Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 809-934-0 | CAS number: 627034-93-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria
The test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, test material had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.
In vitro micronucleus study
Under the experimental conditions of the study, the test material did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or presence of a rat liver metabolising system.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 February 2013 to 23 February 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium
TA1537: his C 3076; rfa-; uvrB- (frame shift mutations)
TA98: his D 3052; rfa-; uvrB-; R-factor (frame shift mutations)
TA1535: his G 46; rfa-; uvrB- (base-pair mutations)
TA100: his G 46; rfa-; uvrB-; R-factor (base-pair mutations)
Escherichia coli
WP2 uvrA: trpE; uvrA- (base-pair substitution) - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames. - Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary Range Finding Test: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate
Initial Mutation Test and Confirmatory Mutation Test: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the same concentrations were used. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test material was examined using distilled water, dimethyl sulfoxide (DMSO) and acetone. The test material was soluble in acetone and DMSO at 100 mg/mL concentration, partial dissolution was observed in distilled water at the same concentration. Due to the better biocompatibility to the test system, DMSO was selected for vehicle of the study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO; distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene; 4-nitro-1,2-phenylene-diamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
A standard plate incorporation procedure was performed, as an Initial Mutation Test.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes was as follows: top agar 2000 μL, solvent or test material solution (or reference controls) 50 μL, overnight culture of test strain 100 μL, phosphate buffer (pH 7.4) or S9 mix 500 μL.
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no clear positive effect was observed. Before the overlaying, the test material formulation (or vehicle/solvent or reference control), the bacterial culture, and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test material (in its vehicle/solvent).
The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.
NUMBER OF REPLICATIONS: 3
EVALUATION OF EXPERIMENTAL DATA
The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using Microsoft Excel™ software.
VALIDITY CRITERIA
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analysable concentrations were presented in all strains of the main tests. - Evaluation criteria:
- A test material was considered mutagenic if:
- a dose-related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the spontaneous reversion rate of the negative (solvent) control plates.
A test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY RANGE FINDING TEST
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Preliminary Range Finding Test, the numbers of revertant colonies were mostly in the normal range. Minor differences were detected in some cases, but they were without biological significant, thus they were considered as biological variability of the test system.
No insolubility or signs of cytotoxicity was observed in the preliminary experiment.
INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test using the plate incorporation method, the highest revertant rate was observed in the in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at the concentration of 1581 μg/plate. The mutation factor value was 2.40, slightly above the respective biological threshold value of 2. Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain. However, there was no dose response, and the numbers of revertant colonies were within the historical control range. Additionally, higher numbers of revertant colonies compared to the DMSO solvent control were detected for untreated control (MF: 1.47) and distilled water control (MF: 1.20) in this strain.
In the Confirmatory Mutation Test using the pre-incubation method, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain with metabolic activation at 500 μg/plate concentration. The observed mutation factor value was 1.48. However, no dose-dependent relationship was observed, and the observed mutation factor value was below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range.
Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some other cases. However, no dose-dependence was observed in any cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in all cases, so they were considered as reflecting the biological variability of the test.
The sporadic increases in the number of revertant colonies were examined for consistency; none of them were repeatable when comparing the performed main experiments. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between test material treated samples and negative (solvent) controls.
Sporadically, lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
No insolubility or signs of cytotoxicity was observed in the main tests.
VALIDITY OF THE TESTS
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests.
The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid. - Conclusions:
- The test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, test material had no mutagenic activity on the growth of the applied bacterium tester strains under the test conditions used in this study.
- Executive summary:
The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, EPA OPPTS 870.5100, under GLP conditions. The method followed also conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.
The study was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test, an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).
Based on the results of the Solubility Test, the test material was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Test, most of the observed revertant colony numbers were below the respective biological threshold value. The observed sporadic increases in the number of revertant colonies were examined for consistency; none of them were repeatable when comparing the performed main experiments. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between test material treated samples and solvent controls; Thus, they were considered as biological variability of the test system.
The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.
The findings of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test material had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 November 2015 to 18 december 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).
- Suitability of cells: L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei.
- Cell cycle length, doubling time or proliferation index: The average cell cycle time is approximately 10-12 hours.
- Methods for storage: The cells were stored in a cryoprotective medium (10% horse serum and 10 % dimethylsulfoxide (DMSO)) at -80 °C and each batch of frozen cells was checked for the absence of mycoplasma.
CULTURE CONDITIONS
Cell cultures were grown at 37°C in a humidified atmosphere of 5 % CO2/95 % air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and sodium pyruvate (200 μg/mL). This medium was supplemented by heat-inactivated horse serum at 10 % (v/v). - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- - Preliminary toxicity test: 0.02, 0.2, 1, 2, 5, 10 mM
- First experiment: 0.63, 1.25, 2.5, 5, 10 mM
- Second experiment: 1.25, 2.5, 5, 7.5, 10 mM - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle was Dimethylsulfoxide (DMSO). - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Colchicine
- Details on test system and experimental conditions:
- PRELIMINARY TOXICITY TEST
To assess the cytotoxicity of the test material, six dose-levels (one culture/dose-level) were tested both with and without metabolic activation, as follows:
- With metabolic activation: 3 h treatment + 24 h recovery
- Without metabolic activation: 3 h treatment + 24 h recovery and 24 h treatment + 0 h recovery
The treatments of this preliminary test were performed as described below for the main experiments. Assessment of cytotoxicity was performed by evaluation of Population Doubling. After the final cell counting, the cells were not harvested and no slides were prepared.
MAIN EXPERIMENTS
In two independent experiments, five dose-levels of the test material were tested in duplicate (two cultures/dose-level), with or without metabolic activation, using treatment duration as follows:
- With metabolic activation: 3 h treatment + 24 h recovery (first experiment); 3 h treatment + 24 h recovery (second experiment)
- Without metabolic activation: 3 h treatment + 24 h recovery and 24 h treatment + 0 h recovery (first experiment)
> Treatment
On the day of treatment, cells were counted and suspended in order to reach approximately 3 x 10^5 cells/mL (final concentration = N0) in the final treatment medium (culture medium containing 5 % inactivated horse serum).
Cells were exposed in 24-well plates to the test or control materials, with or without S9 mix, at 37 °C in a humidified atmosphere of 5 % CO2/95 % air.
At the end of the treatment period, the cells were washed twice. Cells were suspended in culture medium containing 10% inactivated horse serum and the plates were incubated for the recovery period (if any), at 37 °C in a humidified atmosphere of 5 % CO2/95 % air.
At the end of the recovery period (if any), the cells were counted to determine the final count at the time of harvesting (N) in order to assess the cytotoxicity by the evaluation of PD.
> Assessment of cytotoxicity
For each culture, the Population Doubling (PD) (d) was calculated and used relative to that of the vehicle control. The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.
PD = [log (N/N0)] / log 2
Mean PD as % of control = ( Mean PD treated / Mean PD vehicle control) x 100
The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).
Decrease in PD (%) = 100 - Mean PD as % of control
> Cell harvesting and slides preparation
After the final cell counting, the cells were washed with culture medium containing 10 % inactivated horse serum and 1 % pluronic acid. The cells were suspended in 49.5 % culture medium containing 10 % inactivated horse serum, 50 % PBS and 0.5 % pluronic acid, before being fixed.
Following the fixation, the cells were kept at 4 °C for at least an overnight period.
Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), at least three dose-levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5 % Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.
> Analysis of the slides
All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring).
For each main experiment (with or without S9 mix), micronuclei were analysed for at least three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).
Appropriate test material dose-levels for scoring of micronuclei were selected mainly on the basis of the achieved reduction of PD and on the presence of precipitate.
Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
- micronuclei should be clearly surrounded by a nuclear membrane,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- non-refractility of the micronuclei,
- micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should be located within the cytoplasma of the cell,
- only mononucleated cells with a number of micronuclei ≤ 5 should be scored to exclude apoptosis and nuclear fragmentation.
Number of cells with micronuclei and number of micronuclei per cell were given separately for each treated and control culture. - Evaluation criteria:
- > Acceptance criteria
Each main experiment was considered valid if the following criteria were met:
- the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone mitotis),
- the mean frequency of micronucleated cells in the vehicle control should be consistent with (but not necessary within) control historical data of the Laboratory. In any case, this frequency should be ≤ 5 %,
- a statistically significant increase in the frequency of micronucleated cells had to be obtained in the positive controls over the background frequency of the vehicle control cultures.
> Evaluation criteria
A test material is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.
A test material is considered negative if none of the criteria for a positive response was met. - Statistics:
- For each condition of each cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the X² test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose-levels. This statistical analysis was performed using SAS Enterprise Guide software. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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
- Additional information on results:
- PRELIMINARY CYTOTOXICITY TEST
Based on available solubility data, the test material was dissolved in DMSO, at 324.36 mg/mL. Therefore, using this stock solution and a treatment volume of 0.5 % (v/v) in the culture medium, the highest recommended dose-level of 10 mM (corresponding to 1621.8 μg/mL) was achievable. Thus, the dose-levels selected for the treatment of the preliminary test were 0.02, 0.2, 1, 2, 5 and 10 mM.
At the highest dose-level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was equal to 344 mOsm/kg H2O (379 mOsm/kg for the vehicle control).
Therefore, none of the selected dose-levels was considered to produce extreme culture conditions and the highest recommended dose-level of 10 mM could be selected as the high dose-level for the main experiments.
No precipitate was observed in the culture medium at the end of the treatment periods.
Following the 3- and 24-hour treatments with or without S9 mix, no noteworthy toxicity was observed at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
MAIN EXPERIMENTS
Since the test material was found to be freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 10 mM, according to the criteria specified in the international regulations.
The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.
> Main experiment without S9 mix
With a treatment volume of 0.5 % (v/v) in culture medium, the selected dose-levels were 0.63, 1.25, 2.5, 5 and 10 mM for both 3- and 24-hour treatments.
No precipitate was observed in the culture medium at the end of the treatment periods.
- Cytotoxicity
Following the 3- and 24-hour treatments, no noteworthy toxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
- Micronucleus analysis
The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 mM for both 3- and 24-hour treatments, the latter corresponding to the highest recommended dose-level.
Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analysed dose-levels in comparison to the vehicle controls. Moreover, none of the analysed dose-levels showed frequency of micronucleated cells of both replicate cultures above the vehicle control historical range.
These results met the criteria of a negative response.
> Main experiment with S9 mix
With a treatment volume of 0.5 % (v/v) in culture medium, the selected dose-levels were as follows:
- 0.63, 1.25, 2.5, 5 and 10 mM for the first experiment,
- 1.25, 2.5, 5, 7.5 and 10 mM for the second experiment.
No precipitate was observed in the culture medium at the end of the treatment periods.
- Cytotoxicity
No noteworthy toxicity was induced in either experiment, at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
- Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
- 2.5, 5 and 10 mM for the first experiment, the latter corresponding to the highest recommended dose-level,
- 5, 7.5 and 10 mM for the second experiment, the latter corresponding to the highest recommended dose-level.
In the first experiment, a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test. Moreover, a statistically significant difference in comparison to the corresponding vehicle control was obtained at the highest tested dose-level of 10 mM (p < 0.05). However, only the frequency of micronucleated cells of one out of two replicate cultures was above the corresponding vehicle historical range (10 ‰ versus [0-6 ‰] for the historical data). Since the criteria of a positive response were only partially met, a second confirmatory experiment was undertaken in order to check the reliability of this increase, under the same experimental conditions but using a narrower range of dose-levels.
In the second experiment, neither statistically nor dose-related increase in the frequency of micronucleated cells was noted at any of the analysed dose-levels. Moreover, the mean frequencies of micronucleated cells remained within the corresponding vehicle historical range, as well as the individual frequencies of each replicate culture.
Since the increase noted in the first experiment (mainly in one out of two cultures) was not reproduced in the second experiment, performed under the same experimental conditions, despite using a narrower range of dose-levels, it was considered as non-biologically relevant. Consequently, the overall results with S9 mix were considered as a negative response. - Conclusions:
- Under the experimental conditions of the study, the test material did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or presence of a rat liver metabolising system.
- Executive summary:
The potential of the test material to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- was investigated in accordance with the standardised guideline OECD 487, under GLP conditions.
After a preliminary toxicity test, the test material TD-115, dissolved in DMSO, was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. In the first experiment cells were treated without S9 mix for treatment times of 3 hours followed by a 24 hour recovery of for 24 hours with 0 hour recovery. In the first experiment with S9 mix, cells were treated for 3 hours followed by a 24 hour recovery. In the second experiment cells with S9 mix, cells were treated for 3 hours with a 24 hour recovery.
Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5 % Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analysed for three dose-levels of the test material, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
Since the test material was found to be freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 10 mM, according to the criteria specified in the international regulations.
The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.
In the main experiment without S9 mix, with treatment volume of 0.5 % (v/v) in culture medium, the selected dose-levels were 0.63, 1.25, 2.5, 5 and 10 mM for both 3- and 24-hour treatments. No precipitate was observed in the culture medium at the end of the treatment periods.
Following the 3- and 24-hour treatments, no noteworthy toxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 mM for both 3- and 24-hour treatments, the latter corresponding to the highest recommended dose-level.
Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analysed dose-levels in comparison to the vehicle controls. Moreover, none of the analysed dose-levels showed frequency of micronucleated cells of both replicate cultures above the vehicle control historical range. These results met the criteria of a negative response.
In the main experiments with S9 mix, with a treatment volume of 0.5 % (v/v) in culture medium, the selected dose-levels were 0.63, 1.25, 2.5, 5 and 10 mM for the first experiment and 1.25, 2.5, 5, 7.5 and 10 mM for the second experiment. No precipitate was observed in the culture medium at the end of the treatment periods.
No noteworthy toxicity was induced in either experiment, at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 mM for the first experiment, the latter corresponding to the highest recommended dose-level, and 5, 7.5 and 10 mM for the second experiment, the latter corresponding to the highest recommended dose-level.
In the first experiment, a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test. Moreover, a statistically significant difference in comparison to the corresponding vehicle control was obtained at the highest tested dose-level of 10 mM (p < 0.05). However, only the frequency of micronucleated cells of one out of two replicate cultures was above the corresponding vehicle historical range.
Since this increase was not reproduced in the second experiment, performed under the same experimental conditions, despite using a narrower range of dose-levels, it was considered as non-biologically relevant.
Consequently, the overall results with S9 mix were considered as a negative response.
Under the experimental conditions of the study, the test material did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the absence or presence of a rat liver metabolising system.
Referenceopen allclose all
Table 1: Summary Table of Results of the Initial Mutation Test
Concentrations (µg/plate) |
Mean values of revertants/ Mutation factor (MF) |
Tester strain |
|||||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||
Untreated control |
Mean |
23.0 |
35.0 |
91.0 |
119.7 |
7.3 |
10.0 |
12.7 |
11.0 |
35.3 |
37.7 |
||
MF |
0.99 |
1.02 |
0.99 |
1.09 |
1.47 |
0.94 |
1.09 |
1.00 |
0.88 |
1.47 |
|||
DMSO control |
Mean |
23.3 |
34.3 |
91.7 |
109.3 |
5.0 |
10.7 |
11.7 |
11.0 |
40.0 |
25.7 |
||
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|||
Distilled water control |
Mean |
─ |
─ |
97.7 |
─ |
6.0 |
─ |
─ |
─ |
47.0 |
─ |
||
MF |
─ |
─ |
1.07 |
─ |
1.20 |
─ |
─ |
─ |
1.18 |
─ |
|||
5000 |
Mean |
29.7 |
26.3 |
94.7 |
121.3 |
8.0 |
11.7 |
10.3 |
8.7 |
40.0 |
45.3 |
||
MF |
1.27 |
0.77 |
1.03 |
1.11 |
1.60 |
1.09 |
0.89 |
0.79 |
1.00 |
1.77 |
|||
1581 |
Mean |
29.3 |
29.0 |
88.0 |
129.0 |
12.0 |
9.3 |
13.0 |
15.7 |
53.0 |
45.3 |
||
MF |
1.26 |
0.84 |
0.96 |
1.18 |
2.40 |
0.88 |
1.11 |
1.42 |
1.33 |
1.77 |
|||
500 |
Mean |
28.3 |
28.0 |
97.7 |
109.3 |
9.7 |
8.3 |
12.3 |
12.3 |
47.0 |
46.3 |
||
MF |
1.21 |
0.82 |
1.07 |
1.00 |
1.93 |
0.78 |
1.06 |
1.12 |
1.18 |
1.81 |
|||
158.1 |
Mean |
28.7 |
31.3 |
91.3 |
123.0 |
8.0 |
11.3 |
12.3 |
11.0 |
48.3 |
44.3 |
||
MF |
1.23 |
0.91 |
1.00 |
1.13 |
1.60 |
1.06 |
1.06 |
1.00 |
1.21 |
1.73 |
|||
50 |
Mean |
28.7 |
27.0 |
99.0 |
109.7 |
6.7 |
11.3 |
16.3 |
9.7 |
52.0 |
47.7 |
||
MF |
1.23 |
0.79 |
1.08 |
1.00 |
1.33 |
1.06 |
1.40 |
0.88 |
1.30 |
1.86 |
|||
15.81 |
Mean |
27.0 |
27.0 |
101.7 |
115.0 |
9.0 |
10.7 |
11.3 |
12.0 |
33.3 |
48.7 |
||
MF |
1.16 |
0.79 |
1.11 |
1.05 |
1.80 |
1.00 |
0.97 |
1.09 |
0.83 |
1.90 |
|||
5 |
Mean |
25.7 |
31.3 |
103.0 |
116.7 |
9.0 |
7.7 |
9.0 |
12.0 |
39.7 |
47.7 |
||
MF |
1.10 |
0.91 |
1.12 |
1.07 |
1.80 |
0.72 |
0.77 |
1.09 |
0.99 |
1.86 |
|||
NPD (4µg) |
Mean |
320.0 |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
||
MF |
13.71 |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
|||
2AA (2 µg) |
Mean |
─ |
2263.0 |
─ |
2828.0 |
─ |
250.7 |
─ |
253.7 |
─ |
─ |
||
MF |
─ |
65.91 |
─ |
25.87 |
─ |
23.50 |
─ |
23.06 |
─ |
─ |
|||
2AA (50 µg) |
Mean |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
293.0 |
||
MF |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
11.42 |
|||
SAZ (2 µg) |
Mean |
─ |
─ |
1700.7 |
─ |
656.3 |
─ |
─ |
─ |
─ |
─ |
||
MF |
─ |
─ |
17.41 |
─ |
109.39 |
─ |
─ |
─ |
─ |
─ |
|||
9AA (50 µg) |
Mean |
─ |
─ |
─ |
─ |
─ |
─ |
380.0 |
─ |
─ |
─ |
||
MF |
─ |
─ |
─ |
─ |
─ |
─ |
32.57 |
─ |
─ |
─ |
|||
MMS (2 µL) |
Mean |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
988.3 |
─ |
||
MF |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
21.03 |
─ |
|||
Table 2: Summary Table of Results of the Confirmatory Mutation Test
Concentrations (µg/plate) |
Mean values of revertants/ Mutation factor (MF) |
Tester strain |
||||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
||
Untreated control |
Mean |
34.3 |
40.0 |
94.3 |
124.0 |
7.7 |
14.7 |
7.7 |
8.7 |
36.7 |
44.7 |
|
MF |
0.99 |
1.15 |
0.95 |
1.08 |
0.61 |
1.29 |
0.82 |
0.96 |
0.99 |
0.92 |
|
|
DMSO control |
Mean |
34.7 |
34.7 |
99.7 |
115.3 |
12.7 |
11.3 |
9.3 |
9.0 |
37.0 |
48.3 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
|
Distilled water control |
Mean |
─ |
─ |
105.0 |
─ |
9.0 |
─ |
─ |
─ |
43.7 |
─ |
|
MF |
─ |
─ |
1.05 |
─ |
0.71 |
─ |
─ |
─ |
1.18 |
─ |
|
|
5000 |
Mean |
32.7 |
44.0 |
108.3 |
118.3 |
8.3 |
11.0 |
9.7 |
7.7 |
36.3 |
46.3 |
|
MF |
0.94 |
1.27 |
1.09 |
1.03 |
0.66 |
0.97 |
1.04 |
0.85 |
0.98 |
0.96 |
|
|
1581 |
Mean |
33.7 |
37.0 |
96.0 |
112.0 |
6.7 |
9.0 |
10.0 |
6.0 |
37.0 |
45.0 |
|
MF |
0.97 |
1.07 |
0.96 |
0.97 |
0.53 |
0.79 |
1.07 |
0.67 |
1.00 |
0.93 |
|
|
500 |
Mean |
33.7 |
37.3 |
105.3 |
107.7 |
7.0 |
10.7 |
6.7 |
13.3 |
38.0 |
45.0 |
|
MF |
0.97 |
1.08 |
1.06 |
0.93 |
0.55 |
0.94 |
0.71 |
1.48 |
1.03 |
0.93 |
|
|
158.1 |
Mean |
32.3 |
38.7 |
96.0 |
116.0 |
5.3 |
5.3 |
5.7 |
7.3 |
39.0 |
48.0 |
|
MF |
0.93 |
1.12 |
0.96 |
1.01 |
0.42 |
0.47 |
0.61 |
0.81 |
1.05 |
0.99 |
|
|
50 |
Mean |
31.7 |
32.3 |
110.7 |
119.7 |
7.7 |
7.0 |
7.0 |
7.0 |
33.0 |
45.0 |
|
MF |
0.91 |
0.93 |
1.11 |
1.04 |
0.61 |
0.62 |
0.75 |
0.78 |
0.89 |
0.93 |
|
|
15.81 |
Mean |
32.0 |
33.7 |
91.3 |
117.7 |
9.3 |
8.7 |
6.3 |
8.3 |
35.0 |
43.3 |
|
MF |
0.92 |
0.97 |
0.92 |
1.02 |
0.74 |
0.76 |
0.68 |
0.93 |
0.95 |
0.90 |
|
|
5 |
Mean |
27.7 |
34.0 |
100.7 |
118.3 |
8.0 |
11.7 |
6.3 |
7.0 |
31.7 |
43.0 |
|
MF |
0.80 |
0.98 |
1.01 |
1.03 |
0.63 |
1.03 |
0.68 |
0.78 |
0.86 |
0.89 |
|
|
NPD (4µg) |
Mean |
296.0 |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
|
MF |
8.54 |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
|
|
2AA (2 µg) |
Mean |
─ |
2263.3 |
─ |
2501.3 |
─ |
212.7 |
─ |
215.3 |
─ |
─ |
|
MF |
─ |
65.29 |
─ |
21.69 |
─ |
18.76 |
─ |
23.93 |
─ |
─ |
|
|
2AA (50 µg) |
Mean |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
274.7 |
|
MF |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
5.68 |
|
|
SAZ (2 µg) |
Mean |
─ |
─ |
1292.7 |
─ |
1117.3 |
─ |
─ |
─ |
─ |
─ |
|
MF |
─ |
─ |
12.31 |
─ |
124.15 |
─ |
─ |
─ |
─ |
─ |
|
|
9AA (50 µg) |
Mean |
─ |
─ |
─ |
─ |
─ |
─ |
457.3 |
─ |
─ |
─ |
|
MF |
─ |
─ |
─ |
─ |
─ |
─ |
49.00 |
─ |
─ |
─ |
|
|
MMS (2 µL) |
Mean |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
1187.3 |
─ |
|
MF |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
─ |
27.19 |
─ |
|
|
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation study in bacteria
The mutagenic potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, EPA OPPTS 870.5100, under GLP conditions. The method followed also conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The study was carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test, an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).
Based on the results of the Solubility Test, the test material was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.
In the Initial Mutation Test and Confirmatory Mutation Test, most of the observed revertant colony numbers were below the respective biological threshold value. The observed sporadic increases in the number of revertant colonies were examined for consistency; none of them were repeatable when comparing the performed main experiments. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between test material treated samples and solvent controls; Thus, they were considered as biological variability of the test system.
The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.
The findings of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test material had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.
In vitro micronucleus study
The potential of the test material to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/- was investigated in accordance with the standardised guideline OECD 487, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
After a preliminary toxicity test, the test material TD-115, dissolved in DMSO, was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. In the first experiment cells were treated without S9 mix for treatment times of 3 hours followed by a 24 hour recovery of for 24 hours with 0 hour recovery. In the first experiment with S9 mix, cells were treated for 3 hours followed by a 24 hour recovery. In the second experiment cells with S9 mix, cells were treated for 3 hours with a 24 hour recovery.
Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5 % Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analysed for three dose-levels of the test material, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
Since the test material was found to be freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 10 mM, according to the criteria specified in the international regulations.
The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.
In the main experiment without S9 mix, with treatment volume of 0.5 % (v/v) in culture medium, the selected dose-levels were 0.63, 1.25, 2.5, 5 and 10 mM for both 3- and 24-hour treatments. No precipitate was observed in the culture medium at the end of the treatment periods.
Following the 3- and 24-hour treatments, no noteworthy toxicity was induced at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 mM for both 3- and 24-hour treatments, the latter corresponding to the highest recommended dose-level.
Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analysed dose-levels in comparison to the vehicle controls. Moreover, none of the analysed dose-levels showed frequency of micronucleated cells of both replicate cultures above the vehicle control historical range. These results met the criteria of a negative response.
In the main experiments with S9 mix, with a treatment volume of 0.5 % (v/v) in culture medium, the selected dose-levels were 0.63, 1.25, 2.5, 5 and 10 mM for the first experiment and 1.25, 2.5, 5, 7.5 and 10 mM for the second experiment. No precipitate was observed in the culture medium at the end of the treatment periods.
No noteworthy toxicity was induced in either experiment, at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD.
The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 mM for the first experiment, the latter corresponding to the highest recommended dose-level, and 5, 7.5 and 10 mM for the second experiment, the latter corresponding to the highest recommended dose-level.
In the first experiment, a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test. Moreover, a statistically significant difference in comparison to the corresponding vehicle control was obtained at the highest tested dose-level of 10 mM (p < 0.05). However, only the frequency of micronucleated cells of one out of two replicate cultures was above the corresponding vehicle historical range.
Since this increase was not reproduced in the second experiment, performed under the same experimental conditions, despite using a narrower range of dose-levels, it was considered as non-biologically relevant.
Consequently, the overall results with S9 mix were considered as a negative response.
Under the experimental conditions of the study, the test material did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the absence or presence of a rat liver metabolising system.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
