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Gene mutation assay on bacteria

The potential of the test item Diisopropylamine (DIPA) to induce reverse mutation in Salmonella typhimurium was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) (Dechariaux, 1990). DIPA was tested in two independent experiments, with and without a metabolic activation system, both performed according to the preincubation method and to the standard plate incorporation method. Each strain TA1535, TA1537, TA1538, TA98 and TA100 was exposed to the test item at five dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 100, 250, 500, 1000, 2500 µg/plate (TA1535, TA1537, TA1538, TA100) and 50, 100, 250, 1000, 2500 ug/plate (TA98) and then 500, 1000, 2500, 5000, 10000 µg/plate (all strains). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. DIPA did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains. Diisopropylamine did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

This result is confirmed by two supporting assays realized on S. typhimurium (Müller, 1988, and Mortelmans 1986) and E. coli (Müller, 1988), which gave negative results:

The potential of Diisopropylamine (DIPA) to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100) was evaluated in accordance with the OECD guideline 471 and in compliance with the Principles of Good Laboratory Practice (Müller, 1988). DIPA was tested with and without a metabolic activation system, according to the direct plate incorporation method. Salmonellas were exposed to DIPA at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 4, 20, 100, 500, 2500, 5000µg/plate. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. Cytotoxicity was observed for the highest dose (5000 µg/plate and above) with and without metabolic activation in all strains. All the positive control compounds gave the expected increase in the number of revertant colonies. No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation on the 5 tested strains. Under these experimental conditions, DIPA did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

The potential of Diisopropylamine (DIPA) to induce reverse mutation in Escherichia coli WP2uvrA was evaluated according to a protocol similar to the OECD guideline 472 and in compliance with the Principles of Good Laboratory Practice (Müller, 1988). DIPA was tested with and without a metabolic activation system, according to the direct plate incorporation method. Bacterias were exposed to DIPA at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test: 4, 20, 100, 500, 2500, 5000µg/plate. After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. Cytotoxicity was observed for the highest dose (5000 µg/plate and above) with and without metabolic activation. All the positive control compounds gave the expected increase in the number of revertant colonies. No noteworthy increase in the number of revertants was observed for all doses with and without metabolic activation. Under these experimental conditions, DIPA did not show any mutagenic activity in the bacterial reverse mutation test with Escherichia coli WP2uvrA.

The genotoxic activity of Diisopropylamine DIPA is evaluated in the Ames test on Salmonella typhimurium in a protocol similar to the OECD 471 guideline (Mortelmans 1986). Doses of 312.5, 625, 1250, 2500, and 5000µg/plate are tested on five strains (TA 98, TA 100, TA 1535 and TA 1537) according to the preincubation method (20 min). Cytotoxicity was observed for 3333µg/plate with metabolic activation and for 10000µg/plate without metabolic activation in any strain. No genotoxic activity was observed for all doses with and without metabolic activation on the 4 tested strains.

In conclusion, Diisopropylamine did not induce genotoxicity in the Ames test on Salmonella typhimurium and Escherichia coli in the absence and presence of metabolic activation.

Gene mutation assay on mammalian cells

Diisopropylamine was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol (Lloyd, 2010). 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.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 31.63 to 1012 

µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) was 506 µg/mL, which gave 36% and 49% RS in the absence and presence of S-9, respectively.

Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 1012 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 600 µg/mL, which gave 5% and 13% RS in the absence and presence of S-9, respectively.In the absence of S-9, no concentration gave 10 -20% RS (cultures treated at 500 and 600 µg/mL gave 56% and 5% RS, respectively), therefore both concentrations were analysed.

In Experiment 2 ten concentrations, ranging from 100 to 750 µg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 560 µg/mL in the absence of S-9 and 750 µg/mL in the presence of S-9, which gave 3% and 6% RS, respectively.In the absence and presence of S-9, no concentration gave 10 -20% RS. In the absence of S-9, cultures treated at 530 and 560 µg/mL gave 34% and 3% RS, respectively and in the presence of S-9, cultures treated at 650 and 750 µg/mL gave 31% and 6% RS, respectively. Both concentrations were therefore analysed under each treatment condition.

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 normal 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.

No statistically significant increases in mutant frequency were observed following treatment with Diisopropylamine at any concentration tested in the absence and presence of S-9 in Experiments 1 and 2 and there were no significant linear trends. Although concentrations giving <10% RS were analysed in the absence of S-9 in Experiments 1 and 2 and in the presence of S-9 in Experiment 2,there was no evidence of mutagenic activity under any of these three treatment conditions, therefore this did not affect data interpretation.

It is concluded that Diisopropylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Chromosomal aberrations

The potential of Diisopropylamine (DIPA) to induce structural chromosome aberrations in human lymphocytes was evaluated according to OECD 473 and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice (McEnaney, 1990). DIPA was tested in two independent experiments, both with and without a metabolic activation system. In the first experiment lymphocytes cultures were exposed to vehicle or test item (doses: 735 and 1500µg/mL) for 20 hours without S9. Treatment with S9mix was for 3 hours followed by a 17hour recovery period prior to harvest. The second experiment included a delayed sampling time at 600 and 1500µg/mL. Treatment in absence of S9 was continuous for 20 or 44hours. Treatment in presence of S9 was for 3 hours followed by a 17 or 41hour recovery period. Diisopropylamine was unable to induce any significant increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in either experiment or at either harvest time.

Short description of key information:
DIPA has not been found to induce any genotoxic effect on bacteria and mammalian cells in gene mutation assays or chromosomal aberrations in human lymphocytes.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

DIPA has not been found to induce any genotoxic effect on bacteria and mammalian cells in gene mutation assays or chromosomal aberrations in human lymphocytes. DIPA is therefore not classified according to CLP criteria.