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Gene mutation assay

 

The genotoxic activity of ethyldiisopropylamine EDIPA is evaluated in the Ames test on Salmonella typhimurium according to the OECD 471 guideline (Haddouk, 2008). Doses of 312.5, 625, 1250, 2500, and 5000µg/plate are tested on five strains (TA 98, TA 100, TA 102, TA 1535 and TA 1537) according to two methods: direct plate incorporation (the first experiment and the second without S9mix) and the preincubation method (60 min at 37°C - second experiment with S9mix). The number of revertants colonies is evaluated 48 to 72 hours later. Cytotoxicity was observed for the highest doses (2500µg/plate and higher) with and without metabolic activation in any strain. No genotoxic activity was observed for all doses with and without metabolic activation on the 5 tested strains. Ethyldiisopropylamine did not induce genotoxicity in the Ames test on Salmonella typhimurium in the absence and presence of metabolic activation.

Two BASF tests were realised on S. typhymurium and E. coli according to OECD guidelines 471 and 472 respectively (BASF 1997). No genotoxic activity was observed for all doses with and without metabolic activation on the tested strains.

 

N-Ethyldiisopropylamine 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 Lloyd, 2013b). The study consisted of a cytotoxicity Range-Finder Experiment followed by three 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 inacetone. 

The study was conducted in compliance with the Good Laboratory Practice Regulations and in accordance with OECD Guideline 476 (1997).

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 40.38 to 1292 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentrations to provide =10% relative survival (RS) were 646 µg/mL in the absence of S-9 and 1292 µg/mL in the presence of S-9, which gave 57% and 15% RS, respectively.

In Experiment 1 ten concentrations, ranging from 200 to 1292 µg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration analysed was 1292 µg/mL, which gave 22% and 28% RS in the absence and presence of S-9, respectively.

In Experiment 2 ten concentrations, ranging from 150 to 1292 µg/mL, were tested in the absence and presence of S-9. Sevendays after treatment, the highest concentration analysed was 1292 µg/mL, which gave 38% and 40% RS in the absence and presence of S-9, respectively.

As the mutation data for Experiments 1 and 2 were inconclusive, a third, confirmatory experiment was performed.

In Experiment 3 eight concentrations, ranging from 200 to 1292 µg/mL, were tested in the absence and presence of S-9. Sevendays after treatment, the highest concentration analysed was 1292 µg/mL, which gave 42% and 39% 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. Untreated controls (containing culture medium only) were also included as the chosen vehicle (acetone) is not frequently used in this laboratory. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiment 1, no significant increases in mutant frequency (MF), compared to the vehicle control MF, were observed at any concentration analysed and no significant linear trends were noted in the absence and presence of S-9, indicating a negative result.

In Experiment 2 in the absence of S-9, no significant increases in MF, compared to the vehicle control MF, were observed at any concentration analysed and no significant linear trend was noted. In the presence of S-9 a significant increase in MF, compared to the vehicle control MF, was observed at the highest concentration analysed (1292mg/mL) and there was a weakly significant linear trend. However, the MF of 6.62 observed at 1292mg/mL was less than three times the historical vehicle control mean MF value at the time of this experiment (2.29 x 3, i.e. 6.87) and there was no real concentration-dependent increase in MF over the entire range tested. To clarify the observations of Experiment 2, a confirmatory experiment (designated Experiment 3) was performed in the absence and presence of S-9.

In Experiment 3 (as in Experiment 1), there were no significant increases in MF, compared to the vehicle control MF, at any concentration analysed and no significant linear trends were noted in either the absence or presence of S-9, indicating a negative result.

The data from Experiments 1 and 3 confirm that the isolated observation in the presence of S-9 in Experiment 2 may be considered of no biological relevance.

It is concluded thatN-Ethyldiisopropylaminedid not induce biologically relevant increases in mutant frequency at thehprtlocus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to 1292 µg/mL (equivalent to 10 mM) in three independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

 

Chromosomal aberration assay

 

N-Ethyldiisopropylamine was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment (Watters, 2012). 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 acetone and the highest concentrations tested in the Micronucleus Experiment, 1293 µg/mL (equivalent to 10mM), were 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-Ethyldiisopropylamine 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 the table below:

Micronucleus Experiment (48 hour PHA) – Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Control Range Historical(%)#

Statistical significance

 

 

 

 

 

 

3+21 hour -S-9

Vehiclea

-

0.15

0.10 – 0.95

-

 

800.0

4

0.45

 

p < 0.05

 

1000

0

0.35

 

NS

 

1293

0

0.55

 

p < 0.05

 

*MMC, 0.80

ND

9.35

 

p < 0.001

 

 

 

 

 

 

3+21 hour +S-9

Vehiclea

-

0.50

0.00 – 1.10

-

 

800.0

0

0.15

 

NS

 

1000

4

0.35

 

NS

 

1293

2

0.25

 

NS

 

*CPA, 12.50

ND

3.00

 

p < 0.001

 

 

 

 

 

 

24+0 hour -S-9

Vehiclea

-

0.35

0.10 – 1.10

-

 

950.0

8

0.55

 

NS

 

1050

22

0.35

 

NS

 

1150

12

0.60

 

NS

 

1293

12

0.55

 

NS

 

*VIN, 0.02

ND

12.97

 

p < 0.001

 

 

 

 

 

 

a             Vehicle control was acetone

*             Positive control

#             95thpercentile of the observed range

NS          Not significant

ND         Not determined

Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate cells (MNBN) in the vehicle cultures fell within current historical vehicle control (normal) ranges. It was therefore considered not necessary to analyse the untreated controls. 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 valid.

Treatment of cells with N-Ethyldiisopropylamine for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.05) than those observed in concurrent vehicle controls for the highest and lowest concentrations analysed. However, the MNBN cell frequency of all cultures analysed fell within the normal range. Therefore, the statistical significance is considered not biologically relevant.

Treatment of cells with N-Ethyldiisopropylamine for 3+21 hours in the presence and 20+0 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly higher than 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-Ethyldiisopropylamine did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of S-9. Concentrations were tested up to the equivalent of 10 mM, a recommended regulatory maximum concentration for in vitro cytogenetic assays.

 



Short description of key information:
Four studies (two in vitro gene revertion assay in bacteria and one gene mutation in mammalian cells and one in vitro micronucleus assay in mouse lymphoma cells) have been performed according to international guidelines and GLP. All results were negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Four studies (two in vitro gene revertion assay in bacteria and one gene mutation in mammalian cells and one in vitro micronucleus assay in mouse lymphoma cells) have been performed according to international guidelines and GLP. All results were negative. The conclusion for genetic toxicity is therefore that ethyldiisopropylamine does not require classification as mutagenic.