Benzyltriethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay

Test substance did not induce a significant increase in the number of revertant colonies beyond the negative control incidence in any of the tested strains either with or without a metabolic activation system.

In vitro chromosomal abbreviation study

Test substance 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data to 1984-10-25

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study report

Justification for type of information:

Data is from study report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only two strains evaluated (Salmonella typhimurium TA98 and TA100), limited data on the test item and the methodology

Principles of method if other than guideline:

Ames assay was performed to determine the mutagenic potential of test substance.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): T779
- IUPAC name: N-Benzyl-N,N-diethylethanaminium chloride
- Substance type: white powder
- Physical state: solid

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium, other: TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: other: See below

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

With and without metabolic activation, main and repeat experiments: 0, 312.5, 625, 1250, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation at 5 µg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: at 1 µg/plate for TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation: for both strains at 1 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Tests were performed by mixing test article dilutions, or appropriate controls, 1-6 x E8 bacteria from an overnight culture in nutrient broth (0.1 mL /plate +/- 0.5 mL of S9 mix in histidine-biotin supplemented top agar in petri dishes.
After incubation of the plates for 48 hours at 37°C in the dark, the number of macroscopic colonies (his+) revertants were counted.

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
- The viability of the strain was also verfied by plating appropriate dilutions of the bacterial suspension onto histidine rich agar plates.

Rationale for test conditions:

No data

Evaluation criteria:

The results were considered as positive if there was a dose-related increase in the number of revertant colonies with at least a twofold increase over the controls.
Results that did not meet the criteria for determining a positive result were considered to be negative.

Statistics:

no data

Species / strain:

S. typhimurium, other: TA 98 and TA 100

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:

valid

Positive controls validity:

valid

Additional information on results:

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

RANGE-FINDING/SCREENING STUDIES:
- Before conducting the mutagenicity study the test substance was assessed for bacterial cytotoxicity using one of the bacterial strains. The Salmonella strain was inoculated from cultures on master plates into nutrient broth and grown overnight at 37°C. The viability of the strain was tested by plating appropriate (10E-6) dilutions of the bacterial suspension onto histidine rich agar plates. Appropriate dilutions of test substance, bacterial strain (10E-6) and top agar were mixed and poured on agar plates. The number of surviving colonies were counted and the toxic dose level was determined. The test substance was tested for bacterial cytotoxicity at doses up to 5000 µg/plate. At doses of 100, 500, 1000 and 5000 µg/plate the cell viability of the bacteria was only slightly reduced. 5000 µg/plate was selected as the highest dose for the plate incorporation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive controls, 5 µg 2-nitrofluorene/plate, 1 µg sodiumazide/plate and 1 µg 2-aminoanthracene/plate, induced a significant increase in the number of revertant colonies

ADDITIONAL INFORMATION ON CYTOTOXICITY: See range finding/screening study data

Remarks on result:

other: No mutagenic potential

Conclusions:

Test substance did not induce a significant increase in the number of revertant colonies beyond the negative control incidence in any of the tested strains either with or without a metabolic activation system.

Executive summary:

Ames assay was performed to determine the mutagenic potential of test substance. The test chemical was dissolved in water and used at dose levels of 0, 312.5, 625, 1250, 2500, and 5000 µg/plate in the presence and absence of S9 metabolic activation system in the main and repeat experiments performed. After incubation of the plates for 48 hours at 37°C in the dark, the number of macroscopic colonies (his+) revertants were counted. Concurrent solvent and positive control chemicals were also included in the study. The results were considered as positive if there was a dose-related increase in the number of revertant colonies with at least a twofold increase over the controls. Results that did not meet the criteria for determining a positive result were considered to be negative. Test substance did not induce a significant increase in the number of revertant colonies beyond the negative control incidence in any of the tested strains either with or without a metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-07-06 to 2006-11-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Data is from study report

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:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000008753
- Expiration date of the lot/batch: 2008-03-01
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, light protected
- Stability under test conditions:Not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: The final concentration of deionised water in the culture medium was 10 % (v/v). No data on stability is provided..
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human lymphocytes

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

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:

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 cytogenetic
evaluation, 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.

Vehicle / solvent:

- 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.

Untreated negative controls:

yes

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

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

Details on test system and experimental conditions:

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 technique

NUMBER 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 data

Rationale for test conditions:

No data

Evaluation criteria:

- 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).

Statistics:

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.

Species / strain:

lymphocytes: human

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

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:

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 observed

RANGE-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).

Remarks on result:

other: No mutagenic potential

Conclusions:

Test substance 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.

Executive summary:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test substance. 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, test substance 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for the various publication were observed for test chemical. Test chemical was reviewed to determine the mutagenic nature of N-benzyl-N,N-diethylethanaminium chloride (56-37-1). The studies are as mentioned below:

 

Ames assay

Ames assay was performed to determine the mutagenic potential of test substance. The test chemical was dissolved in water and used at dose levels of 0, 312.5, 625, 1250, 2500, and 5000 µg/plate in the presence and absence of S9 metabolic activation system in the main and repeat experiments performed. After incubation of the plates for 48 hours at 37°C in the dark, the number of macroscopic colonies (his+) revertants were counted. Concurrent solvent and positive control chemicals were also included in the study. The results were considered as positive if there was a dose-related increase in the number of revertant colonies with at least a twofold increase over the controls. Results that did not meet the criteria for determining a positive result were considered to be negative. Test substance did not induce a significant increase in the number of revertant colonies beyond the negative control incidence in any of the tested strains either with or without a metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro chromosomal abbreviation study

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test substance. 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, test substance 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.

Based on the data summarized, N-benzyl-N,N-diethylethanaminium chloride (56-37-1) did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria test substance N-benzyl-N,N-diethylethanaminium chloride (56-37-1) did not induce gene mutation .Hence it is not likely to be classified as mutagenic in vitro.