m 3 ; respirable dust = 0.63 mg/m 3 ; total endotoxin 1,589 EU/m 3 (0.16 μg/m 3 ); respirable endotoxin = 58.9 EU/m 3 (0.006 μg/m 3 ) 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/m 3 ), 2.4 mg/m 3 total dust, 0.16 mg/m 3 respirable dust, and 614 EU/m 3 endotoxin (0.614 μg/m 3 ) 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/m 3 ) over the 12 tasks; total dust (0.4– 5.1 mg/m 3 ), fungal spores (0.02–

2.0 10 6 /m 3 ), bacteria (0.2–48 10 6 /m 3 ), endotoxin (0.5– 28/10 3 EU/m 3 [0.05– 2.8 μg/m 3 ) 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/m 3 ) in late summer, but was not detected in winter; levels of endotoxin were highest during late summer (15 ng/m 3 ) while levels of 1,3-β-glucan (85 ng/m 3 ) and horse allergen (18,300 U/m 3 ) 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 midexpi ratory flow



## Controlled Human Inhalation Exposure Studies

Controlled exposure studies conducted in volunteer s to evaluate irritation effects and changes in lung function following acute inhalation exposure to ammonia are summarized in

Table A-10.

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Table A-10. Controlled human exposure studies of a mmonia inhalation

Subjects Exposure conditions Results Reference

Seven male volunteers 500 ppm (354 mg/m 3 ) for

30 min from masked breathing apparatus for nose and throat inhalation; there was no mention of preexposure examinations Hyperventilation (50–250% increase above controls) characterized by increased breathing rate and expiratory minute volume (i.e., volume of air exhaled in 1 min); no coughing was induced, excessive lacrimation occurred in two subjects; two subjects reported nose and throat irritation that lasted 24 hrs after exposure; no changes were reported in nitrogen metabolism or in blood or urine urea, ammonia, or nonprotein nitrogen Silverman et al., 1949 b

Seven male volunteers with an average age of 31 yrs 30, 50, and 90 ppm (21, 35,

and 64 mg/m 3 ) for 10 min in an inhalation chamber; physical and neurological examinations were conducted prior to

exposure Increased eye erythema at 90 ppm (64 mg/m 3 ) compared to 30 and 50 ppm (21 and 35 mg/m 3 ) exposure; 90 ppm (64 mg/m 3 ) 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/m 3 ) than at 90 ppm (64 mg/m 3 ) MacEwen et al., 1970 a

18 healthy servicemen volunteers, 18– 39 yrs old 50–344 mg/m 3 (70–486 ppm)

for a half-day (session day 2); sessions on days 1 and 3 acted as controls; all volunteers underwent a preliminary examination prior to exposure No effect at concentrations of 71 mg/m 3 (100 ppm); reduced expiratory minute volume at concentrations ranging from 106 to 235 mg/m 3 (150–332 ppm) compared to controls (not dose dependent); exercise tidal volume was increased at 106 mg/m 3 (150 ppm), but reduced at higher concentrations in a dose-dependent manner Cole et al.,

1977 a

Six male and female volunteers, 24– 46 yrs old 25, 50, and 100 ppm (18, 35,

and 71 mg/m 3 ) ammonia for 6 hrs/d 1 time/wk over 6 wks; occasional brief exposure to 150–200 ppm (106– 141 mg/m 3 ); 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/m 3 ) became easily tolerated and had no effect on general health after acclimation occurred; brief exposure to 150–200 ppm (106–141 mg/m 3 ) produced lacrimation and transient discomfort Ferguson et al.,

1977 b

15 volunteers, 18– 53 yrs old 50, 80, 110, and 140 ppm (35,

57, 78, and 99 mg/m 3 ) for 2 hrs in an exposure chamber; there was no mention of preexposure examinations No effect on vital capacity or FEV 1 ; 140 ppm (99 mg/m 3 ) caused severe irritation and could not be tolerated; reported eye irritation increased with concentration Verberk, 1977 b

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Table A-10. Controlled human exposure studies of a mmonia inhalation

Subjects Exposure conditions Results Reference 20 male volunteers; groups of four were exposed to ammonia at various concentrations and durations Group 1: exposed to 2 mg/m 3

(3.0 ppm) for 37 d; Group 2: exposed to 5 mg/m 3 (7.2 ppm) for 17 d; Group 3: exposed to 2 mg/m 3(3.0 ppm) for 35 d with shortterm increases to 10 mg/m 3 (14 ppm); Groups 4 and 5: exposed to 2 and 5 mg/m 3 (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/m 3 (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/m 3 (7.2 ppm); increased temperature and humidity resulted inincreased urine adrenalin, urine 7-oxycorticosteroids and free 11-oxycorticosteroid levels in plasma at 5.1 mg/m 3 (7.2 ppm) Kalandarov et al., 1984 b

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/m 3 ) and bronchoconstriction was seen at 85 ppm (60.1 mg/m 3 ) Douglas and Coe, 1987 b

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/m 3 ) 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 FEV 1 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., 2004 a

12 healthy volunteers (7 females, 5 males) 21–28 yrs old 5 and 25 ppm (4 and 18

mg/m 3 ) 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/m 3 ); there were no changes in lung function or exhaled nitric oxide levels in exposed individuals; exposure did not result in upper-airway inflammation or bronchial responsiveness Sundblad et al.,

2004 a

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Table A-10. Controlled human exposure studies of a mmonia 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/m 3 ) by dynamic olfactometry; there was no mention of preexposure examinations Mean odor detection threshold <20 ppm (14 mg/m 3 ), mean irritation (lateralization) threshold well above 20 ppm (14 mg/m 3 ), dose-response for odor annoyance and irritation; strong olfactory and moderate to strong irritating sensations at >15 ppm (11 mg/m 3 ) Altmann et al.,

2006 b

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/m 3 ) 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/m 3 ), there were no significant differences in symptoms reported between the groups; the perceivedintensity of symptoms was concentrationdependent in both groups Ihrig et al.,

2006 a

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/m 3 )

(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/m 3 ); ocular irritation threshold = 175 ppm (124 mg/m 3 ); there were no changes in lung function (FEV 1 ) for subjects in either group Petrova et al.,

2008 a

24 healthy female volunteers age 18– 45 yrs (mean age 29.9 yrs) 0.03–615.38 ppm (0.02–

435 mg/m 3 ) (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; m ean odor detection threshold of 2.6 ppm (2 mg/m 3 ) (both static and dynamic) and mean irritation thresholds of 31.7 or 60.9 ppm (22 or 43 mg/m 3 ) for static and dynamic methods, respectively Smeets et al.,

2007 a

a Investigators reported that informed consent by vol unteers and/or study approval by local boards/offic ials regarding ethical conduct was obtained.

b This controlled-exposure study did not provide info rmation on the human subjects research ethics proce dures undertaken in the study; however, there is no evide nce that the conduct of the research was fundamenta lly unethical or significantly deficient relative to th e ethical standards prevailing at the time the rese arch was conducted.



### Altmann et al. (2006) showed a dose-dependent incr ease 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 ir ritation sensations occurred at concentrations >15 ppm (11 mg/m 3 

### ), with odor detection thresholds at <20 ppm (14 mg /m 3 

### ). In another study,

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12 healthy volunteers exposed to 5 and 25 ppm (4 an d 18 mg/m 3 

### ) ammonia on three different occasions for 1.5 hours in an exposure chamber whil e exercising on a stationary bike reported discomfort in the eyes and odor detection at 5 ppm (4 mg/m 3 

### ) (Sundblad et al., 2004). Eye

irritation was also shown to increase in a concentr ation-dependent manner in 15 volunteers exposed to ammonia for 2 hours in an exposure chamb er at concentrations of 50, 80, 110, and 140 ppm (35, 57, 78, and 99 mg/m 3 

### ); ammonia concentrations of 140 ppm (99 mg/m 3 

### ) caused severe and intolerable irritation (Verberk, 1977). The lachrymatory threshold was determined to be 55 ppm (39 mg/m 3 

### ) 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/m 3 

### ) ammonia gas did not produce severe lacrimation in seven volunteers after 10 minutes in an exposure chamber, although increas ed eye erythema was reported (MacEwen et al., 1970). Exposure to 500 ppm (354 mg/m 3 

### ) of ammonia gas for 30 minutes through a masked nose and throat inhalation apparatus resulted in tw o of seven volunteers reporting lacrimation, and two of seven reporting nose and throat irritati on that lasted up to 24 hours after exposure (Silverman et al., 1949). Petrova et al. (2008) investigated irritation thre shold differences between 25 healthy volunteers and 15 mild-to-moderate persistent asthm atic volunteers exposed to ammonia via the eyes and nose at concentrations ranging from 2 to 5 00 ppm (1–354 mg/m 3 

### ) for durations lasting up to 2.5 hours. Irritation threshold, odor intens ity, and annoyance were not significantly different between the two groups. The nasal and ey e irritation thresholds were reported to be 129 ppm (91 mg/m 3 

### ) and 175 ppm (124 mg/m 3 

### ), 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 (0.02–4 35 mg/m 3 

### ). This study found a mean odor detection threshold of 2.6 ppm (2 mg/m 3 

### ) and a mean irritation threshold of 31.7 or 60.9 p pm (22 or 43 mg/m 3 

### ), depending on the olfactometry methodology follow ed (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 reg ular workplace exposure to ammonia were exposed to ammonia vapors at concentrations of 0, 1 0, 20, and 50 ppm (0, 7, 14, and 35 mg/m 3 

### ) 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 t he nonhabituated group, but at a concentration of 20 ppm (14 mg/m 3 

### ), there were no differences in perceived symptoms between the groups. However, the perceived intensity of sy mptoms was concentration-dependent in both groups, but was only significant in the group of vo lunteers not familiar with ammonia exposure (Ihrig et al., 2006). Ferguson et al. (1977) repor ted habituation to eye, nose, and throat irritation

in six male and female volunteers after 2–3 weeks o f exposure to ammonia concentrations of 25, 50, and 100 ppm (18, 35, and 71 mg/m 3 

### ) during a 6-week study (6 hours/day, 1 time/week).

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Continuous exposure to even the highest concentrati on 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/m 3 

### ) (Douglas and Coe, 1987); however, there were no bronchial symptoms re ported in seven volunteers exposed to ammonia at concentrations of 30, 50, or 90 ppm (21, 35, and 64 mg/m 3 

### ) 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/m 3 

### ) for 1.5 hours in an exposure chamber while exercising on a stationary b ike 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 vo lume in 1 second (FEV 1 

### ) (Sundblad et al., 2004). In another study, 18 healthy servicemen vol unteers were placed in an exposure chamber for 3 consecutive half-day sessions. Exposure to a mmonia at concentrations of 50–344 mg/m 3 

### (70–486 ppm) occurred on the second session, with s essions 1 and 3 acting as controls (Cole et al., 1977). The no-effect concentration was determ ined to be 71 mg/m 3 

### (100 ppm). Exercise tidal volume was increased at 106 mg/m 3 

### (150 ppm), but then decreased at higher concentrat ions in a concentration-dependent manner (Cole et al., 1 977). Decreased FEV 1 

### 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/m 3 

### ) in swine confinement buildings for 4 hours at 1-week intervals; however, swine confinement buildi ngs also include confounding exposures to dust, bacteria, endotoxin, and molds, thereby makin g measurement of effects due to ammonia uncertain in this study (Cormier et al., 2000). Differences in lung function between healthy and a sthmatic volunteers exposed to

ammonia were evaluated in several studies. There w ere no changes in lung function as measured by FEV 1 

### in 25 healthy volunteers and 15 mild/moderate pers istent asthmatic volunteers after ocular and nasal exposure to 2–500 ppm (1–354 mg/m 3 

### ) 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/m 3 

### ) 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 FEV 1 

### in the healthy volunteers for any exposure. A decrease in FEV 1 

### was reported in asthmatics exposed to dust and ammonia, but not to ammonia alone; similarly, incre ased bronchial hyperreactivity was reported in asthmatics after exposure to dust and ammonia, b ut not to ammonia alone. Exposure to dust alone caused similar effects, suggesting that dust was responsible for decreased lung function (Sigurdarson et al., 2004).

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In summary, volunteer studies demonstrate that eye irritation can occur following acute exposure to ammonia at concentrations as low as 5 p pm (4 mg/m 3 

### ). 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 expos ed to ammonia do not suggest that asthmatics are more sensitive to the lung effects of ammonia.



## Case Studies and Reports of Human Exposure to Ammon ia

Oral exposure to ammonia most commonly involved in gestion of household cleaning solutions or biting into the capsules of ammonia sm elling 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 effec ts included drooling, erythematous and edematous lips, reddened and blistered tongues, dys phagia, 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 in toxication characterized by lethargy, restlessness, irritability, and confusion in a 37-y ear-old man following surgery. Most other cases of ammonia ingestion involved household cleaning so lutions 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 sud sy ammonia (ammonia 3.6%; pH 11.5), respectively. The first case resulted in a white a nd 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 complicatio ns in either case. Christesen (1995) reported that of the 11 cases involving accidental or intent ional 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 di fficulty 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

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ammonia was involved that appropriate treatment, fo llowed by a satisfactory outcome, was

achieved. Inhalation is the most frequently reported route o f exposure and cause of morbidity and fatality, and often occurs in conjunction with derm al 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 perceiv ed 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; mo derate dyspnea; rapid, shallow breathing; cyanosis; some vomiting; transient bronchospasm; ed ema and some evidence of burns to the lips and oral mucosa, and localized to general rhonchi i n the lungs (Weiser and Mackenroth, 1989; Yang, 1987; O’Kane, 1983; Ward et al., 1983; Countu rier et al., 1971; Caplin, 1941). Severe effects include second- and third-degree burns to t he nasal passages, soft palate, posterior pharyngeal wall, and larynx, upper airway obstructi on, loss of consciousness, bronchospasm, dyspnea, persistent, productive cough, bilateral di ffuse rales and rhonchi, production of large amounts of mucous, pulmonary edema, marked hypoxemi a, local necrosis of the lung, deterioration of the whole lung, and fatality. Del ayed effects of acute exposure to high concentrations of ammonia include bronchiectasis, b ronchitis, bronchospasm/asthma, dyspnea upon exertion and chronic productive cough, bronchi olitis, 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., 1 983; Stroud, 1981; Close, 1980; Taplin, et al., 1976; Walton, 1973; Kass et al., 1972; Slot, 1938). Respiratory effects were also observed following c hronic occupational exposure to ammonia. After 18 months and 1 year on the job, re spectively, 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, progress ive deterioration of the clinical condition of a 68-year-old male was documented for 4 years, and de velopment of diffuse interstitial and severe restrictive lung disease was reported following lon g-term repetitive occupational exposure to ammonia at or above the odor recognition level (Bra utbar 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 silverwa re. 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 us ing this product was found to be 8–15 ppm (6–11 mg/m 3 

### ). Acute dermal exposure to anhydrous (liquid) ammoni a 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 ammoni a in which first-, second-, and third-degree

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burns occurred over as much as 50% of the total bod y surface (Lalic et al., 2009; Pirjavec et al., 2009; Arwood et al., 1985). Frostbite injury has a lso 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., 198 5). 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. (197 2) 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 trai n in the vicinity. Several victims at or near

the scene of an overturned truck that had been carr ying 8,000 gallons of anhydrous ammonia were reported as having second- and third-degree bu rns 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 i n 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), immedi ate effects include temporary blindness, blepharospasm, conjunctivitis, corneal burns, ulcer ation, edema, chemosis, and loss of corneal epithelium (George et al., 2000; Helmers et al., 19 71; Highman, 1969; McGuinness, 1969; Levy et al., 1964; Abramovicz, 1924). The long-term eff ects included photophobia, progressive loss of sensation, formation of bilateral corneal opacit ies 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; Lev y et al., 1964; Abramovicz, 1924). White et al. (2007) reported a case with acute bila teral corneal injury that developed into bilateral

uveitis with stromal vascularization and stromal ha ze and scarring, and pigmented keratic precipitates that resulted in legal blindness. An increase in intraocular pressure, resembling acute-angle closure glaucoma, was reported by Highm an (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 th at the bacterium Helicobacter pylori

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

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etiology of chronic atrophic gastritis. Male Sprag ue-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 measuremen ts of the thickness of the mucosa about

175 μ M from the pyloric ring in the antral mucosa. The parietal cell number per gland was determined at three locations in the oxyntic glandu lar area. Mucosal lesions were not observed macro- or microscopically. There was a statistical ly significant decrease in mean antral mucosal thickness with increasing dose and duration of expo sure (Table A-11). Parietal cell number per oxyntic gland decreased in a statistically signific ant 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 i ndex was significantly lower only for the antral mucosa with 0.01% ammonia. The authors sugg ested that administration of ammonia in drinking water causes gastric mucosal atrophy. Bas ed 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 ± 40 a

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

Body mucosa

2 wks 574 ± 116 568 ± 159 591 ± 183

4 wks 618 ± 154 484 ± 123 440 ± 80 a,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 produ ced 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, o r 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 th e initial body weight (150 g) and estimated

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daily water intake (50 mL), the daily dose at a dri nking water concentration of 0.01% ammonia was estimated to be 33 mg/kg-day. Cellular migrati on was measured by labeling cells with 5-bromo-2-deoxyuridine (BrDU) at different time per iods and measuring the incorporation of

this modified nucleoside with a histochemical techn ique using anti BrDU monoclonal antibodies. Antral and body mucosa thickness was measured as de scribed in Kawano et al. (1991). The measurement of cell proliferation in the gastric mu cosa was estimated using the labeling index in gastric pits (ratio of labeled nuclei to total nucl ei 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) tow ard 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 conclu ded that 0.01% ammonia increased epithelial cell migration in the antrum leading to mucosal atr ophy. The EPA identified a LOAEL of 33 mg/kg-day based on decreased thickness of the ga stric 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 admini stered 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 ± 29 b 531 ± 32

8 Wks 168 ± 26 b 508 ± 29

a Extracted from Figure 3 of Tsujii et al. (1993); me an ± 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 5 1 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 st andard 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 6 1–110 and 120–230 mg/kg-day, respectively. The exact duration of the study is n ot reported, but it is clear from the data that by the end of the experiment, some rabbits received on ly three or four doses before dying as a result of intoxication in 5.5 days and other rabbits recei ved 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 th e 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 s imilar age and body weight were also determined. The average weight of adrenals from th ese 41 control rabbits was 400.0 ± 13.4 mg. Fazekas (1939) reported that differences in mean ad renal weight in ammonium hydroxide-treated animals were significant, althoug h there was no description of the statistical analysis performed in this study. Chemical evaluat ion of the adrenals from treated rabbits revealed fat content 4.5 times greater and choleste rol content 6.5 times greater than controls. At the beginning of the experiment, a greater weight l oss was observed among those rabbits receiving ammonium hydroxide more frequently (daily ) at higher doses. Body weights fluctuated among treated rabbits and generally decr eased 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 increa ses 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 exposu re (during the first 5–10 minutes that lasted up to 7 hours) to 20–30 mm Hg. Following several m onths 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 d ecreases 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 blo od pressure and body weight could be

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attributed to hypertrophy of the adrenal cortex. T his study is limited by lack of reporting detail and inadequate study design. The EPA did not ident ify a NOAEL or LOAEL from this study.

Toth (1972) Toth (1972) evaluated whether hydrazine, methylhydr azines, 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 amm onium hydroxide (0.1, 0.2, and 0.3%) were administered continuously in the drinking wate r of 5- and 6-week-old randomly bred Swiss mice (50/sex) for their entire lifetime. For ammon ium 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, a nd 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 approxi mate 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 C 3 

### H mice (40/sex) were exposed to ammonium hydroxide in the drinking water at a concentration of 0.1% for t heir 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 m g/kg-day for males and females, respectively. Data were not reported for a concurr ent control group. Mice were monitored weekly for changes in body weights and gross pathol ogical changes were recorded. The animals

were either allowed to die or were killed when foun d 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% ammoniu m hydroxide concentration was as follows: malignant lymphomas: 3/50 (males), 9/50 (f emales); and lung adenoma or adenocarcinoma: 7/50 (males), 4/50 (females). Tumo r 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 (fema les); and breast tumors: 4/50 (females). Tumor incidence at the 0.1% ammonium hydroxide conc entration was: malignant lymphomas: 4/50 (males), 10/50 (females); lung adenoma or aden ocarcinoma: 5/50 (males), 12/50 (females); and breast tumors: 1/50 (females). The denominator s were not adjusted for survival, and concurrent control data were not provided. For a s econd strain of mice (C 3 

### H) 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 bre ast 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 n ot carcinogenic in either strain of mouse.

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Because concurrent control tumor incidence was not provided other than the incidence of breast

tumors in C 3 

### H female mice, the incidence of tumors in treated m ice cannot be independently compared to control tumor incidence.

Tsujii et al. (1995, 1992a) Tsujii et al. (1995, 1992a) evaluated the role of a mmonia in H. pylori -related gastric carcinogenesis. H. pylori is a bacterium that produces a potent urease, whic h generates ammonia from urea in the stomach, and has been implicated i n the development of gastric cancer. Tsujii et al. (1995, 1992a) pretreated groups of 40–44 male S prague-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 d uring 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 eac h 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 h istologically for evidence of ulcers, lesions, and tumors. Tsujii et al. (1995) also eva luated serum gastrin levels from blood collected at 30 and 46 weeks and mucosal cell proliferation i n 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 inde x (fraction of the gastric pit occupied by the proliferation zone). Tsujii et al. (1995, 1992a) observed a significantl y greater incidence of gastric cancers among rats receiving ammonia after pretreatment wit h MNNG compared to rats receiving only MNNG and tap water (

p < 0.01, χ

2 

### test). Seventy percent of MNNG+ammonia-treated ra ts 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 co ntrol rats (Tsujii et al., 1995). The numbers of gastric tumors per rat in this study were also s ignificantly higher among MNNG+ammoniaexposed rats than controls ( p < 0.001, Mann-Whitney test), suggesting that ammon ia was a promoter. In the absence of an ammonia-only treatm ent group, however, it is not possible to distinguish with certainty between possible promoti on and initiator activity. The degree of

differentiation of adenocarcinomas in control and a mmonia-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

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muscularis propria or deeper ( p < 0.01, 22% compared to controls 12%). In this st udy, the labeling index and the proliferation zone index wer e statistically significantly elevated in ammonia exposed rats compared to controls in the fu ndic mucosa and antral mucosa. Tsujii et al. (1995) explored the hypothesis that a mmonia 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 (1 4 mg/m 3 

### ) 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/m 3 

### ) 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 s elected tissues (lungs, trachea, turbinates, liver, and spleen) were examined for gross and micr oscopic pathological changes. No significant effects were observed in animals exposed for up to 4 weeks, but exposure to 20 ppm (14 mg/m 3 

### ) for 6 weeks caused darkening, edema, congestion, an d hemorrhage in the lung. Exposure of guinea pigs to 50 ppm (35 mg/m 3 

### ) ammonia for 6 weeks caused grossly enlarged and c ongested spleens, congested livers and lungs, and pulmonary edema. NOAEL and LOAEL values were not identified in this study because only two anima ls per group were examined at a single time period for the 20 ppm (14 mg/m 3 

### ) exposure groups (mice and guinea pigs).

Coon et al. (1970) Coon et al. (1970) exposed groups of male and femal e 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, o r 770 mg/m 3 

### ammonia (0, 219, or 1,089 ppm) 8 hours/day, 5 days/week for 6 weeks (an hydrous 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 be fore and after the exposures for determination of hemoglobin concentration, packed e rythrocyte volume, and total leukocyte counts. Animals were routinely checked for clinica l 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

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monkeys. Sections of the brain, spinal cord, and a drenals 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/m 3 

### ammonia did not result in any deaths or adverse cl inical signs of toxicity in any of the animals. Hematological valu es 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/m 3 

### caused initial mild to moderate lacrimation and dy spnea in rabbits and dogs. However, these clinical signs disappeared by the se cond week of exposure. No significant alterations were observed in hematology tests or up on gross or microscopic examinations at the highest dose. However, consistent nonspecific infl ammatory changes (not further described) were observed in the lungs from rats and guinea pig s in the high-dose group that were more extensive than in control animals (incidence not re ported). 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/m 3 

### (0, 57, 180, 371, 644, or 665 ppm) for 90–114 days. Fifteen guinea pigs, three rabbit s, two dogs, and three monkeys were also exposed continuously under similar conditions to am monia at 40 mg/m 3 

### (57 ppm) or 470 mg/m 3 

### (665 ppm). No significant effects were reported in any animals exposed to 40 mg/m 3 

### and nonspecific inflammatory changes in the lungs and k idneys (not further described) were seen in rats exposed to 127 mg/m 3 

### (180 ppm) ammonia. Exposure to 262 mg/m 3 

### (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/m 3 

### (644 ppm) was observed due to high mortality in th e rats (50/51). Thirty-two of 51 rats died by day 25 of exposure; no histopatholo gical examinations were conducted in these rats. Exposure to 470 mg/m 3 

### (665 ppm) caused death in 13/15 rats and 4/15 guin ea pigs and marked eye irritation in dogs and rabbits. Dogs experienced heavy lacrim ation and nasal discharge, and corneal opacity was noted in rabbits . Hematological values did not differ significantly from controls in animals exposed to 4 70 mg/m 3 

### (665 ppm) ammonia. Histopathological evaluation of animals exposed to 470 mg/m 3 

### (665 ppm) consistently showed focal or diffuse interstitial pneumonitis in all an imals and also alterations in the kidneys (calcification and proliferation of tubular epithel ium), heart (myocardial fibrosis), and liver (fatty change) in several animals of each species (inciden ce not reported). The study authors did not

determine a NOAEL or LOAEL concentration from this study. EPA identified a NOAEL of 40 mg/m 3 

### (57 ppm) and a LOAEL of 127 mg/m 3 

### (180 ppm) based on nonspecific inflammatory changes in the lungs and kidneys in rats exposed to ammonia for 90 days.

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Stombaugh et al. (1969) Stombaugh et al. (1969) exposed groups of Duroc pig s (9/group) to measured concentrations of 12, 61, 103, or 145 ppm ammonia ( 8, 43, 73, or 103 mg/m 3 

### ) continuously for 5 weeks (anhydrous ammonia, purity not reported). Endpoints evaluated included clinical signs, food consumption (measured 3 times/week), weight ga in (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 impossibl e to determine whether this reduction was statistically significant. Food efficiency (food c onsumed/kg body weight gain) was not affected. Exposure to ≥ 103 ppm (>73 mg/m 3 

### ) ammonia appeared to cause excessive nasal, lacrim al, and mouth secretions and increased the frequency of cou gh (incidence data for these effects were not reported). Examination of the respiratory tract di d 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 sp ecific-pathogen-free derived Yorkshire Landrace pigs to 0 or 100 ppm ammonia (0 or 71 mg/m 3 

### ) continuously for up to

6 weeks. The mean concentration of ammonia in the control chamber was 8 ppm (6 mg/m 3 

### ). Additional groups of pigs were exposed to similar l evels of ammonia as well as to 0.3 mg/ft 3 

### of ground corn dust to simulate conditions on commerci al farms. Pigs were monitored daily for clinical signs and changes in behavior. Initial an d terminal body weights were measured to determine body weight gain during the exposure peri od. Blood samples were collected prior to the start of each experiment and at study terminati on 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 we ekly intervals, and tracheal swabs for bacterial and fungal culture were taken. Histologi cal examination was conducted on tissue samples from the lung, trachea, and bronchial lymph nodes. During the first week of exposure, exposed pigs exh ibited slight signs of conjunctival irritation including photophobia and excessive lacr imation. These irritation effects were not apparent beyond the first week. Measured air conce ntrations in the exposure chambers increased to more than 150 ppm (106 mg/m 3 

### ) on two occasions. Doig and Willoughby (1971) rep orted that at this concentration, the signs of conjunctiv al irritation were more pronounced in all pigs. No adverse effects on body weight gain were apparen t. Hematological parameters and gross pathology were comparable between exposed and contr ol pigs. Histopathology revealed epithelial thickening in the trachea of exposed pig s and a corresponding decrease in the numbers

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of goblet cells as shown in Table A-13. Tracheal t hickening was characterized by thinning and irregularity of the ciliated brush border and an in creased number of cell layers. Changes in bronchi and bronchioles characterized as lymphocyti c cuffing were comparable between exposed and control pigs. Similarly, intraalveolar hemorrh age and lobular atelectasis were common findings in both exposed and control pigs. Pigs ex posed to both ammonia and dust exhibited similar reactions as those pigs exposed only to amm onia, 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 trachea l epithelium of pigs exposed only to ammonia.

Table A-13. Summary of histological changes observ ed 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/m 3 NH 3 Control 71 mg/m 3 NH 3 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 e xposure at 71 mg/m 3 

### may be detrimental to young pigs. The authors suggested t hat although the structural damage to the upper respiratory epithelium was slight, such chang es 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/m 3 

### (100 ppm), based on damage to the upper respirator y epithelium. A NOAEL could not be identified from t his single-concentration study.

Broderson et al. (1976) Broderson et al. (1976) exposed groups of Sherman r ats (5/sex/dose) continuously to 10 or 150 ppm ammonia (7 or 106 mg/m 3 

### , respectively) for 75 days (anhydrous ammonia, pur ity not reported). The 10-ppm exposure level represent ed the background ammonia concentration resulting from cage bedding that was changed 3 time s/week. The 150-ppm concentration resulted from cage bedding that was replaced occasi onally, but never completely changed. F344 rats (6/sex/group) were exposed to ammonia in an in halation chamber at concentrations of 0 or 250 ppm (177 mg/m 3 

### ) continuously for 35 days. Rats were sacrificed a t the end of the exposure

### A-50 DRAFT – DO NOT CITE OR QUOTE

period, and tissues were prepared for histopatholog ical examination of nasal passages, middle ear, trachea, lungs, liver, kidneys, adrenal, pancr eas, testicle, mediastinal lymph nodes, and spleen. Histopathological changes were observed in the nasa l passage of rats exposed to 150 ppm (106 mg/m 3 

### ) for 75 days (from bedding) or 250 ppm (177 mg/m 3 

### ) 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 respir atory 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 glandu lar crypts were observed and neutrophils were located in the epithelial layer, the lumina of subm ucosal glands, and the nasal passages. Dilation

of small blood vessels and edema were observed in t he submucosa of affected areas. Collagen replacement of submucosal glands and the presence o f lymphocytes and neutrophils were also observed. No histopathological alterations were se en in control rats (10 ppm from bedding or 0 ppm from the inhalation chamber). Broderson et a l. (1976) did not identify a NOAEL or LOAEL from this study. EPA identified a NOAEL of 1 0 ppm (7 mg/m 3 

### ) and a LOAEL of 150 ppm (106 mg/m 3 

### ) 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 mic e 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 a lbino mice under unspecified conditions to ammonia vapor derived from a 12% ammo nia solution (air concentrations were not reported) 15 minutes/day, 6 days/week for up to 8 w eeks. 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 a nd examined histologically. Frozen sections of the nasal mucosa were subjected to hist ochemical analysis (succinic dehydrogenase, nonspecific estrase, acid phosphatase, and alkaline phosphatase [ALP]). Histological examination revealed a progression of changes in th e 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 a t 6 weeks, and dysplasia in the nasal epithelium at 7 weeks. Similar changes were exagge rated 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 p olarity of the epithelium, hyperchromatism and mitotic figures with an intact basement membrane, a nd an invasive adenocarcinoma in one rat

sacrificed at 8 weeks. Histochemical results revea led changes in succinic dehydrogenase, acid

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phosphatase, and ALP in exposed mice compared to co ntrols (magnitude of change not reported), especially in areas of the epithelium ch aracterized by dysplasia. Succinic dehydrogenase and acid phosphatase changes were lar gest 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 st rongest in the goblet cells from the basal part of the epithelium and basement membrane. In summary, Gaafar et al. (1992) observed that amm onia exposure induces histological changes in the nasal mucosa of male mice that incre ase in severity over longer exposure periods. Corresponding abnormalities in histochemistry sugge st altered cell metabolism and energy production, cell injury, cell proliferation, and po ssible chronic inflammation and neoplastic transformation. The study authors did not determin e 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/m 3 

### ) and 1.2, 2.7, 5.1, or 9.9 mg/m 3 

### inhalable dust for 5 weeks (16 treatment combinations). The concentrations of ammonia and d ust used were representative of those found commercially. A split-plot design was used in whic h 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 c oncentrations × 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 expe riment and tested for porcine reproductive and respiratory syndrome virus and swine influenza. Fi ve sentinel pigs were sacrificed at the start of each batch and lung, nasal cavity, and trachea, tog ether with material from any lesions, were examined postmortem and subjected to bacteriologica l examination. Postmortem examination involved examining the pigs’ external surfaces for condition and abnormalities, examination of the abdomen for peritonitis and lymph node size, in ternal gross examination of the stomach for abnormalities, and gross examination of the nasal t urbinates, thorax, larynx, trachea, tracheobronchial lymph nodes, and lung. Pigs were monitored for clinical signs (daily), growth rate, feed consumption, and feed conversion efficie ncy (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 remain ing 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 re covery and the remaining 10 pigs were sacrificed and examined postmortem. The pigs in this study demonstrated signs of respi ratory 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 am monia 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/m 3 

### (37 ppm), based on the lack of respiratory or othe r disease following exposure to ammonia in the presen ce 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/m 3 

### ) 6 hours/day, 5 days/week for up to 18 weeks (anhy drous ammonia, purity not reported). The actual concentr ation measured in the exposure chamber varied between 140 (99 mg/m 3 

### ) and 200 ppm (141 mg/m 3 

### ). 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 contr ol guinea pigs) and the heart, lungs, liver, stomach and small intestine, spleen, kidneys, and a drenal glands were removed for microscopic examination; the upper respiratory tract was not ex amined. No exposure-related effects were observed in guinea pigs sacrificed after 6 or 12 we eks 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 adre nal 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 ki dney tubules showed cloudy swelling with precipitated albumin in the lumens and some urinary casts (cylindrical structures indicative of disease). The incidence of histopathological lesio ns was not reported. EPA identified the ammonia concentration of 170 ppm (120 mg/m 3 

### ) to be a LOAEL based on congestion of the spleen, liver, and kidneys and early degenerative c hanges in the adrenal gland. A NOAEL could not be identified in this single-concentration stud y.

Curtis et al. (1975) Curtis et al. (1975) exposed groups of crossbred p igs (4–8/group) to 0, 50, or 75 ppm ammonia (0, 35, or 53 mg/m 3 

### ) continuously for up to 109 days (anhydrous ammoni a, >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

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respiratory tract, the eye and its associated struc tures, 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 evalu ations of tissues and organs were unremarkable. The turbinates, trachea, and lungs o f all pigs were classified as normal. The study authors did not identify a NOAEL or LOAEL fro m this study. EPA identified a NOAEL of 75 ppm (53 mg/m 3 

### ) 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 (y oung female pigs) in a conventional grower from 2 to 4.5 months of age; pigs were expos ed 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 conc entration of ammonia of 7 ppm (range, 4– 12 ppm) (5 mg/m 3 

### ; range, 3–8.5 mg/m 3 

### ) or 35 ppm (range, 26–45 ppm) (25 mg/m 3 

### ; range, 18– 32 mg/m 3 

### ) for 6 weeks (Diekman et al., 1993). A control gr oup was not included in this study. The low concentration of ammonia was obtained by th e flushing of manure pits weekly and the higher concentration of ammonia was maintained by a dding anhydrous ammonia (purity not reported) to manure pits that were not flushed. Af ter 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 w ere weighed. The remaining 20 gilts/group were mated with mature boars and continued being ex posed to ammonia until gestation day 30, at which time they were sacrificed. Fetuses were e xamined for viability, weight, and length, and the number of copora lutea were counted. Gilts exposed to 35 ppm (25 mg/m 3 

### ) ammonia gained less weight than gilts exposed to 7 ppm (5 mg/m 3 

### ) during the first 2 weeks of exposure (7% decrease

, p < 0.01), but growth rate

recovered thereafter. Mean scores for lesions in t he 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/m 3 

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

### ). In gilts that were mated, conception rates were similar between the two groups (94.1 versus 100% in low ver sus high exposure). At sacrifice on day 30 of gestation, body weights were not significantly d ifferent between the two groups. In addition, there were no significant differences between the t wo groups regarding percentage lung tissue with lesions and mean snout grade. Number of corpo ra lutea, number of live fetuses, and weight and length of the fetuses on day 30 of gestation we re not significantly different between

### A-54 DRAFT – DO NOT CITE OR QUOTE

treatment groups. Diekman et al. (1993) did not id entify NOAEL or LOAEL concentrations for maternal or fetal effects in this study. The EPA d id not identify NOAEL or LOAEL values from this study due to the absence of a control group an d 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 NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceRatsFemale Porton rats(16/group)0 or 141(0 or 200 ppm)Continuousexposure for 4,8, or 12 dHistology of the trachea 4 d: transitional-stratified appearance of theepithelium8 d: gross change with disappearance of ciliaand stratification on luminal surface12 d: increased epithelial thicknessGamble and Clough,1976Male OFA rats(27/group)0 or 354(0 or 500 ppm)Continuousexposure for 1–8 wksBody weight, organweights, airway structure,cell population, alveolarmacrophagesNo deaths occurred; decreased foodconsumption and body weight gain; increasedlung and kidney weights; at 3 wks, nasalirritation and upper respiratory tractinflammation, but no effect on lower airways;slight decrease in alveolar macrophages; nohistopathological effects seen at 8 wks,suggesting adaptation to exposureRichard et al., 1978aMale and femaleWistar rats(5/sex/group)9,898–37,825(14,000–53,500 ppm);no mention of controlgroup10, 20, 40, or60 minClinical signs, pathology,LC50Eye irritation, eye and nasal discharge,dyspnea; hemorrhagic lungs on necropsy;10-min LC50= 28,492 mg/m3(40,300 ppm)20-min LC50= 20,217 mg/m3(28, 595 ppm)40-min LC50= 14,352 mg/m3(20,300 ppm)60-min LC50= 11,736 mg/m3(16,600 ppm)Appelman et al., 1982Male Crl:COBS CD(Sprague-Dawley)rats (8/group)11, 23, 219, and 818(15, 32, 310, and1,157 ppm); arterialblood collected priorto exposure served ascontrol24 hrs Clinical signs, histology,blood pH, blood gasmeasurementNo clinical signs of toxicity, no histologicdifferences in tracheal or lung sections, nochange in blood pH or pCO2, minor changes inpO2Schaerdel et al., 1983Male Crl:COBS CD(Sprague-Dawley)rats (14/group)3, 17, 31, 117, and505 (4, 24, 44, 165,and 714 ppm); arterialblood collected priorto exposure served ascontrol3 and 7 d Hepatic cytochrome P450content andethylmorphineN-demethylase activityNo dose-related change in P450 content orenzyme activitySchaerdel et al., 1983A-56 DRAFT – DO NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMale Long-Evansrats (4/group)70 and 212(100 and 300 ppm);results werecompared to“control”, but it wasnot clear if theauthors werereferring to historicalor concurrent controls6 hrs Clinical signs, behavioralobservationDecreased running, decreased activity Tepper et al., 1985Female Wistar rats(5/group)0, 18, or 212(0, 25, or 300 ppm)6 hrs/d for 5, 10,or 15 dBlood ammonia, urea,glutamine, and pH; brainammonia, glutamine;histopathology of lungs,heart, liver, and kidneys(light and electronmicroscopy)Brain and blood glutamine increased; slightacidosis (i.e., decreased blood pH) 212 mg/m3(300 ppm); lung hemorrhage observed in someexposed ratsManninen et al., 1988Female Wistar rats(5/group)0, 18, or 212(0, 25, or 300 ppm)6 hrs/d for 5 d Plasma and brainammonia and amino acidanalysisIncrease in brain and plasma glutamineconcentrations, increased brain/plasma ratio ofthreonineManninen andSavolainen, 1989Female albino rats(8/group)0, 848–1,068 (1,200–1,510 ppm)3 hrs Mortality, respiratorymovement, and O2consumptionNo deaths reported; inhibition of externalrespiration and decreased O2consumptionRejniuk et al., 2007Male SpragueDawley rats(number/group notgiven)Air concentration notgiven; ammonia vaporadded to inspiratoryline of ventilator;controls exposed tosame volume of roomair20 sec Activity of upper thoracicspinal neuronsLower airway irritation, activation of vagalpulmonary afferents and upper thoracic spinalneurons receiving pulmonary sympathetic inputQin et al., 2007a, bA-57 DRAFT – DO NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMale Wistar rats(4/group)0, 92–1,243 (130–1,758 ppm); thepreexposure periodwas used as thecontrol for eachanimal45 min Airway reflexes by thechanges in respiratorypatterns elicited byammonia in either dry,steam-humidified, oraqueous aerosolcontaining atmospheresAmmonia-induced upper respiratory tractsensory irritation is not affected to anyappreciable extent by wet atmospheres (withor without aerosol) up to 1,243 mg/m3Li and Pahluhn, 2010Male rats(10/group)0, 848–1,068 (0,1,200–1,510 ppm) atthe beginning and endof the exposureperiod)3 hrs Oxygen consumption Decreased O2consumption Rejniuk et al., 2008MiceMice (20/group,species, sex notspecified)6,080–7,070(8,600–10,000 ppm);no controls10 min LC50LC50= 7,056 mg/m3(9,980 ppm)Silver and McGrath,1948Male Swiss albinomice (4/group)5,050–20,199(7,143–28,571 ppm);no controls30–120 min LC50LC50(30 min) = 15,151 mg/m3(21,430 ppm) Hilado et al., 1977Albino mice (sex notspecified; 6/dose)Air concentration notmeasured; resultswere compared to“control”, but it wasnot clear if theauthors werereferring to historicalor concurrent controlsContinuously for2 or 5 dRegional brain metabolism(cerebral cortex,cerebellum, brainstem);MAO, enzymes ofglutamate and GABAmetabolism, and (Na+-K+)-ATPase; amino acid levelsin the brainAltered activities of MAO, glutamatedecarboxylase, ALT, GABA-transaminase, and(Na+-K+)-ATPase; increased alanine anddecreased glutamateSadasivudu et al.,1979; Sadasivudu andMurthy, 1978A-58 DRAFT – DO NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMale Swiss-Webstermice(4/group)Concentrations notgiven; baseline levelsestablished prior toexposure10 min Reflex decrease inrespiratory rate was usedas an index of sensoryirritation; RD50= theconcentration associatedwith a 50% decrease in therespiratory rateRD50= 214 mg/m3(303 ppm)Kane et al., 1979Male albino ICRmice (12/dose)0–3,436(0–4,860 ppm)1 hr (14-dfollowup)Clinical signs, body weight,organ weight,histopathology, LC50Eye and nose irritation, dyspnea, ataxia,seizures, coma, and death; decreased bodyweight and increased liver to body weight ratioin mice surviving to 14 d; effects in the lungincluded focal pneumonitis, atelectasis, andintralveolar hemorrhage; liver effects includedhepatocellular swelling and necrosis, vascularcongestion; LC50= 2,990 mg/m3(4,230 ppm)Kapeghian et al., 1982Male Swiss-Webstermice (16–24/group)0 or 216(0 or 305 ppm)6 hrs/d for 5 d Respiratory tracthistopathologyLesions in the nasal respiratory epithelium(moderate inflammation, minimal necrosis,exfoliation, erosion, or ulceration); no lesions intrachea or lungsBuckley et al., 1984Male albino ICRmice (12/dose)0, 954, 3,097, or 3,323(0, 1,350, 4,380, or4,700 ppm)4 hrs Hexobarbitol sleepingtime, microsomal proteincontent, liver microsomalenzyme activityIncreased hexobarbitol sleeping time(3,097 mg/m3), increased microsomal proteincontent and aminopyrene-N-deethylase andaniline hydroxylase activities (3,323 mg/m3)Kapeghian et al., 1985Male albino ICRmice (12/dose)0, 81, or 233(0, 115, or 330 ppm)4 hrs/d for 4 d Microsomal proteincontent, liver microsomalenzyme activityNo dose-dependent effects on microsomalenzymesKapeghian et al., 1985Male Swiss mice(6/dose)71 and 212(100 and 300 ppm);data collected duringthe 2 d separatingeach ammoniaexposure served asthe control baseline6 hrs Clinical signs, behavioralobservationDecreased running, decreased activity Tepper et al., 1985A-59 DRAFT – DO NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMice (4/group) 3, 21, 40, or 78 (4, 30,56, or 110 ppm),lowest measuredconcentration was thenominal control group2 d Responses to atmosphericammonia in anenvironmental preferencechamber with fourchambers of differentconcentrations ofammoniaNo distinguishable preference for, or aversionto, different NH3concentrationsGreen et al., 2008Male OF1 mice(4/group)0, 92–1,243 (130–1,758 ppm); thepreexposure periodwas used as thecontrol for eachanimal45 min Airway reflexes by thechanges in respiratorypatterns elicited byammonia in either dry,steam-humidified, oraqueous aerosolcontaining atmospheresAmmonia-induced upper respiratory tractsensory irritation is not affected to anyappreciable extent by wet atmospheres (withor without aerosol) up to 1,243 mg/m3Li and Pahluhn, 2010RabbitsFemale NewZealand Whiterabbits (7–9/dose)0, 35, or 71(0, 50, or 100 ppm)2.5–3.0 hrsLung functionDecreased respiratory rate at bothconcentrationsMayan and Merilan,1972Rabbits (species,sex, number/dosenot specified)0, 707–14,140(0, 1,000–20,000 ppm)15–180 min Lung function, death Bradycardia at 1,768 mg/m3(2,500 ppm);arterial pressure variations and blood gasmodifications (acidosis indicated by decreasedpH and increased pCO2) at 3,535 mg/m3(5,000 ppm); death occurred at 4,242 mg/m3(6,000 ppm)Richard et al., 1978bNew Zealand Whiterabbits (16 total;8/dose)Peak concentrations:24,745–27,573 mg/m3(35,000–39,000 ppm);concurrent controlstested4 min Lung function, heart rate,blood pressure, bloodgasesLung injury was evident after 2–3 min(decreased pO2increased airway pressure)Sjöblom et al., 1999A-60 DRAFT – DO NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceCatsMixed breed straycats (5/group)0 or 707(0 or 1,000 ppm)10 min Lung function, lunghistopathology on 1, 7, 21,and 35 d postexposureLungfunction deficits were correlated with lunghistopathology; acute effects were followed bychronic respiratory dysfunction (secondarybronchitis, bronchiolitis, andbronchopneumonia)Dodd and Gross, 1980PigsYoung pigs(2/group)0, 35, 71, or 106 (0,50, 100, or 150 ppm)Continuousexposure for4 wksClinical signs, foodconsumption, bodyweight, gross necropsy,organ weight,histopathologyLethargy and histopathological alterations inthe tracheal and nasal epithelium wereobserved at 71 and 106 mg/m3; decreased bodyweight occurred at all concentrations (7–19%decrease from control)Drummond et al.,1980Male and femaleBelgian Landracepigs (4/group)0, 18, 35, or 71(0, 25, 50, or100 ppm)6 d Clinical signs, body weight,lung functionLethargy and decreased body weight gain (allconcentrations); no effect on lungmicrovascular hemodynamics and permeabilityGustin et al., 1994Belgian Landracepigs (4/group)0, 18, 35, or 71(0, 25, 50, or100 ppm)6 d Clinical signs, body weight,neutrophil count, andalbumin in nasal lavagefluidNasal irritation (increased neutrophils in nasallavage fluid) and decreased body weight gain atall concentrationsUrbain et al., 1994Landrace-Yorkshirepigs (4/group)0 or 42 (0 or 60 ppm) 15 min/d for8 wksThromboxane A2 (TXA2),leukotriene C4 (LTC4), andprostaglandin (PGI2)productionSignificant increases in TXA2 and LTC4, nosignificant effect on PGI2 productionChaung et al., 2008Hybrid gilts (Whitesynthetic Pietrain,white Duroc,Landrace, LargeWhite)(14 pigs/group)<4 (control) or14 (<5 or 20 ppm)15 wks Salivary cortisol, adrenalmorphometry, bodyweight, food conversionefficiency, general healthscores, play behavior;reaction to light and noiseintensity testedconcurrentlyDecreased salivary cortisol, larger adrenalcortices, less play behavior, no measurableimpact on productivity or physiologicalparametersO’Connor et al., 2010A-61 DRAFT – DO NOT CITE OR QUOTETable A-14. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceCattleMale Holesteincalves(number/group notspecified)0, 35, or 71(0, 50, or 100 ppm)2.5 hrs Respiration rate, clinicalchemistryNo significant effect on respiration, BUN, pH,pO2, or pCO2Mayan and Merilan,1976Brahman/Charolais(group size notreported)<6 (control), 11, 23, or34 (<8 [control], 16,32, or 48 ppm)12 d Behavioral activity, bodyweight, analysis ofbronchioalveolar lavage(BAL) fluid, hematologicalvariables (hemoglobin,mean cell volume, plateletvolume, eosinophils,neutrophils, total whitecell count, monocytes)Increased lacrimation, nasal secretions,coughing, increased standing (as opposed tolying down), dose-related increases inmacrophage activity and neutrophil percentagein BAL fluid indicating lung inflammation, noeffect on hematological variables or bodyweightPhillips et al., 2010Holstein Friesianand Brown Swiss (10of each breed)~0, 4, and 15,(0.3 × 10-6, 6, and21 ppm)10 d at eachconcentrationRespiration and pulse rate,blood gas parametersRespiration and pulse rates were higher ininadequately ventilated barns (elevatedammonia and CO2)Sabuncuoglu et al.,2008

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## Genotoxicity Studies

Table A-15. Summary of in vitro studies of ammonia genotoxicity

End point Test system Concentration a Results b

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/m 3 ) 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 org anisms

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 fract ions from male Sprague- Dawley rats pretreated with pentachlorobiphenyl (KC500).

d Only positive in treatments using toxic levels of N H 3 (98% lethality).



### Table A-16. Summary of in vivo studies of ammonia genotoxicity

End point Test system Dose/ concentration a Results b Comments Reference Genotoxicity studies in mammalian systems in vivo

Chromosomal aberrations Human

lymphocytes 88.28 μg/m 3 + c 22 healthy workers occupationally exposed to ammonia in an Indian fertilizer factory (ambient concentration of 0.0883 mg/m 3 ); 42 nonexposed factory staff served as control subjects Yadav and Kaushik, 1997

Sister chromatid exchange Human

lymphocytes 88.28 μg/m 3 + 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/ concentration a Results b Comments Reference

Micronucleus formation Swiss albino mice 12.5–50 mg/kg + Intraperitoneal i njections 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 expo sure 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/m 3 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 chro matid exchanges, and mitotic index all increased wi th increased duration of exposure. This study is diff icult to interpret because of small samples sizes a nd confounding by smoking and alcohol consumption. In addition, t he levels of ammonia in the plant seemed low compar ed to other fertilizer plant studies (see, for example, S ection 1.1; Rahman et al., 2007; Ali, 2001; Ballal et al., 1998); the accuracy and reliability of the sampling and measur ement 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 regulator y 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/m 3 ), based on lack of significant alterations in lung function in chronically exposed workers (Holness et al., 1989) and a composite unce rtainty 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/m 3 ) for exposures ranging from 10 minutes to 8 hours to protect against mild irritati on, based on mild irritation in human subjects AEGL-2 (disabling) – 220 ppm (154 mg/m 3 ) for a 10-minute exposure to 110 ppm (77 mg/m 3 ) for an 8-hour exposure, based on irritation in hu man subjects AEGL-3 (lethal) – 2,700 ppm (1,888 mg/m 3 ) for a 10-minute exposure to 390 ppm (273 mg/m 3 ) 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/m 3 ) 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/m 3 ) 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 indust ry – 50 ppm (35 mg/m 3 ) 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 reco gnized as safe when used in accordance with good manufacturing or feeding pr actices (21 CFR 582.1139)

### C-1 DRAFT – DO NOT CITE OR QUOTE 

# APPENDIX C: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS AND DISPOSITION

To be added