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Gene mutation assay

 

In an OECD Guideline 471/472 study (BASF, 1997), Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvr A were exposed to the N,N-dimethylisopropylamine in water at 0, 20, 100, 500, 2500 and 5000 µg/plate in the standard plate test with and without S9-mix, and at 0, 4, 20, 100, 500 and 2500 µg/plate (Salmonella strains) or 0, 20, 100, 500, 2500 and 5000 µg/plate (E. coli WP2 uvrA) in the preincubation test with and without S9-mix (Aroclor-induced rat liver S9 mix). Positive and negative (vehicle) controls were included in the study. N,N-dimethylisopropylamine did not induce revertants in any bacterial strains tested both with and without metabolic activation, acytotoxic effect was seen in some strains in the highest concentration.

 

The genotoxic activity of N,N-dimethylisopropylaminewas evaluated in the Ames test on Salmonella typhimurium according to the OECD 471 guideline (Bichet, 1988). Doses of 50, 100, 500, 750, 1000 and 2500 µg/plate are tested on five strains (TA98, TA100, TA1535, TA1537 and TA1538) in presence or absence of metabolic activation according to the preincubation method (20 min at 37°C). The number of revertants colonies is evaluated after 48hours incubation at 37°C. Appropriate positive and negative controls are used to validate the study. Cytotoxicity was observed for the highest dose (2500 µg/plate) with and without metabolic activation. No genotoxic activity was observed for all doses with and without metabolic activation on the 5 tested strains used.

 

In conclusion, N,N-Dimethylisopropylamine did not induce genotoxicity in the Ames test on Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli strainWP2 uvr Ain the absence and presence of metabolic activation.

 

N,N-Dimethylisopropylamine 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, 2013). 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.

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

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 27.24 to 871.7 mg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration, 871.7 mg/mL, gave 34% and 55% relative survival (RS) in the absence and presence of S-9, respectively.

In Experiment 1, twelve concentrations, ranging from 20 to 871.1 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 871.7 µg/mL, which gave 62% and 65% RS in the absence and presence of S-9, respectively.

In Experiment 2, six concentrations, ranging from 100 to 871.7 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 871.7 µg/mL, which gave 72% and 58% 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 1-oxide (without S-9) and benzo(a)pyrene (with S-9). The study was accepted as valid.

In Experiments 1 and 2, no significant increases in mutant frequency(MF), compared to the vehicle controls, were observedfollowing treatment withN,N-Dimethylisopropylamineat any concentration analysed in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that N,N-Dimethylisopropylamine did not induce mutation at thehprt 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).

Chromosomal aberration assay

 

N,N-dimethylisopropylamine was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in two independent experiments both in the absence and presence of metabolic activation (S-9), according to the OECD Guideline 473 (Whitwell, 2000). In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17 hour recovery period prior to harvest (3+17). Chromosome aberrations were analysed at 489.4, 652.5, 870.0µg/mL. The highest concentrations chosen for analysis, 870.0µg/mL, induced approximately 14% and 0% mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively. In Experiment 2, treatment in the absence of S-9 was continuous for 20hours (doses: 445.4, 556.8, 696.0 µg/mL) and treatment in presence of S-9 was for 3hours only followed by a 17-hour recovery period prior to harvest (3+17) (doses: 556.8, 696.0, 870.0 µg/mL. The highest concentrations chosen for analysis, 696.0 and 870.0 µg/mL, induced approximately 54% and 17% mitotic inhibition respectively.Appropriate negative control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline-1-oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Cultures treated with N,N-dimethylisopropylamine in the absence and presence of S-9 (Experiment 1 and 2) resulted in frequencies of cells with structural aberrations, which were similar to those seen in concurrent negative controls. All cultures receiving the test article had numbers of cells with structural aberrations (excluding gaps) that were within historical negative (normal) control ranges.

In conclusion, N,N-dimethylisopropylamine did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to 10mM in the absence and presence of S-9mix.


Justification for selection of genetic toxicity endpoint
None selected, all in vitro assays are negative.

Short description of key information:
N,N-dimethylisopropylamine did not induce gene mutation in Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537 and TA1538) and Escherichia coli WP2 uvrA as well as at the hprt locus of L5178Y mouse lymphoma cells. N,N-dimethylisopropylamine did not induce chromosome aberrations in cultured human peripheral blood lymphocytes.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to CLP criteria, no classification is warranted for germ cells mutagenicity.