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In vitro

An OECD guideline 471 study was conducted to determine the mutagenic potential of amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl). In the dose range finding test withSalmomenlla typhimuriumstrain TA100 andEscherichia colistrain WP2 uvrA, the substance was tested up to concentrations of 5,000 µg/plate in triplicate in both the presence and absence of 5% (v/v) S9-mix. No precipitation was observed on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100, 333, 1,000, 3,330 and 5,000 µg/plate in the absence of 5% (v/v) S9-mix and at 333, 1,000, 3,330 and 5,000 µg/plate in the presence of 5% (v/v) S9-mix. InEscherichia colistrain WP2 uvrA, toxicity was observed at the dose level of 5,000 µg/plate in the presence of 5% (v/v) S9-mix. Based on the preliminary test, the substance was evaluated at concentrations of 3, 10, 33, 100, 333 and 1,000 µg/plate in the presence and absence of 5% (v/v) S9-mix in strains TA1535, TA1537 and TA98 in the first mutation assay. Toxicity was observed in all strains. In the second mutation assay, the substance was tested up to concentrations of 1,000 µg/plate in the presence and absence of 5% (v/v) S9-mix in strains TA 100, TA1535, TA1537 and TA98 and up to concentrations of 5,000 µg/plate in strain WP2 uvrA. Toxicity was observed in all strains except WP2 uvrA. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independent repeat. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study, it was concluded that the test substance is not mutagenic in theSalmonella typhimuriumandEscherichia colireverse mutation assay (Verspeek-Rip CM, 2014).

An OECD guideline 471 compliant study was conducted to determine the mutagenic potential of amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl).Salmonella typhimuriumstrains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance using the Ames plate incorporation method at up to seven dose levels for each bacterial strain, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 500 µg/plate (-S9 -mix) and 1.5 to 1,500 µg/plate (+S9 -mix) in the first experiment. The experiment was repeated on a separate day using a similar dose range to experiment 1, fresh cultures of the bacterial strains and fresh test substance formulations. Extra doses were incorporated into each experiment to allow for test substance toxicity. The vehicle (ethanol) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 -mix. The test substance caused a visible reduction in the growth of bacterial lawn to all of the strains of Salmonella tested both with and without metabolic activation. The test substance was, therefore, tested up to its toxic limit. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains at any dose of the test substance, either with or without metabolic activation (Thompson PW, 1996).

Amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) was evaluated at 5,000 µg/plate for mutagenicity in an Ames test using the bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation with rat liver S9 mix. The test was negative (Schöberl, 1991).

A study was conducted to assess the mutagenicity of the structurally similar lauric acid diethanolamine condensate (LDEA, CAS No.120-40-1)in the mouse lymphoma cell line L5178Y. The mouse lymphoma cells were treated at 2.5, 5, 10, 20, 30, 40 and 50 μg/mL with and without metabolic activation and at 5, 10, 20, 30, 40, 50 and 60 μg/mL in trial 1 for 4 hours. Cells were treated with the substance at 5, 10, 20, 30, 40 μg/mL with metabolic activation and 5, 10, 20, 30 without metabolic activation in trial 2 for 4 hours. After the 48 hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells and cells were plated in non-selective medium and soft agar to determine cloning efficiency. No increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to the test substance, with or without S9 (NTP report 480, 1999).

A study was conducted to determine the potential of the structurally similar lauric acid diethanolamine condensate (LDEA, CAS No.120-40-1) to inducechromosomal aberrations in Chinese Hamster Ovary cells. The doses studied were 4.99, 15.00, 49.90 and 100.00 µg/mL (without S9); 1.50, 4.99, 15.00 and 30.00 µg/mL (with S9). Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. A single flask per dose was used and two hundred first-division metaphase cells were scored at each dose level. The test substance did not induce an increase in the number of chromosomal aberrations (NTP report 480, 1999).

In vivo

The chromosome-damaging effect of amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) was investigated in 70 NMRI mice, according to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) and Method No. 431, Annex V of EEC Directive 79/831. The animals were divided into 5 groups, of which 3 were test groups, one a negative control group and one a positive control group. The test groups were given a single oral gavage dose of 15,000 mg test substance/kg bw (which was the maximum tolerated dose), the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 or 72 hours after treatment. From bone marrow smears, micronucleus counts were made per 1,000 polychromatic erythrocytes. Amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) did not induce an increase in the frequency of micronucleated normochromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen TH, 1985).

The mouse peripheral blood micronucleus test was conducted to assess the clastogenic potential of amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl). The test substance (in ethanol) was applied dermally for 14 weeks with the frequency of 5 exposures/week at 0, 25, 50, 100, 200, 400 and 800 mg/kg bw. Peripheral blood samples were obtained from male and female mice, and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of the five animals per dose group. Significant increases in the frequencies of micronucleated normochromatic erythrocytes (NCEs) were seen in peripheral blood of both male and female mice at the end of 14 weeks. Statistical analysis of the data showed positive trends for both data sets as well as significantly elevated micronucleus frequencies at the highest dose tested (NTP report 479, 2001).

The genotoxicity potential (induction of chromosomal aberrations) of the structurally similar lauric acid diethanolamine condensate (LDEA, CAS No.120-40-1) using the mouse peripheral blood micronucleus test was investigated. The test substance was applied dermally for 14 weeks with a frequency of 5 exposures/week. Peripheral blood samples were obtained from male and female mice, and smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. No increase in the frequency of micronucleated normochromatic erythrocytes was observed at any dose level tested (NTP report 480, 1999).


Justification for selection of genetic toxicity endpoint
All data contribute to the overall endpoint conclusion.

Short description of key information:
Amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) was negative in vitro and negative in all in vivo micronucleus tests except one in which significantly elevated micronucleus frequencies was detected at the highest dose (800 mg/kg bw) in male and female mice. However, considering that the in vivo study was the most reliable (Klimisch score 1) and was negative at a maximum tolerated oral dose of 15,000 mg/kg bw, amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) was not considered have any genotoxic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) was negative in in vitro and overall in vivo genetic toxicity tests. Although there was one in vivo study which showed amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) to have clastogenic potential at the highest dose tested (800 mg/kg bw), based on the overall weight of evidence amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) is not expected to have any genotoxic potential. Therefore no classification is required for this endpoint according to EC (67/548/EEC) and CLP (EC 1272/2008) criteria.