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EC number: 218-658-4 | CAS number: 2212-32-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A battery of in vitro genetic toxicity tests have been conducted with the submission substance. 2-[[2 -(Dimethylamino)ethyl] methylamino]ethanol was negative in an Ames bacterial mutation assay, a mammalian mutation assay and a chromosome aberration assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Test material is Halb- PMDETA
Batch number: Vers. 98/044-10
CAS number: 2212-32-0
Purity: 99.4% (GC)
Appearance: Yellowish liquid
Storage: Room termperature - Target gene:
- Histidine (S. typhimurium) and tryptophan (E. Coli) operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- 1 volume of Aroclor 1254 induced rat liver S-9 fraction mixed with 9 volumes of cofactors mix (MgCl2, KCl, Glu-6-p, NADP, phosphate buffer, NaH2PO4, Na2HPO2.2H2O)
- Test concentrations with justification for top dose:
- 0, 20, 100, 500, 2,500 and 5,000 µg/plate (both experiments)
- Vehicle / solvent:
- Due to the good solubility of the test substance in water, water was selected as the vehicle.
- Untreated negative controls:
- yes
- Remarks:
- (sterility control)
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: 2-aminoanthracene, (2-AA) in the presence of S-9; N-methyl-N'-nitro-N -nitrosoguanidine (MNNG) and 4-nitro-o-phenylene-diamine (4-NOPD) in the absence of S-9
- Remarks:
- 60 µg 2-AA/plate for E. Coli and 2.5 µg 2-AA/plate (remaining strains); 5 µg MNNG/plate for TA 1535 and TA 100; 10 µg 4-NOPD/plate for TA 98, 100 µg 9-AAC/plate for TA 1537, 5 µg 4-NQO/plate for E. Coli. All positive controls were dissolved in DMSO.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: - Experiment 1 (range finder): in agar (plate incorporation test); Experiment 2: pre-incubation test
DURATION- Preincubation period (Exp. 2 only): ca. 20 min- Exposure duration: 48 - 72 hours at 37°C
NUMBER OF REPLICATIONS: 2 experiments; triplicate cultures/experiment
DETERMINATION OF CYTOTOXICITY - Method: reduction in the number of revertants or a clearing of the bacterial background lawn, reduction of the titer.The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments. - Evaluation criteria:
- Acceptance criteria of study: The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.- The sterility controls revealed no indication of bacterial contamination.- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data- The titer of viable bacteria was equal or greater than 1E+09 per milliliter.Evaluation criteria:Positive result when; A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.Negative result when; The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
- Statistics:
- Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
- Species / strain:
- S. typhimurium TA 98
- 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
- Species / strain:
- S. typhimurium 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
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Reduction in the number of revertants
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- 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- Precipitation: No precipitation of the test item was observed up to the highest concentration tested.
- Conclusions:
- No evidence of mutagenicity was seen in any of the tester strains, in the absence and presence of metabolic activation.
- Executive summary:
A bacterial reverse mutation test (Ames test) was conducted with Halb-PMDETA according to OECD 471 and EEC 92 -69 B14 and B13. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA were tested in triplicate the absence and in the presence of metabolic activation at concentrations up to 5000 µg/plate in two independent experiments. Plate incorporation methodology was used in Test 1 and pre-incubation methodology was used in Test 2 . There was some evidence of toxicity in strains TA1535 and TA1537 in the form of a marked reduction in the number of revertant colonies. There was no precipitation of the test article or increases in the number of revertants when compared to the concurrent controls. The overall conclusion is that the test material
Halb-PMDETA is not mutagenic when tested up to 5000 µg/plate in the absence and presence of metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 12 November 2015 to 21 Sep 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- other: The Japanese Ministry of Health, Labour and Wefare (MHLW), Ministry of Economy Trade and Industry (METI) and Ministry of the Environmenatl (MOE) Guidelines of 31 March 2011
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian chromosome aberration assay
- Specific details on test material used for the study:
- CAS number: 2212-32-0
Appearance: yellow liquid
Batch number: 1721964
Purity: 99%
Retest date: 08 April 2016
Storage conditions: Room temperature protected from light. - Species / strain / cell type:
- lymphocytes:
- Metabolic activation:
- with and without
- Metabolic activation system:
- 2% S9 - Phenobarbital/β-napthaflavone induced
- Test concentrations with justification for top dose:
- 45.70, 91.39, 182.79, 365.58, 548.37, 731.15, 1462.3 µg/mL for 4 hour treatments in the absence and presence of S-9 with 20 hours recovery and 24 hours in the absence of S-9 with no recovery.
The maximum concentration tested was 10 mM (based on a molecular weight of 146.23). - Vehicle / solvent:
- Minimum Essential Media (MEM)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using B dU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approx mately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 35 years
Main Experiment: male, aged 31 years
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation, the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.
For the 4(20)-hour exposure in the absence of S9 - After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
For the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.0 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours. The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.
For the Prelimiary test three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 5.71 to 1462.3 μg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.
For the main experiment, three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 45.70 to 1462.3μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 45.70 to 1462.3μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 45.70 to 1462.3μg/mL.
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation. The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium. The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.The slides were coded using a computerized random number generator. A total of 2000 lymphocyte cell nucleiwere counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. - Evaluation criteria:
- The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.
A test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in the dose groups should be within the range of the laboratory historical control data (normal range)
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level
A test item can be classified as able to induce
chromosomal aberrations in human lymphocytes if the following are all met
1) The number of cells with structural chromosome aberrations is outside the range of the nomal range.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test..
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations were assessed (where necessary) on a case by case basis. - Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No polyploid cells were observed.
There was no marked changed in pH of 1 pH unit or osmolality of 50 mOsm/kg in cultures containing test material at concentrations up to 1462.3 µg/mL - Conclusions:
- 2 -[[2 -(Dimethylamino)ethyl] methylamino]ethanol was considered to be non-clastogenic to human lymphocytes in vitro under the conditions of this study.
- Executive summary:
An in vitro chromosome aberration assay was conducted in accordance with OECD 473. Human peripheral blood lymphocytes were treated with the test material 2 -[[2 -(Dimethylamino)ethyl] methylamino]ethanol [CAS# 2212-32-0] at concentrations up to 1462 µg/mL (10 mM) for 4 hours with 20 hours recovery in the absence and in the presence of metabolic activation (phenobarbital/β-napthaflavone induced S-9) and for 24 hours with no recovery in the absence of metabolic activation. The highest concentration was 10 mM based on a molecular weight of 146.2. The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 12 November 201 to 02 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Kanpoan number 287 - Environment Protection Agency
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Eisei number 127 - Ministry of Health and Welfare
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Heisei 09/10/31 Kikyoku number 2 - Ministry of International Trade and Industry
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian cell mutation study
- Specific details on test material used for the study:
- CAS number: 2212-32-0
Appearance: yellow liquid
Batch number: 1721964
Purity: 99%
Retest date: 08 April 2016
Storage conditions: Room temperature protected from light. - Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- 2% S9 - Phenobarbital/β-napthaflavone induced
- Test concentrations with justification for top dose:
- 91.38, 182.75, 365.5, 731, 1096.5 and 1462 µg for 4 hours in the absence and presence of S-9
22.5, 45, 90, 180, 225, 270, 315 and 360 µg/mL for 24 hours in the absence of S-9.
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level.
The highest concentration for the 4-hour treatments was 10 mM (based on the molecular weight of 146.23). The highest concentration for the 24-hour treatment was based on toxicity. - Vehicle / solvent:
- RPMI 1640 containing no serum (R0 medium)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Several days before starting the mutagenicity experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium insterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at up to eight dose levels of the test item (91.38 to 1462 μg/mL for the 4-hour exposure groups, and 22.5 to 360 μg/mL for the 24-hour exposure group), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group). The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The
numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item. Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells/total wells plated
%V = - ln P(0) x 100/number of cells/well
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = %V/Mean Solvent Control %V
For each culture relative total growth, RTG, was calculated:
RTG = (RCE x RSG)/100
Mutant Frequency (MF) per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium. - Evaluation criteria:
- Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system. - Statistics:
- The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS. The statistical package used indicates the presence of statistically significant increases and linear-trend events.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Remarks:
- Vehicle control (R0 medium)
- Positive controls validity:
- valid
- Additional information on results:
- There was no marked changed in pH of 1 pH unit or osmolality of 50 mOsm/kg in cultures containing test material at concentrations up to 1462.3 µg/mL
- Conclusions:
- 2-[[2-(Dimethylamino)ethyl]methylamino]ethanol was not mutagenic when tested up to 1462 µg/mL (10 mM) for 4 hours in the absence and presence of metabolic activation or 360 µg/mL, a toxic concentration, for 25 hours in the absence of metabolic activation.
- Executive summary:
A mouse lymphoma assay was conducted in accordance with OECD 490. L5178Y cells were assesed in the mammalian mutation assay at the tk locus. In the main experiment, 2-[[2-(Dimethylamino)ethyl]methylamino]ethanol [CAS# 2212-32-0] was tested at concentrations up to 1462 µg/mL (10 mM) for 4 hours in the absence and in the presence of metabolic activation ( Phenobarbital/β-napthaflavone induced S-9) and at concentrations up to 360 µg/mL for 24 hours in the absence of metabolic activation. The highest concentration for the 24-hour treatment was limited by toxicity, as determined in a Range-Finder experiment. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the global evaluation factor (GEF), consequently it is considered to be non-mutagenic in this assay.
Referenceopen allclose all
Results of Experiment I (plate incorporation assay)
|
[C] µg/plate |
Revertants /plate (mean value of 3 plates ± standard deviation) |
|||||||||
TA 1535 |
TA 100 |
TA 1537 |
TA 98 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Water |
0 |
20 ± 1 |
20 ± 3 |
129 ± 4 |
139 ± 11 |
11 ± 2 |
12 ± 2 |
26 ± 3 |
39 ± 1 |
31 ± 6 |
33 ± 4 |
Test substance |
20 |
16 ± 2 |
17 ± 3 |
123 ± 8 |
150 ± 18 |
11 +1 |
10 ± 1 |
24 ± 2 |
33 ± 2 |
27 ± 2 |
40 ± 8 |
100 |
17 ± 1 |
17 ± 3 |
145 ± 11 |
151 ± 19 |
8 ± 1 |
9 ± 3 |
20 ± 2 |
32 ± 2 |
25 ± 4 |
33 ± 6 |
|
500 |
15 ± 1 |
14 ± 2 |
141 ± 13 |
131 ± 6 |
8 ± 1 |
9 ± 1 |
24 ± 3 |
30 ± 2 |
27 ± 5 |
38 ± 2 |
|
2500 |
17 ± 1 |
16 ± 2 |
131 ± 9 |
121 ± 9 |
8 ± 1 |
6 ± 2 |
27 ± 5 |
30 ± 2 |
31 ± 2 |
29 ± 2 |
|
5000 |
13 ± 2 |
12 ± 2 |
147 ± 7 |
161 ± 3 |
7 ± 2 |
7 ± 1 |
28 ± 3 |
27 ± 6 |
32 ± 3 |
33 ± 3 |
|
|
|||||||||||
MNNG |
5.0 |
605 ± 78 |
/ |
642 ± 21 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
2 – AA |
2.5 |
/ |
133 ± 16 |
/ |
1348 ± 69 |
|
109 ± 12 |
/ |
1073 ± 60 |
/ |
/ |
60.0 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
204 ± 10 |
|
9 – AAC |
100.0 |
/ |
/ |
/ |
/ |
589 ± 34 |
/ |
/ |
/ |
/ |
/ |
NOPD |
10.0 |
|
/ |
/ |
/ |
/ |
|
691 ± 9 |
/ |
/ |
/ |
4-NQO |
2.5 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
943 ± 21 |
/ |
2-aminoanthracene, (2-AA); N-methyl-N'-nitro-N -nitrosoguanidine (MNNG), 4-nitro-o-phenylene-diamine (4-NOPD), 9-aminoacridine 9 - AAC), 4-nitroquinol i ne-N-oxide (4-NQO)
Results of Experiment II (preincubation assay)
|
[C] µg/plate |
Revertants /plate (mean value of 3 plates ± standard deviation) |
|||||||||
TA 1535 |
TA 100 |
TA 1537 |
TA 98 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Water |
0 |
18 ± 3 |
18 ± 2 |
122 ± 9 |
149 ± 11 |
11 ± 2 |
10 ± 2 |
23 ± 5 |
26 ± 3 |
33 ± 4 |
36 ± 2 |
Test substance |
20 |
18 ± 2 |
12 ± 3 |
125 ± 15 |
138 ± 19 |
11 ± 3 |
10 ± 2 |
26 ± 2 |
27 ± 2 |
28 ± 4 |
27 ± 1 |
100 |
18 ± 4 |
17 ± 3 |
104 ± 4 |
120 ± 5 |
11 ± 3 |
14 ± 5 |
19 ± 4 |
24 ± 6 |
27 ± 7 |
31 ± 4 |
|
500 |
16 ± 3 |
12 ± 2 |
101 ± 12 |
122 ± 20 |
8 ± 1 |
10 ± 3 |
23 ± 5 |
22 ± 2 |
26 ± 5 |
33 ± 1 |
|
2500 |
17 ± 3 |
13 ± 3 |
91 ± 10 |
115 ± 20 |
8 ± 2 |
11 ± 3 |
20 ± 4 |
22 ± 7 |
28 ± 5 |
32 ± 6 |
|
5000 |
14 ± 2 |
9 ± 1 |
90 ± 6 |
131 ± 10 |
6 ± 2 |
11 ± 2 |
21 ± 2 |
26 ± 4 |
20 ± 8 |
27 ± 3 |
|
|
|||||||||||
MNNG |
5.0 |
1978 ± 54 |
/ |
1591 ± 159 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
2 – AA |
2.5 |
/ |
155 ± 28 |
/ |
841 ± 11 |
|
114 ± 9 |
/ |
764 ± 69 |
/ |
/ |
60.0 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
234 ± 15 |
|
9 – AAC |
100.0 |
/ |
/ |
/ |
/ |
356 ± 58 |
/ |
/ |
/ |
/ |
/ |
NOPD |
10.0 |
|
/ |
/ |
/ |
/ |
|
994 ± 76 |
/ |
/ |
/ |
4-NQO |
2.5 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
979 ± 67 |
/ |
2-aminoanthracene, (2-AA); N-methyl-N'-nitro-N -nitrosoguanidine (MNNG), 4-nitro-o-phenylene-diamine (4-NOPD), 9-aminoacridine 9 - AAC), 4-nitroquinol i ne-N-oxide (4-NQO)
Mitotic index data for preliminary test
Concentration (µg/mL) |
4-hour in the absence of S-9 |
4-hour in the presence of S-9 |
24 hour in the absence of S-9 |
|||
Mitotic index |
% of control |
Mitotic index |
% of control |
Mitotic index |
% of control |
|
0 |
1.95 |
100 |
1.60 |
100 |
3.70 |
100 |
5.71 |
ND |
ND |
ND |
ND |
ND |
ND |
11.42 |
ND |
ND |
ND |
ND |
ND |
ND |
22.85 |
ND |
ND |
ND |
ND |
ND |
ND |
45.70 |
ND |
ND |
ND |
ND |
ND |
ND |
91.39 |
ND |
ND |
ND |
ND |
ND |
ND |
182.79 |
ND |
ND |
ND |
ND |
3.50 |
95 |
365.58 |
2.85 |
146 |
1.60 |
100 |
3.00 |
81 |
731.15 |
2.15 |
110 |
0.65 |
41 |
1.10 |
30 |
1462.3 |
3.15 |
162 |
1.70 C |
106 C |
NM |
NM |
ND Not
determined
NM No metaphases or
insufficient metaphases suitable for scoring
C Cloudy
precipitate observed at the end of exposure period in blood-free cultures
Main Experiment: 4-hour treatment in the absence of S-9 with 20 hour recovery
Concentration (µg/mL) |
Replicate |
MI (%) |
N |
Number of aberrations |
Total number of aberrations |
Frequency of aberrations (%) |
|||||||
g |
Ctb |
Cte |
Csb |
Cse |
Others |
With gaps |
Without gaps |
With gaps |
Without gaps |
||||
0 (Vehicle control) |
A |
5.25 |
150 |
2 |
0 |
0 |
0 |
0 |
0 |
2 |
0 |
2 |
0 |
B |
4.40 |
150 |
1 |
2 |
0 |
0 |
0 |
0 |
3 |
2 |
3 |
2 |
|
TOTAL |
(100) |
300 |
3 |
2 |
0 |
0 |
0 |
0 |
5 |
2 |
5 (1.7) |
2 (0.7) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
548.37 |
A |
4.35 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.20 |
150 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
1 |
1 |
|
TOTAL |
(89) |
300 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 |
1 (0.3) |
1 (0.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
731.15 |
A |
4.25 |
150 |
0 |
3 |
0 |
0 |
0 |
0 |
3 |
3 |
3 |
3 |
B |
4.10 |
150 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
1 |
2 |
1 |
|
TOTAL |
(87) |
300 |
1 |
4 |
0 |
0 |
0 |
0 |
5 |
4 |
5 (1.7) |
4 (1.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1462.3 |
A |
2.25 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
3.25 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
TOTAL |
(57) |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 (0.3) |
1 (0.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Positive control |
A |
1.45 |
150 |
2 |
2 |
2 |
3 |
0 |
0 |
9 |
7 |
5 |
3 |
B |
2.55 |
50a |
1 |
10 |
8 |
1 |
0 |
0 |
20 |
19 |
15 |
15 |
|
TOTAL |
(41) |
200 |
3 |
12 |
10 |
4 |
0 |
0 |
29 |
26 |
20 (10.0) |
18 (9.0) *** |
|
a Slide
evaluation terminated when 15 cells with aberrations (excluding gaps)
had been observed
*** p<0.01
MI Mitotic
index
N Number
of cells scored
g gaps
Ctb Chromatid
breaks
Cte Chromatid
exchanges
Csb Chromosome
breaks
Cse Chromosome
exchanges
Main Experiment: 4-hour treatment in the presence of S-9 with 20 hour recovery
Concentration (µg/mL) |
Replicate |
MI (%) |
N |
Number of aberrations |
Total number of aberrations |
Frequency of aberrations (%) |
|||||||
g |
Ctb |
Cte |
Csb |
Cse |
Others |
With gaps |
Without gaps |
With gaps |
Without gaps |
||||
0 (Vehicle control) |
A |
5.00 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.05 |
150 |
2 |
1 |
0 |
0 |
0 |
0 |
3 |
1 |
3 |
1 |
|
TOTAL |
(100) |
300 |
2 |
1 |
0 |
0 |
0 |
0 |
3 |
1 |
3 (1.0) |
1 (0.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
548.37 |
A |
4.15 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.30 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
|
TOTAL |
(93) |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 (0.3) |
1 (0.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
731.15 |
A |
3.90 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
4.60 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
TOTAL |
(94) |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1462.3 |
A |
3.60 |
150 |
0 |
1 |
0 |
2 |
0 |
0 |
3 |
3 |
3 |
3 |
B |
4.15 |
150 |
3 |
3 |
0 |
1 |
0 |
0 |
7 |
4 |
6 |
4 |
|
TOTAL |
(86) |
300 |
3 |
4 |
0 |
3 |
0 |
0 |
10 |
7 |
9 (3.0) |
7 (2.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Positive control |
A |
4.15 |
150 |
1 |
11 |
0 |
3 |
0 |
0 |
15 |
14 |
15 |
14 |
B |
2.70 |
146a |
5 |
11 |
2 |
5 |
0 |
0 |
23 |
18 |
18 |
15 |
|
TOTAL |
(76) |
296 |
6 |
22 |
2 |
8 |
0 |
0 |
38 |
32 |
33 (11.1) |
29 (9.8) *** |
|
a Slide
evaluation terminated when 15 cells with aberrations (excluding gaps)
had been observed
*** p<0.01
MI Mitotic
index
N Number
of cells scored
g gaps
Ctb Chromatid
breaks
Cte Chromatid
exchanges
Csb Chromosome
breaks
Cse Chromosome
exchanges
Main Experiment: 24-hour treatment in the absence of S-9 with no recovery
Concentration (µg/mL) |
Replicate |
MI (%) |
N |
Number of aberrations |
Total number of aberrations |
Frequency of aberrations (%) |
|||||||
g |
Ctb |
Cte |
Csb |
Cse |
Others |
With gaps |
Without gaps |
With gaps |
Without gaps |
||||
0 (Vehicle control) |
A |
5.40 |
150 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
B |
5.75 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
TOTAL |
(100) |
300 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
1 (0.3) |
1 (0.3) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
365.38 |
A |
5.00 |
150 |
1 |
4 |
0 |
1 |
0 |
0 |
6 |
5 |
5 |
5 |
B |
3.95 |
150 |
1 |
0 |
0 |
0 |
1 |
0 |
2 |
1 |
2 |
1 |
|
TOTAL |
(80) |
300 |
2 |
4 |
0 |
1 |
1 |
0 |
8 |
6 |
7 (2.3) |
6 (2.0) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
548.37
|
A |
4.10 |
150 |
2 |
2 |
0 |
1 |
0 |
0 |
5 |
3 |
4 |
2 |
B |
3.40 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
TOTAL |
(67) |
300 |
2 |
2 |
0 |
1 |
0 |
0 |
5 |
3 |
4 (1.3) |
2 (0.7) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
731.15 |
A |
2.70 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
3.40 |
150 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
TOTAL |
(55) |
300 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0 (0.0) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Positive control |
A |
2.65 |
70a |
2 |
12 |
6 |
2 |
0 |
0 |
22 |
20 |
15 |
15 |
B |
2.35 |
87a |
0 |
10 |
5 |
2 |
0 |
0 |
17 |
17 |
15 |
15 |
|
TOTAL |
(45) |
157 |
2 |
22 |
11 |
4 |
0 |
0 |
39 |
37 |
30 (19.1) |
30 (19.1) *** |
|
a Slide
evaluation terminated when 15 cells with aberrations (excluding gaps)
had been observed
*** p<0.01
MI Mitotic
index
N Number
of cells scored
g gaps
Ctb Chromatid
breaks
Cte Chromatid
exchanges
Csb Chromosome
breaks
Cse Chromosome
exchanges
The qualitative assessment of the slides determined that precipitate and toxicity was similar to that observed in the Preliminary Toxicity Test. In the absence and presence of metabolic activation (S9), the maximum dose level of the test item with metaphases suitable for scoring was 1462.3 μg/mL. In the continuous exposure group the maximum dose level of the test item with metaphases suitable for scoring was 731.15 μg/mL.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at 1462.3 μg/mL levels tested in in the presence of S9 only.
The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 43% mitotic inhibition was
achieved at 1462.3 μg/mL. In the presence of S9, a slight dose-related inhibition of mitotic index of 14% mitotic inhibition was observed at 1462.3 μg/mL. An inhibition of mitotic index of 45% was noted at 731.15 μg/mL in the 24-hour continuous exposure group. Therefore in the maximum dose levels selected for metaphase analysis were 1462.3 μg/mL and 731.15 μg/mL in the 4(20)-hour exposure without S9 and 24-hour continuous exposure groups respectively where near optimum toxicity was achieved. In the 4(20)-hour exposure groups the maximum dose selected for metaphase analysis was 1462.3 μg/mL where 14% mitotic inhibition was achieved.
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The positive control chemical concentrations assessed for metaphase analysis were those that were closest to the 55±5% value given as optimum toxicity in the test guideline.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
Range-finding study results
Dose (µg/mL) |
%RSG (-S9) 4-hour exposure |
%RSG (+S9) 4-hour exposure |
%RSG (-S9) 24-hour exposure |
0 |
100 |
100 |
100 |
5.71 |
102 |
109 |
103 |
11.42 |
100 |
101 |
87 |
22.84 |
112 |
105 |
114 |
45.69 |
106 |
99 |
87 |
91.38 |
110 |
99 |
91 |
182.75 |
105 |
110 |
64 |
365.5 |
95 |
97 |
6 |
731 |
53 |
81 |
0 |
1462 |
25 |
33 |
0 |
Results of mutagenicity test (4 hours in the absence of S-9)
Concentration (µg/mL) |
Suspension Growth |
Relative suspension growth (%) |
Viability (%) |
Relative total growth |
Mutant frequency |
0 |
10.97 |
100 |
79.38 |
1.00 |
125.77 |
91.38 |
13.07 |
103 |
75.99 |
0.99 |
115.86 |
182.75 |
10.13 |
87 |
74.81 |
0.83 |
130.26 |
365.5 |
9.50 |
79 |
74.81 |
0.75 |
132.37 |
731 |
8.57 |
64 |
69.31 |
0.56 |
136.04 |
1096.5 |
6.34 |
35 |
69.31 |
0.31 |
118.12 |
1462 |
6.18 |
25 |
72.54 |
0.23 |
145.33 |
Positive control |
8.77 |
72 |
47.33 |
0.43 |
1556.26 |
Threshold for a positive response is 251.77 (sum of mutant frequency for vehicle control and the GEF of 126).
Results of mutagenicity test (4 hours in the presence of S-9)
Concentration (µg/mL) |
Suspension Growth |
Relative suspension growth (%) |
Viability (%) |
Relative total growth |
Mutant frequency |
0 |
10.99 |
100 |
88.43 |
1.00 |
116.50 |
91.38 |
11.25 |
108 |
91.18 |
1.11 |
112.11 |
182.75 |
10.70 |
100 |
81.66 |
0.93 |
137.03 |
365.5 |
10.23 |
97 |
80.34 |
0.88 |
127.23 |
731 |
10.57 |
84 |
101.93 |
0.98 |
132.67 |
1096.5 |
8.06 |
57 |
77.20 |
0.50 |
151.31 |
1462 |
6.41 |
34 |
93.66 |
0.36 |
126.48 |
Positive control |
8.28 |
70 |
67.78 |
0.55 |
683.69 |
Threshold for a positive response is 242.50 (sum of mutant frequency for vehicle control and the GEF of 126).
Results of mutagenicity test (24 hours in the absence of S-9)
Concentration (µg/mL) |
Suspension Growth |
Relative suspension growth (%) |
Viability (%) |
Relative total growth |
Mutant frequency |
0 |
84.56 |
100 |
81.33 |
1.00 |
114.98 |
22.5 |
81.71 |
94 |
NP |
NP |
NP |
45 |
77.46 |
87 |
85.83 |
0.97 |
108.04 |
90 |
79.36 |
94 |
77.81 |
0.90 |
127.28 |
180 |
65.19 |
64 |
86.56 |
0.82 |
116.25 |
225 |
49.24 |
41 |
77.81 |
0.55 |
123.21 |
270 |
35.12 |
25 |
82.33 |
0.41 |
105.06 |
315 |
19.40 |
10 |
69.84 |
0.20 |
112.85 |
360 |
13.30 |
5 |
NP |
NP |
NP |
Positive control |
41.38 |
36 |
55.72 |
0.33 |
1121.86 |
NP: Not plated for viability or 5-TFT resistance
Threshold for a positive response is 240.98 (sum of mutant frequency for vehicle control and the GEF of 126).
There was evidence of marked toxicity following exposure to the test item in all three of the exposure groups, as indicated by the %RSG and RTG values. There was also evidence of a modest reduction in viability (%V) in the 24-hour exposure group in the absence of metabolic activation, indicating that residual toxicity had occurred in this exposure group. Based on the RTG values, optimum or near optimum levels of toxicity were achieved in the 4-hour and the 24-hour exposure groups in the absence of metabolic activation. The excessive toxicity observed at 360 μg/mL in the 24-hour exposure group in the absence of metabolic activation, resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, including the 10 mM limit concentration and concentrations that achieved optimum or near optimum levels of toxicity. Precipitation of the test item was not observed at the end of the exposure period in any of the three exposure groups.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
There is no indication of genetic toxicity in a battery of in vitro tests: testing in vivo is not required. Based on the negative studies in vitro with 2 -[[2 -(Dimethylamino)ethyl] methylamino]ethanol, there is no requirement for classification for germ cell mutagenicity according to the CLP Regulation.
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