Tris[(2-hydroxyethyl)ammonium] citrate

Endpoint summary

Additional information

Tris[(2 -hydroxyethyl)ammonium] citrate is a salt of monoethanolamine and citric acid and is expected to dissociated into the respective monoethanolammonium cation and dihydrogen citrate anion (which may subsequently undergo (partial) dissociation to monohydrogen citrate and citrate anions) upon uptake by the body. Therefore it is considered to be acceptable to derive lacking information on toxicological properties of tris[(2 -hydroxyethyl)ammonium] citrate by read-across from its starting materials. Citric acid is an important intermediate of the Krebs cycle (also known as citric acid cycle), and therefore occurs naturally as a metabolite in virtually all living organisms. It is also used as a natural food preservative and a food additive. The average daily intake (ADI) is not limited according to the evaluation ofJoint FAO/WHO Expert Committee on Food Additives (1973), which concluded that the substance is non-hazardous to men. Therefore the toxicological behavior of tris[(2-hydroxyethyl)ammonium] citrate is expected to be governed primarily by the toxicity of monoethanolamine. Nevertheless available data on citric acid have also been taken into account.

In vitro studies

Gene mutations in bacteria

Monoethanolamine gave negative results in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538 strains, and Escherichia coli (WP2 tyr-) strains both in the presence and the absence of metabolic activation at concentration levels up to 2000 µg/plate (Dean et al., 1985a). Concentrations >= 2000 µg/plate of the test substance were cytotoxic.

For citric acid, an Ames test with Salmonella typhimurium strains TA1535, TA100, TA98, TA1537, TA92 and TA94, performed according to protocol similar to OECD Guideline 471 (Ishidate et al., 1984), with and without metabolic activation is available. Concentrations up to 5000 µg/plate were tested. Negative results were observed in all strains both with and without metabolic activation.

Chromosome aberrations in mammalian cells

The ability of monoethanolamine to induce chromosome aberrations in mammalian cells in vitro was studied in rat hepatocytes, in the absence of metabolic activations, at concentration levels 100 - 400 µg/mL (Dean et al., 1985b). Monoethanolamine gave negative results in the study. No information on cytotoxicity was reported.

Citric acid was tested in an in vitro micronucleus test (Yilmaz et a., 2008) in peripheral human lymphocytes at concentration levels 50, 100, 200 and 3000 µg/mL in the absence of metabolic activation, using 72 hours exposure duration. The study was performed according to a protocol similar to OECD Guideline 487 (draft). Cytotoxicity was observed at the highest concentration. A statistically significant, dose-dependent increase in the percentage of binucleate cells with micronuclei was observed. It is concluded that the test substance is positive for the induction of micronuclei in cultured human lymphocytes under the conditions of this study.

Gene mutations in mammalian cells

The ability of monoethanolamine to induce gene mutations in mammalian cells was studied in the mouse lymphoma assay, performed according to OECD Guideline 476 and under GLP (Dow, 1999). In this study, the concentrations of 38.1, 76.3, 152.5, 305 and 610 µg/mL were used, both with and without metabolic activation, with the highest concentration representing the limit dose of 10mM. No cytotoxicity was observed. Monoethanolamine did not induce an increase in the mutation frequency both with and without metabolic activation at any concentration level.

No data on citric acid are available. According to the endpoint specific REACH guidance on mutagenicity (chapter R.7A p374-402) and as indicated in Table R.7.7-5 of this chapter, no in vitro gene mutation study is needed in case the following test results are obtained: 1) negative gene mutation test in bacteria (Ames test), 2) positive in vitro chromosome aberration test, and 3) negative in vivo chromosome aberration test (see point 9 of the Table R.7.7-5). Since for citric acid a negative Ames test (gene mutation), a positive in vitro chromosome aberration test, and a negative in vivo chromosome aberration test are available, the performance of an in vitro gene mutation test is not required under REACH.

In vivo studies

One micronucleus test with mice with monoethanolamine, performed according to OECD Guideline 474 and under GLP, was available for assessment (BASF AG, 1995). Monoethanolamine was administered orally at a single dose of375, 750 or 1500 mg/kg bw to groups of 5 male and female mice. Animals were sacrificed 24 or 48 h post-dosing and bone marrow slides were prepared.Signs of toxicity were observed in the mid and high dose level groups. There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups or between the two sacrifice intervals. Based on the results of the study, it was concluded that monoethanolamine gave negative results in the study.

For citric acid, in vivo dominant lethal assay and chromosome aberration assay are available (Litton, 1975). In the dominant lethal assay, performed according to a protocol similar to EU Method B.22, 10 treated male rats were mated with two virgin female rats each week for 7 (test 1) or 8 (test 2) weeks. Two weeks after mating, female rats were sacrificed and the fertility index, preimplantation loss and lethal effects were determined and compared with those same parameters from negative and positive control animals. The animals received either a single application of the test substance by intraperitoneal injection, or 5 consecutive daily intraperitoneal injections. The dose levels were 1.2, 12.0 and 120 mg/kg bw in test 1, and 300, 500 and 3500 mg/kg bw/day in test 2. A statistically significant increase in resorptions was noted in a single week of the 7 (single dose) or 8 (repeated doses) studies in test 1 in the acute study. Test 2, which was run consequently, showed no evidence of any effects. It is concluded that citric acid is negative for the induction of dominant lethals under the conditions of the study.

In the chromosome aberration assay with male rats, performed according to a protocol similar to OECD Guideline 475, animals received either a single intraperitoneal injection of the test substance, or 5 consecutive daily injections of the test substance. After a single administration, animals were sacrificed at 6, 24 and 48 hours past administration. After 5 consecutive injections, 4 hours after the final administration, 4 mg/kg Colcemid was given intraperitoneally. Animals were sacrificed 2 hours later. Two tests were performed, test 1 and test 2. The dose levels were 1.2, 12.0, 120 mg/kg bw in test 1 and 300, 500, 3000, 3500 mg/kg bw in test 2. No treatment-related increase in the number of cells with aberrations was observed. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in vivo under the conditions of the test.

In summary, neither monoethanolamine nor citric acid are genotoxic in vivo. Based on this, tris[(2 -hydroxyethyl)ammonium] citrate is also considered to be not genotoxic.

Short description of key information:
Based on the read-across with the starting materials monoethanolamine and citric acid, tris[(2-hydroxyethyl)ammonium] citrate is considered to be not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the read-across with the starting materials monoethanolamine and citric acid, classification of tris[(2-hydroxyethyl)ammonium] citrate for genotoxicity is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and EU Directive 67/548/EEC.