2-dibutylaminoethanol

Endpoint summary

Additional information

1) In vitro data

- Gene mutation in bacterial cells:

In the first bacterial reverse mutation assay conducted by the Ministry of Health Japan (2011) according to OECD Guideline 471 and GLP, the test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvrA in a preincubation test. Doses of up to 625 µg/plate were evaluated with metabolic activation (S9: rat liver-mix, induced with phenobarbital and 5,6-benzoflavone) and doses up to 5000 µg/plate without metabolic activation (both approaches for all strains). DMSO was included as vehicle control and appropriate positive control substances were also evaluated. Growth inhibition was observed at and above 2500 μg/plate without S9 mix, and at 625 μg/plate (highest dose) with S9 mix in all four S. typhimurium strains. For E.coli, growth inhibition was observed without S9 mix at 5000 μg/plate (highest dose) and at 625 μg/plate (highest dose) in the presence of S9 mix. No twice or more increase of his+ or trp+ revertants compared to the negative control value or dose-dependency. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium and E.coli mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.

In a second bacterial reverse mutation assay, conducted similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay but without E.coli as marker for cross-linking mutagenicity), the test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 (NTP, 1984). The following dose levels were evaluated: 0, 33, 100, 333, 1000 and isolated also concentrations of 1600, 2222, 3333, 5000 and 6666 µg/plate (preincubation test with and without S9 mix (fraction from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver); all strains). A vehicle control was included in the test, but not further specified. Appropriate positive control substances were also evaluated. Slight toxicity was observed in TA 100 / 1535 / 1537 at 1600 and in TA 98 at 2222 µg/plate onwards. No dose-related biologically relevant increase or doubling in the number of his+ revertants was observed in the test in any of the tested strains with or without metabolic activation. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.

The study of Zeiger et al. (1987) was also conducted similar to OECD Guideline 471: Bacterial Reverse Mutation Assay, but without E.coli as marker for cross-linking mutagenicity. The test substance was evaluated for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 at dose levels up to 5000 µg/plate in a preincubation test with and without metabolic activation (Aroclor 1254-induced rat and hamster liver homogenate). DMSO was included as vehicle control and appropriate positive control substances were also evaluated. Slight or complete clearing of background lawn was observed in all strains from 1600 µg/plate onwards. No dose-related increase over the corresponding solvent control was seen as well as a reproducible, dose-related increase in his-revertants in replicate trials. The positive controls yielded the expected results.

- Chromosome aberration test in mammalian cells:

The test substance was assessed for its potential to induce structural chromosome aberrations in CHL/IU cells in vitro in an OECD guideline study (OECD 473) which followed GLP requirements (Ministry of Health Japan, 2011). The following dose levels were evaluated: without S9 mix: 0, 0.43, 0.85, 1.7 mg/mL, with S9 mix (S9 rat liver-mix, induced with phenobarbital and 5,6-benzoflavone): 0, 0.16, 0.33, 0.65, 1.3 mg/mL. DMSO was included as vehicle control and appropriate reference mutagens were used as positive controls (-S9 mix: Mitomycin C; +S9 mix: Cyclophosphamide). Exposure duration was 6 h and the recovery period 18 h. Colcemid (ultimate dose: 0.1μg/mL) was added 2 hrs before completion of culture. Cells with structural chromosomal aberrations (23.5 %) increased significantly by the high dose (1.7 mg/mL) after treatment without metabolic activation. After treatment with metabolic activation, structural chromosomal aberrations (2.5 - 96 %) were induced dose-dependently. Polyploidy was statistically significantly increased at the middle (0.85 mg/mL) and high (1.7 mg/mL) doses after treatment without metabolic activation (4.3 % and 2.3 %), and the low dose (0.33 mg/mL) after treatment with metabolic activation (the 13.5 %). Cell growth and mitotic index decreased dose dependent with and without S9 mix. Due to the results of this study, the test substance induced chromosome aberration in CHL/IU cells under the present test conditions.

- Micronucleus test in mammalian cells:

An in vitro micronucleus test was conducted according to OECD Guideline 487 and GLP (BASF SE, 2013). The test substance was assessed in V79 cells in vitro both in the absence and the presence of a metabolizing system. The following concentrations were tested and evaluated: 1st Experiment (- / + S9 mix; 4 h exposure): 450.00, 900.00, 1800.00 μg/mL; 2nd Experiment (- S9 mix, 24 h exposure): 56.25, 112.50, 225.00 µg/mL; 2nd Experiment (+ S9 mix, 4 h exposure): 675.00, 1350.00, 1800.00 μg/mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. Cytotoxicity indicated by clearly reduced relative increase in cell count was observed in the 2nd Experiment in the absence of S9 mix after 24 hours continuous treatment from 900 μg/mL onward. Additionally, in the absence of metabolic activation cell attachment and/or cell morphology was adversely effected from 450 μg/mL onward after 4 hours exposure in the 1st Experiment and from 900 μg/mL onward after 24 hours exposure in the 2nd Experiment, respectively. Besides, in the 2nd Experiment in the presence of metabolic activation cell attachment and/or cell morphology was adversely influenced from 1350 μg/mL onward. The test substance did not cause any relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. Statistical significance observed in several test groups in both experiments in the presence of metabolic activation has to be regarded as biologically irrelevant because all values were clearly within historical negative control data range. Thus, the test substance is considered not to have a chromosome-damaging (clastogenic) effect or to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and presence of metabolic activation.

 

- Gene mutation in mammalian cells:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster lung fibroblasts (V79) in vitro (BASF SE, 2012). The GLP study was conducted according to OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test; HPRT locus). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. The following doses were evaluated in this study: 1st Experiment (-S9, 4 h): 108.8, 217.5, 435, 652.5, 870.0 µg/mL); 1st Experiment (+S9, 4 h) / 2nd Experiment (-S9, 24 h / +S9, 4 h): 108.8, 217.5, 435, 870.0, 1740 µg/mL). After an attachment period of 24 hours and the respective treatment period an expression phase of 7 days and a selection period of 7 – 10 days followed. The colonies of each test group were fixed, stained with10% methylene blue in 0.01% KOH solutionand counted. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the second experiment at 870.0 µg/mL and above without metabolic activation following 24 hours treatment. The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency I or relative cell density was covered without metabolic activation. No relevant cytotoxic effect was noted up to a maximum concentration of 1740 µg/mL in the presence of metabolic activation.No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed up to the maximumconcentration.Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

2) In vivo data

Dibutylethanolamine was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD 474 (BASF SE, 2013). For this purpose, the test substance, dissolved in corn oil, was administered once orally to male animals at levels of 100, 200 and 400 mg/kg bw/d.

As vehicle control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range.

Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the present study, the single oral administration of Dibutylethanolamine did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

Thus, under the experimental conditions of this study, the test substance Dibutylethanolamine does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data are reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the data, classification is not warranted.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification is not warranted.