N,N'-ethylenebis[N-acetylacetamide]

Endpoint summary

Additional information

Bacterial Mutation Tests:

TAED was assessed for its potential to induce gene mutations in a bacterial reverse mutation assay (Timm, 1988) according to OECD Guideline 471 and GLP. The test was performed using the plate incorporation method. The Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and in addition the Escherichia coli strain WP2 with and without S9-mix were examined applying test concentrations of 10, 33.3, 100, 333.3 and 500 µg TAED/plate. 500 µg TAED/plate was selected as the highest test concentration, since the test substance was partially insoluble at this concentration. No distinct toxic effects occurred in the test groups. Up to the highest dose, no significant and reproducible increase in revertant colony numbers was obtained in any of the strains used.

This result is supported by a further study performed according to the respective EPA OPPTS guideline (Insley, 1982). As this study was not done under GLP it represents supporting information. TAED was tested at concentrations of 10, 100, 1000 and 10000 µg/plate with and without S9-mix using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537 and TA 1538. No indication was given whether TAED was soluble or remained undissolved at the various concentrations. The test substance did neither cause toxic effects nor an increase in revertant colony numbers in the Salmonella typhimurium strains used.

 

Chromosome Aberration Test in Vitro

The potential of TAED to induce structural chromosome aberrations was assessed in V79 cells of the Chinese hamster (Heidemann, 1989) and in human lymphocytes in vitro (Wright, 1995). Both studies were conducted according to the OECD Guideline 473 and GLP.

Duplicate human lymphocyte cultures were treated with the test substance and evaluated for chromosome aberrations, together with vehicle (PEG 400) and positive controls. Four treatment conditions were used: 4 hours of exposure in the presence of S9-mix with cell harvest 20 hours and 44 hours after start of treatment, and a 20 and 44 hour continuous exposure in the absence of activation. Two independent experiments were performed. In experiment 1 (20 hour harvest) 570, 1140 and 2280 µg/mL (equivalent to 10 mM) were selected for chromosomal analysis. In experiment 2, cultures were exposed to TAED at 570, 1140 and 2280 µg/mL (20 hour harvest), or 2280 µg/mL (44 hour harvest). There was no real evidence of toxicity of TAED at the 20 hour harvest, but at the 44 hour harvest mean mitotic indices were reduced to approximately 65% of the vehicle control value, both in the presence and absence of S9 -mix. The solubility of TAED at the given concentrations in the culture medium was not reported. Chromosomal analysis revealed that TAED induced no biologically or reproducible, statistically significant increases in the frequency of cells with aberrations.

Duplicate cultures of V79 cells were treated with TAED for 4 hours in the presence and absence of S9-mix, harvested at 7 hours, 18 hours and 28 hours after start of treatment and evaluated for chromosome aberrations. Solvent and positive controls were included in the study. TAED concentrations tested were 500 µg/mL in cultures harvested after 7 or 28 hours and 20, 200 and 500 µg/mL in cultures harvested after 18 hours. 500 µg/mL was chosen as the maximum concentration, because higher concentrations could not be dissolved in the culture medium. The mitotic index was only slightly reduced at 500 µg/mL at the 7 h harvest in the presence and absence of S9-mix. There was no relevant increase in cells with structural aberrations.
TAED was non-clastogenic to V79 cells of the Chinese hamster and human lymphocytes in vitro under the experimental conditions used.

 

Mammalian Cell Gene Mutation Test (HPRT test)

The test item TAED was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The main experiments were evaluated at the following concentrations (the maximum concentration of 2280.0 µg/mL is equal to approximately 10 mM):

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	71.3	142.5	285.0	570.0^P	1140.0^P
4 hours	+	71.3	142.5	285.0	570.0^P	1140.0^P
		Experiment II
24 hours	-	142.5	285.0	570.0	1140.0^P	2280.0^P
4 hours	+	142.5	285.0	570.0	1140.0^P	2280.0^P

P precipitation of the test item visible at the end of treatment

Precipitation of the test item visible to the naked eye at the end of treatment was noted in experiment I at 570.0 µg/mL and above with and without metabolic activation. In experiment II precipitation occurred at 1140.0 µg/mL and above with and without metabolic activation.

There was no relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% up to the maximum concentration with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls.

 A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental parts.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.1 up to 19.6 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.3 up to 25.5 mutants per 10⁶cells.

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, TAED is considered to be non-mutagenic in this HPRT assay.

Micronucleus Test in Vivo

In a micronucleus test performed according to OECD Guideline 474 and GLP (Durward, 1995), groups of 10 albino CD-1 mice (5 males and 5 females) were given a single oral dose of TAED at 312.5, 625 and 1250 mg/kg bw. Negative and positive control groups receiving the vehicle, arachis oil, or cyclophosphamide were also included. Control and high dose animals were killed 24, 48 and 72 hours after dosing; animals of intermediate and low dosage groups as well as the positive control group were killed 24 hours after dosing. Bone marrow was extracted from the femurs and smear preparations were made and stained. Three premature deaths were observed after dosing with 1250 mg/kg bw. There were no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in animals dosed with TAED when compared to the concurrent vehicle control groups. No significant change in the ratio of polychromatic/normochromatic erythrocytes was observed, but the presence of clinical observations and premature deaths at the high dose level indicated that systemic absorption had occurred.
In conclusion, TAED was considered to be non-genotoxic in the micronucleus test in vivo.

This conclusion is supported by the outcome of a further micronucleus test (Wallat, 1984). The test followed the principles of OECD Guideline 474, but is not in accordance with GLP and represents therefore supporting information. Groups of mice (7 males and 7 females) received TAED at dose levels of 250, 1250 and 2500 mg/kg bw given twice, separated by an interval of 24 hours, by oral gavage. Negative and positive control groups receiving the vehicle, carboxymethyl cellulose (1%)/cremophor (0.5%), or cyclophosphamide were also included. The animals were sacrificed 30 hours after the first substance application and bone marrow smears from both femurs were prepared. No premature deaths occurred. No significant change in the ratio of polychromatic/normochromatic erythrocytes was observed, but the presence of clinical observations indicated that systemic absorption had occurred. At all dose levels the group mean micronucleated cell count of TAED was comparable with the concurrent negative control value.

Justification for selection of genetic toxicity endpoint
Endpoint selection:
KEY_471/472_1988_CCR_89.0054,
KEY_473_1995_Safepharm_ProjectNo.167/063,
KEY_476_HPRT_2014_Harlan_1578800,
KEY_474_MN in vivo_1995_Safepharm_167/064

Short description of key information:
TAED was non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and the Escherichia coli strain WP2 with and without S9-mix , non-castogenic in V79 cells and human lymphocyte cultures, did not induce gene mutations at the HPRT locus in V79 cells and was non-mutagenic in the in vivo micronucleus test in mice.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

TAED was non-mutagenic in the bacterial reverse mutation assays and in the in vitro mammalian cell gene mutaion assay (HPRT), non-castogenic in V79 cells and human lymphocyte cultures and non-mutagenic in the micronucleus test in vivo. Based on these data it is concluded that TAED is not subject to classification and labelling according to Directive 67/548/EEC and Regulation 1272/2008/EC regarding genetic toxicity.