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Gene mutation assays

The method meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and No. 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay) (Bowles, 2009). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2 uvrA were treated with N-(3-aminopropyl) iminodiethanol "APDEA" using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (1 0% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. APDEA caused no visible reduction in the growth of the bacterial background lawn at any dose level. APDEA was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. APDEA was considered to be non-mutagenic the conditions of this test.

N-(3-aminopropyl) iminodiethanol "APDEA" 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 (Massip, 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). APDEA was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for all experiments. The study was conducted in accordance with OECD Guideline 476 (1997) and the principles on Good Laboratory Practice. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 50.63 to 1620 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed (1620 mg/mL) gave 76% and 91% relative survival (RS) in the absence and presence of S-9, respectively.

In Experiment 1 ten concentrations, ranging from 100 to 1620 µg/mL, were tested in the absence and presence of S-9. Seven days after treatment the highest concentration analysed (1620 mg/mL) to determine viability and 6TG resistance gave 49% and 65% RS in the absence and presence of S-9, respectively. In Experiment 2 seven concentrations, ranging from 150 to 1620 µg/mL, were tested in the absence and presence of S-9. Seven days after treatment the highest concentration analysed (1620 mg/mL) to determine viability and 6TG resistance gave 75% and 81% 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 (MF) in vehicle 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 Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with APDEA at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. It is concluded that APDEA did not induce mutation at thehprtlocus of L5178Y mouse lymphoma cells when tested up to 10 mM (an acceptable maximum concentration for this type of study according to current regulatory guidelines) in two independent experiments in the absence and presence of arat liver metabolic activation system (S-9).

Chromosomal aberration assays

This report describes the effect of N-(3-Aminopropyl) diethanolamine “APDEA “ on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix) (Buskens, 2011a). The possible clastogenicity of APDEA was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EC guidelines. All concentrations reported were corrected for the purity. A correction factor of 1.1025 was used. APDEA was dissolved in RPMI 1640 medium. In the first cytogenetic assay, APDEA was tested up to 1622µg/ml (= 0.01 M) for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest dose level recommended in the guidelines. In the second cytogenetic assay, APDEA was tested up to 1622µg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix APDEA was also tested up to 1622µg/ml for a 3 h exposure time with a 48 h fixation time. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. APDEA did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of APDEA on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that APDEA does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. Finally, it is concluded that this test is valid and that APDEA is not clastogenic in human lymphocytes under the experimental conditions described in this report.

N-(3-Aminopropyl) diethanolamine “APDEA “ was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow (Buskens, 2011b). The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch TG70L02N of APDEA was a clear slightly yellow liquid with a purity of 90.7%. All concentrations reported were corrected for the purity. A correction factor of 1.1025 was used. The test substance was dissolved in physiological saline. In the dose range finding study 3 males and 3 females were dosed once via oral gavage with 2000 mg APDEA per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no differences in toxicity between sexes only males were used in the main limit study. In the main study male animals were dosed once via oral gavage with vehicle or with 2000 mg APDEA per kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 4 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with APDEA or control animals receiving vehicle or cyclophosphamide. Bone marrow of the groups treated with APDEA was sampled 24 or 48 hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. All animals treated with APDEA exhibited both group mean and individual micronucleated polychromatic erythrocytes frequencies which were comparable with both the concurrent vehicle control and the laboratory’s historical vehicle control data for both time points. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the laboratory’s historical vehicle control data range. All CP-treated animals exhibited marked increases in micronucleated polychromatic erythrocytes such that the frequency of micronucleated polychromatic erythrocytes in the positive control group was significantly (p=0.01) greater than the observed frequency in the concurrent vehicle control group. Hence, both criteria for an acceptable assay were met. The groups that were treated with APDEA showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis. It is concluded that APDEA is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.


Justification for selection of genetic toxicity endpoint
In vivo study performed according to the OECD guideline 474 (Notox 2011, 496697) supporting the results of three in vitro studies performed according to the OECD 471/472, 473 and 476, respectively. All three studies are GLP compliant.

Short description of key information:
Four studies (in vitro gene mutation assay in bacteria and mammalian cells, in vitro chromosomal aberration in human lymphocytes and in vivo micronucleus in mouse) 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 (in vitro gene mutation assay in bacteria and in mammalian cells, in vitro chromosomal aberration in human lymphocytes and in vivo micronucleus in mouse) have been performed according to international guidelines and GLP. All results were negative. The conclusion for genetic toxicity is therefore that N-(3-Aminopropyl)diethanolamine does not require classification as mutagenic.

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