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Genetic toxicity in vitro

Description of key information

No genetic toxicity study with diammonium hydrogen citrate is available, thus the genetic toxicity will be addressed with existing data on the individual moieties ammonium and citric acid/citrates.

Diammonium hydrogen citrate is not expected to be genotoxic, since the two moieties ammonium and citric acid/citrates have not shown gene mutation potential in bacteria.

Additional information

Substance specific information on acute oral toxicty are not available for diammonium hydrogen citrate. Hence, a read-across concept was developed:

Read-across - diammonium hydrogen citrate:

The target substancediammonium hydrogen citrate(CAS # 221-146-3, EC # 221-146-3) is a mono-constituent substance that consists of two monovalent ammonium cation and a divalent hydrogencitrate anion, designated as inorganic salt (IUPAC 2005). Upon dissolution,diammonium hydrogen citrateliberates ammonium cations and (depending on pH) citrate anions, which represent the sub-categories. In the following, “citrate anions” represents the anions citrate, hydrogencitrate and dihydrogencitrate as well as citric acid.Citrate, hydrogencitrate, dihydrogencitrate and citric acid are considered as common anion based on theequilibrium between those anions in aqueous solutions under physiological/environmental conditions depending on pH value which is clearly described in published literature and summarised in the following equations:

Diammonium hydrogen citrate consists of two ammonium [NH4]+ ions and one hydrogen citrate ion [C6H6O7]2-. Based on the solubility of diammonium hydrogen citrate in water (1,000 g/L at 20°C), a complete dissociation of diammonium hydrogen citrate, resulting in ammonium (NH4+) and (hydrogen) citrate, may be assumed.

 

Depending on solution pH, citrate anions exist as citrate, hydrogencitrate, dihydrogencitrate and citric acid  species in aqueous solutions under physiologically/environmentally relevant conditions as summarised in the following equations:

 

C6H8O7+ H2O <->(C6H7O7)- + H3O+                       [pka1: 3.13; citric acid             <->dihydrogencitrate]

(C6H7O7)- + H2O <-> H3O++(C6H6O7)2-                 [pKa2: 4.76;dihydrogencitrate <-> hydrogencitrate]

(C6H6O7)2-+ H2O <->(C6H5O7)3-+H3O+                [pKa3:6.40; hydrogencitrate    <->citrate]

Ammonium cations are formed by an acid-base reaction of e.g. H2O (or other, stronger aids) and NH3as follows:

NH4++ OH-<->NH3+ H2O                                        [pka:9.25; ammonium            <-> ammonia] 

NH4+and NH3coexist in aqueous solution in adynamic pH-dependent equilibrium. Under basic conditions (pH >10), ammonia (NH3) redominates whereas theammoniumion (NH4+) is the dominant species in weakly basic to neutral (environmental) conditions. With decreasing pH, the ammonium cation becomes the only species. A Hägg-graph representing the described equilibrium in solution is provided in Section 13 (Read-across assessment report) using the above given pka[1]value at 25°C

[1]Weast, R.C. (ed.) (1974) CRC Handbook of Chemistry and Physics, 55th ed. CRC Press.

Since the target substance and the source substance release the (eco-)toxicological relevant units under environmental/physiological relevant conditions, the overall ecotoxicity/toxicity of the dissociated diammonium hydrogen citrate can be interpolated by assessing the (eco-)toxicity of the individual moieties. The category hypothesis, i.e. release of the common (eco-)toxicological units, applies to the target substance and the source substances.Thus, the category consists of ammonium salts for which the (eco-)toxicity is either governed by the ammonium cation (sub-category 1) or the citric acid anion (sub-category 2).

Genetic toxicity in vitro - ammonium:

Ammonium ion is endogenously produced in the human digestive tract, much of it arising from the bacterial degradation of nitrogenous compounds from ingested food. About 4,200 mg/day are produced, greater than 70% of which is synthesized or liberated within the colon and its fecal contents. The total amount absorbed is about 4,150 mg/day, or 99% of the amount produced (Summerskill and Wolpert 1970); absorption after oral loading of NH4 + is similarly complete (Fürst et al. 1969). Evidence fromCastell and Moore (1971) and Mossberg and Ross (1967) suggests that absorption of NH4 + increases as the pH of the contents of the lumen increases, and that the ammonium ion is actively transported at the lower pH levels (pH 5 was lowest detected absorption). Ammonium ion absorbed from the gastrointestinal tract travels via the hepatic portal vein directly to the liver, where in healthy individuals, most of it is converted to urea and glutamine. Human and animal data show that little of it reaches the systemic circulation as ammonia or ammonium compounds, but that it is a normal constituent of plasma at low levels (Brown et al. 1957; Pitts 1971; Salvatore et al. 1963; Summerskill and Wolpert 1970). Analysis of plasma drawn from 10 healthy young male subjects yielded endogenously derived NH4+ concentrations ranging from 30 to 55μg NH3/100 mL, with a mean of 39μg/100 mL (Brown et al. 1957).

Ammonia/ammonium is an essential mammalian metabolite for DNA, RNA, and protein synthesis and is necessary for maintaining acid-base balance. Ammonia is produced and used endogenously in all mammalian species. It has been estimated that up to 17 grams of ammonia are produced in humans daily. Of these 17 grams, approximately 4 grams are produced in the gut by intestinal bacteria, where it enters the portal circulation and is metabolized rapidly in the liver to urea. Ammonia is excreted primarily as urea and urinary ammonium compounds through the kidneys. Levels of ammonia in the blood from healthy humans range from 0.7 to 2 mg/L (ATSDR,2004). 

 

Both ammonium chloride and ammonium sulphate gave negative results when tested in bacterial reverse mutation assays (OECD, 2003, 2004). Overall, based on this information ammonium shows no genotoxic potential.

 

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.

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.

Summerskill WHJ, Wolpert E. 1978. Ammonia metabolism in the gut.Am J Clin Nutr 23:633-639

Fürst P, Josephson B, Maschio G, et al. 1969.Nitrogen balance after intravenous and oral administration of ammonium salts to man. J Appl Phys 26:13-22.

Mossberg SM, Ross G. 1967. Ammonia, movement in the small intestine: Preferential transport by the ileum. J Clin Invest 46(4):490-498.

Brown RH, Duda GD, Korkes S, et al. 1957. A colorimetric micromethod for determination of ammonia; the ammonia content of rat tissues and human plasma. Arch Biochem Biophys 66:301-309.

Genetic toxicity - citric acid/citrates:

Genetic toxicity in vitro

Two publications could be identified that investigated the mutagenic potential of citric acid, as described below:

Ishidate et al. (1984) evaluated the mutagenic potential of citric acid in a bacterial reverse mutation assay conducted equivalent or similar to OECD 471. The substance in phosphate buffer was tested using S. typhimurium strains TA1535, TA1537, TA98, TA100, TA92 and TA94 with metabolic activation. Six concentrations were tested (max. concentration was 5.0 mg/plate; no further information on concentrations were given). A negative or solvent control was used concurrently. The preincubation method was used and the concentrations were tested in duplicates. Citric acid was found to be non-mutagenic under the given experimental conditions.

Al-Ani and Al-Lami (1988) investigated the mutagenic potential of citirc acid anhydrate in a bacterial reverse mutation assay conducted equivalent or similar to OECD 471. The substance in distilled water was tested using S. typhimurium strains TA97, TA98, TA100 and TA104 with and without metabolic activation. Citric acid was applied at concentrations of 500, 1000 and 2000 µg/plate using the plate incorporation method and the concentrations were tested in triplicates. A solvent control and a positive control were run concurrently. Citric acid anhydrate was found to be non-mutagenic under the given experimental conditions. No cytotoxicity was observed.

Furthermore, two publications were identified, which evaluated the mutagenic potential of potassium citrate and sodium citrate, which could be used to analyse the potential of mutagenicity of citric acid.

Anonymous (1975) examined the mutagenic potential of potassium citrate in a bacterial reverse mutation assay conducted equivalent or similar to OECD 471. The substance in DMSO was tested using S. typhimurium strains TA1535, TA1537 and TA1538 with and without metabolic activation. Potassium citrate was applied at concentrations of 0.001, 0.002 and 0.004 % using the plate incorporation method and the concentrations were tested in duplicates. A solvent control and positive controls were run concurrently. Potassium citrate was found to be non-mutagenic and no cytotoxicity was observed during the study.

Anonymous (1975) examined the mutagenic potential of sodium citrate in a bacterial reverse mutation assay conducted equivalent or similar to OECD 471. The substance in DMSO was tested using S. typhimurium strains TA1535, TA1537 and TA1538 with and without metabolic activation. Sodium citrate was applied at concentrations of 0.000625, 0.001250 and 0.002500 % using the plate incorporation method and the concentrations were tested in duplicates. A solvent control and positive controls were run concurrently. Sodium citrate was found to be non-mutagenic and cytotoxicity was only observed in the TA1537 strain with metabolic activation at 0.002500 %.

Although these studies had some experimental and reporting deficiencies (e.g. individual data missing; historical data missing; number of strains too low; number of tested concentrations too low or concentrations were not clearly defined; confirmatory test missing), combined these studies enable to conclude on the mutagenic potential of citric acid. All mutagenicity tests showed the non-mutagenic potential of the substances and it can be concluded that citric acid should be considered to be non-mutagenic. Furthermore, HERA (2005) also concluded that "citric acid is not mutagenic in vitro and in vivo".

Reference:

- Hera (2005) Substance:Citric acid and salts (CAS# 77 -92 -9; 5949 -29 -1; 6132 -04 -3) Edition 1.0 - April 2005. p. 1- 6.

Genetic toxicity in vitro - diammonium hydrogen citrate:

Diammonium hydrogen citrate is not expected to be genotoxic, since the two moieties ammonium and citric acid/citrates have not shown gene mutation potential in bacteria. Further testing is not required. Thus, diammonium hydrogen citrate is not to be classified according to regulation (EC) 1272/2008 as genetic toxicant. For further information on the toxicity of the individual constituents, please refer to the relevant sections in the IUCLID.  

Justification for classification or non-classification

Diammonium hydrogen citrate is not expected to be genotoxic, since the two moieties ammonium and citric acid/citrates have not shown gene mutation potential in bacteria. Thus, diammonium hydrogen citrate is not to be classified according to regulation (EC) 1272/2008 as genetic toxicant.