Tetrylammonium bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical N,N,N-triethylethanaminium bromide (CAS no 71-91-0) did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

The test chemical N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) did not induce structural chromosomal aberrations in the cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

other: Target data

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver enzyme (RLE) mix

Test concentrations with justification for top dose:

1. 0.01, 1, 5 or 20 mg/plate2. 100- 10000 µg/plate

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: No data- Justification for choice of solvent/vehicle: No data2. - Vehicle(s)/solvent(s) used: Yes, no detailed data available - Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

yes

Remarks:

Sterile distilled water and DMSO

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminofluorene (TA98)

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

No data

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data- Exposure duration: 48 hrs- Expression time (cells in growth medium): 48 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data2. Details on test system and conditionsMETHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data available- Exposure duration: 48 h - Expression time (cells in growth medium): 48 h- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1. A chemical is considered to be mutagenic if the number of induced revertants is two or more times greater than the number of spontaneous revertants.(1) a significant relationship between the number of revertant colonies and dose concentration and (2) a two-fold or more increase in the number of revertant colonies over the number of spontaneous revertants seen in the control and (3) a Mutagenicity Index (MI) value greater than 2.0 or a Mutagenicity Activity Ratio (MAR) greater than 2.5.2. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Statistics:

1. No data

Species / strain:

S. typhimurium, other: TA98 and TA100

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Both the positive control chemicals yielded 20–100 times more revertants than the number of spontaneous revertants, indicating a high level of mutagenicity

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: Preliminary data was obtained from a rapid spot-test screening experiment indicating that the ILs tested were non-mutagenic at quantities below 1 mg/ plate.COMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data2. RANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical N,N,N-triethylethanaminium bromide (CAS no 71-91-0) did not induce mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0). The studies are as metnioned below:

Gene mutation toxicity study (Ames assay) was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strain TA98 and TA100 with and without S9 metabolic activation system at dose levels of 0.01, 1, 5, 20 mg/plate. The doses were selected on the basisrapid spot-test screening experimentindicating that the test chemical wasnon-mutagenic at quantities below 1 mg/ plate. Sodium azide (TA100) and 2-aminofluorene (TA98) were used at positive controls and sterile distilled water and DMSO was used as negative controls. The plates were incubated for 48 hrs and the number of induced revertants was counted. The test chemical did not induce gene mutation in Salmonella typhimurium strain TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study, ames mutagenicity test was conducted for chemical Tetraethyl orthosilicate (Ethyl silicate) to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with dose concentration of 100-10000 µg/plate in plate incorporation assay. Based on the preliminary study conducted, the test compound was used at a five dose level from 100- 10000 µg/plate. Tetraethyl orthosilicate (Ethyl silicate) failed to induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 both in the presence and absence of S9 activation system and hence is not likely to be a gene mutant.

Based on the data available, N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) is not likely to classify as a gene mutant as per the criteria mentioned in CL Pregulation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Ninth Addendum, adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Annex 4A Directive 2000/32/EC: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test"

Deviations:

no

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

1. not applicable2. Thymidime kinase

Species / strain / cell type:

lymphocytes: human lymphocytes

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; containing 10 % FCS (fetal calf serum). The antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL, NATTERMANN, D-50829 Köln), and HEPES (final concentration 10 mM)- Properly maintained: no data- Periodically checked for Mycoplasma contamination: no data- Periodically checked for karyotype stability: no data- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / 2

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.- Properly maintained: No data available - Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1. Concentrations dosed:- Experiment I: 4 hour exposure without metabolic activation: 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis.)- Experiment I: 4 hour exposure with metabolic activation: 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis.)- Experiment II: 22 hour continuous exposure without metabolic activation: 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis.)The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. With respect to the molecular weight of the test item, 2280 μg/mL of T779 (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 14.8 and 2280 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed before start of treatment. Since the cultures fulfilled the requirements for cytogeneticevaluation, this preliminary test was designated Experiment I.Using reduced mitotic indices as an indicator for toxicity in the pre-test, no clear toxic effects were observed after 4 hrs treatment up to the highest applied concentration, in the absence and presence of S9 mix. Therefore, 2280 μg/mL was chosen as top treatment concentration for continuous exposure in Experiment II.2. 250 -1300 µg/mL

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: deionized water- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.2. No data available

Untreated negative controls:

yes

Remarks:

1

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation: at a concentration of 770 µg/mL (Experiment I) and 550 µg/mL (Experiment II)

Untreated negative controls:

yes

Remarks:

1

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation: at a concentration of 37.5 µg/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.

Details on test system and experimental conditions:

1. METHOD OF APPLICATION:- in medium- About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation.- In Experiment II, the culture medium was replaced with complete medium (with 10 % FCS) containing the test substance. The culture medium at continuous treatment was not changed until preparation of the cells.All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).DURATION- Exposure duration: 4 hours (Experiment I); 22 hours (Experiment II - continuous exposure)- Expression time: 15 hours (Experiment I); 19 hours (Experiment II) (Start of exposure until introduction of spindle inhibitor)- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours (Experiments I and II)SPINDLE INHIBITOR (cytogenetic assays): Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 3°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide.STAIN (for cytogenetic assays): Giemsa or fluorescent plus Giemsa techniqueNUMBER OF REPLICATIONS:- Replicates consisting of two primary cultures were tested.NUMBER OF CELLS EVALUATED:- One-hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis. DETERMINATION OF CYTOTOXICITY - Method: mitotic index (1000 cells per culture were counted.)OTHER EXAMINATIONS: - Determination of polyploidy: yes. The number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.- Determination of endoreplication: no data2. METHOD OF APPLICATION: in mediumCells at start: 6000000 cellsDURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium):48 h- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selectionSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200cells/plate for viable count determinations DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated culturesOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1. - The test substance was classified as non-mutagenic if: the number of induced structural chromosome aberrations in all evaluated dose groups was in the range of the laboratory historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and no significant increase of the number of structural chromosome aberrations is observed.- A test item is classified as mutagenic if: the number of induced structural chromosome aberrations was not in the range of the laboratory historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.A test item can be classified as aneugenic if:- the number of induced numerical aberrations is not in the range of the laboratory historical control data (0.0 – 1.5 % polyploid cells).2. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Statistics:

1. Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test substance was not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.2. No data

Species / strain:

lymphocytes: human

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Remarks:

1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the mitotic index was clearly reuced at the highest concentration evaluated.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH and osmolality: No relevant increase in the pH and osmolality (Exp. I: solvent control: 276 mOsm, pH 7.3 versus 298 mOsm and pH 7.4 at 2280 µg/mL)- Evaporation from medium: no data- Water solubility: 630 g/L- Precipitation: In both experiments, no visible precipitation of the test item in the culture medium could be observedRANGE-FINDING/SCREENING STUDIES: - The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times were 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 ug/mL) to reassure the replication time of the cultured lymphocytes. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. COMPARISON WITH HISTORICAL CONTROL DATA: - EMS (550 and 770 µg/mL, respectively) and CPA (37.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations- The proliferation index of the lymphocytes in solvent control cultures in the 22 hrs preparation interval with and without S9 mix (4 hrs treatment; both 1.00), in the 22 hours preparation interval without S9 mix (continuous treatment; 1.35), was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cellsand by a clear clastogenicity observed after treatment with the positive control substances. ADDITIONAL INFORMATION ON CYTOTOXICITY:- In experiment I in the presence and absence of S9 mix, no cytotoxicity was observed up to the highest applied concnetration. In Experiment II, after continuous exposure, in the absence of S9 mix, the mitotic index was clearly reduced at the highest concentration evaluated (44.9% of the control).2. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: No data availableCOMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) did not induce structural chromosomal aberrations in the cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0). The studies are as metnioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using human lymphocytes in the presence and absence of metabolic activation system in experiment 1 of 4 hrs duration and in the absence of metabolic activation sytem in experiment 2 of 22 hours continuous duration. The test chemical was dissolved with deionized water and used at dose levels of 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 1 and 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 2. About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation. In Experiment II, the culture medium was replaced with complete medium (with 10 % FCS) containing the test substance. The culture medium at continuous treatment was not changed until preparation of the cells. All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air). Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 3°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. One-hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis. The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. In experiment I in the presence and absence of  S9 mix, no cytotoxicity was observed up to the highest applied concnetration. In Experiment II, after continuous exposure, in the absence of S9 mix, the mitotic index was clearly reduced at the highest concentration evaluated (44.9% of the control). No chromosome aberration was however observed in both the experiments. Based on the observations made, the test chemical did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 250 - 1300 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not inducea doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data available for the test chemical, the test chemical N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) did not induce structural chromosomal aberrations in the cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Ames test:

Data available for the test chemicals was reviewed to determine the mutagenic nature of N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0). The studies are as metnioned below:

Gene mutation toxicity study (Ames assay) was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strain TA98 and TA100 with and without S9 metabolic activation system at dose levels of 0.01, 1, 5, 20 mg/plate. The doses were selected on the basisrapid spot-test screening experimentindicating that the test chemical wasnon-mutagenic at quantities below 1 mg/ plate. Sodium azide (TA100) and 2-aminofluorene (TA98) were used at positive controls and sterile distilled water and DMSO was used as negative controls. The plates were incubated for 48 hrs and the number of induced revertants was counted. The test chemical did not induce gene mutation in Salmonella typhimurium strain TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study, ames mutagenicity test was conducted for chemical Tetraethyl orthosilicate (Ethyl silicate) to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with dose concentration of 100-10000 µg/plate in plate incorporation assay. Based on the preliminary study conducted, the test compound was used at a five dose level from 100- 10000 µg/plate. Tetraethyl orthosilicate (Ethyl silicate) failed to induce mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 both in the presence and absence of S9 activation system and hence is not likely to be a gene mutant.

Based on the data available, N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) is not likely to classify as a gene mutant as per the criteria mentioned in CL Pregulation.

Chromosome aberration in vitro:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using human lymphocytes in the presence and absence of metabolic activation system in experiment 1 of 4 hrs duration and in the absence of metabolic activation sytem in experiment 2 of 22 hours continuous duration. The test chemical was dissolved with deionized water and used at dose levels of 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 1 and 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 2. About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation. In Experiment II, the culture medium was replaced with complete medium (with 10 % FCS) containing the test substance. The culture medium at continuous treatment was not changed until preparation of the cells. All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air). Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 3°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. One-hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis. The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. In experiment I in the presence and absence of  S9 mix, no cytotoxicity was observed up to the highest applied concnetration. In Experiment II, after continuous exposure, in the absence of S9 mix, the mitotic index was clearly reduced at the highest concentration evaluated (44.9% of the control). No chromosome aberration was however observed in both the experiments. Based on the observations made, the test chemical did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

In another study, the gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 250 - 1300 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not inducea doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data available for the test chemical, the test chemical N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) did not induce structural chromosomal aberrations in the cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

Based on the weight of evidence approach and the existing data available, N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the weight of evidence approach and the existing data available, N,N,N-triethylethanaminium bromide (CAS no 71 -91 -0) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.