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Genetic toxicity in vitro (weight of evidence)

Two reliable Ames tests with the source chemical 2 -Aminoethanol are available, performed according to protocols similar to OECD guideline 471 and using test substance concentrations up to 5000 μg/plate (JETOC, 1996) and up to 2000 μg/plate (Dean et al., 1985a). In the first study, mutagenicity of 2 -Aminoethanol was tested in Salmonella typhimurium (TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538) strains and Escherichia coli (WP2uvrA and WP2uvrA/pKM101) strains; in the second study, its mutagenicity was tested in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Escherichia coli WP2 tyr strains. In both tests, the test substance was found to be not mutagenic, both in the presence and the absence of metabolic activation. Concentrations >= 2000 µg/plate of the test substance were cytotoxic. The substance 2 -Aminoethanol was also negative in a chromosome aberration test in rat hepatocytes, performed according to the protocol similar to OECD guideline 473, at concentrations of 100 -400 μg/mL in the absence of a metabolic activation system (Dean et al., 1985b). No cytotoxicity was observed, but the highest concentration corresponded to the limit dose of 10 mM. In a reliable in vitro gene mutation study in mammalian cells (Chen et al., 1984), 2 -Aminoethanol was tested in a HGPRT assay with Chinese hamster lung fibroblasts (V79) in the absence of metabolic activation. Again, negative results were observed.

The source chemical Sodium nitrate was tested for mutagenicity in the Ames test with the S. typhimurium strains TA 92, TA 94, TA 98, TA 100, 1535 and TA 1537 (Ishidate et al., 1984a). The potential of Sodium nitrate to induce reverse mutation was tested at 6 concentrations up to 5000 µg/plate using the preincubation test method, both with and without a mammalian metabolic activation system (S9 -mix). The treatment with the test substance did not induce a relevant increase in the number of revertant colonies over background at any of the tested concentrations with or without S9 -mix. No bacteriotoxic effects were observed up to 5000 µg/plate. Based on this result, the test substance was not mutagenic under the chosen experimental conditions. In the same publication (Ishidate et al, 1984b), information on a chromosome aberration assay with Sodium nitrate was provided. In this assay, which was considered to be substantially less reliable as compared with the chromosome aberration assay conducted by NOTOX BV (2010; 493631; see below), incubation with the test substance for 24 and 48 hours resulted in structural chromosomal aberrations in a Chinese hamster fibroblast cell line (CHL cells). Of note, no metabolic activation system was used, the selected exposure durations were relatively long with 24 and 48 hours and cytotoxicity was not assessed in this study. In a recent GLP guideline study by NOTOX B.V. (2010; 493631) the ability of Sodium nitrate to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated. The study consisted of a cytotoxicity range-finder followed by two independent cytogenetic tests, each conducted in the absence or presence of a mammlian metabolic activation system (S9 -mix). Concentrations of 100, 333 and 850 µg/mL were used for the main experiments. In the first cytogenetic test cells were exposed for 3 hours and fixed after 24 hours with and without S9 -mix. In the second cytogenetic test 24 hour exposure with 24 hour fixation and 48 hour exposure with 48 hour fixation were tested without S9 -mix, and 3 hour exposure with 48 hour fixation was tested with S9-mix. Per culture, 100 metaphase chromosome spreads were evaluated. Under the experimental conditions described, Sodium nitrate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations when tested with and without S9 -mix. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed. The number of cells with chromosome aberrations in the solvent control was within the laboratory historical control data range. The positive control substances induced the expected increases in chromosome aberration, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Genetic toxicity in vivo (weight of evidence)

In an in vivo micronucleus test performed with the source chemical 2 -Aminoethanol according to OECD guideline 474 and GLP, 375, 750 and 1500 mg/kg bw of the test substance was administered orally by gavage to groups of 5 NMRI mice/sex (BASF AG, 1995; 26MO221/944213). Signs of toxicity were observed in the mid and high dose level groups. After single application of the test substance, the animals were sacrificed 24 or 48 hours post-dosing and bone marrow slides were prepared. 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 2 -Aminoethanol showed no chromosome-damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis.

The source chemical Sodium nitrate was tested in vivo for its potential to induce heritable translocations in germ cells of male C3H mice (Alantić et al., 1988a). For this purpose, the test substance, dissolved in distilled water, was administered once daily to parental males by oral gavage for a period of 14 days at dose levels of 600 and 1200 mg/kg bw/day. Water was used as the vehicle control, administered by the same route. Ten days after the last treatment, each male was mated with two virgin females (7 -day mating period). Three weeks after birth, F1 males were taken out and left until maturation (10 -12 weeks). Heritable translocation, cytogenetic changes and sperm abnormalities were assessed. The germ cell stages analysed were spermatids (for the heritable effects) and differentiating and stem-cell spermatogonia (for direct effects). Under the conditions of this study, a lack of heritable translocations, sperm abnormalities and morphological changes was demonstrated in F1 males originating from treated P males. Significant effects were restricted to the subacutely treated parental males, which displayed increased sex chromosomal univalency at both dose levels and a slightly increased number of abnormal sperm heads at the high dose level. In this study, Sodium nitrate was not mutagenic. The same authors (Alantić et al., 1988b) investigated potential effects of Sodium nitrate on spermatids of C3H mice teated by oral gavage at dose levels of 600 or 1200 mg/kg bw/day for a period of 3 days by measuring unscheduled DNA synthesis (UDS). The vehicle distilled water served as negative control item and Methyl methanesulfonate (MMS, 75 mg/kg bw i.p.) as positive control item. UDS was determined 17 days after the end of treatment and sperm head abnormality was assessed 11 and 17 days after treatment. Sodium nitrate did not induce the UDS response in early to mid spermatids (17 days) and was thus considered to be non-mutagenic. The positive control substance MMS induced a marked UDS response, which was considered to confirm sensitivity of the test system. In addition, treatment with Sodium nitrate did not result in an increased number of abnormal sperm heads under the conditions of this study. In a study by Luca et al. (1985) the mutagenic activity of Sodium nitrate was assessed in vivo by evaluating its ability to induce micronuclei and chromosomal aberrations in bone marrow cells of Swiss mice and Wistar rats, respectively, following acute (two gavage treatments in 24 hours; mice and rats) or subacute exposure (daily gavage treatments for 2 weeks; rats only) at dose levels of 108, 323, 969 or 2906 mg/kg bw/day. After acute exposure to Sodium nitrate, no significant difference between the frequency of chromosomal aberrations in the exposed and control groups was detected in either rats or mice with the exception of mice receiving 969 mg/kg bw. Rats treated repeatedly for two weeks with a long sampling time (24 hours) displayed significant increases in aberrant metaphases. This repeated dosing protocol combined with the long sampling time was not compliant with testing requirements, and since settings with acute dosing did not result in the induction of chromosome aberrations, this positive finding was considered not reliable. In the micronucleus study in mice, two acute doses of Sodium nitrate resulted in an induction of micronuclei. However, there were indications for cytotoxic effects of Nitrate on bone marrow cells.


Justification for selection of genetic toxicity endpoint
No endpoint selection was conducted, since several reliable studies with the relevant source chemicals were identified to assess the genetic toxicity hazard displayed by the target chemical (weight-of-evidence).

Short description of key information:
The reliable in vitro and in vivo studies on genetic toxicity conducted with the source chemicals 2-Aminoethanol and Sodium nitrate demonstrated no relevant genotoxic hazard in aqueous formulations. The few positive/ambiguous results obtained for Sodium nitrate were considered incidental and at least in part related to methodological deficiencies. On the basis of the overall weight-of-evidence, the target chemical (2-Hydroxyethyl)ammonium nitrate, which is the 1:1 salt of the source chemicals, is identified as a substance without genotoxic activity.
Please refer also to the read-across and weight-of-evidence statement provided in section 13.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of reliable studies obtained in vitro and in vivo for the source chemicals 2 -Aminoethanol and Sodium nitrate (weight-of-evidence), the target chemical (2 -Hydroxyethyl)ammonium nitrate is not considered to be subject to classification for genetic toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (GHS/CLP).