Registration Dossier
Registration Dossier
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EC number: 938-572-7 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
An Ames test, an in vitro chromosome aberration study and in vitro mammalian mutation study are available for the submission substance DMP Technical [reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3 -diol and 2 -ethylpropane-1,3 -diol].
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 14 September 2009 to 01 March 2010
- 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:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- other: in vitro chromosome aberration assay
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- The cell line used was Chinese hamster ovary (CHO 10 B4) cells, obtained from the University of Leiden in 1987. The cell line is mycoplasma tested (in house) on a regular basis. The cells were grown as monolayers, and have a generation time of approximately 12 h. The modal chromosome number has been determined for these cells to be 21. The cells were incubated at 37°C.
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Colcemid was added to all cultures at a final concentration of 0.1 μg/mL.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9
- Test concentrations with justification for top dose:
- The highest concentration tested was 10 mM (1041 µg/mL). This showed no change in osmolality or changes to the colour of the media. No observations of precipitation were made.
- Vehicle / solvent:
- Ham's F-10 medium
- Untreated negative controls:
- yes
- Remarks:
- Ham's F-10 medium
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- Cells were trypsinised from stock flasks at passage numbers 15 (Test 1) and 12 (Test 2), and resuspended in fresh culture medium at densities of 0.1 x 10^6 or 0.05 x 10^6 cells/mL. These cells, in 5 mL volumes, were dispensed into 25 cm2 tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment respectively. Test cultures were established from the stock flask about 20 h before testing.
Tests were conducted both in the presence and absence of S9 mix. Treatments with test item or vehicle control substances were performed on duplicate cell cultures. Several concentrations of the positive controls were tested using single cultures.
Test 1 comprised treatments in the absence and presence of S-9 for 6 hours with recovery and harvest at 24 hours.
Test 2 comprised treatments in the presence of S-9 for 6 hours with recovery and harvest at 24 hours and treatments in the absence of S-9 for 22 hours with no recovery and harvest at 24 hours or included a recovery period with harvest at 48 hours.
Cultures to be treated in the presence of S9 mix were washed before treatment with serum free medium. Exposure medium was prepared, immediately before dosing, in sterile containers.
After treatment, cells were washed twice with serum free medium, then full growth medium added, for the recovery period and colcemid treatment. The volume of medium for the recovery period was 5 mL. S-9 was included at 10% v/v, where applicable.
Living cultures were examined for evidence of changes to cell morphology, once at the end of the treatment period and again before harvesting of cultures.
Colcemid was added to all cultures at a final concentration of 0.1 μg/mL. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase; the stage of cell division at which chromosomes can be examined using light microscopy. Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approximately 190 g), and treated with hypotonic solution (1% trisodium citrate) for 15 min at room temperature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 3:1). Two further changes (after sedimentation as before) of fixative were made.
Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity.
For both experiments, 3 slides per culture were made. All slides were marked with the study number and assigned a unique, coded number from a computer generated sequence. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with 5% Giemsa, then made permanent by mounting coverslips with DPX mountant.
Slides were examined for evidence of metaphase cells and signs of cellular necrosis.
Three concentration levels were selected for assessment of chromosomal aberrations.
From 2 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells per culture, were examined where possible. Slides were scored in order of coded number. A reduced number of metaphases were scored if a high proportion (≥40%) of metaphase cells were found to be damaged. A microscope was used for this assessment, the magnification used being x 1000 or x 1250, achieved with x 10 or x 12.5 eyepieces and x 100 objective. The number of chromosomes in each metaphase cell and all abnormalities, using the nomenclature of Gebhart (1970) was recorded. The types of structural and numerical aberrations recorded are listed in abbreviations page. The positions on the slides of any structurally aberrant cells were recorded using the Vernier scale on the microscope stage.
As cultures harvested at both culture times were negative with regards to structural aberrations, a further assessment of polyploidy was made. The Study Director selected the later harvest time as being more appropriate for this analysis. This decision was based on results from the previous assessments. For this assessment, approximately 300 metaphase cells were cursorily examined at a magnification of x 400 or x 500 and deemed to be either diploid, polyploid or endoreduplicated. In this assessment no metaphase cell in a field was rejected.
This assessment was considered more objective, avoiding scorer selection of either normal or polyploid cells. In addition, the larger sample size gave a more accurate frequency of such cells in the population.
From the cell counts, the number of cells recovered per culture, was calculated. This was then compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures to give a measure of toxicity.
From the results, 5 parameters were calculated, and judged as negative, suspicious or positive.
These parameters were:
1. Lesions per cell
2. Percentage of aberrant cells including cells with gaps only
3. Percentage of aberrant cells excluding cells with gaps only
4. Percentage of aneuploid cells
5 Percentage of polyploid cells (normal and endoreduplicated) from additional assessment of polyploidy
The third parameter is considered the most important in judging the true clastogenicity of a test item.
The results obtained were compared with the historical control data.
The experiments in this study were deemed to be valid because they fulfilled the following criteria:
There was no evidence of contamination
Cells in vehicle control cultures had normal growth
The results of vehicle and positive control cultures were typical
The test item had 3 acceptable dose levels for assessment - Evaluation criteria:
- A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.
The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historica negative control data. The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% conf dence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.
A test was positive if the response in at least one acceptable dose level was significant by the criterion described above. A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of abe rant cells (between 95% and 99% confidence limits). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive. - Statistics:
- No statistical analysis was performed.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- 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 applicable
- Positive controls validity:
- valid
- Conclusions:
- There was no evidence of clastogenicity with DMP Tech under the conditions of this study
- Executive summary:
An in vitro chromosome aberration study was performed with DMP Tech (Dimethylolpropane tech) using Chinese Hamster Ovary (CHO) cells according to OECD test guideline 473. Testing was conducted in the absence and in the presence of S-9 for 6 hours with a harvest at 24 hours after the start of treatment, and in the absence of S-9 for 22 hours with a harvest at 24 and 48 hours. Toxicity was measured based on cell counts. No toxicity was observed in treated cultures. Testing was conducted in duplicate, except for the positive control where single cultures were employed. 100 metaphases were scored for each vehicle and positive control culture and for the highest three concentrations. There were no increases in the number of structural aberrations when compared to the concurrent vehicle control. It can be concluded, therefore, that there is no evidence for the clastogenicity of DMP Tech based on the results of this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 25 November 2009 to 07 May 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- other: Mammalian cell mutation assay
- Target gene:
- Thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The cells used were from the tk+tk- -3.7.2C mouse lymphoma L5178Y cell line obtained from Dr D Clive, Burroughs Wellcome & Company, Research Triangle Park, NC27709, USA, in December 1982. The cells grow in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium.
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced-1254 S9 from male rat liver
- Test concentrations with justification for top dose:
- The highest concentration tested was 1041 µg/mL (equivalent to 10mM).
- Vehicle / solvent:
- RPMI 1640 supplemented with penciliin (100 units/mL), streptomycin (100 µg/mL), sodium bicarbonate (1.125 g/L) and pluronic acid (0.05% w/v), termed R0P.
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- ethylmethanesulphonate
- methylmethanesulfonate
- Details on test system and experimental conditions:
- The basic culture medium (R0P) was RPMI 1640 medium, supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), sodium bicarbonate (1.125 g/L) and pluronic acid (0.05% w/v). For cell growth, heat-inactivated horse serum (10% v/v) was added to R0P to give R10P.
The medium used during treatment for 4 h was R0P supplemented with 5% v/v heat-inactivated horse serum (R5P). The medium used during treatment for 24 h was R10P. For colony formation, cloning medium was used, consisting of R0P supplemented with heat-inactivated horse serum (20% v/v), sodium pyruvate (1.9 mM), and amphotericin B (fungizone) (2.5 μg/mL). For selection of tk-tk- cells, cloning medium was supplemented with trifluorothymidine (TFT) at 3 μg/mL.
For the four hour exposure, on the day of the test (Day 0), samples of cell culture (in 5 mL R10P) were dispensed to sterile tubes. Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by 4 mL of test solution. Vehicle control cultures received 4 mL R0P. Positive control cultures received 0.1 mL of the appropriate solution plus 3.9 mL R0P. The final reaction mixture in all cultures contained 10 mL of cells, at a population density of ca 6.0 x 105 cells/mL, in R5P medium. All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 4 h. After this, the cells were gently sedimented by centrifugation at ca 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated to give a cell density of ca 3 x 105/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 cells/mL on Day 1. An experiment is conducted using an extended 24 h exposure period, when the results of the first experiment in the absence of S9 mix are negative. The extended exposure period
facilitates continuous exposure to the test item through >1 cell cycle.
For the 24 hour exposure, on the day of the test (Day 0), samples of cell culture (in 10 mL R10P) were dispensed to sterile tubes. R50P (R0P:serum, 50:50) (2 mL) was added to each tube followed by 8 mL of the test solution. Vehicle control cultures received 8 mL R0P. Positive control cultures received 0.2 mL of the appropriate solution plus 7.8 mL R0P. The final reaction mixture in all cultures contained 20 mL of cells, at a population density of ca 3 x 105 cells/mL, in R10P medium. (The larger volumes allow the same numbers of cells to be treated as in the experiments conducted at 4 h exposure, but at half the density. The lower density is required to allow cell growth during the exposure period. The serum concentration is not lowered, as some essential nutrients can become exhausted during the exposure period.) All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give ca 3 x 105 cells/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 cells/mL on Day 2.
On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined. The cell counts over the 2 or 3 days of the experiments provided a measure of suspension growth. This in turn provided a measure of RSG. In this study all treated cultures were selected for assessment. The cultures were then assessed for expression of genetic damage. This was determined by performing two parallel cloning assays: the cloning efficiency assay and the mutant selection assay. For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well.
For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 104 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well. All dishes were incubated at ca 37°C in an atmosphere of 5% CO2:95% air (v/v) until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).
The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well (Moore et al (2000)). In addition, there are morphological differences. Large colonies tend to be similar to those found on the cloning efficiency plates, being generally flat. Small colonies tend to look dense in comparison. Any wells containing both colony types were scored as a large type. (The total number of empty wells is required for the calculation of mutant fraction, so each well can only be scored once).
For treated cultures, this value includes initial cell loss to toxicity and subsequent recovery during the expression period. The measure is used to assess the results of the preliminary toxicity test, and to determine acceptable dose levels for assessment in the mutation tests.
Total suspension growth (SG) is calculated as follows, for experiments with a 4 h exposure period:
[Day 1 count/Concentration on Day 0] x [Day 2 count/Final concentration on Day 1]
For experiments with a 24 h exposure period, the calculation is:
[Day 1 count/Concentration on Day 0] x [Day 2 count/Final concentration on Day 1] x [Day 3 count/Final concentration on Day 2]
The total suspension growth values are then expressed as percentages of the vehicle control mean value to give the relative suspension growth (RSG).
The recommended endpoint for assessing cytotoxicity in mouse lymphoma mutation tests is Relative Total Growth (RTG) (Moore et al (2002)). RTG combines the suspension growth above with the cloning efficiency (CE) of the non-mutants at the end of the expression period, again expressing individual values as percentages of the vehicle control mean. The CE is calculated from the zero term of the Poisson distribution using the formula:
Cloning efficiency (CE) = -In(P(o)/number of cells per well where P(o) = empty wells/total wells
Total Growth = SG x CE (non-mutants)
Relative Total Growth (RTG) (%) = [individual total growth /mean vehcile control total growth] x 100
The number of empty wells from the non-mutant cloning efficiency assay and the number of empty wells from the TFT-resistance assay were used to calculate the mutant fraction. The mutant fraction per viable
cell was calculated as
Mutant fraction per viable cell = CE in medium containing TFT/CE in non-selective medium = CE of mutats/CE of non mutants
Each mutant fraction was expressed per 10^6 viable cells.The ratio of small type mutant colonies to large type mutant colonies was expressed for each culture.
For 4 h experiments, the mean vehicle control suspension growth was required to be between 8 and 32 (Moore et al (2003)). For experiments with a 24 h exposure period, a value between 32 and 180 was required (Moore et al (2007)).
Errors in cell dilution or very high mutant fractions may result in plates with no negative wells. The Poisson distribution analysis does not generate data for such scores and so any pair of plates containing 0% empty wells was rejected. Plates containing 100% empty wells may arise from either errors in cell dilution or severe toxicity. Any such data was given consideration before acceptance.
The mean vehicle control cloning efficiency was required to be greater than 65% and less than 120%. (Experience shows that the acceptance or rejection of an occasional culture giving >120% CE makes no difference to the overall results, and therefore such instances were accepted.)
Results for any one treatment/concentration were inadequate if there were less than 2 acceptable cultures. Where results were obtained from a single culture, they may have been included as supporting evidence.
Analysable results should have been obtained from at least 4 concentrations of DMP Tech in any experiment. These may have included results extending into the toxic range of less than 10% RTG. The highest concentration of DMP Tech should have been limited by solubility or toxicity, or, in the absence of these, should have been the maximum practical concentration of test item, based on the recommendations in current guidelines, ie 5000 μg/mL or 10 mM, whichever is lower. The negative control mean mutant fraction range recommended by the IWGT mouse lymphoma workgroup for the microwell assay is 50-170 mutants per million (Moore et al (2006)). At Charles River, the range is just slightly lower, being ca 40-160 mutants per million. Concern over accepting results in the range 40-50 mutants per million centres on the possibility of reduced recovery of the less viable mutants. Any experiments with a vehicle mean mutant fraction between 40 and 50 mutants per million were accepted, providing the positive control results were unequivocally acceptable. In any experiment, MMS and at least one concentration of 3-MC should have yielded an induced mutant fraction (IMF = No. mutants per million for treated group, minus No. mutants per million for vehicle control) of at least 300 mutants per million, with a ratio of small/large colonies of greater than 0.67 (>40% small). The mean RTG values for all positive control treatments should have been >10%. EMS, a large colony inducer, was included at a low dose to monitor the sensitivity of the system to weak mutagens. Appendix 4 reflects the current historical control database. Variations between batches of positive control chemicals, media, serum and S9 preparations can all result in large differences in positive control responses. There is therefore no requirement for the positive control mutant fractions obtained in this study to fall within the historical ranges. There should have been an absence of confounding technical problems, eg, contamination, outliers, excessive toxicity, osmolality and pH changes. - Evaluation criteria:
- An experiment was considered positive if one or more concentrations were biologically significant and there was a significant linear trend. An experiment may also have been classed as positive in the absence of a linear trend if there was mitigating evidence. This may have been, for example, the presence of a similar level of toxicity at all concentrations assessed. In such a case, the confirmatory experiment would have been expected to assess concentrations covering different levels of toxicity, o establish a linear trend.
Additional comparisons that can aid interpretation of results include:
a comparison of the induced mutant fraction with the historical maximum for difference between vehicle controls
b comparison of the mutant fraction of a treated group with the historical range of vehicle control values
A test item was positive if 2 positive experiments out of 2 were recorded within the same activation condition. Test items that gave a negative response in the standard exposure in the absence of S9 mix, but gave a positive response in the extended exposure, were liable to a confirmatory experiment with the extended exposure.
The IWGT recommends that biological significance be attached to increases in IMF that exceed a value based on the global background mutant fraction. This value, the g obal evaluation factor (GEF), is defined as the mean of the global vehicle control distribution plus one standard deviation. For the microwell cloning version of the assay this value is 126 mutants per million. Biological significance was therefore assumed to apply to treatments that gave an IMF value >126 mutants per million. - Statistics:
- The results for each experiment were subjected to statistical analysis by the recommended UKEMS method
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Toxic in 24 hour exposure in the absence of S-9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- A combined statistical analysis of Assays 2 and 4 (where all treatment conditions and concentrations of test item were identical) was made. There were no statistically significant differences between the DMP Tech-treated groups and the vehicle controls. The test for linear trend was not significant (P = 0.40).
- Conclusions:
- No evidence of mutagenicity was seen in this mouse lymphoma assay performed with DMP Tech in the absence or presence of metabolic activation.
- Executive summary:
An in vitro mammalian mutation study was performed with DMP Tech using mouse lymphoma L5178Y cells according to OECD test guideline 476. Testing was conducted in the absence and in the presence of S-9 for 4 hours and in the absence of S-9 for 24 hours. Testing was conducted at concentrations up to 1041 µg/mL (10 mM). Relative total growth was used to indicate the level of toxicity. Toxicity of 10 -20% was noted in the 24 hour exposure in the absence of S-9 only. Slight toxicity was noted in the 4-hour treatments, though testing was conducted up to a regulatory maximum of 10 mM. The induced mutant frequency did not exceed the global evaluation factor (126 mutants per million). It can be concluded, therefore, that there is no evidence for the mutagenicity of DMP Tech based on the results of this study.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 15 September - 2 October 2017
- 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:
- July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 'Reverse mutation test using bacteria’ described in Council Regulation (EC) No. 440/2008, as amended.
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Various: reversion to histidine / tryptophan dependence. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system. Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- SD rat liver S9 fraction (phenobarbital/5,6-benzoflavone induced)
- Test concentrations with justification for top dose:
- Tested up to the limit concentration (5 uL/plate)
- Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Remarks:
- Untreated plate
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
Two main assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test
point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish
the number of bacteria in the cultures. The first main assay was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The second main assay was performed using a pre-incubation method. The components were added in turn to an empty test-tube. The incubate was vortexed and placed at 37°C for 30 minutes. 2 mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates from the main assays were held at 4°C for 24 hours while plates from the preliminary toxicity test were immediately scored by counting the number of revertant colonies on each plate. - Rationale for test conditions:
- Standard test conditions: highest concentration not limited by toxicity or solubility.
- Evaluation criteria:
- The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100-500 million for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. - Statistics:
- Not required
- Key result
- 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
- Key result
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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
- Key result
- Species / strain:
- S. typhimurium TA 1537
- 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
- Key result
- 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:
- Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100-500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid. The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.
- Conclusions:
- No evidence of mutagenicity was seen under the conditions of this assay, either in the absence or presence of metabolic activation.
- Executive summary:
The submission substance DMP technical [reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3-diol and 2-ethylpropane-1,3-diol] was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy (Ames test). The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used; experiments were performed both in the absence and presence of metabolic activation (liver S9 fraction from rats SD pre-treated with phenobarbital and 5,6-benzoflavone). The test item was used as a solution in DMSO. On the basis of a preliminary toxicity test Main Assay I (plate incorporation method) used concentrations of 5.00, 2.50, 1.25, 0.625 and 0.313 µL/plate. No toxicity was observed with any tester strain at any concentration in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers were observed at any concentration tested, Main Assay II was performed (pre-incubation) using the same concentrations. No increases in revertant numbers and no toxicity was observed at any concentrationin any tester strain in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any concentration, in any tester strain, in the absence or presence of S9 metabolism. It is therefore concluded that the submission substance [reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3 -diol and 2 -ethylpropane-1,3 -diol] does not induce reverse mutation under the conditions of this assay.
Referenceopen allclose all
Results: Experiment 1 - 6 hour treatment in the presence of S-9, 24 hour harvest
Concentration (µg/mL) |
Replicate |
Index (% of mean vehicle control cell count) |
N |
Number of structural aberrations |
Frequency of structural aberrations (%) |
% of cells with |
||||||||||
Ctg |
Ctb |
Ctf |
Csg |
Csb |
Csf |
Cse |
Other |
With gaps |
Without gaps |
AE |
ER |
PP |
||||
0 (vehicle control) |
A |
96 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
B |
104 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0.5 |
|
260 |
A |
102 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
B |
96 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
|
521 |
A |
91 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
B |
95 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1.5 |
0 |
|
1041 |
A |
91 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
0 |
B |
87 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2.5 |
0 |
|
Cyclophosphamide 40 µg/mL |
A |
- |
100 |
5 |
3 |
0 |
0 |
0 |
0 |
8 |
0 |
13 |
9 |
2 |
0 |
0 |
Cyclophosphamide 50 µg/mL |
A |
- |
100 |
2 |
13 |
1 |
0 |
0 |
0 |
11 |
0 |
21 |
21 |
7 |
0 |
0 |
Results: Experiment 1 - 6 hour treatment in the absence of S-9, 24 hour harvest
Concentration (µg/mL) |
Replicate |
Index (% of mean vehicle control cell count) |
N |
Number of structural aberrations |
Frequency of structural aberrations (%) |
% of cells with |
||||||||||
Ctg |
Ctb |
Ctf |
Csg |
Csb |
Csf |
Cse |
Other |
With gaps |
Without gaps |
AE |
ER |
PP |
||||
0 (vehicle control) |
A |
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
100 |
100 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0 |
0 |
0 |
0 |
|
260 |
A |
97 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
97 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0 |
0 |
|
521 |
A |
97 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
87 |
100 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0 |
0 |
0 |
|
1041 |
A |
73 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
B |
77 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
|
Methyl methane-sulphonate 30 µg/mL |
A |
- |
100 |
2 |
4 |
0 |
0 |
0 |
0 |
2 |
0 |
8 |
6 |
0 |
0 |
1 |
Methyl methane-sulphonate 40 µg/mL |
A |
- |
100 |
1 |
4 |
3 |
0 |
0 |
0 |
2 |
0 |
9 |
8 |
0 |
0 |
0 |
Results: Experiment 2 - 6 hour treatment in the presence of S-9, 24 hour harvest
Concentration (µg/mL) |
Replicate |
Index (% of mean vehicle control cell count) |
N |
Number of structural aberrations |
Frequency of structural aberrations (%) |
% of cells with |
||||||||||
Ctg |
Ctb |
Ctf |
Csg |
Csb |
Csf |
Cse |
Other |
With gaps |
Without gaps |
AE |
ER |
PP |
||||
0 (vehicle control) |
A |
96 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
104 |
100 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
0 |
1 |
1 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0.5 |
0 |
0.5 |
0.5 |
0 |
|
260 |
A |
93 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
B |
100 |
100 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
0 |
|
Total |
- |
200 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0.5 |
0.5 |
0 |
0 |
|
521 |
A |
93 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
98 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
1041 |
A |
102 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
0 |
1 |
B |
81 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1.5 |
0 |
0 |
|
Methyl methane-sulphonate 30 µg/mL |
A |
- |
100 |
2 |
10 |
2 |
0 |
2 |
0 |
6 |
0 |
17 |
16 |
9 |
0 |
2 |
Methyl methane-sulphonate 40 µg/mL |
A |
- |
100 |
3 |
14 |
3 |
0 |
2 |
5 |
9 |
0 |
25 |
24 |
9 |
0 |
0 |
Results: Experiment 2 - 22 hour treatment in the absence of S-9, 24 hour harvest
Concentration (µg/mL) |
Replicate |
Index (% of mean vehicle control cell count) |
N |
Number of structural aberrations |
Frequency of structural aberrations (%) |
% of cells with |
||||||||||
Ctg |
Ctb |
Ctf |
Csg |
Csb |
Csf |
Cse |
Other |
With gaps |
Without gaps |
AE |
ER |
PP |
||||
0 (vehicle control) |
A |
98 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
102 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
260 |
A |
91 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
105 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
521 |
A |
108 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
106 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
1041 |
A |
89 |
100 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
1 |
B |
92 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0.5 |
0.5 |
0 |
0.5 |
|
Methyl methane-sulphonate 30 µg/mL |
A |
- |
100 |
4 |
9 |
1 |
0 |
0 |
0 |
6 |
0 |
15 |
14 |
0 |
0 |
0 |
Methyl methane-sulphonate 40 µg/mL |
A |
- |
50 |
3 |
19 |
2 |
0 |
2 |
1 |
10 |
0 |
44 |
44 |
0 |
0 |
0 |
Results: Experiment 2 - 22 hour treatment in the absence of S-9, 48 hour harvest
Concentration (µg/mL) |
Replicate |
Index (% of mean vehicle control cell count) |
N |
Number of structural aberrations |
Frequency of structural aberrations (%) |
% of cells with |
||||||||||
Ctg |
Ctb |
Ctf |
Csg |
Csb |
Csf |
Cse |
Other |
With gaps |
Without gaps |
AE |
ER |
PP |
||||
0 (vehicle control) |
A |
98 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
102 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
260 |
A |
95 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
112 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
521 |
A |
111 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
99 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
1041 |
A |
101 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
B |
115 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Total |
- |
200 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Methyl methane-sulphonate 20 µg/mL |
A |
- |
100 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
1 |
0 |
0 |
Methyl methane-sulphonate 40 µg/mL |
A |
- |
100 |
0 |
9 |
2 |
0 |
0 |
0 |
7 |
0 |
14 |
14 |
3 |
0 |
0 |
N: Number
of cells scored
Ctg: Chromatid gaps
Ctb: Chromatid
breaks
Ctf: Chromatid
fragment
Csg: Chromosome gap
Csb: Chromosome break
Csf: Chromosome
fragment
Cse: Chromosome exchange
AE: Aneuploidy
ER: Endoreduplication
PP: Polyploidy
Experiment 1 – 4 hour exposure in the absence of S-9
Concentration (µg/mL) |
RTG (%) |
Mutant fraction (x 10-6) |
IMF |
Ratio large:small colonies |
Statistical significance |
0 vehicle control |
100 |
69 |
N/A |
1.07 |
N/A |
125 |
99 |
62 |
≤0 |
1.88 |
NS |
250 |
85 |
61 |
≤0 |
0.74 |
NS |
500 |
85 |
56 |
≤0 |
1.70 |
NS |
1041 |
64 |
87 |
18 |
1.70 |
NS |
EMS, 250 µg/mL |
60 |
525 |
466 |
0.47 |
* |
MMS, 10 µg/mL |
36 |
939 |
870 |
2.43 |
* |
Experiment 2 – 4 hour exposure in the presence of S-9
Concentration (µg/mL) |
RTG (%) |
Mutant fraction (x 10-6) |
IMF |
Ratio large:small colonies |
Statistical significance |
0 vehicle control |
100 |
67 |
N/A |
1.11 |
N/A |
125 |
83 |
81 |
13 |
1.94 |
NS |
250 |
80 |
79 |
12 |
1.03 |
NS |
500 |
65 |
77 |
10 |
1.90 |
NS |
1041 |
41 |
86 |
19 |
0.50 |
NS |
3-MC, 2.5 µg/mL |
67 |
523 |
455 |
1.15 |
* |
3-MC, 10 µg/mL |
48 |
718 |
651 |
1.23 |
* |
Experiment 3 – 24 hour exposure in the absence of S-9
Concentration (µg/mL) |
RTG (%) |
Mutant fraction (x 10-6) |
IMF |
Ratio large:small colonies |
Statistical significance |
0 vehicle control |
100 |
49 |
N/A |
1.21 |
N/A |
125 |
109 |
41 |
≤0 |
1.58 |
NS |
250 |
84 |
45 |
≤0 |
4.10 |
NS |
500 |
87 |
75 |
26 |
0.96 |
NS |
1041 |
16 |
88 |
40 |
0.86 |
NS |
EMS, 250 µg/mL |
33 |
1439 |
1390 |
0.39 |
* |
MMS, 10 µg/mL |
21 |
1786 |
1737 |
1.93 |
* |
Experiment 4 – 4 hour exposure in the presence of S-9
Concentration (µg/mL) |
RTG (%) |
Mutant fraction (x 10-6) |
IMF |
Ratio large:small colonies |
Statistical significance |
0 vehicle control |
100 |
57 |
N/A |
1.46 |
N/A |
125 |
100 |
68 |
10 |
2.02 |
NS |
250 |
87 |
70 |
12 |
2.39 |
NS |
500 |
90 |
47 |
≤0 |
1.90 |
NS |
1041 |
60 |
70 |
13 |
1.50 |
NS |
3-MC, 2.5 µg/mL |
49 |
968 |
911 |
1.62 |
* |
3-MC, 10 µg/mL |
30 |
1134 |
1077 |
2.00 |
* |
IMF: Induced
mutant frequency fraction 10-6; mutant fraction of treatment
minus mutant fraction of vehicle control group
N/A: Not applicable
NS: Not
significant
* Significant
difference in log mutant fraction compared with vehicle control (P<0.05)
Summary of results
Mean revertant numbers |
||||||||||
Treatment |
EXPERIMENT 1 (PLATE INCORPORATION) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Untreated |
27 |
36 |
138 |
149 |
17 |
14 |
16 |
20 |
28 |
39 |
DMSO |
28 |
34 |
126 |
125 |
17 |
14 |
16 |
18 |
27 |
33 |
0.313 µL |
30 |
29 |
167 |
161 |
13 |
15 |
18 |
19 |
26 |
39 |
0.625 µL |
26 |
29 |
154 |
182 |
18 |
16 |
16 |
18 |
28 |
36 |
1.25 µL |
28 |
27 |
148 |
177 |
15 |
14 |
18 |
21 |
25 |
36 |
2.50 µL |
27 |
34 |
152 |
160 |
17 |
13 |
17 |
18 |
25 |
30 |
5.00 µL |
27 |
30 |
154 |
153 |
16 |
15 |
14 |
20 |
26 |
32 |
2-NF |
127 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
NaN3 |
- |
- |
569 |
- |
405 |
- |
- |
- |
- |
- |
9AA |
- |
- |
- |
- |
- |
- |
- |
158 |
- |
- |
MMS |
- |
- |
- |
- |
- |
- |
- |
- |
208 |
- |
2AA |
- |
771 |
- |
1421 |
- |
178 |
- |
146 |
- |
228 |
Treatment |
EXPERIMENT 2 (PREINCUBATION) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Untreated |
31 |
40 |
130 |
133 |
15 |
17 |
17 |
20 |
28 |
33 |
DMSO |
34 |
36 |
116 |
122 |
17 |
18 |
17 |
20 |
25 |
36 |
0.313 µL |
33 |
31 |
117 |
119 |
15 |
16 |
15 |
20 |
24 |
34 |
0.625 µL |
30 |
32 |
125 |
160 |
14 |
14 |
14 |
21 |
23 |
32 |
1.25 µL |
30 |
34 |
141 |
121 |
16 |
15 |
20 |
21 |
24 |
30 |
2.50 µL |
31 |
36 |
133 |
123 |
14 |
16 |
17 |
18 |
31 |
35 |
5.00 µL |
32 |
36 |
125 |
124 |
17 |
14 |
16 |
17 |
29 |
31 |
2-NF |
197 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
NaN3 |
- |
- |
487 |
- |
406 |
- |
- |
- |
- |
- |
9AA |
- |
- |
- |
- |
- |
- |
17 |
- |
- |
- |
MMS |
- |
- |
- |
- |
- |
- |
- |
- |
148 |
- |
2AA |
- |
753 |
- |
1469 |
- |
116 |
- |
100 |
- |
231 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test
The submission substance DMP technical [reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3-diol and 2-ethylpropane-1,3-diol] was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy (Ames test). The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used; experiments were performed both in the absence and presence of metabolic activation (liver S9 fraction from rats SD pre-treated with phenobarbital and 5,6-benzoflavone). The test item was used as a solution in DMSO. On the basis of a preliminary toxicity test Main Assay I (plate incorporation method) used concentrations of 5.00, 2.50, 1.25, 0.625 and 0.313 µL/plate. No toxicity was observed with any tester strain at any concentration in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers were observed at any concentration tested, Main Assay II was performed (pre-incubation) using the same concentrations. No increases in revertant numbers and no toxicity was observed at any concentrationin any tester strain in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any concentration, in any tester strain, in the absence or presence of S9 metabolism. It is therefore concluded that the submission substance [reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3 -diol and 2 -ethylpropane-1,3 -diol] does not induce reverse mutation under the conditions of this assay.
Mammalian cell clastogenicity assay
An in vitro chromosome aberration study was performed with DMP Tech (Dimethylolpropane tech) using Chinese Hamster Ovary (CHO) cells according to OECD test guideline 473. Testing was conducted in the absence and in the presence of S-9 for 6 hours with a harvest at 24 hours after the start of treatment, and in the absence of S-9 for 22 hours with a harvest at 24 and 48 hours. Toxicity was measured based on cell counts. No toxicity was observed in treated cultures. Testing was conducted in duplicate, except for the positive control where single cultures were employed. 100 metaphases were scored for each vehicle and positive control culture and for the highest three concentrations. There were no increases in the number of structural aberrations when compared to the concurrent vehicle control. It can be concluded, therefore, that there is no evidence for the clastogenicity of DMP Tech based on the results of this study.
Mammalian cell mutation assay
An in vitro mammalian mutation study was performed with DMP Tech using mouse lymphoma L5178Y cells according to OECD test guideline 476. Testing was conducted in the absence and in the presence of S-9 for 4 hours and in the absence of S-9 for 24 hours. Testing was conducted at concentrations up to 1041 µg/mL (10 mM). Relative total growth was used to indicate the level of toxicity. Toxicity of 10 -20% was noted in the 24 hour exposure in the absence of S-9 only. Slight toxicity was noted in the 4-hour treatments, though testing was conducted up to a regulatory maximum of 10 mM. The induced mutant frequency did not exceed the global evaluation factor (126 mutants per million). It can be concluded, therefore, that there is no evidence for the mutagenicity of DMP Tech based on the results of this study.
Justification for classification or non-classification
The submission substance DMP Technical [reaction mass of 2-ethyl-2-(methoxymethyl)-propane-1,3 -diol and 2 -ethylpropane-1,3 -diol] is not classified for germ cell mutagenicity according to CLP based on the negative results seen in the three available in vitro genotoxicity tests.
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