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Key value for chemical safety assessment

Additional information

There are three in vitro GLP guideline studies available for N-butylaminoethanol.

Gene mutation in bacteria:

In a reverse gene mutation assay in bacteria (BASF, 1997, Report No. 40M0579/964373), strains [TA 1535, TA 1537, TA 98, TA 100] of S. typhimurium and E. coli [E. coli WP2] were exposed to the test substance dissolved in water at concentrations of 20, 100, 500, 2500, 5000 µg/plate in the absence of mammalian metabolic activation and in the presence of Aroclor 1254-induced rat liver S9 mix, respectively (the preincubation method and the standard plate test was used). A bacteriotoxic effect was observed in the plate incorporation test depending on the test conditions from about 2500 µg/plate onward. No significant increase in mutant frequency was observed, either with or without metabolic activation.The positive controls induced the appropriate responses in the corresponding strains.

The study is classified as reliable without restriction and satisfies the requirements for test guideline OECD 471 (Ames test) for in vitro mutagenicity in bacterial cells.

Gene mutation in mammalian cells

N-Butylaminoethanol was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol (OECD 476; Covance Laboratories Ltd., 2012a; Report No. 8260615). The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in purified water. A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 36.63 to 1172 μg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration, 1172 μg/mL, gave 42% and 38% relative survival (RS) in the absence and presence of S-9, respectively.

In Experiment 1 six concentrations, ranging from 200 to 1172 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 1172 μg/mL, which gave 48% and 81 % RS in the absence and presence of S-9, respectively.

In Experiment 2 seven concentrations, ranging from 200 to 1172 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 1172 μg/mL, which gave 55% and 60% RS in the absence and presence of S-9, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline-N-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiment 1 in the absence and presence of S-9 and Experiment 2 in the presence of S-9, no significant increases in mutant frequency (MF), compared to the vehicle control MF, were observed at any concentration analysed and there were no statistically significant linear trends. In the presence of S-9 in Experiment 2, the vehicle control MF of 8.30 mutants/10E6 viable cells was less than three times the current historical mean MF value at the time of the experiment (2.87 x 3 = 8.61) and was considered acceptable. The mean MF values at the intermediate concentrations of 400 and 1050 μg/mL in this experiment (9.04 and 11.60, respectively) were more than three times the current historical mean MF va lue but were not significantly different from the mean vehicle control value of 8.30. Furthermore, there was clearly no evidence of a concentration-related response and these elevated MF values were considered not to have affected data interpretation, i.e. there were no mutagenic effects.

In Experiment 2 in the absence of S-9, a significant increase in MF, compared to the vehicle control MF, was observed at the second highest concentration tested (1050 μg/mL), giving a mean MF value of 11.73 compared to the concurrent mean vehicle MF of 4.07. The upper limit for MF for the vehicle control (three times the historical mean value of 2.47) in this case was 7.41 , therefore the mean MF value at 1050 μg/mL exceeded this value. The individual MF values at 1050 μg/mL were 20.12 and 4.73, therefore only one exceeded three times the historical mean value and there was marked heterogeneity for mutation between replicates such that the data could have been excluded, but were included for comparative purposes. Furthermore, the mean MF values at 200, 750 and 900 μg/mL also exceeded three times the historical mean MF value. However, apart from at 750 μg/mL, only one of the two individual MF values exceeded three times the historical mean value at each concentration. Again, there was clearly no evidence of a concentration-related effect and no significant linear trend was observed.

The significant increase observed at a single concentration in the absence of S-9 in Experiment 2 was not reproduced within or between experiments and was not concentration-related, therefore was considered not biologically relevant.

It is concluded that N-Butylaminoethanol did not induce biologically relevant increases in mutant frequency at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to a concentration equivalent to 10 mM in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Cytogenicity study

N-Butylaminoethanol was tested in an in vitro micronucleus assay (OECD 487) using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment (Covance Laboratories Ltd., 2012b; Report No. 8260614).

Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 1172 μg/mL (equivalent to 10 mM), was determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of N-Butylaminoethanol on the replication index (RI). In the Micronucleus Experiment, micronuclei were analysed at three or four concentrations and a summary of the micronucleus data is presented in Table 1:

Table 1. Micronucleus Experiment - Results summary

Treatment

Concentration (µg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency(%)

Historical Control Range (%) #

Statistical significance

3+21 hour-S-9

Vehiclea

0.20

0.10-1.00

800.0

0

0.35

NS

1000

4

0.25

NS

1172

6

0.40

NS

*MMC, 0.80

ND

8.10

p ≤ 0.001

3+21 hour+S-9

Vehiclea

-

0.70

0.10-1.10

-

800.0

5

0.40

NS

1000

10

0.65

NS

1172

4

0.25

NS

*CPA, 12.50

ND

2.20

p ≤ 0.001

24+0 hour -S-9

Vehiclea

-

0.50

0.10-1.40

-

800.0

7

0.50

NS

1050

28

0.35

NS

1125

40

0.65

NS

1172

34

0.35

NS

*VIN. 0.02

ND

9.81

p ≤ 0.001

aVehicle control was purified water

* Positive control

# 95thpercentile of the observed range

NS =Not significant

ND = Not determined

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within current historical vehicle control (nominal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were met and the study was therefore considered as valid.

Treatment of cells with N-Butylaminoethanol in the absence and presence of S-9 resulted in frequencies of MNBN cells, which were similar to and not significantly higher from those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all treated cultures fell within normal ranges.

It is concluded that N-Butylaminoethanol did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of S-9 when tested up to 1172 μg/mL (equivalent to 10 mM, a recommended regulatory maximum concentration for in vitro cytogenetic assays).


Justification for selection of genetic toxicity endpoint
No study is selected since all studies are negative.

Short description of key information:
Genetic toxicity:
- in vitro: negative (Ames test, OECD 471; BASF, 1997);
- in vitro: negative ± S9 mix (MNT, human lymphocytes; OECD 487; Covance Laboratories Ltd., 2012);
- in vitro: negative ± S9 mix (Mammalian cell gene mutation assay, mouse lymphoma L5178Y cells, OECD 476; Covance Laboratories Ltd., 2012);

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

N-butylaminoethanol was negative in bacterial strains (OECD 471), in the Mammalian Cell Gene Mutation Assay (OECD 476) and in the Micronucleus Test (OECD 487). Based on these data, in accordance with Regulation (EC) No. 1272/2008, BEA does not need to be classified and labelled as a genotoxic substance.