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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Additional information

Except for a bacterial reverse mutation assay, no other genotoxicity data has been identified on amides, C16-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl). However, a substantial amount of reliable and good quality and validin vitroandin vivostudies are available on other DEA-derived FAA , particularly on amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl), amides, C12, N,N-bis(2-hydroxyethyl), and amides, C18-unsatd., N,N-bis(hydroxyethyl) which have been thoroughly investigated as part of NTP programme. The use of data from the FAA category for the assessment of the carcinogenicity of amides, C16-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) by means of read-across (analogue approach) is justified due to their structural similarities as indicated by a common functional group (i. e., FAA), common breakdown products (e. g., hydroxylation and oxidation of alkyl chains). The structural similarity of C16-18 and C18-unsatd. DEA and C8-18 and C18-unsatd. N, N-bis(hydroxyethyl), C12 N,N-bis(hydroxyethyl), or C18-unsatd., N,N-bis(hydroxyethyl) (“DEA-FAA”) results in similar physico-chemical, human health and environmental properties.

In vitro

A bacterial reverse mutation assay was conducted to determine the mutagenic potential of amides, C16-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) 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 study was according to EU Method B.13/14. The test concentration was 5,000 µg/plate. Under the conditions of the study, the test substance was found to be non-mutagenic (Schöberl, 1991).

A bacterial reverse mutation assay was conducted by NTP to determine the mutagenic potential of the structurally similar test substance, amides, C8-18 (even-numbered) and C18 -unsatd., N,N-bis(hydroxyethyl) inSalmonella typhimurium.Strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method at up to eight dose levels for each bacterial strain, in triplicate, both with and without the addition of S9 mix (i.e., Aroclor induced rat and hamster liver homogenate metabolising system). The dose range was 0.1 to 6.7 µg/plate in the absence of S9 mix and from 3.3 to 200 µg/plate in the presence of S9 mix. Cytotoxicity was observed at 6.7 µg/plate without metabolic activation in the strain TA 1535 and at ≥100 and 200 µg/plate with metabolic activation in the strains TA 97 and 100 respectively. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. The vehicle (ethanol) control or the negative 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. Under the test conditions, the test substance was not mutagenic inSalmonella typhimuriumstrain TA97, TA98, TA100 or TA1535, with or without S9 metabolic activation (NTP report 479, 1999).

A chromosomal aberration assay was conducted by the NTP to determine the genotoxic potential of the structurally similar test substance, amides C8-18 (even-numbered) and C18 -unsatd. N,N-bis(hydroxyethyl) by the induction of chromosomal aberrations in Chinese Hamster Ovary cells. The doses studied were16, 30 and 50 μg/mL with and without S9 mix. 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 a significant increase in the number of chromosomal aberrations. The vehicle (ethanol) control or the negative control flasks induced an increase in the number of chromosomal aberrations within the normal range. All the positive control chemicals used in the test induced a marked increase in the number of chromosomal aberrations both with and without the S9 -mix. Under the conditions of the study, the test substance was found to be non-clastogenic in the presence or absence of metabolic activation (NTP report 479, 1999).

A mouse lymphoma assay was conducted by the NTP to determine the genotoxic potential of the structurally similar test substance, amides C8-18 (even-numbered) and C18 -unsatd. N,N-bis(hydroxyethyl) by inducing the mutations in the L5178Y cell line. The test substance was tested at the following concentrations : Without metabolic activation (-S9): Trial 1: 0, 1.25, 2.5, 5, 6, 10, 12 nL/mL; Trial 2: 0, 4, 5, 6, 8, 10, 12 nL/mL; Trial 3: 0, 1.5, 3, 6, 8, 10, 12, 15 nL/mL; With metabolic activation (+S9): Trial 1: 0, 1.25, 2.5, 5, 10, 15 nL/mL; Trial 2: 0, 4, 5, 6, 8, 10, 12 nL/mL; Trial 3: 0, 6, 8, 10, 12, 15, 20 nL/mL; Trial 4: 0, 5, 10, 15, 20, 30, 40, 50 nL/mL. 6 x106cells in triplicate cultures were exposed to the test substance (either in the presence or absence of metabolic activation), positive control and solvent control for 4 h. After the 48 hour expression period, cells were plated for selection of TFT-resistant cells and for cloning efficiency.The plates were scored after an incubation period of 10 to 12 days at 37±1°C in 5% CO2. Under the test conditions, the test substance did not produce any increase in mutant L5178Y mouse lymphoma cell in the presence and absence of metabolic activation (NTP report 479, 1999).

A sister chromatid exchange assay was conducted by NTP to determine the DNA damage/repair potential of structurally similar amides, C8-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) by the induction of frequencies of sister chromatid exchanges in Chinese Hamster Ovary cells. The doses studied were 0.5, 1.6, 5.0 and 16 μg/mL without metabolic activation (-S9); 0.5, 5 and 16 μg/mL in trail-1 and 5, 10, 16 and 30 μg/mL in trial-2 with metabolic activation (+S9). Concurrent solvent and positive controls (i.e., mitomycin-C (without S9 mix) and cyclophosphamide (with S9 mix)) were also included. A single flask per dose was used and fifty second-division metaphase cells were scored at each dose level. The test substance did not induce any significant increase in the frequencies of sister chromatid exchanges. The solvent control and the positive controls induced exchanges within the normal range indicating the test conditions to be valid. Under the test conditions, the test substance did not induce significant sister chromatid exchanges in the Chinese Hamster Ovary cells both in the presence and absence of S9 (NTP report 479, 1999).

 In vivo

A micronucleus assay was conducted to determine the chromosome-damaging effect of structurally similar amides, C8-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) in 70 NMRI mice, according to OECD guideline 474 (mammalian erythrocyte micronucleus test) and EU Method 431. The animals were divided into five groups, including three test groups, one negative control and one positive control. The test groups were given a single oral gavage dose of 15 g test material/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 and 72 hours respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. The test substance 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).

A micronucleus assay was conducted with the structurally similar amides, C8-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) in B6C3F1 mice. 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 in 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).

A micronucleus assay was also investigated with the structurally similar LDEA. The test substance was applied dermally for 14 weeks with a frequency of five 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).

A micronucleus assay was conducted with structurally similar amides, C18-unsatd., N,N-bis(hydroxyethyl). The test substance was applied dermally for 13 weeks at 0, 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. Under the conditions of the test, the substance did not increase the frequency of micronucleated normochromatic erythrocytes (NCEs) in peripheral blood of both male and female mice at the end of 13 weeks (NTP report 481, 2001).

Justification for selection of genetic toxicity endpoint

No study was selected, since results from most of the in vitro and in vivo studies were negative, except for one in vivo micronucleus study conducted with structurally similar C8-18 and C18-unsatd. DEA.

Short description of key information:

Results from amides, C16-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl) and structurally similar substances were negative in all in vitro and in vivo tests except in one in vivo micronucleus assay conducted with amides, C8 -18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) in which significantly elevated micronucleus frequencies were detected at the highest dose (i. e., 800 mg/kg bw) in male and female mice which was attributed to the presence of free DEA in the solution tested.However, amides, C16-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl) was negative when tested in an in vivo study at a maximum tolerated oral dose of 15g/kg bw. Based on an overall weight of evidence, amides, C16 -18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl) is not expected to have any genotoxic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Data with amides, C16-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) and structurally similar substances were negative in allin vitroandin vivotests except onein vivomicronucleus assay conducted with amides, C8 -18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) in which significantly elevated micronucleus frequencies were detected at the highest dose (i. e., 800 mg/kg bw) in male and female mice.which was attributed to the presence of free DEA in the solution tested.Based on the overall weight of evidence, amides, C16-18(even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl) is not expected to have any genotoxic potential. Therefore no classification is required for genotoxicity according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).