Ammonia, anhydrous

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published paper; investigative study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

Rats with surgically implanted aortic cannulas were exposed to varying environmental ammonia concentrations (15-1157 ppm). Blood pH, pCO2 (partial pressure of CO2), PO2 (partial pressure of O2) and blood ammonia concentration were measured.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

other: Crl:COBS CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

The animals were male Crl:COBS CD(SD) rats weighing 300 to 400 g, obtained from Charles River Laboratories (MA). The rats were quarantined for 5 days, in groups of 6 in stainless steel wire-bottom suspended cages. The quarantine cubicles had an environmental temperature of 21±0.5°C, 50±20% relative humidity, 29 fresh air changes per hour, and a 12-hour light/dark photoperiod.
The rats received a commercial rodent diet (RMH 3000) and water ad libitum. Urine and faeces were removed from the cages daily to reduce endogenous production of ammonia and other gaseous contaminants. No ammonia was detected in the quarantine cubicles throughout the course of the study.

Route of administration:

inhalation

Vehicle:

unchanged (no vehicle)

Details on exposure:

The ammonia exposure was conducted in a clear rigid plastic isolator fitted with two sets of rubber gloves. The atmosphere inside the chamber was produced by mixing compressed air and anhydrous ammonia. The total gas flow, measured at the outflow port of the chamber with a thermal anemometer, was set to 35 litres/minute (providing 3 complete air changes per hour).
The ammonia concentration within the chamber was measured for each exposure group by the gas impingement technique. Daily measurements of ammonia concentration, temperature and relative humidity inside the chamber were made during each exposure period.

Duration and frequency of treatment / exposure:

Phase 1: single exposure of 24 hours
Phase 2: 3 or 7 day continuous exposure

Remarks:

Doses / Concentrations:
The rats were exposed to varying environmental ammonia concentrations (15-1157 ppm).

No. of animals per sex per dose / concentration:

Phase 1: 3 males/dose
Phase 2: 14 males/dose, divided into two subgroups of 7/dose

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

A positive control was not included.

Details on study design:

The purpose of the study was to determine if environmental ammonia was absorbed through the lungs of the rats into the blood and exerted an effect on blood pH, blood gases and hepatic drug metabolizing enzyme activity. In phase 1 of the study, rats were exposed to varying environmental ammonia concentrations (15-1157 ppm). Blood pH, pCO2, pO2 and blood ammonia concentrations were measured at 0, 8, 12 and 24 h post exposure. In phase 2, hepatic microsomal enzyme activity (ethylmorphine-N-demethylase and cytochrome P-450) was determined after a 3 or 7 day exposure to varying environmental ammonia concentrations (4-714 ppm).

Details on dosing and sampling:

In phase 1 of the study, rats were exposed to varying environmental ammonia concentrations (15-1157 ppm). Blood pH, pCO2, pO2 and blood ammonia concentrations were measured at 0, 8, 12 and 24 h post exposure. In phase 2, hepatic microsomal enzyme activity (ethylmorphine-N-demethylase and cytochrome P-450) was determined after a 3 and 7 day exposure to varying environmental ammonia concentrations (4-714 ppm).
All arterial blood samples were collected through a permanently implanted aortic cannula (fitted under general anaesthesia). The animals were placed in individual metabolism cages within the chamber. Blood samples were collected by connecting a 3 ml heparinised syringe to the adaptor needle on the cannula and withdrawing 1 ml of blood. Blood gas and pH determinations were done on 0.6 ml samples of blood, using a pH/blood gas analyser. Blood ammonia concentrations were measured on 0.3 ml samples using the glutamate dehydrogenase procedure.
On the 3rd and 7th days of exposure in phase 2, one subgroup was removed from the isolator, and the animals decapitated. The liver microsomal fraction was isolated from the right lateral and left liver lobes, frozen in liquid nitrogen, and stored at -70°C. At the time of analysis, the final protein concentration of the microsomal fraction was adjusted to 0.75 mg of microsomal proten per ml of solution.
The trachea and lungs were also removed following phase 2 exposure and perfused with 10% neutral buffered formalin for 1 week. Sections were prepared, then embedded and stained with haematoxylin and eosin for histological examination.

Statistics:

Two way ANOVA with repeated measures of one factor was applied to phase 1 data. A three way factorial analysis was used to analyse phase 2 data. Comparisons were made using the Newman-Keul technique. In phase 2, significant differences between daily values were determined by the orthogonal components technique. A linear regression was done on each time period in phase 1.

Preliminary studies:

No preliminary studies conducted.

Details on absorption:

No significant changes were found in the blood pH and pCO2. The pO2 and the microsomal enzymes had only minor changes. The blood ammonia concentration increased significantly in a linear fashion with increasing environmental ammonia concentrations indicating pulmonary absorption of ammonia.

Details on distribution in tissues:

No changes in the histologic appearance of the lungs or trachea.

Details on excretion:

No parameter measured.

Metabolites identified:

not measured

Details on metabolites:

No further information

No significant changes were found in the blood pH and pCO2. The pO2 and the microsomal enzymes had only minor changes. The blood ammonia concentration increased significantly in a linear fashion with increasing environmental ammonia concentrations indicating pulmonary absorption of ammonia. These levels also declined over time at higher concentrations suggesting that compensation occurred. There was no evidence of toxicity of ammonia during the exposure periods.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
No significant changes were found in the blood pH and pCO2. The pO2 and the microsomal enzymes had only minor changes. The blood ammonia concentration increased significantly in a linear fashion with increasing environmental ammonia concentrations indicating pulmonary absorption of ammonia.

Executive summary:

The purpose of the study was to determine if environmental ammonia was absorbed through the lungs of the rats into the blood and exerted an effect on blood pH, blood gases and hepatic drug metabolizing enzyme activity. In phase 1 of the study, rats were exposed to varying environmental ammonia concentrations (15-1157 ppm). Blood pH, pCO2, pO2 and blood ammonia concentrations were measured at 0, 8, 12 and 24 h post exposure. In phase 2, hepatic microsomal enzyme activity (ethylmorphine-N-demethylase and cytochrome P-450) was determined after a 3 and 7 day exposure to varying environmental ammonia concentrations (4-714 ppm). No significant changes were found in the blood pH and pCO2. The pO2 and the microsomal enzymes had only minor changes. The blood ammonia concentration increased significantly in a linear fashion with increasing environmental ammonia concentrations indicating pulmonary absorption of ammonia.

Description of key information

Gaseous ammonia is rapidly absorbed through the lungs. Significant dermal absorption is not considered likely. Ammonia is generated in the gastrointestinal tract by the bacterial flora and is readily absorbed. Ammonia is metabolised to urea in mammalian species and is excreted in the urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

100

Additional information

Oral absorption

Ammonia is generated by the bacterial flora of the gastrointestinal tract (~4 g/day) and as a very small water-soluble molecule, is likely to be rapidly and extensively absorbed. Based upon the ionisable/hydrophilic properties of Anhydrous ammonia, oral absorption is considered to be possible. This is also supported by the observed oral toxicity.

 

Inhalation absorption

The rather high vapor pressure of anhydrous ammonia promotes deposition in the deeper airways so that absorption by inhalation is expected. The results of a study in the rat (Schaerdel et al, 1983) demonstrate that the gaseous substance is absorbed into the bloodstream following inhalation exposure; this is consistent with the water solubility and small molecular size of the substance.

 

Dermal absorption

Significant dermal absorption is not considered to be likely under exposure scenarios where the integrity of the skin barrier is maintained. If the skin is compromised (e.g. in cases involving burns), dermal absorption may be more extensive.

 

Distribution

Ammonia is distributed to all tissues in the body and is capable of crossing the blood-brain barrier. Target organs were demonstrated in toxicity studies after oral and inhalation dosing (demonstrating mortality, diarrhea, increased absolute and relative kidney weights, relative liver weights increased).

 

Metabolism

The physiological role of ammonia as a product of normal metabolism (protein catabolism) is very well characterised. Ammonia is rapidly detoxified in the liver by the urea or glutamine cycle (see Context below).

 

The urea cycle consists of five enzymes: carbamoylphosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase. The initial reaction of the urea cycle is the formation of carbamoyl phosphate from ammonia and bicarbonate, a reaction catalysed by CPS I, which requires N-acetylglutamate as an allosteric cofactor. Condensation of carbamoyl phosphate with ornithine yields citrulline (by OTC); this in turn condenses with aspartate to give argininosuccinate (by ASS), a reaction that requires the cleavage of two further high-energy phosphate bonds. Argininosuccinate is hydrolysed to fumarate and arginine (by argininosuccinase). Arginine is cleaved by arginase to give urea and ornithine. OTC, like CPS I, is also a major mitochondrial protein; the remaining enzymes are in the cytoplasm of hepatocytes. Urea synthesis cannot therefore be saturated at realistic substrate concentrations. The urea cycle is high capacity and the capacity also increases short-term and long-term in response to increased demand (e.g. in response to dietary protein intake). It is estimated that some 7 to 25% of the ammonia delivered via the portal vein escapes periportal urea synthesis and is used for glutamine synthesis

 

Excretion

Excretion via the urine is expected because Anhydrous ammonia is well soluble in water. Ammonia is rapidly detoxified in mammals by conversion to urea by the urea cycle in liver cells and is subsequently excreted (as urea) in urine following glomerular filtration. Ammonium ions (NH4 +) are also excreted by the kidney. Hepatic excretion of urea (15 -30% of that generated) results in the generation of ammonia by the gastrointestinal flora, and subsequent reabsoprtion.

 

Context

The body excretes approximately 30 g urea/day; urea is synthesised by the hepatic urea cycle from ammonia generated by protein catabolism. Therefore it can be calculated that the body typically produces 16 g/day ammonia , although this figure is influenced by the amount of dietary protein. Therefore it can be concluded that exposure to ammonia at quantities in this range will be without toxicological effect. Ammonia is toxic and therefore exposures greatly exceeding the normal production may overwhelm the normal detoxification mechanisms.

 

The normal serum level of ammonia is 0.15 -100 ug/dL ( In healthy adults the physiological ammonium level in blood is typically below 35μmol/L (0.67 mg/L = 67 µg/dL) (EFSA, 2012)). Assuming a blood volume of 5L (55% serum), this is equivalent to approximately 0.4 -2.75 mg ammonia present in the blood of a normal adult at any point in time. However it is notable that, due to the generation of ammonia by gastrointestinal tract bacteria, the concentration of ammonia in the hepatic portal circulation is much higher than that in the rest of the systemic circulation.

 

A health risk assessment of ammonium released from water filters was performed by EFSA (2012). The European Commission asked the European Food Safety Authority (EFSA) for scientific assistance regarding the possible impact on human health of exposure to ammonium released from water filter cartridges.

Ammonium is rapidly absorbed via the gastrointestinal (GI) tract (Conn, 1972; ATSDR, 2004). Several studies in human volunteers orally administered 15N-labelled ammonium indicated an almost complete absorption with subsequent excretion in the urine or retention in the organism, where the nitrogen is used for the synthesis of biomolecules or enters in the urea cycle (Richards et al., 1975; Patterson et al., 1995; Metges et al., 1999). In the GI tract, the bacterial degradation of nitrogenous compounds present in food or of secreted urea is estimated to produce about 3 to 4 g/day of endogenous ammonium (ATSDR, 2004; EFSA, 2011). The majority of the endogenously produced ammonium in the GI tract is then absorbed and transported to the liver.

 

Endogenous ammonium is ubiquitously present in humans and animals. In healthy adults, the physiological ammonium levels in blood are normally below 35 µmol/L (corresponding approximately to 0.67 mg/L) (Häussinger, 2007). In humans, most of the ammonium absorbed in the GI tract is metabolised in the liver and it has only limited influence upon the systemic levels of ammonium. For instance, only small, transient increases in blood levels were observed 15 minutes after the oral administration of 44.4 mg/kg bw of ammonium chloride (corresponding to 15 mg ammonium/kg bw) to male and female healthy volunteers (Conn, 1972). Due to its positive charge, ammonium entering the systemic circulation has a limited mobility in the organism. However, under physiological pH of 7.4 ammonium is in equilibrium with its neutral form ammonia, which can diffuse through cell membranes including the blood-brain barrier and distribute into tissues (ATSDR, 2004). The fraction of ammonia in equilibrium with ammonium is less than 5 % under physiological pH.11. However, toxic ammonia concentrations in the body can be reached in subjects with reduced ammonia metabolism or urea excretion (e.g. enzyme deficiencies due to genetic disorders, or severely impaired hepatic or renal functions) (Van de Poll et al., 2008; Ryan and Shawcross, 2011).

 

In the liver, ammonium is metabolised either via the formation of urea (which is considered the main metabolic pathway) or via the formation of glutamine (ATSDR, 2004; Häussinger, 2007). While the metabolism to urea mainly occurs in the liver, formation of glutamine from ammonium is observed also in other tissues, notably in the brain (ATSDR, 2004). Evidence shows that the hepatic metabolism can efficiently control the physiological ammonium levels even under partial hepatic functionality or high exogenous uptake (Häussinger, 1990; ATSDR. 2004; Van de Poll et al., 2008).

 

In humans, urinary excretion of urea is the main elimination pathway of both exogenous and endogenous ammonium. Oral administration of ¹⁵N-labelled ammonium salts to human volunteers led to a urinary excretion up to approximately 70 % of the administered ammonium dose as urea (Richards et al., 1975; ATSDR, 2004)

 

References:

ATSDR (Agency for Toxic Substances and Disease Registry), 2004. Toxicological profile for ammonia. U.S. Department of Health and Human Services, Atlanta, Georgia, 269 pp.

Conn HO, 1972. Studies of the sources and significance of blood ammonia IV. Early ammonia peaks after ingestion of ammonium salts. Yale Journal of Biology and Medicine, 45, 543-549.

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids, 2011. Scientific Opinion on Flavouring Group Evaluation 46 Revision 1 (FGE.46Rev1). Ammonia and three ammonium salts from chemical group 30. EFSA Journal 2011; 9(2):1925, 35 pp.

EFSA Health risk of ammonium released from water filters, European Food Safety Authority. EFSA Journal 2012;10(10):2918

Häussinger D, 2007. Ammonia, urea production and pH regulation. In: The Textbook of Hepatology: from basic science to clinical practice, 3rd Edition. Eds Rodes J, Benhamou J-P, Blei A, Reichen J and Rizzetto M. Wiley-Blackwell, 181-192.

Metges CC, Petzke KJ, El-Khoury AE, Henneman L, Grant I, Bedri S, Regan MM, Fuller MF and Young VR, 1999. Incorporation of urea and ammonia nitrogen into ileal and fecal microbial proteins and plasma free amino acids in normal men and ileostomates. American Journal of Clinical Nutrition, 70, 1046-1058.Patterson BW, Carraro F, Klein S and Wolfe RR, 1995. Quantification of incorporation of [¹⁵N]ammonia into plasma amino acids and urea. American Journal of Physiology – Endocrinology and Metabolism, 269, E508-E515.

Richards P, Brown CL, Houghton BJ and Wrong OM, 1975. The incorporation of ammonia nitrogen into albumin in man: the effects of diet, uremia and growth hormone. Clinical Nephrology, 3, 172-179.

Ryan J and Shawcross DL, 2011 Hepatic encephalopathy. Medicine, 39, 617-620.

Van de Poll MCG, Ligthart-Melis GC, Olde Damink SWM, van Leeuwen PAM, Beets-Tan RGH, Deutz NEP, Wigmore SJ, Soeters PB and Dejong CHC, 2008. The gut does not contribute to systemic ammonia release in humans without portosystemic shunting. American Journal of Physiology – Gastrointestinal and Liver Physiology, 295, G760-G765.