Reaction mass of 2-(methylamino)ethanol, compound with sulphur dioxide and bis[(2-hydroxyethyl)ammonium] sulphite

Endpoint summary

Additional information

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.In a second available studyperformed according to OECD guideline 476 and GLP, 2 -Aminoethanol was tested in the mouse lymphoma (L5178Y TK+/-) forward mutation assay, with and without metabolic activation, using test substance concentrations up to 610 μg/ml (Dow Chemical Company, 1999). No cytotoxicity was observed, but the highest concentration corresponded to the limit dose of 10 mM.

In the bacterial gene mutation assay, N-Methylethanolamine was tested in the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 up to a concentration of 3333 µg/plate with and without metabolic activation (Zeiger, 1987). No mutagenic effects were detected. Additionally, a chromosome-aberration test was performed using V79 cells at concentrations ranging from 50 to 800 µg/mL. Methylethanolamine did not lead to a relevant increase in the number of structural chromosomal aberrations in the absence and presence of metabolic activation (BASF AG, 2008). The types and frequencies of structural chromosome aberrations were close to the range of the concurrent negative control values at both sampling times and clearly within in the range of the historical negative control data. A mammalian gene mutation assay according to OECD 476 was performed under GLP. Here, mouse lymphoma L5178Y cells were treated with concentrations from 50 to 600 µg/mL in the absence of S-9 and from 100 to 750 µg/mL in the presence of S-9 mix. Methylethanolamine did also not induce mutation at thehprtlocus up to highly toxic concentrations tested (BASF AG, 2010).

The source chemical Sodium sulfite was tested for mutagenicity in the Ames test with the S. typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 (BASF AG, 1989). The bacterial strains were exposed to 20 - 5000 µg/plate with and without S9-mix. The cytotoxicity was determined. No bacteriotoxic effects were observed. An increase in the number of his+ revertants was not observed without or with S9 -mix. The test substance was not mutagenic under the chosen experimental conditions.

The source chemical Sodium metabisulfite was assayed for mutagenicity in the Ames test with the S. typhimurium strains TA 1535, TA 100, TA 1537 and TA 98. The bacterial strains were exposed to 20 - 5000 µg/plate in the standard and preincubation test, both with and without S9 –mix (BASF AG, 1989). The cytotoxicity was determined. No bacteriotoxic effects were observed. An increase in the number of his+ revertants was not observed, without or with S-9 mix. Sodium metabisulfite was also tested for the ability to induce mutation at the Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells (Covance Laboratories Ltd, 2010). The study consisted of a cytotoxicity range-finder experiment followed by three independent experiments. A 3-hour treatment incubation period was used for all experiments. In the range-finder experiment, concentrations were tested in the absence and presence of S9 mix, ranging from 59.44 to 1902 µg/mL (equivalent to 10 mM at the highest concentration tested). At 1902 µg/mL, 37 % and 50 % relative survival (RS) was detected in the absence and presence of S9 mix, respectively. In Experiment I, concentrations ranging from 200 to 1902 µg/mL were tested in the absence and presence of S9 mix. The highest concentration analysed gave 43 % and 65 % RS with and without S9 mix, respectively.In Experiment II, 1902 µg/mL gave 30 % and 75 % RS with and without S9-mix. In Experiment III, 1902 µg/mL gave 43 % RS with S9-mix. In Experiment I in the presence of S9 mix, statistically significant increases in mutant frequency were observed at 1600 and 1902 µg/mL. However, there was no significant linear trend. These data did not fulfil all the criteria for a positive result and were therefore considered equivocal. In Experiment I without S9 mix and Experiment II in the absence and presence of S9 mix, no significant increases in mutant frequency were observed at any concentration, indicating a negative result. In order to confirm this, a third experiment was performed. No statistically significant increases in mutant frequency were observed, indicating a negative result.

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). 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 sulfite was tested for chromosomal damage induction (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method (BASF SE, 2008). For this purpose, the test substance, dissolved in purified water, was administered once subcutaneously to groups of male animals at dose levels of 250, 500 or 1000 mg/kg bw in a volume of 10 mL/kg bwe in each case. Purified water was used as the vehicle control, administered by the same route. Cyclophosphamide and Vincristine sulfate were used as positive control substances. The animals were sacrificed and the bone marrow of the 2 femora was prepared 24 and 48 hours after administration in the highest dose group of 1000 mk/kg bw and in the vehicle controls. In the test groups at 500 and 250 mg/kg bw and in the positive control groups, the 24 -hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes (PCEs) were evaluated per animal and investigated for micronuclei. The normocytes (NCEs) with and without micronuclei occurring per 2000 PCEs were also recorded. According to the results of this study, the single subcutaneous administration of the test substance did not lead to any relevant increase in the number of PCEs containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Under the experimental conditions, the test substance had no chromosome damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Justification for selection of genetic toxicity endpoint
Several reliable studies with the relevant source chemicals were identified to assess the genetic toxicity hazard displayed by the target chemical (weigh-of-evidence).

Short description of key information:
The in vitro and in vivo studies on genetic toxicity conducted with the source chemicals Methylethanolamine, 2-Aminoethanol or Sodium sulfite/Disodium disulfite demonstrated that none of the source chemicals displayed a genotoxic hazard in aqueous formulations. On the basis of the weight-of-evidence, the target chemical Bis[(2-hydroxyethyl)ammonium] sulfite, which is the salt of the source chemicals, is identified as a substance without genotoxic activity.

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 Methylethanolamine, 2-Aminoethanol and Sodium sulfite/Disodium disulfite (weight-of-evidence), the target chemical Bis[(2 -hydroxyethyl)ammonium] sulfite 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)