Registration Dossier
Registration Dossier
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EC number: 215-410-7 | CAS number: 1325-87-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
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:
- 14 Jun - 14 Jul 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- 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:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Identification: Lumière Blue PTM 0154N
- Physical state/Appearance: Blue powder
- Batch: 981108
- Purity: Preparation containing ≥90% UVCB (treated as 100%)
- Expiry Date: 28 April 2022
- Storage Conditions: Room temperature in the dark
The vehicle control used was as follows:
Identity: Dimethyl sulphoxide
Batch number (purity): 1690734 (>99%), Expiry: 03/2022 (Experiment 1) 1710280 (>99%), Expiry: 05/2022 (Experiment 2)
Identity: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
Expiry date: 18 September 2017
Solvent: DMSO
Concentration: 2 μg/plate for WP2uvrA 3 μg/plate for TA100 5 μg/plate for TA1535
Identity: 9-Aminoacridine (9AA)
Purity: 99.9%
Expiry date: 01 October 2017
Solvent: DMSO
Concentration: 80 μg/plate for TA1537
Identity: 4-Nitroquinoline-1-oxide (4NQO)
Purity: 100%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 0.2 μg/plate for TA98
Identity: 2-Aminoanthracene (2AA)
Purity: 97.5%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 1 μg/plate for TA100 2 μg/plate for TA1535 and TA1537 10 μg/plate for WP2uvrA
Identity: Benzo(a)pyrene (BP)
Purity: 96%
Expiry date: 12 October 2017
Solvent: DMSO
Concentration: 5 μg/plate for TA98 - Target gene:
- histidine synthesis
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction
- Test concentrations with justification for top dose:
- 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO;
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-Aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Exposure duration: 48h
NUMBER OF REPLICATIONS: 3 - Evaluation criteria:
- The test material may be considered positive in this test system if it induces a reproducible, dose-related
and statistically significant increase in the revertant count in at least one strain of bacteria. - Statistics:
- Dunnetts Regression Analysis (* = p < 0.05)
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item was considered to be non-mutagenic under the conditions of this test.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- This study was conducted between 30 June 2017 and 02 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study is considered to be Reliability 1 as it has been conducted according to OECD Test Guideline 487 and in compliance with GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- Information as provided by the Sponsor.
Identification: Lumière Blue PTM 0154N
CAS Number : 1325-87-7
EC Number : 215-410-7
Physical state/Appearance: Purple powder
Batch : 981108
Purity : Preparation containing ≥90% UVCB* (treat as 100%)
Expiry Date : 28 April 2022
Storage Conditions : Room temperature in the dark
Intended use/Application : Dyestuff/pigment
No correction for purity was made. - Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability
- Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 Microsomal fractions
- Test concentrations with justification for top dose:
- The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL. The test item was insoluble in MEM at 25 and 50 mg/mL and in dimethyl sulphoxide at 500 mg/mL but was suspendable in MEM at both concentrations listed previously in solubility checks performed in house. The test item produced a better suspension at 25 mg/mL, so this was chosen as the maximum practical dose level. Prior to each experiment, the test item was accurately weighed, suspended in MEM and serial dilutions prepared.
- Vehicle / solvent:
- Prior to each experiment, the test item was accurately weighed, dissolved/ suspended in MEM and serial dilutions prepared
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- The vehicle control used was as follows: Identity: Eagle's minimal essential medium with HEPES buffer (MEM) Supplier: Sigma Aldrich Batch number (purity): RNBF9655 (Not Applicable)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Identity: Demecolcine (DEME-C) CAS No.: 477-30-5 Supplier: Sigma Aldrich Batch Number: SLBM6528V Purity: 100% Expiry Date: 01 May 2019 Solvent: Sterile distilled water Concentration: 0.2 µg/mL for 24-hour continuous exposure
- Details on test system and experimental conditions:
- Test System and Supporting Information
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test : male, aged 29 years
Main Experiment: male, aged 29 years
Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/NF S9 30/06/17 was used in this study.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
Experimental Design and Study Conduct
Test Item Preparation
The test item was considered to be a UVCB* and, therefore, the maximum recommended dose was initially set at 5000 µg/mL.
The test item was insoluble in MEM at 25 and 50 mg/mL and in dimethyl sulphoxide at 500 mg/mL but was suspendable in MEM at both concentrations listed previously in solubility checks performed in house. The test item produced a better suspension at 25 mg/mL, so this was chosen as the maximum practical dose level. Prior to each experiment, the test item was accurately weighed, suspended in MEM and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991). However, precipitate was observed at each formulation during the pH and osmolality check so the maximum dose level was limited to 80 µg/mL in the preliminary toxicity test in order to achieve non precipitating dose levels.
The pH and osmolality readings are presented in the following table:
Dose Concentration (µg/mL) 0 9.77 19.53 39.06 78.13 156.25 312.5 625 1250 2500
pH 7.24 7.27 7.26 7.27 7.27 7.30 7.29 7.29 7.29 7.24
Osmolality mOsm 273 275 - 277 - 274 - 278 - 285
- = Not performed for this dose concentration
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood
4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.
4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.
24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.
Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 0.313, 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.
Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 0.5, 1, 1.5, 2, 2.5, 3 and 4 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0.125, 0.25, 0.5, 1, 1.5, 2, 2.5 and 3 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 0.25, 0.5, 1, 1.5, 2, 2.5 and 3 µg/mL.
Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Assessments
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.
Coding
The slides were coded before analysis using a computerized random number generator.
Cytokinesis Block Proliferation Index (CBPI)
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
Scoring of Micronuclei
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter. - Evaluation criteria:
- Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9 mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed. - Statistics:
- Statistical Analysis
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible - Key result
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0.313 to 80 µg/mL. The maximum dose was based around the potential lowest precipitating dose level due to the nature of the test item.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 1.25 µg/mL, in the 4-hour exposure groups and at and above 10 µg/mL in the 24-hour continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2.5 µg/mL absence of metabolic activation (S9) and at and above 5 µg/mL in the presence of S9-mix. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on both toxicity and the lowest precipitating dose level because the onset of both coincided in the 4-hour exposure groups and was based on toxicity only in the 24-hour continuous exposure group. - Remarks on result:
- other: The test item was toxic to human lymphocytes but did not induce significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced ca 50% cytostasis and/or was the lowest precipitating dose level.
- Conclusions:
- The test item, Lumière Blue PTM 0154N, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non aneugenic to human lymphocytes in vitro.
- Executive summary:
This report describes the results of anin vitrostudy for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.
Methods
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate and toxicity, depending on the exposure group. The dose levels selected for the Main Test were as follows:
Group
Final concentration of test item
LumièreBlue PTM 0154N(µg/mL)4-hour without S9
0, 0.5, 1, 1.5, 2, 2.5, 3, 4
4-hour with S9 (2%)
0, 0.125, 0.25, 0.5, 1, 1.5, 2., 2.5, 3
24-hour without S9
0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3
Results
All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% cytostasis and/or was the lowest precipitating dose level.
Conclusion
The test item,LumièreBlue PTM 0154Nwas considered to be non-clastogenic and non‑aneugenic to human lymphocytesin vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 October to 07 December 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SOURCE SPONSOR AND BATCH NO 981108
- Expiration date of the lot/batch: 28 April 2022
- Purity test date: not specified
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
the test item was accurately weighed and formulated in MEM medium prior to serial dilutions being prepared. The test item was considered to be a UVCB* and, therefore, the maximum recommended dose was initially set at 5000 µg/mL and no correction for purity was applied to the formulations. However, the test item produced a better suspension at 2500 µg/mL, so this was chosen as the maximum practical dose level.
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: suitable
- Cell cycle length, doubling time or proliferation index: 12-16H
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: 12-16h
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM with 10% FBS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction
- Test concentrations with justification for top dose:
- In the preliminary test, as the test item was considered to be a UVCB, therefore, the maximum recommended dose was initially set at 5000 µg/mL and no correction for purity was applied to the formulations. However, the test item produced a better suspension at 2500 µg/mL, so this was chosen as the maximum practical dose level.
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.
The concentration range of test item was 0.004 to 0.5 µg/mL in the absence of metabolic activation, and 0.002 to 2 µg/mL in the presence of metabolic activation. - Vehicle / solvent:
- The test substance was formulated in MEM medium
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
The concentration range of test item was 0.004 to 0.5 µg/mL in the absence of metabolic activation, and 0.002 to 2 µg/mL in the presence of metabolic activation.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes. - Rationale for test conditions:
- Standard test conditions
- Evaluation criteria:
- i) The average absolute cloning efficiency of the Day7 negative controls should exceed 50%. All assays <50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.
iii) The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a statistically significant increase compared with the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended concentration (10 mM, 2 mg/mL or 2 µL/mL whichever is lower, or 5 mg/mL for a UVCB*), or include the lowest precipitating concentration. Treatments that reduce relative clonal survival to less than ten percent will not be scored for mutant frequency in the assay.
v) Adequate numbers of cells and concentrations are analyzable. Mutant frequencies are normally derived from sets of ten dishes/flasks for the mutant colony count and three dishes for cloning colony counts. To allow for contamination losses it is acceptable to score a minimum of eight mutant selection dishes and two cloning efficiency flasks.
vi) Five concentrations of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analyzed duplicate concentrations is considered necessary in order to accept a single assay for evaluation of the test item. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Referenceopen allclose all
Spontaneous Mutation Rates (Concurrent Negative Controls)
Experiment 1 (Plate Incorporation)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
91 |
11 |
35 |
24 |
11 |
|||||
114 |
(98) |
9 |
(9) |
35 |
(36) |
18 |
(20) |
14 |
(13) |
90 |
8 |
39 |
18 |
14 |
|||||
Experiment 2 (Pre-Incubation)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
96 |
28 |
21 |
16 |
9 |
|||||
77 |
(88) |
14 |
(22) |
24 |
(24) |
23 |
(17) |
12 |
(12) |
90 |
25 |
26 |
11 |
14 |
|||||
Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 27 June 2017 |
To: 30 June 2017 |
||||||||||||||||||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||||||||||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||||||||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
|||||||||||||||||||||||
Solvent Control (DMSO) |
107 81 108 |
(99) 15.3# |
10 9 11 |
(10) 1.0 |
34 31 32 |
(32) 1.5 |
19 21 25 |
(22) 3.1 |
18 18 19 |
(18) 0.6 |
|
|||||||||||||||||
1.5 μg |
92 111 103 |
(102) 9.5 |
11 11 11 |
(11) 0.0 |
35 40 40 |
(38) 2.9 |
20 15 20 |
(18) 2.9 |
10 19 20 |
(16) 5.5 |
|
|||||||||||||||||
5 μg |
94 92 91 |
(92) 1.5 |
9 8 10 |
(9) 1.0 |
45 34 30 |
(36) 7.8 |
21 22 24 |
(22) 1.5 |
19 24 20 |
(21) 2.6 |
|
|||||||||||||||||
15 μg |
104 122 114 |
(113) 9.0 |
11 10 10 |
(10) 0.6 |
35 36 33 |
(35) 1.5 |
23 24 23 |
(23) 0.6 |
10 18 13 |
(14) 4.0 |
|
|||||||||||||||||
50 μg |
118 98 103 |
(106) 10.4 |
11 11 10 |
(11) 0.6 |
32 41 39 |
(37) 4.7 |
24 28 19 |
(24) 4.5 |
19 17 18 |
(18) 1.0 |
|
|||||||||||||||||
150 μg |
108 105 102 |
(105) 3.0 |
10 12 7 |
(10) 2.5 |
42 35 34 |
(37) 4.4 |
25 13 23 |
(20) 6.4 |
19 19 22 |
(20) 1.7 |
|
|||||||||||||||||
500 μg |
113 104 113 |
(110) 5.2 |
10 12 9 |
(10) 1.5 |
30 32 32 |
(31) 1.2 |
22 25 28 |
(25) 3.0 |
20 17 23 |
(20) 3.0 |
|
|||||||||||||||||
1500 μg |
32 PSI 30 PSI 21 PSI |
(28) 5.9 |
0 PVI 0 PVI 0 PVI |
(0) 0.0 |
24 PSI 22 PSI 18 PSI |
(21) 3.1 |
17 PSI 15 PSI 11 PSI |
(14) 3.1 |
14 PSI 12 PSI 10 PSI |
(12) 2.0 |
|
|||||||||||||||||
5000 μg |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
8 PSI 7 PSI 9 PSI |
(8) 1.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
|
|||||||||||||||||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|
|||||||||||||||||||||
3 μg |
5 μg |
2 μg |
0.2 μg |
80 μg |
|
|||||||||||||||||||||||
797 742 887 |
(809) 73.2 |
419 511 605 |
(512) 93.0 |
210 192 228 |
(210) 18.0 |
182 235 220 |
(212) 27.3 |
345 306 376 |
(342) 35.1 |
|
||||||||||||||||||
Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 27 June 2017 |
To: 30 June 2017 |
|||||||||||||||||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||||||||||||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||||||||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||||||||||||||||||
Solvent Control (DMSO) |
90 93 100 |
(94) 5.1# |
11 11 10 |
(11) 0.6 |
33 34 34 |
(34) 0.6 |
31 18 24 |
(24) 6.5 |
12 10 14 |
(12) 2.0 |
|||||||||||||||||
1.5 μg |
112 95 106 |
(104) 8.6 |
9 8 12 |
(10) 2.1 |
42 41 13 |
(32) 16.5 |
25 26 21 |
(24) 2.6 |
13 13 13 |
(13) 0.0 |
|||||||||||||||||
5 μg |
106 101 97 |
(101) 4.5 |
14 15 14 |
(14) 0.6 |
33 34 34 |
(34) 0.6 |
26 21 21 |
(23) 2.9 |
18 9 10 |
(12) 4.9 |
|||||||||||||||||
15 μg |
102 107 107 |
(105) 2.9 |
12 8 11 |
(10) 2.1 |
40 39 41 |
(40) 1.0 |
21 24 17 |
(21) 3.5 |
14 10 13 |
(12) 2.1 |
|||||||||||||||||
50 μg |
106 92 97 |
(98) 7.1 |
12 8 6 |
(9) 3.1 |
38 34 33 |
(35) 2.6 |
18 21 19 |
(19) 1.5 |
10 9 8 |
(9) 1.0 |
|||||||||||||||||
150 μg |
107 103 94 |
(101) 6.7 |
11 12 12 |
(12) 0.6 |
34 36 44 |
(38) 5.3 |
19 24 18 |
(20) 3.2 |
10 12 6 |
(9) 3.1 |
|||||||||||||||||
500 μg |
100 105 106 |
(104) 3.2 |
10 13 9 |
(11) 2.1 |
43 38 34 |
(38) 4.5 |
12 8 12 |
(11) 2.3 |
12 18 12 |
(14) 3.5 |
|||||||||||||||||
1500 μg |
28 PSI 35 PSI 25 PSI |
(29) 5.1 |
6 PSI 4 PSI 4 PSI |
(5) 1.2 |
22 PSI 30 PSI 21 PSI |
(24) 4.9 |
4 PSI 7 PSI 11 PSI |
(7) 3.5 |
9 PSI 6 PSI 8 PSI |
(8) 1.5 |
|||||||||||||||||
5000 μg |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
8 PSI 6 PSI 9 PSI |
(8) 1.5 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
|||||||||||||||||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
|||||||||||||||||||||
1 μg |
2 μg |
10 μg |
5 μg |
2 μg |
|||||||||||||||||||||||
1770 2076 1950 |
(1932) 153.8 |
298 332 339 |
(323) 21.9 |
483 389 400 |
(424) 51.4 |
193 189 220 |
(201) 16.9 |
468 519 532 |
(506) 33.8 |
||||||||||||||||||
Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 11 July 2017 |
To: 14 July 2017 |
|||||||||||||||||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||||||||||||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||||||||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||||||||||||||||||
Solvent Control (DMSO) |
88 117 108 |
(104) 14.8# |
12 21 17 |
(17) 4.5 |
21 31 27 |
(26) 5.0 |
19 20 15 |
(18) 2.6 |
15 10 9 |
(11) 3.2 |
|||||||||||||||||
0.5 μg |
109 80 110 |
(100) 17.0 |
19 13 14 |
(15) 3.2 |
30 20 25 |
(25) 5.0 |
14 17 28 |
(20) 7.4 |
16 16 13 |
(15) 1.7 |
|||||||||||||||||
1.5 μg |
63 116 129 |
(103) 35.0 |
18 16 11 |
(15) 3.6 |
24 30 42 |
(32) 9.2 |
14 17 22 |
(18) 4.0 |
11 13 11 |
(12) 1.2 |
|||||||||||||||||
5 μg |
94 123 103 |
(107) 14.8 |
19 17 16 |
(17) 1.5 |
26 32 43 |
(34) 8.6 |
12 19 9 |
(13) 5.1 |
12 19 14 |
(15) 3.6 |
|||||||||||||||||
15 μg |
114 108 86 |
(103) 14.7 |
20 18 10 |
(16) 5.3 |
30 30 27 |
(29) 1.7 |
12 6 16 |
(11) 5.0 |
14 15 15 |
(15) 0.6 |
|||||||||||||||||
50 μg |
83 76 71 |
(77) 6.0 |
10 17 13 |
(13) 3.5 |
30 31 24 |
(28) 3.8 |
17 17 21 |
(18) 2.3 |
15 7 13 |
(12) 4.2 |
|||||||||||||||||
150 μg |
62 61 64 |
(62) 1.5 |
24 S 14 S 17 S |
(18) 5.1 |
16 30 29 |
(25) 7.8 |
13 12 14 |
(13) 1.0 |
6 S 12 S 16 S |
(11) 5.0 |
|||||||||||||||||
500 μg |
36 S 27 S 24 S |
(29) 6.2 |
0 V 0 V 0 V |
(0) 0.0 |
14 13 18 |
(15) 2.6 |
7 S 11 S 7 S |
(8) 2.3 |
0 V 0 V 0 V |
(0) 0.0 |
|||||||||||||||||
1500 μg |
0 PVI 0 PVI 0 PVI |
(0) 0.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
15 PSI 13 PSI 15 PSI |
(14) 1.2 |
11 PSI 7 PSI 9 PSI |
(9) 2.0 |
0 PTI 0 PTI 0 PTI |
(0) 0.0 |
|||||||||||||||||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||||||||||||||||||
3 μg |
5 μg |
2 μg |
0.2 μg |
80 μg |
|||||||||||||||||||||||
690 753 862 |
(768) 87.0 |
2001 2252 2346 |
(2200) 178.4 |
483 503 553 |
(513) 36.1 |
295 332 330 |
(319) 20.8 |
178 166 176 |
(173) 6.4 |
||||||||||||||||||
Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)
Test Period |
From: 11 July 2017 |
To: 14 July 2017 |
|||||||||||||||||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||||||||||||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||||||||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||||||||||||||||||
Solvent Control (DMSO) |
101 91 105 |
(99) 7.2# |
27 21 25 |
(24) 3.1 |
41 30 33 |
(35) 5.7 |
31 18 22 |
(24) 6.7 |
20 15 15 |
(17) 2.9 |
|||||||||||||||||
0.5 μg |
64 76 63 |
(68) 7.2 |
21 21 19 |
(20) 1.2 |
37 36 33 |
(35) 2.1 |
16 26 21 |
(21) 5.0 |
6 15 13 |
(11) 4.7 |
|||||||||||||||||
1.5 μg |
67 72 74 |
(71) 3.6 |
19 18 21 |
(19) 1.5 |
32 41 37 |
(37) 4.5 |
28 19 26 |
(24) 4.7 |
19 20 21 |
(20) 1.0 |
|||||||||||||||||
5 μg |
81 73 78 |
(77) 4.0 |
18 15 17 |
(17) 1.5 |
36 36 23 |
(32) 7.5 |
23 24 20 |
(22) 2.1 |
9 8 13 |
(10) 2.6 |
|||||||||||||||||
15 μg |
69 65 73 |
(69) 4.0 |
17 24 25 |
(22) 4.4 |
36 44 31 |
(37) 6.6 |
15 22 24 |
(20) 4.7 |
9 14 16 |
(13) 3.6 |
|||||||||||||||||
50 μg |
72 71 65 |
(69) 3.8 |
21 16 12 |
(16) 4.5 |
39 27 27 |
(31) 6.9 |
27 23 24 |
(25) 2.1 |
15 11 12 |
(13) 2.1 |
|||||||||||||||||
150 μg |
65 54 51 |
(57) 7.4 |
14 25 18 |
(19) 5.6 |
31 28 15 |
(25) 8.5 |
16 28 19 |
(21) 6.2 |
15 22 23 |
(20) 4.4 |
|||||||||||||||||
500 μg |
41 48 49 |
(46) 4.4 |
21 S 24 S 23 S |
(23) 1.5 |
42 39 29 |
(37) 6.8 |
29 17 22 |
(23) 6.0 |
9 14 18 |
(14) 4.5 |
|||||||||||||||||
1500 μg |
17 PSI 19 PSI 17 PSI |
(18) 1.2 |
0 PVI 0 PVI 0 PVI |
(0) 0.0 |
17 PSI 19 PSI 17 PSI |
(18) 1.2 |
15 PSI 8 PSI 12 PSI |
(12) 3.5 |
3 PSI 3 PSI 2 PSI |
(3) 0.6 |
|||||||||||||||||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
|||||||||||||||||||||
1 μg |
2 μg |
10 μg |
5 μg |
2 μg |
|||||||||||||||||||||||
671 645 650 |
(655) 13.8 |
271 252 283 |
(269) 15.6 |
110 118 111 |
(113) 4.4 |
141 125 131 |
(132) 8.1 |
388 307 393 |
(363) 48.3 |
||||||||||||||||||
Micronucleus Test – Main Experiment
The dose levels of the controls and the test item are given in the table below:
Group |
Final concentration of test item |
4-hour without S9 |
0*, 0.5, 1*, 1.5*, 2*, 2.5, 3, 4, MMC0.2* |
4-hour with S9 (2%) |
0*, 0.125, 0.25, 0.5, 1*, 1.5*, 2*, 2.5, 3, CP5* |
24-hour without S9 |
0*, 0.25, 0.5*, 1*, 1.5*, 2, 2.5, 3,DC0.075* |
* = Dose levels selected for analysis of micronucleus frequency in binucleate cells
MMC= Mitomycin C
CP = Cyclophosphamide
DC = Demecolcine
The qualitative assessment of the slides determined that the toxicity and precipitate was similar to that observed in the Preliminary Toxicity Test and there were binucleate cells suitable for scoring at the maximum dose level of test item for all three exposure groups.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 2 µg/mL, in the 4-hour exposure groups and at and above 2.5 µg/mL in the 24-hour continuous exposure group.
The CBPI data for the short exposure groups and for the 24-hour exposure group confirm the qualitative observations in that a dose-related inhibition of CBPI was observed in all three exposure groups.
In the 4-hour group in the absence of S9, 37%, 48% and 63% inhibition of cell proliferation was achieved at 1, 1.5 and 2 µg/mL, respectively, where 2µg/mL was also the onset of precipitate. Additionally, few binucleates were present at the maximum concentration tested (4 µg/mL). Therefore the maximum dose level selected for analysis of binucleate cells was 2 µg/mL.
In the presence of S9, a plateau of toxicity was noted where 50%, 46% and 60% inhibition of cell proliferation was achieved at 1, 1.5 and 2 µg/mL, respectively, with 2 µg/mL being the onset of precipitate, too. Therefore the maximum dose level selected for analysis of binucleate cells was 2 µg/mL.
In the 24-hour continuous exposure group, 18%, 38%, 62%, 72% and 83% inhibition of cell proliferation was achieved at 0.5, 1, 1.5, 2 and 2.5 µg/mL, respectively. 2.5 µg/mL was also the dose level which saw the onset of precipitate and there were few binucleates were present at the maximum concentration tested (3 µg/mL). Therefore the maximum dose level selected for analysis of binucleate cells was 1.5 µg/mL.
The micronucleus data indicated that the vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
The Ames test was negative (non-mutagenic), the Micronucleus test was negative (non-clastogenic and non-aneugenic) and the V79 HPRY Gene mutation assay was negiative (non-mutagenic) therefore classification not required.
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