2-[(1-methylpropyl)amino]ethanol

Endpoint summary

Additional information

Gene mutation assays

The potential of 2-[(1-methylpropyl)amino]ethanol to induce reverse mutation inSalmonella typhimuriumwas evaluated according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice (Sire, 2010). A preliminary toxicity test was performed to define the dose-levels of 2-[(1-methylpropyl)amino]ethanol to be used for the mutagenicity study. The test item was then tested in three independent experiments, without and/or with a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. All experiments were performed according to the direct plate incorporation method except for the second and third tests with S9 mix, which were performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.2-[(1-methylpropyl)amino]ethanol was dissolved in water for injections.

The dose-levels of the positive controls were as follows:

without S9 mix

.           1 µg/plate of sodium azide (NaN₃): TA 1535 and TA 100 strains,

.           50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

.           0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

.           0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

 with S9 mix

.           2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537 and TA 98 strains,

.           5 µg/plate of Benzo(a)pyrene (BAP): TA 100 strain,

.           10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

ince the test item was freely soluble and non-severely toxic, the highest dose-level selected was 5000 µg/plate, according to the criteria specified in the international guidelines.

Experiments without S9 mix

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

A moderate toxicity was noted at 5000 µg/plate in the TA 100 strain. No noteworthy toxicity was observed at any dose-levels, in the other tester strains.

The test item did not induce any noteworthy increase in the number of revertants in any of the five strains.

 Experiments with S9 mix

The selected treatment-levels were:

.           312.5, 625, 1250, 2500 and 5000 µg/plate for the first and second experiments,

.           625, 1250, 1875, 2500, 3750 and 5000 µg/plate for the third experiment.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

No noteworthy toxicity was observed in any tester strains in the first experiment (direct plate incorporation method). In the second and third experiments (preincubation method), a moderate to strong toxicity was induced at dose-levels>2500 µg/plate in all tester strains.

Noteworthy increases in the number of revertants in comparison to the vehicle control were noted in the second experiment in the TA 98 strain (up to 2.0-fold the vehicle control value) and in the TA 102 strain (up to 2.2-fold the vehicle control value). Since these slight increases were not observed in the first experiment (using the direct plate incorporation method) and were not reproduced in the third confirmatory experiment (using the preincubation method), despite the closer range of dose-levels used, they were not considered as biologically relevant.

The test item did not induce any noteworthy increase in the number of revertants in any of the other tester strains.

2-[(1-methylpropyl)amino]ethanol did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.

The potential of the test item, 2-[(1-methylpropyl)amino]ethanol, to induce mutations was evaluated at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells. The study was performed according to the international guidelines (OECD No 476 and Council Regulation (EC) No. 440/2008) and in compliance with the Principles of Good Laboratory Practice (Sarlang, 2011b).

After a preliminary toxicity test, 2-[(1-methylpropyl)amino]ethanol, was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 10^5 cells/mL (3 -hour treatment) or cultures of 50 mL at 2 x 10^5 cells/mL (24 -hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. The dose-levels for the positive controls were as follows:

. without S9 mix: Methylmethane sulfonate (MMS), used at a final concentration of 25μg/mL, (3-hour treatment) or 5μg/mL (24-hour treatment),

. with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3μg/mL.

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. The mutation frequencies of the positive controls also met the acceptance criteria of a study. The study was therefore considered as valid.

The test item 2-[(1-methylpropyl)amino]ethanolwas dissolved in the vehicle which was the culture medium RPMI. In the final culture medium, the dose-level of 10 mM showed no precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. Thus, the dose-level of 10 mM was retained as the top-dose to be tested in the preliminary toxicity test. Since the test item was toxic in the preliminary test, the choice of the highest dose-level to be used in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

Experiments without S9 mix

Using a treatment volume of 0.5% in culture medium, the selected dose-levels were as follows:

. 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for the first experiment (3-hour treatment),

. 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 mM for the second experiment (24-hour treatment).

Following the 3-hour treatment, a moderate toxicity was induced at 10 mM, as shown by a 42% decrease in Adj. RTG. Following the 24-hour treatment, a marked to severe toxicity was induced at dose-levels ≥ 0.313 mM, as shown by a 79-100% decrease in Adj. RTG.

Following the 3-hour treatment, no noteworthy increase in the mutation frequency was observed at up to the highest tested dose-level of 10 mM. Following the 24-hour treatment, no noteworthy increase in the mutation frequency was induced at dose-levels up to 0.625 mM which showed a 94% decrease in Adj. RTG. At the higher tested dose-levels of 1.25 and 2.5 mM, noteworthy increases in the mutation frequency were observed (+127 and +246 x 10^-6 compared to vehicle control mean value, respectively). These increases exceeded the Global Evaluation Factor of +126 x 10^-6 but they were only observed at dose-levels inducing a very high level of toxicity (low CE2 values and decrease in Adj. RTG ≥ 99%). Moreover, it is important to notice that, these high values of mutation frequency did not directly arise from an higher number of wells containing small and/or large colonies. Since these high mutation frequency values were not linked to the ability of the cells to grow in presence of TFT in the mutant plates but to the low value of CE2, they were considered not to be biologically relevant.

Experiments with S9 mix

Using a treatment volume of 0.5% in culture medium, the selected dose-levels were 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for both experiments (3-hour treatments).

In the first and second experiments, a moderate toxicity was induced at 10 mM, as shown by a 57% and 59% decreases in Adj.RTG, respectively.

No noteworthy increase in the mutation frequency was noted in comparison to the negative control in either experiment.These results met the criteria for a negative response.

Under the experimental conditions of this study, the test item, 2-[(1-methylpropyl)amino]ethanol, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Chromosomal aberration assays

The potential of test item, 2-[(1-methylpropyl)amino]ethanol to induce chromosome aberrations was evaluated in cultured human lymphocytes. The study was performed according to the international guidelines (OECD 473 and Council Regulation (EC) No. 440/2008) and in compliance with the Principles of Good Laboratory Practice (Sarlang, 2011b).

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at, for 48 hours.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:

.           without S9 mix, cells were exposed continuously to the test or control items until harvest,

.           with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

The dose-levels of the positive controls were as follows

. without S9 mix, Mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

. with S9 mix, Cyclophosphamide: 12.5 or 25 µg/mL.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered to be valid.

The test item 2-[(1-METHYLPROPYL)AMINO]ETHANOL was dissolved in the vehicle which was the culture medium RPMI. In the final culture medium, the dose‑level ofshowed no precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. Thus, the dose-level ofwas retained as the top-dose to be tested in the first main experiment.

With a treatment volume of 0.5% in culture medium, the dose-levels used for treatment were as follows:

. 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 andfor the first experiment with and without S9 mix,

. 0.313, 0.625, 1.25, 2.5, 5 andfor the second experiment with and without S9 mix

Experiments without S9 mix

No noteworthy decrease in the Mitotic Index was noted at any of the tested dose-levels following the 3-hour treatment in the first experiment.Following the 20-hour treatment in the second experiment, a severe decrease in the Mitotic Index was observed atas shown by a 82% decrease in MI. Following the 44-hour treatment in the second experiment, a moderate to severe decrease in the Mitotic Index was observed at dose-levels ≥as shown by a 41 to 85% decrease in MI.

The dose-levels selected for metaphase analysis were as follows:

. 2.5, 5 andfor the 3-hour treatment, the latter being the highest recommended dose‑level,

. 1.25, 2.5 andfor the 20-hour treatment, the higher dose-level being too cytotoxic,

. 5 mM for the 44-hour treatment, this dose-level inducing a 41% decrease in MI and the higher dose-level being too cytotoxic.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3-, 20- or the 44-hour treatments without S9 mix. Thus, these results met the criteria for a negative response.

Experiments with S9 mix

No noteworthy decrease in the Mitotic Index was noted at any of the tested dose-levels, in either experiment and at any of the harvest time .

The dose-levels selected for the metaphases analysis were as follows:

. 2.5, 5 and 10 mM for the 20-hour harvest time in both experiments, the latter being the highest recommended dose-level,

. 10 mM for the 44 -hour harvest time, this dose-level being the highest recommended dose‑level.

The test item did not induce chromosome aberrations in cultured human lymphocytes, in the presence or the absence of a rat metabolizing system.

The potential of the test item, 2-[(1-METHYLPROPYL)AMINO]ETHANOL , to induce damage to the chromosomes or the mitotic apparatus was evaluated in bone marrow cells of rats.The study was performed according to the international guidelines (OECD No 474 and Council Regulation (EC) No. 440/2008) and in compliance with the Principles of Good Laboratory Practice (Sarlang, 2011c). 

Two preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study.In the main study, three groups of five male and five female Sprague-Dawley rats received two oral treatments of 2-[(1-methylpropyl)amino]ethanol at dose-levels of 100, 200 and 400 mg/kg/day, at a 24‑hour interval.One group of five males and five females received the vehicle (drinking water treated by reverse osmosis) under the same experimental conditions, and acted as control group.One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 Erythrocytes (PE + NE).

Neither mortality nor clinical signs were observed in the animals of either sex from the vehicle and positive control groups.Also, neither mortality nor clinical signs were observed in the animals of both sexes at any of the tested dose-levels.The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions.The study was therefore considered to be valid.

The mean values of the PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group, showing there is no evidence of test article related bone marrow toxicity.The mean values of MPE in the groups treated with the test item (1.0, 1.1, 1.0 MPE/1000 PE for the male groups; 1.0, 1.4 and 0.6 MPE/1000 PE for the female groups at 100, 200 and 400 mg/kg bw, respectively) were equivalent to those of the vehicle control group (0.8 and 0.9 MPE/1000 PE for the males and females, respectively). There are no statistically significant increases in the frequency of MPE for the test item-treated groups when compared to the concurrent vehicle control group.Therefore, the criteria for a positive response are not met, thus it is concluded that, in this study, the test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells.

Under the experimental conditions of the study, the test item, 2-[(1-METHYLPROPYL)AMINO]ETHANOL , did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 100, 200 and 400 mg/kg/day.

In the first experiment, a statistically significant increase (p < 0.05) in the frequency of cells with structural chromosomal aberrations was noted. This increase was not dose-related and the mean frequency of aberrant cells obtained at 5 mM remained within the historical data range for the vehicle control (3.0%versus0 to 5.0% for the historical data). Moreover, in a second independent experiment performed under the same experimental conditions, no statistically significant increase in the frequency of cells with structural or numerical chromosomal aberrations was noted at either the 20- or the 44-hour harvest time. Therefore, since this increase was neither dose-related nor reproducible, it was considered to be non-biologically significant.

 

Justification for selection of genetic toxicity endpoint
All tests were negative.

Short description of key information:
2-[(1-methylpropyl)amino]ethanol did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells and clastogenic activity in cultured human lymphocytes and in the bone marrow cells of rats.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is warranted according to CLP and DSD criteria.