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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames test (OECDTG 471), the in vitro chromosome aberration test (OECDTG 473) as well as the gene mutation test in mammalian cells (OECDTG 490) all provided negative results.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 October 1999 - 5 November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
92/69/EEC
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Liver S9-mix from rats induced with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Doses for the plate incorporation assay were determined based on the solubility of the test item at 5000 µg/plate. If no limited solubility was observed, this dose was used as the top dose with addition of at least five additional doses.
- Plate incorporation assay (all strains, with and without S9): 16, 50, 160, 500, 1600 and 5000 µg/plate
Doses in the preincubation assay were determined based on the results of the first assay.
- Preincubation assay (all strains, with and without S9): 16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
Sufficient data was available in the literature and from own experience, indicating that this solvent has no influence on the spontaneous mutant count of the used strains.
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: nitrofurantoin (for TA100, without S9), 4-nitro-1,2-phenylene diamine (for TA1537 and TA98, without S9); 2-aminoanthracene (all strains, with S9)
Remarks:
Positive controls were dissolved in DSMO
Details on test system and experimental conditions:
Two individual experiments were performed, one plate incorporation assay and one preincubation assay to confirm the results of the plate incorporation assay.

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes (only applicable to the second experiment)
- Exposure duration: 48 hours (in both experiments)

NUMBER OF REPLICATIONS: one plate with S9 and one plate without S9 per concentration

METHODS:
Plate incorporation: 0.1 mL of the test item, 0.1 mL tester strain (bacteria), 0.5 mL S9 mix or buffer and 2.0 mL soft agar were added to a petri dish with solid agar after 30 seconds in a waterbath (45 °C). The plates were incubated at 37 °C for 48 hours.
Preincubation: 0.1 mL tester strain (bacteria), 0.1 mL of the test item and 0.5 mL S9 mix or buffer was preincubated for 20 minutes in a 37 °C water bath. After the preincubation, 2.0 mL soft agar was added and the solutions were added to plates with solid agar. The plates were incubated at 37 °C for 48 hours.

DETERMINATION OF CYTOTOXICITY
- Method: colony counting was performed by an automatic counter.
Cytotoxicity was determined by assessing the background growth, determination of a dose-dependent reduction in the mutant count per plate and titer determination.
- Other: the dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased five-fold to permit the complete growth of the bacteria.

Evaluation criteria:
EVALUATION CRITERIA:
- A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
- For TA1535, TA100 and TA98 this increase should be about twice that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA102, an increase of about 100 mutants should be reached.
- If otherwise, the result is considered to be negative.


ACCEPTABILITY CRITERIA:
- The negative controls have to be within the expected range, as defined by published data and/or the laboratories' own historical data.
- The positive controls have to show sufficient effects, as defined by the laboratories' experience.
- Titer determinations have to demonstrate sufficient bacterial density in the suspension.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the preincubation test at 5000 µg/plate only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments, at 5000 µg/plate only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments, at 5000 µg/plate only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments at and above 1600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- None of the five strains showed a dose-related and biologically relevant increase in mutant counts compared to the negative controls in the plate incorporation test, both with and without S9. These results were confirmed in the preincubation assay.
- No precipitation was observed in any of the strains, at any dose level, with and without S9.
- The positive controls showed relevant increases in mutant counts showing that the test system functioned properly.
Conclusions:
Based on the results of this AMES test, in which no biologically relevant effects of the test item on five Salmonella strains were observed compared to the negative controls, N,N-Bis-(hydroxyethyl)-m-toluidine was determined to be non-mutagenic.
Executive summary:

An Ames test was performed, according to OECD guideline 471 and GLP principles, to assess the mutagenic potential of N,N-Bis-(hydroxyethyl)-m-toluidine in five Salmonella strains (TA1535, TA1537, TA98, TA100 and TA102). Two individual experiments were carried out, a plate incorporation assay and a preincubation assay, in which the test item was tested up to a concentration of 5000 µg/plate in the absence and in the presence of metabolic activation (S9 -mix). Toxicity of the test item on the bacterial strains was observed only at the highest doses. No increases in mutant counts compared to the solvent controls were seen. The results of the positive controls were within the historical data range of the test facility and showed that the test system functioned properly. Based on these results, N,N-Bis-(hydroxyethyl)-m-toluidine, was determined to be non-mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2017 - 08 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- pH: 8-9 at concentration of 1% w/w
- Specific gravity: 1.115 kg/L
Species / strain / cell type:
lymphocytes: peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: cells from healthy, adult, non smoking humans were used as recommended in international guidelines (e.g. OECD).
- Age of donors and Average Generation Time (AGT)
* dose-range finding study: age 31, AGT = 13.8 h
* first cytogenetic assay: age 35, AGT = 13.9 h
* second cytogenetic assay: age 28, AGT = 13.4 h
* additional second cytogenetic assay: age 22, AGT = 13.2 h
- Whole blood treated with heparin was used


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
Dose-range finding test (without and with S9): 63, 125, 250, 500, 1000 and 1953 μg the test item/mL
First cytogenic assay (without and with S9): 500, 1000 and 1953 μg/mL
Second cytogenic assay (without S9): 50, 250, 350, 400, 500 and 600 μg/mL
Additional second cytogenic assay (without S9): 50, 200, 400, 500, 600, 700 and 800 μg/mL
Vehicle / solvent:
- Vehicle use: dimethyl sulfoxide
- Justification for choice of vehicle: according to OECD guideline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24 h and 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity (set to maintain): 80-100% (actual: 48-90%)
- Temperature (set to maintain): 37.0 ± 1.0°C (actual: 34.8-37.1 °C)

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: at least 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: the test item was not cytotoxic or difficult to dissolve in aqueous solutions, the highest concentration analyzed was the recommended dose level of 0.01 M or had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).
Statistics:
Fisher's exact test, one-sided, was used to compare the incidence of abberant cells cells with the concurrent negative control.
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Since no appropriate cytotoxicity was observed, the second assay was repeated.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE-RANGE FINDING STUDY:
- Precipitation: the test item showed no precipitation at 1953 μg/mL which was therefore selected at the highest dose in the dose-range finding study.
- Mitotic index: without S9, for the 3 hour exposure the number of metaphases ranged from 69-93% of the control, for the 24 hour exposure the number of metaphases ranged from 15-91% of the control and for the 48 hour exposure the number of metaphases ranged from 0-100% of the control; With S9: the number of metaphases ranged from 90-98% of the control.

FIRST CYTOGENETIC ASSAY:
- Doses selected for scoring: 500, 1000 and 1953 μg/mL (all tested doses)
- Mitotic index: without S9: number of metaphases was 93-99% of control; with S9: number of metaphases was 93-100% of control
- Mutagenicity: both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix, the test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY:
- Doses selected for scoring: no appropriate cytotoxicity was observed at the concentrations tested, therefore no dose levels could be selected for scoring of chromosome aberrations
- Mitotic index: 24 hour exposure: number of metaphases spread was 58-86% of control; 48 hour exposure: number of metaphases spread was 66-99% of control.
- Mutagenicity: since no appropriate cytotoxicity was observed at the concentrations tested, the experiment was repeated in cytogenetic assay 2A.

CYTOGENETIC ASSAY 2A:
- Doses selected for scoring: 50, 400 and 600 μg/mL
- Mitotic index: 24 hour exposure: number of metaphases spread was 38-96% of control; 48 hour exposure: number of metaphases spread was 39-89% of control.
- Mutagenicity: the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations and did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

OTHER:
- pH of 1953 μg/mL: 7.625
- Osmolarity of 1953 μg/mL: 0.439 mOsm/kg
- pH and osmolarity in the solvent control were 7.626 and 0.454 mOsm/kg, respectively.

ACCEPTABILITY:
- The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.
- The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.
- The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells and the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
Remarks on result:
other: First cytogenetic assay
Conclusions:
A chromosome aberration study with Ethanol, 2,2’-[(3-methylphenyl)imino]bis- was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Ethanol, 2,2’-[(3-methylphenyl)imino]bis- is not clastogenic in human lymphocytes.
Executive summary:

A chromosome aberration study was performed according to OECD guideline 473 and GLP principles to assess the clastogenic potential of Ethanol, 2,2’-[(3-methylphenyl)imino]bis-in human lymphocytes, either in the absence or in the presence of a metabolic activation system (S9 -mix). In the dose-range finding study precipitation of the test item was observed at 1953 μg/mL and this was chosen as the highest dose in the first cytogenetic assay, which consisted of a exposure time of 3 hours and a fixation time of 24 hours in the absence and presence of S9 -mix. In the second cytogenetic assay the test item was tested up to 600 µg/ml for both a 24 hour and a 48 hour exposure time, both with a fixation time of 24 and 48 hours, respectively, in the absence of S9 -mix. In both assays, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations and did not increase the number of polyploid cells and cells with endoreduplicated chromosomes. Therefore it can be concluded that Ethanol, 2,2’-[(3-methylphenyl)imino]bis- does not disturb mitotic processes and cell cycle progression and that it is not clastogenic in human lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 June 2018 - 21 Augustus 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 63, 125, 250, 500, 1000, and 1953 μg/mL

Experiment 1
3 hour treatment (without S9): 31, 63, 125, 250, 500, 1000, 1500, 1800 and 1953 μg/mL
3 hour treatment (with S9): 31, 63, 125, 250, 500, 1000, 1500 and 1953 μg/mL

Experiment 1a
3 hour treatment (with S9): 63, 125, 250, 500, 1000, 1250, 1500, 1600, 1700, 1800 and 1953 µg/ mL

Experiment 2
24 hour treatment (without S9): 16, 31, 63, 125, 250, 500, 700, 800, 900 and 1000 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification of Vehicle: Based on a solubility test, DMSO was selected as vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9; 7.5 µg/mL in Hanks’ balanced salt solution without calcium and magnesium.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

TEST ITEM PREPARATION
The test item was dissolved in DMSO. Test item concentrations were used within 1 hour after preparation.


DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium.
The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

OTHER EXAMINATIONS
pH and the osmolarity of the culture medium containing the highest, non-precipitating concentration were recorded.
Evaluation criteria:
DATA EVALUATION
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.

- The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
-pH and osmolarity: The pH and osmolarity at a concentration of 1953 μg/mL were 7.39 and 0.416 Osm/kg respectively (compared to 7.39 and 0.445 Osm/kg in the solvent control).

RANGE-FINDING/SCREENING STUDIES
The 3 hour exposure showed a relative suspension growth of 31 and 29% at the test item concentration of 1953 μg/mL compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively.
The 24 hour exposure showed relative suspension growth of 10% at the test item concentration of 1000 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 1953 μg/mL.

EXPERIMENT 1
A repeat experiment with S9 mix was performed (1a). The first experiment was rejected because no dose level with a cell surival below 52% was observed.

In the absence of S9-mix, the dose levels of 31 to 500 μg/mL showed no cytotoxicity. Therefore, the dose level of 31 µg/mL was not regarded relevant for mutation frequency measurement.
The dose levels of 1500 and 1700 μg/mL and 1600 and 1800 μg/mL with S9 showed similar cytotoxicity. Therefore, the dose levels of 1500 and 1600 µg/mL were not regarded relevant for mutation frequency measurement. The dose level of 1953 μg/mL with S9 mix was not used for mutation frequency measurement, since this dose level was too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 63, 125, 250, 500, 1000, 1500, 1800 and 1953 μg/mL exposure medium.
With S9-mix: 63, 125, 250, 500, 1000, 1250, 1700 and 1800 μg/mL exposure medium.

In the absence of S9-mix the relative total growth of the highest test item concentration of 1953 µg/mL was 5% compared to the total growth of the solvent controls. Whereas one dose level below (1800 µg/mL) showed a RTG of 26%.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 16% compared to the total growth of the solvent controls.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


EXPERIMENT 2

The dose levels of 900 to 1000 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were: 16, 31, 63, 125, 250, 500, 700 and 800 µg/mL exposure medium.
The relative total growth of the highest test item was 15% compared to the total growth of the solvent controls.

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

ACCEPTANCE:
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The suspension growth over the two-day expression period for cultures treated with DMSO was between 19 and 20 (3 hour treatment) and 105 and 114 (24 hour treatment).
Conclusions:
An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles with ethanol, 2,2’-[(3-methylphenyl)imino]bis-. Based on the results, ethanol, 2,2’-[(3-methylphenyl)imino]bis- is not mutagenic with and without metabolic activation in the TK mutation test system under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames

An Ames test was performed, according to OECD guideline 471 and GLP principles, to assess the mutagenic potential of N,N-Bis-(hydroxyethyl)-m-toluidine in five Salmonella strains (TA1535, TA1537, TA98, TA100 and TA102). Two individual experiments were carried out, a plate incorporation assay and a preincubation assay, in which the test item was tested up to a concentration of 5000 µg/plate in the absence and in the presence of metabolic activation (S9-mix). Toxicity of the test item on the bacterial strains was observed only at the highest doses. No increases in mutant counts compared to the solvent controls were seen. The results of the positive controls were within the historical data range of the test facility and showed that the test system functioned properly. Based on these results, N,N-Bis-(hydroxyethyl)-m-toluidine, was determined to be non-mutagenic.

Chromosome aberration study

A chromosome aberration study was performed according to OECD guideline 473 and GLP principles to assess the clastogenic potential of Ethanol, 2,2’-[(3-methylphenyl)imino]bis-in human lymphocytes, either in the absence or in the presence of a metabolic activation system (S9 -mix). In the dose-range finding study precipitation of the test item was observed at 1953 μg/mL and this was chosen as the highest dose in the first cytogenetic assay, which consisted of a exposure time of 3 hours and a fixation time of 24 hours in the absence and presence of S9 -mix. In the second cytogenetic assay the test item was tested up to 600 µg/ml for both a 24 hour and a 48 hour exposure time, both with a fixation time of 24 and 48 hours, respectively, in the absence of S9 -mix. In both assays, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations and did not increase the number of polyploid cells and cells with endoreduplicated chromosomes. Therefore it can be concluded that Ethanol, 2,2’-[(3-methylphenyl)imino]bis-does not disturb mitotic processes and cell cycle progression and that it is not clastogenic in human lymphocytes.

Mouse lymphoma assay

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of the substance to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The test item was tested in the absence of S9 in a 3 and 24 hour treatment period and in the presence of S9 in a 3 hour treatment period. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test item did not induce an increase in the mutation frequency. Therefore, the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the results of the Ames test, the in vitro chromosome aberration study and the mouse lymphoma study, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 (CLP).