Registration Dossier
Registration Dossier
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EC number: 282-029-0 | CAS number: 84082-82-6 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Salix alba, Salicaceae.
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A bacterial genetic toxicity study according to OECD Guideline 471 has been performed with White willow bark extract under GLP conditions. The test item had no mutagenic activity on the bacterial strains under the test conditions used in this study.
An in vitro genetic toxicity study in mammalian cells (HPRT) according to OECD guideline 476 has been performed with White willow bark extract under GLP conditions. The test item had no mutagenic acitivity on the test cells in this study.
An in vitro genetic toxicity study in mammalian cells (Micronucleus test) according to OECD guideline 487 has been performed with White willow bark extract under GLP conditions. The test item
did not induce any chromosome damage, or damage to the cell division apparatus in the cells in this study
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:
- 12.02.2019 - 28.06.2019
- 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:
- version from 21 July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- trp/his
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- plate incorporation method: 5000, 1581, 500, 158.1, 50, 15.81 and 0 μg/plate. 5000 µg/plate is the highest recommended dose according to the guideline.
pre-incubation method: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0 μg/plate. Doses higher than 500 showed excessive cytotoxicity. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF), Acetone, Ethanol and n-Hexane. The highest solubility was achieved using DMSO, at a concentration of 50 mg/mL.
- Justification for percentage of solvent in the final culture medium: highest soluble concentration - Untreated negative controls:
- yes
- Remarks:
- With and without S9
- Negative solvent / vehicle controls:
- yes
- Remarks:
- With and without S9
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With and without S9
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylene-diamine (NPD) 2-Aminoanthracene (2-AA)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1 plate-incorporation, 1 pre-incubation
METHOD OF TREATMENT/ EXPOSURE:
plate incorporation:
Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
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 item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 µL
vehicle or test item formulation (or reference controls) 100 (50)* µL
overnight culture of test strain 100 µL
phosphate buffer (pH 7.4) or S9 mix 500 µL
*Note: Treatment volume was 100 µL for test item formulations and its solvent; treatment volume was 50 µL for positive control substances and their solvents.
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 (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
preincubation :
Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 5.3.6.) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
- Rationale for test conditions:
- Guideline
- Evaluation criteria:
- The colony numbers on the untreated / negative (solvent) / positive control and test item 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 (mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test item and for the controls.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analyzable concentrations are presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a Negative Response:
The test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation - Statistics:
- No statistical testing was performed, as the results were unambigious according to the evaluation criteria.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- for the pre-incubation assay, concentrations above 500 µg/plate showed excessive toxicity. For the plate incorporation method, the highest recommended test item concentration according to the guideline was used.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
- Executive summary:
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD TG 471 and in compliance with GLP.
The experiments were 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/beta-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-Incubation Method) and an Assay 3 (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/mL. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA98 and TA100 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate and in the Assay 3 were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
In the Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in all tested bacterial strains, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains with and without metabolic activation was repeated in Assay 3 using a modified concentration range. Results of the invalid experiment were not reported; however, all data will be kept and archived in the raw data binder.
No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effects of the test item (reduced / slightly reduced background lawn development) was observed in the Assay 3 in all tested bacterial strains with and without metabolic activation at higher concentrations.
In the Assay 1 and Assay 3, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.
The mean values of revertant colonies of the negative (vehicle/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 analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the bacterial strains under the test conditions used in this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13.02.2019 - 17.07.2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes) (migrated information)
- Version / remarks:
- Version from 29 July 2016
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Version from 31.05.2008
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT
- Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- 5000, 2000, 1000, 500, 250, 125 and 62.5 µg/mL for all types of tests. Top dose is the maximum recommended dose for a UVCB.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: DMSO and distilled water were considered as solvents. The test item had better solubility in distilled water.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- vehicle for positive controls was DMSO, solvent controls with DMSO were tested as well.
- Positive control substance:
- 9,10-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 5 h exposure with S9: 2,
5 h exposure without S9: 1,
24 h exposure without S9: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2x10^6 per 100 mm dish
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h (with and without S9), 24 h (without S9)
- Harvest time after the end of treatment: 5 h experiments: 19 h, 24h experiments: directly after end of treatment
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days, with subcultivation
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 20 µg/mL
6-thioguanine (abbreviation: 6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 µg/mL)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
viability assessment: 3 dishes with 200 cells/dish. mutation frequency assessment: 5 dishes with 4x10^5 cells each. Manual counting of colonies.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival by cloning efficiency - Rationale for test conditions:
- OECD TG 476
- Evaluation criteria:
- The test item was considered to be mutagenic in this assay if the following criteria are met:
- The assay is valid.
- The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
- Increase of the mutant frequency is reproducible.
- There is a dose-response relationship.
- The historical control range is considered when deciding if the result is positive.
Results which only partially meet the criteria are judged on a case-by-case basis, taking into consideration historical control data. - Statistics:
- The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The homogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- No mutagenic effect of the test material was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.
- Executive summary:
An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to evaluate the potential of the tes tmaterial to cause gene mutation (according to Guideline OECD 476, performed under GLP). Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix).
Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1
5-hour treatment in the presence of S9-mix:
5000, 2000, 1000, 500, 250, 125 and 62.5 µg/mL
5-hour treatment in the absence of S9-mix:
5000, 2000, 1000, 500, 250, 125 and 62.5 µg/mL
Assay 2
5-hour treatment in the presence of S9-mix:
5000, 2000, 1000, 500, 250, 125 and 62.5 µg/mL
24-hour treatment in the absence of S9-mix:
5000, 2000, 1000, 500, 250, 125 and 62.5 µg/mL
In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of
6-thioguanine as a selective agent) was determined.
In Assay 1, insolubility (precipitate/ minimal amount of precipitate) was detected at higher concentrations, in Assay 2 no insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In Assay 1 and 2, in the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. In Assay 1 with metabolic activation, statistically significant increase of mutant frequencies (at p<0.05 level or p<0.01 level) was observed at 5000, 2000 and 125 µg/mL concentration (see Table 5), although the observed values were in harmony with all historical control data. Furthermore, the observed mutant frequency values (11.6 x 10-6 at 5000 µg/mL; 7.8 x 10^-6 at 2000 µg/mL and 12.2 x 10^-6 at 125 µg/mL) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10^-6). There was no evidence for a dose-response relationship due to the treatment (a trend analysis showed no effect of treatment).
In Assay 2 with metabolic activation no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence for a dose-response relationship due to the treatment (a trend analysis showed no effect of treatment). These experiments are considered to be negative.
In Assay 1, in the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence for a dose-response relationship due to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.
In Assay 2, in the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed, thus an evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no evidence for a dose-response relationship due to the treatment (a trend analysis showed no effect of treatment). This experiment is considered to be negative.
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with all historical control data in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. Seven evaluated concentrations of the test substance were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested in accordance with the recommendation of the OECD No. 476 guideline in each test). The overall study was considered to be valid.
In conclusion, no mutagenic effect of the test material was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 4.3.2019 - July 2019
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- version from 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Additional strain / cell type characteristics:
- other: TK+/-
- Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- In a preliminary test, dose levels included the highest recommended dose for UVCBs:
10, 100, 500, 1000, 2500 and 5000 µg/mL.
At the end of the treatment periods, a precipitate was observed in the culture medium at dose levels >= 1000 µg/mL, therefore the highest concentration was not included in the main cytogenetic expriment.
In the main experiments, with a treatment volume of 5% (v/v) in culture medium, the dose levels were:
125, 250, 500, 750, 1000, 1500 and 2000 µg/mL for the 3-hour treatment without S9 mix,
125, 250, 500, 1000, 1500 and 2000 µg/mL for the 24-hour treatment without S9 mix and for the 3 hour treatment with S9 mix.
The dose level of 1000 µg/mL corresponded to the lowest dose level with minimal precipitate visible in the culture medium at the end of the treatment periods. Therefore the dose levels selected for micronucleus analysis were:
500, 750 and 1000 µg/mL for the 3-hour treatment without S9 mix,
250, 500 and 1000 µg/mL for the 24-hour treatment without S9 mix and for the 3-hour treatment with S9 mix. - Vehicle / solvent:
- According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was saline solution (0.9% NaCl).
The test item was dissolved in the vehicle at the following concentrations:
100 mg/mL for the preliminary cytotoxicity test and 40 mg/mL for the main experiment.
In the final treatment solution (cell culture medium) solubility decreased and precipitation was observed at concentrations >= 1000 µg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine (migrated information)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
In a single experiment, up to seven dose-levels of the test item were tested in duplicate (two cultures/dose level), both with and without metabolic activation, using treatment duration as follows:
Without S9 mix 3 h treatment + 24 h recovery, 24 h treatment + 0 h recovery.
With S9 mix 3 h treatment + 24 h recovery
METHOD OF TREATMENT/ EXPOSURE:
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 items, with or without S9 mix, at 37°C in a humidified atmosphere of 5% CO2/95% air.
TREATMENT AND HARVEST SCHEDULE:
At the end of the treatment period (for schedule see above), 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, at 37°C in a humidified atmosphere of 5% CO2/95% air.
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
No cytokinesis block was used. 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 5°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), four 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.
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 cytogenetic experiment (with or without S9 mix), micronuclei were analyzed for 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 item 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 by microscopic evaluation, on the basis of the recommendations of Miller et al. (1995) Environ. Mol. Mutagen. 26, 240-247, according to the following criteria:
micronuclei should be located within the cytoplasma of the cell,
micronuclei should be clearly surrounded by a nuclear membrane,
micronuclei should be round or oval in shape,
the micronucleus area should be less than one-third of the area of the main nucleus,
micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
micronuclei should have similar staining intensity to that of the main nuclei,
micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
only mononucleated cells with a number of micronuclei <= 5 should be scored to exclude apoptosis and nuclear fragmentation.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
For each culture, the Population Doubling (PD) 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 to the starting count, divided by the log of 2. 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%). - Rationale for test conditions:
- OECD Guideline 487
- Evaluation criteria:
- Acceptance criteria
Each main cytogenetic experiment was considered valid if the following criteria were met:
adequate number of cells and concentrations were analyzable,
the mean PD of the vehicle control had to be ≥ 1 (indicating that cells have undergone mitosis),
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 and this frequency should be greater than or equal to the minimum value of the historical data.
Evaluation criteria
The biological relevance of the results was always taken into account when evaluating results.
Evaluation of a positive response: a test item 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.
Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met. - Statistics:
- For each condition of the 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 chi-squared 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 frequencies of micronucleated cells and 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, but tested up to precipitating concentrations
- Remarks:
- cytotoxicity was observed at concentrations where precipitation was observed (>= 1500 µg/mL)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- All the acceptance criteria were met, the study was therefore considered to be valid.
The highest dose level selected for the main cytogenetic experiment was based on the level of precipitate, according to the criteria specified in the international regulations.
With a treatment volume of 5% (v/v) in culture medium, the dose levels selected were:
125, 250, 500, 750, 1000, 1500 and 2000 µg/mL for the 3-hour treatment without S9 mix,
125, 250, 500, 1000, 1500 and 2000 µg/mL for the 24-hour treatment without S9 mix and for the 3 hour treatment with S9 mix.
At the end of the treatment periods, a precipitate was observed in the culture medium at dose levels ≥ 1000 µg/mL.
Cytotoxicity
Following the 3- and the 24-hour treatments without S9 mix, a severe cytotoxicity was induced at dose levels ≥ 1500 µg/mL.
Following the 3 hour treatment with S9 mix, no cytotoxicity was induced at any of the tested dose levels.
Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
500, 750 and 1000 µg/mL for the 3-hour treatment without S9 mix,
250, 500 and 1000 µg/mL for the 24-hour treatment without S9 mix and for the 3-hour treatment with S9 mix.
The dose level of 1000 µg/mL corresponded to the lowest dose level with minimal precipitate visible in the culture medium at the end of the treatment period.
Following the 3- and 24-hour treatments with and without S9 mix, neither statistically significant, nor dose related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control. Moreover, none of the analyzed dose levels showed frequencies of micronucleated cells of each replicate culture above the corresponding vehicle control historical range.
Therefore, the results met the criteria of a negative response.
Detailed result tables are attached - Conclusions:
- Under the experimental conditions of the study, the test item 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 presence or absence of a rat liver metabolizing system.
- Executive summary:
The objective of this study was to evaluate the potential of the test item, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-. The test was performed according to OECD Guideline 487 under GLP conditions.
Method
After a preliminary cytotoxicity test, the test item dissolved in saline solution (i.e. 0.9% NaCl), was tested in a single experiment, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:
Without S9 mix 3 h treatment + 24 h recovery; 24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h 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 four 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 analyzed for 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).
Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
Results
All the acceptance criteria were met, the study was therefore considered to be valid.
The highest dose level selected for the main cytogenetic experiment was based on the level of precipitate, according to the criteria specified in the international regulations.
With a treatment volume of 5% (v/v) in culture medium, the dose levels selected were:
125, 250, 500, 750, 1000, 1500 and 2000 µg/mL for the 3-hour treatment without S9 mix,
125, 250, 500, 1000, 1500 and 2000 µg/mL for the 24-hour treatment without S9 mix and for the 3 hour treatment with S9 mix.
At the end of the treatment periods, a precipitate was observed in the culture medium at dose levels ≥ 1000 µg/mL.
Cytotoxicity
Following the 3- and the 24-hour treatments without S9 mix, a severe cytotoxicity was induced at dose levels ≥ 1500 µg/mL.
Following the 3 hour treatment with S9 mix, no cytotoxicity was induced at any of the tested dose levels.
Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
500, 750 and 1000 µg/mL for the 3-hour treatment without S9 mix,
250, 500 and 1000 µg/mL for the 24-hour treatment without S9 mix and for the 3-hour treatment with S9 mix.
The dose level of 1000 µg/mL corresponded to the lowest dose level with minimal precipitate visible in the culture medium at the end of the treatment period.
Following the 3- and 24-hour treatments with and without S9 mix, neither statistically significant, nor dose related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control. Moreover, none of the analyzed dose levels showed frequencies of micronucleated cells of each replicate culture above the corresponding vehicle control historical range.
Therefore, the results met the criteria of a negative response.
Conclusion
Under the experimental conditions of the study, the test item 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 presence or absence of a rat liver metabolizing system.
Referenceopen allclose all
Summary Table of the plate incorporation assay (Assay 1)
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
16.3 |
20.7 |
98.7 |
100.0 |
13.3 |
12.7 |
9.3 |
7.7 |
47.0 |
49.3 |
|
MF |
0.96 |
1.02 |
1.05 |
1.02 |
1.21 |
1.23 |
1.00 |
0.82 |
1.02 |
0.98 |
||
DMSO control 50µL |
Mean |
17.7 |
20.3 |
-- |
97.7 |
-- |
13.0 |
6.7 |
8.0 |
-- |
48.7 |
|
MF |
1.04 |
1.00 |
-- |
1.00 |
-- |
1.26 |
0.71 |
0.86 |
-- |
0.97 |
||
Distilled water control |
Mean |
-- |
-- |
97.0 |
-- |
11.7 |
-- |
-- |
-- |
47.3 |
-- |
|
MF |
-- |
-- |
1.03 |
-- |
1.06 |
-- |
-- |
-- |
1.03 |
-- |
||
DMSO 100µL control |
Mean |
17.0 |
20.3 |
94.3 |
98.0 |
11.0 |
10.3 |
9.3 |
9.3 |
46.0 |
50.3 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
19.3 |
22.3 |
83.7 |
101.7 |
17.0 |
14.7 |
8.0 |
8.3 |
44.0 |
53.3 |
|
MF |
1.14 |
1.10 |
0.89 |
1.04 |
1.55 |
1.42 |
0.86 |
0.89 |
0.96 |
1.06 |
||
1581 |
Mean |
18.7 |
24.3 |
91.7 |
105.3 |
11.3 |
14.0 |
6.7 |
9.3 |
46.0 |
50.7 |
|
MF |
1.10 |
1.20 |
0.97 |
1.07 |
1.03 |
1.35 |
0.71 |
1.00 |
1.00 |
1.01 |
||
500 |
Mean |
16.3 |
23.7 |
97.3 |
104.3 |
11.0 |
12.3 |
7.3 |
8.0 |
46.3 |
49.7 |
|
MF |
0.96 |
1.16 |
1.03 |
1.06 |
1.00 |
1.19 |
0.79 |
0.86 |
1.01 |
0.99 |
||
158.1 |
Mean |
17.3 |
23.3 |
93.0 |
100.3 |
11.3 |
9.7 |
7.3 |
9.0 |
46.3 |
50.0 |
|
MF |
1.02 |
1.15 |
0.99 |
1.02 |
1.03 |
0.94 |
0.79 |
0.96 |
1.01 |
0.99 |
||
50 |
Mean |
17.0 |
19.0 |
89.7 |
98.7 |
10.7 |
10.7 |
7.3 |
9.0 |
47.3 |
49.0 |
|
MF |
1.00 |
0.93 |
0.95 |
1.01 |
0.97 |
1.03 |
0.79 |
0.96 |
1.03 |
0.97 |
||
15.81 |
Mean |
15.7 |
19.7 |
99.0 |
96.7 |
12.3 |
11.7 |
8.3 |
9.0 |
46.7 |
50.0 |
|
MF |
0.92 |
0.97 |
1.05 |
0.99 |
1.12 |
1.13 |
0.89 |
0.96 |
1.01 |
0.99 |
||
NPD (4mg) |
Mean |
405.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
22.94 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2409.3 |
-- |
2488.0 |
-- |
204.0 |
-- |
302.0 |
-- |
-- |
|
MF |
-- |
118.49 |
-- |
25.47 |
-- |
15.69 |
-- |
37.75 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
249.3 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.12 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1036.0 |
-- |
1129.3 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
10.68 |
-- |
96.80 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
409.3 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
61.40 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1098.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
23.21 |
-- |
||
Summary Table of the pre-incubation assay (Assay 3)
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
|||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||
Untreated control |
Mean |
18.7 |
20.7 |
95.0 |
93.3 |
12.3 |
14.0 |
7.7 |
10.7 |
46.7 |
50.0 |
||
MF |
0.98 |
1.00 |
1.21 |
1.05 |
0.90 |
1.05 |
1.00 |
1.33 |
1.06 |
1.01 |
|||
DMSO control 50µL |
Mean |
17.7 |
20.0 |
-- |
91.0 |
-- |
14.3 |
7.3 |
7.7 |
-- |
49.0 |
||
MF |
0.93 |
0.97 |
-- |
1.02 |
-- |
1.08 |
0.96 |
0.96 |
-- |
0.99 |
|||
Distilled water control |
Mean |
-- |
-- |
86.7 |
-- |
14.0 |
-- |
-- |
-- |
44.7 |
-- |
||
MF |
-- |
-- |
1.11 |
-- |
1.02 |
-- |
-- |
-- |
1.02 |
-- |
|||
DMSO 100µL control |
Mean |
19.0 |
20.7 |
78.3 |
89.0 |
13.7 |
13.3 |
7.7 |
8.0 |
44.0 |
49.3 |
||
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|||
500 |
Mean |
13.3 |
21.7 |
70.7 |
98.3 |
10.7 |
13.0 |
8.3 |
10.3 |
40.0 |
49.3 |
||
MF |
0.70 |
1.05 |
0.90 |
1.10 |
0.78 |
0.98 |
1.09 |
1.29 |
0.91 |
1.00 |
|||
158.1 |
Mean |
15.3 |
21.0 |
83.7 |
97.7 |
11.7 |
11.3 |
5.3 |
10.0 |
41.0 |
49.3 |
||
MF |
0.81 |
1.02 |
1.07 |
1.10 |
0.85 |
0.85 |
0.70 |
1.25 |
0.93 |
1.00 |
|||
50 |
Mean |
20.0 |
21.7 |
88.3 |
91.7 |
11.7 |
12.0 |
9.3 |
8.3 |
42.7 |
49.3 |
||
MF |
1.05 |
1.05 |
1.13 |
1.03 |
0.85 |
0.90 |
1.22 |
1.04 |
0.97 |
1.00 |
|||
15.81 |
Mean |
18.7 |
19.7 |
86.3 |
92.0 |
9.0 |
12.7 |
8.7 |
9.7 |
44.3 |
51.0 |
||
MF |
0.98 |
0.95 |
1.10 |
1.03 |
0.66 |
0.95 |
1.13 |
1.21 |
1.01 |
1.03 |
|||
5 |
Mean |
19.3 |
26.3 |
88.3 |
89.3 |
12.0 |
11.7 |
8.3 |
10.7 |
40.0 |
50.0 |
||
MF |
1.02 |
1.27 |
1.13 |
1.00 |
0.88 |
0.88 |
1.09 |
1.33 |
0.91 |
1.01 |
|||
1.581 |
Mean |
16.7 |
23.3 |
84.3 |
94.0 |
12.3 |
12.3 |
8.3 |
8.0 |
40.3 |
49.7 |
||
MF |
0.88 |
1.13 |
1.08 |
1.06 |
0.90 |
0.93 |
1.09 |
1.00 |
0.92 |
1.01 |
|||
0.5 |
Mean |
16.0 |
23.7 |
89.7 |
92.7 |
12.0 |
10.7 |
8.0 |
11.3 |
40.7 |
48.0 |
||
MF |
0.84 |
1.15 |
1.14 |
1.04 |
0.88 |
0.80 |
1.04 |
1.42 |
0.92 |
0.97 |
|||
NPD (4mg) |
Mean |
406.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
MF |
23.02 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|||
2AA (2mg) |
Mean |
-- |
2430.7 |
-- |
2460.0 |
-- |
219.3 |
-- |
208.0 |
-- |
-- |
||
MF |
-- |
121.53 |
-- |
27.03 |
-- |
15.30 |
-- |
27.13 |
-- |
-- |
|||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
258.0 |
||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.27 |
|||
SAZ (2mg) |
Mean |
-- |
-- |
1065.3 |
-- |
1234.7 |
-- |
-- |
-- |
-- |
-- |
||
MF |
-- |
-- |
12.29 |
-- |
88.19 |
-- |
-- |
-- |
-- |
-- |
|||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
410.7 |
-- |
-- |
-- |
||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
56.00 |
-- |
-- |
-- |
|||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1109.3 |
-- |
||
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
24.84 |
-- |
|||
Summarized Survival Results of Assay 1
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total |
Cloning |
Relative |
+ |
5 |
A1 |
5000 µg/mL |
1171 |
0.976 |
101 |
2000 µg/mL |
1224 |
1.020 |
105 |
|||
1000 µg/mL |
1211 |
1.009 |
104 |
|||
500 µg/mL |
1257 |
1.048 |
108 |
|||
250 µg/mL |
1166 |
0.972 |
100 |
|||
125 µg/mL |
1086 |
0.905 |
93 |
|||
62.5 µg/mL |
1191 |
0.993 |
102 |
|||
Negative control |
1165 |
0.971 |
100 |
|||
Negative control for DMBA (1% (v/v) DMSO) |
1150 |
0.958 |
99 |
|||
Untreated control |
1134 |
0.945 |
97 |
|||
Positive control (DMBA) |
76 |
0.063 |
7 |
|||
- |
5 |
A1 |
5000 µg/mL |
1073 |
0.894 |
92 |
2000 µg/mL |
1293 |
1.078 |
111 |
|||
1000 µg/mL |
1144 |
0.953 |
98 |
|||
500 µg/mL |
1209 |
1.008 |
104 |
|||
250 µg/mL |
1226 |
1.022 |
106 |
|||
125 µg/mL |
1215 |
1.013 |
105 |
|||
62.5 µg/mL |
1194 |
0.995 |
103 |
|||
Negative control |
1162 |
0.968 |
100 |
|||
Negative control for EMS (1% (v/v) DMSO) |
1264 |
1.053 |
109 |
|||
Untreated control |
1214 |
1.012 |
104 |
|||
Positive control (EMS) |
968 |
0.807 |
83 |
A1 = Assay 1
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
- = in the absence of S9-mix EMS = Ethylmethanesulfonate, 0.4 µL/mL
Negative (vehicle) control = 2.5% (v/v) Distilled water
DMSO = Dimethyl sulfoxide
Summarized Survival Results of Assay 2
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total |
Cloning |
Relative |
+ |
5 |
A2 |
5000 µg/mL |
1231 |
1.026 |
113 |
2000 µg/mL |
1130 |
0.942 |
104 |
|||
1000 µg/mL |
1154 |
0.962 |
106 |
|||
500 µg/mL |
1204 |
1.003 |
111 |
|||
250 µg/mL |
1082 |
0.902 |
99 |
|||
125 µg/mL |
1103 |
0.919 |
101 |
|||
62.5 µg/mL |
1081 |
0.901 |
99 |
|||
Negative control |
1088 |
0.907 |
100 |
|||
Negative control for DMBA (1% (v/v) DMSO) |
1036 |
0.863 |
95 |
|||
Untreated control |
1061 |
0.884 |
98 |
|||
Positive control (DMBA) |
79 |
0.066 |
7 |
|||
- |
24 |
A2 |
5000 µg/mL |
1016 |
0.847 |
83 |
2000 µg/mL |
1100 |
0.917 |
90 |
|||
1000 µg/mL |
1196 |
0.997 |
98 |
|||
500 µg/mL |
1187 |
0.989 |
98 |
|||
250 µg/mL |
1164 |
0.970 |
96 |
|||
125 µg/mL |
1132 |
0.943 |
93 |
|||
62.5 µg/mL |
1187 |
0.989 |
98 |
|||
Negative control |
1217 |
1.014 |
100 |
|||
Negative control for EMS (1% (v/v) DMSO) |
1211 |
1.009 |
100 |
|||
Untreated control |
1247 |
1.039 |
102 |
|||
Positive control (EMS) |
368 |
0.307 |
30 |
A2 = Assay 2
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
- = in the absence of S9-mix EMS = Ethylmethanesulfonate, 0.4 µL/mL
Negative (vehicle) control = 2.5% (v/v) Distilled water
DMSO = Dimethyl sulfoxide
Summarized Viability Results of Assay 1
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Cloning Efficiency |
+ |
5 |
A1 |
5000 µg/mL |
1176 |
0.980 |
2000 µg/mL |
1083 |
0.903 |
|||
1000 µg/mL |
1109 |
0.924 |
|||
500 µg/mL |
1117 |
0.931 |
|||
250 µg/mL |
1067 |
0.889 |
|||
125 µg/mL |
1110 |
0.925 |
|||
62.5 µg/mL |
1168 |
0.973 |
|||
Negative control |
1121 |
0.934 |
|||
Negative control for DMBA (1% (v/v) DMSO) |
1144 |
0.953 |
|||
Untreated control |
1096 |
0.913 |
|||
Positive control (DMBA) |
1111 |
0.926 |
|||
- |
5 |
A1 |
5000 µg/mL |
1129 |
0.941 |
2000 µg/mL |
1215 |
1.013 |
|||
1000 µg/mL |
1109 |
0.924 |
|||
500 µg/mL |
1116 |
0.930 |
|||
250 µg/mL |
1158 |
0.965 |
|||
125 µg/mL |
1116 |
0.930 |
|||
62.5 µg/mL |
1067 |
0.889 |
|||
Negative control |
1119 |
0.933 |
|||
Negative control for EMS (1% (v/v) DMSO) |
1089 |
0.908 |
|||
Untreated control |
1124 |
0.937 |
|||
Positive control (EMS) |
770 |
0.642 |
A1 = Assay 1
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
- = in the absence of S9-mix EMS = Ethylmethanesulfonate, 0.4 µL/mL
Negative (vehicle) control = 2.5% (v/v) Distilled water
DMSO = Dimethyl sulfoxide
Summarized Viability Results of Assay 2
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Cloning Efficiency |
+ |
5 |
A2 |
5000 µg/mL |
1098 |
0.915 |
2000 µg/mL |
1060 |
0.883 |
|||
1000 µg/mL |
1104 |
0.920 |
|||
500 µg/mL |
1080 |
0.900 |
|||
250 µg/mL |
1065 |
0.888 |
|||
125 µg/mL |
1135 |
0.946 |
|||
62.5 µg/mL |
1121 |
0.934 |
|||
Negative control |
1086 |
0.905 |
|||
Negative control for DMBA (1% (v/v) DMSO) |
1073 |
0.894 |
|||
Untreated control |
1105 |
0.921 |
|||
Positive control (DMBA) |
1009 |
0.841 |
|||
- |
24 |
A2 |
5000 µg/mL |
954 |
0.795 |
2000 µg/mL |
1081 |
0.901 |
|||
1000 µg/mL |
1071 |
0.893 |
|||
500 µg/mL |
1161 |
0.968 |
|||
250 µg/mL |
1167 |
0.973 |
|||
125 µg/mL |
1183 |
0.986 |
|||
62.5 µg/mL |
1147 |
0.956 |
|||
Negative control |
1127 |
0.939 |
|||
Negative control for EMS (1% (v/v) DMSO) |
1105 |
0.921 |
|||
Untreated control |
1166 |
0.972 |
|||
Positive control (EMS) |
359 |
0.299 |
A2 = Assay 2
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
- = in the absence of S9-mix EMS = Ethylmethanesulfonate, 0.4 µL/mL
Negative (vehicle) control = 2.5% (v/v) Distilled water
DMSO = Dimethyl sulfoxide
Summarized Mutagenicity Results of Assay 1
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Mutant |
+ |
5 |
A1 |
5000 µg/mL |
46 |
11.6** |
2000 µg/mL |
28 |
7.8* |
|||
1000 µg/mL |
20 |
5.4 |
|||
500 µg/mL |
26 |
6.9 |
|||
250 µg/mL |
31 |
8.7 |
|||
125 µg/mL |
45 |
12.2** |
|||
62.5 µg/mL |
16 |
4.1 |
|||
Negative control |
24 |
6.5 |
|||
Negative control for DMBA (1% (v/v) DMSO) |
23 |
6.0 |
|||
Untreated control |
24 |
6.6 |
|||
Positive control (DMBA) |
1927 |
519.3** |
|||
- |
5 |
A1 |
5000 µg/mL |
26 |
6.9 |
2000 µg/mL |
33 |
8.0 |
|||
1000 µg/mL |
19 |
5.1 |
|||
500 µg/mL |
20 |
5.4 |
|||
250 µg/mL |
17 |
4.4 |
|||
125 µg/mL |
24 |
6.6 |
|||
62.5 µg/mL |
24 |
6.8 |
|||
Negative control |
19 |
5.1 |
|||
Negative control for EMS (1% (v/v) DMSO) |
19 |
5.2 |
|||
Untreated control |
23 |
6.1 |
|||
Positive control (EMS) |
1360 |
530.5** |
* = Statistically significant increase (at p< 0.05) compared to the relevant vehicle control
** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control
In the statistical analysis, negative trends were not considered significant.
A1 = Assay 1
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
- = in the absence of S9-mix EMS = Ethylmethanesulfonate, 0.4 µL/mL
Negative (vehicle) control = 2.5% (v/v) Distilled water
DMSO = Dimethyl sulfoxide
Mutant frequencies refer to 106clonable cells.
Summarized Mutagenicity Results of Assay 2
S9 mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Total number of colonies |
Mutant |
+ |
5 |
A2 |
5000 µg/mL |
23 |
6.3 |
2000 µg/mL |
28 |
8.0 |
|||
1000 µg/mL |
20 |
5.4 |
|||
500 µg/mL |
23 |
6.4 |
|||
250 µg/mL |
20 |
5.6 |
|||
125 µg/mL |
23 |
6.1 |
|||
62.5 µg/mL |
18 |
4.8 |
|||
Negative control |
22 |
6.1 |
|||
Negative control for DMBA (1% (v/v) DMSO) |
30 |
8.4 |
|||
Untreated control |
25 |
6.8 |
|||
Positive control (DMBA) |
1910 |
567.3** |
|||
- |
24 |
A2 |
5000 µg/mL |
19 |
6.0 |
2000 µg/mL |
24 |
6.7 |
|||
1000 µg/mL |
18 |
5.0 |
|||
500 µg/mL |
33 |
8.5 |
|||
250 µg/mL |
17 |
4.4 |
|||
125 µg/mL |
22 |
5.6 |
|||
62.5 µg/mL |
25 |
6.5 |
|||
Negative control |
23 |
6.1 |
|||
Negative control for EMS (1% (v/v) DMSO) |
17 |
4.6 |
|||
Untreated control |
33 |
8.5 |
|||
Positive control (EMS) |
817 |
680.9** |
** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control
In the statistical analysis, negative trends were not considered significant.
A2 = Assay 2
+ = in the presence of S9-mix DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL
- = in the absence of S9-mix EMS = Ethylmethanesulfonate, 0.4 µL/mL
Negative (vehicle) control = 2.5% (v/v) Distilled water
DMSO = Dimethyl sulfoxide
Mutant frequencies refer to 106clonable cells.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
No in vivo study on genetic toxicity is available for White willow bark extract
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
WWBE was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, performed according to OECD Guideline 471 under GLP conditions with strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/beta-naphthoflavone-induced rats.
In the pre-incubation assay, cytotoxic effects of the test item (reduced / slightly reduced background lawn development) were observed in all tested bacterial strains with and without metabolic activation at higher concentrations.
For both the plate-incorporation assay as well as the pre-incubation assay, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.
The validity criteria of the study were met. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
WWBE was further tested in an in vitro mammalian cell assay in CHO K1 Chinese hamster ovary cells at the hprt locus with and without metabolic activation (S9 mix). The test was performed according to OECD Guideline 476 under GLP conditions. Three tests with 5 h exposure were performed, two in the presence of S9 and one without metabolic activation. One test with 24 h exposure without S9 was performed. After 5 h and 24 h exposure without S9 -Mix, no change in mutant frequencies were observed. After 5h exposure in the presence of S9-Mix, mutant frequencies were increased at some test concentrations, but without a dose-response relationship and within the historical range for negative control samples. The validity criteria of the study were met.
In conclusion, no mutagenic effect of White willow bark extract was observed either in the presence or absence of a metabolic activation system under the conditions of the HPRT assay.
WWBE was also tested in an in vitro Micronucleus assay using the mouse cell line L5178Y TK+/-. The test was performed according to OECD Guideline 487 under GLP conditions.
Following the 3- and 24-hour treatments with and without S9 mix, neither statistically significant, nor dose related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control. Moreover, none of the analyzed dose levels showed frequencies of micronucleated cells of each replicate culture above the corresponding vehicle control historical range. All the acceptance criteria were met, the study was therefore considered to be valid.
Justification for classification or non-classification
Based on the result of the genetic toxicity studies on bacteria (OECD 471) and mammalian cells (OECD 476) and a micronucleus assay on mammalian cells (OECD 487), white willow bark extract is not classified for genetic toxicity according to Regulation (EC) No. 1272/2008.
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