N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine

Endpoint summary

Additional information

Gene mutation assays

The potential of N'-(3-aminopropyl) -N, N-dimethylpropane-1,3-diamine (DMAPAPA) to induce reverse mutation in bacteriaSalmonella typhimuriumwas evaluated during an Ames test according to the 471 OECD guideline (Molinier, 1993). Two experiments have been realized, according to two methods, the direct plate incorporation method (both experiments without S9 mix, first experiment with S9 mix) and the preincubation method (1 hour, 37°C, second experiment with S9 mix). Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. The concentrations tested were 156.25, 312.5, 625, 1250 and 2500 µg/plate in the first test and 125, 250, 500, 1000 and 2000 µg/plate in the second test, 2500 µg/plate being the concentration which showed moderate toxicity and being the limit of solubility in the molten agar. The negative and solvent control results were equivalent to historical controls. The number of revertants induced by the positive controls was statistically higher than the controls, indicating the sensitivity of the test system. The test substance DMAPAPA did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 strains. In conclusion, DMAPAPA did not show mutagenic activity in the Ames test.

Two tests were realised on S. typhymurium and E. coli according to OECD guidelines 471 and 472 respectively (BASF 1998). No genotoxic activity was observed for all doses of DMAPAPA with and without metabolic activation on the tested strains.

N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine 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, 2012). 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 (1997). In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S‑9, ranging from 49.78 to 1593 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentrations to provide >10% relative survival (RS) were 99.56 mg/mL in the absence of S-9 and 398.3 mg/mL in the presence of S-9, which gave 28% and 19% RS, respectively. In Experiment 1 eleven concentrations, ranging from 30 to 250 µg/mL in the absence of S‑9 and ten concentrations, ranging from 50 to 500 mg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 200mg/mL in the absence of S‑9 and 500 mg/mL in the presence of S‑9, which gave 14% and 31% RS, respectively. In Experiment 2 eleven concentrations, ranging from 20 to 250 µg/mL in the absence of S-9 and from 100 to 750 mg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 100mg/mL in the absence of S‑9 and 600 mg/mL in the presence of S‑9, which gave 15% and 18% RS, respectively. In the presence of S-9, no concentration gave 10%-20% RS in Experiment 1 when tested up to a maximum of 500 µg/mL (which was based on the Range-Finder toxicity data). In Experiment 2, the highest concentration analysed was raised to 600 µg/mL, which gave 18% RS. It was therefore considered that suitable levels of cytotoxicity were achieved during the course of the study under this treatment condition and the data were considered acceptable. 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). Therefore the study was accepted as valid. In the absence of S-9 in Experiment 1, a significant increase in mutant frequency (MF), compared to the vehicle control, was observed at one intermediate concentration (80 µg/mL), giving a mean MF value of 6.62 mutants/10⁶viable cells compared to the concurrent mean vehicle MF of 1.99. The upper limit for MF for the vehicle control should be no more than three times the historical mean value which, in the absence of S-9 at the time of Experiment 1, was 2.21, thus the upper limit for a vehicle control would be 6.63. The mean MF value at 80 mg/mL was therefore marginally lower than this value and of the individual MF values at 80 µg/mL (6.37 and 6.87), one was above and one below the upper limit for a vehicle control. There was also no significant linear trend in Experiment 1, indicating the lack of a concentration-related response. In the absence of S-9 in Experiment 2, there were no significant increases in MF, compared to the vehicle control, at any concentration analysed and no significant linear trend was observed. In the presence of S-9 in Experiment 1, there were no significant increases in MF, compared to the vehicle control, at any concentration analysed and no significant linear trend was observed. In the presence of S-9 in Experiment 2, a significant increase in MF, compared to the vehicle control, was observed at one intermediate concentration (300 µg/mL), giving a mean MF value of 6.16 compared to the concurrent mean vehicle MF of 2.50. The upper limit for MF for the vehicle control (three times the historical mean value) in this case was 7.56, therefore the mean MF value at 300 mg/mL was lower than this value and the individual MF values at 300 µg/mL were 10.91 and 2.53, therefore only one exceeded three times the historical mean value. A significant linear trend was observed in Experiment 2 but there was no clear indication of a concentration-related increase in MF over the entire range analysed. The small, sporadic increases observed during the course of this study at individual concentrations in the absence and presence of S-9 were therefore not reproduced within or between experiments, therefore they were not considered biologically relevant. It is concluded that N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine did 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 toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S‑9).

Chromosomal aberration assay

N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine was tested in anin vitromicronucleus 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 study was conducted in compliance with the Good Laboratory Practice Regulations and in accordance with OECD Guideline 476 (1997). N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine was formulated in water for irrigation (purified water) and the highest concentration tested in the Micronucleus Experiment, 1250 mg/mL (limited by toxicity), 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'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine on the replication index (RI). In the Micronucleus Experiment, micronuclei were analysed at three, four or five concentrations and a summary of the micronucleus data is presented in the following table:

Table: Micronucleus Experiment – Results summary

Treatment	Concentration (mg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Historical Control Range (%)^#	Statistical significance
3+21 hour -S-9	Vehicle^a	-	0.30	0.10–0.95	-
	900.0	0	0.50		NS
	950.0	8	0.55		NS
	1000	46	0.65		NS
	*MMC, 0.80	ND	5.35		p < 0.001
3+21 hour +S-9	Vehicle^a	-	0.55	0.00–1.10	-
	700.0	0	0.25		NS
	750.0	10	0.30		NS
	800.0	46	0.45		NS
	850.0	54	0.60		NS
	*CPA, 12.50	ND	1.75		p < 0.001
24+0 hour -S-9	Vehicle^a	-	0.55	0.10–1.10	-
	600.0	14	0.20		NS
	650.0	41	0.20		NS
	700.0	41	0.15		NS
	750.0	57	0.93		NS
	850.0	44	0.00		NS
	*VIN, 0.02	ND	13.61		p < 0.001
a        Vehicle control was purified water *        Positive control #        95^thpercentile 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 (normal) 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 valid. Treatment of cells with N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine in the absence and presence 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 the normal ranges. It is concluded that N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.

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

Short description of key information:
N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine did not induce reverse mutation in Salmonella typhimurium and Escherichia coli, gene mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells and chromosomal aberration in an in vitro micronucleus assay in human lymphocyte cultures

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to REGULATION (EC) No 1272-2008 and Annex VI of Commission Directive 2001/59/EC:

Not classified, based on the battery of negative genetic toxicology studies that have been conducted with N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine.